BLOOD PRESSURE REGULATION BY THE ENDOCANNABINOID SYSTEM IN
CONDITIONS ASSOCIATED WITH HYPERTENSION
By
CHRISTOPHER LEE SCHAICH
A Dissertation Submitted to the Graduate Faculty of
WAKE FOREST GRADUATE SCHOOL OF ARTS AND SCIENCES
In Partial Fulfillment of the Requirements for the Degree of
DOCTOR OF PHILOSOPHY
Integrative Physiology and Pharmacology
May 2014
Winston-Salem, North Carolina
Approved By:
Debra I. Diz, Ph.D., Advisor
Wayne E. Pratt, Ph.D., Chair
Allyn C. Howlett, Ph.D.
Hossam A. Shaltout, Ph.D.
Brian F. Thomas, Ph.D.
ACKNOWLEDGEMENTS
I must begin by thanking my wonderful advisor Dr. Debra Diz, for her personal and
professional guidance throughout my graduate career. I have grown tremendously as a
scientist under Dr. Diz and will carry her influence with me the rest of my life. I also
thank all members of the Diz Lab, in particular Ellen Tommasi for her assistance with
experiments as well as her availability to commiserate during more frustrating days. I
give my deepest appreciation to all faculty and students of the Hypertension & Vascular
Research Center, and to Katie Atkins, Pam Dean and Robert Lanning of the Hypertension
and Molecular Core Laboratories for their assistance with my project.
I must individually thank all members of my dissertation committee, as each has
uniquely contributed to my project.
Dr. Wayne Pratt, for teaching me how to do
intracranial injections in his lab during my first semester; Dr. Allyn Howlett, for drawing
me into endocannabinoid research and inspiring much of my research; Dr. Hossam
Shaltout, for his support since my first day in lab and later for his assistance with
measuring conscious baroreflex function; and Dr. Brian Thomas, for his commitment to
institutional collaboration and, with Dr. Megan Grabenauer, the mass spectrometry
measurements that are a crucial part of this dissertation. Thank you all for your guidance,
support and direction throughout my project.
Finally, I would like to thank my family and friends who have given me immense
support and love during my time as a graduate student. I am so proud and lucky to call
Winston-Salem my home because of all the amazing people I have met here who quickly
accepted me into the community. And to my parents Rosemarie and Bob, brother Danny,
ii
Aunt Linda, Uncle Paul, cousins Michael and David, and grandparents Jimmy and Maria:
this is all for you. Thank you for always challenging me to do better.
iii
TABLE OF CONTENTS
LIST OF FIGURES AND TABLES................................................................................ vi
LIST OF ABBREVIATIONS........................................................................................... ix
ABSTRACT....................................................................................................................... x
CHAPTER ONE:
INTRODUCTION
1. The Autonomic Nervous System....................................................................... 3
2. The Classical and Brain RAS............................................................................ 9
3. The Endocannabinoid System.......................................................................... 17
4. The Use of Transgenic and Aged Animals to Investigate the Role of the
Endocannabinoid System in Blood Pressure Regulation..................................... 32
5. Rationale and Aims.......................................................................................... 41
6. References........................................................................................................ 45
CHAPTER TWO:
MEDULLARY ENDOCANNABINOIDS CONTRIBUTE TO
THE DIFFERENTIAL RESTING BAROREFLEX SENSITIVITY
IN RATS WITH ALTERED BRAIN RENIN-ANGIOTENSIN
SYSTEM EXPRESSION............................................................ 69
In revision to Hypertension
CHAPTER THREE:
ALTERATIONS IN THE MEDULLARY ENDOCANNABINOID
SYSTEM CONTRIBUTE TO AGE-RELATED IMPAIRMENT
OF BAROREFLEX SENSITIVITY.......................................... 107
Submitted to Hypertension
iv
CHAPTER FOUR:
ACUTE AND CHRONIC SYSTEMIC CB1 CANNABINOID
RECEPTOR BLOCKADE IMPROVES BLOOD PRESSURE
REGULATION AND METABOLIC PROFILE IN
HYPERTENSIVE (mRen2)27 RATS....................................... 137
In revision to American Journal of Physiology –
Regulatory, Integrative and Comparative Physiology
CHAPTER FIVE:
SUMMARY AND CONCLUSIONS
1. Summary of Results....................................................................................... 175
2. Endocannabinoid Modulation of Baroreflex Sensitivity during Hypertension
and Aging........................................................................................................... 178
3. Mechanisms for Endocannabinoid Modulation of Baroreflex Sensitivity.... 183
4. CB1 Receptor Blockade and Blood Pressure Regulation in Ang II-Dependent
Hypertension...................................................................................................... 189
5. Chronic CB1 Receptor Blockade in Metabolic Syndrome............................. 191
6. Interactions between the Endocannabinoid System and the Brain RAS....... 195
7. Viability of CB1 Receptor Blockade as a Therapeutic Strategy.................... 200
8. General Limitations of Studies...................................................................... 204
9. Concluding Remarks...................................................................................... 209
10. References.................................................................................................... 212
SCHOLASTIC VITA.................................................................................................... 225
v
LIST OF FIGURES AND TABLES
CHAPTER ONE
Figure 1-1:
Effect of CB1 Receptor Agonist in NTS of Sprague-Dawley
Rats.............................................................................................. 42
Figure 1-2:
Effect of CB1 Receptor Agonist in NTS of (mRen2)27 Rats...... 43
CHAPTER TWO
Figure 2-1:
Histological Analysis of Microinjection Sites............................. 75
Table 2-1:
Values of MAP, HR and BRS in Response to NTS Microinjection
of Vehicle..................................................................................... 76
Figure 2-2:
Baseline BRS for Control of HR in SD, (mRen2)27 and
ASrAOGEN Rats......................................................................... 79
Figure 2-3:
Baseline BRS for Control of HR in SD, (mRen2)27 and
ASrAOGEN Rat Treatment Groups............................................ 80
Figure 2-4:
Effect of NTS Microinjection of CB1 Receptor Antagonist
SR141716A on BRS for Control of HR Evoked by PE in
SD Rats........................................................................................ 81
Figure 2-5:
Effect of NTS Microinjection of SR141716A on BRS for Control
of HR Evoked by PE in (mRen2)27 Rats.................................... 82
Figure 2-6:
Effect of NTS Microinjection of SR141716A on BRS for Control
of HR Evoked by PE in ASrAOGEN Rats.................................. 83
Table 2-2:
Values of MAP and HR in Response to NTS Microinjection of
SR141716A.................................................................................. 85
Figure 2-7:
Baseline MAP and HR of SD, (mRen2)27 and
ASrAOGEN Rats......................................................................... 86
vi
Figure 2-8:
MAP Responsiveness in SD, (mRen2)27 and ASrAOGEN Rats at
Baseline and in Response to NTS Microinjection of
SR141716A.................................................................................. 87
Figure 2-9:
2-AG and Anandamide Content in Dorsal Medulla of SD,
(mRen2)27 and ASrAOGEN Rats................................................ 88
Figure 2-10:
CB1, CB2 and CRIP1a mRNA in Dorsal Medulla of SD,
(mRen2)27 and ASrAOGEN Rats............................................... 89
Figure 2-11:
Effects of Chemoreflex Activation on MAP and HR Induced by
Intravenous Phenylbiguanide in SD, (mRen2)27 and ASrAOGEN
Rats.............................................................................................. 94
CHAPTER THREE
Table 3-1:
Mean Age and Values of MAP and HR in Response to NTS
Microinjection of SR141716A................................................... 116
Figure 3-1:
Baseline BRS for Control of HR in Response to Increases in AP in
Aged SD Rats............................................................................. 116
Figure 3-2:
Effect of NTS Microinjection of SR141716A (36 pmol) in Aged
SD Rats with Normal or Impaired Baseline BRS for Control of
HR.............................................................................................. 117
Figure 3-3:
MAP Responsiveness to Intravenous PE in Aged SD Rats with
Normal or Impaired Baseline BRS, Before and After NTS
Microinjection of SR141716A................................................... 118
Figure 3-4:
2-AG and Anandamide Content in the Dorsal Medulla of 15-, 25and 70-Week-Old SD Rats......................................................... 119
Figure 3-5:
Expression of CB1, CB2 and CRIP1a mRNA in the Dorsal Medulla
of 15- and 70-Week-Old SD Rats.............................................. 120
Figure 3-6:
Effects of Chemoreflex Activation on MAP and HR Induced by
Intravenous PBG in Aged SD Rats............................................ 127
vii
CHAPTER FOUR
Figure 4-1:
Design and Timeline of Experiments........................................ 144
Table 4-1:
Acute Systemic SR141716A or Vehicle + Overnight FR in
(mRen2)27 and SD Rats............................................................ 148
Figure 4-2:
Acute Systemic SR141716A or Vehicle + Overnight FR in
(mRen2)27 and SD Rats............................................................ 149
Figure 4-3:
Effect of Chronic Systemic SR141716A on Body Weight and Fat
Mass in (mRen2)27 Rats............................................................ 150
Figure 4-4:
Effect of Chronic Systemic SR141716A on Food and Water
Intake, and Urine Volume in (mRen2)27 Rats.......................... 152
Table 4-2:
Chronic Systemic SR141716A or Vehicle in (mRen2)27 Rats.. 153
Figure 4-5:
Effect of Chronic Systemic SR141716A on Blood Pressure and
HR in (mRen2)27 Rats............................................................... 154
Figure 4-6:
Effect of Chronic Systemic SR141716A on Leptin, Insulin and
Fasting Blood Glucose in (mRen2)27 Rats............................... 155
Figure 4-7:
Effect of Chronic Systemic SR141716A on Conscious Baroreflex
Function, HRV and BPV........................................................... 156
viii
LIST OF ABBREVIATIONS
2-AG…………...
ACE……………
Ang I…...............
Ang II…..............
Ang-(1-7)………
AT1…………….
AT2…………….
AV3V…………..
cAMP…………..
Ca-NAT………...
CNS…………….
CVLM………….
DAG……………
DAGL………….
dmnX…………..
ERK……………
FAAH………….
GABA………….
MAGL………….
MAPK………….
NAE…………....
NAPE…………..
NAPE-PLD…….
NMDA…………
NTS………….....
PKA……………
PI3K……………
PLC………….....
PTX…………….
RAS…………….
RVLM………….
SFO…………….
SHR…………....
THC……………
2-arachidonoylglycerol
angiotensin converting enzyme
angiotensin I
angiotensin II
angiotensin-(1-7)
Ang II type I receptor
Ang II type II receptor
anteroventral third ventricle area
cyclic adenosine monophosphate
Ca2+-dependent N-acyltransferase
central nervous system
caudal ventrolateral medulla
diacylglycerol
DAG lipase
dorsal motor nucleus of the vagus
extracellular signal-regulated kinase
fatty acid amide hydrolase
γ-aminobutyric acid
monoacylglycerol lipase
mitogen-activated protein kinase
N-acylethanolamine
N-acylphosphotidylethanolamine
NAPE phospholipase D
N-methyl-D-aspartate
solitary tract nucleus
protein kinase A
phosphoinositide-3 kinase
phospholipase C
pertussis toxin
renin-angiotensin system
rostral ventrolateral medulla
subfornical organ
spontaneously hypertensive rat
Δ9-tetrahydrocannabinol
ix
ABSTRACT
Christopher Lee Schaich
BLOOD PRESSURE REGULATION BY THE ENDOCANNABINOID SYSTEM IN
CONDITIONS ASSOCIATED WITH HYPERTENSION
Dissertation under the Direction of
Debra I. Diz, Ph.D., Professor
Hypertension is associated with various conditions including obesity, type II diabetes and
aging. Despite decades of research, the mechanisms involved in the development of
hypertension are still poorly understood. Emerging evidence suggests hypertension is
mediated by an imbalance in autonomic nervous system activity. Baroreflex sensitivity
for control of heart rate, an important index of vagus nerve function as well as general
morbidity and mortality, is often impaired in conditions associated with hypertension.
Restoration of baroreflex function is now recognized as a therapeutic strategy for
prevention of cardiovascular diseases. Therefore, it is crucial to identify key factors that
regulate brainstem regions controlling autonomic outflow in conditions associated with
baroreflex dysfunction.
Mounting evidence suggests a functional role for the endocannabinoid system in
the impairment of baroreflex sensitivity during hypertension, which may be related to
interactions with the renin-angiotensin system (RAS). Our goal is to determine if the
brain endocannabinoid system contributes to baroreflex dysfunction and elevated blood
pressure in conditions associated with altered RAS activity.
x
Specifically, we determine whether CB1 cannabinoid receptor tone is present at
the level of the brainstem solitary tract nucleus (NTS) in transgenic rats with high or low
brain RAS activity. Data from these studies demonstrate that blockade of NTS CB1
receptors in anesthetized hypertensive (mRen2)27 rats with high brain RAS expression
dose-dependently improves the blunted baroreflex sensitivity in this strain. However,
blockade of CB1 receptors in the NTS of anesthetized ASrAOGEN rats with low brain
RAS expression has opposite effects, dose-dependently reducing the enhanced baroreflex
sensitivity in this strain.
In contrast, there is no effect of NTS CB1 blockade in
normotensive Sprague-Dawley rats.
Biochemical data indicate higher medullary
production of the endocannabinoid 2-arachidonoylglycerol (2-AG) in (mRen2)27 rats
compared to lower 2-AG production in ASrAOGEN rats. Together, these observations
suggest a differential role for the endocannabinoid system in the modulation of baroreflex
sensitivity in conditions associated with altered brain RAS activity.
We then determine whether altered endocannabinoid tone is present at the level of
the NTS of aged Sprague-Dawley rats.
Microinjection studies in older animals
demonstrate that blockade of NTS CB1 receptors normalizes baroreflex sensitivity only in
animals that exhibit age-related impaired baseline baroreflex function.
However,
biochemical data indicate significantly higher 2-AG content in the dorsal medulla of aged
relative to young adult Sprague-Dawley rats, and also show altered expression of CB1
and CB2 receptor mRNA expression with age in this region. These studies suggest a role
for the endocannabinoid system in the impairment of baroreflex sensitivity associated
with age.
xi
Finally, we evaluate the hemodynamic and metabolic effects of systemic
administration of a CB1 receptor antagonist in (mRen2)27 rats, which feature increased
body weight and insulin resistance in addition to angiotensin II-dependent hypertension.
Results from these studies indicate that systemic blockade of CB1 receptors significantly
reduces blood pressure in this strain while having no effect on blood pressure in
normotensive control rats.
The blood pressure-lowering effect is associated with
significant improvement in indices of conscious autonomic outflow following chronic
treatment with the CB1 antagonist. Furthermore, animals receiving chronic systemic CB1
blockade exhibit an improved metabolic profile compared to animals treated with the
vehicle.
In summary, the present studies suggest that upregulation of the endocannabinoid
system contributes to the maintenance of hypertension, impaired baroreflex sensitivity,
and symptoms of metabolic syndrome associated with overactivity of the RAS. These
results are consistent with an interaction between the endocannabinoid system and RAS
that may promote the development of hypertension associated with aging and obesity.
Therefore, we conclude that targeting the endocannabinoid system for blockade may
confer therapeutic benefits during hypertension associated with an activated RAS.
xii
CHAPTER ONE
INTRODUCTION
Cardiovascular disease remains the leading cause of morbidity and mortality for both
men and women in the United States. Hypertension, diagnosed when systolic blood
pressure is greater than 140 mmHg and/or diastolic blood pressure is greater than 90
mmHg, is the single most important risk factor for the development of cardiovascular
disease1 and currently affects 1 in 3 U.S. adults.2 The incidence of hypertension rises
substantially with increases in body mass index,3 a unit of relative body fat calculated
from the weight and height of patients. For example, values of body mass index greater
than 40 kg/m2, a level classified as “extreme obesity” and representing 6-8% of U.S.
adults,4 are associated with a 7-fold increase in the incidence of hypertension compared
to the lean population.5 In addition, the incidence of hypertension rises with age, in part
due to the increased prevalence of obesity in aged populations.6 However, while there
are several effective drug therapies to treat patients with hypertension, many
antihypertensive medications have reduced efficacy and produce undesirable side effects
in aged and obese patients.7-9
Therefore, research into the relationships among
hypertension, aging and obesity is needed to gain better insights into therapeutic
treatments for these conditions and their related pathologies.
Despite decades of research, the mechanisms involved in the development of
hypertension are not fully understood. While several interacting factors likely contribute
to its onset and progression, emerging evidence suggests that an imbalance in autonomic
1
nervous system activity is integral to the development of hypertension associated with
aging, obesity and type II diabetes.10,
11
The autonomic imbalance observed in these
conditions is linked to alterations in various biochemical factors associated with the onset
of hypertension, including the metabolic hormones insulin12 and leptin,13,
catecholamine signaling in the brain,15,
16
14
and components of the RAS.17 The studies
contained in this volume present evidence that altered central endocannabinoid system
signaling is an additional mechanism that may contribute to autonomic imbalance in
conditions associated with hypertension, providing novel insights for the preservation of
autonomic function and blood pressure in hypertension, aging and obesity.
2
1. The Autonomic Nervous System
The autonomic nervous system is a part of the peripheral nervous system that controls
cardiovascular, digestive and respiratory homeostasis, as well as a multitude of other
functions associated with the viscera.18 The autonomic nervous system is divided into
two opposing branches, the sympathetic and parasympathetic nervous systems, which
regulate visceral functioning through reflex arcs between sensory and motor neurons in
the periphery and autonomic brainstem nuclei. The cardiovascular system is regulated by
a balance of output from both the sympathetic and parasympathetic nervous systems,
which must work together to maintain cardiovascular homeostasis and correct short-term
disturbances in the circulation.
Activation of the sympathetic nervous system is
associated with energy mobilization in response to environmental stress. For control of
the cardiovascular system, the sympathetic nervous system mediates increases in cardiac
output and vasoconstriction, primarily through noradrenergic neurotransmission acting at
β1 receptors in the heart and α1 receptors in arterioles to elevate cardiac output, heart rate
and thus blood pressure.18
Opposing the sympathetic nervous system, the parasympathetic nervous system
regulates vegetative and restorative functions. During cardiovascular regulation, the
parasympathetic nervous system exerts tonic inhibitory control over the heart to lower
heart rate to decrease cardiac output and blood pressure.19 These actions are mediated
primarily by cholinergic neurotransmission through efferent neurons of the vagus nerve
that originate in the brain and innervate nodal pacemaker cells in the heart.
Parasympathetic activity is often assessed by activity of the vagus nerve,19 which is
considered a marker of general morbidity and mortality.20
3
Heart rate variability, a
measure of vagal tone to the heart, and baroreflex sensitivity for control of heart rate in
response to increases in arterial pressure are often reported as indices of vagus nerve
function.19
The Arterial Baroreflex
Arterial blood pressure is regulated by autonomic feedback control systems that work in
concert over varying time frames to determine the prevailing level of pressure and its
variability.21 The arterial baroreflex is the primary control system for regulation of blood
pressure in the short-term time frame.
The effectors of the baroreflex are stretch-
sensitive baroreceptors concentrated in the walls of the aortic arch, carotid sinus, and
auricles of the heart and vena cava that are capable of detecting peripheral changes in
blood pressure or volume.
Although baroreflexes also exist for control over the
vasculature, renal function and fluid intake,22 the studies presented in this volume focus
on the baroreflex for control of heart rate. The baroreflex for control of heart rate is a
negative feedback loop in which changes in blood pressure produce immediate, transient
compensatory changes in heart rate to return blood pressure back to the operating point of
the system.
High pressure baroreceptors located in the aortic arch and carotid sinus are
innervated by the vagus and glossopharyngeal nerves, respectively, and initiate
autonomic feedback loops when activated by increases in pressure.
Baroreceptor
stimulation produces excitation along vagal and glossopharyngeal afferents which make
their first synapse in the NTS of the dorsomedial medulla. The NTS is a major site of
autonomic integration and receives input from chemosensitive neurons23 and the gut,24 in
addition to baroreceptor neurons. Lesions to this region eliminate baroreflex responses,25
4
illustrating the importance of the NTS to the integrity of the baroreflex arc.25 The NTS
integrates information from afferent baroreceptor fibers and sends input to second order
brainstem nuclei involved in the modulation of sympathetic and parasympathetic nervous
system activity.25
To effectively minimize variability in blood pressure during its activation, the
baroreflex concomitantly modulates outflowing sympathetic and parasympathetic
pathways,19, 26 illustrated in the figure below. Excitation of baroreceptor afferents causes
the NTS to transmit excitatory glutamatergic input to the caudal ventrolateral medulla
(CVLM), a vasodepressor region that inhibits sympathetic activity, and preganglionic
neurons in the dorsal motor nucleus of the vagus (dmnX) and nucleus ambiguus.
Concurrent stimulation of these brain sites represents the diverging sympathetic and
parasympathetic branches of the baroreflex arc.
Neurons of the CVLM transmit
inhibitory γ-aminobutyric acid (GABA) input to the rostral ventrolateral medulla
(RVLM), a pressor region containing pre-motor neurons that project to sympathetic
preganglionic neurons along the interomediolateral cell column of the spinal cord.
Inhibition of the RVLM by the CVLM thus reduces sympathetic outflow to the heart and
vasculature to lower heart rate and blood pressure. Concurrently, activated neurons of
the dmnX are joined by efferent neurons from the nucleus ambiguus to reduce blood
pressure by lowering heart rate and cardiac output. Alternatively, decreases in blood
pressure silence baroreceptor activity, leading to disinhibition of the RVLM and
inactivation of the dmnX and nucleus ambiguus, to elevate blood pressure back to
temporal operating levels.
5
Adapted from Arnold (2009).27 Glu refers to glutamate; BP to blood pressure; HR to
heart rate; + indicates excitatory input via glutamate; - indicates inhibitory input via
GABA.
Two distinct components of the baroreflex that are independently regulated are
the set-point and the sensitivity (occasionally called the baroreflex gain). 28, 29 The setpoint represents the pressure at which the baroreflex operates, typically near the level of
resting blood pressure, but will change or reset itself rapidly following sustained changes
in cardiac output, as during exercise.30 Baroreflex sensitivity represents the effectiveness
for correction of heart rate or sympathetic nerve activity. Baroreflex sensitivity for
control of heart rate influences the ability to maintain arterial pressure within a narrow
range by increasing heart rate variability. It is also an important index of vagus nerve
function and is often impaired in conditions associated with hypertension including
aging, obesity and type II diabetes.31, 32
Autonomic Imbalance
Activity of the sympathetic and parasympathetic nervous systems is normally in dynamic
balance and rapidly adjusts based on changing environmental or physiological
6
circumstances.18 However, an imbalance in the autonomic nervous system, typically in
which sympathetic nervous system predominates due to increased activity or decreased
parasympathetic activity, is associated with various pathological conditions and may be
an important mechanism promoting the development of age- and obesity-related
hypertension.11, 33, 34
Independent of hypertension, aging and obesity are associated with increased
activation of the sympathetic nervous system in both humans and animals.11,
34, 35
Although increased sympathetic activation is hypothesized to be a beneficial
compensatory mechanism in obesity to increase resting energy expenditure,36 elevations
in sympathetic outflow are also associated with the development of hypertension.18,
37
The observation that pharmacological blockade of adrenergic receptors yields greater
decreases in blood pressure in obese relative to lean hypertensive patients confirms the
importance of sympathetic activation to obesity-related hypertension.38
Reduced vagus nerve-mediated parasympathetic function also contributes to the
pathogenesis of hypertension, independently of increased sympathetic activity.19 In fact,
all established risk factors for cardiovascular disease are associated with impaired indices
of parasympathetic function, including heart rate variability and baroreflex sensitivity for
control of heart rate.19, 32 Reduced baroreflex sensitivity is associated with cardiovascular
diseases including stroke, atherosclerosis and end organ damage, and is believed to
precede and contribute to the onset of hypertension.19, 32, 39 For this reason, identification
of factors that contribute to reductions in baroreflex sensitivity will be critical for
understanding the development of hypertension associated with various conditions.
7
To date, the majority of research focuses on the contribution of the RAS in
maintaining baroreflex function and other indices of autonomic activity, but additional
factors are being discovered. For instance, a role for leptin in the modulation of central
nervous system (CNS) activity and impairment of baroreflex sensitivity has recently
emerged,40,
41
following previous demonstrations of insulin-mediated sympathetic
activation and baroreflex impairment.42, 43 There are a few studies evaluating the role of
the endocannabinoid system in sympathetic baroreflexes.
Cannabinoid receptor
activation at brain sites involved in autonomic regulation elicit cardiodepressor effects
and prolong baroreflex-evoked sympathetic nerve inhibition,44, 45 implicating the ability
to modulate parasympathetic function. Curiously, the latter effect is absent in an animal
model of chronic hypertension,46 suggesting differential modulation of autonomic
reflexes in hypertensive versus normal states.
However, the contribution of the
endocannabinoid system to modulation of parasympathetic activity in either the normal
or hypertensive state has yet to be considered.
8
2. The Classical and Brain RAS
The RAS is the predominant biological
signaling system for regulation of blood
pressure and volume homeostasis in
vertebrates.
The description of the
circulating, or classical, RAS is of an
endocrine cascade in which active peptide hormones, synthesized from enzymes and
precursors secreted from various systemic tissues, bind to receptors in heart, vasculature
and kidney to exert their actions.47
As illustrated above, angiotensinogen, an inert
glycoprotein derived from the liver, is the precursor of the RAS and is cleaved by the
kidney-derived enzyme renin to form the inactive decapeptide angiotensin (Ang) I. Ang I
may then be processed by lung-derived angiotensin converting enzyme (ACE) into the
octapeptide hormone Ang II. Ang I is also cleaved by various endopeptidases including
neprilysin to form the heptapeptide hormone Ang-(1-7).48 Alternatively, Ang II may be
cleaved by an ACE homologue, ACE2, to form Ang-(1-7).
Ang II is the primary effector of the RAS and exerts physiological actions by
binding to G protein-coupled Ang II type I (AT1) or type II (AT2) receptors. The
ubiquitously expressed AT1 receptor mediates the majority of pressor actions attributed
to Ang II, which include vasoconstriction, renal sodium and water reabsorption, adrenal
aldosterone and epinephrine release, production of reactive oxygen species, cell
proliferation and fibrosis, angiogenesis and suppression of baroreflex function.47 ACE
inhibitors or AT1 receptor antagonists abolish these actions by preventing the formation
or receptor-mediated actions of Ang II, respectively. As a receptor coupled to Gq/11,
9
activation of AT1 is typically associated with inhibition of adenylyl cyclase and
activation of phospholipase C (PLC) to initiate membrane-derived phosphoinositide
turnover and intracellular calcium mobilization.49 Most of the actions mediated by the
AT2 receptor are depressor effects in opposition to the AT1 receptor, and include
vasodilation and inhibition of cellular proliferation.50
The hormone Ang-(1-7) opposes the pressor actions of Ang II. Primary actions of
Ang-(1-7) include vasodilation, antiproliferation and facilitation of baroreflex sensitivity
for control of heart rate.47 However, Ang II and Ang-(1-7) can both elicit similar pressor
or depressor actions in certain brain nuclei51 and stimulate vasopressin release through
actions in the hypothalamus.52 The actions of Ang-(1-7) are blocked by [D-Ala7]-Ang(1-7), but not AT1 or AT2 receptor antagonists, indicating a unique receptor through
which Ang-(1-7) exerts its effects. In 2003, Santos and colleagues identified Ang-(1-7)
as an endogenous ligand of the G protein-coupled mas receptor,53 which stimulates
prostaglandin and nitric oxide release in target cells,54 among other actions.
The classical RAS plays an essential role in blood pressure regulation through
actions on the cardiovascular system and various brain nuclei lacking a blood-brain
barrier, called circumventricular organs. Historically, it was presumed that the endocrine
RAS was physically separated from the brain by the blood-brain barrier.55 The discovery
of the brain circumventricular organs, which have direct access to cerebrospinal fluid and
contain fenestrated capillaries to allow access to large molecular weight molecules,
challenged this presumption and sparked debate and research into the actions of
circulating Ang II inside the brain.56 Even before their discovery it was already known
that central infusion of Ang II increases blood pressure,57 suggesting the presence of
10
specific receptors for Ang II in the brain. Further observations of normal or even low
plasma renin and ACE activity in animal models of hypertension, coupled with clinical
data showing beneficial effects of ACE inhibitors and AT1 blockers in hypertensive
patients with low plasma Ang II levels, suggests the existence of an extra-circulatory
RAS.56 All major components of the RAS are now known to coexist in various tissues,
including the heart, adipose tissue, vasculature and brain.58 Thus, both the classical and
brain RAS participate in blood pressure regulation by exerting actions on specific brain
nuclei.
The Brain RAS and Blood Pressure Regulation
As reviewed in detail by others,56, 59 Ang II and Ang-(1-7) are produced in the brain60 and
AT1, AT2 and mas receptors are expressed beyond the blood-brain barrier in many brain
regions known to influence cardiovascular function.61 Ang II alters blood pressure when
microinjected into various CNS sites, elevating it from within the hypothalamic
paraventricular nucleus,62 NTS63 and interomediolateral spinal cord column.64
In
contrast, Ang II decreases blood pressure when microinjected into the dmnX,65 CVLM66
and RVLM.67 Importantly, microinjection of AT1 receptor antagonists into several of
these listed regions produces changes in blood pressure that are opposite to those elicited
by Ang II.62,
68
Pressor effects of peripheral or central administration of Ang II are
abolished by sympathetic nerve incision, implying that sympathetic activation underlies
central Ang II-mediated increases in blood pressure.69
Indeed, AT1 receptors are
abundant at each synaptic relay of the sympathetic and parasympathetic nervous systems,
as well as in sympathetic preganglionic neurons of the interomediolateral cell column,
paravertebral sympathetic ganglia, sympathetic nerve terminals and brain sites involved
11
in control of sympathetic outflow such as the hypothalamus, dmnX, CVLM and
RVLM.70
Collectively, Ang II mediates changes in blood pressure through actions at
many specific brain sites, although the molecular and neurotransmitter mechanisms
involved in the alteration of neuronal activity at these sites is unknown. The central
pressor actions of Ang II may be mediated in part by interactions with other
neurotransmitter systems. For example, Ang II stimulates release of dopamine, substance
P and norepinephrine from CNS neurons, all of which can contribute to elevations in
blood pressure.51, 71
Similar to Ang II, Ang-(1-7) is synthesized in the circumventricular organs and
other brain nuclei involved in blood pressure regulation.52, 72-74 Microinjection of Ang(1-7) into these sites generally produces blood pressure effects that are opposite to those
of Ang II, with the notable exceptions of in the dmnX, CVLM and RVLM. In these
regions Ang II and Ang-(1-7) elicit similar responses, decreasing blood pressure in the
dmnX and CVLM, and increasing pressure in the RVLM.51,
75
The mechanisms
underlying these actions appear to differ, however, as Ang-(1-7)-mediated reductions in
blood pressure are attenuated by endothelial and neuronal nitric oxide synthase blockers,
which have no effect on Ang II responses.76 Furthermore, in contrast to Ang II, Ang-(17) reduces neuronal catecholamine release.77, 78
The distribution of AT1 and mas receptors is consistent with the ability of Ang II
and Ang-(1-7) to modulate baroreflex function. Intracerebroventricular infusion or NTS
microinjection of Ang II impairs baroreflex sensitivity for control of heart rate in
response to increases in arterial pressure,79, 80 demonstrating an inhibitory central effect of
the peptide on vagal tone. Furthermore, NTS administration of a selective AT1 receptor
12
antagonist improves baroreflex sensitivity in anesthetized Sprague-Dawley rats,
suggesting that endogenous Ang II in this region exerts tonic influence over baroreflex
function.68 The mechanism of Ang II impairment of baroreflex sensitivity is unclear, but
likely involves facilitation of sympathetic outflow based on the ability of Ang II to
modulate release of neurotransmitters that mediate sympathetic responses.51, 71
In contrast to Ang II, central infusion or NTS microinjection of Ang-(1-7)
improves baroreflex sensitivity for control of heart rate in normotensive and hypertensive
rats, in addition to eliciting depressor and bradycardic responses.75, 81 The mechanism by
which Ang-(1-7) enhances baroreflex sensitivity may be related to its ability to reduce
sympathetic tone and modulate local effects of norepinephrine in the brain, as Ang-(1-7)
decreases norepinephrine release and Ang II-mediated norepinephrine release from
neurons.78, 82 An alternative mechanism may involve synergistic effects with bradykinin,
another peptide known to increase baroreflex sensitivity, suggesting these two peptides
can interact in the NTS to modulate baroreflex function.83 In addition, [D-Ala7]-Ang-(17) alone in the NTS impairs baroreflex sensitivity, revealing that like Ang II, endogenous
Ang-(1-7) in the brain exerts a tonic influence within the NTS to modulate baroreflex
function. Together, these observations suggest that a balance in RAS peptide actions in
brain nuclei mediating the baroreflex is important to maintaining normal baroreflex
function. Since the prevailing level of baroreflex sensitivity in part depends on the
endogenous balance of these two peptides, an increase in Ang II or a decrease in Ang-(17) activity in the NTS may result in baroreflex dysfunction and subsequent blood
pressure instability.
13
There is substantial evidence to associate dysfunction of the brain RAS with the
development and maintenance of hypertension.59
For instance, animal models of
hypertension such as the spontaneously hypertensive rat (SHR), DOCA and Dahl saltsensitive rats and the transgenic (mRen2)27 rat, exhibit hyperactivity of the brain RAS
with elevations in sympathetic nerve activity.84
Blockade of central ACE or AT1
receptors by RAS inhibitors reduces blood pressure in SHR and (mRen2)27 rats at doses
that have no effect in their normotensive controls,85, 86 supporting a neural basis for the
development of hypertension in these animals. Central and systemic blockade of Ang II
also improves the set-point and sensitivity of the baroreflex in hypertensive patients and
animal.59 These data suggest that disruption of Ang II signaling within the brain restores
autonomic balance and control of blood pressure in hypertensive populations.
Finally, altered central Ang-(1-7) signaling may also contribute to impaired
baroreflex function and the progression of hypertension. Microinjection of [D-Ala7]Ang-(1-7) into the CVLM improves the blunted sympathetically-mediated baroreflex
sensitivity found in two-kidney one-clip hypertensive rats, while AT1 blockade in this
area has no effect, suggesting that Ang-(1-7) in the CVLM may contribute to impaired
baroreflex function in hypertensive animals.87 Furthermore, restoration of Ang-(1-7) in
the NTS, but not blockade of AT1 receptors, improves baroreflex sensitivity in aged
Sprague-Dawley and young (mRen2)27 rats.88, 89
Collectively, these observations confirm the importance of the balance between
central Ang II and Ang-(1-7) for normal baroreflex modulation.
The mechanisms
involved in modulation of baroreflex sensitivity and blood pressure by the brain RAS are
still unclear, especially in light of additional factors that are known to influence blood
14
pressure regulation through central mechanisms. Therefore, the studies contained in this
volume examine the contribution of one potential factor, the endocannabinoid system, to
central blood pressure regulation.
Central Ang II Pathways in Hypertension
Systemic and centrally generated Ang II activates AT1 receptors located along specific
brain pathways that contribute to hypertension, including in the circumventricular organs
and brainstem nuclei involved in regulating sympathetic outflow.
Three of the
circumventricular organs straddling the third ventricle—the subfornical organ (SFO),
median preoptic nucleus and organum vasculosum of lamina terminalis—form a
continuum of AT1 receptor-expressing neurons called the anteroventral third ventricular
(AV3V) area that plays an important role in mediating effects of the brain RAS on fluid
homeostasis and blood pressure regulation. Lesions to the AV3V area dramatically
attenuate the development of Ang II-dependent hypertension in the two-kidney oneclip,90 renal wrap,91 and chronic Ang II infusion92 animal models, indicating that elevated
systemic Ang II acts through this region to promote the development of hypertension.
The area postrema, situated on the fourth ventricle in close association with the NTS, is
an additional circumventricular organ implicated in the central actions of Ang II. Lesions
to the area postrema attenuate pressor effects of chronic systemic Ang II93 as well as the
development of hypertension in young SHR.94
Direct efferent projections from the SFO terminate in the anterior hypothalamus,
the paraventricular nucleus and the RVLM. Electrophysiological studies demonstrate
that AT1 receptor antagonists inhibit activation of these nuclei in response to stimulation
of the SFO, suggesting that some SFO neurons use Ang II as an excitatory
15
neurotransmitter to activate downstream regions involved in modulation of sympathetic
nerve activity. Furthermore, microinjection of AT1 receptor antagonists into each of
these nuclei lowers resting blood pressure in hypertensive animals.95-98 In addition to the
SFO, the RVLM also receives input from the paraventricular nucleus,99 and
cardiovascular effects of paraventricular nucleus stimulation in anesthetized rats are
blocked by AT1 receptor blockade in the RVLM.100 Activation of the RVLM directly
stimulates the interomediolateral cell column of the spinal cord and thus enhances
sympathetic outflow to promote or maintain hypertension.
In summary, these observations provide a multisynaptic neuroanatomical model
for the actions of central and circulating Ang II in the development of hypertension
driven by increased sympathetic activity. However, despite a known role for Ang II, the
molecular mechanisms of chronic sympathetic stimulation in hypertension are unknown,
creating the need for research into factors that mediate this Ang II-related activity.
16
3. The Endocannabinoid System
The discovery of an endogenous receptor as the primary site of action for Δ 9tetrahydrocannabinol (THC), the principal psychoactive compound in cannabis, has
intensified research into the endocannabinoids.
As reviewed by others101-103, the
endocannabinoid system is a paracrine cellular signaling system comprising the
biologically active endocannabinoids anandamide and 2-AG, the G protein-coupled
receptors mediating their effects, and their respective biosynthetic and degradative
enzymes. The actions of the endocannabinoids are exerted predominantly through CB1
and CB2 cannabinoid receptors. The CB1 receptor is widely expressed in the CNS and
peripherally, whereas the CB2 receptor is primarily concentrated in immune tissues. CB1
receptors in the CNS are predominantly expressed on presynaptic neuronal terminals.
Their activation at these sites by anandamide or 2-AG synthesized and released from
postsynaptic neurons directly modulates voltage-gated ion channels to repolarize the
cellular membrane and decrease intracellular Ca2+ concentrations, temporarily reducing
release of neurotransmitter into the synaptic cleft.
Thus, a primary function of
endocannabinoid signaling in the CNS is to modulate neurotransmission at the level of
the synapse.104
Cannabinoid receptors in the periphery, quiescent under normal conditions,
become activated and upregulated during cardiovascular disease and its metabolic risk
factors.105 Two distinct cannabinoid receptors, CB1 and CB2, sharing approximately 44%
sequence homology, have been identified to date.101, 106 Like the RAS, the near ubiquity
of the endocannabinoid system makes it inevitable that its physiological actions are
complex and often tissue-, context- or even ligand-specific. The emergence of a role for
17
the endocannabinoid system in the pathogenesis of cardiovascular diseases and metabolic
syndrome105, 107, 108 makes it critical to understand its integrative signaling mechanisms in
order to gain novel insights into the relationship between the endocannabinoid system
and these pathological conditions.
The relevance of the CB2 receptor to autonomic, cardiovascular and metabolic
homeostasis is still emerging. Expression was once thought to be restricted to immune
tissues and glial cells, although there is now some conflicting evidence of its expression
in the brainstem.109,
110
Further recent evidence implicates a cardioprotective role for
peripherally upregulated CB2 receptor expression in atherosclerosis and ischemia.111-113
As more emphasis is placed on the inflammatory process in chronic disease states, a
functional role for the CB2 receptor in cardiovascular or metabolic disease is likely to
emerge.
However, due to the present lack of clarity surrounding the functional
significance of brainstem CB2 receptors,114 as well as preliminary data illustrating CB1mediated responses in the NTS, the studies in this volume primarily focus on actions of
the CB1 receptor.
Biosynthesis and Degradation
Anandamide and 2-AG, the two best characterized endocannabinoid ligands, are
synthesized upon demand from membrane phospholipids and then degraded shortly after
release.115-117 Endocannabinoids in the CNS contrast with classical neurotransmitters
because they are not stored in vesicles, but rather released immediately upon synthesis
from postsynaptic neurons to retrogradely activate CB1 receptors expressed on
presynaptic terminals. In the periphery, endocannabinoids act as paracrine and autocrine
mediators. The biosynthesis and degradation of both molecules is governed by multiple
18
parallel enzymatic pathways to ensure tight temporal and spatial control over their
signaling functions. The biosynthetic pathways of anandamide and 2-AG illustrate this
concept.
Anandamide is a lipid signaling molecule belonging to a class of naturally
occurring fatty acids known as the N-acylethanolamines (NAEs).
The precise
biosynthetic pathway of anandamide is not without controversy,118 but the direct
synthesis pathway includes plasma membrane phospholipid precursors, the intermediate
substrate N-acylphosphatidylethanolamine (NAPE), and the enzymes Ca2+-dependent Nacyltransferase (Ca-NAT) and NAPE-hydrolyzing phospholipase D (NAPE-PLD).117
Typically, Ca-NAT will catalyze the transfer of arachidonic acid from a
glycerophospholipid to the ethanolamine headgroup of a membrane phospholipid to form
NAPE.
NAPE-PLD then hydrolyzes NAPE to anandamide.
The degradation of
anandamide is governed by the integral membrane enzyme fatty acid amide hydrolase
(FAAH), as well as by NAE-hydrolyzing acid amidase, to yield arachidonic acid and
ethanolamine.117
In contrast to anandamide, three major biosynthetic pathways for 2-AG are
proposed.119 The first, which appears to dominate in the CNS,120 is through a two-step
process involving conversion of the membrane phospholipid phosphotidylinositol 4,5bisphosphate to a diacylglycerol (DAG) intermediate by PLC. DAG is then catalyzed by
one of two isoforms of DAG lipase (DAGL) into 2-AG. The second pathway involves
the conversion of a phosphatidyl lipid to the intermediate 2-arachidonoyl-lyso
phosphatidyl inositol by the action of phosphatidyl lipase, and then to 2-AG by a lysoPLC.121 The third pathway involves lysophosphatidic acid hydrolysis by a phosphatase
19
to form 2-AG.122 The involvement of the latter two pathways in the production of 2-AG
in the CNS has not been evaluated in detail, but may explain reports of 2-AG-mediated
actions that are insensitive to DAGL inhibitors.123
A more diverse assortment of
enzymes participate in the metabolism of 2-AG than anandamide.
For example,
monoacylglycerol lipase (MAGL) is accepted as the dominant enzyme in the degradation
of 2-AG, but a role has been found for others depending on context including FAAH,
cyclooxygenase-2 and the serine hydrolases ABHD6 and ABHD12.124 The physiological
relevance of these enzymes in 2-AG metabolism is still emerging.
CB1 Receptor Signaling
The CB1 receptor is primarily a Gi/o protein-coupled receptor that is widely expressed
throughout the CNS, and also found in the myocardium, adipose tissue, hepatic tissue,
and endothelial and smooth muscle cells of the vasculature and gastrointestinal tract.125
CB1 receptor agonists include the endocannabinoids anandamide and 2-AG, the plantderived THC and its analogs, and synthetic compounds such as CP55,940, HU210, and
WIN55,212-2.
Common antagonists of the CB1 receptor include rimonabant
(SR141716A), AM251 and AM281.
Prior to the characterization of the cannabinoid receptors in 1988,126 it was
believed that the lipophilicity of THC enabled it to interact with the plasma membrane to
disrupt enzyme and ion channel activity.127
Howlett and Fleming first observed
functional inhibition of adenylyl cyclase and subsequent reduction in intracellular cAMP
following application of THC to neuroblastoma cells.128 Adenylyl cyclase inhibition by
THC was blocked by pertussis toxin (PTX), indicating the involvement of a Gi/o
protein.129 The CB1 receptor was cloned from rat cerebral cortex in 1990.130
20
The sensitivity of most intracellular cannabinoid responses to PTX demonstrates
the preference of CB1 receptors for the Gi/o protein family.131,
132
However, strong
evidence exists that CB1 receptors can stimulate cAMP accumulation under conditions
that prevent interaction of the receptor with Gi/o proteins, such as during PTX treatment,
perhaps due to disinhibition of adenylyl cyclase.133,
134
Others suggest that G protein
coupling to CB1 may be an agonist-specific event,133, 135 or dependent upon concurrent
activation of co-expressed G protein-coupled receptors.134, 136 It is also possible that nonreceptor proteins associated with the CB1 receptor can direct the probability of switching
signaling pathways.137
Activation of presynaptic CB1 receptors in neuronal cells temporarily reduces the
amount of neurotransmitter released into the synaptic cleft.120,
138-140
The principal
mechanism underlying cannabinoid-induced inhibition of neurotransmitter release is the
direct modulation of voltage-gated membrane ion channels by presynaptic CB1 receptors.
During bursts of activity, postsynaptic neurons produce and release 2-AG or anandamide,
which then bind to presynaptic CB1 receptors. The Gβγ subunits associated with CB1
receptors then directly interact with presynaptic membrane-bound ion channels.
Cumulatively, this results in hyperpolarization of the plasma membrane, and reduction of
the local intracellular Ca2+ concentration necessary for neurotransmitter release.
High intracellular concentration of Ca2+ is required for proper neurotransmitter
vesicle fusion to the plasma membrane and subsequent exocytosis, and is supplied locally
by the voltage-operated Ca2+ channels that open when the membrane is depolarized.
Anandamide, WIN55,212-2 and CP55,940 each inhibit Ca2+ channels via CB1 receptors
in cell preparations and brain tissue slices, effects blocked by SR141716A or PTX.141, 142
21
This inhibition is independent of other signaling enzymes known to interact with voltagegated ion channels, such as the cyclic adenosine monophosphate (cAMP)/protein kinase
A (PKA) pathway,141 therefore likely resulting from direct interaction with dissociated
Gβγ subunits.
Inwardly rectifying K+ currents stabilize the membrane potential following
release of neurotransmitter. WIN55,212-2 enhances inward K+ currents carried by G
protein-coupled inwardly rectifying K+ channels, and is sensitive to low concentrations of
Ba2+, a K+ channel inhibitor.142
It was later demonstrated that WIN55,212-2- and
CP55,940-mediated inhibition of glutamatergic signaling in mouse nucleus accumbens is
blocked by both Ba2+ and SR141716A,143 suggesting CB1 receptor activation of inwardly
rectifying K+ channels as a mechanism for the inhibition of neurotransmitter release. Atype K+ channels, by contrast, are rapidly activating voltage-gated ion channels that
mediate the deactivating inward K+ currents as part of the repolarization process
following cell depolarization. WIN55,212-2 increases this current in a concentrationdependent and SR141716A- and PTX-sensitive manner.144
In addition to voltage-gated ion channels and neurotransmission, the CB1 receptor
modulates several other intracellular processes essential to normal cell function. Since
Howlett and colleagues’ demonstration of CB1 receptor-mediated suppression of cAMP
production in neuroblastoma cells,127,
128
functional inhibition of adenylyl cyclase by
cannabinoid agonists has been identified in many other settings. These responses are
generally PTX- and CB1 antagonist-sensitive and occur independently of forskolin, the
universal adenylyl cyclase activator. In the CNS, cannabinoid-mediated attenuation of
forskolin-stimulated cAMP accumulation has been demonstrated in slices of rat
22
hippocampus, striatum, cerebral cortex and cerebellum,145 as well as in vivo in rat
striatum.146
The complexity of cannabinoid adenylyl cyclase regulation was recognized
following several reports of CB1-mediated increases in cAMP accumulation.134, 147-149 It
is now known that adenylyl cyclase has nine distinct isozymes, activated by different
stimuli, organized into six classes based on functional similarities and tissue
expression.150 In addition to the potential dual coupling of CB1 to Gi/o and Gs, contrasting
effects of cannabinoid agonists on functional adenylyl cyclase activity is attributed to the
specific isoform present in different cellular preparations.151
Altered cAMP
concentration can yield major changes in cellular activity due to its downstream
consequences (i.e., PKA phosphorylation, A-type K+ channel regulation, and gene
expression via cAMP response elements). Therefore, modulation of adenylyl cyclase
activity must be carefully considered when interpreting intracellular cannabinoid
signaling events.152, 153
The mitogen-activated protein kinase (MAPK) pathway is a major signaling
mechanism that governs many cellular functions including cell growth, differentiation,
adhesion and apoptosis.154-156 Activation of the MAPK signaling cascade is typically
initiated by activation of a tyrosine kinase- or G protein-coupled receptor. CB1 receptors
are almost universally positively linked to the MAPK pathway.102, 157, 158 In vitro, CB1
agonists increase phosphorylation of the MAPK extracellular signal-regulated kinase
(ERK)1/2 in cells expressing both endogenous and recombinant CB1 receptors.158-161
These effects are SR141716A- and PTX-sensitive.
The mechanisms for MAPK
activation by CB1 receptors are not yet fully known; multiple pathways could result in
23
CB1-mediated MAPK induction, and these likely depend on cell type and stimulus. For
example, astrocytoma cells utilize a pathway involving phosphoinositide-3 kinase (PI3K)
and Akt, which then activates Raf-1 to initiate the MAPK cascade to subsequently
increase glucose metabolism.161 The same PI3K-Akt-Raf pathway is activated in CHO
cells transfected with recombinant CB1 receptors in response to anandamide.162
MAPK activity may be regulated indirectly via CB1 receptor stimulation through
its effects on cAMP accumulation. For example, MAPK activation in MCF-7 cancer
cells by anandamide is significantly attenuated by forskolin and 8-Bromo-cAMP, an
analogue of cAMP.163 A similar effect following anandamide and 2-AG exposure was
observed in rat hippocampal slices pretreated with 8-Bromo-cAMP,164 suggesting that a
decrease in cAMP accumulation may facilitate the stimulatory effects of CB1 activation
on the MAPK pathway. In vivo, administration of cannabinoid agonists produces a
progressive and transient activation of ERK1/2 in several rat and mouse brain regions
that is blocked by SR141716A, suggesting involvement of the CB1 receptor.165-167
Furthermore, the effect of cannabinoid agonists on ERK1/2 is absent in CB1-/- knockout
mice.166
Role in Cardiovascular Regulation
Chronic marijuana use in humans and prolonged treatment in animals can lead to postural
hypotension and bradycardia,168-175 providing the first evidence of a role for cannabinoid
modulation of the cardiovascular system. In 1996, Varga and colleagues reported a
prolonged blood pressure depressor effect of systemically administered anandamide in
anesthetized rats.176
This depressor response was accompanied by bradycardia and
reduced cardiac contractility, and these effects were sensitive to CB1 antagonists. The
24
expression of CB1 in central autonomic sites,177 myocardium178 and vascular walls179 may
account for the direct cardiovascular actions of cannabinoid ligands.
In conscious normotensive animals the cardiovascular actions of CB1 ligands are
less pronounced or absent entirely, leading some to conclude that the endocannabinoid
system plays a limited role in normal cardiovascular regulation.105 Under anesthesia, for
example, autonomic function is compromised,180 potentially contributing to the loss of
normal baroreflex function in the presence of CB1 receptor activation. However, the
importance of the endocannabinoid system in cardiovascular regulation is much more
apparent when studied in experimental models of hypertension. In models of both acute
and chronic hypertension, and across anesthetized and conscious animals, the
cardiovascular effects of systemically administered cannabinoids are amplified compared
to normotensive controls.181-183 Specifically, greater CB1-mediated reductions in heart
rate and blood pressure, as well as associated increases in vasodilation, are reported
across these hypertensive models compared with normotensive animals.
The
mechanisms of these hypertension-specific CB1 effects are not fully understood, but may
involve modulation of sympathetic outflow from the brain by CB1 receptors, in addition
to direct vasodilator and inotropic effects in the vasculature and heart, respectively.184, 185
Others find increased CB1 receptor expression in the heart, aorta and coronary arteries in
hypertension and other cardiovascular diseases.186, 187 It is also possible that central CB1
coupling to Gi proteins is enhanced in hypertension, as recently reported in the SHR
brain.188 Upregulation of Giα protein isoforms is documented in the heart and vasculature
of these and other hypertensive animals.189-191 The CB1 receptor thus participates in
25
autonomic cardiovascular regulation, with greater importance to the homeostatic
adjustments under pathological conditions.
The endocannabinoid system could be an attractive therapeutic target for the
treatment of hypertension and other cardiovascular diseases.
In fact, the use of
marijuana-like substances as antihypertensive medications was contemplated as early as
the 1970s because of observed long-lasting decreases in blood pressure associated with
chronic marijuana use in humans and animals.171-174 However, the inability to separate
the neurobehavioral, metabolic and cardiovascular effects of cannabinoid agonists led to
the abandonment of this strategy. Interest in pursuing the endocannabinoid system as a
target of antihypertensive therapy has since reemerged with the discovery of amplified
CB1-mediated cardiovascular effects in hypertension, suggesting a role as a
compensatory mechanism in some animal models.181
CB1 cannabinoid receptors are densely expressed in the NTS and are associated
with vagal afferent neurons of the nodose ganglion.192, 193 It was observed as early as
1975 that prolonged THC ingestion impaired circulatory reflexes to standing and exercise
in humans.168
Later tissue bathing studies show that NTS neurons are sensitive to
cannabinoid ligands,194, 195 implying that the endocannabinoid system can modulate the
arterial baroreflex. Importantly, both increases and decreases in the frequency of action
potentials are observed in response to cannabinoid receptor stimulation in the NTS,194
suggesting the presence of CB1 receptors on both glutamate- and GABA-releasing
neurons. However, the precise mechanism of neuronal modulation within the NTS by the
endocannabinoid system appears complex.
26
A series of studies over the past decade examined the effects of cannabinoid
receptor activation within the NTS on modulation of the sympathetic branch of the
baroreflex, and report CB1-mediated disinhibition of baroreceptive NTS neurons.
Specifically, they demonstrate that microinjection of cannabinoid agonists, including
anandamide and WIN55,212-2, into the NTS prolongs baroreflex-evoked renal
sympathetic nerve inhibition in anesthetized Sprague-Dawley and Wistar-Kyoto rats. 45,
46, 196
This effect is absent in the SHR strain,46 suggesting differential regulation of
baroreflex function in hypertensive relative to normotensive conditions. The same study
also reports reduced CB1 receptor density in the NTS of SHR, which may account for the
differences in the baroreflex-evoked sympathetic response. However, these studies did
not assess effects on the parasympathetically-mediated vagal branch of the baroreflex for
control of heart rate.
Differential modulation of glutamate and GABA neurotransmitters in the NTS
may be responsible for these effects. In fact, a preponderance of data links the altered
balance of NTS neurotransmission shifted toward excessive GABAergic tone to several
models of hypertension, including SHR197, 198 and transgenic (mRen2)27 rats.199, 200 The
origin of this altered inhibitory tone in hypertension and the factors that mediate it remain
unclear. We propose that altered endocannabinoid system tone within the NTS may be
one contributing factor based on the data contained in the present studies. Another
possibility involves interactions with other factors known to regulate autonomic or
cardiovascular function, including the brain RAS.
27
Metabolic Effects and Cardiovascular Risk Factors
An emerging model of metabolic syndrome has identified central and peripheral
overactivity of the endocannabinoid system as a key participant in its pathogenesis.
Metabolic syndrome refers to a cluster of risk factors for cardiovascular disease including
obesity, glucose intolerance, insulin resistance, leptin resistance and dyslipidemia.201-203
Excess sympathetic nervous system activity is also typical of these conditions.11, 26 A
dynamic relationship exists between the endocannabinoid system and cardiometabolic
regulation as revealed by peripheral and central CB1 receptor blockade in obese humans
and in animal models of metabolic syndrome.
Animals administered cannabinoid agonists gain a significant amount of weight,
and this weight gain is attributable not only to increased food intake204 but also to
alterations in peripheral metabolic processes. Specifically, activation of CB1 receptors in
adipose tissue in animals fed a high-fat diet directly stimulates adipogenesis and
attenuates secretion of adiponectin,205 while CB1 activation in the liver directly increases
gene expression of lipogenic transcription factors and their enzymatic targets, increasing
the release of free fatty acids.206-208 Attenuation of adiponectin release has significant
downstream consequences for metabolic balance, as it is an important modulator of
numerous processes including glucose management and fatty acid catabolism.
The
actions of weight-independent CB1 receptor overstimulation in fat and liver cells can lead
to impaired glucose utilization accompanied by peripheral insulin resistance due to direct
inhibition of insulin receptor activation,207-209 which in turn may contribute to the onset of
type II diabetes. Increased CB1-mediated hepatic free fatty acid generation has also been
shown to disrupt the balance of cholesterol, decreasing high density lipoprotein while
28
increasing low density lipoprotein and triglycerides in the circulation.210 Additionally,
central CB1 activation dampens insulin signaling in the brain and periphery, and is
associated with altered central leptin receptor expression.211-213 Therefore, while CB1
agonists may acutely reduce blood pressure in hypertension through actions in the heart
and vasculature,214-216 they may adversely affect important metabolic parameters to
induce a condition resembling metabolic syndrome.
CB1 blockade thus has potential as a therapy for some metabolic pathologies.108,
217
Indeed, CB1 antagonists are now well-associated with reversal of diet-induced obesity
in animals, as well as with normalization or improvement of glucose tolerance, insulin
resistance, leptin resistance, and obesity-related inflammation in obese or diabetic
animals.218-223 Human clinical trials confirmed that chronic treatment with SR141716A is
effective for the management of obesity and type II diabetes,224-229 with a modest
secondary improvement to blood pressure in hypertensive patients.230 Unfortunately,
further clinical trials were halted due to reports of negative psychiatric effects of
increased anxiety and depression associated with chronic CB1 receptor blockade.231
Despite its withdrawal from clinical development, SR141716A remains an important
pharmacological tool to study the role of the endocannabinoid system in metabolic,
cardiovascular and autonomic dysfunction. It remains unclear how CB1 antagonists will
alter the cardiovascular system in the face of metabolic syndrome or hypertension.
Recent data, however, may reveal an important mechanism for the exacerbation of
hypertension and metabolic syndrome involving endocannabinoid system activation.
29
Interactions with the RAS
The direct effects of SR141716A on blood pressure are not clearly predictable. Reports
suggest that reductions in blood pressure observed in obese humans and animals
following treatment with SR141716A are not independent of general weight loss.221, 230
However, emerging evidence suggests an important interaction between the
endocannabinoid system and the RAS in hypertension and other cardiovascular diseases,
which may potentially be a primary mechanism underlying the pathogenic properties of
Ang II or endocannabinoids.
SR141716A treatment in apolipoprotein E-deficient mice fed a high-fat diet yields
decreased vascular AT1 receptor expression and reduced arterial constriction with
improved endothelial function.232 At the cellular and molecular levels, a recent study by
Rozenfeld and colleagues indicates that CB1-AT1 receptor interaction via dimerization
enhances Ang II-mediated ERK1/2 signaling in Neuro2A and HEK293 cells expressing
CB1 and AT1 receptors.233 Furthermore, administration of SR141716A prevents Ang IImediated mitogenic signaling and fibrogenic gene expression in these cells. In vivo, CB1
receptors mediate the enhancement of ERK1/2 phosphorylation and cAMP accumulation
by Ang II in activated hepatic stellate cells harvested from rats treated with ethanol for 8
months. Additional CB1-AT1 receptor interactions may be through a transactivation
mechanism involving the production and release of 2-AG following stimulation of AT1Gq protein signaling in target cells.234, 235
The maintenance of hypertension in several animal models of the disease and in
humans is often dependent in part on Ang II acting at AT1 receptors in vasculature and
kidney to modulate vascular tone and fluid balance, as well as in the brain for impairment
30
of baroreflex sensitivity and facilitation of sympathetic outflow.49, 199, 236, 237 Thus, it is
possible that CB1 receptor blockade may lower blood pressure independently of weight
loss or improvement in metabolic profile. In hypertension resulting from excess action of
Ang II, CB1 receptor blockade may serve as a potentially beneficial therapy. In addition,
long-term AT1 receptor blockade has a beneficial metabolic profile in older animals and
elderly human patients.238,
239
While the improvement of metabolic profiles with AT1
blockade may be interpreted as directly interfering with Ang II-mediated attenuation of
signaling pathways for metabolic hormones such as insulin and leptin, we must now
consider that effects on the endocannabinoid system may also contribute.
In summary, the resulting dyslipidemia, insulin resistance and obesity that can
arise due to perturbations of the endocannabinoid system or the RAS are components of
metabolic syndrome that represent significant risk factors for hypertension and other
cardiovascular diseases. The endocannabinoid system will require more intensive study
of its potential negative metabolic and cardiovascular risk factor profile, including the
long term effects on baroreflex function after systemic administration, before
endocannabinoid-targeted therapeutic strategies can progress.
31
4. The Use of Transgenic and Aged Animals to Investigate the Role of the
Endocannabinoid System in Blood Pressure Regulation
Transgenic rodents with systemic alterations in components of the RAS are an important
tool to study the contribution of various physiological factors to the development and
maintenance of hypertension. The importance of the RAS to cardiovascular regulation is
illustrated by the observation that mice with targeted deletion of renin, angiotensinogen,
ACE or AT1 receptors all have decreased resting blood pressure. 59
Similarly,
overexpression of angiotensinogen increases blood pressure.240 Recent studies using
transgenic rodents highlight the crucial role of the brain RAS in the pathogenesis of
hypertension through alterations in cardiovascular and autonomic regulation, consistent
with the central distribution of angiotensin receptors in brain regions that regulate these
functions.56, 59 For example, central administration of RAS blockers that have no effect
in normotensive animals reduces blood pressure in hypertensive SHR and transgenic
(mRen2)27 rats.82 Studies in transgenic ASrAOGEN rats with low brain RAS expression
extend these findings to show differential sources of angiotensinogen for Ang II and
Ang-(1-7) within the brain that are involved in baroreflex modulation,241 consistent with
mice overexpressing glial versus neuronal angiotensinogen or renin.242
Additional
studies in aged Sprague-Dawley rats find a similar role for the brain RAS in the
progression of increasing blood pressure and reduced baroreflex sensitivity with age.88
Although the contribution of the brain RAS to modulation of blood pressure and
baroreflex function is well established, less is known about how the RAS may interact
with various other factors present in the brain, such as the endocannabinoid system, to
produce its physiological actions.
32
In addition to influence over blood pressure and baroreflex modulation, recent
studies using transgenic rodents provide evidence that the brain RAS is involved in the
regulation of energy balance and metabolic profile. For instance, (mRen2)27 rats with
overactive brain RAS are heavier and exhibit insulin and leptin resistance in response to
an oral glucose load relative to age-matched Sprague-Dawley rats.243,
244
In contrast,
ASrAOGEN rats with low brain RAS activity are lighter and have enhanced insulin and
leptin responsiveness for metabolic actions.243 Evidence linking the RAS to metabolic
dysfunction includes the observations that RAS blockade in Fischer-344 rats or mice fed
a high-fat diet reduces body weight and improves insulin sensitivity.245,
246
In
hypertensive patients, ACE inhibitors and AT1 blockers are associated with positive
metabolic effects including reduced plasma insulin and leptin levels and reduced risk for
new-onset diabetes.247-249 Together, these reports suggest that disruption of brain Ang II
is associated with overall improved metabolic profile with increased sensitivity to insulin
and leptin.
While sensitivity to metabolic hormones is beneficial for energy and glucose
homeostasis, some metabolic actions of CB1 receptor blockade are attributed to increased
stimulation of the sympathetic nervous system,250,
251
which may potentially elevate
arterial pressure at central sites of action. Whether CB1 receptor blockade also results in
altered baroreflex sensitivity, blood pressure or metabolic profile in transgenic or aged
animals with differential brain RAS activity has yet to be determined. Therefore, we
employed transgenic ASrAOGEN and (mRen2)27 rats with low or high brain RAS
expression, respectively, and aged Sprague-Dawley rats to determine interactions
33
between the brain RAS and the endocannabinoid system with respect to autonomic,
cardiovascular or metabolic regulation.
Transgenic (mRen2)27 Rats
The transgenic (mRen2)27 rat strain was created in 1990 by insertion of the mouse
submandibular gland Ren2 renin gene into the genome of the Hannover Sprague-Dawley
rat.252 Rats homozygous for the Ren2 gene exhibit fulminant hypertension beginning
around 4 to 5 weeks of age that results in a high mortality rate at a young age, making
antihypertensive treatment necessary for survival.253 Heterozygous (mRen2)27 rats are
used for most research studies and exhibit hypertensive systolic blood pressure levels
ranging from 170 to 190 mmHg, similar to SHR.252, 254 This strain has shown a stable
expression and phenotype for approximately 24 years and is used extensively by our
laboratory to evaluate the role of the brain RAS in baroreflex modulation.89, 255, 256
In (mRen2)27 rats, the renin transgene is highly expressed in the brain and
adrenal gland with reduced expression in the kidney, in contrast to non-transgenic
animals which exhibit low renin expression in the brain and adrenal gland.257 Whether
circulating levels of renin, angiotensinogen, Ang I, Ang II or Ang-(1-7) are altered is
unclear as studies report reduced, similar, or modestly elevated plasma levels of these
components relative to Sprague-Dawley rats.252, 258, 259 However, an 18-fold increase in
brain tissue Ang II levels is reported in (mRen2)27 rats compared to Sprague-Dawley
rats.260 Chronic oral treatment with ACE inhibitors or AT1 receptor blockers lowers
blood pressure in (mRen2)27 rats,261, 262 indicating that the hypertension they exhibit is
dependent on actions of Ang II at AT1 receptors. Mounting evidence suggests that
hypertension in (mRen2)27 rats is of central origin. For example, intracerebroventricular
34
administration of AT1 blockers reduce blood pressure and heart rate in these animals, 86
suggesting that central Ang II is responsible for the maintenance of hypertension, with
potential concomitant disturbances in autonomic balance.
In addition to hypertension, (mRen2)27 rats display several indices of autonomic
imbalance, including increased sympathetic transmission in response to Ang II relative to
Sprague-Dawley rats.263 This strain also exhibits an inverted circadian rhythm of blood
pressure264 and reduced resting blood pressure under urethane-chloralose anesthesia,
similar to the resting level of Sprague-Dawley rats, providing further evidence of
enhanced sympathetic nervous system activity. Despite lower resting pressure under
anesthesia, (mRen2)27 rats still exhibit marked impairments in baroreflex sensitivity for
control of heart rate in response to increases in blood pressure,265 indicating reduced
parasympathetic tone. The impaired baroreflex sensitivity in this strain is likely related to
low brain Ang-(1-7) because microinjection of [D-Ala7]-Ang-(1-7) in the NTS has no
effect on baroreflex sensitivity in these animals,89 unlike the inhibitory effect of mas
receptor blockade in Sprague-Dawley and ASrAOGEN rats.241
Moreover, NTS
microinjection of an ACE inhibitor, but not an AT1 receptor antagonist, improves
baroreflex sensitivity in (mRen2)27 rats,83 providing further evidence that reduced Ang(1-7) tone contributes to impaired cardiovascular regulation in this strain.
(mRen2)27 rats exhibit an anxiogenic behavioral profile,266,
267
Finally,
potentially due to
hyperactivity of the hypothalamic-pituitary-adrenal axis that mediates stress responses.268,
269
The anxiety-like behaviors of these rats are abolished by chronic systemic ACE
inhibition,266 suggesting that they too are mediated by an overactive RAS underlying
autonomic imbalance.
35
Hypertensive (mRen2)27 rats have increased body weight and higher fat mass
relative to ASrAOGEN and Sprague-Dawley rats.243,
270
Although plasma levels of
insulin, leptin and glucose are similar to those in ASrAOGEN and Sprague-Dawley rats
at 15 weeks of age, (mRen2)27 rats display whole body and skeletal muscle insulin
resistance in response to oral glucose tests. Therefore, these animals are studied as a
model of both hypertension and metabolic syndrome.271 While our understanding of the
cardiovascular sensitivity to metabolic hormones in this strain is still emerging, the
observation that they exhibit metabolic dysfunction may imply that these animals have
altered endocannabinoid tone associated with upregulated RAS expression. Determining
factors that contribute to baroreflex and metabolic dysfunction in (mRen2)27 rats will be
necessary to further understand the contribution of the endocannabinoid system to obesity
and hypertension in the face of an overactivated brain RAS.
The (mRen2)27 rat showcases the powerful role of increased extrarenal renin
expression in the regulation of cardiovascular homeostasis, yet many new questions have
been generated as a result of this model.
While several other models of chronic
hypertension exist, the advantage of the (mRen2)27 rat is that a well-defined single
genetic manipulation is the primary initiator of a series of consequences yielding the
development of hypertension with metabolic dysfunction.272
Thus, it is possible to
determine interactions between experimental manipulations in this strain and the genetic
background against which they occur, in contrast to models with a broad and elusive
pathophysiology, such as the SHR, exhibiting a variety of features that cannot be easily
compared to outbred normotensive controls.273
36
Transgenic ASrAOGEN Rats
The second model of altered brain RAS we use is the transgenic ASrAOGEN rat, a
unique rodent model for under-expression of the endogenous brain RAS via targeted gene
silencing created by Ganten and colleagues in 1999. Transgenic ASrAOGEN rats were
created by transfection of Sprague-Dawley rats with an antisense oligonucleotide to
angiotensinogen driven by a glial fibrillary acid protein promoter274 to target expression
specifically to astrocytes.275 Since astrocytes are the primary source of angiotensinogen
in the brain,276 the antisense oligonucleotide yields a 90% reduction in angiotensinogen
protein levels in brain regions including the medulla and hypothalamus.274, 275 Therefore,
as expected, ASrAOGEN rats have decreased brain tissue levels of Ang I, Ang II and
Ang-(1-7) compared to Sprague-Dawley rats, but upregulated brain AT1 receptor
expression.277, 278 The brain specificity of the antisense oligonucleotide is confirmed by
the unaltered levels of circulating angiotensinogen in ASrAOGEN relative to SpragueDawley rats.274,
275
ASrAOGEN rats have maintained a stable phenotype over the 15
years since their creation and, like (mRen2)27 rats, are used extensively by our laboratory
to investigate the contribution of the brain RAS to cardiovascular and metabolic
regulation in pathologies related to hypertension and aging.241, 255, 279, 280
Conscious ASrAOGEN rats exhibit lower resting blood pressure and heart rate
relative to Sprague Dawley rats, with systolic pressures typically between 80 and 100
mmHg.275 The hypotension in this strain is generally attributed to reduced glia-derived
angiotensin peptides, although it may also be due in part to reduced plasma vasopressin
levels and resulting diabetes insipidus-like syndrome in these animals.275 Similar to
(mRen2)27 rats, ASrAOGEN rats display altered circadian rhythm of blood pressure,
37
suggestive of autonomic imbalance. However, baroreflex sensitivity for control of heart
rate and heart rate variability are significantly enhanced in these animals relative to
Sprague-Dawley rats,241,
255
indicating higher resting parasympathetic activity. In line
with this interpretation, ASrAOGEN rats exhibit a paradoxical elevation of resting
arterial pressure while under the influence of urethane-chloralose anesthesia.
This
anesthesia is reported to increase sympathetic neurotransmission in the brain,180, 281 which
in ASrAOGEN rats may be mediated by increased expression of AT1 receptors in
brainstem regions involved in descending sympathetic pathways including the NTS and
RVLM.277 Elevated blood pressure under anesthesia notwithstanding, blockade of either
NTS AT1 or mas receptors decreases pressure in these animals, while only mas receptor
blockade decreases baroreflex sensitivity.241
This implies a non-glial source for
angiotensinogen involved in maintaining Ang-(1-7) tone over baroreflex function,
providing further evidence for and possible mechanism of independent modulation of
baroreflex sensitivity and blood pressure by RAS peptides. In addition to differences in
blood pressure regulation, ASrAOGEN rats feature lower body weight with enhanced
insulin sensitivity and increased food consumption.243, 279 Collectively, the reduced brain
RAS activity exhibited by ASrAOGEN rats provides an ideal animal model in which to
compare and contrast influence of the endocannabinoid system over cardiovascular and
autonomic regulation relative to (mRen2)27 rats with an overactive brain RAS.
Aged Sprague-Dawley Rats
Aging is associated with gradual increases in systolic blood pressure, as well as
reductions in heart rate variability and baroreflex sensitivity for control of heart rate
which may facilitate the progression of hypertension and related diseases.88,
38
282
Autonomic imbalance with increased sympathetic tone is a primary cause of these
cardiovascular deficits associated with aging.283 Treatment with ACE inhibitors or AT1
receptor blockers reduces age-related cardiovascular and metabolic dysfunction and
improves lifespan in humans and animals,284 suggesting that Ang II plays a role in the
development of adverse cardiovascular consequences during aging.
In transgenic
ASrAOGEN rats, blood pressure remains low while baroreflex and metabolic function
are preserved over lifespan,277, 280 implicating the brain RAS in age-related cardiovascular
decline. Furthermore, as in young adult (mRen2)27 rats, NTS microinjection of Ang-(17) but not an AT1 receptor antagonist restores baroreflex sensitivity for control of heart
rate in older Sprague-Dawley rats.88 Together, the body of evidence suggests that aging
is an additional context associated with altered RAS activity that contributes to
autonomic imbalance and subsequent reduced baroreflex sensitivity. The role of the
endocannabinoid system in age-related cardiovascular pathologies has not yet been
investigated. Therefore, older Sprague-Dawley rats represent an additional setting in
which to evaluate the contribution of endocannabinoids to blood pressure regulation in a
condition associated with hypertension with altered brain RAS activity.
In summary, transgenic rats with high or low brain RAS activity and aged
Sprague-Dawley rats exhibit alterations in autonomic cardiovascular regulation, and thus
may be powerful model systems in which to evaluate potential interactions between the
RAS and the endocannabinoid system in the face of hypertension and related pathologies.
The table below summarizes the features of animals used in the present studies relative to
young adult Hannover Sprague-Dawley rats from our colony:
39
Aged
Sprague-Dawley
ASrAOGEN
(mRen2)27
Hannover
Sprague-Dawley
Hannover
Sprague-Dawley
—
Hypotensive
Hypertensive
Hypertensive
↑Parasympathetic
↑Sympathetic
↑Sympathetic
Enhanced
Impaired
Impaired
Light
Heavy
Heavy
Sensitivity to Insulin
and Leptin:
Enhanced
Resistant
Resistant
Brain RAS Activity:
Low
High
High
?
?
?
Parent Strain:
Conscious Blood Pressure:
Autonomic Balance:
Baroreflex Sensitivity:
Body Weight:
NTS Endocannabinoid
Tone:
40
5. Rationale and Aims
Ongoing studies in our laboratory focus on understanding factors that alter baroreflex
sensitivity for control of heart rate at the level of the NTS. The majority of evidence to
date indicates a key role for RAS peptides in modulating baroreflex function in which the
endogenous balance of Ang II and Ang-(1-7) within this brain region determines the
prevailing level of baroreflex sensitivity. However, additional factors, such as leptin, 41
have been recently identified to influence resting baroreflex sensitivity, suggesting that
alterations in endogenous hormone or signaling molecule levels during conditions related
to hypertension may contribute to baroreflex dysfunction.
Previous studies by others show that exogenous cannabinoids alter neuronal cell
firing in the NTS194 as well as baroreflex-evoked sympathetic nerve activity.45, 285 One
report notes that the enhanced sympathoinhibition elicited by cannabinoid agonists in the
NTS of normotensive animals is absent in SHR, suggesting differential effects of
cannabinoids on baroreflex modulation in hypertensive relative to normotensive animals.
However, the effect of exogenous cannabinoids in the NTS on baroreflex sensivity for
control of heart rate in response to increases in arterial pressure, a vagally-mediated
autonomic response, is unknown.
Therefore, we evaluated the effect of NTS
microinjection of a non-selective cannabinoid receptor agonist, CP55,940, on baroreflex
sensitivity for control of heart rate in anesthetized young adult Sprague-Dawley rats.
Preliminary studies demonstrate a dose-related biphasic effect of NTS
cannabinoid receptor activation on baroreflex sensitivity,286 in which lower doses of
CP55,940 (0.12 and 1.2 pmol/120 nL) facilitate while a higher dose (12 pmol/120 nL)
impairs baroreflex sensitivity (Figure 1-1).
41
These responses are fully prevented or
reversed by co-administration of the selective CB1 receptor antagonist SR141716A
(Figure 1-1), suggesting that effects on the baroreflex are mediated by the CB1 rather than
CB2 receptor. We then tested whether the lower doses of CP55,940, which increase
baroreflex sensitivity in Sprague-Dawley rats, would similarly improve baroreflex
sensitivity in hypertensive (mRen2)27 with impaired baseline baroreflex function.287
Similar to baroreflex-evoked sympathetic nerve activity in SHR,46 there is no effect of
NTS cannabinoid receptor activation on baroreflex sensitivity for control of heart rate in
(mRen2)27 rats (Figure 1-2), consistent with differential modulation of the baroreflex by
cannabinoids in hypertension.
Baseline
2.5
**
+CP55,940
+SR141716A
(36 pmol)
Evoked BRS
(ms/mmHg)
2.0
1.5
1.0
**
0.5
SR
+
ol
pm
pm
1.
2
12
ol
+
SR
SR
ol
pm
0.
12
12
+
pm
ol
ol
pm
1.
2
pm
0.
12
Ve
hi
cl
e
ol
0.0
Figure 1-1. Effect of CB1 Agonist in NTS of Sprague-Dawley Rats.
Microinjection of CP55,940 into the NTS of Sprague-Dawley rats produced a dose-related biphasic effect
on baroreflex sensitivity for control of heart rate in response to increases in arterial pressure after 10
minutes. Subsequent microinjection of SR141716A (36 pmol/120 nL) reversed changes in baroreflex
sensitivity elicited by CP55,940. Pooled baseline is represented in the figure (N = 16). **P < 0.01 vs.
respective baselines; n = 5-6.
42
1.0
Baseline
+CP55,940
Evoked BRS
(ms/mmHg)
0.8
0.6
0.4
0.2
Figure 1-2. Effect of CB1 Agonist in
NTS of (mRen2)27 Rats.
There was no effect of 0.12- or 1.2-pmol
of CP55,940 microinjected into the NTS
of (mRen2)27 rats after 10 minutes.
Pooled baseline is represented in the
figure; n = 3 both groups.
ol
pm
1.
2
0.
12
pm
ol
0.0
These data raise additional questions. Neither endocannabinoid expression nor
activity has been investigated in (mRen2)27 rats, so it is unknown whether
endocannabinoid actions in the NTS contribute to the suppressed baseline baroreflex
sensitivity in this strain. However, the central and peripheral endocannabinoid system is
overactive in other animal models of hypertension and metabolic syndrome.181,
288
Moreover, signaling interactions between CB1 and AT1 receptors may function to
enhance the pathogenic properties of Ang II,233 previously shown to transactivate CB1
receptors via AT1 receptor-mediated stimulation of 2-AG production.234, 289 Therefore,
we hypothesize differential roles for the endocannabinoid system in modulation of
baroreflex sensitivity at the level of the NTS in rats with altered brain RAS activity, and
employ transgenic (mRen2)27 and ASrAOGEN as well as aged Sprague-Dawley rats to
investigate the functional role of endocannabinoids in influencing baroreflex function
during these conditions. We further hypothesize a role for the endocannabinoid system in
the maintenance of the Ang II-dependent hypertension and symptoms of metabolic
syndrome exhibited by (mRen2)27 rats.243 To evaluate this hypothesis, we assess effects
of acute and chronic systemic CB1 receptor blockade on blood pressure, metabolic profile
43
and conscious baroreflex function in this hypertensive strain. Accordingly, the specific
aims of the present studies are as follows:
Aim 1:
Determine if endocannabinoid tone at the level of the NTS contributes to
the differential resting baroreflex sensitivity for control of heart rate in rats
with high or low brain RAS activity using transgenic and older animals.
Aim 2:
Determine if systemic CB1 receptor blockade alters conscious blood
pressure and autonomic function, and improves metabolic profile in
hypertensive (mRen2)27 rats.
44
References
1.
Lenfant C, Chobanian AV, Jones DW, Roccella EJ. Seventh report of the Joint
National Committee on the Prevention, Detection, Evaluation, and Treatment of
High Blood Pressure (JNC 7): resetting the hypertension sails. Hypertension.
2003;41:1178-1179.
2.
Go AS, Mozaffarian D, Roger VL, et. a. Heart disease and stroke statistics--2014
update: a report from the american heart association. Circulation. 2014;129:e28e292.
3.
Brown CD, Higgins M, Donato KA, Rohde FC, Garrison R, Obarzanek E, Ernst
ND, Horan M. Body mass index and the prevalence of hypertension and
dyslipidemia. Obes Res. 2000;8:605-619.
4.
Flegal KM, Carroll MD, Ogden CL, Curtin LR. Prevalence and trends in obesity
among US adults, 1999-2008. JAMA. 2010;303:235-241.
5.
Wilson PW, D'Agostino RB, Sullivan L, Parise H, Kannel WB. Overweight and
obesity as determinants of cardiovascular risk: the Framingham experience. Arch
Intern Med. 2002;162:1867-1872.
6.
Wilson PW, Kannel WB. Obesity, diabetes, and risk of cardiovascular disease in
the elderly. Am J Geriatr Cardiol. 2002;11:119-123,125.
7.
Wofford MR, Smith G, Minor DS. The treatment of hypertension in obese
patients. Curr Hypertens Rep. 2008;10:143-150.
8.
Beckett NS, Peters R, Fletcher AE, Staessen JA, Liu L, Dumitrascu D,
Stoyanovsky V, Antikainen RL, Nikitin Y, Anderson C, Belhani A, Forette F,
Rajkumar C, Thijs L, Banya W, Bulpitt CJ. Treatment of hypertension in patients
80 years of age or older. N Engl J Med. 2008;358:1887-1898.
9.
Stokes GS. Management of hypertension in the elderly patient. Clin Interv Aging.
2009;4:379-389.
10.
Brook RD, Julius S. Autonomic imbalance, hypertension, and cardiovascular risk.
Am J Hypertens. 2000;13:112S-122S.
11.
Hall JE, Brands MW, Hildebrandt DA, Kuo J, Fitzgerald S. Role of sympathetic
nervous system and neuropeptides in obesity hypertension. Braz J Med Biol Res.
2000;33:605-618.
12.
Takatori S, Zamami Y, Hashikawa-Hobara N, Kawasaki H. Insulin resistanceinduced hypertension and a role of perivascular CGRPergic nerves. Curr Protein
Pept Sci. 2013;14:275-281.
45
13.
Thorens B. Glucose sensing and the pathogenesis of obesity and type 2 diabetes.
Int J Obes (Lond). 2008;32 Suppl 6:S62-71.
14.
Xiong XQ, Chen WW, Zhu GQ. Adipose afferent reflex: sympathetic activation
and obesity hypertension. Acta Physiol (Oxf). 2013
15.
Mollace V, Donato Di Paola E, Gratteri S, Nistico G. Age-related alterations in
cardiovascular responsiveness to clonidine infused into the nucleus tractus
solitarii in rats. Neuropharmacology. 1989;28:37-42.
16.
Itoh H, Bunag RD. Age-related reduction of reflex bradycardia in conscious rats
by catecholaminergic nucleus tractus solitarius lesions. Mech Ageing Dev.
1993;67:47-63.
17.
Reid IA. Interactions between ANG II, sympathetic nervous system, and
baroreceptor reflexes in regulation of blood pressure. Am J Physiol.
1992;262:E763-778.
18.
De Freitas AF. Cardiovascular regulation by the autonomic nervous system: a
paradigm of self-organization, complexity and chaos. Rev Port Cardiol.
2000;19:161-188.
19.
Thayer JF, Lane RD. The role of vagal function in the risk for cardiovascular
disease and mortality. Biol Psychol. 2007;74:224-242.
20.
Weber CS, Thayer JF, Rudat M, Wirtz PH, Zimmermann-Viehoff F, Thomas A,
Perschel FH, Arck PC, Deter HC. Low vagal tone is associated with impaired post
stress recovery of cardiovascular, endocrine, and immune markers. Eur J Appl
Physiol. 2010;109:201-211.
21.
Di Rienzo M, Parati G, Radaelli A, Castiglioni P. Baroreflex contribution to blood
pressure and heart rate oscillations: time scales, time-variant characteristics and
nonlinearities. Philos Trans A Math Phys Eng Sci. 2009;367:1301-1318.
22.
Nafz B, Persson PB. Renal arterial pressure variability. A role in blood pressure
control? Ann N Y Acad Sci. 2001;940:407-415.
23.
Sapru HN. Carotid chemoreflex. Neural pathways and transmitters. Adv Exp Med
Biol. 1996;410:357-364.
24.
Naslund E, Hellstrom PM. Appetite signaling: from gut peptides and enteric
nerves to brain. Physiol Behav. 2007;92:256-262.
25.
Andresen MC, Kunze DL. Nucleus tractus solitarius--gateway to neural
circulatory control. Annu Rev Physiol. 1994;56:93-116.
26.
Hart EC, Charkoudian N. Sympathetic neural mechanisms in human blood
pressure regulation. Curr Hypertens Rep. 2011;13:237-243.
46
27.
Arnold AC. Modulators of Baroreflex Sensitivity in Conditions Associated with
Hypertension. Department of Physiology and Pharmacology. 2009;Doctor of
Philosophy
28.
Head GA. Baroreflexes and cardiovascular regulation in hypertension. J
Cardiovasc Pharmacol. 1995;26 Suppl 2:S7-16.
29.
Osborn JW, Jacob F, Guzman P. A neural set point for the long-term control of
arterial pressure: beyond the arterial baroreceptor reflex. Am J Physiol Regul
Integr Comp Physiol. 2005;288:R846-855.
30.
Mancia G, Grassi G, Ferrari A, Zanchetti A. Reflex cardiovascular regulation in
humans. J Cardiovasc Pharmacol. 1985;7 Suppl 3:S152-159.
31.
Shibao C, Gamboa A, Diedrich A, Ertl AC, Chen KY, Byrne DW, Farley G,
Paranjape SY, Davis SN, Biaggioni I. Autonomic contribution to blood pressure
and metabolism in obesity. Hypertension. 2007;49:27-33.
32.
Thayer JF, Yamamoto SS, Brosschot JF. The relationship of autonomic
imbalance, heart rate variability and cardiovascular disease risk factors. Int J
Cardiol. 2010;141:122-131.
33.
Hall JE. Pathophysiology of obesity hypertension. Curr Hypertens Rep.
2000;2:139-147.
34.
Ferrari AU, Radaelli A, Centola M. Invited review: aging and the cardiovascular
system. J Appl Physiol (1985). 2003;95:2591-2597.
35.
Hotta H, Uchida S. Aging of the autonomic nervous system and possible
improvements in autonomic activity using somatic afferent stimulation. Geriatr
Gerontol Int. 2010;10 Suppl 1:S127-136.
36.
Landsberg L, Young JB. Fasting, feeding and regulation of the sympathetic
nervous system. N Engl J Med. 1978;298:1295-1301.
37.
Malpas SC. Sympathetic nervous system overactivity and its role in the
development of cardiovascular disease. Physiol Rev. 2010;90:513-557.
38.
Wofford MR, Anderson DC, Jr., Brown CA, Jones DW, Miller ME, Hall JE.
Antihypertensive effect of alpha- and beta-adrenergic blockade in obese and lean
hypertensive subjects. Am J Hypertens. 2001;14:694-698.
39.
Liu AJ, Ma XJ, Shen FM, Liu JG, Chen H, Su DF. Arterial baroreflex: a novel
target for preventing stroke in rat hypertension. Stroke. 2007;38:1916-1923.
40.
Rahmouni K. Differential Control of the Sympathetic Nervous System by Leptin:
Implications for Obesity. Clin Exp Pharmacol Physiol Suppl. 2007;34 Suppl:S8S10.
47
41.
Arnold AC, Shaltout HA, Gallagher PE, Diz DI. Leptin impairs cardiovagal
baroreflex function at the level of the solitary tract nucleus. Hypertension.
2009;54:1001-1008.
42.
McKernan AM, Calaresu FR. Insulin microinjection into the nucleus tractus
solitarii of the rat attenuates the baroreceptor reflex. J Auton Nerv Syst.
1996;61:128-138.
43.
Scherrer U, Sartori C. Insulin as a vascular and sympathoexcitatory hormone:
implications for blood pressure regulation, insulin sensitivity, and cardiovascular
morbidity. Circulation. 1997;96:4104-4113.
44.
Niederhoffer N, Schmid K, Szabo B. The peripheral sympathetic nervous system
is the major target of cannabinoids in eliciting cardiovascular depression. Naunyn
Schmiedebergs Arch Pharmacol. 2003;367:434-443.
45.
Seagard JL, Dean C, Patel S, Rademacher DJ, Hopp FA, Schmeling WT, Hillard
CJ. Anandamide content and interaction of endocannabinoid/GABA modulatory
effects in the NTS on baroreflex-evoked sympathoinhibition. Am J Physiol Heart
Circ Physiol. 2004;286:H992-1000.
46.
Brozoski DT, Dean C, Hopp FA, Hillard CJ, Seagard JL. Differential
endocannabinoid regulation of baroreflex-evoked sympathoinhibition in
normotensive versus hypertensive rats. Auton Neurosci. 2009;150:82-93.
47.
Lavoie JL, Sigmund CD. Minireview: overview of the renin-angiotensin system-an endocrine and paracrine system. Endocrinology. 2003;144:2179-2183.
48.
Welches WR, Brosnihan KB, Ferrario CM. A comparison of the properties and
enzymatic activities of three angiotensin processing enzymes: angiotensin
converting enzyme, prolyl endopeptidase and neutral endopeptidase 24.11. Life
Sci. 1993;52:1461-1480.
49.
Mehta PK, Griendling KK. Angiotensin II cell signaling: physiological and
pathological effects in the cardiovascular system. Am J Physiol Cell Physiol.
2007;292:C82-97.
50.
Carey RM, Jin XH, Siragy HM. Role of the angiotensin AT2 receptor in blood
pressure regulation and therapeutic implications. Am J Hypertens. 2001;14:98S102S.
51.
Diz DI, Pirro NT. Differential actions of angiotensin II and angiotensin-(1-7) on
transmitter release. Hypertension. 1992;19:II41-48.
52.
Schiavone MT, Santos RA, Brosnihan KB, Khosla MC, Ferrario CM. Release of
vasopressin from the rat hypothalamo-neurohypophysial system by angiotensin(1-7) heptapeptide. Proc Natl Acad Sci U S A. 1988;85:4095-4098.
48
53.
Santos RA, Simoes e Silva AC, Maric C, Silva DM, Machado RP, de Buhr I,
Heringer-Walther S, Pinheiro SV, Lopes MT, Bader M, Mendes EP, Lemos VS,
Campagnole-Santos MJ, Schultheiss HP, Speth R, Walther T. Angiotensin-(1-7)
is an endogenous ligand for the G protein-coupled receptor Mas. Proc Natl Acad
Sci U S A. 2003;100:8258-8263.
54.
Sampaio WO, Souza dos Santos RA, Faria-Silva R, da Mata Machado LT,
Schiffrin EL, Touyz RM. Angiotensin-(1-7) through receptor Mas mediates
endothelial nitric oxide synthase activation via Akt-dependent pathways.
Hypertension. 2007;49:185-192.
55.
Schelling P, Hutchinson JS, Ganten U, Sponer G, Ganten D. Impermeability of
the blood-cerebrospinal fluid barrier for angiotensin II in rats. Clin Sci Mol Med
Suppl. 1976;3:399s-402s.
56.
Xu P, Sriramula S, Lazartigues E. ACE2/ANG-(1-7)/Mas pathway in the brain:
the axis of good. Am J Physiol Regul Integr Comp Physiol. 2011;300:R804-817.
57.
Bickerton RK, Buckley JP. Evidence for a central mechanism in angiotensininduced hypertension. Proc Soc Exp Biol Med. 1961;106:836-843.
58.
Paul M, Poyan Mehr A, Kreutz R. Physiology of local renin-angiotensin systems.
Physiol Rev. 2006;86:747-803.
59.
O'Callaghan EL, Choong YT, Jancovski N, Allen AM. Central angiotensinergic
mechanisms associated with hypertension. Auton Neurosci. 2013;175:85-92.
60.
Ganten D, Hermann K, Bayer C, Unger T, Lang RE. Angiotensin synthesis in the
brain and increased turnover in hypertensive rats. Science. 1983;221:869-871.
61.
Song K, Allen AM, Paxinos G, Mendelsohn FA. Mapping of angiotensin II
receptor subtype heterogeneity in rat brain. J Comp Neurol. 1992;316:467-484.
62.
Bains JS, Potyok A, Ferguson AV. Angiotensin II actions in paraventricular
nucleus: functional evidence for neurotransmitter role in efferents originating in
subfornical organ. Brain Res. 1992;599:223-229.
63.
Rettig R, Healy DP, Printz MP. Cardiovascular effects of microinjections of
angiotensin II into the nucleus tractus solitarii. Brain Res. 1986;364:233-240.
64.
Suter C, Coote JH. Intrathecally administered angiotensin II increases
sympathetic activity in the rat. J Auton Nerv Syst. 1987;19:31-37.
65.
Diz DI, Barnes KL, Ferrario CM. Hypotensive actions of microinjections of
angiotensin II into the dorsal motor nucleus of the vagus. J Hypertens Suppl.
1984;2:S53-56.
49
66.
Allen AM, Mendelsohn FA, Gierobat ZJ, Blessing WW. Vasopressin Release
Following Microinjection of Angiotensin II into the Caudal Ventrolateral Medulla
Oblongata in the Anaesthetized Rabbit. J Neuroendocrinol. 1990;2:867-873.
67.
Andreatta SH, Averill DB, Santos RA, Ferrario CM. The ventrolateral medulla. A
new site of action of the renin-angiotensin system. Hypertension. 1988;11:I163166.
68.
Matsumura K, Averill DB, Ferrario CM. Angiotensin II acts at AT1 receptors in
the nucleus of the solitary tract to attenuate the baroreceptor reflex. Am J Physiol.
1998;275:R1611-1619.
69.
Allen AM. Inhibition of the hypothalamic paraventricular nucleus in
spontaneously hypertensive rats dramatically reduces sympathetic vasomotor
tone. Hypertension. 2002;39:275-280.
70.
Averill DB, Diz DI. Angiotensin peptides and baroreflex control of sympathetic
outflow: pathways and mechanisms of the medulla oblongata. Brain Res Bull.
2000;51:119-128.
71.
Vatta MS, Bianciotti LG, Papouchado ML, Locatelli AS, Polidoro-Arena J,
Fernandez BE. Modulation of noradrenergic neurotransmission by angiotensin II
and angiotensin III. Pharmacodyn. Therap. (Life Sci.). 1990;9:177-185.
72.
Block CH, Santos RA, Brosnihan KB, Ferrario CM. Immunocytochemical
localization of angiotensin-(1-7) in the rat forebrain. Peptides. 1988;9:1395-1401.
73.
Krob HA, Vinsant SL, Ferrario CM, Friedman DP. Angiotensin-(1-7)
immunoreactivity in the hypothalamus of the (mRen-2d)27 transgenic rat. Brain
Res. 1998;798:36-45.
74.
Santos RA, Brosnihan KB, Chappell MC, Pesquero J, Chernicky CL, Greene LJ,
Ferrario CM. Converting enzyme activity and angiotensin metabolism in the dog
brainstem. Hypertension. 1988;11:I153-157.
75.
Campagnole-Santos MJ, Diz DI, Santos RA, Khosla MC, Brosnihan KB, Ferrario
CM. Cardiovascular effects of angiotensin-(1-7) injected into the dorsal medulla
of rats. Am J Physiol. 1989;257:H324-329.
76.
Alzamora AC, Santos RA, Campagnole-Santos MJ. Hypotensive effect of ANG II
and ANG-(1-7) at the caudal ventrolateral medulla involves different
mechanisms. Am J Physiol Regul Integr Comp Physiol. 2002;283:R1187-1195.
77.
Gironacci MM, Vatta M, Rodriguez-Fermepin M, Fernandez BE, Pena C.
Angiotensin-(1-7) reduces norepinephrine release through a nitric oxide
mechanism in rat hypothalamus. Hypertension. 2000;35:1248-1252.
50
78.
Gironacci MM, Valera MS, Yujnovsky I, Pena C. Angiotensin-(1-7) inhibitory
mechanism of norepinephrine release in hypertensive rats. Hypertension.
2004;44:783-787.
79.
Campagnole-Santos MJ, Diz DI, Ferrario CM. Baroreceptor reflex modulation by
angiotensin II at the nucleus tractus solitarii. Hypertension. 1988;11:I167-171.
80.
Campagnole-Santos MJ, Heringer SB, Batista EN, Khosla MC, Santos RA.
Differential baroreceptor reflex modulation by centrally infused angiotensin
peptides. Am J Physiol. 1992;263:R89-94.
81.
Oliveira DR, Santos RA, Santos GF, Khosla M, Campagnole-Santos MJ. Changes
in the baroreflex control of heart rate produced by central infusion of selective
angiotensin antagonists in hypertensive rats. Hypertension. 1996;27:1284-1290.
82.
Gironacci MM, Yujnovsky I, Gorzalczany S, Taira C, Pena C. Angiotensin-(1-7)
inhibits the angiotensin II-enhanced norepinephrine release in coarcted
hypertensive rats. Regul Pept. 2004;118:45-49.
83.
Isa K, Arnold AC, Westwood BM, Chappell MC, Diz DI. Angiotensin-converting
enzyme inhibition, but not AT(1) receptor blockade, in the solitary tract nucleus
improves baroreflex sensitivity in anesthetized transgenic hypertensive
(mRen2)27 rats. Hypertens Res. 2011;34:1257-1262.
84.
Veerasingham SJ, Raizada MK. Brain renin-angiotensin system dysfunction in
hypertension: recent advances and perspectives. Br J Pharmacol. 2003;139:191202.
85.
Hutchinson JS, Mendelsohn FA, Doyle AE. Blood pressure responses of
conscious
normotensive
and
spontaneously hypertensive
rats
to
intracerebroventricular and peripheral administration of captopril. Hypertension.
1980;2:546-550.
86.
Szczepanska-Sadowska E, Paczwa P, Lon S, Ganten D. Increased pressor
function of central vasopressinergic system in hypertensive renin transgenic rats.
J Hypertens. 1998;16:1505-1514.
87.
Cangussu LM, de Castro UG, do Pilar Machado R, Silva ME, Ferreira PM, dos
Santos RA, Campagnole-Santos MJ, Alzamora AC. Angiotensin-(1-7) antagonist,
A-779, microinjection into the caudal ventrolateral medulla of renovascular
hypertensive rats restores baroreflex bradycardia. Peptides. 2009;30:1921-1927.
88.
Sakima A, Averill DB, Gallagher PE, Kasper SO, Tommasi EN, Ferrario CM, Diz
DI. Impaired heart rate baroreflex in older rats: role of endogenous angiotensin(1-7) at the nucleus tractus solitarii. Hypertension. 2005;46:333-340.
51
89.
Diz DI, Garcia-Espinosa MA, Gallagher PE, Ganten D, Ferrario CM, Averill DB.
Angiotensin-(1-7) and baroreflex function in nucleus tractus solitarii of
(mRen2)27 transgenic rats. J Cardiovasc Pharmacol. 2008;51:542-548.
90.
Haywood JR, Fink GD, Buggy J, Boutelle S, Johnson AK, Brody MJ. Prevention
of two-kidney, one-clip renal hypertension in rat by ablation of AV3V tissue. Am
J Physiol. 1983;245:H683-689.
91.
Buggy J, Fink GD, Johnson AK, Brody MJ. Prevention of the development of
renal hypertension by anteroventral third ventricular tissue lesions. Circ Res.
1977;40:I110-117.
92.
Marvar PJ, Thabet SR, Guzik TJ, Lob HE, McCann LA, Weyand C, Gordon FJ,
Harrison DG. Central and peripheral mechanisms of T-lymphocyte activation and
vascular inflammation produced by angiotensin II-induced hypertension. Circ
Res. 2010;107:263-270.
93.
Fink GD, Bruner CA, Mangiapane ML. Area postrema is critical for angiotensininduced hypertension in rats. Hypertension. 1987;9:355-361.
94.
Mangiapane ML, Skoog KM, Rittenhouse P, Blair ML, Sladek CD. Lesion of the
area postrema region attenuates hypertension in spontaneously hypertensive rats.
Circ Res. 1989;64:129-135.
95.
Chen AD, Zhang SJ, Yuan N, Xu Y, De W, Gao XY, Zhu GQ. Angiotensin AT1
receptors in paraventricular nucleus contribute to sympathetic activation and
enhanced cardiac sympathetic afferent reflex in renovascular hypertensive rats.
Exp Physiol. 2011;96:94-103.
96.
Kubo T, Yamaguchi H, Tsujimura M, Hagiwara Y, Fukumori R. An angiotensin
system in the anterior hypothalamic area anterior is involved in the maintenance
of hypertension in spontaneously hypertensive rats. Brain Res Bull. 2000;52:291296.
97.
Kubo T, Yamaguchi H, Tsujimura M, Hagiwara Y, Fukumori R. Blockade of
angiotensin receptors in the anterior hypothalamic preoptic area lowers blood
pressure in DOCA-salt hypertensive rats. Hypertens Res. 2000;23:109-118.
98.
McKinley MJ, Allen AM, May CN, McAllen RM, Oldfield BJ, Sly D,
Mendelsohn FA. Neural pathways from the lamina terminalis influencing
cardiovascular and body fluid homeostasis. Clin Exp Pharmacol Physiol.
2001;28:990-992.
99.
Shafton AD, Ryan A, Badoer E. Neurons in the hypothalamic paraventricular
nucleus send collaterals to the spinal cord and to the rostral ventrolateral medulla
in the rat. Brain Res. 1998;801:239-243.
52
100.
Tagawa T, Dampney RA. AT(1) receptors mediate excitatory inputs to rostral
ventrolateral medulla pressor neurons from hypothalamus. Hypertension.
1999;34:1301-1307.
101.
Howlett AC, Barth F, Bonner TI, Cabral G, Casellas P, Devane WA, Felder CC,
Herkenham M, Mackie K, Martin BR, Mechoulam R, Pertwee RG. International
Union of Pharmacology. XXVII. Classification of cannabinoid receptors.
Pharmacol Rev. 2002;54:161-202.
102.
Pertwee RG. The pharmacology of cannabinoid receptors and their ligands: an
overview. Int J Obes (Lond). 2006;30 Suppl 1:S13-18.
103.
Turu G, Hunyady L. Signal transduction of the CB1 cannabinoid receptor. J Mol
Endocrinol. 2010;44:75-85.
104.
Castillo PE, Younts TJ, Chavez AE, Hashimotodani Y. Endocannabinoid
signaling and synaptic function. Neuron. 2012;76:70-81.
105.
Pacher P, Mukhopadhyay P, Mohanraj R, Godlewski G, Batkai S, Kunos G.
Modulation of the endocannabinoid system in cardiovascular disease: therapeutic
potential and limitations. Hypertension. 2008;52:601-607.
106.
Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral
receptor for cannabinoids. Nature. 1993;365:61-65.
107.
Despres JP. The endocannabinoid system: a new target for the regulation of
energy balance and metabolism. Crit Pathw Cardiol. 2007;6:46-50.
108.
Pacher P, Batkai S, Kunos G. The endocannabinoid system as an emerging target
of pharmacotherapy. Pharmacol Rev. 2006;58:389-462.
109.
Van Sickle MD, Duncan M, Kingsley PJ, Mouihate A, Urbani P, Mackie K, Stella
N, Makriyannis A, Piomelli D, Davison JS, Marnett LJ, Di Marzo V, Pittman QJ,
Patel KD, Sharkey KA. Identification and functional characterization of brainstem
cannabinoid CB2 receptors. Science. 2005;310:329-332.
110.
Gong JP, Onaivi ES, Ishiguro H, Liu QR, Tagliaferro PA, Brusco A, Uhl GR.
Cannabinoid CB2 receptors: immunohistochemical localization in rat brain. Brain
Res. 2006;1071:10-23.
111.
Montecucco F, Lenglet S, Braunersreuther V, Burger F, Pelli G, Bertolotto M,
Mach F, Steffens S. CB(2) cannabinoid receptor activation is cardioprotective in a
mouse model of ischemia/reperfusion. J Mol Cell Cardiol. 2009;46:612-620.
112.
Zhao Y, Yuan Z, Liu Y, Xue J, Tian Y, Liu W, Zhang W, Shen Y, Xu W, Liang
X, Chen T. Activation of cannabinoid CB2 receptor ameliorates atherosclerosis
associated with suppression of adhesion molecules. J Cardiovasc Pharmacol.
2010;55:292-298.
53
113.
Steffens S, Pacher P. Targeting cannabinoid receptor CB(2) in cardiovascular
disorders: promises and controversies. Br J Pharmacol. 2012;167:313-323.
114.
Atwood BK, Mackie K. CB2: a cannabinoid receptor with an identity crisis. Br J
Pharmacol. 2010;160:467-479.
115.
Di Marzo V. Endocannabinoids: synthesis and degradation. Rev Physiol Biochem
Pharmacol. 2008;160:1-24.
116.
Di Marzo V. Endocannabinoid signaling in the brain: biosynthetic mechanisms in
the limelight. Nat Neurosci. 2011;14:9-15.
117.
Ueda N, Tsuboi K, Uyama T. Enzymological studies on the biosynthesis of Nacylethanolamines. Biochim Biophys Acta. 2010;1801:1274-1285.
118.
Leung D, Saghatelian A, Simon GM, Cravatt BF. Inactivation of N-acyl
phosphatidylethanolamine phospholipase D reveals multiple mechanisms for the
biosynthesis of endocannabinoids. Biochemistry. 2006;45:4720-4726.
119.
Murataeva N, Straiker A, Mackie K. Parsing the players: 2-AG synthesis and
degradation in the CNS. Br J Pharmacol. 2013
120.
Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M, Watanabe M.
Endocannabinoid-mediated control of synaptic transmission. Physiol Rev.
2009;89:309-380.
121.
Higgs HN, Glomset JA. Identification of a phosphatidic acid-preferring
phospholipase A1 from bovine brain and testis. Proc Natl Acad Sci U S A.
1994;91:9574-9578.
122.
Nakane S, Oka S, Arai S, Waku K, Ishima Y, Tokumura A, Sugiura T. 2Arachidonoyl-sn-glycero-3-phosphate,
an
arachidonic
acid-containing
lysophosphatidic acid: occurrence and rapid enzymatic conversion to 2arachidonoyl-sn-glycerol, a cannabinoid receptor ligand, in rat brain. Arch
Biochem Biophys. 2002;402:51-58.
123.
Zhang L, Wang M, Bisogno T, Di Marzo V, Alger BE. Endocannabinoids
generated by Ca2+ or by metabotropic glutamate receptors appear to arise from
different pools of diacylglycerol lipase. PLoS One. 2011;6:e16305.
124.
Blankman JL, Simon GM, Cravatt BF. A comprehensive profile of brain enzymes
that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem Biol.
2007;14:1347-1356.
125.
Van Laere K, Koole M, Sanabria Bohorquez SM, Goffin K, Guenther I, Belanger
MJ, Cote J, Rothenberg P, De Lepeleire I, Grachev ID, Hargreaves RJ, Bormans
G, Burns HD. Whole-body biodistribution and radiation dosimetry of the human
54
cannabinoid type-1 receptor ligand 18F-MK-9470 in healthy subjects. J Nucl
Med. 2008;49:439-445.
126.
Devane WA, Dysarz FA, 3rd, Johnson MR, Melvin LS, Howlett AC.
Determination and characterization of a cannabinoid receptor in rat brain. Mol
Pharmacol. 1988;34:605-613.
127.
Martin BR. Cellular effects of cannabinoids. Pharmacol Rev. 1986;38:45-74.
128.
Howlett AC, Fleming RM. Cannabinoid inhibition of adenylate cyclase.
Pharmacology of the response in neuroblastoma cell membranes. Mol Pharmacol.
1984;26:532-538.
129.
Howlett AC, Qualy JM, Khachatrian LL. Involvement of Gi in the inhibition of
adenylate cyclase by cannabimimetic drugs. Mol Pharmacol. 1986;29:307-313.
130.
Matsuda LA, Lolait SJ, Brownstein MJ, Young AC, Bonner TI. Structure of a
cannabinoid receptor and functional expression of the cloned cDNA. Nature.
1990;346:561-564.
131.
Howlett AC, Gray AJ, Hunter JM, Willardson BM. Role of molecular chaperones
in G protein beta5/regulator of G protein signaling dimer assembly and G protein
betagamma dimer specificity. J Biol Chem. 2009;284:16386-16399.
132.
Howlett AC. Efficacy in CB1 receptor-mediated signal transduction. Br J
Pharmacol. 2004;142:1209-1218.
133.
Bonhaus DW, Chang LK, Kwan J, Martin GR. Dual activation and inhibition of
adenylyl cyclase by cannabinoid receptor agonists: evidence for agonist-specific
trafficking of intracellular responses. J Pharmacol Exp Ther. 1998;287:884-888.
134.
Glass M, Felder CC. Concurrent stimulation of cannabinoid CB1 and dopamine
D2 receptors augments cAMP accumulation in striatal neurons: evidence for a Gs
linkage to the CB1 receptor. J Neurosci. 1997;17:5327-5333.
135.
Mukhopadhyay S, Howlett AC. Chemically distinct ligands promote differential
CB1 cannabinoid receptor-Gi protein interactions. Mol Pharmacol.
2005;67:2016-2024.
136.
Jarrahian A, Watts VJ, Barker EL. D2 dopamine receptors modulate Galphasubunit coupling of the CB1 cannabinoid receptor. J Pharmacol Exp Ther.
2004;308:880-886.
137.
Howlett AC, Blume LC, Dalton GD. CB(1) cannabinoid receptors and their
associated proteins. Curr Med Chem. 2010;17:1382-1393.
138.
Freund TF, Katona I, Piomelli D. Role of endogenous cannabinoids in synaptic
signaling. Physiol Rev. 2003;83:1017-1066.
55
139.
Lovinger DM. Presynaptic modulation by endocannabinoids. Handb Exp
Pharmacol. 2008:435-477.
140.
Marsicano G, Lutz B. Neuromodulatory functions of the endocannabinoid system.
J Endocrinol Invest. 2006;29:27-46.
141.
Mackie K, Devane WA, Hille B. Anandamide, an endogenous cannabinoid,
inhibits calcium currents as a partial agonist in N18 neuroblastoma cells. Mol
Pharmacol. 1993;44:498-503.
142.
Mackie K, Lai Y, Westenbroek R, Mitchell R. Cannabinoids activate an inwardly
rectifying potassium conductance and inhibit Q-type calcium currents in AtT20
cells transfected with rat brain cannabinoid receptor. J Neurosci. 1995;15:65526561.
143.
Robbe D, Alonso G, Duchamp F, Bockaert J, Manzoni OJ. Localization and
mechanisms of action of cannabinoid receptors at the glutamatergic synapses of
the mouse nucleus accumbens. J Neurosci. 2001;21:109-116.
144.
Deadwyler SA, Hampson RE, Mu J, Whyte A, Childers S. Cannabinoids
modulate voltage sensitive potassium A-current in hippocampal neurons via a
cAMP-dependent process. J Pharmacol Exp Ther. 1995;273:734-743.
145.
Bidaut-Russell M, Devane WA, Howlett AC. Cannabinoid receptors and
modulation of cyclic AMP accumulation in the rat brain. J Neurochem.
1990;55:21-26.
146.
Wade MR, Tzavara ET, Nomikos GG. Cannabinoids reduce cAMP levels in the
striatum of freely moving rats: an in vivo microdialysis study. Brain Res.
2004;1005:117-123.
147.
Felder CC, Joyce KE, Briley EM, Glass M, Mackie KP, Fahey KJ, Cullinan GJ,
Hunden DC, Johnson DW, Chaney MO, Koppel GA, Brownstein M. LY320135,
a novel cannabinoid CB1 receptor antagonist, unmasks coupling of the CB1
receptor to stimulation of cAMP accumulation. J Pharmacol Exp Ther.
1998;284:291-297.
148.
Maneuf YP, Brotchie JM. Paradoxical action of the cannabinoid WIN 55,212-2 in
stimulated and basal cyclic AMP accumulation in rat globus pallidus slices. Br J
Pharmacol. 1997;120:1397-1398.
149.
Busch L, Sterin-Borda L, Borda E. Expression and biological effects of CB1
cannabinoid receptor in rat parotid gland. Biochem Pharmacol. 2004;68:17671774.
150.
Patel TB, Du Z, Pierre S, Cartin L, Scholich K. Molecular biological approaches
to unravel adenylyl cyclase signaling and function. Gene. 2001;269:13-25.
56
151.
Rhee MH, Bayewitch M, Avidor-Reiss T, Levy R, Vogel Z. Cannabinoid receptor
activation differentially regulates the various adenylyl cyclase isozymes. J
Neurochem. 1998;71:1525-1534.
152.
Hampson RE, Evans GJ, Mu J, Zhuang SY, King VC, Childers SR, Deadwyler
SA. Role of cyclic AMP dependent protein kinase in cannabinoid receptor
modulation of potassium "A-current" in cultured rat hippocampal neurons. Life
Sci. 1995;56:2081-2088.
153.
Childers SR, Deadwyler SA. Role of cyclic AMP in the actions of cannabinoid
receptors. Biochem Pharmacol. 1996;52:819-827.
154.
Wetzker R, Bohmer FD. Transactivation joins multiple tracks to the ERK/MAPK
cascade. Nat Rev Mol Cell Biol. 2003;4:651-657.
155.
Roux PP, Blenis J. ERK and p38 MAPK-activated protein kinases: a family of
protein kinases with diverse biological functions. Microbiol Mol Biol Rev.
2004;68:320-344.
156.
Rose BA, Force T, Wang Y. Mitogen-activated protein kinase signaling in the
heart: angels versus demons in a heart-breaking tale. Physiol Rev. 2010;90:15071546.
157.
Dalton GD, Bass CE, Van Horn CG, Howlett AC. Signal transduction via
cannabinoid receptors. CNS Neurol Disord Drug Targets. 2009;8:422-431.
158.
Wartmann M, Campbell D, Subramanian A, Burstein SH, Davis RJ. The MAP
kinase signal transduction pathway is activated by the endogenous cannabinoid
anandamide. FEBS Lett. 1995;359:133-136.
159.
Bouaboula M, Poinot-Chazel C, Bourrie B, Canat X, Calandra B, RinaldiCarmona M, Le Fur G, Casellas P. Activation of mitogen-activated protein
kinases by stimulation of the central cannabinoid receptor CB1. Biochem J.
1995;312 ( Pt 2):637-641.
160.
Sanchez C, Galve-Roperh I, Rueda D, Guzman M. Involvement of sphingomyelin
hydrolysis and the mitogen-activated protein kinase cascade in the Delta9tetrahydrocannabinol-induced stimulation of glucose metabolism in primary
astrocytes. Mol Pharmacol. 1998;54:834-843.
161.
Galve-Roperh I, Rueda D, Gomez del Pulgar T, Velasco G, Guzman M.
Mechanism of extracellular signal-regulated kinase activation by the CB(1)
cannabinoid receptor. Mol Pharmacol. 2002;62:1385-1392.
162.
Gomez del Pulgar T, Velasco G, Guzman M. The CB1 cannabinoid receptor is
coupled to the activation of protein kinase B/Akt. Biochem J. 2000;347:369-373.
57
163.
Melck D, Rueda D, Galve-Roperh I, De Petrocellis L, Guzman M, Di Marzo V.
Involvement of the cAMP/protein kinase A pathway and of mitogen-activated
protein kinase in the anti-proliferative effects of anandamide in human breast
cancer cells. FEBS Lett. 1999;463:235-240.
164.
Derkinderen P, Valjent E, Toutant M, Corvol JC, Enslen H, Ledent C, Trzaskos J,
Caboche J, Girault JA. Regulation of extracellular signal-regulated kinase by
cannabinoids in hippocampus. J Neurosci. 2003;23:2371-2382.
165.
Valjent E, Pages C, Rogard M, Besson MJ, Maldonado R, Caboche J. Delta 9tetrahydrocannabinol-induced MAPK/ERK and Elk-1 activation in vivo depends
on dopaminergic transmission. Eur J Neurosci. 2001;14:342-352.
166.
Derkinderen P, Ledent C, Parmentier M, Girault JA. Cannabinoids activate p38
mitogen-activated protein kinases through CB1 receptors in hippocampus. J
Neurochem. 2001;77:957-960.
167.
Rubino T, Forlani G, Vigano D, Zippel R, Parolaro D. Modulation of extracellular
signal-regulated kinases cascade by chronic delta 9-tetrahydrocannabinol
treatment. Mol Cell Neurosci. 2004;25:355-362.
168.
Benowitz NL, Jones RT. Cardiovascular effects of prolonged delta-9tetrahydrocannabinol ingestion. Clin Pharmacol Ther. 1975;18:287-297.
169.
Dewey WL, Harris LS, Howes JF, Kennedy JS, Granchelli FE, Pars HG, Razdan
RK. Pharmacology of some marijuana constituents and 2 heterocyclic analogues.
Nature. 1970;226:1265-1267.
170.
Hardman HF, Domino EF, Seevers MH. General pharmacological actions of
some synthetic tetrahydrocannabinol derivatives. Pharmacol Rev. 1971;23:295315.
171.
Graham JD, Li DM. Cardiovascular and respiratory effects of cannabis in cat and
rat. Br J Pharmacol. 1973;49:1-10.
172.
Cavero I, Solomon T, Buckley JP, Jandhyala BS. Studies on the hypotensive
activity of (-)-delta9-trans-tetrahydrocannabinol in anesthetized dogs. Res
Commun Chem Pathol Pharmacol. 1973;6:527-540.
173.
Perez-Reyes M, Lipton MA, Timmons MC, Wall ME, Brine DR, Davis KH.
Pharmacology of orally administered 9 -tetrahydrocannabinol. Clin Pharmacol
Ther. 1973;14:48-55.
174.
Vollmer RR, Cavero I, Ertel RJ, Solomon TA, Buckley JP. Role of the central
autonomic nervous system in the hypotension and bradycardia induced by (-)delta 9-trans-tetrahydrocannabinol. J Pharm Pharmacol. 1974;26:186-192.
58
175.
Adams MD, Earnhardt JT, Dewey WL, Harris LS. Vasoconstrictor actions of
delta8- and delta9-tetrahydrocannabinol in the rat. J Pharmacol Exp Ther.
1976;196:649-656.
176.
Varga K, Lake KD, Huangfu D, Guyenet PG, Kunos G. Mechanism of the
hypotensive action of anandamide in anesthetized rats. Hypertension.
1996;28:682-686.
177.
Ibrahim BM, Abdel-Rahman AA. Role of brainstem GABAergic signaling in
central cannabinoid receptor evoked sympathoexcitation and pressor responses in
conscious rats. Brain Res. 2011;1414:1-9.
178.
Bonz A, Laser M, Kullmer S, Kniesch S, Babin-Ebell J, Popp V, Ertl G, Wagner
JA. Cannabinoids acting on CB1 receptors decrease contractile performance in
human atrial muscle. J Cardiovasc Pharmacol. 2003;41:657-664.
179.
Stanley C, O'Sullivan SE. Vascular targets for cannabinoids: animal and human
studies. Br J Pharmacol. 2013
180.
Shimokawa A, Kunitake T, Takasaki M, Kannan H. Differential effects of
anesthetics on sympathetic nerve activity and arterial baroreceptor reflex in
chronically instrumented rats. J Auton Nerv Syst. 1998;72:46-54.
181.
Batkai S, Pacher P, Osei-Hyiaman D, Radaeva S, Liu J, Harvey-White J,
Offertaler L, Mackie K, Rudd MA, Bukoski RD, Kunos G. Endocannabinoids
acting at cannabinoid-1 receptors regulate cardiovascular function in
hypertension. Circulation. 2004;110:1996-2002.
182.
Wheal AJ, Bennett T, Randall MD, Gardiner SM. Cardiovascular effects of
cannabinoids in conscious spontaneously hypertensive rats. Br J Pharmacol.
2007;152:717-724.
183.
Ho WS, Gardiner SM. Acute hypertension reveals depressor and vasodilator
effects of cannabinoids in conscious rats. Br J Pharmacol. 2009;156:94-104.
184.
Niederhoffer N, Szabo B. Effect of the cannabinoid receptor agonist WIN55212-2
on sympathetic cardiovascular regulation. Br J Pharmacol. 1999;126:457-466.
185.
Pacher P, Batkai S, Kunos G. Cardiovascular pharmacology of cannabinoids.
Handb Exp Pharmacol. 2005:599-625.
186.
Sugamura K, Sugiyama S, Nozaki T, Matsuzawa Y, Izumiya Y, Miyata K,
Nakayama M, Kaikita K, Obata T, Takeya M, Ogawa H. Activated
endocannabinoid system in coronary artery disease and antiinflammatory effects
of cannabinoid 1 receptor blockade on macrophages. Circulation. 2009;119:2836.
59
187.
Naito Y, Tsujino T, Akahori H, Ohyanagi M, Mitsuno M, Miyamoto Y,
Masuyama T. Augmented cannabinoid receptors expression in human aortic valve
stenosis. Int J Cardiol. 2010;145:535-537.
188.
Godlewski G, Alapafuja SO, Batkai S, Nikas SP, Cinar R, Offertaler L, OseiHyiaman D, Liu J, Mukhopadhyay B, Harvey-White J, Tam J, Pacak K,
Blankman JL, Cravatt BF, Makriyannis A, Kunos G. Inhibitor of fatty acid amide
hydrolase normalizes cardiovascular function in hypertension without adverse
metabolic effects. Chem Biol. 2010;17:1256-1266.
189.
Marcil J, Thibault C, Anand-Srivastava MB. Enhanced expression of Gi-protein
precedes the development of blood pressure in spontaneously hypertensive rats. J
Mol Cell Cardiol. 1997;29:1009-1022.
190.
Kost CK, Jr., Herzer WA, Li PJ, Jackson EK. Pertussis toxin-sensitive G-proteins
and regulation of blood pressure in the spontaneously hypertensive rat. Clin Exp
Pharmacol Physiol. 1999;26:449-455.
191.
Ge C, Garcia R, Anand-Srivastava MB. Enhanced expression of Gialpha protein
and adenylyl cyclase signaling in aortas from 1 kidney 1 clip hypertensive rats.
Can J Physiol Pharmacol. 2006;84:739-746.
192.
Mailleux P, Vanderhaeghen JJ. Distribution of neuronal cannabinoid receptor in
the adult rat brain: a comparative receptor binding radioautography and in situ
hybridization histochemistry. Neuroscience. 1992;48:655-668.
193.
Burdyga G, Varro A, Dimaline R, Thompson DG, Dockray GJ. Expression of
cannabinoid CB1 receptors by vagal afferent neurons: kinetics and role in
influencing neurochemical phenotype. Am J Physiol Gastrointest Liver Physiol.
2010;299:G63-69.
194.
Himmi T, Perrin J, El Ouazzani T, Orsini JC. Neuronal responses to cannabinoid
receptor ligands in the solitary tract nucleus. Eur J Pharmacol. 1998;359:49-54.
195.
Seagard JL, Hopp FA, Hillard CJ, Dean C. Effects of endocannabinoids on
discharge of baroreceptive NTS neurons. Neurosci Lett. 2005;381:334-339.
196.
Brozoski DT, Dean C, Hopp FA, Seagard JL. Uptake blockade of
endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition.
Brain Res. 2005;1059:197-202.
197.
Catelli JM, Sved AF. Enhanced pressor response to GABA in the nucleus tractus
solitarii of the spontaneously hypertensive rat. Eur J Pharmacol. 1988;151:243248.
198.
Mei L, Zhang J, Mifflin S. Hypertension alters GABA receptor-mediated
inhibition of neurons in the nucleus of the solitary tract. Am J Physiol Regul
Integr Comp Physiol. 2003;285:R1276-1286.
60
199.
Ribeiro JM, Santos RA, Pesquero JB, Bader M, Krieger EM. Autonomic control
in rats with overactivity of tissue renin-angiotensin or kallikrein-kinin system.
Regul Pept. 2005;129:155-159.
200.
Zhang Q, Yao F, O'Rourke ST, Qian SY, Sun C. Angiotensin II enhances
GABA(B) receptor-mediated responses and expression in nucleus tractus solitarii
of rats. Am J Physiol Heart Circ Physiol. 2009;297:H1837-1844.
201.
Gupta S, Gupta BM. Metabolic syndrome: diabetes and cardiovascular disease.
Indian Heart J. 2006;58:149-152.
202.
Pacholczyk M, Ferenc T, Kowalski J. [The metabolic syndrome. Part I:
definitions and diagnostic criteria for its identification. Epidemiology and
relationship with cardiovascular and type 2 diabetes risk]. Postepy Hig Med Dosw
(Online). 2008;62:530-542.
203.
Barth RJ. Insulin resistance, obesity and the metabolic syndrome. S D Med.
2011;Spec No:22-27.
204.
Romero-Zerbo SY, Bermudez-Silva FJ. Cannabinoids, eating behaviour, and
energy homeostasis. Drug Test Anal. 2014;6:52-58.
205.
Vettor R, Pagano C. The role of the endocannabinoid system in lipogenesis and
fatty acid metabolism. Best Pract Res Clin Endocrinol Metab. 2009;23:51-63.
206.
Cota D, Marsicano G, Tschop M, Grubler Y, Flachskamm C, Schubert M, Auer
D, Yassouridis A, Thone-Reineke C, Ortmann S, Tomassoni F, Cervino C, Nisoli
E, Linthorst AC, Pasquali R, Lutz B, Stalla GK, Pagotto U. The endogenous
cannabinoid system affects energy balance via central orexigenic drive and
peripheral lipogenesis. J Clin Invest. 2003;112:423-431.
207.
Osei-Hyiaman D, DePetrillo M, Pacher P, Liu J, Radaeva S, Batkai S, HarveyWhite J, Mackie K, Offertaler L, Wang L, Kunos G. Endocannabinoid activation
at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to dietinduced obesity. J Clin Invest. 2005;115:1298-1305.
208.
Osei-Hyiaman D, Liu J, Zhou L, Godlewski G, Harvey-White J, Jeong WI, Batkai
S, Marsicano G, Lutz B, Buettner C, Kunos G. Hepatic CB1 receptor is required
for development of diet-induced steatosis, dyslipidemia, and insulin and leptin
resistance in mice. J Clin Invest. 2008;118:3160-3169.
209.
Kim W, Lao Q, Shin YK, Carlson OD, Lee EK, Gorospe M, Kulkarni RN, Egan
JM. Cannabinoids induce pancreatic beta-cell death by directly inhibiting insulin
receptor activation. Sci Signal. 2012;5:ra23.
210.
Poirier B, Bidouard JP, Cadrouvele C, Marniquet X, Staels B, O'Connor SE,
Janiak P, Herbert JM. The anti-obesity effect of rimonabant is associated with an
improved serum lipid profile. Diabetes Obes Metab. 2005;7:65-72.
61
211.
Thanos PK, Ramalhete RC, Michaelides M, Piyis YK, Wang GJ, Volkow ND.
Leptin receptor deficiency is associated with upregulation of cannabinoid 1
receptors in limbic brain regions. Synapse. 2008;62:637-642.
212.
Song D, Bandsma RH, Xiao C, Xi L, Shao W, Jin T, Lewis GF. Acute
cannabinoid receptor type 1 (CB1R) modulation influences insulin sensitivity by
an effect outside the central nervous system in mice. Diabetologia. 2011;54:11811189.
213.
O'Hare JD, Zielinski E, Cheng B, Scherer T, Buettner C. Central endocannabinoid
signaling regulates hepatic glucose production and systemic lipolysis. Diabetes.
2011;60:1055-1062.
214.
Hiley CR. Endocannabinoids and the heart. J Cardiovasc Pharmacol.
2009;53:267-276.
215.
Batkai S, Pacher P. Endocannabinoids and cardiac contractile function:
pathophysiological implications. Pharmacol Res. 2009;60:99-106.
216.
Lipez-Miranda V, Herradon E, Martin MI. Vasorelaxation caused by
cannabinoids: mechanisms in different vascular beds. Curr Vasc Pharmacol.
2008;6:335-346.
217.
Kunos G, Tam J. The case for peripheral CB(1) receptor blockade in the treatment
of visceral obesity and its cardiometabolic complications. Br J Pharmacol.
2011;163:1423-1431.
218.
Janiak P, Poirier B, Bidouard JP, Cadrouvele C, Pierre F, Gouraud L, Barbosa I,
Dedio J, Maffrand JP, Le Fur G, O'Connor S, Herbert JM. Blockade of
cannabinoid CB1 receptors improves renal function, metabolic profile, and
increased survival of obese Zucker rats. Kidney Int. 2007;72:1345-1357.
219.
Maynadier M, Basile I, Gary-Bobo M. Adiponectin normalization: a clue to the
anti-metabolic syndrome action of rimonabant. Drug Discov Today. 2009;14:192197.
220.
Irwin N, Hunter K, Frizzell N, Flatt PR. Antidiabetic effects of sub-chronic
administration of the cannabinoid receptor (CB1) antagonist, AM251, in obese
diabetic (ob/ob) mice. Eur J Pharmacol. 2008;581:226-233.
221.
Mingorance C, Alvarez de Sotomayor M, Jimenez-Palacios FJ, Callejon Mochon
M, Casto C, Marhuenda E, Herrera MD. Effects of chronic treatment with the
CB1 antagonist, rimonabant on the blood pressure, and vascular reactivity of
obese Zucker rats. Obesity (Silver Spring). 2009;17:1340-1347.
222.
Russell JC, Kelly SE, Diane A, Wang Y, Mangat R, Novak S, Vine DF, Proctor
SD. Rimonabant-mediated changes in intestinal lipid metabolism and improved
62
renal vascular dysfunction in the JCR:LA-cp rat model of prediabetic metabolic
syndrome. Am J Physiol Gastrointest Liver Physiol. 2010;299:G507-516.
223.
Bell-Anderson KS, Aouad L, Williams H, Sanz FR, Phuyal J, Larter CZ, Farrell
GC, Caterson ID. Coordinated improvement in glucose tolerance, liver steatosis
and obesity-associated inflammation by cannabinoid 1 receptor antagonism in fat
Aussie mice. Int J Obes (Lond). 2011;35:1539-1548.
224.
Despres JP, Golay A, Sjostrom L. Effects of rimonabant on metabolic risk factors
in overweight patients with dyslipidemia. N Engl J Med. 2005;353:2121-2134.
225.
Van Gaal LF, Rissanen AM, Scheen AJ, Ziegler O, Rossner S. Effects of the
cannabinoid-1 receptor blocker rimonabant on weight reduction and
cardiovascular risk factors in overweight patients: 1-year experience from the
RIO-Europe study. Lancet. 2005;365:1389-1397.
226.
Scheen AJ, Finer N, Hollander P, Jensen MD, Van Gaal LF. Efficacy and
tolerability of rimonabant in overweight or obese patients with type 2 diabetes: a
randomised controlled study. Lancet. 2006;368:1660-1672.
227.
Pi-Sunyer FX, Aronne LJ, Heshmati HM, Devin J, Rosenstock J. Effect of
rimonabant, a cannabinoid-1 receptor blocker, on weight and cardiometabolic risk
factors in overweight or obese patients: RIO-North America: a randomized
controlled trial. JAMA. 2006;295:761-775.
228.
Van Gaal LF, Scheen AJ, Rissanen AM, Rossner S, Hanotin C, Ziegler O. Longterm effect of CB1 blockade with rimonabant on cardiometabolic risk factors: two
year results from the RIO-Europe Study. Eur Heart J. 2008;29:1761-1771.
229.
Christopoulou FD, Kiortsis DN. An overview of the metabolic effects of
rimonabant in randomized controlled trials: potential for other cannabinoid 1
receptor blockers in obesity. J Clin Pharm Ther. 2011;36:10-18.
230.
Ruilope LM, Despres JP, Scheen A, Pi-Sunyer X, Mancia G, Zanchetti A, Van
Gaal L. Effect of rimonabant on blood pressure in overweight/obese patients
with/without co-morbidities: analysis of pooled RIO study results. J Hypertens.
2008;26:357-367.
231.
Sam AH, Salem V, Ghatei MA. Rimonabant: From RIO to Ban. J Obes.
2011;2011:432607.
232.
Tiyerili V, Zimmer S, Jung S, Wassmann K, Naehle CP, Lutjohann D, Zimmer A,
Nickenig G, Wassmann S. CB1 receptor inhibition leads to decreased vascular
AT1 receptor expression, inhibition of oxidative stress and improved endothelial
function. Basic Res Cardiol. 2010;105:465-477.
63
233.
Rozenfeld R, Gupta A, Gagnidze K, Lim MP, Gomes I, Lee-Ramos D, Nieto N,
Devi LA. AT1R-CB(1)R heteromerization reveals a new mechanism for the
pathogenic properties of angiotensin II. EMBO J. 2011;30:2350-2363.
234.
Turu G, Simon A, Gyombolai P, Szidonya L, Bagdy G, Lenkei Z, Hunyady L.
The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptorstimulated cannabinoid CB1 receptor activity. J Biol Chem. 2007;282:7753-7757.
235.
Turu G, Varnai P, Gyombolai P, Szidonya L, Offertaler L, Bagdy G, Kunos G,
Hunyady L. Paracrine transactivation of the CB1 cannabinoid receptor by AT1
angiotensin and other Gq/11 protein-coupled receptors. J Biol Chem.
2009;284:16914-16921.
236.
Grisk O, Rettig R. Interactions between the sympathetic nervous system and the
kidneys in arterial hypertension. Cardiovasc Res. 2004;61:238-246.
237.
Johns EJ. Angiotensin II in the brain and the autonomic control of the kidney. Exp
Physiol. 2005;90:163-168.
238.
Janiak P, Bidouard JP, Cadrouvele C, Poirier B, Gouraud L, Grataloup Y, Pierre
F, Bruneval P, O'Connor SE, Herbert JM. Long-term blockade of angiotensin
AT1 receptors increases survival of obese Zucker rats. Eur J Pharmacol.
2006;534:271-279.
239.
Rodriguez-Garcia JL, Paule A, Dominguez J, Garcia-Escribano JR, Vazquez M.
Changes in plasma norepinephrine and endothelin levels and metabolic profile
after AT1-receptor blockade in human hypertension. Am J Cardiol.
2000;85:1147-1150, A1110.
240.
Yang G, Merrill DC, Thompson MW, Robillard JE, Sigmund CD. Functional
expression of the human angiotensinogen gene in transgenic mice. J Biol Chem.
1994;269:32497-32502.
241.
Sakima A, Averill DB, Kasper SO, Jackson L, Ganten D, Ferrario CM, Gallagher
PE, Diz DI. Baroreceptor reflex regulation in anesthetized transgenic rats with
low glia-derived angiotensinogen. Am J Physiol Heart Circ Physiol.
2007;292:H1412-1419.
242.
Yang G, Gray TS, Sigmund CD, Cassell MD. The angiotensinogen gene is
expressed in both astrocytes and neurons in murine central nervous system. Brain
Res. 1999;817:123-131.
243.
Kasper SO, Carter CS, Ferrario CM, Ganten D, Ferder LF, Sonntag WE,
Gallagher PE, Diz DI. Growth, metabolism, and blood pressure disturbances
during aging in transgenic rats with altered brain renin-angiotensin systems.
Physiol Genomics. 2005;23:311-317.
64
244.
Sloniger JA, Saengsirisuwan V, Diehl CJ, Dokken BB, Lailerd N, Lemieux AM,
Kim JS, Henriksen EJ. Defective insulin signaling in skeletal muscle of the
hypertensive TG(mREN2)27 rat. Am J Physiol Endocrinol Metab.
2005;288:E1074-1081.
245.
Gilliam-Davis S, Payne VS, Kasper SO, Tommasi EN, Robbins ME, Diz DI.
Long-term AT1 receptor blockade improves metabolic function and provides
renoprotection in Fischer-344 rats. Am J Physiol Heart Circ Physiol.
2007;293:H1327-1333.
246.
Weisinger RS, Stanley TK, Begg DP, Weisinger HS, Spark KJ, Jois M.
Angiotensin converting enzyme inhibition lowers body weight and improves
glucose tolerance in C57BL/6J mice maintained on a high fat diet. Physiol Behav.
2009;98:192-197.
247.
Weber MA. Comparison of type 1 angiotensin II receptor blockers and
angiotensin converting enzyme inhibitors in the treatment of hypertension. J
Hypertens Suppl. 1997;15:S31-36.
248.
Scheen AJ. Prevention of type 2 diabetes mellitus through inhibition of the ReninAngiotensin system. Drugs. 2004;64:2537-2565.
249.
Koh KK, Park SM, Quon MJ. Leptin and cardiovascular disease: response to
therapeutic interventions. Circulation. 2008;117:3238-3249.
250.
Quarta C, Bellocchio L, Mancini G, Mazza R, Cervino C, Braulke LJ, Fekete C,
Latorre R, Nanni C, Bucci M, Clemens LE, Heldmaier G, Watanabe M, LesteLassere T, Maitre M, Tedesco L, Fanelli F, Reuss S, Klaus S, Srivastava RK,
Monory K, Valerio A, Grandis A, De Giorgio R, Pasquali R, Nisoli E, Cota D,
Lutz B, Marsicano G, Pagotto U. CB(1) signaling in forebrain and sympathetic
neurons is a key determinant of endocannabinoid actions on energy balance. Cell
Metab. 2010;11:273-285.
251.
Bellocchio L, Soria-Gomez E, Quarta C, Metna-Laurent M, Cardinal P, Binder E,
Cannich A, Delamarre A, Haring M, Martin-Fontecha M, Vega D, Leste-Lasserre
T, Bartsch D, Monory K, Lutz B, Chaouloff F, Pagotto U, Guzman M, Cota D,
Marsicano G. Activation of the sympathetic nervous system mediates hypophagic
and anxiety-like effects of CB(1) receptor blockade. Proc Natl Acad Sci U S A.
2013;110:4786-4791.
252.
Mullins JJ, Peters J, Ganten D. Fulminant hypertension in transgenic rats
harbouring the mouse Ren-2 gene. Nature. 1990;344:541-544.
253.
Bader M, Zhao Y, Sander M, Lee MA, Bachmann J, Bohm M, Djavidani B,
Peters J, Mullins JJ, Ganten D. Role of tissue renin in the pathophysiology of
hypertension in TGR(mREN2)27 rats. Hypertension. 1992;19:681-686.
65
254.
Okamoto K, Aoki K, Nosaka S, Fukushima M. Cardiovascular Diseases in the
Spontaneously Hypertensive Rat. Jpn Circ J. 1964;28:943-952.
255.
Diz DI, Jessup JA, Westwood BM, Bosch SM, Vinsant S, Gallagher PE, Averill
DB. Angiotensin peptides as neurotransmitters/neuromodulators in the
dorsomedial medulla. Clin Exp Pharmacol Physiol. 2002;29:473-482.
256.
Nautiyal M, Shaltout HA, de Lima DC, do Nascimento K, Chappell MC, Diz DI.
Central angiotensin-(1-7) improves vagal function independent of blood pressure
in hypertensive (mRen2)27 rats. Hypertension. 2012;60:1257-1265.
257.
Zhao Y, Bader M, Kreutz R, Fernandez-Alfonso M, Zimmermann F, Ganten U,
Metzger R, Ganten D, Mullins JJ, Peters J. Ontogenetic regulation of mouse Ren2d renin gene in transgenic hypertensive rats, TGR(mREN2)27. Am J Physiol.
1993;265:E699-707.
258.
Bachmann S, Peters J, Engler E, Ganten D, Mullins J. Transgenic rats carrying the
mouse renin gene--morphological characterization of a low-renin hypertension
model. Kidney Int. 1992;41:24-36.
259.
Tokita Y, Franco-Saenz R, Reimann EM, Mulrow PJ. Hypertension in the
transgenic rat TGR(mRen-2)27 may be due to enhanced kinetics of the reaction
between mouse renin and rat angiotensinogen. Hypertension. 1994;23:422-427.
260.
Campbell DJ, Rong P, Kladis A, Rees B, Ganten D, Skinner SL. Angiotensin and
bradykinin peptides in the TGR(mRen-2)27 rat. Hypertension. 1995;25:10141020.
261.
Lantelme P, Lo M, Mullins JJ, Gharib C, Bizollon CA, Sassard J. Comparison
between chronic converting enzyme inhibition and AT1 blockade in mRen2
transgenic rats. J Cardiovasc Pharmacol. 1996;27:476-481.
262.
Bohm M, Lee M, Kreutz R, Kim S, Schinke M, Djavidani B, Wagner J, Kaling
M, Wienen W, Bader M, et al. Angiotensin II receptor blockade in
TGR(mREN2)27: effects of renin-angiotensin-system gene expression and
cardiovascular functions. J Hypertens. 1995;13:891-899.
263.
Aileru AA, Logan E, Callahan M, Ferrario CM, Ganten D, Diz DI. Alterations in
sympathetic ganglionic transmission in response to angiotensin II in (mRen2)27
transgenic rats. Hypertension. 2004;43:270-275.
264.
Witte K, Lemmer B. Development of inverse circadian blood pressure pattern in
transgenic hypertensive TGR(mREN2)27 rats. Chronobiol Int. 1999;16:293-303.
265.
Borgonio A, Pummer S, Witte K, Lemmer B. Reduced baroreflex sensitivity and
blunted endogenous nitric oxide synthesis precede the development of
hypertension in TGR(mREN2)27 rats. Chronobiol Int. 2001;18:215-226.
66
266.
Wilson W, Voigt P, Bader M, Marsden CA, Fink H. Behaviour of the transgenic
(mREN2)27 rat. Brain Res. 1996;729:1-9.
267.
Krskova L, Vrabcova M, Talarovicova A, Zeman M. Influence of up-regulated
renin-angiotensin system on the exploration, anxiety-related behavior and object
recognition. Acta Biol Hung. 2009;60:369-383.
268.
Sander M, Bader M, Djavidani B, Maser-Gluth C, Vecsei P, Mullins J, Ganten D,
Peters J. The role of the adrenal gland in hypertensive transgenic rat
TGR(mREN2)27. Endocrinology. 1992;131:807-814.
269.
Sander M, Ganten D, Mellon SH. Role of adrenal renin in the regulation of
adrenal steroidogenesis by corticotropin. Proc Natl Acad Sci U S A. 1994;91:148152.
270.
Lee MA, Bohm M, Paul M, Bader M, Ganten U, Ganten D. Physiological
characterization of the hypertensive transgenic rat TGR(mREN2)27. Am J
Physiol. 1996;270:E919-929.
271.
Manrique C, Lastra G, Habibi J, Pulakat L, Schneider R, Durante W, Tilmon R,
Rehmer J, Hayden MR, Ferrario CM, Whaley-Connell A, Sowers JR. Nebivolol
improves insulin sensitivity in the TGR(Ren2)27 rat. Metabolism. 2011;60:17571766.
272.
Engler S, Paul M, Pinto YM. The TGR(mRen2)27 transgenic rat model of
hypertension. Regul Pept. 1998;77:3-8.
273.
Louis WJ, Howes LG. Genealogy of the spontaneously hypertensive rat and
Wistar-Kyoto rat strains: implications for studies of inherited hypertension. J
Cardiovasc Pharmacol. 1990;16 Suppl 7:S1-5.
274.
Schinke M, Bohm M, Bricca G, Ganten D, Bader M. Permanent inhibition of
angiotensinogen synthesis by antisense RNA expression. Hypertension.
1996;27:508-513.
275.
Schinke M, Baltatu O, Bohm M, Peters J, Rascher W, Bricca G, Lippoldt A,
Ganten D, Bader M. Blood pressure reduction and diabetes insipidus in transgenic
rats deficient in brain angiotensinogen. Proc Natl Acad Sci U S A. 1999;96:39753980.
276.
Deschepper CF, Bouhnik J, Ganong WF. Colocalization of angiotensinogen and
glial fibrillary acidic protein in astrocytes in rat brain. Brain Res. 1986;374:195198.
277.
Monti J, Schinke M, Bohm M, Ganten D, Bader M, Bricca G. Glial
angiotensinogen regulates brain angiotensin II receptors in transgenic rats
TGR(ASrAOGEN). Am J Physiol Regul Integr Comp Physiol. 2001;280:R233240.
67
278.
Jackson L, Sakima A, Oden SD, Ganten D, Ferrario CM, Gallagher P, Diz DI.
Angiotensin receptor density and messenger RNA levels in brain medulla of
(mRen2)27 and (ASrAogen) transgenic rats during aging. Hypertension.
2004;44:516 (abstract).
279.
Kasper SO, Ferrario CM, Ganten D, Diz DI. Rats with low brain angiotensinogen
do not exhibit insulin resistance during early aging. Endocrine. 2006;30:167-174.
280.
Arnold AC, Sakima A, Ganten D, Ferrario CM, Diz DI. Modulation of reflex
function by endogenous angiotensins in older transgenic rats with low glial
angiotensinogen. Hypertension. 2008;51:1326-1331.
281.
Shimokawa A, Jin QH, Ishizuka Y, Kunitake T, Takasaki M, Kannan H. Effects
of anesthetics on norepinephrine release in the hypothalamic paraventricular
nucleus region of awake rats. Neurosci Lett. 1998;244:21-24.
282.
Monahan KD. Effect of aging on baroreflex function in humans. Am J Physiol
Regul Integr Comp Physiol. 2007;293:R3-R12.
283.
Hajduczok G, Chapleau MW, Johnson SL, Abboud FM. Increase in sympathetic
activity with age. I. Role of impairment of arterial baroreflexes. Am J Physiol.
1991;260:H1113-1120.
284.
Diz DI, Varagic J, Groban L. Aging and the brain renin-angiotensin system:
relevance to age-related decline in cardiac function. Future Cardiol. 2008;4:237245.
285.
Rademacher DJ, Patel S, Hopp FA, Dean C, Hillard CJ, Seagard JL.
Microinjection of a cannabinoid receptor antagonist into the NTS increases
baroreflex duration in dogs. Am J Physiol Heart Circ Physiol. 2003;284:H15701576.
286.
Schaich CL, Howlett AC, Diz DI. Dose-Related Biphasic Modulation of
Baroreflex Sensitivity by the CB1 Receptor in Rat Solitary Tract Nucleus.
Hypertension. 2011;58:e105 (Abstract).
287.
Schaich CL, Howlett AC, Diz DI. Contribution of Endocannabinoids via CB1
Receptors to the Impaired Baroreflex Sensitivity in (mRen2)27 Hypertensive
Rats. Hypertension. 2012;60:A117 (Abstract).
288.
Silvestri C, Di Marzo V. The endocannabinoid system in energy homeostasis and
the etiopathology of metabolic disorders. Cell Metab. 2013;17:475-490.
289.
Szekeres M, Nadasy GL, Turu G, Soltesz-Katona E, Toth ZE, Balla A, Catt KJ,
Hunyady L. Angiotensin II Induces Vascular Endocannabinoid Release, Which
Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors. J Biol
Chem. 2012;287:31540-31550.
68
CHAPTER TWO
MEDULLARY ENDOCANNABINOIDS CONTRIBUTE TO THE
DIFFERENTIAL RESTING BAROREFLEX SENSITIVITY IN RATS WITH
ALTERED BRAIN RENIN-ANGIOTENSIN SYSTEM EXPRESSION
By
Chris L. Schaich,1 Megan Grabenauer,1, 2 Brian F. Thomas,1, 2 Patricia E. Gallagher,1
Allyn C. Howlett,1 Debra I. Diz1
1
Department of Physiology and Pharmacology; and Hypertension & Vascular Research
Center, Wake Forest School of Medicine, Winston-Salem, NC, USA
2
Analytical Chemistry and Pharmaceutics, RTI International, Research Triangle Park,
NC, USA
Short title: Endocannabinoids and Baroreflex Sensitivity
The following manuscript is in revision for Hypertension and represents the efforts of the
first author. Differences in formatting and organization reflect the requirements of the
journal.
69
Abstract
CB1 cannabinoid receptors are expressed on vagal afferent fibers and neurons within the
solitary tract nucleus, implicating a role in modulation of the arterial baroreflex.
Hypotensive ASrAOGEN rats with low glial angiotensinogen have enhanced bradycardic
baroreflex sensitivity compared to Sprague-Dawley rats, while (mRen2)27 rats, a model
of angiotensin II-dependent hypertension, feature impaired baroreflex sensitivity. Mass
spectrometry revealed higher levels of the endocannabinoid 2-arachidonoylglycerol in
(mRen2)27 rats compared to ASrAOGEN rats (2.70 ± 0.28 vs. 1.17 ± 0.09 ng/mg tissue;
P < 0.01), while Sprague-Dawley rats had intermediate content (1.85 ± 0.27 ng/mg
tissue). Microinjection of the CB1 receptor antagonist SR141716A into the solitary tract
nucleus did not change baroreflex sensitivity in anesthetized Sprague-Dawley rats.
However, SR141716A in (mRen2)27 rats dose-dependently improved baroreflex
sensitivity: 0.36 pmol/120 nL of SR141716A increased baroreflex sensitivity from 0.43 ±
0.03 to 0.71 ± 0.04 ms/mmHg (P < 0.001), and 36 pmol of SR141716A increased
baroreflex sensitivity from 0.47 ± 0.02 to 0.94 ± 0.10 ms/mmHg (P < 0.01) in this strain.
In contrast, both the 0.36-pmol (1.50 ± 0.12 versus 0.86 ± 0.08 ms/mmHg; P < 0.05) and
36-pmol (1.38 ± 0.16 versus 0.46 ± 0.003 ms/mmHg; P < 0.01) SR141716A
microinjections reduced baroreflex sensitivity in ASrAOGEN rats. These observations
reveal differential regulatory functions of the brain endocannabinoid system for
modulating baroreflex function during conditions associated with altered brain reninangiotensin system expression that may involve interactions with neurotransmitters
known to influence cardiovagal baroreflex sensitivity.
70
Introduction
Tonic overactivation of the endocannabinoid system is implicated in the maintenance of
cardiovascular diseases, including hypertension and atherosclerosis, and the metabolic
syndrome.1,
2
These adverse conditions are associated with arterial baroreflex
dysfunction and reduced heart rate variability, themselves indices of general morbidity
and mortality.3 Baroreflex sensitivity (BRS) for control of heart rate (HR), measured as
the bradycardia evoked in response to increases in mean arterial pressure (MAP), is an
important indicator of parasympathetic activity and vagus nerve function, and its
impairment often precedes the development of cardiovascular disease.3
The G protein-coupled CB1 and CB2 cannabinoid receptors mediate the biological
actions of the endocannabinoid system in target cells. CB1 is widely expressed in the
central nervous system (CNS) and in peripheral tissues, enabling the activated receptor to
regulate a variety of physiological functions including blood pressure and heart rate.4 In
contrast, CB2 receptors are primarily restricted to immune tissues and are expressed in the
CNS at levels many-fold lower than CB1.5 CB1 receptors in the CNS are predominantly
expressed on presynaptic neuronal terminals, where their activation by the
endocannabinoids anandamide or 2-arachidonoylglycerol (2-AG), released from
postsynaptic neurons, directly modulates voltage-gated ion channels to repolarize the
cellular membrane, temporarily reducing the release of neurotransmitter into the synaptic
cleft (see Castillo et al. (2012)6 for review).
Presynaptic CB1 receptors are densely expressed in the solitary tract nucleus
(NTS) of the dorsomedial medulla, the primary site for termination of baroreceptor
afferent neurons relaying sensory information from the periphery, as well as in vagal
71
afferent neurons originating in the nodose ganglion.7,
8
Several studies show that
anandamide within the NTS modulates baroreflex-evoked suppression of renal
sympathetic nerve activity in rats by altering release of the inhibitory neurotransmitter
GABA.9,
10
Furthermore, blockade of CB1 receptors in the medial prefrontal cortex, a
limbic structure involved in the modulation of autonomic responses exerting influence on
the cardiovascular system, enhances spontaneous BRS for control of HR in conscious
rats.11 Therefore, the brain endocannabinoid system likely influences BRS through the
modulation of neurotransmitter release in brain sites regulating effects on the baroreflex.
There
is
growing
evidence
for
signaling
interactions
between
the
endocannabinoid system and the pathogenic actions of the renin-angiotensin system
(RAS). For instance, blockade of CB1 receptors in Sprague-Dawley (SD) rats treated
chronically with ethanol prevents angiotensin (Ang) II-mediated mitogenic signaling and
profibrogenic gene expression in hepatic stellate cells.12 In obese Zucker rats, longterm
CB1 receptor blockade reduces the vasoconstrictor effect of acutely administered Ang
II.13 However, no studies to date have investigated the endocannabinoid-RAS interaction
in animals with altered brain RAS expression.
Transgenic (mRen2)27 rats, a monogenetic model of Ang II-dependent
hypertension in which the mouse Ren2 renin gene was transfected into the genome of the
normotensive SD rat, have a phenotype of chronic hypertension with markedly impaired
BRS for control of HR compared to their normotensive genetic controls.14, 15 In contrast,
transgenic ASrAOGEN rats with low brain angiotensinogen, resulting from glial
overexpression of an angiotensinogen antisense oligonucleotide, exhibit chronic
hypotension with significantly enhanced resting BRS compared to SD rats.16, 17 These
72
strains are frequently utilized to investigate the contribution of the brain RAS and its
interacting factors to impaired BRS during hypertension.18, 19 In this study, we provide
functional and biochemical evidence for altered endocannabinoid tone at the level of the
dorsomedial medulla and NTS in rats with altered brain RAS activity.
73
Methods
Animals
Experiments were performed in 15- to 20-week-old male Hannover SD and transgenic
(mRen2)27 and ASrAOGEN rats obtained from the Hypertension & Vascular Research
Center colony at Wake Forest School of Medicine. Animals were housed two per cage in
a humidity- and temperature-controlled room with ad libitum access to food (standard
chow) and water. The colony maintained a 12-hour light/dark cycle (lights on at 06:00).
The Institutional Animal Care and Use Committee approved all experimental procedures.
Surgical Procedures and Hemodynamic Measures
As described previously,18,
20
rats were anesthetized with combination urethane-
chloralose (750 and 35 mg/kg, respectively) via intraperitoneal injections with
supplemental intravenous doses given as needed (diluted to 3:7 with saline). Animals
were instrumented with femoral artery and vein catheters and placed in a stereotaxic
frame with the head tilted at a 45° downward angle for surgical exposure of the dorsal
brain medulla. Rats were allowed a 30 minute resting and equilibration period before
testing began. Pulsatile arterial pressure (AP) was recorded and digitized by a data
acquisition system (AcqKnowledge software version 3.8.1, BIOPAC Systems, Goleta,
CA), and heart rate (HR) was determined from the AP wave. After obtaining stable
measures of MAP and HR, baseline BRS was established by sequential intravenous bolus
injection of 3 doses of phenylephrine (PE; 2, 5 and 10 μg/kg in 0.9% NaCl) to determine
the bradycardic BRS response to increases in AP. BRS for bradycardia was defined for
each animal as the slope of the relationship between changes in MAP (ΔMAP; mmHg)
and the pulse interval (ΔPI; ms) generated from the 3 doses of PE (mean R2 = 0.95 for all
74
animals; N = 34).
Reflex testing was repeated 10 and 60 minutes after bilateral
microinjection of the CB1 receptor-selective antagonist SR141716A into the NTS, and
was completed within 15 minutes, with each animal serving as its own control.
Maximum transient changes in MAP and HR in response to NTS microinjection of
SR141716A were also recorded.
NTS Microinjections
SR141716A (0.36 or 36 pmol in a 120
nL volume of 10% DMSO in artificial
cerebrospinal fluid vehicle) or vehicle
(120 nL) was microinjected bilaterally
into the NTS (0.4 mm rostral, 0.4 mm
lateral to the calamus scriptorius, and
0.4 mm below the dorsal surface) using
a glass micropipette connected to a
hand-held
syringe,
described.18,
20
were
as
previously
Doses of SR141716A
determined
based
on
the
literature21 and previous experience with
the drug.22
NTS microinjection of
vehicle solution had no effect on evoked
Figure 2-1.
Histological Analysis of
Microinjection Sites
Representative
photomicrography
(5X
magnification) of an unstained rat medullary section
(30 µm) at approximately -13.8 mm caudal to
bregma showing a typical NTS microinjection site
(0.4 mm rostral, 0.4 mm lateral to the calamus
scriptorius [caudal tip of the area postrema] and 0.4
mm below the dorsal surface). Only data from
injections within the medial NTS at rostrocaudal
level -13.3 to -14.0 mm caudal to bregma were used
in this study. AP = area postrema; C = central canal;
dmnX = dorsal motor nucleus of the vagus; NG =
nucleus gracilis; NTS = solitary tract nucleus.
BRS, MAP or HR in SD, (mRen2)27 or ASrAOGEN rats (Table 2-1). At the end of
experiments, brains were removed, frozen, and sectioned (30 μm) for localization of
microinjection sites (Figure 2-1). Only data from injections within the medial NTS at the
75
rostrocaudal level -13.3 to -14.0 mm caudal to bregma were used in the analysis. The
accuracy rate for injections was >90%.
Table 2-1. Values of MAP, HR and BRS in Response to NTS Microinjection of Vehicle
Group
N
Vehicle in SD rats
Baseline
Values at 10 minutes
5
Vehicle in (mRen2)27 rats
Baseline
Values at 10 minutes
3
Vehicle in ASrAOGEN rats
Baseline
Values at 10 minutes
4
MAP
(mmHg)
HR
(bpm)
BRS
(ms/mmHg)
93 ± 2
92 ± 3
330 ± 12
319 ± 15
1.02 ± 0.12
1.02 ± 0.10
96 ± 8
92 ± 4
294 ± 20
296 ± 9
0.44 ± 0.04#
0.45 ± 0.06#
119 ± 8#
121 ± 8#
360 ± 16
358 ± 9
1.43 ± 0.09*
1.46 ± 0.14*
Values are mean ± SEM and represent MAP, HR and BRS values at baseline and 10 minutes after NTS
microinjection of vehicle; N = number of animals; MAP = mean arterial pressure; HR = heart rate.
*
P < 0.05 vs. SD; #P < 0.01.
Mass Spectrometry Detection of Anandamide and 2-AG
In separate groups of naïve 15-week-old SD, (mRen2)27 and ASrAOGEN rats, brains
were removed and frozen on dry ice for excision of 3-mm3 dorsal medullary sections.
For the analysis of the two major endocannabinoids anandamide and 2-AG, an extraction
technique was developed for rat medulla tissue. Optimal extraction conditions for the
tissue were obtained using HPLC-grade acetonitrile and tissue homogenization with
stainless steel beads (2.3 mm, BioSpec Products, Bartlesville, OK) that reduced
enzymatic degradation and attenuated 2-AG isomerization. Tissue homogenates were
fortified with isotopically-labeled internal standards for 2-AG and anandamide 2-[2H5]2-
76
AG (AG-d5) and [2H4]anandamide (AEA-d4) on a per-mg basis for sample integrity and
robustness of the method. The chromatography solvents were Honeywell B&J HPLCgrade ammonium acetate and acetic acid–double distilled (Sigma-Aldrich). Stock
solutions of anandamide and 2-AG and their deuterated analogs were freshly prepared
and stored as recommended by the manufacturer. The study samples, quality control
samples, and standards (targeted analysis) were processed using automated liquid
handling to ensure the reproducibility and consistency of the sample preparation method.
A Waters Acuity ultraperformance liquid chromatography (UPLC) system
(Waters, Milford, MA) equipped with an Acquity UPLC column was utilized for the
separation of endocannabinoids prior to quantitative analysis by selected reaction
monitoring (LC-MS/MS). A triple quadrupole mass spectrometer (API-5000; AB
SCIEX, Framingham, MA) was coupled to the UPLC for quantitative analysis, and
operating parameters were optimized for anandamide and 2-AG. Calibration curves
spanning the range of quantitation were produced through least-squares linear regression
analysis of response (analyte integrated area/internal standard integrated area) versus
concentration, and weighted to improve fit, accuracy and precision. Sample content of
anandamide or 2-AG is expressed as ng analyte per mg tissue wet weight.
Quantification of CB1, CB2, and CRIP1a mRNA
RT-qPCR was used to measure mRNA levels of various effector components of the brain
endocannabinoid system, including CB1 and CB2 receptor, and the cannabinoid receptor
interacting protein-1a (CRIP1a)23 in dorsal medullary tissue from the same naïve 15week-old rats (n = 4-6 each strain). Brains were removed and frozen on dry ice as
described for endocannabinoid measurements. Isolated RNA from excised tissue was
77
assessed for concentration and stability. Total RNA (1 µg) was reverse transcribed using
AMV reverse transcriptase in a 20-µL reaction mixture containing deoxyribonucleotides,
random hexamers, and RNase inhibitor in reverse-transcriptase buffer, as described
previously.18,
20, 24
For RT-qPCR, 2 µL of resultant cDNA were added to TaqMan
Universal PCR Master Mix with the appropriate gene-specific primer/probe set for CB1
and CB2 receptors, and CRIP1a (Applied Biosystems), and amplification was performed.
All reactions were performed in triplicate. 18S ribosomal RNA served as the internal
control. Results were quantified as Ct values, in which Ct is the threshold cycle of PCR
at which an amplified product is first detected, and was defined as relative gene
expression (ratio of target:control).
Analysis of Data
Values are presented as mean ± SEM. Comparisons of changes in BRS over time in
response to SR141716A or vehicle in the different strains were assessed using a repeated
measures two-way ANOVA. Where appropriate on the basis of the two-way ANOVA,
one-way ANOVA and post-hoc Tukey multiple comparisons elucidated further
differences between strains or time points. Multiple regression analysis was performed
on BRS scatterplots to determine slopes of fit lines.
The criterion for statistical
significance was P < 0.05. Statistical tests were performed using Prism 5.0 (GraphPad
Software, San Diego, CA).
78
Results
Baseline BRS for Control of HR in Response to Increases in MAP in SD, (mRen2)27
and ASrAOGEN Rats
The pooled bradycardic BRS for control of HR at baseline for ASrAOGEN rats was
significantly higher compared to SD rats (1.43 ± 0.07 vs. 1.03 ± 0.06 ms/mmHg; P <
0.001; n = 11 and 10, respectively; Figure 2-2), while baseline BRS in (mRen2)27 rats
was significantly lower than in SD rats (0.44 ± 0.02 ms/mmHg; P < 0.001; n = 13; Figure
2-2). These observations are fully consistent with previous studies from our laboratory.18,
19
Furthermore, there were no differences in baseline BRS values of SD, (mRen2)27 or
ASrAOGEN rat treatment groups receiving vehicle or various doses of SR141716A
(Figure 2-3).
A
2.0
Baseline BRS
(ms/mmHg)
‡
1.5
1.0
‡
0.5
0.0
ASrAOGEN
SD
(mRen2)27
B
PI (ms)
150
ASrAOGEN
SD
(mRen2)27
100
50
0
0
20
40
60
MAP (mmHg)
80
100
79
Figure 2-2. Baseline BRS for Control of
HR in SD, (mRen2)27 and ASrAOGEN
Rats
A, Pooled baseline BRS for control of HR in
response to increases in AP evoked by PE
prior to NTS microinjection of 120 nL of
vehicle or SR141716A in anesthetized SD (n
= 10), (mRen2)27 (n = 13) and ASrAOGEN
(n = 11) rats. B, Scatterplot illustrates
significantly different pooled baseline BRS
reflex testing regression lines among
ASrAOGEN (1.41 ± 0.15 ms/mmHg), SD
(0.90 ± 0.09 ms/mmHg) and (mRen2)27
(0.48 ± 0.09 ms/mmHg) rats (P < 0.0001).
‡P < 0.001 vs. SD.
A
B
(mRen2)27 Baseline BRS
(ms/mmHg)
SD Baseline BRS
(ms/mmHg)
2.0
1.5
1.0
0.5
0.0
Vehicle
36 pmol
2.0
1.5
1.0
0.5
0.0
Vehicle
0.36 pmol
36 pmol
ASrAOGEN Baseline BRS
(ms/mmHg)
C
2.0
1.5
1.0
0.5
0.0
Vehicle
0.36 pmol
36 pmol
Figure 2-3. Baseline BRS for Control of HR in SD, (mRen2)27 and ASrAOGEN Rat Treatment
Groups
There were no differences in baseline BRS for control of HR in response to increases in AP evoked by PE
among treatment groups of SD (n = 5), (mRen2)27 (n = 3-5) or ASrAOGEN (n = 3-4) rats receiving
vehicle or various doses of SR141716A.
Effect of CB1 Receptor Antagonist on BRS for Control of HR in SD, (mRen2)27 and
ASrAOGEN Rats
In SD rats, NTS microinjection of 36 pmol of SR141716A had no effect on BRS for
control of HR in response to increases in MAP produced by PE at 10 or 60 minutes after
microinjection (Figure 2-4). However, microinjection of 0.36 and 36 pmol doses of
SR141716A into the NTS of hypertensive (mRen2)27 rats significantly and dosedependently increased BRS by 65% (P < 0.001) and 100% (P < 0.01), respectively, after
80
10 minutes (Figure 2-5). These effect are fully consistent with previous experiments by
our laboratory showing improvement in spontaneous BRS following systemically
administered SR141716A in conscious (mRen2)27 rats.25
Timecourse experiments
showed that the BRS-enhancing effect of each dose in (mRen2)27 rats was maintained at
60 minutes post-microinjection (Figure 2-5).
In contrast to the potentiating effect found in (mRen2)27 rats, NTS microinjection
of 0.36 pmol SR141716A in hypotensive ASrAOGEN rats significantly reduced BRS for
control of HR by 43% (P < 0.05), while 36 pmol of SR141716A reduced BRS by 67%
after 10 minutes (P < 0.01; Figure 2-6).
These changes suggest a dose-dependent
response to NTS microinjection of SR141716A, although the differences between the
effects of the two doses did not reach statistical significance after post-hoc analysis. As
in (mRen2)27 rats, timecourse experiments showed that the attenuating effect of each
dose on BRS was maintained at 60 minutes after microinjection (Figure 2-6).
A
Evoked BRS
(ms/mmHg)
2.0
1.5
1.0
0.5
0.0
Baseline
10 min
60 min
B
80
Baseline
+SR141716A (36 pmol)
60 min
PI (ms)
60
40
20
0
0
20
40
60
MAP (mmHg)
80
81
Figure
2-4.
Effect
of
NTS
Microinjection of CB1 Receptor
Antagonist SR141716A on BRS for
Control of HR Evoked by PE in SD
Rats. A, NTS microinjection of 36
pmol of SR141716A in SD rats did not
significantly change BRS after 10
minutes, nor after 60 minutes in the
same animals (n = 5). B, The slope of
the relationship between the increases in
MAP produced by PE and the
corresponding reflex bradycardia (ΔPI)
does not show change from baseline in
the linear regression slope 10 or 60
minutes following NTS microinjection
of 36 pmol of SR141716A (1.00 ± 0.08
ms/mmHg baseline; 1.13 ± 0.11
ms/mmHg after 10 minutes; 1.14 ± 0.14
ms/mmHg after 60 minutes; R2 = 0.84
to 0.92 for pooled data).
A
B
36 pmol SR141716A
2.0
1.5
1.5
‡
1.0
Evoked BRS
(ms/mmHg)
Evoked BRS
(ms/mmHg)
0.36 pmol SR141716A
2.0
‡
0.5
0.0
†
†
10 min
60 min
1.0
0.5
0.0
Baseline
10 min
60 min
Baseline
C
80
Baseline
0.36 pmol SR141716A
PI (ms)
60
36 pmol SR141716A
40
20
0
0
20
40
60
MAP (mmHg)
80
100
Figure 2-5. Effect of NTS Microinjection of SR141716A on BRS for Control of HR Evoked by PE in
(mRen2)27 Rats. A-B, In (mRen2)27 rats, NTS microinjection of 0.36- (n = 5) and 36-pmol (n = 5) of
SR141716A significantly improved BRS after 10 and 60 minutes. C, The 0.36- and 36- pmol doses
produced graded, though not statistically significant, increases in the slope of the regression line in
(mRen2)27 rats (0.47 ± 0.10 ms/mmHg baseline; 0.69 ± 0.10 ms/mmHg 10 minutes after 0.36 pmol of
SR141716A; 0.79 ± 0.12 ms/mmHg 10 minutes after 36 pmol SR141716A; R2 = 0.44 to 0.79 for pooled
data). †P < 0.01 vs. baseline; ‡P < 0.01.
82
A
B
36 pmol SR141716A
2.0
1.5
1.5
*
*
1.0
Evoked BRS
(ms/mmHg)
Evoked BRS
(ms/mmHg)
0.36 pmol SR141716A
2.0
0.5
0.0
1.0
†
†
10 min
60 min
0.5
0.0
Baseline
10 min
60 min
Baseline
C
120
Baseline
PI (ms)
100
+SR141716A (0.36 pmol)
+SR141716A (36 pmol)
80
60
40
20
0
0
20
40
60
MAP (mmHg)
80
100
Figure 2-6. Effect of NTS Microinjection of SR141716A on BRS for Control of HR Evoked by PE in
ASrAOGEN Rats. A-B, In ASrAOGEN rats, NTS microinjection of 0.36- (n = 4) and 36-pmol (n = 3) of
SR141716A significantly attenuated BRS after 10 and 60 minutes. C, The 0.36- and 36- pmol doses of
SR141716A produced equivalent, statistically significant reductions in the slope of the regression line in
ASrAOGEN rats (1.42 ± 0.15 ms/mmHg baseline; 0.58 ± 0.27 ms/mmHg 10 minutes after 0.36 pmol of
SR141716A; 0.42 ± 0.04 ms/mmHg 10 minutes after 36 pmol SR141716A; P < 0.001; R2 = 0.32 to 0.93 for
pooled data). *P < 0.05 vs. baseline; †P < 0.01.
MAP and HR Responses to NTS Microinjection of CB1 Receptor Antagonist
There were no significant differences in baseline MAP or HR within groups of SD,
(mRen2)27 or ASrAOGEN rats receiving vehicle or SR141716A microinjections, nor
were there significant differences between pooled baseline HR of SD and (mRen2)27 rats
receiving vehicle or SR141716A (Table 2-1 and 2-2; Figure 2-7). However, as reported
previously,18 the pooled baseline MAP of anesthetized ASrAOGEN rats was significantly
higher compared to SD rats (115 ± 4 vs. 94 ± 2 mmHg, respectively; P < 0.01; Figure 283
7A), possibly due to an anesthesia-induced activation of the sympathetic nervous system
observed in these animals.18 The pooled baseline HR was also significantly higher in
ASrAOGEN rats compared to SD and (mRen2)27 rats (347 ± 7 vs. 313 ± 8 and 312 ± 6
bpm, respectively; P < 0.01; Figure 2-7B). The pooled baseline MAP of anesthetized
(mRen2)27 rats (108 ± 4 ms/mmHg) was also significantly higher compared to SD rats (P
< 0.05; Figure 2-7A).
Acute NTS microinjection of either 0.36 or 36 pmol of SR141716A did not
significantly change resting MAP in SD or ASrAOGEN rats, nor HR in SD, (mRen2)27
or ASrAOGEN rats at the time of reflex testing 10 and 60 minutes after microinjection.
While 0.36 pmol of SR141716A did not have a significant effect on resting MAP in
(mRen2)27 rats over the duration of testing, the 36 pmol dose produced a modest yet
statistically significant decrease in MAP at 10 and 60 minutes post-microinjection (P <
0.001 vs. baseline; Table 2-2). There were no significant effects of NTS microinjection
of vehicle on MAP or HR in any strain (Table 2-1). These responses indicate that
differences in BRS after administration of SR141716A are generally not attributable to
differences in resting hemodynamics, confirming that the set point of the baroreflex is
regulated independently from the sensitivity. Differences in MAP or HR within groups
of transgenic animals relative to SD rats at individual timepoints are noted in Table 2-1
and Table 2-2.
Microinjection of 36 pmol of SR141716A yielded a modest yet statistically
significant potentiation of intravenous PE-induced MAP increases in SD rats after 10 and
60 minutes (P < 0.05; Figure 2-8), consistent with sympathetic activation associated with
SR141716A.26
However, there were no differences in MAP responsiveness to PE
84
injections among groups of (mRen2)27 or ASrAOGEN rats receiving various doses of
SR141716A.
Table 2-2. Values of MAP and HR in Response to NTS Microinjection of SR141716A
Group
N
SD 36 pmol SR141716A
Baseline
Values at 10 minutes
Values at 60 minutes
5
(mRen2)27 0.36 pmol SR141716A
Baseline
Values at 10 minutes
Values at 60 minutes
5
(mRen2)27 36 pmol SR141716A
Baseline
Values at 10 minutes
Values at 60 minutes
5
ASrAOGEN 0.36 pmol SR141716A
Baseline
Values at 10 minutes
Values at 60 minutes
4
ASrAOGEN 36 pmol SR141716A
Baseline
Values at 10 minutes
Values at 60 minutes
3
MAP
(mmHg)
HR
(bpm)
94 ± 3
90 ± 3
91 ± 4
297 ± 6
280 ± 9
291 ± 11
103 ± 5
99 ± 6
96 ± 4
319 ± 5
308 ± 5
326 ± 9*
119 ± 6#
109 ± 7‡
105 ± 7‡
316 ± 5
318 ± 10*
325 ± 10*
115 ± 7*
113 ± 8#
104 ± 4
347 ± 12#
361 ± 14§
360 ± 5§
107 ± 5
104 ± 7
98 ± 6
335 ± 7
344 ± 16#
345 ± 15#
Values are mean ± SEM and represent baseline MAP and HR and values at 10 or 60 minutes after the
initial SR141716A NTS microinjection; N = number of animals.
‡P < 0.001 vs. baseline.
*
P < 0.05 vs. SD; #P < 0.01; §P < 0.001.
85
A
Baseline MAP
(mmHg)
150
†
*
100
50
0
ASrAOGEN
SD
(mRen2)27
SD
(mRen2)27
B
Baseline HR
(bpm)
400
†#
300
200
100
0
ASrAOGEN
Figure 2-7. Baseline MAP and HR of SD, (mRen2)24 and ASrAOGEN Rats
A-B, Pooled baseline MAP (A) and HR (beats per minute [bpm]) (B) prior to NTS microinjection of 120
nL of vehicle or SR141716A (0.36 or 36 pmol) in anesthetized SD (n = 10), (mRen2)27 (n = 13) and
ASrAOGEN (n = 11) rats. *P < 0.05 vs. SD; †P < 0.01 vs. SD; #P < 0.01 vs. (mRen2)27.
86
A
B
Baseline
SD Rats
80
40
*
60
MAP
(mmHg)
60
MAP
(mmHg)
80
ASrAOGEN
SD
(mRen2)27
*
20
40
20
0
0
0
5
PE (g/kg)
10
0
C
5
PE (g/kg)
10
D
ASrAOGEN Rats
(mRen2)27 Rats
80
80
Baseline
0.36 pmol SR141716A
36 pmol SR141716A
40
20
Baseline
0.36 pmol SR141716A
36 pmol SR141716A
60
MAP
(mmHg)
60
MAP
(mmHg)
Baseline
36 pmol SR141716A
(10 min)
36 pmol SR141716A
(60 min)
40
20
0
0
0
5
PE (g/kg)
10
0
5
PE (g/kg)
10
Figure 2-8. MAP Responsiveness in SD, (mRen2)27 and ASrAOGEN Rats at Baseline and in
Response to NTS Microinjection of SR141716A
Changes in MAP in responsiveness to intravenous graded doses of PE were assessed in anesthetized SD,
(mRen2)27 and ASrAOGEN rats at baseline and in response to NTS microinjection of 0.36 or 36 pmol of
SR141716A. A, Baseline MAP responsiveness to 2 µg of PE was significantly greater in ASrAOGEN and
(mRen2)27 rats compared to SD rats (P < 0.05). B, 36 pmol of SR141716A in SD rats significantly
potentiated PE-induced increases in MAP at 10 and 60 minutes after NTS microinjection (P < 0.05; n = 5).
C-D, There were no significant differences in PE-induced increases in MAP within groups of (mRen2)27
(C; n = 5 per group) or ASrAOGEN rats (D; n = 3-4 per group).
Dorsal Medullary 2-AG and Anandamide Content in SD, (mRen2)27 and
ASrAOGEN Rats
Mass spectrometry revealed that levels of 2-AG were lowest in dorsal medulla of
ASrAOGEN rats and highest in (mRen2)27 rats (1.17 ± 0.09 vs. 2.70 ± 0.28 ng/mg
tissue, respectively; P < 0.01; n = 4-5; Figure 2-9A). SD rats had intermediate medullary
2-AG content (1.85 ± 0.27 ng/mg tissue; n = 5), falling just short of statistical
significance compared to (mRen2)27 rats (P = 0.069) and ASrAOGEN rats (P = 0.052).
87
Of note, the relative 2-AG levels parallel the trend in resting conscious MAP and are
inversely related to the baseline BRS for control of HR across rat strains.
Dorsal
medullary anandamide was found in levels 1000-fold lower than 2-AG and there were no
significant differences among rat strains (Figure 2-9B).
A
Figure 2-9. 2-AG and Anandamide
Content in Dorsal Medulla of SD,
(mRen2)27 and ASrAOGEN Rats.
A, Mass spectrometry revealed levels
of 2-AG were significantly higher in
the dorsal medullary tissue of 15week-old (mRen2)27 rats (n = 5)
relative to ASrAOGEN rats (n = 4),
and trended higher (P = 0.069) in
(mRen2)27 rats compared to SD rats
(n = 5). In addition, there was a trend
(P = 0.052) for higher 2-AG levels in
dorsal medulla of SD rats relative to
ASrAOGEN rats. B, Anandamide
was detected at levels 1000-fold lower
than 2-AG in the same tissue samples,
and there were no significant
differences among strains.
Representative chromatograms of 2AG (A) and anandamide (B) are
shown next to respective figures. †P
< 0.01 vs. ASrAOGEN.
4
2-AG
(ng/mg tissue)
†
3
2
1
0
ASrAOGEN
SD
(mRen2)27
ASrAOGEN
SD
(mRen2)27
B
Anandamide
(pg/mg tissue)
4
3
2
1
0
CB1, CB2 and CRIP1a mRNA in SD, (mRen2)27 and ASrAOGEN Rats
Relative gene expression of CB1 and CB2 receptors, and CRIP1a was measured in dorsal
medullary tissue of naïve SD (n = 5), (mRen2)27 (n = 4) and ASrAOGEN (n = 6) rats at
15 weeks of age (Figure 2-10). CB1 receptor mRNA was approximately 0.25-fold lower
in the dorsal medulla of ASrAOGEN rats relative to SD (P < 0.01) and (mRen2)27 (P <
88
0.05) rats (Figure 2-10A). There were no differences in relative mRNA levels of CB2
receptor or CRIP1a among the same rat groups (Figure 2-10B and 2-10C).
B
1.5
1.0
CB2 Receptor
Relative Gene Expression
CB1 Receptor
Relative Gene Expression
A
†*
0.5
0.0
ASrAOGEN
SD
(mRen2)27
ASrAOGEN
SD
(mRen2)27
1.5
1.0
0.5
0.0
ASrAOGEN
SD
(mRen2)27
CRIP1a
Relative Gene Expression
C
1.5
1.0
0.5
0.0
Figure 2-10. CB1, CB2 and CRIP1a mRNA in Dorsal Medulla of SD, (mRen2)27 and ASrAOGEN
Rats. A, Relative gene expression of CB1 receptor was lower in the dorsal medullary tissue of 15-week-old
ASrAOGEN rats (n = 6) compared to SD (n = 5) and (mRen2)27 (n = 4) rats. B-C, No significant
differences in relative gene expression of CB 2 receptor (B) or CRIP1a (C) were found among strains. *P <
0.05 vs. (mRen2)27; †P < 0.01 vs. SD.
89
Discussion
In this study, we report for the first time the endocannabinoid content and relative mRNA
levels of proteins associated with the endocannabinoid system in the dorsal medulla of
(mRen2)27 and ASrAOGEN rats compared to SD rats. Of note, the relative levels of
dorsal medullary 2-AG paralleled brain RAS expression and resting conscious blood
pressure, and were inversely related to baseline BRS for control of HR across the
hypotensive, normotensive and hypertensive rat strains. We also determined the effects
of exogenous CB1 cannabinoid receptor blockade by SR141716A on cardiovagal BRS, an
index of parasympathetic activity, at the level of the NTS in anesthetized rats with
differential brain RAS expression profiles. The improvement in BRS in response to
SR141716A in (mRen2)27 rats was associated with increased 2-AG content in the dorsal
medulla, suggesting that long-term increases in brain angiotensin peptides may be related
to upregulation of 2-AG release that contributes to the blunted baseline BRS in this
hypertensive strain, consistent with Ang II-mediated vascular endocannabinoid release.27
In ASrAOGEN rats, however, medullary 2-AG content was significantly lower compared
to (mRen2)27 rats, and CB1 receptor mRNA was significantly lower relative to SD and
(mRen2)27 rats. Therefore, the BRS-suppressing effect of SR141716A in the NTS of
ASrAOGEN rats suggests differential regulatory functions of the brain endocannabinoid
system in rats with high or low brain RAS expression.
Evidence suggests that exogenous cannabinoids in the NTS modulate baroreflex
function by interacting with local excitatory or inhibitory neurotransmitter systems.
Exogenous anandamide in the NTS of anesthetized SD rats prolongs PE-evoked
inhibition of renal sympathetic nerve activity,9 indicating an increase in sympathetic
90
BRS. In addition, anandamide shortens the duration of sympathetic nerve inhibition
following blockade of NTS GABA receptors,9 revealing that inhibition of excitatory
glutamate release by anandamide is obscured by a more dominant GABA-mediated
effect. The synthetic cannabinoid agonists WIN55,212-2 and CP55,940 in the NTS of
anesthetized SD rats suppress sympathetic nerve discharge.21 These effects are prevented
by NTS microinjection of the CB1 receptor-selective antagonist AM281, which had no
effect alone in the NTS.
Therefore, our current study investigating the role of
endocannabinoids in the parasympathetically-mediated cardiovagal branch of the
baroreflex potentially reveals evidence for altered NTS glutamate or GABA
neurotransmission through CB1 receptor activation in the context of altered brain RAS
activity.
Results showing improved BRS in (mRen2)27 rats after SR141716A
microinjection in the NTS are bolstered by observations of direct and indirect inhibition
of GABA release in rat arcuate nucleus by SR141716A.28 In ASrAOGEN rats with
increased NTS glutamate content,29 suppression of glutamate may be the predominant
effect of NTS CB1 receptor blockade, which may explain the inhibition of BRS by CB1
receptor blockade in these animals. More research is needed to clarify this mechanism,
for which a magnetic resonance spectroscopy technique previously used in our laboratory
to assess glutamate and GABA content in the NTS of these rats may be useful.29
Whole-cell patch clamp recordings in rat brainstem slices reveal that WIN55,2122 and exogenous anandamide in the NTS suppress synaptic input to neurons in the dorsal
motor nucleus of the vagus (dmnX),30 an important second-order nucleus in the
parasympathetic baroreflex arc.
Therefore, upregulation of NTS or dmnX
endocannabinoids or cannabinoid receptors would likely contribute to the suppression of
91
visceral autonomic responses, such as BRS for control of HR.
However, transient
receptor potential vanilloid type 1 (TRPV1) channels in the NTS and dmnX31 activated
by anandamide32 may also regulate synaptic activity in brainstem nuclei involved
baroreflex mediation. Activation of TRPV1 channels triggers spontaneous glutamate
release in the NTS, opposing the actions of CB1 receptors,33 while exerting a tonic
influence over inputs to the dmnX in rats and mice.34,
35
The presence of TRPV1 in
brainstem nuclei in the baroreflex arc thus provides an additional role for
endocannabinoids in modulating baroreflex function, and may contribute to the actions of
exogenous anandamide and WIN55,212-2, reported to inhibit neuronal TRPV1,36 in the
studies discussed above.9, 30 Although brainstem endocannabinoids may modulate BRS
via TRPV1 channels, we show only the contribution of endocannabinoid modulation of
BRS through actions at CB1 receptors because SR141716A is a selective CB1 receptor
antagonist that is not reported to interact with TRPV1 or other receptors, including CB2
receptors.37, 38
Our present study presents evidence for a direct action of endocannabinoids to
modulate baroreflex function in transgenic rats over- or underexpressing components of
the brain RAS. Microinjection of the selective CB1 receptor antagonist SR141716A
(0.36 and 36 pmol) into the NTS differentially altered BRS measured as the bradycardic
response to PE-evoked increases in AP in (mRen2)27 rats with Ang II-dependent
hypertension and in hypotensive Ang-deficient ASrAOGEN rats. In (mRen2)27 rats, the
0.36-pmol dose of SR141716A significantly improved BRS, and the 36-pmol dose
improved BRS from baseline to a greater degree, indicating a dose-response relationship.
In fact, the 36-pmol dose improved BRS in these animals to ≈1.05 ms/mmHg, a level
92
comparable with baseline BRS of SD rats and suggesting normalization of BRS for
control of HR in (mRen2)27 rats.
In ASrAOGEN rats, however, SR141716A
significantly impaired BRS to ≈0.46 ms/mmHg, a level often observed in hypertension
and comparable to baseline BRS of (mRen2)27 rats.19 The differential BRS responses to
CB1 receptor antagonism in the NTS of these strains with altered brain RAS expression
support the interpretation that the brain endocannabinoid system exerts a tonic influence
over baroreflex function via NTS CB1 receptors which may contribute to suppressed
baseline cardiovagal BRS in (mRen2)27 rats and enhanced baseline BRS in ASrAOGEN
rats.
In contrast to the transgenic animals, there was no effect of CB1 receptor blockade
by 36 pmol of SR141716A in the NTS on BRS for control of HR in SD rats. This is in
line with previous observations by others that NTS microinjection of SR141716A alone
in dogs did not affect BRS,39 nor did the CB1-selective antagonist AM281 by itself alter
sympathetic nerve discharge in SD rats.21 This may indicate minimal or absent tonic
influence of the endocannabinoid system over baroreflex function in these normotensive
animals, or that baseline CB1 receptor modulation of the baroreflex in SD rats reflects
balanced effects on GABA and glutamate release. It is also possible that this reflects a
compensatory balance of additional factors in the NTS, such as endovanniloids,34 leptin,24
serotonin,40 and nonesterified fatty acids41 that may potentially influence baroreflex
function.
We did not evaluate the effect of NTS CB1 receptor blockade on the
sympathetically-mediated tachycardic BRS response to reductions in AP, so we cannot
yet determine if the endocannabinoid system selectively influences the parasympathetic
over the sympathetic baroreflex in our study paradigm. However, NTS microinjection of
93
0.36- or 36-pmol of SR141716 in SD, (mRen2)27 and ASrAOGEN rats did not alter
depressor and bradycardic responses to cardiac chemosensitive vagal fiber activation
induced by intravenous phenylbiguanide (Figure 2-11). The absence of alterations in
CVA responses supports the specificity of SR141716A actions on BRS because these
responses are mediated by chemoreceptor fibers that converge with baroreceptor inputs
within the NTS.42
A
PBG in SD Rats
Baseline
PBG in SD Rats
36 pmol
Baseline
0
HR (bpm)
-20
MAP (mmHg)
36 pmol
0
-40
-60
-50
-100
-150
-80
+SR141716A
-100
B
-200
PBG in (mRen2)27 Rats
PBG in (mRen2)27 Rats
Baseline
0.36 pmol
36 pmol
Baseline
0
-40
-60
-100
-150
-80
-100
Baseline
0.36 pmol
PBG in ASrAOGEN Rats
36 pmol
Baseline
-20
-80
-100
0.36 pmol
36 pmol
0
HR (bpm)
MAP (mmHg)
0
-60
+SR141716A
-200
PBG in ASrAOGEN Rats
-40
36 pmol
-50
HR (bpm)
MAP (mmHg)
-20
C
0.36 pmol
0
-50
-100
-150
-200
+SR141716A
Figure 2-11. Effects of Chemoreflex Activation on MAP and HR Induced by Intravenous
Phenylbiguanide in SD, (mRen2)27 and ASrAOGEN Rats
No change in depressor or bradycardic responses to chemosensitive vagal fiber activation following NTS
microinjection of SR141716A in SD, (mRen2)27 or ASrAOGEN rats (n = 3-5 all treatment groups; pooled
baselines are represented in figures).
94
We found that 2-AG content was highest in dorsal brainstem of (mRen2)27 rats
and lowest in ASrAOGEN rats, which may be a potential mechanism to explain
differential resting BRS among SD, (mRen2)27 and ASrAOGEN rats. Anandamide may
also play an important role in this region, but its detection at 1000-fold lower levels
without significant differences among strains indicates that 2-AG is likely the more
functionally significant endocannabinoid with respect to BRS modulation at the level of
the NTS in the face of altered brain RAS activity. We further observed a significantly
lower expression of CB1 receptor mRNA in dorsal medullary tissue of ASrAOGEN rats
relative to SD and (mRen2)27 rats. Together, these observations suggest differential
regulation of the endocannabinoid system in dorsal medulla of (mRen2)27 and
ASrAOGEN rats, respectively, paralleling the relative expression of RAS components in
these animals. In (mRen2)27 rats, the higher dorsal medullary 2-AG levels may reflect
increased release,27 increased activity of enzymes involved in its biosynthesis, such as
diacylglycerol lipase, or downregulation of its primary degradative enzyme
monoacylglycerol lipase43 as the principal drivers of upregulated CB1 receptor tone in
this region. Our data are consistent with evidence for upregulated endocannabinoid tone
that is widely reported in acute and chronic animal models of cardiovascular disease,
including atherosclerosis and hypertension.44-46
Although dorsal medullary gene expression of CB1 receptor in (mRen2)27 rats
did not differ from SD rats, enhanced sensitivity of the receptor cannot be ruled out as an
additional mechanism for upregulated CB1 receptor tone in this strain because Ang IIinduced increased expression of Gi protein isoforms is reported in SHR47 and one-kidney
one-clip hypertension.48 It is also possible that a signaling interaction between CB1 and
95
Ang II type 1 (AT1) receptors enhances the pathogenic signaling of Ang II, as recently
described.12 In ASrAOGEN rats, lower levels of 2-AG and CB1 receptor mRNA suggest
a more general downregulation of the dorsal medulla endocannabinoid system that may
contribute to the enhanced baseline BRS for control of HR in these animals. Whether the
differential expression of the CB1 receptor and production of 2-AG in the dorsal
brainstem of (mRen2)27 and ASrAOGEN rats is attributable to a direct interaction with
the RAS or an indirect effect is currently unknown; however, transactivation of CB1 by
AT1 receptors and direct stimulation of 2-AG production by Ang II has been observed in
vitro.49, 50 While we detected CB2 receptor mRNA in the NTS, it is likely not involved in
BRS modulation because previous studies in our laboratory with the non-selective
cannabinoid receptor agonist CP55,940 show all effects on BRS in SD rats are
completely blocked or reversed by the CB1 antagonist SR141716A.22 It is also unlikely
that the CB1-associated protein CRIP1a, believed to serve an autoinhibitory function of
CB1 receptors,23 is a primary contributor to differential modulation of BRS because we
did not detect significant differences in mRNA expression among the three rat strains.
We cannot exclude the possibility that the spread of the SR141716A injection
may have accessed neighboring brainstem nuclei, such as the area postrema or the dmnX,
for effects on BRS. However, microinjection of
125
I-Sar-Thr Ang II at this volume was
mostly confined to the NTS,51 and functional assessments showed that 50 nL of an AT1
receptor antagonist injected into the dmnX did not alter responses to NTS microinjection
of Ang II.52 The injection of SR141716A accessed NTS neuronal cell bodies as well as
presynaptic vagal afferents and other terminals for projecting pathways within the NTS,
so it is not clear which elements mediate the effects on BRS. However, Ang II peptides
96
are believed to exert actions on BRS at neuronal cell fibers, glial and vascular elements in
the NTS.53
We also cannot exclude the possibility that weak inverse agonist properties of
SR141716A are reflected in our present data.54 Maximal inhibition of constitutive Gprotein activity by SR141716A ranged from 20-40% at micromolar concentrations
depending on brain region in rats, although effects in brainstem were not assessed.55
However, it is unclear if this property of SR141716A yields functionally significant
actions in vivo.
For example, SR141716A by itself only slightly reduced neuronal
discharge in rat locus coeruleus.56 In the NTS of dogs SR141716A had no effect on
baseline AP or resting sympathetic BRS,39 fully consistent with our present data in SD
rats. Finally, in the periphery SR141617A did not alter basal constriction of rat gracilis
arterioles.27 Potential inverse agonism effects notwithstanding, the increased levels of 2AG we detected in dorsal brainstem of (mRen2)27 rats are consistent with a competitive
antagonist effect of SR141716A in NTS to improve BRS, while the impairments to BRS
in ASrAOGEN rats by SR141716A are too large to attribute to an inverse agonist effect.
We investigated changes in cardiovagal BRS, MAP and HR in response to acute,
site-specific CB1 receptor blockade in normotensive, hypertensive, and hypotensive rat
strains.
Our results support our hypothesis for differential expression of brain
endocannabinoid system components paralleling the trend for brain RAS expression in
these animals. The novel finding that NTS CB1 receptor blockade produces opposite
effects in rats over- or under-expressing components of the brain RAS may have
implications for understanding the mechanisms of autonomic reflex control of HR and
blood pressure in pathological conditions that are in part dependent on an activated brain
97
RAS, such as hypertension.
Effects of chronic peripheral or central CB1 receptor
blockade will need to be studied to further evaluate the role of endocannabinoid-mediated
impairments to BRS in pathophysiologies associated with elevated or diminished
circulating, cerebrospinal fluid, or brain tissue RAS.
98
Perspectives
The impairment of BRS for control of heart rate, an index of vagus nerve function, often
precedes the onset of hypertension and stroke3 and is a common feature of cardiovascular
risk factors, including obesity and aging.57 Identifying factors that modulate baroreflex
function may therefore be important for understanding predisposition or progression of
these negative conditions.
The present data suggest an upregulated brain
endocannabinoid system in Ang II-dependent hypertension may contribute to the
impaired BRS typical of these conditions, and that blockade of CB1 receptors improves
BRS. Therefore, CB1 receptor-mediated impairments in BRS may contribute to the
progression of increasing AP in populations with elevated Ang II or endocannabinoid
production. The positive metabolic effects of chronic systemic CB1 receptor blockade in
obese or diabetic humans and animals are well known.58 The results of our present study
suggest that chronic systemic administration of a CB1 receptor antagonist may have
additional, positive autonomic or cardiovascular effects in hypertension accompanied by
impaired metabolic function. Understanding the consequences and mechanisms of an
upregulated endocannabinoid system in hypertension will be important for therapeutic
targeting of all features of cardiovascular disease.
99
Acknowledgements
We thank Ellen Tommasi, Robert Lanning, and Alex Kovach for technical assistance.
Sources of Funding
This work was supported by the National Heart, Lung, and Blood Institute grant HL51952, the National Institute on Drug Abuse grant DA-03690, and RTI International
(Research Triangle Park, NC).
Support from the Farley-Hudson Foundation
(Jacksonville, NC) is gratefully acknowledged.
Disclosures
None.
100
References
1.
Pacher P, Batkai S, Kunos G. The endocannabinoid system as an emerging target
of pharmacotherapy. Pharmacol Rev. 2006;58:389-462.
2.
Despres JP. The endocannabinoid system: a new target for the regulation of
energy balance and metabolism. Crit Pathw Cardiol. 2007;6:46-50.
3.
Thayer JF, Lane RD. The role of vagal function in the risk for cardiovascular
disease and mortality. Biol Psychol. 2007;74:224-242.
4.
Pertwee RG. The pharmacology of cannabinoid receptors and their ligands: an
overview. Int J Obes (Lond). 2006;30 Suppl 1:S13-18.
5.
Van Sickle MD, Duncan M, Kingsley PJ, Mouihate A, Urbani P, Mackie K, Stella
N, Makriyannis A, Piomelli D, Davison JS, Marnett LJ, Di Marzo V, Pittman QJ,
Patel KD, Sharkey KA. Identification and functional characterization of brainstem
cannabinoid CB2 receptors. Science. 2005;310:329-332.
6.
Castillo PE, Younts TJ, Chavez AE, Hashimotodani Y. Endocannabinoid
signaling and synaptic function. Neuron. 2012;76:70-81.
7.
Mailleux P, Vanderhaeghen JJ. Distribution of neuronal cannabinoid receptor in
the adult rat brain: a comparative receptor binding radioautography and in situ
hybridization histochemistry. Neuroscience. 1992;48:655-668.
8.
Burdyga G, Varro A, Dimaline R, Thompson DG, Dockray GJ. Expression of
cannabinoid CB1 receptors by vagal afferent neurons: kinetics and role in
influencing neurochemical phenotype. Am J Physiol Gastrointest Liver Physiol.
2010;299:G63-69.
9.
Seagard JL, Dean C, Patel S, Rademacher DJ, Hopp FA, Schmeling WT, Hillard
CJ. Anandamide content and interaction of endocannabinoid/GABA modulatory
effects in the NTS on baroreflex-evoked sympathoinhibition. Am J Physiol Heart
Circ Physiol. 2004;286:H992-1000.
10.
Brozoski DT, Dean C, Hopp FA, Seagard JL. Uptake blockade of
endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition.
Brain Res. 2005;1059:197-202.
11.
Ferreira-Junior NC, Fedoce AG, Alves FH, Correa FM, Resstel LB. Medial
prefrontal cortex endocannabinoid system modulates baroreflex activity through
CB(1) receptors. Am J Physiol Regul Integr Comp Physiol. 2012;302:R876-885.
12.
Rozenfeld R, Gupta A, Gagnidze K, Lim MP, Gomes I, Lee-Ramos D, Nieto N,
Devi LA. AT1R-CB(1)R heteromerization reveals a new mechanism for the
pathogenic properties of angiotensin II. EMBO J. 2011;30:2350-2363.
101
13.
Janiak P, Poirier B, Bidouard JP, Cadrouvele C, Pierre F, Gouraud L, Barbosa I,
Dedio J, Maffrand JP, Le Fur G, O'Connor S, Herbert JM. Blockade of
cannabinoid CB1 receptors improves renal function, metabolic profile, and
increased survival of obese Zucker rats. Kidney Int. 2007;72:1345-1357.
14.
Bader M, Zhao Y, Sander M, Lee MA, Bachmann J, Bohm M, Djavidani B,
Peters J, Mullins JJ, Ganten D. Role of tissue renin in the pathophysiology of
hypertension in TGR(mREN2)27 rats. Hypertension. 1992;19:681-686.
15.
Borgonio A, Pummer S, Witte K, Lemmer B. Reduced baroreflex sensitivity and
blunted endogenous nitric oxide synthesis precede the development of
hypertension in TGR(mREN2)27 rats. Chronobiol Int. 2001;18:215-226.
16.
Schinke M, Baltatu O, Bohm M, Peters J, Rascher W, Bricca G, Lippoldt A,
Ganten D, Bader M. Blood pressure reduction and diabetes insipidus in transgenic
rats deficient in brain angiotensinogen. Proc Natl Acad Sci U S A. 1999;96:39753980.
17.
Kasper SO, Carter CS, Ferrario CM, Ganten D, Ferder LF, Sonntag WE,
Gallagher PE, Diz DI. Growth, metabolism, and blood pressure disturbances
during aging in transgenic rats with altered brain renin-angiotensin systems.
Physiol Genomics. 2005;23:311-317.
18.
Sakima A, Averill DB, Kasper SO, Jackson L, Ganten D, Ferrario CM, Gallagher
PE, Diz DI. Baroreceptor reflex regulation in anesthetized transgenic rats with
low glia-derived angiotensinogen. Am J Physiol Heart Circ Physiol.
2007;292:H1412-1419.
19.
Diz DI, Garcia-Espinosa MA, Gallagher PE, Ganten D, Ferrario CM, Averill DB.
Angiotensin-(1-7) and baroreflex function in nucleus tractus solitarii of
(mRen2)27 transgenic rats. J Cardiovasc Pharmacol. 2008;51:542-548.
20.
Arnold AC, Sakima A, Ganten D, Ferrario CM, Diz DI. Modulation of reflex
function by endogenous angiotensins in older transgenic rats with low glial
angiotensinogen. Hypertension. 2008;51:1326-1331.
21.
Durakoglugil MS, Orer HS. Cannabinoid receptor activation in the nucleus tractus
solitaries produces baroreflex-like responses in the rat. Int J Biomed Sci.
2008;4:229-237.
22.
Schaich CL, Howlett AC, Diz DI. Dose-Related Biphasic Modulation of
Baroreflex Sensitivity by the CB1 Receptor in Rat Solitary Tract Nucleus.
Hypertension. 2011;58:e105 (Abstract).
23.
Niehaus JL, Liu Y, Wallis KT, Egertova M, Bhartur SG, Mukhopadhyay S, Shi S,
He H, Selley DE, Howlett AC, Elphick MR, Lewis DL. CB1 cannabinoid
receptor activity is modulated by the cannabinoid receptor interacting protein
CRIP 1a. Mol Pharmacol. 2007;72:1557-1566.
102
24.
Arnold AC, Shaltout HA, Gallagher PE, Diz DI. Leptin impairs cardiovagal
baroreflex function at the level of the solitary tract nucleus. Hypertension.
2009;54:1001-1008.
25.
Schaich CL, Shaltout HA, Howlett AC, Diz DI. Chronic Systemic CB1 Receptor
Blockade Reduces Blood Pressure and Weight Gain and Improves Baroreflex
Function and Heart Rate Variability in Hypertensive (mRen2)27 Rats.
Hypertension. 2013;62:A183 (Abstract).
26.
Bellocchio L, Soria-Gomez E, Quarta C, Metna-Laurent M, Cardinal P, Binder E,
Cannich A, Delamarre A, Haring M, Martin-Fontecha M, Vega D, Leste-Lasserre
T, Bartsch D, Monory K, Lutz B, Chaouloff F, Pagotto U, Guzman M, Cota D,
Marsicano G. Activation of the sympathetic nervous system mediates hypophagic
and anxiety-like effects of CB(1) receptor blockade. Proc Natl Acad Sci U S A.
2013;110:4786-4791.
27.
Szekeres M, Nadasy GL, Turu G, Soltesz-Katona E, Toth ZE, Balla A, Catt KJ,
Hunyady L. Angiotensin II Induces Vascular Endocannabinoid Release, Which
Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors. J Biol
Chem. 2012;287:31540-31550.
28.
Menzies JR, Ludwig M, Leng G. Direct and indirect effects of cannabinoids on in
vitro GABA release in the rat arcuate nucleus. J Neuroendocrinol. 2010;22:585592.
29.
Garcia-Espinosa MA, Shaltout HA, Olson J, Westwood BM, Robbins ME, Link
K, Diz DI. Proton magnetic resonance spectroscopy detection of
neurotransmitters in dorsomedial medulla correlate with spontaneous baroreceptor
reflex function. Hypertension. 2010;55:487-493.
30.
Derbenev AV, Stuart TC, Smith BN. Cannabinoids suppress synaptic input to
neurones of the rat dorsal motor nucleus of the vagus nerve. J Physiol.
2004;559:923-938.
31.
Peters JH, McDougall SJ, Fawley JA, Smith SM, Andresen MC. Primary afferent
activation of thermosensitive TRPV1 triggers asynchronous glutamate release at
central neurons. Neuron. 2010;65:657-669.
32.
Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sorgard M, Di Marzo V,
Julius D, Hogestatt ED. Vanilloid receptors on sensory nerves mediate the
vasodilator action of anandamide. Nature. 1999;400:452-457.
33.
Andresen MC, Fawley JA, Hofmann ME. Peptide and lipid modulation of
glutamatergic afferent synaptic transmission in the solitary tract nucleus. Front
Neurosci. 2012;6:191.
103
34.
Derbenev AV, Monroe MJ, Glatzer NR, Smith BN. Vanilloid-mediated
heterosynaptic facilitation of inhibitory synaptic input to neurons of the rat dorsal
motor nucleus of the vagus. J Neurosci. 2006;26:9666-9672.
35.
Anwar IJ, Derbenev AV. TRPV1-dependent regulation of synaptic activity in the
mouse dorsal motor nucleus of the vagus nerve. Front Neurosci. 2013;7:238.
36.
Wang W, Cao X, Liu C, Liu L. Cannabinoid WIN 55,212-2 inhibits TRPV1 in
trigeminal ganglion neurons via PKA and PKC pathways. Neurol Sci.
2012;33:79-85.
37.
Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B, Congy C,
Martinez S, Maruani J, Neliat G, Caput D, et al. SR141716A, a potent and
selective antagonist of the brain cannabinoid receptor. FEBS Lett. 1994;350:240244.
38.
Rinaldi-Carmona M, Barth F, Heaulme M, Alonso R, Shire D, Congy C, Soubrie
P, Breliere JC, Le Fur G. Biochemical and pharmacological characterisation of
SR141716A, the first potent and selective brain cannabinoid receptor antagonist.
Life Sci. 1995;56:1941-1947.
39.
Rademacher DJ, Patel S, Hopp FA, Dean C, Hillard CJ, Seagard JL.
Microinjection of a cannabinoid receptor antagonist into the NTS increases
baroreflex duration in dogs. Am J Physiol Heart Circ Physiol. 2003;284:H15701576.
40.
Nalivaiko E, Sgoifo A. Central 5-HT receptors in cardiovascular control during
stress. Neurosci Biobehav Rev. 2009;33:95-106.
41.
Shaltout HA, Abdel-Rahman AA. Mechanism of fatty acids induced suppression
of cardiovascular reflexes in rats. J Pharmacol Exp Ther. 2005;314:1328-1337.
42.
Paton JF. Convergence properties of solitary tract neurones driven synaptically by
cardiac vagal afferents in the mouse. J Physiol. 1998;508 ( Pt 1):237-252.
43.
Di Marzo V. Endocannabinoids: synthesis and degradation. Rev Physiol Biochem
Pharmacol. 2008;160:1-24.
44.
Pacher P, Batkai S, Kunos G. Blood pressure regulation by endocannabinoids and
their receptors. Neuropharmacology. 2005;48:1130-1138.
45.
Sugamura K, Sugiyama S, Nozaki T, Matsuzawa Y, Izumiya Y, Miyata K,
Nakayama M, Kaikita K, Obata T, Takeya M, Ogawa H. Activated
endocannabinoid system in coronary artery disease and antiinflammatory effects
of cannabinoid 1 receptor blockade on macrophages. Circulation. 2009;119:2836.
104
46.
Naito Y, Tsujino T, Akahori H, Ohyanagi M, Mitsuno M, Miyamoto Y,
Masuyama T. Augmented cannabinoid receptors expression in human aortic valve
stenosis. Int J Cardiol. 2010;145:535-537.
47.
Marcil J, Thibault C, Anand-Srivastava MB. Enhanced expression of Gi-protein
precedes the development of blood pressure in spontaneously hypertensive rats. J
Mol Cell Cardiol. 1997;29:1009-1022.
48.
Ge C, Garcia R, Anand-Srivastava MB. Enhanced expression of Gialpha protein
and adenylyl cyclase signaling in aortas from 1 kidney 1 clip hypertensive rats.
Can J Physiol Pharmacol. 2006;84:739-746.
49.
Turu G, Simon A, Gyombolai P, Szidonya L, Bagdy G, Lenkei Z, Hunyady L.
The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptorstimulated cannabinoid CB1 receptor activity. J Biol Chem. 2007;282:7753-7757.
50.
Turu G, Varnai P, Gyombolai P, Szidonya L, Offertaler L, Bagdy G, Kunos G,
Hunyady L. Paracrine transactivation of the CB1 cannabinoid receptor by AT1
angiotensin and other Gq/11 protein-coupled receptors. J Biol Chem.
2009;284:16914-16921.
51.
Campagnole-Santos MJ, Diz DI, Ferrario CM. Baroreceptor reflex modulation by
angiotensin II at the nucleus tractus solitarii. Hypertension. 1988;11:I167-171.
52.
Fow JE, Averill DB, Barnes KL. Mechanisms of angiotensin-induced hypotension
and bradycardia in the medial solitary tract nucleus. Am J Physiol.
1994;267:H259-266.
53.
Barnes KL, DeWeese DM, Andresen MC. Angiotensin potentiates excitatory
sensory synaptic transmission to medial solitary tract nucleus neurons. Am J
Physiol Regul Integr Comp Physiol. 2003;284:R1340-1353.
54.
Pertwee RG. Inverse agonism and neutral antagonism at cannabinoid CB1
receptors. Life Sci. 2005;76:1307-1324.
55.
Sim-Selley LJ, Brunk LK, Selley DE. Inhibitory effects of SR141716A on Gprotein activation in rat brain. Eur J Pharmacol. 2001;414:135-143.
56.
Muntoni AL, Pillolla G, Melis M, Perra S, Gessa GL, Pistis M. Cannabinoids
modulate spontaneous neuronal activity and evoked inhibition of locus coeruleus
noradrenergic neurons. Eur J Neurosci. 2006;23:2385-2394.
57.
Thayer JF, Yamamoto SS, Brosschot JF. The relationship of autonomic
imbalance, heart rate variability and cardiovascular disease risk factors. Int J
Cardiol. 2010;141:122-131.
58.
Van Gaal LF, Rissanen AM, Scheen AJ, Ziegler O, Rossner S. Effects of the
cannabinoid-1 receptor blocker rimonabant on weight reduction and
105
cardiovascular risk factors in overweight patients: 1-year experience from the
RIO-Europe study. Lancet. 2005;365:1389-1397.
Novelty and Significance: 1) What Is New, 2) What Is Relevant?
What Is New?

Blockade of NTS CB1 receptors produces opposite effects on baroreflex
sensitivity for control of heart rate in animals with differential expression of the brain
RAS.

Transgenic (mRen2)27 rats have increased production of 2-AG in dorsal medulla
compared to ASrAOGEN rats; ASrAOGEN rats have decreased CB1 receptor gene
expression relative to Sprague-Dawley and (mRen2)27 rats.
What Is Relevant?

Results indicate novel mechanisms for preservation of baroreflex function
involving the endocannabinoid system in conditions associated with altered brain RAS
activity.
Summary
Altered brain RAS activity is associated with differential expression and function of the
dorsal medullary endocannabinoid system with respect to blood pressure regulation.
Increased endocannabinoid production in this region may maintain impaired resting
baroreflex sensitivity in conditions associated with upregulated brain RAS expression.
106
CHAPTER THREE
ALTERATIONS IN THE MEDULLARY ENDOCANNABINOID SYSTEM
CONTRIBUTE TO AGE-RELATED IMPAIRMENT OF BAROREFLEX
SENSITIVITY
By
Chris L. Schaich,1 Megan Grabenauer,1, 2 Brian F. Thomas,1, 2 Patricia E. Gallagher,1
Allyn C. Howlett,1 Debra I. Diz1
1
Department of Physiology and Pharmacology; and Hypertension & Vascular Research
Center, Wake Forest School of Medicine, Winston-Salem, NC, USA
2
Analytical Chemistry and Pharmaceutics, RTI International, Research Triangle Park,
NC, USA
Short title: Endocannabinoids, Aging and Baroreflex Sensitivity
The following manuscript was submitted to Hypertension and represents the efforts of the
first author. Differences in formatting and organization reflect the requirements of the
journal.
107
Abstract
As they age, Sprague-Dawley (SD) rats develop elevated systolic blood pressure
associated with impaired baroreflex sensitivity (BRS) for control of heart rate and
metabolic dysfunction. An imbalance in the angiotensin (Ang) peptides Ang II and Ang(1-7) in the brain solitary tract nucleus (NTS) also emerges with age and contributes to
the age-related decline in BRS.
We previously demonstrated in young (mRen2)27
hypertensive rats featuring low medullary Ang-(1-7) relative to Ang II that impaired BRS
in this strain is restored by CB1 cannabinoid receptor blockade in the NTS, consistent
with elevated content of the endocannabinoid 2-arachidonoylglycerol (2-AG) in dorsal
medulla relative to SD rats. We now report that in older SD rats, dorsal medullary 2-AG
levels were two-fold higher at 70 versus 15 weeks of age (3.89 ± 0.66 vs. 1.85 ± 0.27
ng/mg tissue; P < 0.05; n = 4-6). Furthermore, relative expression of CB1 receptor
mRNA was significantly lower in aged rats, while CB2 mRNA was significantly higher.
Microinjection of the CB1 receptor antagonist SR141716A (36 pmol) into the NTS of
older SD rats normalized BRS only in animals that exhibited impaired baseline BRS
relative to younger animals (0.56 ± 0.06 ms/mmHg baseline vs. 1.06 ± 0.05 ms/mmHg
after 60 min; P < 0.05; n = 4). In contrast, there was no effect of SR141716A in older SD
rats with normal baseline BRS (1.14 ± 0.17 ms/mmHg baseline vs. 1.29 ± 0.23
ms/mmHg after 60 min; n = 4), consistent with previous observations in younger SD rats.
Therefore, the present study provides evidence for altered endocannabinoid tone within
the NTS of older SD rats that may contribute to age-related impairment of BRS.
108
Introduction
Aging is associated with changes in autonomic regulation of blood pressure that may
facilitate the progression or maintenance of hypertension and related diseases.1
An
imbalance in the autonomic nervous system, typically in which the sympathetic nervous
system predominates over the parasympathetic, is a primary cause of increased systolic
blood pressure in aged individuals with hypertension.2 Declining baroreflex sensitivity
(BRS) for control of heart rate (HR), an index of vagus nerve function and general
mortality,3 is permissive toward reduced heart rate variability, increased sympathetic
outflow, and impaired ability to correct subsequent elevations in blood pressure during
aging.4 Therefore, identification of central nervous system factors that contribute to agerelated reductions in BRS is required to gain a greater understanding of the development
of hypertension in the worldwide aging population.
Alterations in the brain endocannabinoid system that accompany age-related
cognitive decline,5 cardiovascular diseases and metabolic disorders6 may contribute to the
modulation of baroreflex function during aging. The endocannabinoid system is a central
and peripheral cellular signaling system that comprises the endogenous cannabinoids
anandamide and 2-arachidonoylglycerol (2-AG), their G protein-coupled CB1 and CB2
receptor subtypes, and enzymes regulating their biosynthesis and degradation.
Upregulated endocannabinoid tone is reported in hypertension and its metabolic risk
factors,7, 8 suggesting a pathogenic or compensatory role in these negative conditions.
Mounting evidence links upregulated endocannabinoid tone with imbalances in
the two active peptides of the renin-angiotensin system (RAS), angiotensin (Ang) II and
Ang-(1-7).
13, 14
Ang II exerts a widespread influence over the sympathetic nervous
109
system and impairs BRS during aging,9,
10
and may stimulate production of
endocannabinoids through actions at Ang II type 1 (AT1) receptors.11 We previously
demonstrated that blockade of CB1 receptors within the solitary tract nucleus (NTS) of
the dorsomedial medulla, the primary site for termination of baroreceptor afferent
neurons, improves the blunted BRS for control of HR in young adult (mRen2)27 rats
with Ang II-dependent hypertension, with no effect in normotensive Sprague-Dawley
(SD) rats with normal baroreflex function.12, 13 The impaired BRS in (mRen2)27 rats is
also associated with loss of Ang-(1-7) facilitation of the BRS,14 resembling the brain
RAS profile of aged SD rats.15 To date there has been no investigation into the role of
the NTS endocannabinoid system in impaired BRS associated with aging.
In the present study, we evaluate the effect of the CB1 receptor antagonist
SR141716A on BRS for control of HR when microinjected into the NTS of older SD rats.
We also employ mass spectrometry to measure levels of dorsal medullary
endocannabinoids, and RT-qPCR to measure mRNA levels of cannabinoid receptors
(CB1 and CB2), and cannabinoid receptor interacting protein (CRIP)1a16 in the NTS of
younger and older SD rats. Based on evidence for decreased NTS Ang-(1-7) tone in
aging15 and the association between augmented Ang II and endocannabinoid tone in
hypertensive rats with impaired BRS,12,
endocannabinoid levels in aged rats.
13
we hypothesized a similar trend for
We now provide functional and biochemical
evidence that components of the endocannabinoid system within the NTS change during
normal aging and may contribute to age-related declining baroreflex function.
110
Methods
Animals
Experiments were performed in 15-, 25-, and 60- to 78-week-old male Hannover SD rats
obtained from the Hypertension & Vascular Research Center colony at Wake Forest
School of Medicine. Rats were housed two-per-cage in a temperature- and humiditycontrolled room that maintained a 12-hour light/dark cycle (lights on at 06:00), with free
access to food (standard chow) and water.
All procedures were approved by the
Institutional Animal Care and Use Committee.
Surgical Procedures and Hemodynamic Measures
As described previously,15 aged SD rats (60- to 69-weeks-old; mean age = 64 ± 1 weeks
for all rats; N = 8) were anesthetized with combination urethane-chloralose (750 and 35
mg/kg, respectively) via intraperitoneal injections with supplemental doses given
intravenously as needed (diluted to 3:7 with saline).
Polyethylene catheters (Clay
Adams) were inserted into the femoral artery and vein, with the venous catheter
positioned near the right atrium. Rats were then placed in a stereotaxic frame with the
head tilted at a 45° downward angle for surgical exposure of the dorsal medulla, and were
provided a mixture of room air and oxygen to breathe.
A 30 minute resting and
equilibration period was allowed before testing began. Pulsatile arterial pressure (AP)
and mean AP (MAP) were recorded by data acquisition software (AcqKnowledge version
3.8.1, BIOPAC Systems, Inc., Goleta, CA), and HR was calculated from the AP wave.
After obtaining stable measures of MAP and HR, baseline BRS was established by
sequential bolus intravenous injection of 3 doses (2, 5 and 10 μg/kg in 0.9% NaCl) of
phenylephrine (PE) to determine the bradycardic BRS response for increases in AP. BRS
111
for bradycardia was defined as the slope of the relationship between changes in MAP
(ΔMAP; mmHg) and the pulse interval (ΔPI; ms) generated from the 3 doses of PE
(mean R2 = 0.92 for all animals) in units of ms/mmHg. Reflex testing was repeated 60
minutes after bilateral microinjection of the CB1 receptor-selective antagonist
SR141716A (obtained from the National Institute on Drug Abuse) into the NTS, and was
completed within 15 minutes, with each rat serving as its own control. The doses and
timecourse of the study were chosen based on previous experience with SR141716A
microinjections in our laboratory12,
13
in which the effect of the drug on BRS was
immediate and sustained over 60 minutes without changing resting MAP or HR.
NTS Microinjections
SR141716A (36 pmol in a 120 nL volume of 10% DMSO in artificial cerebrospinal fluid
vehicle) was bilaterally microinjected into the NTS (0.4 mm rostral, 0.4 mm lateral to the
calamus scriptorius [caudal tip of the area postrema], and 0.4 mm below the dorsal
surface) using a glass micropipette connected to a hand-held syringe, as reported
previously.15 Previous studies in older SD rats from a similar age range in our laboratory
revealed NTS microinjection of the vehicle solution has no significant effect on evoked
BRS, MAP or HR at any timepoint studied.15,
17
At the end of testing, brains were
removed, frozen, and sectioned (30 μm) for localization of microinjection sites. Only
data from injections within the medial NTS at the rostrocaudal level -13.3 to -14.0 mm
caudal to bregma were used in the analysis.
Mass Spectrometry Detection of Anandamide and 2-AG
A detailed protocol can be found in Chapter Two of this dissertation. Medullary tissue
was obtained from separate age groups of naïve SD rats: 15-, 25-, and 67- to 78-weeks112
old (mean age of older animals = 70 ± 2 weeks; n = 4-6; P < 0.05 vs. age of rats utilized
in the microinjection studies). Brains were removed and frozen on dry ice for excision of
3-mm3 dorsal medullary sections. For analysis of the endocannabinoids anandamide and
2-AG, an extraction technique was developed for rat medulla tissue as previously
described. Study samples, quality control samples, and standards (targeted analysis) were
processed using automated liquid handling to ensure the reproducibility and consistency
of the sample preparation method. Sample content of anandamide or 2-AG is expressed
as ng analyte per mg tissue wet weight.
Quantification of CB1, CB2, and CRIP1a mRNA
RT-qPCR was used to measure mRNA levels of various effector components of the brain
endocannabinoid system, including CB1 and CB2 receptor, and CRIP1a, in dorsal
medullary tissue from the same 15- and 70-week-old rats in which anandamide and 2-AG
were measured. Brains were removed and frozen on dry ice as described above. Isolated
RNA from excised tissue was assayed for concentration and stability. As described
previously,15 total RNA (1 µg) was reverse transcribed using AMV reverse transcriptase
in a 20-µL reaction mixture containing deoxyribonucleotides, random hexamers, and
RNase inhibitor in reverse-transcriptase buffer. For RT-qPCR, 2 µL of the resultant
cDNA was added to TaqMan Universal PCR Master Mix with the appropriate genespecific primer/probe set for CB1 and CB2 receptors, and CRIP1a (Applied Biosystems),
and amplification performed. All reactions were performed in triplicate. 18S ribosomal
RNA served as the internal control. Results were quantified as Ct values, in which Ct is
the threshold cycle of PCR at which an amplified product is first detected, and was
defined as relative gene expression (ratio of target:control).
113
Analysis of Data
Values are presented as mean ± SEM. Comparisons of changes in BRS from baseline in
response to SR141716A in older SD rats were analyzed by Student’s paired t-test.
Multiple regression analysis was performed on BRS scatterplots to determine slopes of fit
lines. One-way ANOVA and post-hoc Tukey multiple comparisons were used to analyze
differences in medullary endocannabinoid content between age groups. Differences in
relative mRNA expression between young and older rats were analyzed by unpaired ttests. The criterion for statistical significance was P < 0.05. Statistical tests were
performed using Prism 5.0 (GraphPad Software, San Diego, CA).
114
Results
CB1 Receptor Blockade in the NTS of Aged SD Rats: BRS for Control of HR, MAP,
HR, and MAP Responsiveness
In 60- to 69-week-old SD rats used in our study for assessment of cardiovagal baroreflex
function, we divided animals into two groups based on baseline BRS values. One group
of animals exhibited normal baseline BRS for control of HR similar to younger SD rats 15
whereas the others exhibited lower baseline BRS typical of hypertensive rats in our
laboratory14 (1.14 ± 0.17 ms/mmHg Normal vs. 0.56 ± 0.06 ms/mmHg Impaired; P <
0.05; n = 4; Figure 3-1). The cutoff for assignment was 0.80 ms/mmHg and the mean
ages of the Normal and Impaired baseline BRS groups were both 64 ± 1 weeks (Table 31). There was no effect of SR141716A (36 pmol) microinjection in aged SD rats with
normal baseline BRS (Figure 3-2A and 3-2B). However, the CB1 antagonist improved
BRS by approximately 89% to 1.06 ± 0.05 ms/mmHg in aged SD rats with impaired
baseline BRS (P < 0.05; Figure 3-2C and 3-2D). There was a significant negative
correlation between baseline BRS and change in BRS after SR141716A microinjection
(R2 = 0.54; P < 0.05; Figure 3-2E).
There were no differences in baseline MAP or HR between Normal and Impaired
baseline BRS groups, nor were values significantly changed by NTS microinjection of
SR141716A after 60 minutes (Table 3-1). In addition, there were no differences in MAP
responsiveness to intravenous PE injections during reflex testing by SR141716A in either
group (Figure 3-3).
115
A
Baseline BRS in Aged SD Rats
2.0
ms/mmHg
1.5
1.0
*
0.5
0.0
Normal
Impaired
B
80
Normal BRS
Impaired BRS
PI (ms)
60
40
20
Figure 3-1. Baseline BRS for Control
of HR in Response to Increases in AP
in Aged SD Rats. A, Assessment of
cardiovagal BRS in 64-week-old rats
revealed one group that exhibited
baseline BRS values similar to young
SD rats (Normal; n = 4) and another
group that exhibited significantly lower
baseline BRS typical of aging or
hypertension (Impaired; n = 4). B, The
slope of the relationship between
increases in MAP produced by PE and
the corresponding reflex bradycardia
(ΔPI) for aged SD rats with normal
baseline BRS was significantly greater
than for rats with impaired baseline
BRS (1.04 ± 0.21 ms/mmHg Normal
vs. 0.50 ± 0.11 ms/mmHg Impaired; P <
0.05; R2 = 0.67 to 0.71 for pooled data).
*P < 0.05 vs. Normal.
0
0
20
40
MAP (mmHg)
60
Table 3-1. Mean Age and Values of MAP and HR in Response to NTS Microinjection of SR141716A
Group
Mean Age
(weeks)
Aged SD Rats with Normal BRS
Baseline
Values at 60 minutes
64 ± 1
Aged SD Rats with Impaired BRS
Baseline
Values at 60 minutes
64 ± 1
MAP
(mmHg)
HR
(bpm)
81 ± 3
80 ± 4
279 ± 7
267 ± 12
89 ± 8
84 ± 4
292 ± 10
283 ± 13
Values are mean ± SEM and represent MAP and HR values at baseline and 60 minutes after NTS
microinjection of 36 pmol of SR141716A; MAP = mean arterial pressure; HR = heart rate; n = 4 both
groups.
116
A
B
2.0
80
1.5
60
PI (ms)
ms/mmHg
Normal Baseline BRS
1.0
0.5
Baseline
+SR141716A
40
20
0.0
0
Baseline
+SR141716A
0
20
40
60
MAP (mmHg)
C
D
Impaired Baseline BRS
80
2.0
60
*
PI (ms)
ms/mmHg
1.5
Baseline
+SR141716A
1.0
0.5
40
20
0.0
0
Baseline
+SR141716A
0
20
40
60
MAP (mmHg)
E
Effect of SR141716A
(% Change in BRS)
300
200
100
0
-100
0.0
0.5
1.0
1.5
Baseline BRS (ms/mmHg)
2.0
Figure 3-2. Effect of NTS Microinjection of SR141716A (36 pmol) in Aged SD Rats with Normal or
Impaired Baseline BRS for Control of HR. A-B, SR141716A had no effect in aged SD rats with normal
baseline BRS. C, In aged SD rats with impaired baseline BRS, SR141716A significantly improved BRS to
a level comparable with normal baseline BRS. D, SR141716A increased the slope of the BRS regression
line in rats with impaired baseline BRS (0.50 ± 0.11 ms/mmHg baseline; 1.00 ± 0.13 ms/mmHg 60 minutes
after 36 pmol of SR141716A; P < 0.01; R2 = 0.67 to 0.87 for pooled data). E, Magnitude of SR141716A
effect on BRS was negatively associated with baseline BRS among all animals (R2 = 0.54; P < 0.05; N = 8).
*P < 0.05 vs. baseline.
117
A
Baseline MAP Responsiveness
80
Normal BRS
Impaired BRS
MAP
(mmHg)
60
40
20
0
0
5
PE (g/kg)
10
C
B
Normal Baseline BRS
Impaired Baseline BRS
80
40
20
Baseline
+SR141716A
60
MAP
(mmHg)
60
MAP
(mmHg)
80
Baseline
+SR141716A
40
20
0
0
0
5
PE (g/kg)
10
0
5
PE (g/kg)
10
Figure 3-3. MAP Responsiveness to Intravenous PE in Aged SD Rats with Normal or Impaired
Baseline BRS, Before and After NTS Microinjection of SR141716A
A, There were no differences in MAP responsiveness between Normal and Impaired BRS groups of aged
SD rats at baseline reflex testing. B-C, There was not a significant change 60 minutes after NTS
microinjection of SR141716A in either group (n = 4 per group).
118
Dorsal Medullary 2-AG and Anandamide Content in Young Adult, Middle Aged
and Older SD Rats
Levels of 2-AG were lowest in 15-week-old and highest in 70-week-old SD rats (1.17 ±
0.09 vs. 3.89 ± 0.66 ng/mg tissue, respectively; P < 0.001; n = 4; Figure 3-4A). Middle
aged SD rats (25 weeks) had intermediate medullary 2-AG content (3.13 ± 0.46 ng/mg
tissue; n = 4), falling just short of statistical significance compared to 15-week-old rats (P
= 0.052; Figure 3-4A). Of note, the relative 2-AG levels parallel the trend in resting
conscious MAP and are inversely related to the baseline BRS for control of HR reported
across the SD rat lifespan. In contrast, dorsal medullary anandamide was found in levels
1000-fold lower than 2-AG and there were no significant differences among age groups
(Figure 3-4B).
A
*
2-AG
(ng/mg tissue)
5
4
3
2
1
0
15 weeks
25 weeks
70 weeks
15 weeks
25 weeks
70 weeks
B
Anandamide
(pg/mg tissue)
10
8
6
4
2
0
119
Figure 3-4. 2-AG and Anandamide
Content in the Dorsal Medulla of 15, 25- and 70-Week-Old SD Rats. A,
Mass spectrometry revealed levels of
2-AG were significantly higher in the
dorsal medullary tissue of 70-week-old
relative to 15-week-old SD rats. In
addition, 2-AG levels trended higher in
25-week-old relative to 15-week-old
rats (P = 0.052). B, Anandamide was
detected at levels 1000-fold lower than
2-AG in the same tissue samples, and
there were no significant differences
among age groups. *P < 0.05 vs. 15
weeks; n = 4-6 all groups.
CB1, CB2 and CRIP1a mRNA in Young and Older SD Rats
CB1 receptor mRNA was approximately 0.45-fold lower in the dorsal medulla of 70week-old SD rats relative to 15-week-old rats (P < 0.001; n = 5-6; Figure 3-5A), while
medullary CB2 receptor mRNA was approximately 0.73-fold higher in the older rats
compared to young rats (P < 0.05; n = 5-6; Figure 3-5B). There was no difference in
relative mRNA levels of CRIP1a between the same age groups (Figure 3-5C).
B
*
2.0
CB2 Receptor
Relative Gene Expression
CB1 Receptor
Relative Gene Expression
A
1.5
1.0
***
0.5
0.0
15 weeks
70 weeks
15 weeks
70 weeks
2.0
1.5
1.0
0.5
0.0
15 weeks
70 weeks
CRIP1a
Relative Gene Expression
C
2.0
1.5
1.0
0.5
0.0
Figure 3-5. Expression of CB1, CB2 and CRIP1a mRNA in the Dorsal Medulla of 15- and 70-WeekOld SD Rats. A, RT-qPCR determined that relative gene expression of CB 1 receptor was significantly
lower in dorsal medullary tissue of 70- versus 15-week-old SD rats. B, Relative gene expression of CB 2
receptor was significantly higher in the same tissue of 70- versus 15-week-old rats. C, There was not a
significant difference in relative gene expression of CRIP1a in the dorsal medulla between 15- and 70week-old rats. *P < 0.05 vs. 15 weeks; ***P < 0.001; n = 5-6 all groups.
120
Discussion
In this study we report the following: (1) blockade of CB1 receptors in the NTS of aged
SD rats normalizes cardiovagal BRS for control of HR only in animals that exhibit agerelated impairment in baseline BRS but has no effect in aged animals with normal BRS;
(2) 2-AG content in the dorsal medulla increases over the lifespan of SD rats; and (3)
aging in SD rats is associated with significantly decreased CB1 receptor mRNA
expression and significantly increased CB2 receptor mRNA expression in the dorsal
medulla relative to younger animals. Collectively, these results suggest that alterations in
the components of the dorsal medullary endocannabinoid system may contribute to agerelated decline in baroreflex function.
Aging, independent of resting blood pressure or weight gain, is associated with
physiological changes that can reduce baroreflex function over the lifespan of humans
and animals.1,
18
Progressive autonomic imbalance with blunted sympathetic or
parasympathetic reflex control over the heart and vasculature is accepted as a primary
contributor to elevated blood pressure during aging.19 Several factors may play a major
role in the development of age-related autonomic dysfunction, including the brain reninangiotensin system (RAS),9, 10 and metabolic hormones insulin and leptin.20 The balance
of factors involved in impaired baroreflex function during aging remains unclear.
However, reported signaling interactions between the endocannabinoid system and the
RAS,21 insulin22 and leptin23 may illuminate some of the mechanisms underlying agerelated reductions in BRS for control of HR.
The RAS peptides Ang II and Ang-(1-7) have opposite effects on baroreflex
modulation, with Ang II in the NTS reducing and Ang-(1-7) facilitating BRS,24 but the
121
provenance of alterations in the components of the RAS associated with aging are
unknown. AT1 receptor mRNA in rat brain either declines with age25 or does not
change,26 and Ang-(1-7) mas receptor mRNA expression is no different in older versus
younger rats.15
However, a shift in balance between the content or actions of the
counterbalancing Ang peptides occurs during aging that results in predominance of Ang
II tone in the NTS, leading to age-related impairment of BRS.27 This is illustrated by
previous studies in our laboratory in which AT1 blockade in the NTS by candesartan
improved BRS for control of HR in both young and older SD rats, but mas receptor
blockade by d-Ala7-Ang-(1-7) following candesartan treatment impaired BRS only in
young rats.15 The same study also reported that mRNA expression of neprilysin, an
endopeptidase that cleaves angiotensinogen to form Ang-(1-7),28 was reduced in dorsal
medulla of older rats,15 in line with earlier reports of decreased neprilysin activity in
forebrain and plasma of older animals.29, 30 Collectively, the role of the BRS-enhancing
peptide Ang-(1-7) in the tonic regulation of baroreflex function in the NTS is abrogated
via downregulated formation to the BRS-attenuating peptide Ang II during aging.
We previously reported increased dorsal medullary 2-AG content in young
transgenic (mRen2)27 hypertensive rats,31 which feature upregulated brain Ang II tone
with markedly impaired BRS for control of HR,32 compared to young SD rats and
transgenic Ang-deficient ASrAOGEN rats with enhanced baseline BRS. The dorsal
medulla of aged SD rats therefore represents the second in vivo setting associated with
increased Ang II-to-Ang-(1-7) RAS imbalance with impaired BRS in which we found
increased levels of 2-AG.
Functional data from our current study support our
hypothesis that enhanced NTS endocannabinoid tone contributes to defective baroreflex
122
function in aging because blockade of NTS CB1 receptors in aged SD rats normalized
BRS for control of HR only in animals with impaired baseline BRS; there was no effect
of NTS CB1 blockade in aged rats with preserved BRS. These results are similar to our
previous observations that CB1 blockade in the NTS of younger SD rats had no effect on
BRS but dose-dependently improved BRS in (mRen2)27 rats. We further previously
demonstrated a dose-related biphasic effect of NTS CB1 receptor activation on BRS, in
which low doses of a CB1 agonist enhanced whereas a high dose reduced BRS in young
SD rats,33 revealing that elevated CB1 receptor signaling in the NTS can impair BRS in
healthy animals. Half of the aged animals used in the microinjection experiments of our
current study had normal baseline BRS whereas the others exhibited impaired BRS at
baseline. This may be a reflection of the younger age range of rats used in our current
study for the microinjection experiments compared to previous studies in our
laboratory,15 as well as those exhibiting elevated 2-AG. Therefore, rats from our current
microinjection studies may have been aged close to the transitional period when agerelated impairment in BRS first manifests.
Evidence of signaling interactions between the endocannabinoid system and RAS
is mounting11, 21, 34 and may be a potential mechanism of action for the effects of NTS
CB1 receptor blockade on blunted BRS for control of HR associated with aging. In
addition to direct endocannabinoid-RAS interactions in the NTS, it is conceivable that
CB1 receptors may facilitate the AT1 receptor-mediated activation of sympathetic
baroreflex pathways from the hypothalamus by disinhibition of hypothalamic neurons.35,
36
Although our current study does not directly address this intriguing interaction, it
123
would be interesting to investigate whether co-microinjection of CB1 and AT1 or mas
ligands in the NTS yields additive or synergistic effects on BRS.
In addition to the RAS, endocannabinoids are also known to interact with central
insulin22 and leptin23 signaling pathways. Insulin resistance in the periphery and central
nervous system is a cardinal risk factor for age-related diseases including Alzheimer’s
dementia, obesity, type II diabetes, and cardiovascular disease.37 Direct CB1 receptormediated inhibition of insulin receptor signaling has been shown in the periphery,38 and
endocannabinoids modulate insulin signaling pathways in the brain.22 Defective leptin
signal transduction in the brain is associated with aging and age-related obesity,39 and
accompanies overactive endocannabinoid signaling independent of obesity or aging.23
Importantly, deficient central leptin receptor signaling during aging directly influences
CB1 receptor mRNA expression in the dorsal medulla.40 Both insulin41 and leptin42
decrease BRS for control of HR through actions in the NTS, so it is certainly possible
that endocannabinoids may interact with either hormone to suppress BRS in hypertension
or aging. Other factors involved in BRS modulation at the level of the dorsal medulla
during aging and known to interact with the endocannabinoid system include serotonin,43
catecholamines,44 TRPV1 ion channels,45 and the inhibitory neurotransmitter γaminobutyric acid (GABA).46 We cannot discount that endocannabinoid interactions
with any of these factors are reflected in our present functional data, particularly
interactions with GABA because GABAA receptor-mediated inhibitory responses in
neurons become less sensitive to cannabinoids with age.47
Expression of CB1 receptors in dorsal medullary sites along the baroreflex arc,
including in terminals of nodose ganglion vagal afferent neurons,48 the NTS,49 and dorsal
124
motor nucleus,50 has been previously documented in younger rats. Reduced CB1 mRNA
in this region is consistent with increased 2-AG production, although we did not
previously detect a difference in CB1 mRNA expression in young (mRen2)27 rats that
also exhibited higher medullary 2-AG relative to SD rats.51 In contrast to receptor
mapping studies over lifespan in humans, which find no changes in CB2 receptor
distribution with age in healthy individuals,52 we report that dorsal medullary CB2 mRNA
is increased in aged relative to younger SD rats. Peripheral expression of CB2 receptors
is upregulated in atherosclerotic and ischemic animals, and their activation confers
cardioprotective effects in these settings.53,
54
The relevance of the CB2 receptor to
autonomic, cardiovascular, and metabolic homeostasis is still emerging,55 however, and
less is known about its role in aging. Furthermore, the presence of CB 2 receptors on
neurons in this region is disputed because previous studies, including at least one that
utilized RT-qPCR, failed to detect evidence of CB2 receptors in the dorsal medulla.50, 56
Nevertheless, on the basis of our own data we cannot exclude a role for upregulated CB2
receptor expression in the modulation of cardiovagal BRS in aged rats. However, our
current study examines the functional role of only CB1 receptors in age-related BRS
impairment because SR141716A is a selective antagonist of CB1 that is not reported to
interact with any other receptors, including CB2.57
Peripheral functional or structural changes in the vasculature associated with
aging likely further contributes to diminished BRS.58 Older SD rats have an attenuated
vascular response to intravenous PE, an index of sympathetic activity at vascular α1adrenergic receptors, as well as a lower resting HR compared to younger rats.15 The
attenuated pressor response to PE in older rats may elicit inadequate carotid sinus stretch,
125
which in turn may reduce baroreceptor afferent input to the NTS to yield reduced BRS.
Lower HR in aging may be associated with altered cardiac pacemaker cell function or
density or responsiveness of cardiomyocyte β-adrenergic receptors, both of which would
reduce BRS for control of HR.59 The extent to which either of these factors is reflected in
animals with impaired baseline BRS in our current study is unknown, although
normalized BRS following NTS CB1 receptor blockade in these animals suggests their
contribution is minimal.
We did not evaluate changes in the sympathetically-mediated tachycardic BRS
before and after NTS CB1 blockade, so we do not know if the endocannabinoid system
selectively influences the parasympathetic bradycardic over the tachycardic BRS for
control of HR in aged SD rats. However, NTS microinjection of SR141716A did not
alter depressor or bradycardic responses to intravenous phenylbiguanide while assessing
cardiac chemosensitive vagal fiber activation (CVA), nor were there differences in CVA
responses between normal and impaired BRS rat groups at baseline (Figure 3-6).
Responsiveness of carotid chemoreceptors can be either attenuated24 or enhanced60 by
Ang II actions in the brain or periphery. Thus, signaling differences in central and
peripheral autonomic pathways may be reflected in the responses of animals used in our
study. However, unaltered CVA responses after NTS microinjection of SR141716A
indicates specificity of actions on BRS because these responses are mediated by sensory
inputs that converge with baroreceptor inputs in the NTS.61
At present, the actions of the endocannabinoid system during aging are
understudied relative to its body of literature.
An understanding of how the
endocannabinoid system contributes to age-related cardiovascular diseases is lacking.
126
However, evidence we present for altered endocannabinoid tone in the dorsal medulla of
aged SD rats suggests that upregulated endocannabinoid signaling in this region
contributes to impaired cardiovagal BRS for control of HR during aging. More research
is necessary to determine if endocannabinoids influence BRS via direct regulation of
NTS neurotransmitter pathways or indirectly through interactions with other autonomic
regulatory systems such as the brain RAS.
A
PBG in Aged SD Rats at Baseline
-40
-80
-200
PBG in Aged SD Rats
with Normal Baseline BRS
PBG in Aged SD Rats
with Normal Baseline BRS
+SR141716A
0
HR (bpm)
-20
-40
-80
-200
PBG in Aged SD Rats
with Impaired Baseline BRS
PBG in Aged SD Rats
with Impaired Baseline BRS
-20
-40
Baseline
+SR141716A
-100
-150
0
Baseline
-50
-60
C
Impaired
-100
-150
Baseline
Normal
-50
-60
0
MAP (mmHg)
0
HR (bpm)
-20
B
MAP (mmHg)
PBG in Aged SD Rats at Baseline
Impaired
+SR141716A
0
HR (bpm)
MAP (mmHg)
0
Normal
Baseline
+SR141716A
-50
-100
-60
-150
-80
-200
Figure 3-6. Effects of Chemoreflex Activation on MAP and HR Induced by Intravenous PBG in
Aged SD Rats
A, No difference in depressor or bradycardic responses to CVA between aged SD rats with normal or
impaired baseline BRS. B-C, No changes in either group following NTS microinjection of SR141716A (n
= 3-4 all groups).
127
Perspectives
Baroreflex function declines with age in healthy human populations, which can
contribute to the self-perpetuating progression of hypertension target organ damage and
other cardiovascular diseases.62
The importance of addressing blunted BRS during
treatment for hypertension is now recognized,1 creating the need for research into factors
that may influence BRS.
Blockade of the endocannabinoid system in conditions
associated with upregulated RAS activity, including in hypertension,63 obesity,64 and
during aging,10,
27
with co-administration with a RAS inhibitor may yield positive
synergistic effects in these conditions, with a concomitant positive influence over
autonomic reflexes. If further research uncovers more evidence for endocannabinoidmediated contributions to age-related pathologies, our current study may advance support
for the endocannabinoid system as a therapeutic target in older populations with impaired
cardiovagal BRS for control of HR.
128
Acknowledgements
We thank Ellen Tommasi, Robert Lanning, and Alex Kovach for technical assistance.
Sources of Funding
This work was supported by grants from the National Heart, Lung, and Blood Institute
(P01-HL51952), the National Institute on Drug Abuse (R01-DA03690), and RTI
International (Research Triangle Park, NC). Support from the Farley-Hudson Foundation
(Jacksonville, NC) is gratefully acknowledged.
Disclosures
None.
129
References
1.
Monahan KD. Effect of aging on baroreflex function in humans. Am J Physiol
Regul Integr Comp Physiol. 2007;293:R3-R12.
2.
Hajduczok G, Chapleau MW, Johnson SL, Abboud FM. Increase in sympathetic
activity with age. I. Role of impairment of arterial baroreflexes. Am J Physiol.
1991;260:H1113-1120.
3.
Thayer JF, Yamamoto SS, Brosschot JF. The relationship of autonomic
imbalance, heart rate variability and cardiovascular disease risk factors. Int J
Cardiol. 2010;141:122-131.
4.
Dauphinot V, Kossovsky MP, Gueyffier F, Pichot V, Gosse P, Roche F,
Barthelemy JC. Impaired baroreflex sensitivity and the risks of new-onset
ambulatory hypertension, in an elderly population-based study. Int J Cardiol.
2013;168:4010-4014.
5.
Bilkei-Gorzo A. The endocannabinoid system in normal and pathological brain
ageing. Philos Trans R Soc Lond B Biol Sci. 2012;367:3326-3341.
6.
Paradisi A, Oddi S, Maccarrone M. The endocannabinoid system in ageing: a new
target for drug development. Curr Drug Targets. 2006;7:1539-1552.
7.
Batkai S, Pacher P, Osei-Hyiaman D, Radaeva S, Liu J, Harvey-White J,
Offertaler L, Mackie K, Rudd MA, Bukoski RD, Kunos G. Endocannabinoids
acting at cannabinoid-1 receptors regulate cardiovascular function in
hypertension. Circulation. 2004;110:1996-2002.
8.
Engeli S, Bohnke J, Feldpausch M, Gorzelniak K, Janke J, Batkai S, Pacher P,
Harvey-White J, Luft FC, Sharma AM, Jordan J. Activation of the peripheral
endocannabinoid system in human obesity. Diabetes. 2005;54:2838-2843.
9.
Averill DB, Diz DI. Angiotensin peptides and baroreflex control of sympathetic
outflow: pathways and mechanisms of the medulla oblongata. Brain Res Bull.
2000;51:119-128.
10.
Diz DI, Varagic J, Groban L. Aging and the brain renin-angiotensin system:
relevance to age-related decline in cardiac function. Future Cardiol. 2008;4:237245.
11.
Turu G, Simon A, Gyombolai P, Szidonya L, Bagdy G, Lenkei Z, Hunyady L.
The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptorstimulated cannabinoid CB1 receptor activity. J Biol Chem. 2007;282:7753-7757.
130
12.
Schaich CL, Howlett AC, Diz DI. Contribution of Endocannabinoids via CB1
Receptors to the Impaired Baroreflex Sensitivity in (mRen2)27 Hypertensive
Rats. Hypertension. 2012;60:A117 (Abstract).
13.
Schaich CL, Howlett AC, Chappell MC, Nautiyal M, Diz DI. Enhanced CB1
Cannabinoid Receptor Tone Contributes to Impaired Baroreflex Sensitivity in
Hypertensive (mRen2)27 Transgenic Rats. FASEB J. 2013;27:926.913 (Abstract).
14.
Diz DI, Garcia-Espinosa MA, Gallagher PE, Ganten D, Ferrario CM, Averill DB.
Angiotensin-(1-7) and baroreflex function in nucleus tractus solitarii of
(mRen2)27 transgenic rats. J Cardiovasc Pharmacol. 2008;51:542-548.
15.
Sakima A, Averill DB, Gallagher PE, Kasper SO, Tommasi EN, Ferrario CM, Diz
DI. Impaired heart rate baroreflex in older rats: role of endogenous angiotensin(1-7) at the nucleus tractus solitarii. Hypertension. 2005;46:333-340.
16.
Niehaus JL, Liu Y, Wallis KT, Egertova M, Bhartur SG, Mukhopadhyay S, Shi S,
He H, Selley DE, Howlett AC, Elphick MR, Lewis DL. CB1 cannabinoid
receptor activity is modulated by the cannabinoid receptor interacting protein
CRIP 1a. Mol Pharmacol. 2007;72:1557-1566.
17.
Arnold AC, Sakima A, Ganten D, Ferrario CM, Diz DI. Modulation of reflex
function by endogenous angiotensins in older transgenic rats with low glial
angiotensinogen. Hypertension. 2008;51:1326-1331.
18.
Tanabe S, Bunag RD. Age-related central and baroreceptor impairment in female
Sprague-Dawley rats. Am J Physiol. 1989;256:H1399-1406.
19.
Taylor JA, Tan CO. BP regulation VI: elevated sympathetic outflow with human
aging: hypertensive or homeostatic? Eur J Appl Physiol. 2013
20.
Canale MP, Manca di Villahermosa S, Martino G, Rovella V, Noce A, De
Lorenzo A, Di Daniele N. Obesity-related metabolic syndrome: mechanisms of
sympathetic overactivity. Int J Endocrinol. 2013;2013:865965.
21.
Rozenfeld R, Gupta A, Gagnidze K, Lim MP, Gomes I, Lee-Ramos D, Nieto N,
Devi LA. AT1R-CB(1)R heteromerization reveals a new mechanism for the
pathogenic properties of angiotensin II. EMBO J. 2011;30:2350-2363.
22.
Labouebe G, Liu S, Dias C, Zou H, Wong JC, Karunakaran S, Clee SM, Phillips
AG, Boutrel B, Borgland SL. Insulin induces long-term depression of ventral
tegmental area dopamine neurons via endocannabinoids. Nat Neurosci.
2013;16:300-308.
23.
Di Marzo V, Goparaju SK, Wang L, Liu J, Batkai S, Jarai Z, Fezza F, Miura GI,
Palmiter RD, Sugiura T, Kunos G. Leptin-regulated endocannabinoids are
involved in maintaining food intake. Nature. 2001;410:822-825.
131
24.
Campagnole-Santos MJ, Heringer SB, Batista EN, Khosla MC, Santos RA.
Differential baroreceptor reflex modulation by centrally infused angiotensin
peptides. Am J Physiol. 1992;263:R89-94.
25.
Kalinyak JE, Hoffman AR, Perlman AJ. Ontogeny of angiotensinogen mRNA
and angiotensin II receptors in rat brain and liver. J Endocrinol Invest.
1991;14:647-653.
26.
Jackson L, Sakima A, Oden SD, Ganten D, Ferrario CM, Gallagher P, Diz DI.
Angiotensin receptor density and messenger RNA levels in brain medulla of
(mRen2)27 and (ASrAogen) transgenic rats during aging. Hypertension.
2004;44:516 (abstract).
27.
Arnold AC, Gallagher PE, Diz DI. Brain renin-angiotensin system in the nexus of
hypertension and aging. Hypertens Res. 2013;36:5-13.
28.
Welches WR, Brosnihan KB, Ferrario CM. A comparison of the properties and
enzymatic activities of three angiotensin processing enzymes: angiotensin
converting enzyme, prolyl endopeptidase and neutral endopeptidase 24.11. Life
Sci. 1993;52:1461-1480.
29.
Iwata N, Takaki Y, Fukami S, Tsubuki S, Saido TC. Region-specific reduction of
A beta-degrading endopeptidase, neprilysin, in mouse hippocampus upon aging. J
Neurosci Res. 2002;70:493-500.
30.
Apelt J, Ach K, Schliebs R. Aging-related down-regulation of neprilysin, a
putative beta-amyloid-degrading enzyme, in transgenic Tg2576 Alzheimer-like
mouse brain is accompanied by an astroglial upregulation in the vicinity of betaamyloid plaques. Neurosci Lett. 2003;339:183-186.
31.
Schaich CL, Howlett AC, Thomas BF, Grabenauer MA, Diz DI. Medullary
endocannabinoid content contributes to the differential resting baroreflex
sensitivity in rats with altered brain renin-angiotensin system expression.
Hypertension. 2013;62:A182 (Abstract).
32.
Lee MA, Bohm M, Paul M, Bader M, Ganten U, Ganten D. Physiological
characterization of the hypertensive transgenic rat TGR(mREN2)27. Am J
Physiol. 1996;270:E919-929.
33.
Schaich CL, Howlett AC, Diz DI. Dose-Related Biphasic Modulation of
Baroreflex Sensitivity by the CB1 Receptor in Rat Solitary Tract Nucleus.
Hypertension. 2011;58:e105 (Abstract).
34.
Szekeres M, Nadasy GL, Turu G, Soltesz-Katona E, Toth ZE, Balla A, Catt KJ,
Hunyady L. Angiotensin II Induces Vascular Endocannabinoid Release, Which
Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors. J Biol
Chem. 2012;287:31540-31550.
132
35.
Tagawa T, Dampney RA. AT(1) receptors mediate excitatory inputs to rostral
ventrolateral medulla pressor neurons from hypothalamus. Hypertension.
1999;34:1301-1307.
36.
Cristino L, Busetto G, Imperatore R, Ferrandino I, Palomba L, Silvestri C,
Petrosino S, Orlando P, Bentivoglio M, Mackie K, Di Marzo V. Obesity-driven
synaptic remodeling affects endocannabinoid control of orexinergic neurons. Proc
Natl Acad Sci U S A. 2013;110:E2229-2238.
37.
Steculorum SM, Solas M, Bruning JC. The paradox of neuronal insulin action and
resistance in the development of aging-associated diseases. Alzheimers Dement.
2014;10:S3-S11.
38.
Kim W, Lao Q, Shin YK, Carlson OD, Lee EK, Gorospe M, Kulkarni RN, Egan
JM. Cannabinoids induce pancreatic beta-cell death by directly inhibiting insulin
receptor activation. Sci Signal. 2012;5:ra23.
39.
Scarpace PJ, Matheny M, Tumer N. Hypothalamic leptin resistance is associated
with impaired leptin signal transduction in aged obese rats. Neuroscience.
2001;104:1111-1117.
40.
Jelsing J, Larsen PJ, Vrang N. The effect of leptin receptor deficiency and fasting
on cannabinoid receptor 1 mRNA expression in the rat hypothalamus, brainstem
and nodose ganglion. Neurosci Lett. 2009;463:125-129.
41.
McKernan AM, Calaresu FR. Insulin microinjection into the nucleus tractus
solitarii of the rat attenuates the baroreceptor reflex. J Auton Nerv Syst.
1996;61:128-138.
42.
Arnold AC, Shaltout HA, Gallagher PE, Diz DI. Leptin impairs cardiovagal
baroreflex function at the level of the solitary tract nucleus. Hypertension.
2009;54:1001-1008.
43.
Itoh H, Bunag RD. Aging reduces cardiovascular and sympathetic responses to
NTS injections of serotonin in rats. Exp Gerontol. 1992;27:309-320.
44.
Itoh H, Bunag RD. Age-related reduction of reflex bradycardia in conscious rats
by catecholaminergic nucleus tractus solitarius lesions. Mech Ageing Dev.
1993;67:47-63.
45.
Anwar IJ, Derbenev AV. TRPV1-dependent regulation of synaptic activity in the
mouse dorsal motor nucleus of the vagus nerve. Front Neurosci. 2013;7:238.
46.
Mollace V, De Francesco EA, Fersini G, Nistico G. Age-dependent changes in
cardiovascular responses induced by muscimol infused into the nucleus tractus
solitarii and nucleus parabrachialis medialis in rats. Br J Pharmacol.
1991;103:1802-1806.
133
47.
Kang-Park MH, Wilson WA, Kuhn CM, Moore SD, Swartzwelder HS.
Differential sensitivity of GABA A receptor-mediated IPSCs to cannabinoids in
hippocampal slices from adolescent and adult rats. J Neurophysiol. 2007;98:12231230.
48.
Burdyga G, Varro A, Dimaline R, Thompson DG, Dockray GJ. Expression of
cannabinoid CB1 receptors by vagal afferent neurons: kinetics and role in
influencing neurochemical phenotype. Am J Physiol Gastrointest Liver Physiol.
2010;299:G63-69.
49.
Mailleux P, Vanderhaeghen JJ. Distribution of neuronal cannabinoid receptor in
the adult rat brain: a comparative receptor binding radioautography and in situ
hybridization histochemistry. Neuroscience. 1992;48:655-668.
50.
Derbenev AV, Stuart TC, Smith BN. Cannabinoids suppress synaptic input to
neurones of the rat dorsal motor nucleus of the vagus nerve. J Physiol.
2004;559:923-938.
51.
Schaich CL, Grabenauer MA, Thomas BF, Gallagher PE, Howlett AC, Diz DI.
Medullary endocannabinoids contribute to the differential resting baroreflex
sensitivity in rats with altered brain renin-angiotensin system expression.
Hypertension. 2014;In revision
52.
Ahmad R, Koole M, Evens N, Serdons K, Verbruggen A, Bormans G, Van Laere
K. Whole-body biodistribution and radiation dosimetry of the cannabinoid type 2
receptor ligand [11C]-NE40 in healthy subjects. Mol Imaging Biol. 2013;15:384390.
53.
Steffens S, Veillard NR, Arnaud C, Pelli G, Burger F, Staub C, Karsak M,
Zimmer A, Frossard JL, Mach F. Low dose oral cannabinoid therapy reduces
progression of atherosclerosis in mice. Nature. 2005;434:782-786.
54.
Montecucco F, Lenglet S, Braunersreuther V, Burger F, Pelli G, Bertolotto M,
Mach F, Steffens S. CB(2) cannabinoid receptor activation is cardioprotective in a
mouse model of ischemia/reperfusion. J Mol Cell Cardiol. 2009;46:612-620.
55.
Atwood BK, Mackie K. CB2: a cannabinoid receptor with an identity crisis. Br J
Pharmacol. 2010;160:467-479.
56.
Tsou K, Brown S, Sanudo-Pena MC, Mackie K, Walker JM.
Immunohistochemical distribution of cannabinoid CB1 receptors in the rat central
nervous system. Neuroscience. 1998;83:393-411.
57.
Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B, Congy C,
Martinez S, Maruani J, Neliat G, Caput D, et al. SR141716A, a potent and
selective antagonist of the brain cannabinoid receptor. FEBS Lett. 1994;350:240244.
134
58.
Chapleau MW, Cunningham JT, Sullivan MJ, Wachtel RE, Abboud FM.
Structural versus functional modulation of the arterial baroreflex. Hypertension.
1995;26:341-347.
59.
Friberg P, Nordlander M, Lundin S, Folkow B. Effects of ageing on cardiac
performance and coronary flow in spontaneously hypertensive and normotensive
rats. Acta Physiol Scand. 1985;125:1-11.
60.
Allen AM. Angiotensin AT1 receptor-mediated excitation of rat carotid body
chemoreceptor afferent activity. J Physiol. 1998;510 ( Pt 3):773-781.
61.
Paton JF. Convergence properties of solitary tract neurones driven synaptically by
cardiac vagal afferents in the mouse. J Physiol. 1998;508 ( Pt 1):237-252.
62.
Thayer JF, Lane RD. The role of vagal function in the risk for cardiovascular
disease and mortality. Biol Psychol. 2007;74:224-242.
63.
Schaich CL, Shaltout HA, Howlett AC, Diz DI. Chronic Systemic CB1 Receptor
Blockade Reduces Blood Pressure and Weight Gain and Improves Baroreflex
Function and Heart Rate Variability in Hypertensive (mRen2)27 Rats.
Hypertension. 2013;62:A183 (Abstract).
64.
Muller-Fielitz H, Hubel N, Mildner M, Vogt FM, Barkhausen J, Raasch W.
Chronic blockade of angiotensin AT1 receptors improves cardinal symptoms of
metabolic syndrome in diet-induced obesity in rats. Br J Pharmacol.
2014;171:746-760.
135
Novelty and Significance: 1) What Is New, 2) What Is Relevant?
What Is New?

Blockade of NTS CB1 receptors in aged Sprague-Dawley rats enhances the
parasympathetic baroreflex sensitivity only in rats that exhibit impaired baseline
baroreflex function.

Older Sprague-Dawley rats have increased production of 2-AG and altered
cannabinoid receptor expression in dorsal medulla compared to younger animals.
What Is Relevant?

Results indicate novel mechanisms for preservation of baroreflex function
involving the endocannabinoid system during normal aging.
Summary
Normal aging is associated with upregulated expression and tone of the dorsal medullary
endocannabinoid system with respect to central blood pressure regulation. Increased
endocannabinoid production in this region may contribute to impaired resting baroreflex
sensitivity in older rats.
136
CHAPTER FOUR
ACUTE AND CHRONIC SYSTEMIC CB1 CANNABINOID RECEPTOR
BLOCKADE IMPROVES BLOOD PRESSURE REGULATION AND
METABOLIC PROFILE IN HYPERTENSIVE (mRen2)27 RATS
By
Chris L. Schaich,1 Hossam A. Shaltout,1, 2 K. Bridget Brosnihan,1 Allyn C. Howlett,1 and
Debra I. Diz1
1
Department of Physiology and Pharmacology; and Hypertension & Vascular Research
Center, Wake Forest School of Medicine, Winston-Salem, NC, USA
2
Department of Obstetrics & Gynecology, Wake Forest School of Medicine, WinstonSalem, NC, USA
Short title: CB1 Receptor Blockade in Hypertension
The following manuscript is in revision for American Journal of Physiology –
Regulatory, Integrative and Comparative Physiology and represents the efforts of the first
author. Differences in formatting and organization reflect the requirements of the
journal.
137
Abstract
We investigated acute and chronic effects of CB1 cannabinoid receptor blockade in reninangiotensin system-dependent hypertension using rimonabant (SR141716A), an orally
active antagonist with central and peripheral actions. In transgenic (mRen2)27 rats, a
model of angiotensin II-dependent hypertension with increased body mass and insulin
resistance, acute systemic blockade of CB1 receptors significantly reduced blood pressure
within 90 minutes but had no effect in Sprague-Dawley rats. No changes in metabolic
hormones occurred with the acute treatment. During chronic CB1 receptor blockade,
(mRen2)27 rats received daily oral administration of SR141716A (10 mg/kg/day) for 28
days. Systolic blood pressure was significantly reduced within 24 hours, and at Day 21
of treatment values were 173 mmHg in vehicle- vs. 149 mmHg in drug-treated rats (P <
0.01). This accompanied lower cumulative weight gain (22 vs. 42 g vehicle; P < 0.001),
fat mass (2.0 vs. 2.9% of body weight; P < 0.05), and serum leptin (2.8 vs. 6.0 ng/mL; P
< 0.05) and insulin (1.0 vs. 1.9 ng/mL; P < 0.01), without sustained changes in food or
water consumption.
Conscious hemodynamic recordings indicate two-fold increases
occurred in spontaneous baroreflex sensitivity (P < 0.05) and heart rate variability (P <
0.01), measures of cardiac vagal tone. The beneficial actions of CB1 receptor blockade in
(mRen2)27 rats support the interpretation that an upregulated endocannabinoid system
contributes to hypertension and impaired autonomic function in this angiotensin IIdependent model. We conclude that systemic CB1 receptor blockade may be an effective
therapy for angiotensin II-dependent hypertension and associated metabolic syndrome.
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Introduction
Hypertension, diagnosed when arterial pressure (AP) exceeds 140/90 mmHg, is the single
most important risk factor for the development of cardiovascular disease and currently
affects 1 in 3 U.S. adults.1 While the mechanisms involved in the development of
hypertension are still not fully understood, the widespread use of medications that inhibit
the formation of the peptide hormone angiotensin (Ang) II or its actions at the
ubiquitously expressed Ang II type 1 (AT1) receptor as first-line treatments implicates
disturbances in the renin-angiotensin system (RAS) in the maintenance of the disease.2
RAS blockers have a beneficial profile in hypertension associated with metabolic
syndrome, unlike many antihypertensive drugs that have reduced efficacy in obese or
diabetic patients3 or produce undesirable metabolic side effects.4 However, there is need
for further research into the mechanisms linking the RAS with the metabolic syndrome in
hypertension.
The central and peripheral endocannabinoid system has emerged as a target for
the treatment of conditions associated with the metabolic syndrome, which include the
cluster of risk factors for cardiovascular disease, obesity and insulin resistance.5 The
endocannabinoid system comprises a spectrum of fatty acids, including the endogenous
cannabinoids anandamide and 2-arachidonoylglycerol (2-AG), which exert their
paracrine-mediating effects through the widely expressed G protein-coupled CB1 and
CB2 receptor subtypes.6
Systemic blockade of CB1 receptors by selective, brain-
penetrating antagonists such as rimonabant (SR141716A) confers beneficial antiobesity
and antidiabetic effects in humans and animals with metabolic syndrome [see Kirilly et
al. (2012)7 for review], implicating overactivity of the endocannabinoid system in the
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pathogenesis or maintenance of these conditions.
The precise role of the
endocannabinoid system in promoting cardiovascular diseases associated with the
metabolic syndrome, including hypertension, is less clear in part due to the inability to
differentiate direct effects on AP from indirect effects mediated by weight loss or
normalization of metabolic factors such as insulin or leptin sensitivity.8
Evidence for signaling interactions between the endocannabinoid system and the
pathogenic actions of Ang II is mounting. For example, Ang II transactivates CB1
receptors by stimulating production of the endocannabinoid 2-arachidonoylglycerol (2AG) in cell culture9 and in rat arteriole beds.10 Blockade of CB1 receptors in SpragueDawley (SD) rats exposed to chronic ethanol treatment prevented Ang II-mediated
mitogenic signaling and profibrogenic gene expression in hepatic stellate cells,11 and in
obese Zucker rats long-term systemic CB1 blockade normalized the pressor effect of
acutely administered Ang II.12 However, no studies to date have investigated the effect
of systemic CB1 receptor blockade on blood pressure in animals with altered brain RAS
expression.
Transgenic (mRen2)27 rats, a monogenetic model of Ang II-dependent
hypertension in which the mouse Ren2 renin gene was transfected into the genome of the
SD rat, have a phenotype of chronic hypertension with markedly impaired baroreflex
control over heart rate (HR), increased body weight, and reduced insulin and leptin
sensitivity by 16 weeks of age compared to their normotensive genetic controls.13-15 In
anesthetized (mRen2)27 rats blockade of CB1 receptors in the brain nucleus of the
solitary tract (NTS) via microinjection of SR141716A dose-dependently improves
baroreflex sensitivity (BRS) for control of HR,16 an index of parasympathetic vagus
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nerve function often impaired in hypertension.17 These results prompted us to study the
effects of acute and chronic systemic CB1 blockade by SR141716A on blood pressure,
metabolic profile and conscious baroreflex function in (mRen2)27 rats. Based on the
evidence we found for an upregulated endocannabinoid system in the brain of this Ang
II-dependent model of hypertension with metabolic syndrome,16 we hypothesized that
chronic systemic blockade of CB1 receptors would improve cardiometabolic function in
this strain, possibly by disrupting the interactions between the endocannabinoid system
and RAS.
141
Methods
Animals
All experiments were performed in male 15- to 20-week-old hypertensive (mRen2)27
rats or normotensive SD rats obtained from the Hypertension & Vascular Research
Center colony at Wake Forest School of Medicine. Animals were housed two per cage in
a humidity- and temperature-controlled room with free access to standard chow (ProLab,
PMI Nutrition International, Brentwood, MO) and water. The colony maintained a 12hour light/dark cycle (lights on at 06:00). All experimental procedures were approved by
the Institutional Animal Care and Use Committee.
Experimental Protocol
Acute experiments were performed in (mRen2)27 or SD rats aged 18- to 20-weeks.
Animals were trained for per os (p.o.) daily oral gavage treatment with the vehicle (0.1%
Tween-80 in double-distilled water) for seven days prior to testing, and acclimated to
metabolic cages (Allentown Caging Equipment, Allentown, NJ) and the tail cuff blood
pressure monitoring system (NIBP-8, Columbus Instruments, Columbus, OH) 48 hours
prior to testing. On the testing day, baseline systolic blood pressure (SBP) and HR (in
beats per minute [bpm]) were measured by tail cuff and recorded as the average of a
minimum of 10 individual readings over a period of approximately 15 minutes per
animal. Animals were then treated with either SR141716A (10 mg/kg p.o.; obtained
from RTI International [Durham, NC] via the National Institute on Drug Abuse; n = 5 per
strain) or its vehicle (n = 5 per strain). After 90 minutes SBP and HR were reassessed in
the same manner. Animals were then placed individually in metabolic cages overnight.
Rats treated with SR141716A had ad libitum access to food and water, while rats treated
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with vehicle were food restricted (FR) to 13 g of food, based on previous observations of
the acute hypophagic effect of SR141716A in both strains in our preliminary studies and
by others,18 to control for changes in food consumption that may contribute to acute
differences in SBP.19 Twenty-four hours after dosing, SBP and HR were again measured.
Animals were then sacrificed by decapitation and trunk blood collected for analysis of
RAS components, insulin and leptin levels. All SPB recordings were obtained between
13:00 and 16:00.
Chronic experiments were performed in separate groups of (mRen2)27 rats
beginning at 15 weeks of age. As in our acute studies, animals were trained for p.o.
injections with daily administration of the vehicle for 7 days and were acclimated to the
tail cuff apparatus and metabolic cages 48 hours prior to the commencement of testing.
Baseline values for SBP, HR, and food and water consumption were obtained on Day 0
of the study when animals were 16-weeks-old. Daily oral treatment with SR141716A (10
mg/kg/day; n = 7) or vehicle (n = 8) began on Day 1 and continued for 28 days. Body
weight was recorded daily through Day 25, while SPB, HR, food and water consumption,
urine volume and fasting blood glucose were measured on treatment Days 7, 14 and 21
(see Figure 4-1). Overnight food and water intake and SBP were measured in subgroups
of SR141716A-treated (n = 3) and vehicle-treated (n = 4) animals on Day 2 to assess
acute feeding effects of systemic CB1 receptor blockade.
Fasting blood glucose
measurements were taken using a Freestyle glucose meter (Abbott Diabetes Care Inc.,
Alameda, CA) from a ~10 µl blood sample obtained from tail pricks at least 60 minutes
after tail cuff recordings, between 15:00 and 17:00. Urine was flash-frozen over dry ice
on Days 0 and 21 and collected for analysis of osmolality and vasopressin content. On
143
Day 25 animals were surgically instrumented with a catheter in the femoral artery to
record conscious hemodynamic measures including BRS, HR variability (HRV) and
blood pressure variability (BPV) on Day 28. After testing was completed on Day 28,
animals were sacrificed by decapitation and trunk plasma and serum collected for
analysis of RAS components, insulin, and leptin levels. Fat mass index was calculated
from white adipose tissue collected from retroperitoneal, inguinal and epididymal stores
and weighed as a percentage of total body weight. As in our acute studies, all tail cuff
SBP, HR, and conscious hemodynamic recordings were obtained between 13:00 and
16:00 and approximately 90 minutes after dosing.
Figure 4-1. Design and timeline of experiments investigating the effect of daily systemic CB 1 receptor
blockade in (mRen2)27 rats.
Surgical Procedures and Conscious Hemodynamic Measures
As described previously,20 rats were anesthetized under 2.5% to 4.0% isofluorane and
instrumented with a femoral artery catheter on the afternoon of Day 25 of the study. Rats
were allowed two days to recover during which they were gavaged with water to prevent
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dehydration in addition to receiving daily drug or vehicle treatment. Pulsatile pressure in
conscious rats was acquired on Day 28 between 13:00 and 15:00 via strain gauge
transducer connecting the arterial catheter to a data acquisition system (AcqKnowledge
software version 3.8.1, BIOPAC Systems, Goleta, CA). HR was calculated from the
arterial pressure wave. Indices of sympathovagal activity were calculated by spectral
analysis of the time and frequency domains using software designed for rats (Nevrokard
SA-BRS, Medistar, Houston, TX), as previously described.20
Consistent with the
duration of recordings used in previous human and rodent studies,20, 21 conscious BRS
was determined from a minimum of 10 minutes of AP recordings obtained within 90
minutes of SR141716A or vehicle dosing. Conscious BRS was calculated in the time
(Sequence [Seq] Up, Seq Down, and Seq All; in units of milliseconds per mmHg) and
frequency (low-frequency [LF] and high-frequency [HF] α indices) domains.
Time
domain analysis was used to calculate differences in HRV, measured as the standard
deviation of the beat-to-beat interval (SDRR) in milliseconds. BPV was measured in the
time domain as the standard deviation of the mean AP (SDMAP) in mmHg.
Biochemical Measurements in Plasma, Serum and Urine
Plasma angiotensin peptides [Ang I, Ang II, Ang-(1-7)] were measured as previously
reported.22
Serum angiotensin converting enzyme (ACE), insulin, leptin, and urine
vasopressin were measured using radioimmunoassays specific for rats according to the
manufacturer’s instructions (Linco, Santa Fe Springs, CA).14
Urine osmolality was
measured using the 5004 Micro-Osmette osmometer (Precision Systems, Natick, MA)
and expressed as milliOsmols per liter (mOsm/L).
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Analysis of Data
Values are presented as mean ± SEM. Comparisons of changes in body weight, SPB,
HR, food and water consumption, and fasting blood glucose over time between drug and
vehicle groups were analyzed by repeated measures two-way ANOVA, with Bonferroni
post-hoc comparisons made between drug and vehicle groups where appropriate. Within
group comparisons were made by repeated measures one-way ANOVA, with post-hoc
Tukey tests used to elucidate further comparisons between timepoints.
Student’s
unpaired two-tailed t-tests were used to analyze comparisons of fat mass index,
biochemical measurements, conscious hemodynamic parameters on Day 28, and
differences in food and water intake, SBP, and HR after Day 1 of chronic treatment in
subgroups. Paired two-tailed t-tests were used to compare 24-hour changes in body
weight from baseline in acute studies. The criterion for statistical significance was P <
0.05. Statistical tests were performed using Prism 5.0 (GraphPad Software, San Diego,
CA).
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Results
Acute systemic CB1 receptor blockade in (mRen2)27 and SD rats
There were no significant differences in baseline SBP, HR (Figure 4-2) or body weight
(Table 4-1) between treatment groups of (mRen2)27 or SD rats. In (mRen2)27 rats, p.o.
injection of SR141716A (n = 5) lowered SBP by approximately 24%, from 176 ± 3
mmHg at baseline to 134 ± 3 mmHg, after 90 minutes (P < 0.001; Figure 4-2A). SPB
remained lower 24 hours after SR141716A administration, with evidence of partial
recovery to 146 ± 5 mmHg (P < 0.01 vs. baseline; Figure 4-2A).
SR141716A in
(mRen2)27 rats also caused a transient reduction in HR, which fell from 409 ± 17 bpm at
baseline to 363 ± 15 bpm after 90 minutes (P < 0.05), but fully recovered within 24 hours
(Figure 4-2B). Furthermore, (mRen2)27 rats treated with SR14171A consumed a similar
amount of food overnight as those treated with vehicle and restricted to 13 g of food,
which produced similar decreases in body weight in both groups (P < 0.01 vs. respective
baseline body weights; Table 4-1). However, (mRen2)27 rats treated with SR141716A
excreted significantly less urine overnight compared to those that received vehicle + FR
(8 ± 1 vs. 14 ± 1 mL; P < 0.05), despite consuming similar amounts of water (Table 4-1).
Vehicle did not significantly alter SBP or HR in (mRen2)27 rats after 90 minutes, nor did
overnight FR change SBP or HR in (mRen2)27 rats 24 hours after receiving vehicle
(Figure 4-2).
In contrast to (mRen2)27 rats, acute administration of SR141716A in SD rats did
not significantly change SBP or HR after 90 minutes or 24 hours (Figure 4-2). As in
(mRen2)27 rats, SD rats treated with SR141716A consumed similar quantities of food
and water overnight as those who received vehicle + FR, producing similar changes in
147
body weight (P < 0.05 vs. respective baseline body weights; Table 4-1). Overnight water
intake was similar between SD groups, but there was a trend for lower urine excretion in
SR141716A-treated SD rats compared to vehicle + FR-treated rats (6 ± 1 vs. 11 ± 2 mL;
P = 0.08; Table 4-1).
As with the SR141716A-treated group, SBP and HR was
unaffected at 90 minutes and 24 hours in SD rats treated with vehicle + FR (Figure 4-2).
There were no differences in circulating levels of RAS peptides or insulin or leptin
between treatment groups in (mRen2)27 or SD rats (Table 4-1).
Table 4-1. Acute Systemic SR141716A or Vehicle + Overnight FR in (mRen2)27 and SD Rats
Parameter
SD
Vehicle
+FR
SD
SR
10 mg/kg
(mRen2)27
Vehicle
+FR
(mRen2)27
SR
10 mg/kg
Baseline Body Weight (g)
388 ± 12
406 ± 22
555 ± 19
555 ± 30
Body Weight at 24 h (g)
379 ± 12†
394 ± 19*
534 ± 16†
530 ± 27†
-9 ± 1
-12 ± 3
-21 ± 4
-25 ± 5
13
12 ± 1
13
13 ± 2
Water Intake (mL)
25 ± 3
22 ± 1
28 ± 1
22 ± 5
Urine Volume (mL)
11 ± 2
6 ± 1#
14 ± 1
8 ± 1#
Plasma Ang I (pg/mL)
139 ± 7
163 ± 19
139 ± 7
163 ± 19
Plasma Ang II (pg/mL)
102 ± 36
113 ± 43
102 ± 36
113 ± 43
Plasma Ang-(1-7) (pg/mL)
67 ± 10
58 ± 12
67 ± 10
58 ± 12
Serum ACE (ng/mL)
35 ± 8
31 ± 10
23 ± 2
24 ± 1
Serum Insulin (ng/mL)
2.9 ± 0.4
2.7 ± 0.3
2.9 ± 0.4
2.7 ± 0.3
Serum Leptin (ng/mL)
5.9 ± 1.0
7.1 ± 1.5
5.9 ± 1.0
7.1 ± 1.5
ΔBody Weight (g)
Food Intake (g)
Values are mean ± SEM. SR refers to SR141716A.
*P < 0.05 vs. baseline; †P < 0.01 vs. baseline; #P < 0.05 vs. (mRen2)27 Vehicle + FR; n = 5 all groups.
148
A
200
SBP (mmHg)
180
160
#
†
140
(mRen2)27 Vehicle + FR
(mRen2)27 SR 10 mg/kg
SD Vehicle + FR
SD SR 10 mg/kg
120
100
0
0
1.5
B
12
16
20
24
Time (h)
Figure 4-2.
Acute systemic
administration of CB1 receptor
antagonist SR141716A lowers SBP
(A) and transiently lowers HR (B)
in (mRen2)27 rats but has no effect
in SD rats. *P < 0.05 vs. baseline;
#P < 0.01; †P < 0.001; n = 5 all
groups. SR refers to SR141716A;
FR = food restriction.
500
HR (bpm)
450
400
350
*
300
0
0
1.5
12
16
20
24
Time (h)
Chronic systemic CB1 receptor blockade in (mRen2)27 rats
Body weight and fat mass
There were no baseline differences in body weight between (mRen2)27 rats in the vehicle
(469 ± 10 g; n = 8) and SR141716A (469 ± 21 g; n = 7) treatment groups. By Day 25 of
treatment body weights in both groups had significantly increased relative to their Day 1
baselines, to 513 ± 12 g (P < 0.001) in vehicle-treated rats and 498 ± 21 g (P < 0.001) in
drug-treated rats (Figure 4-3A). The body weight values on Day 25 did not significantly
differ between treatment groups.
However, rats treated with SR141716A had
approximately 31% lower fat composition, measured as the mass of white adipose tissue
as a percentage of body weight, after Day 28 than rats treated with vehicle over the
duration of the study (P < 0.05; Figure 4-3B). Furthermore, SR141716A-treated rats on
average gained approximately half as much weight as rats treated with vehicle through
149
Day 25 of treatment (44 ± 3 g vs. 22 ± 3 g; P < 0.001; Figure 4-3C). Drug-treated rats
lost approximately 12 g of body weight overnight after the Day 1 dose of SR141716A
(Figure 4-3C) but thereafter steadily gained weight through Day 25 of treatment. Further
statistical analysis of the weight gain curves starting on Day 2 revealed a significant
interaction between treatment groups with respect to cumulative daily weight gain over
the treatment period (P < 0.01), indicating a lower rate of weight gain over the duration of
treatment after the initial overnight weight loss in rats that received SR141716A
(regression slopes after Day 1 = 1.82 ± 0.07 g/day Vehicle vs. 1.29 ± 0.09 g/day
SR141716A; P < 0.0001).
A
B
†
†
4
Fat Mass
(% Body Weight)
Body Weight (g)
600
400
200
Vehicle
SR 10 mg/kg/day
3
*
2
1
0
0
Baseline
Day 25
Cumulative
Weight Gain (g)
C
50
Vehicle
40
SR 10 mg/kg/day
30
20
§
10
0
-10
-20
#
0
5
10
15
Day of Treatment
20
25
Figure 4-3. (mRen2)27 rats treated with chronic CB1 receptor blockade maintained similar raw body
weight (A), but had significantly lower fat mass (B) and gained significantly less weight over the treatment
period (C). Fat mass reflects the sum of white adipose tissue collected after animals were sacrificed on Day
28 and expressed as percent of body weight. †P < 0.001 vs. respective baseline values; *P < 0.05 vs.
Vehicle group; #P < 0.01; §P < 0.0001; n = 6-8.
150
Food and water intake, and urine volume
There were no differences between vehicle and SR141716A treatment groups in food
intake, water intake, or urine volume at baseline. Over the duration of treatment there
were no sustained differences in food intake between treatment groups; however, the
weight loss exhibited after Day 1 by (mRen2)27 rats treated with SR141716A was
associated with a transient reduction in food intake measured in subgroups of animals (13
± 2 g food drug-treated vs. 26 ± 1 g food vehicle-treated on Day 2; n = 3-4; P < 0.01) that
fully recovered by Day 7 of treatment (Figure 4-4A), in agreement with previous
reports.18, 23 There were no significant transient or sustained differences in water intake
between treatment groups (Figure 4-4B). Urine volume trended lower in rats treated with
SR141716A over the duration of treatment compared to vehicle-treated rats, but there
was not a significant treatment effect (Figure 4-4C).
151
A
Food Intake (g)
40
Vehicle
SR 10 mg/kg/day
30
20
10
#
0
0
7
14
Day of Treatment
21
B
C
20
Urine Volume (mL)
Water Intake (mL)
50
40
30
20
10
0
15
10
5
0
0
7
14
Day of Treatment
21
0
7
14
Day of Treatment
21
Figure 4-4. Chronic systemic CB1 receptor blockade produced a transient reduction in food intake (A)
without sustained changes, but not in water intake (B) or urine volume (C) in (mRen2)27 rats. #P < 0.01
vs. Vehicle on Day 2; n = 7-8 on Days 0, 7, 14 and 21; n = 3-4 on Day 2.
Blood pressure, HR and RAS components
There were no baseline differences in SBP or HR between treatment groups. On Day 7,
SBP in SR141716A-treated (mRen2)27 rats was significantly reduced from a baseline of
174 ± 3 mmHg to a new value of 151 ± 3 (P < 0.01) and remained lower through Day 21
of treatment (149 ± 6 mmHg; P < 0.01 vs. baseline; Figure 4-5A). Measurements
conducted in a subgroup of rats (n = 3) on Day 2 of treatment indicated that SBP was
reduced within 24 hours after receiving the Day 1 injection of SR141716A (146 ± 5
152
mmHg; P < 0.05 vs. baseline; Figure 4-5A). There was no effect of vehicle on SBP, nor
was there a significant treatment effect on HR, over the duration of treatment (Figure 45A and 4-5B). Furthermore, no differences between treatment groups were found in
levels of the RAS components Ang I, Ang II, Ang-(1-7) or ACE analyzed from blood
collected after animals were sacrificed on Day 28 of the study (Table 4-2).
Table 4-2. Chronic Systemic SR141716A or Vehicle in (mRen2)27 Rats
(mRen2)27
Vehicle
(mRen2)27
SR 10 mg/kg/day
Plasma Ang I (pg/mL)
353 ± 112
302 ± 52
Plasma Ang II (pg/mL)
71 ± 25
69 ± 11
Plasma Ang-(1-7) (pg/mL)
45 ± 7
46 ± 7
Serum ACE (ng/mL)
26 ± 2
30 ± 4
Urine Vasopressin (pg/mL)
Baseline
Day 21
107 ± 12
100 ± 19
104 ± 16
110 ± 31
Urine Osmolality (mOsm/L)
Baseline
Day 21
1471 ± 107
1639 ± 110
1528 ± 77
1809 ± 69#
Parameter
Values are mean ± SEM. SR refers to SR141716A.
#P < 0.01 vs. Baseline; n = 7-8 all groups.
153
A
SPB (mmHg)
200
Vehicle
SR 10 mg/kg/day
180
160
§
140
#
†
†
0
0
B
7
14
Day of Treatment
21
7
14
Day of Treatment
21
Figure 4-5. Chronic systemic CB1
receptor blockade in (mRen2)27 rats
lowered SBP (A) without significantly
changing HR (B) over the duration of
treatment. #P < 0.01 vs. Vehicle; †P <
0.001; §P < 0.0001; n = 7-8 on Days
0, 7, 14 and 21; n = 3-4 on Day 2.
Day 2 values reflect measurements
made 24 hours following the Day 1
treatment.
500
HR (bpm)
450
400
350
300
0
0
Leptin, insulin, fasting blood glucose, and urine vasopressin and osmolality
At the end of the study, serum collected from (mRen2)27 rats that received chronic
treatment with SR141716A had significantly less leptin (P < 0.05; Figure 4-6A) and
insulin (P < 0.05; Figure 4-6B) compared to rats treated with vehicle. There was no
effect on fasting blood glucose over the duration of the study in either group (Figure 46C).
Furthermore, no differences in urine vasopressin levels were found between
treatment groups (Table 4-2).
Urine osmolality increased in both treatment groups
between baseline and Day 21 of the study, reaching statistical significance in
SR141716A-treated rats (P < 0.01), but there was no effect of treatment (Table 4-2).
154
A
Leptin (ng/mL)
8
Vehicle
SR 10 mg/kg/day
6
4
*
2
0
B
C
100
6
4
2
0
#
Glucose (mg/dL)
Insulin (ng/mL)
8
Vehicle
SR 10 mg/kg/day
90
80
70
60
0
7
14
Day of Treatment
21
Figure 4-6. Following 28-day chronic systemic CB1 receptor blockade (mRen2)27 rats had lower serum
leptin (A) and insulin (B) levels than rats treated with vehicle, while fasting blood glucose (C) was
unaffected over the duration of treatment. *P < 0.05 vs. Vehicle; #P < 0.01; n = 7-8.
Conscious baroreflex function, HRV and BPV
Conscious AP was recorded from (mRen2)27 rats treated with vehicle (n = 5) or
SR141716A (n = 5) on Day 28 of the study for spectral analysis of the AP and
corresponding HR waves to calculate indices of sympathovagal function.
Rats that
received chronic systemic treatment with SR141716A displayed a two-fold greater value
in overall conscious baroreflex function (Seq All; 0.82 ± 0.13 vs. 0.41 ± 0.11 ms/mmHg;
P < 0.05; Figure 4-7A), with significantly greater sympathetic (Seq Down; 0.64 ± 0.05
vs. 0.39 ± 0.09 ms/mmHg; P < 0.05; Figure 4-7B) and parasympathetic (Seq Up; 0.94 ±
0.19 vs. 0.42 ± 0.11 ms/mmHg; P < 0.05; Figure 4-7C) BRS for control of HR compared
to rats treated with vehicle. In addition, HRV, a measure of cardiac vagal tone, was
155
significantly higher in SR141716A-treated rats relative to vehicle-treated rats (4.38 ±
0.49 vs. 2.0 ± 0.26 ms; P < 0.01; Figure 4-7D). There was not a significant difference in
BPV, an index of vascular sympathetic tone, between treatment groups (Figure 4-7E).
A
Conscious BRS (ALL)
Seq All (ms/mmHg)
1.5
*
1.0
Vehicle
SR 10 mg/kg/day
0.5
0.0
B
C
Conscious BRS (Sympathetic)
Conscious BRS (Parasympathetic)
1.5
1.0
Seq Up (ms/mmHg)
Seq Down (ms/mmHg)
1.5
*
0.5
*
1.0
0.5
0.0
0.0
D
E
HRV
BPV
6
8
SDMAP (mmHg)
SDRR (ms)
#
4
2
0
6
4
2
0
Figure 4-7. Chronic systemic CB1 receptor blockade for 28 days via SR141716A in (mRen2)27 rats
improved conscious sympathetic and parasympathetic BRS for control of HR (A-C) and HRV (D)
compared to vehicle-treated rats, without significantly changing BPV (E). *P < 0.05 vs. Vehicle; #P < 0.01;
n = 5.
156
Discussion
In this study, we report for the first time effects of acute and chronic systemic blockade
of CB1 cannabinoid receptors on blood pressure, body weight, fat mass, feeding, serum
leptin and insulin content, and conscious baroreflex function in transgenic hypertensive
rats with upregulated RAS activity.
Acute oral administration of the CB1-selective
antagonist SR141716A significantly lowers SBP in (mRen2)27 rats but has no effect in
normotensive SD control rats. The effect is immediate, occurring within 90 minutes of
administration, and sustained for 24 hours. Chronic daily treatment with SR141716A
maintained the SBP-lowering effect at a similar level over the duration of the study
without significantly changing HR, although the acute studies suggest a modest, transient
bradycardia that recovers within 24 hours may contribute to the initial reduction in SBP
after 90 minutes. Acute and chronic effects of systemic CB1 receptor blockade by
SR141716A are not associated with differences in levels of circulating RAS components,
or urinary vasopressin compared to vehicle treatment. Our acute studies demonstrate that
the transient hypophagic effect of SR141716A alone does not significantly contribute to
initial reductions in blood pressure because overnight FR did not change SBP in
(mRen2)27 rats treated with the vehicle. Furthermore, the reduced SBP in rats that
received chronic treatment with SR141716A is associated with significantly improved
sympathovagal baroreflex function after 28 days.
In addition to these beneficial hemodynamic effects, chronic systemic
SR141716A treatment significantly reduces cumulative weight gain over the treatment
period, and this is associated with reduced fat mass as a percentage of body weight, and
lower leptin and insulin levels in (mRen2)27 rats after the 4-week study. Taken together,
157
data from our current study support our hypothesis that systemic blockade of central and
peripheral CB1 receptors has direct beneficial effects on blood pressure with a
concomitant positive influence over metabolic profile in transgenic (mRen2)27 rats with
Ang II-dependent hypertension and features of metabolic syndrome.
The metabolic effects of chronic systemic SR141716A administration in
(mRen2)27 rats are consistent with the reported literature in humans and animals with
metabolic syndrome. In obese Zucker rats, a model of obesity with leptin receptor
deficiency and insulin resistance,24 chronic treatment with SR141716A dose-dependently
reduces weight gain, fat mass, and improves insulin sensitivity.12, 25 Similar effects of
SR141716A accompanied by an improved serum lipid profile are found in diet-induced
obese mice,23 and in obese and diabetic patients.26
Although we did not directly
investigate insulin sensitivity via glucose tolerance tests, lower circulating insulin levels
coupled with unchanged fasting blood glucose we observed implies that insulin
sensitivity improved in rats that received chronic SR141716A treatment. However, it is
unclear whether the reduced insulin levels in our drug-treated rats are a direct cause or
result of the decreased weight gain and fat mass associated with SR141716A treatment.
Activation of CB2 receptors in pancreatic beta cells reduces insulin release while CB1
receptors are restricted primarily to glucagon-secreting alpha cells.27 Therefore, it is
conceivable that blockade of CB1 receptors could produce a shift in endocannabinoid
signaling within the pancreas and other organs to favor CB2 over CB1 receptor activation.
Peripheral expression of CB2 receptors is upregulated in models of cardiovascular disease
including atherosclerosis and ischemia,28, 29 and their activation confers cardioprotective
effects in these settings. Thus, there may be a role for CB2 receptors in the treatment of
158
the RAS-associated hypertension and metabolic syndrome in (mRen2)27 rats, which
remains unaddressed by our current study.
The reduced weight gain and fat mass in (mRen2)27 rats treated with SR141716A
cannot be attributed to reduced food consumption because feeding was only transiently
reduced and fully recovered within 7 days, in line with other descriptions of the
hypophagic effect of SR141716A.12, 25 A significant statistical interaction between the
vehicle and drug treatment groups further precludes differences in weight gain and fat
mass from being wholly attributed to overnight weight loss associated with hypophagia
experienced by SR141716A-treated rats after Day 1. Several alternative mechanisms
may contribute to the beneficial metabolic actions of CB1 blockade. For instance, obesity
is associated with defective leptin signaling and hyperleptinemia,30 both of which are
exhibited by (mRen2)27 rats14 and may contribute to increased body weight and fat mass
in this strain.
Defective central leptin signaling is also associated with enhanced
endocannabinoid tone independent of obesity.31 Animals that received SR141716A in
our study had significantly lower serum leptin levels with reduced weight gain and lower
body fat after chronic treatment, suggesting improvement in leptin sensitivity or
signaling.
In addition to leptin abnormalities, metabolic syndrome is associated with
depressed plasma adiponectin levels32 and dyslipidemia.33 Adiponectin released from
adipocytes induces fatty acid β-oxidation and increases lipoprotein lipase activity34 with
central regulatory effects on energy expenditure that complement brain actions of
leptin.35 Adipocyte CB1 receptors directly regulate plasma levels of adiponectin,36 and
SR141716A is reported to increase release of adiponectin in obese humans,37 Zucker
159
rats,12 and diet-induced obese mice.38 Therefore, we cannot rule out that the reduced
adiposity and weight gain we observed in (mRen2)27 rats given chronic SR141716A may
in part be attributed to induced adipose tissue lipolysis or increased energy expenditure
through the actions of adiponectin.
Similarly, dyslipidemia in the form of elevated triglycerides is reported in
(mRen2)27 rats and may contribute to defective insulin signaling in this strain.15
Hepatocytes express CB1 receptors that promote fatty acid synthesis through induction of
the lipogenic transcription factor SREBP-1c and its downstream target enzyme fatty acid
synthase.39 Mice fed a high-fat diet had upregulated hepatic CB1 receptor expression and
increased anandamide levels.39 Moreover, insulin resistance associated with a high-fat
diet is linked to hepatic CB1 receptor-mediated synthesis of long chain ceramides.40 All
of these effects are reversed or attenuated by SR141716A39 or by JD5037,40 a
peripherally-restricted CB1 antagonist, as was low density lipoprotein (LDL)/high density
lipoprotein cholesterol ratio by SR141716A in diet-induced obese mice.23 Therefore it is
likely that SR141716A in our study exerted some of its metabolic effects through actions
in the liver.
A similar physiological provenance of metabolic disturbance in (mRen2)27 rats
and other animal models of metabolic syndrome, likely involving upregulated
endocannabinoid tone, may be inferred because of the shared effect profile of CB1
receptor blockade among these different models. Dysfunctional RAS signaling may be
another shared mechanism of metabolic disturbance, as chronic blockade of AT1
receptors lowers AP and improves the principal symptoms of metabolic syndrome in
obese humans,41 Zucker rats,42 spontaneously hypertensive rats (SHR) made obese by
160
high-fat diet,43 and (mRen2)27 rats.44 In fact, mounting evidence suggests the RAS and
the endocannabinoid system may actually work in tandem to promote cardiometabolic
diseases through signaling interactions that may augment the pathogenic effects of AT1
or CB1 receptor activation.
Several observations imply that blocking the
endocannabinoid system can attenuate the actions of an activated RAS. For example,
chronic treatment with SR141716A significantly reduces Ang II-mediated fibrosis in
mouse liver11 and the enhanced pressor response to intravenous Ang II exhibited by
Zucker rats.12 Chronic SR141716A treatment also reduces vascular expression of AT1
receptors in apolipoprotein E-deficient mice.45
However, no previous studies have
investigated interactive effects of CB1 receptor blockade in the context of chronic Ang IIdependent hypertension.
A compelling description of potential heteromerization mechanisms between CB1
and AT1 receptors11 accompanying reduced fibrosis in mouse liver led us to hypothesize
that CB1 receptor blockade would disrupt the Ang II-dependent hypertension in
(mRen2)27 rats, and results from our current study are congruent with this hypothesis.
Orally administered SR141716A reduced SBP in (mRen2)27 rats within 90 minutes by
≈43 mmHg to a level 17 mmHg (~15%) higher than baseline SBP of SD rats, whose SPB
was unaltered by SR141716A.
The fast onset of the effect precludes changes in
overnight food intake or the long-term metabolic effects of SR141716A from
contributing to the reduction of SBP in the acute setting, suggesting the effect on SBP is a
direct consequence of CB1 antagonism. However, whether blockade of CB1 receptors
directly interfered with vascular or central Ang II-AT1 receptor signaling, or reduced
SBP through an alternative mechanism cannot be determined from our study. CB1
161
receptor blockade in Zucker rats is associated with reduced sympathetic activity and
improved renal function,12, 46 which could contribute to reduced SBP in (mRen2)27 rats
because of the Ang II-driven increased sympathetic tone47 and impaired renal function48
in this strain. Even direct interference of AT1 receptor signaling by CB1 blockade may
have additional indirect effects on blood pressure because inhibition of the sympathetic
nervous system following RAS blockade is documented.49 The transiently slowed HR
we observed 90 minutes after treatment, which may indicate enhanced cardiac vagal tone,
likely contributed to an initial reduction in SBP as well.
Spectral analysis methods for measuring indices of blood pressure regulation and
autonomic tone at the conclusion of the chronic study reveal significant improvement in
both sympathetic (Seq Down, Seq All) and parasympathetic (Seq Up, Seq All) conscious
BRS for control of HR in SR141716A-treated rats compared to those that received
vehicle.
These data are fully consistent with our previous studies demonstrating
increased BRS in response to increases in AP, a measure of parasympatheticallymediated cardiovagal baroreflex function, after acute NTS microinjection of SR141716A
in anesthetized (mRen2)27 rats.16 Similar to conscious BRS measurements, HRV was
significantly higher in drug-treated compared to vehicle-treated rats, indicating increased
resting vagal tone. In fact, HRV in the drug-treated rats was comparable to values of
resting HRV previously reported in conscious SD rats.50 These indices of enhanced
central blood pressure regulation likely contribute to the maintenance of reduced SBP
during chronic treatment with SR141716A in (mRen2)27 rats, and also provide direct
evidence for central effects of systemic SR141716A in these animals.
162
It remains unknown to what extent these central actions of SR141716A contribute
to the metabolic or blood pressure effects of the chronic treatment, because distinct
central and peripheral mechanisms of cardiometabolic regulation are reported in the
endocannabinoid literature.51, 52 Therefore it would be interesting to study peripherally
restricted CB1 antagonists in a similar experimental paradigm to distinguish peripheral
from central effects of CB1 receptor blockade in (mRen2)27 rats. Collectively, studies
from our laboratory show SR141716A improves blood pressure regulation in (mRen2)27
rats from an impaired state typical of hypertension and obesity,17 during which the
endocannabinoid sytem and RAS are often overactive, as assessed using several indices
of autonomic function.
In the cardiovascular system, endocannabinoids mediate vasodilation and
cardiomyocyte relaxation through CB1 receptor-specific mechanisms,53 yielding a
hypotensive effect that is enhanced in both acute hypertension and in the chronic SHR
model.54,
55
Indeed, intravenous anandamide normalized while SR141716A further
increased AP in SHR,56 suggesting that upregulated CB1 receptor tone in this model is
likely compensatory rather than pathogenic in nature. The SBP-lowering effect of CB1
receptor blockade in (mRen2)27 rats may therefore seem unexpected without considering
potential endocannabinoid interactions with the RAS or improvement in central blood
pressure control. However, the pathogenesis of hypertension in the SHR is unclear
because unlike (mRen2)27 rats, SHR have a lean phenotype and normal or subnormal
RAS activity.57 Furthermore, there are conflicting reports as to whether SR141716A
corrects modestly elevated, sympathetically-driven SBP in Zucker rats.12,
46
These
reports are further confounded by recent evidence that at least some metabolic effects of
163
SR141716A are mediated by increased sympathetic activation.58 Therefore, specificity of
animal models should be considered carefully when interpreting the blood pressure
effects of systemic cannabinoids.
Although SBP remained suppressed at a consistent level over the chronic
treatment period, it is certainly possible that more slowly manifesting metabolic effects,
such as the reduction in fat mass, or decreased insulin or leptin, may contribute to lower
SBP in the later stages of treatment. The potential restoration of fatty acid oxidation and
reduction of fatty acid synthesis by chronic CB1 blockade could yield a decrease in
sympathetic tone59 and thus blood pressure. Even a RAS-mediated change in SBP could
conceivably be an indirect consequence of chronic CB1 blockade because correction of
hypercholesterolemia may downregulate AT1 receptor expression, which is correlated
with plasma LDL levels in humans and animals,60 and thus reduce intrinsic responses to
Ang II. The blood pressure responses to acute and chronic SR141716A administration in
(mRen2)27 rats in our study are consistent with the interpretation of disrupted Ang II
signaling by CB1 receptor blockade,11 but further study will be needed to elucidate the
precise mechanisms of this effect and to separate direct and indirect effects of CB1
blockade on blood pressure in this strain.
The results of our current study, obtained after acute and chronic systemic
treatment with SR141716A in hypertensive (mRen2)27 rats, contrast with the
interpretation of short-term beneficial effects of CB1 receptor blockade (4-14 days) on
blood pressure and metabolic profile, previously attributed to decreased food
consumption and other transient actions.61
However, altered levels of angiotensin
peptides or metabolic hormones at various timepoints between the timeframe of our acute
164
and chronic studies that we did not evaluate may certainly be a significant factor in the
modulation of blood pressure and metabolism by SR141716A in (mRen2)27 rats.
Nevertheless, our study showcases the (mRen2)27 rat as a unique model in which to
study the hemodynamic and metabolic effects of the endocannabinoid system in a setting
featuring upregulated RAS activity, and also highlights the challenges of treating diseases
as multifarious as hypertension.
165
Perspectives
Preclinical evidence for CB1 receptor blockade as a therapeutic strategy in cardiovascular
and metabolic diseases continues to mount. Recently, SR141716A was found to act
synergistically with an antidiabetic insulin sensitizing agent (BGP-15) to improve insulin
signaling when co-administered in Zucker rats.62 This result invites the possibility that a
lower dose of SR141716A used in combination therapy may yield comparable efficacy to
a higher stand-alone dose, thus anticipating a lower incidence of negative psychiatric side
effects associated with the drug. Combination therapy with SR141716A and a RAS
inhibitor may have a similar synergistic effect in hypertension, with the risk of
psychiatric side effects further lessened because blockade of Ang II formation and
signaling is associated with an antidepressant and anxiolytic behavioral profile.63, 64 We
observed centrally mediated improvements in blood pressure regulation evidenced by
increased BRS and HRV. Thus, peripherally restricted CB1 receptor antagonists may not
provide maximal cardiovascular or metabolic benefits.65 If further research uncovers
more evidence for endocannabinoid-RAS interactions contributing to hypertension or
metabolic syndrome, our current study may advance support for combination CB1-AT1
receptor blockade as additive or synergistic in RAS-dependent hypertension with
potentially mitigated side effects of both treatments.
166
Acknowledgements
We thank Katie Atkins and Pam Dean in the Hypertension Core Assay Laboratory, and
Ellen Tommasi for technical assistance.
Sources of Funding
This work was supported by the National Heart, Lung, and Blood Institute grant P01HL51952, the National Institute on Drug Abuse grant R01-DA03690, and the FarleyHudson Foundation (Jacksonville, NC).
Disclosures
None.
167
References
1.
Go AS, Mozaffarian D, Roger VL, et. a. Heart disease and stroke statistics--2014
update: a report from the american heart association. Circulation. 2014;129:e28e292.
2.
Herichova I, Szantoova K. Renin-angiotensin system: upgrade of recent
knowledge and perspectives. Endocr Regul. 2013;47:39-52.
3.
Wenzel UO, Benndorf R, Lange S. Treatment of arterial hypertension in obese
patients. Semin Nephrol. 2013;33:66-74.
4.
Lamont LS. Beta-blockers and their effects on protein metabolism and resting
energy expenditure. J Cardiopulm Rehabil. 1995;15:183-185.
5.
Grundy SM, Cleeman JI, Daniels SR, Donato KA, Eckel RH, Franklin BA,
Gordon DJ, Krauss RM, Savage PJ, Smith SC, Jr., Spertus JA, Costa F. Diagnosis
and management of the metabolic syndrome: an American Heart
Association/National Heart, Lung, and Blood Institute Scientific Statement.
Circulation. 2005;112:2735-2752.
6.
Pertwee RG, Howlett AC, Abood ME, Alexander SP, Di Marzo V, Elphick MR,
Greasley PJ, Hansen HS, Kunos G, Mackie K, Mechoulam R, Ross RA.
International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid
receptors and their ligands: beyond CB(1) and CB(2). Pharmacol Rev.
2010;62:588-631.
7.
Kirilly E, Gonda X, Bagdy G. CB1 receptor antagonists: new discoveries leading
to new perspectives. Acta Physiol (Oxf). 2012;205:41-60.
8.
Grassi G, Quarti-Trevano F, Seravalle G, Arenare F, Brambilla G, Mancia G.
Blood pressure lowering effects of rimonabant in obesity-related hypertension. J
Neuroendocrinol. 2008;20 Suppl 1:63-68.
9.
Turu G, Varnai P, Gyombolai P, Szidonya L, Offertaler L, Bagdy G, Kunos G,
Hunyady L. Paracrine transactivation of the CB1 cannabinoid receptor by AT1
angiotensin and other Gq/11 protein-coupled receptors. J Biol Chem.
2009;284:16914-16921.
10.
Szekeres M, Nadasy GL, Turu G, Soltesz-Katona E, Toth ZE, Balla A, Catt KJ,
Hunyady L. Angiotensin II Induces Vascular Endocannabinoid Release, Which
Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors. J Biol
Chem. 2012;287:31540-31550.
11.
Rozenfeld R, Gupta A, Gagnidze K, Lim MP, Gomes I, Lee-Ramos D, Nieto N,
Devi LA. AT1R-CB(1)R heteromerization reveals a new mechanism for the
pathogenic properties of angiotensin II. EMBO J. 2011;30:2350-2363.
168
12.
Janiak P, Poirier B, Bidouard JP, Cadrouvele C, Pierre F, Gouraud L, Barbosa I,
Dedio J, Maffrand JP, Le Fur G, O'Connor S, Herbert JM. Blockade of
cannabinoid CB1 receptors improves renal function, metabolic profile, and
increased survival of obese Zucker rats. Kidney Int. 2007;72:1345-1357.
13.
Bader M, Zhao Y, Sander M, Lee MA, Bachmann J, Bohm M, Djavidani B,
Peters J, Mullins JJ, Ganten D. Role of tissue renin in the pathophysiology of
hypertension in TGR(mREN2)27 rats. Hypertension. 1992;19:681-686.
14.
Kasper SO, Carter CS, Ferrario CM, Ganten D, Ferder LF, Sonntag WE,
Gallagher PE, Diz DI. Growth, metabolism, and blood pressure disturbances
during aging in transgenic rats with altered brain renin-angiotensin systems.
Physiol Genomics. 2005;23:311-317.
15.
Sloniger JA, Saengsirisuwan V, Diehl CJ, Dokken BB, Lailerd N, Lemieux AM,
Kim JS, Henriksen EJ. Defective insulin signaling in skeletal muscle of the
hypertensive TG(mREN2)27 rat. Am J Physiol Endocrinol Metab.
2005;288:E1074-1081.
16.
Schaich CL, Howlett AC, Thomas BF, Grabenauer MA, Diz DI. Medullary
endocannabinoid content contributes to the differential resting baroreflex
sensitivity in rats with altered brain renin-angiotensin system expression.
Hypertension. 2013;62:A182 (Abstract).
17.
Thayer JF, Yamamoto SS, Brosschot JF. The relationship of autonomic
imbalance, heart rate variability and cardiovascular disease risk factors. Int J
Cardiol. 2010;141:122-131.
18.
Ravinet Trillou C, Arnone M, Delgorge C, Gonalons N, Keane P, Maffrand JP,
Soubrie P. Anti-obesity effect of SR141716, a CB1 receptor antagonist, in dietinduced obese mice. Am J Physiol Regul Integr Comp Physiol. 2003;284:R345353.
19.
Gradin K, Persson B. Blood pressure and sympathetic activity in spontaneously
hypertensive rats during food restriction. J Neural Transm Gen Sect.
1990;79:183-191.
20.
Shaltout HA, Abdel-Rahman AA. Mechanism of fatty acids induced suppression
of cardiovascular reflexes in rats. J Pharmacol Exp Ther. 2005;314:1328-1337.
21.
Fisher JP, Ogoh S, Junor C, Khaja A, Northrup M, Fadel PJ. Spontaneous
baroreflex measures are unable to detect age-related impairments in cardiac
baroreflex function during dynamic exercise in humans. Exp Physiol.
2009;94:447-458.
22.
Chappell MC, Gallagher PE, Averill DB, Ferrario CM, Brosnihan KB. Estrogen
or the AT1 antagonist olmesartan reverses the development of profound
hypertension in the congenic mRen2. Lewis rat. Hypertension. 2003;42:781-786.
169
23.
Poirier B, Bidouard JP, Cadrouvele C, Marniquet X, Staels B, O'Connor SE,
Janiak P, Herbert JM. The anti-obesity effect of rimonabant is associated with an
improved serum lipid profile. Diabetes Obes Metab. 2005;7:65-72.
24.
Zucker LM, Antoniades HN. Insulin and obesity in the Zucker genetically obese
rat "fatty". Endocrinology. 1972;90:1320-1330.
25.
Vickers SP, Webster LJ, Wyatt A, Dourish CT, Kennett GA. Preferential effects
of the cannabinoid CB1 receptor antagonist, SR 141716, on food intake and body
weight gain of obese (fa/fa) compared to lean Zucker rats. Psychopharmacology
(Berl). 2003;167:103-111.
26.
Van Gaal LF, Rissanen AM, Scheen AJ, Ziegler O, Rossner S. Effects of the
cannabinoid-1 receptor blocker rimonabant on weight reduction and
cardiovascular risk factors in overweight patients: 1-year experience from the
RIO-Europe study. Lancet. 2005;365:1389-1397.
27.
Juan-Pico P, Fuentes E, Bermudez-Silva FJ, Javier Diaz-Molina F, Ripoll C,
Rodriguez de Fonseca F, Nadal A. Cannabinoid receptors regulate Ca(2+) signals
and insulin secretion in pancreatic beta-cell. Cell Calcium. 2006;39:155-162.
28.
Steffens S, Veillard NR, Arnaud C, Pelli G, Burger F, Staub C, Karsak M,
Zimmer A, Frossard JL, Mach F. Low dose oral cannabinoid therapy reduces
progression of atherosclerosis in mice. Nature. 2005;434:782-786.
29.
Montecucco F, Lenglet S, Braunersreuther V, Burger F, Pelli G, Bertolotto M,
Mach F, Steffens S. CB(2) cannabinoid receptor activation is cardioprotective in a
mouse model of ischemia/reperfusion. J Mol Cell Cardiol. 2009;46:612-620.
30.
Maffei M, Halaas J, Ravussin E, Pratley RE, Lee GH, Zhang Y, Fei H, Kim S,
Lallone R, Ranganathan S, et al. Leptin levels in human and rodent: measurement
of plasma leptin and ob RNA in obese and weight-reduced subjects. Nat Med.
1995;1:1155-1161.
31.
Di Marzo V, Goparaju SK, Wang L, Liu J, Batkai S, Jarai Z, Fezza F, Miura GI,
Palmiter RD, Sugiura T, Kunos G. Leptin-regulated endocannabinoids are
involved in maintaining food intake. Nature. 2001;410:822-825.
32.
Arita Y, Kihara S, Ouchi N, Takahashi M, Maeda K, Miyagawa J, Hotta K,
Shimomura I, Nakamura T, Miyaoka K, Kuriyama H, Nishida M, Yamashita S,
Okubo K, Matsubara K, Muraguchi M, Ohmoto Y, Funahashi T, Matsuzawa Y.
Paradoxical decrease of an adipose-specific protein, adiponectin, in obesity.
Biochem Biophys Res Commun. 1999;257:79-83.
33.
Nicholas SB. Lipid disorders in obesity. Curr Hypertens Rep. 1999;1:131-136.
34.
Diez JJ, Iglesias P. The role of the novel adipocyte-derived hormone adiponectin
in human disease. Eur J Endocrinol. 2003;148:293-300.
170
35.
Nedvidkova J, Smitka K, Kopsky V, Hainer V. Adiponectin, an adipocyte-derived
protein. Physiol Res. 2005;54:133-140.
36.
Perwitz N, Fasshauer M, Klein J. Cannabinoid receptor signaling directly inhibits
thermogenesis and alters expression of adiponectin and visfatin. Horm Metab Res.
2006;38:356-358.
37.
Despres JP, Golay A, Sjostrom L. Effects of rimonabant on metabolic risk factors
in overweight patients with dyslipidemia. N Engl J Med. 2005;353:2121-2134.
38.
Tam J, Godlewski G, Earley BJ, Zhou L, Jourdan T, Szanda G, Cinar R, Kunos
G. Role of Adiponectin in the Metabolic Effects of Cannabinoid Receptor-1
Blockade in Mice with Diet-Induced Obesity. Am J Physiol Endocrinol Metab.
2013
39.
Osei-Hyiaman D, DePetrillo M, Pacher P, Liu J, Radaeva S, Batkai S, HarveyWhite J, Mackie K, Offertaler L, Wang L, Kunos G. Endocannabinoid activation
at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to dietinduced obesity. J Clin Invest. 2005;115:1298-1305.
40.
Cinar R, Godlewski G, Liu J, Tam J, Jourdan T, Mukhopadhyay B, Harvey-White
J, Kunos G. Hepatic cannabinoid-1 receptors mediate diet-induced insulin
resistance by increasing de novo synthesis of long-chain ceramides. Hepatology.
2014;59:143-153.
41.
Bramlage P, Schonrock E, Odoj P. Metabolic effects of an AT1-receptor blockade
combined with HCTZ in cardiac risk patients: a non interventional study in
primary care. BMC Cardiovasc Disord. 2008;8:30.
42.
Toblli JE, Munoz MC, Cao G, Mella J, Pereyra L, Mastai R. ACE inhibition and
AT1 receptor blockade prevent fatty liver and fibrosis in obese Zucker rats.
Obesity (Silver Spring). 2008;16:770-776.
43.
Muller-Fielitz H, Hubel N, Mildner M, Vogt FM, Barkhausen J, Raasch W.
Chronic blockade of angiotensin AT1 receptors improves cardinal symptoms of
metabolic syndrome in diet-induced obesity in rats. Br J Pharmacol.
2014;171:746-760.
44.
Sloniger JA, Saengsirisuwan V, Diehl CJ, Kim JS, Henriksen EJ. Selective
angiotensin II receptor antagonism enhances whole-body insulin sensitivity and
muscle glucose transport in hypertensive TG(mREN2)27 rats. Metabolism.
2005;54:1659-1668.
45.
Tiyerili V, Zimmer S, Jung S, Wassmann K, Naehle CP, Lutjohann D, Zimmer A,
Nickenig G, Wassmann S. CB1 receptor inhibition leads to decreased vascular
AT1 receptor expression, inhibition of oxidative stress and improved endothelial
function. Basic Res Cardiol. 2010;105:465-477.
171
46.
Mingorance C, Alvarez de Sotomayor M, Jimenez-Palacios FJ, Callejon Mochon
M, Casto C, Marhuenda E, Herrera MD. Effects of chronic treatment with the
CB1 antagonist, rimonabant on the blood pressure, and vascular reactivity of
obese Zucker rats. Obesity (Silver Spring). 2009;17:1340-1347.
47.
Moriguchi A, Tallant EA, Matsumura K, Reilly TM, Walton H, Ganten D,
Ferrario CM. Opposing actions of angiotensin-(1-7) and angiotensin II in the
brain of transgenic hypertensive rats. Hypertension. 1995;25:1260-1265.
48.
Springate JE, Feld LG, Ganten D. Renal function in hypertensive rats transgenic
for mouse renin gene. Am J Physiol. 1994;266:F731-737.
49.
Fujisawa Y, Nagai Y, Lei B, Nakano D, Fukui T, Hitomi H, Mori H, Masaki T,
Nishiyama A. Roles of central renin-angiotensin system and afferent renal nerve
in the control of systemic hemodynamics in rats. Hypertens Res. 2011;34:12281232.
50.
Arnold AC, Shaltout HA, Gilliam-Davis S, Kock ND, Diz DI. Autonomic control
of the heart is altered in Sprague-Dawley rats with spontaneous hydronephrosis.
Am J Physiol Heart Circ Physiol. 2011;300:H2206-2213.
51.
Cota D, Marsicano G, Tschop M, Grubler Y, Flachskamm C, Schubert M, Auer
D, Yassouridis A, Thone-Reineke C, Ortmann S, Tomassoni F, Cervino C, Nisoli
E, Linthorst AC, Pasquali R, Lutz B, Stalla GK, Pagotto U. The endogenous
cannabinoid system affects energy balance via central orexigenic drive and
peripheral lipogenesis. J Clin Invest. 2003;112:423-431.
52.
Silvestri C, Ligresti A, Di Marzo V. Peripheral effects of the endocannabinoid
system in energy homeostasis: adipose tissue, liver and skeletal muscle. Rev
Endocr Metab Disord. 2011;12:153-162.
53.
Wagner JA, Varga K, Jarai Z, Kunos G. Mesenteric vasodilation mediated by
endothelial anandamide receptors. Hypertension. 1999;33:429-434.
54.
Ho WS, Gardiner SM. Acute hypertension reveals depressor and vasodilator
effects of cannabinoids in conscious rats. Br J Pharmacol. 2009;156:94-104.
55.
Lake KD, Martin BR, Kunos G, Varga K. Cardiovascular effects of anandamide
in anesthetized and conscious normotensive and hypertensive rats. Hypertension.
1997;29:1204-1210.
56.
Batkai S, Pacher P, Osei-Hyiaman D, Radaeva S, Liu J, Harvey-White J,
Offertaler L, Mackie K, Rudd MA, Bukoski RD, Kunos G. Endocannabinoids
acting at cannabinoid-1 receptors regulate cardiovascular function in
hypertension. Circulation. 2004;110:1996-2002.
172
57.
Sokabe H, Kawashima K, Watanabe TX. Blood pressure regulation by
angiotensin in the spontaneously hypertensive rats. Adv Exp Med Biol.
1979;120B:429-435.
58.
Bellocchio L, Soria-Gomez E, Quarta C, Metna-Laurent M, Cardinal P, Binder E,
Cannich A, Delamarre A, Haring M, Martin-Fontecha M, Vega D, Leste-Lasserre
T, Bartsch D, Monory K, Lutz B, Chaouloff F, Pagotto U, Guzman M, Cota D,
Marsicano G. Activation of the sympathetic nervous system mediates hypophagic
and anxiety-like effects of CB(1) receptor blockade. Proc Natl Acad Sci U S A.
2013;110:4786-4791.
59.
Jbilo O, Ravinet-Trillou C, Arnone M, Buisson I, Bribes E, Peleraux A, Penarier
G, Soubrie P, Le Fur G, Galiegue S, Casellas P. The CB1 receptor antagonist
rimonabant reverses the diet-induced obesity phenotype through the regulation of
lipolysis and energy balance. FASEB J. 2005;19:1567-1569.
60.
Nickenig G, Jung O, Strehlow K, Zolk O, Linz W, Scholkens BA, Bohm M.
Hypercholesterolemia is associated with enhanced angiotensin AT1-receptor
expression. Am J Physiol. 1997;272:H2701-2707.
61.
Thornton-Jones ZD, Kennett GA, Benwell KR, Revell DF, Misra A, Sellwood
DM, Vickers SP, Clifton PG. The cannabinoid CB1 receptor inverse agonist,
rimonabant, modifies body weight and adiponectin function in diet-induced obese
rats as a consequence of reduced food intake. Pharmacol Biochem Behav.
2006;84:353-359.
62.
Literati-Nagy Z, Tory K, Literati-Nagy B, Bajza A, Vigh L, Jr., Vigh L, Mandl J,
Szilvassy Z. Synergic insulin sensitizing effect of rimonabant and BGP-15 in
Zucker-obese rats. Pathol Oncol Res. 2013;19:571-575.
63.
Pavel J, Benicky J, Murakami Y, Sanchez-Lemus E, Saavedra JM. Peripherally
administered angiotensin II AT1 receptor antagonists are anti-stress compounds in
vivo. Ann N Y Acad Sci. 2008;1148:360-366.
64.
Kangussu LM, Almeida-Santos AF, Bader M, Alenina N, Fontes MA, Santos RA,
Aguiar DC, Campagnole-Santos MJ. Angiotensin-(1-7) attenuates the anxiety and
depression-like behaviors in transgenic rats with low brain angiotensinogen.
Behav Brain Res. 2013;257:25-30.
65.
Engeli S. Central and peripheral cannabinoid receptors as therapeutic targets in
the control of food intake and body weight. Handb Exp Pharmacol. 2012:357381.
173
Novelty and Significance: 1) What Is New, 2) What Is Relevant?
What Is New?

Acute and chronic systemic CB1 receptor blockade significantly lowers blood
pressure in hypertensive (mRen2)27 rats, with a concomitant positive influence over
conscious autonomic blood pressure regulation and metabolic profile.
What Is Relevant?

Results indicate novel mechanisms for maintenance of hypertension, metabolic
syndrome and impaired autonomic control of blood pressure associated with upregulation
of Ang II signaling.
Summary
The (mRen2)27 rat exhibits Ang II-dependent hypertension with impaired baroreflex
sensitivity and features of metabolic syndrome. We demonstrate for the first time that
treatment with a CB1 receptor antagonist improves all of these parameters in this model,
suggesting a role for the endocannabinoid system in the maintenance of cardiometabolic
pathologies related to overactivity of the RAS.
174
CHAPTER FIVE
SUMMARY AND CONCLUSIONS
1. Summary of Results
Hypertension is the single biggest predictor for future heart attack, stroke or congestive
heart failure1 and currently affects 1 in 3 US adults,2 a figure that is expected to rise as
the population ages and as the prevalence of obesity increases.3 Despite decades of
research, our ability to control chronic high blood pressure is hampered by an incomplete
understanding of the development of the disease. The onset of essential hypertension is
often associated with reductions in baroreflex sensitivity for control of heart rate,4 an
important marker of vagus nerve function, as well as imbalances in RAS activity.
Reduced baroreflex sensitivity and pathogenic RAS signaling is also typical of
established risk factors for hypertension, including aging and obesity.5 This implies the
existence of shared physiological mechanisms contributing to the progression of
cardiovascular diseases, aging, and metabolic dysfunction in part by altering baroreflex
sensitivity. Therefore, the identification of factors that may influence baroreflex function
in these negative conditions is imperative to advance our understanding of hypertension
and its risk factors so that more effective pharmacotherapies can be developed.
The central and peripheral endocannabinoid system plays a critical role in the
modulation of cellular processes governing autonomic, metabolic and cardiovascular
function.6 Over the past decade, the endocannabinoid system has been the subject of
intensive research in part because of its putative contributions to cardiovascular and
175
metabolic diseases.7,
8
Recently, signaling interactions between the endocannabinoid
system and the RAS have been described in several in vitro and in vivo settings.9-13
However, an assessment of the endocannabinoid system in conditions associated with
altered brain RAS activity is lacking. The studies contained in this volume investigate
the contribution of endocannabinoids to regulation of baroreflex sensitivity and blood
pressure under normal conditions and during alterations of the brain RAS. The results of
these studies implicate novel mechanisms for neural control of the circulation in
hypertensive, aged and obese individuals that may provide insight for preservation of
baroreflex function in these conditions.
In
Chapter
Two
we
examine
the
contribution
of
dorsal
medullary
endocannabinoids to the differential resting baroreflex sensitivity observed in rats underand overexpressing components of the brain RAS. Data from these studies provide
functional and biochemical evidence that increased production of 2-AG at the level of the
NTS and surrounding nuclei may underlie the markedly suppressed baroreflex sensitivity
for control of heart rate in hypertensive (mRen2)27 rats with upregulated brain RAS
activity.14 These studies also reveal that hypotensive ASrAOGEN rats underexpressing
glial angiotensinogen have reduced 2-AG and CB1 receptor mRNA expression in the
dorsal medulla that may function to maintain enhanced baroreflex sensitivity in this
strain.15 Thus, we establish a link between the endocannabinoid system and maintenance
of baroreflex sensitivity in animal models featuring altered brain RAS activity.
Normal aging is characterized by progressively rising systolic blood pressure16
with declining baroreflex sensitivity17 due to autonomic imbalance driven in part by
changes in the brain RAS.18,
19
A role for endocannabinoid modulation of age-related
176
impairment of baroreflex function has not yet been demonstrated. Therefore, in Chapter
Three we report altered CB1 and CB2 receptor expression and increasing 2-AG content
across lifespan in the dorsal medulla of young adult, middle aged and older SpragueDawley rats.
We further demonstrate that the medullary endocannabinoid system
functionally contributes to reduced baroreflex sensitivity associated with aging. Thus,
the endocannabinoid system is linked to maintenance of baroreflex sensitivity during
aging, an additional setting associated with altered brain RAS activity and hypertension.
Results from these acute, localized studies prompted us to examine systemic CB1
receptor blockade in the (mRen2)27 rat model of Ang II-dependent hypertension with
metabolic syndrome.20 In Chapter Four we demonstrate positive effects of acute and
chronic systemic treatment with a CB1 receptor antagonist on blood pressure, and
additional positive effects of chronic treatment on conscious baroreflex sensitivity and
metabolic profile. These findings may advance support for CB1 receptor blockade as a
therapeutic strategy for treating RAS-dependent hypertension and metabolic dysfunction.
This study further illustrates an important link between the endocannabinoid system and
hypertension or obesity in the context of an altered brain RAS.
Together, these studies demonstrate important mechanisms for preservation of
baroreflex sensitivity and blood pressure in hypertension, aging and obesity.
The
importance of pharmacological restoration of baroreflex sensitivity in these conditions is
now recognized as a novel method to prevent cardiovascular diseases,21 creating the need
for research into the mechanisms underlying baroreflex dysfunction. Therefore, we
conclude that the endocannabinoid system may be an attractive therapeutic target for the
treatment of RAS-dependent hypertension or metabolic syndrome.
177
2. Endocannabinoid Modulation of Baroreflex Sensitivity during Hypertension and
Aging
Exogenous cannabinoids have long been known to interfere with autonomic homeostasis
in humans. For example, prolonged cannabis use is associated with blunted circulatory
reflexes to standing and exercising, orthostatic intolerance and reduced heart rate
variability.22, 23 The identification of CB1 receptors within the NTS and on terminals of
vagal afferent neurons24-26 and tissue bathing studies showing that NTS neurons are
sensitive to cannabinoid ligands27 suggests a role for the endocannabinoid system in
baroreflex modulation.
The first studies to assess exogenous cannabinoids in baroreflex modulation at the
level of the NTS in normotensive animals generally demonstrate a facilitating effect of
cannabinoid agonists, including anandamide and WIN55,212, on sympathetic nerve
inhibition following increases in blood pressure.28-30 Of note, prolongation of baroreflexevoked sympathetic inhibition observed in Sprague-Dawley and Wistar-Kyoto rats is
absent in SHR,31 providing the first clue for potentially altered regulation of baroreflex
function in hypertension by the endocannabinoid system. However, these studies are
limited in scope and do not assess dose-response relationships or effects on the vagallymediated parasympathetic branch of the baroreflex.
Studies in our laboratory examining the effect of a non-selective synthetic
cannabinoid agonist microinjected into the NTS reveal dose-related biphasic actions of
cannabinoid receptor activation on baroreflex sensitivity for control of heart rate in
anesthetized Sprague-Dawley rats (Figure 1-1).32 Specifically, a low dose of CP55,940
(1.2 pmol/120 nL), comparable to drug concentrations used in the studies mentioned
178
above, enhance baroreflex sensitivity while a 10-fold higher dose (12 pmol) significantly
impairs it to a level typical of hypertension. These effects are fully prevented or reversed
by microinjection of SR141716A before or after the agonist, suggesting they were
mediated by activation of CB1 receptors in the NTS. Enhanced bradycardic baroreflex
sensitivity by lower doses of CP55,940 in the NTS is consistent with prolonged
baroreflex-evoked sympathetic nerve inhibition by cannabinoid agonists found in
normotensive animals.28-30 Furthermore, the biphasic response evoked by increasing
doses of CP55,940 is consistent with biphasic actions of cannabinoids reported at many
areas of the brain, attributable to their ability to modulate various competing
neurotransmitter pathways.33
As we demonstrate in Chapter Two, there is no effect of SR141716A alone in the
NTS of Sprague-Dawley rats, suggesting minimal or absent tonic influence of CB1
receptors in the modulation of baroreflex sensitivity, in line with a lack of alteration in
sympathetic nerve discharge by AM281 alone in the NTS.34 However, the intriguing
result that a higher dose of CP55,940 impairs baroreflex sensitivity makes it conceivable
that overactivation of NTS CB1 receptors by endocannabinoids could contribute to
impaired baroreflex sensitivity during certain contexts, such as onset of hypertension.
Additional studies in anesthetized (mRen2)27 rats with low baseline baroreflex
sensitivity find no effect of NTS microinjection of CP55,940 at the dose that increases
baroreflex sensitivity in Sprague-Dawley rats (Figure 1-2). One interpretation of this
result is that CB1 receptors in the NTS of these animals may be overactivated to suppress
baroreflex function, similar to the effect of the high dose of CP55,940 in Sprague-Dawley
rats.
Indeed, peripheral endocannabinoid tone is increased during hypertension and
179
cardiovascular disease,35 with upregulated CB1 receptor expression in the heart, aorta and
coronary arteries reported in human coronary artery disease and aortic stenosis.36, 37 To
test whether increased endogenous CB1 receptor tone contributes to suppressed
baroreflex sensitivity in hypertension we microinjected SR141716A into the NTS of
(mRen2)27 rats.
These transgenic rats feature overactive sympathetic drive38,
39
in
addition to an imbalance in the brain RAS favoring Ang II signaling.40 Data from
Chapter Two show that NTS CB1 receptor blockade by microinjection of SR141716A
(0.36- and 36-pmol/120 nL) produces dose-dependent improvement in baroreflex
sensitivity for control of heart rate in these animals, increasing it to a level comparable to
resting baroreflex sensitivity of Sprague-Dawley rats at the higher dose tested.
Therefore, we conclude from microinjection studies in (mRen2)27 rats that altered
endogenous CB1 receptor tone at the level of the NTS functionally contributes to
suppressed resting baroreflex sensitivity in this model of Ang II-dependent hypertension.
Normal aging is an additional setting characterized by excessive sympathetic
nervous system activity41 and RAS imbalance18 driving reduced baroreflex sensitivity
with steadily increasing systolic blood pressure.
Previous studies demonstrate that
similar to young (mRen2)27 rats,42, 43 replacement of Ang-(1-7) rather than AT1 receptor
blockade in the NTS restores impaired baroreflex sensitivity in older Sprague-Dawley
rats.44 Furthermore, baroreflex function in older ASrAOGEN rats is preserved relative to
Sprague-Dawley rats. However, mas receptor blockade by [D-Ala7]-Ang-(1-7) in the
NTS impairs baroreflex sensitivity in these transgenic animals45 while having no effect in
older Sprague-Dawley rats.44 Together, these results suggest that loss of Ang-(1-7) rather
180
than upregulation of Ang II tone in the NTS is a common mechanism underlying
impaired baroreflex sensitivity in both hypertension and normal aging.
Based on these physiological similarities between older animals and young
(mRen2)27 rats, we hypothesize that upregulated CB1 receptor tone may also contribute
to low baroreflex sensitivity in aged Sprague-Dawley rats. Data from Chapter Three
support this hypothesis, as NTS microinjection of SR141716A (36 pmol) normalizes
baroreflex sensitivity in anesthetized older Sprague-Dawley rats, but only in rats that
exhibit impaired baseline baroreflex sensitivity.
In aged Sprague-Dawley rats with
preserved baroreflex function, SR141716A in the NTS has no effect. Conscious blood
pressure in these animals was not measured, so it is uncertain if animals with impaired
baseline baroreflex sensitivity also exhibited hypertension. However, these data are fully
consistent with a role for altered brainstem endocannabinoid system activity in the
suppression of baroreflex sensitivity during conditions associated with overactive brain
RAS activity and consequent increased sympathetic outflow.
Effects of exogenous cannabinoids on the cardiovascular system are exaggerated
in anesthetized relative to conscious animals.46 Accordingly, the results we obtained in
anesthetized (mRen2)27 and aged Sprague-Dawley rats are bolstered by data from
Chapter Four in which we assess autonomic function in conscious (mRen2)27 rats
following chronic systemic treatment with SR141716A (10 mg/kg/day) or its vehicle.
Although these data were collected after chronic systemic treatment with the CB1
receptor antagonist, they are fully consistent with the acute microinjection studies and
demonstrate that indices of parasympathetic vagus nerve activity including heart rate
variability and bradycardic baroreflex sensitivity are significantly greater in (mRen2)27
181
rats treated with SR141716A compared to those treated with vehicle. In fact, these
indices in the drug-treated rats are comparable to values of heart rate variability and
bradycardic baroreflex sensitivity recently reported by our laboratory in healthy SpragueDawley animals.47
Furthermore, conscious tachycardic baroreflex sensitivity, a
functional index of the sympathetic branch of the baroreflex for control of heart rate that
was not assessed in the microinjection studies, is also significantly greater in (mRen2)27
rats following chronic systemic CB1 receptor blockade. Therefore, we demonstrate in
three different settings—anesthetized (mRen2)27, aged Sprague-Dawley and conscious
(mRen2)27 rats—that NTS-localized or systemic blockade of CB1 receptors normalizes
impaired baroreflex function in conditions associated with hypertension.
In summary, based on our acute microinjection studies in young (mRen2)27 and
aged Sprague-Dawley rats from Chapters Two and Three, as well as additional studies
from Chapter Four in conscious (mRen2)27 rats following chronic systemic treatment
with SR14171A, we conclude that upregulated NTS endocannabinoid tone contributes to
impaired baseline baroreflex sensitivity in conditions associated with autonomic
imbalance or an overactive brain RAS, including hypertension and normal aging. These
studies indicate novel mechanisms for preservation of baroreflex function during adverse
cardiovascular conditions and suggest that medications that block central CB1 receptors,
such as SR141716A, may improve blood pressure regulation in hypertensive, obese or
elderly patients. However, future studies must elucidate specific mechanisms of action
underlying endocannabinoid modulation of baroreflex sensivity, which may involve
altering neurotransmitter release in the NTS directly or through signaling interactions
with other factors, including the brain RAS.
182
3. Mechanisms for Endocannabinoid Modulation of Baroreflex Sensitivity
A primary role for endocannabinoids in the CNS is to modulate release of
neurotransmitters at the level of the synapse. They do this by activating CB1 receptors
located on presynaptic neuronal terminals, which then stimulate heterotrimeric G proteins
whose subunits directly interact with voltage-gated ion channels to both repolarize the
local plasma membrane and reduce local intracellular calcium concentration.48
We
hypothesize that the immediate effects of cannabinoid ligands on baroreflex sensitivity
demonstrated in the present studies are likely attributable to direct modulation of
neurotransmitter release within the NTS.
Glutamate is accepted as the primary neurotransmitter released from
baroreceptive afferent neurons in the NTS projecting to downstream baroreflex nuclei in
the brainstem,49-57 while GABA interneurons serve a tonic modulatory role over
baroreflex function.57-59 Several lines of evidence link altered NTS glutamate and GABA
balance shifted in favor of increased GABAergic tone to impaired cardiovascular
regulation in hypertension. For example, infusion of GABA-A and -B receptor agonists
into the NTS of conscious normotensive Wistar-Kyoto rats reduces baroreflex sensitivity
for control of heart rate in response to intravenous phenylephrine.60 Altered GABA-A
and -B receptor sensitivity is suggested to be necessary to compensate for or limit
reductions in baroreflex function in Sprague-Dawley rats made hypertensive by onekidney renal-wrap.61 Studies in SHR reveal increased endogenous GABA release and
enhanced GABA receptor sensitivity in the NTS underlying the exaggerated pressor
response to GABA receptor stimulation,62 as well as GABAergic mechanisms within the
NTS acting tonically to suppress baroreflex sensitivity for control of sympathetic nerve
183
activity.63
Finally, Ang II increases GABA-B receptor expression in cultured NTS
neurons64 and enhances GABA-B receptor-mediated depressor responses and expression
in NTS of Sprague-Dawley rats.65 Collectively, these findings suggest a GABAergic
mechanism in the NTS that could contribute to the central nervous system actions of Ang
II, yielding the impairment in baroreflex function and elevated blood pressure found in
various models of hypertension, including the Ang II-dependent (mRen2)27 rat model
used in our present studies.
Although magnetic resonance spectroscopy does not detect significant differences
in the ratio of glutamate-to-GABA in the NTS of (mRen2)27 and Sprague-Dawley rats,66
enhanced NTS GABA receptor expression or sensitivity as a mechanism for blunted
baroreflex sensitivity for control of heart rate in the hypertensive strain cannot be
dismissed. Importantly, the same study reports that the glutamate-to-GABA ratio was
significantly increased in hypotensive ASrAOGEN rats compared to Sprague-Dawley
and (mRen2)27 rats.66
This observation implicates increased NTS glutamatergic
neurotransmission as a mechanism underlying the enhanced baroreflex sensitivity and
hypotension associated with this RAS-deficient strain. Indeed, glutamate released in the
NTS via N-methyl-D-aspartate (NMDA) receptor stimulation or nitric oxide synthesis is
associated with augmented depressor and bradycardic responses in Wistar-Kyoto rats.67
Furthermore, central GABA-A receptor-mediated cardiovascular responses including
changes in arterial pressure, heart rate and renal sympathetic nerve activity are attenuated
in ASrAOGEN rats.68 In summary, upregulated GABAergic neurotransmission within
the NTS is associated with blunted autonomic reflexes including impaired baroreflex
function and hypertension, while increased NTS glutamatergic neurotransmission is
184
associated with enhanced baroreflex function or hypotension. These results support our
hypothesis that glutamate and GABA serve differential modulatory roles in the NTS of
rats with low or high brain RAS expression.
Exogenous cannabinoids in the NTS modulate cardiovascular and baroreflex
function through interactions with the local GABA or glutamate systems. Microinjection
of WIN55,212-2 and CP55,940 into the NTS of anesthetized Sprague-Dawley rats
suppresses sympathetic nerve discharge,34 and this effect is antagonized by NTS
microinjection of the CB1 receptor-selective antagonist AM281 or a non-NMDA
glutamate receptor antagonist, in addition to disruption of the baroreflex arc by sinoaortic
denervation. The same study reports no effect of AM281 alone in the NTS, consistent
with our observations in Sprague-Dawley rats that SR141716A in the NTS does not alter
resting blood pressure, heart rate or baroreflex sensitivity and suggesting absent or
minimal intrinsic CB1 receptor tone in the NTS of Sprague-Dawley rats. Another study
reports that NTS microinjection of anandamide in anesthetized Sprague-Dawley rats
prolongs the phenylephrine-evoked inhibition of renal sympathetic nerve activity,29
indicating an increase in sympathetic baroreflex sensitivity. Furthermore, anandamide
shortens the duration of baroreflex-evoked sympathoinhibition following blockade of
NTS GABA-A receptors,29 revealing the possibility that presynaptic inhibition of
glutamate release by anandamide is obscured by a more dominant GABA-mediated
effect.
Our studies investigating the role of endocannabinoids in modulating the
predominantly parasympathetically-mediated cardiovagal branch of the baroreflex are
therefore consistent with evidence for the maintenance of altered NTS glutamate or
185
GABA neurotransmission in the context of altered brain RAS expression through
activation of CB1 receptors on these neurons.
Electrophysiological studies by others suggest a more complex relationship
between the endocannabinoid and glutamate or GABA systems within the NTS.
Application of WIN55,212-2 to NTS tissue slices in the presence of ionotropic glutamate
receptor antagonists inhibited presynaptic GABA release to anatomically identified
second-order baroreceptive neurons in the NTS, a putative mechanism to enhance
baroreflex signaling in this region,69 although this was not tested functionally.
Importantly, depolarization of second-order baroreceptive neurons decreased the
frequency of miniature inhibitory postsynaptic currents, an effect blocked by the CB 1selective antagonist AM251. This suggests that depolarization of these neurons induces
endocannabinoid release and activation of presynaptic CB1 receptors, which in turn
inhibit the release of GABA.69
However, whole-cell patch clamp recordings in rat
brainstem slices show that WIN55,212-2 and exogenous anandamide in the NTS suppress
synaptic input to neurons in the dmnX,70 an important second-order nucleus in the
parasympathetic baroreflex arc from which vagal efferents to the heart originate.
Therefore, upregulation of dorsal medullary endocannabinoids or CB1 receptors is a
mechanism likely to contribute to the suppression of baroreflex sensitivity for control of
heart rate, consistent with the results of our studies in anesthetized (mRen2)27 and older
Sprague-Dawley rats.
By implication, downregulation of endocannabinoid system
expression in this region would likely increase glutamate release, contributing to
enhanced baroreflex sensitivity as exhibited by young adult ASrAOGEN rats.
186
The contribution of central TRPV1 ion channels to regulation of synaptic activity
in brainstem nuclei involved baroreflex mediation should not escape consideration.
TRPV1 is found on peripheral and central neurons and is activated by endocannabinoids,
among other chemical and noxious stimuli.71 In the brainstem, TRPV1 is expressed in
the NTS and dmnX.72
Functionally, the activation of TRPV1 channels triggers
spontaneous glutamate release in the NTS, opposing the actions of CB1 receptors,73-75
while exerting a tonic influence over excitatory and inhibitory inputs to the dmnX in rats
and mice.76,
77
These actions would likely contribute to modulation of baroreflex
function, but this has not been tested directly. However, the presence of TRPV1 in
brainstem nuclei in the baroreflex arc provides an additional mechanism for modulation
of baroreflex function by the endocannabinoid system. It also may further explain the
actions of exogenous anandamide as well as WIN55,212-2, reported to inhibit TRPV1 in
trigeminal ganglion neurons,78 in the studies discussed above.29, 69 Although brainstem
endocannabinoids may modulate baroreflex sensitivity through actions at both CB1
receptors and TRPV1 channels, in Chapters Two and Three we show only the
contribution of endocannabinoid modulation of baroreflex function through actions at
CB1 receptors in our present study because SR141716A is a selective antagonist of CB1
that is not reported to interact with any other receptors, including TRPV1 and CB2
receptors.79, 80
In summary, data from our acute studies in transgenic rats with altered brain RAS
expression are consistent with differential medullary endocannabinoid tone contributing
to modulation of baroreflex sensitivity in these animals through the tonic control over
excitatory or inhibitory neurotransmission in the NTS. These data are further consistent
187
with the interpretation that acute NTS CB1 blockade is able to abrogate tonic excitatory
or inhibitory neurotransmission in this region. Therefore, we hypothesize that the use of
magnetic resonance spectroscopy in future experiments will detect an increase in
glutamatergic or GABAergic neurotransmission after blockade of CB1 receptors in the
NTS of (mRen2)27 or ASrAOGEN rats, respectively. However, the physiological events
that create the intrinsic balance of neurotransmission within the NTS, and whether the
endocannabinoid system contributes to disruption of this balance during conditions
associated with hypertension, are less clear and will be the focus of future studies.
188
4. CB1 Receptor Blockade and Blood Pressure Regulation in Ang II-Dependent
Hypertension
Systemically administered cannabinoid agonists generally produce a sustained
hypotensive effect accompanied by bradycardia through CB1 receptor-mediated signaling
in the vasculature and heart, respectively.81, 82 These depressor effects are amplified in
models of acute and chronic hypertension,46, 83, 84 suggesting activation of the peripheral
endocannabinoid system in response to elevated blood pressure, perhaps as a
compensatory mechanism. Indeed, intravenous SR141716A further increases pressure in
SHR,35 providing evidence of a compensatory rather than pathogenic role for the
endocannabinoid system in this strain.
However, data from acute microinjection studies showing improvement in
baroreflex sensitivity by CB1 receptor blockade in the NTS prompted us to study effects
of CB1 receptor blockade in a more clinically relevant study design. We wanted to
determine if chronic oral administration of SR141716A improves conscious baroreflex
function in (mRen2)27 rats, in addition to whether it alters blood pressure in the
conscious state.
Therefore, in Chapter Four we report that systemic CB1 receptor
blockade reduces blood pressure in conscious (mRen2)27 rats. The effect occurs within
90 minutes and is sustained after 24 hours, with evidence of partial recovery to baseline.
There is no evidence that tolerance develops to the blood pressure-lowering effect of
SR141716A as pressure remained reduced to a similar level throughout the duration of
the 28-day study. Furthermore, initial reductions in blood pressure cannot be attributed
to changes in food consumption or other short-term metabolic effects. Finally, we report
that at the end of the chronic treatment period, animals that received the drug had
189
significantly better indices of conscious autonomic control of blood pressure, including
conscious baroreflex sensitivity in response to increases or decreases in blood pressure
and heart rate variability, relative to animals treated with vehicle.
Collectively, these results indicate an important role for the endocannabinoid
system in the maintenance of hypertension in (mRen2)27 rats, contravening the blood
pressure effect of systemic CB1 receptor blockade reported in SHR.35 These conflicting
data may indicate differential roles for the endocannabinoid system in different forms of
hypertension.
For instance, endocannabinoids may play a pathogenic role in the
monogenetic Ang II-dependent hypertension of (mRen2)27 rats, versus a protective or
compensatory role in SHR, whose hypertension is of unknown origin.
It is unclear to what extent the centrally-mediated actions of systemic CB1
blockade contribute to reducing blood pressure in (mRen2)27 rats. Evidence suggests
that the hypertension in this strain may be predominantly mediated by central
mechanisms because intracerebroventricular administration of RAS inhibitors normalizes
blood pressure in these animals.85 Future studies will elucidate the relative contributions
of the central and peripheral endocannabinoid systems in the maintenance of
hypertension in (mRen2)27 rats with the use of peripherally restricted CB1 antagonists.
Furthermore, chronic CB1 receptor blockade produced many beneficial metabolic effects
in (mRen2)27 rats, which may contribute to overall enhanced blood pressure regulation
in the later stages of the study. Therefore, additional timecourse experiments will be
performed to clarify if different mechanisms underlie the immediate and sustained blood
pressure reductions over the duration of the present study. Nevertheless, these data
suggest that CB1 receptor blockade may be beneficial in Ang II-dependent hypertension.
190
5. Chronic CB1 Receptor Blockade in Metabolic Syndrome
Obesity is a major risk factor for hypertension and is closely associated with type 2
diabetes. Increased body fat and insulin resistance, both exhibited by (mRen2)27 rats, 86
are together cardinal symptoms of metabolic syndrome, which also includes
hyperglycemia and dyslipidemia. These features of the (mRen2)27 rat may be driven by
overactivity of the RAS in various tissues, as an activated RAS promotes all
characteristic properties of metabolic syndrome.87
Moreover, RAS inhibitors are
generally associated with positive metabolic effects in humans and animals, including in
(mRen2)27 rats.88-90
Like the RAS, the endocannabinoid system is well-associated with the promotion
and maintenance of metabolic syndrome.
Upregulated CB1 receptor tone is widely
reported in obese or diabetic humans and animals.91,
92
In animals, exogenous
cannabinoid agonists directly promote overeating, fat deposition and dyslipidemia
through CB1 receptor-mediated actions in the CNS, white adipose tissue and liver,
respectively.93 Most importantly, systemic CB1 receptor blockade universally normalizes
or improves metabolic profile in obese humans and animals.94-97 Metabolic effects of
systemic CB1 receptor blockade in (mRen2)27 rats are unknown. However, the similar
beneficial metabolic profile of RAS inhibitors and CB1 receptor antagonists in other
models suggests a shared etiology of metabolic syndrome likely involving the RAS and
endocannabinoid system.
The chronic study paradigm utilized in Chapter Four allows the monitoring of
other parameters that may change only over a longer timeframe.
Accordingly, we
evaluated the effects of chronic systemic CB1 receptor blockade on metabolic profile in
191
(mRen2)27 rats. Over the 28-day treatment period we tracked daily changes in body
weight, weekly changes in blood glucose and food and water intake, and cumulative
differences in fat mass and in circulating insulin and leptin at the end of the study. Based
on the similarities between systemic CB1 receptor and RAS blockade during metabolic
syndrome reported in humans and animals, we hypothesized that the metabolic
parameters we studied in (mRen2)27 rats would improve over the duration of chronic
systemic treatment with a CB1 receptor antagonist.
Data from Chapter Four demonstrate that chronic systemic treatment with
SR141716A improves several indices of metabolic function in (mRen2)27 rats. Drugtreated rats gain significantly less weight over the duration of the study compared to rats
treated with vehicle.
This lower weight gain is not attributable to reduced food
consumption alone because food intake is only transiently reduced, recovering fully
within seven days of treatment. Furthermore, the difference in weight gain is associated
with lower fat mass as a percentage of body weight, as well as lower serum leptin and
insulin levels at the end of the study. Lower insulin content is not associated with
changes in weekly blood glucose levels, which do not change over the duration of
treatment and are identical between treatment groups. Although we did not directly test
leptin and insulin signaling actions, reduced circulating levels coupled with lower body
fat and preserved blood glucose implies enhanced sensitivity of these hormones.
Collectively, these results support our hypothesis that the endocannabinoid system
contributes to increased body mass and insulin resistance in the (mRen2)27 rat model of
Ang II-dependent hypertension.
192
Questions remain regarding the timecourse of treatment and the mechanisms
underlying the beneficial metabolic actions of chronic systemic CB1 receptor blockade in
these animals. For example, it is unclear whether lower insulin and leptin levels in drugtreated rats are a cause or result of decreased weight gain or lower fat mass. In addition,
the contribution of other parameters that we did not examine, including plasma
cholesterol and adiponectin content, and central leptin signaling, to the improved
metabolic profile in drug-treated rats is unknown. Systemic CB1 receptor blockade is
associated with an improved serum lipid profile related to effects in the liver,98 as well as
increased release of adiponectin from white adipose tissue.99, 100 Adiponectin and leptin
both regulate energy homeostasis through actions in the CNS,93,
101, 102
so enhanced
actions of these hormones could certainly contribute to the improved metabolic profile in
(mRen2)27 rats treated with SR141716A.
Furthermore, it is unclear whether these
metabolic effects are mediated predominantly by actions in the CNS or periphery.
Peripherally restricted CB1 receptor antagonists are available, but identification of
distinct central and peripheral roles for the endocannabinoid system in maintaining
energy homeostasis raises questions about the relative effectiveness of these compounds
relative to brain-penetrating agents.103 Future studies will be performed to establish a
timecourse of metabolic events in (mRen2)27 rats during chronic systemic CB1 receptor
blockade, as well as investigate effects on other tissue, hormone and signaling systems
that may be altered by treatment. Peripherally restricted CB1 antagonists will also be
evaluated in (mRen2)27 rats in the future.
In summary, the results from Chapter Four suggest a role for the endocannabinoid
system in the pathogenesis or maintenance of hypertension and metabolic syndrome
193
exhibited by (mRen2)27 rats. The effects of chronic systemic CB1 receptor blockade on
metabolic profile in this strain are fully consistent with the clinical and animal literature.
Combined with the novel finding that systemic CB1 receptor blockade in this strain
significantly reduces blood pressure raises the intriguing possibility that combination
treatment with a RAS blocker may produce additive or synergistic effects in certain
forms of hypertension. Moreover, all of the results obtained from these studies, including
data from Chapters Two and Three, are consistent with an interaction between the RAS
and endocannabinoid system that may be an important mechanism contributing to
hypertension and impaired baroreflex function associated with obesity and aging.
194
6. Interactions between the Endocannabinoid System and the Brain RAS
Baroreflex impairment during hypertension, aging and metabolic syndrome is likely the
result of the disruption of a complex balance of factors within the NTS and surrounding
nuclei mediating cardiovagal reflexes.
Alterations in expression of brain RAS
components are well-associated with these conditions19,
104,
105
and may also
independently contribute to suppressed baroreflex function.106 Other factors are known
to impair baroreflex sensitivity when elevated or altered during hypertension associated
with aging and obesity, such as insulin107 and leptin.108 Functional and biochemical data
from Chapters Two, Three and Four advance support for altered endocannabinoid system
expression or activity as an additional contributing factor to declining baroreflex function
during conditions associated with hypertension.
In (mRen2)27 rats with overactive brain RAS we demonstrate dose-dependent
improvement in baroreflex sensitivity for control of heart rate following NTS
microinjection of a CB1 receptor antagonist, as well as normalization of cardiac vagal
nerve function following chronic systemic CB1 receptor blockade. We also demonstrate
that CB1 receptor blockade in the NTS of ASrAOGEN rats with low brain RAS
expression produces the opposite effect, dose-dependently reducing baroreflex
sensitivity. These results are in contrast to effects of NTS CB1 blockade in young adult
Sprague-Dawley rats, which elicits no change in baroreflex sensitivity.
Opposite
baroreflex-modulating effects of CB1 receptor blockade in the NTS of animals with
differential expression of brain RAS components implies differential expression or
activity of medullary endocannabinoid system components in these animals. Indeed,
mass spectrometry reveals that 2-AG content in the dorsal medulla is highest in
195
(mRen2)27 and aged Sprague-Dawley rats and lowest in ASrAOGEN rats. Furthermore,
real-time PCR reveals lower expression of CB1 receptor mRNA in the dorsal medulla of
ASrAOGEN rats relative to young adult Sprague-Dawley and (mRen2)27 rats. Thus, the
pattern of medullary endocannabinoid expression parallels brain RAS activity across
these strains or ages, and CB1 receptor-mediated tonic control over baroreflex sensitivity
is exhibited when endocannabinoid system components are up- or down-regulated.
Therefore, we must consider potential interactions that are reported between these
systems, which may functionally contribute to central regulation of blood pressure.
Signaling interactions between CB1 and AT1 receptors are demonstrated in
numerous in vitro and in vivo settings. Activity of DAGL, the major enzyme involved in
the biosynthesis of 2-AG in the brain, appears to play a critical role in basal or
constitutive CB1 receptor signaling, because its inhibition reduces basal activity of CB1
receptors while increasing their expression in Chinese hamster ovary cells and cultured
hippocampal neurons.9 Importantly, DAGL activity can be stimulated by Ang II via AT1
receptor activation, and Ang II-induced Gi/o protein activation is blocked by CB1 receptor
blockade and inhibition of DAGL.9
This observation implies a CB1 receptor
transactivation mechanism by Ang II stimulation of DAGL activity and subsequent 2-AG
production, a hypothesis confirmed by mass spectroscopy analyses of 2-AG content in
several cell types following application of Ang II.10 Indeed, transactivation of CB1
receptors may hypothetically occur by stimulation of any Gq/11-coupled receptor,
including AT1 receptors, but also demonstrated by M1, M3, and M5 muscarinic, V1
vasopressin, alpha-1 adrenergic, and B2 bradykinin receptors in recombinant systems.10
However, AT1 receptors co-localize and co-precipitate with upregulated CB1 receptor
196
expression in the CNS and periphery during Ang II-mediated pathologies.12 Moreover,
CB1 receptor antagonists and DAGL inhibition enhanced the vasoconstrictor effect of
Ang II in rat and mouse skeletal muscle arteriole beds, demonstrating a functional in vivo
role for Ang II-stimulated production and release of 2-AG to attenuate vasoconstriction
by Ang II.13
In addition to describing a potential mechanism for the differential
expression of dorsal medullary 2-AG content in (mRen2)27 and ASrAOGEN rats, these
data suggest a much broader role for CB1 receptors in brain paracrine-mediating
functions during activation of Gq/11 proteins by AT1 receptors.
Further
evidence
from
bioluminescence
resonant
energy
transfer
and
immunoprecipitation techniques supports direct physical interactions between CB1 and
AT1 receptors that function to augment Ang II-mediated signaling. As demonstrated by
Rozenfeld and colleagues,12 AT1-CB1 heteromers display antibody specificity and appear
to signal through both Gi/o and Gq/11 proteins to augment endocannabinoid- or Ang IIstimulated mitogenic signaling. Furthermore, antagonists of either CB1 or AT1 receptors
are able to block mitogenic and fibrogenic signaling in response to both CB1 and AT1
receptor stimulation in in vitro and in vivo settings.12 A direct role for this proposed
interaction in maintaining hypertension is illustrated by the observation that elevated
blood pressure caused by microinjection of Ang II into the hypothalamic paraventricular
nucleus is completely prevented by prior microinjection of a CB1 receptor antagonist.109
More recent evidence shows that these interactions modulate further actions in the
periphery, including liver fibrosis12 and gastroprotective effects in the gut,110 in addition
to modulation of vasoreactivity.13 Thus, it is possible that effects of disrupting this
interaction by SR141716A are reflected in data from Chapter Four showing metabolic
197
effects of chronic CB1 receptor blockade in (mRen2)27 rats, especially considering
increased extra-renal tissue expression of the Ren2 transgene in these animals.111
Collectively, there is strong evidence for signaling interactions between CB1 and
AT1 receptors through cross-talk, transactivation and heteromerization mechanisms that
likely contribute to the promotion or maintenance of various pathological conditions
associated with upregulated Ang II or endocannabinoid signaling. The influence of these
interactions over blood pressure regulation in our experiments with transgenic animals is
likely significant based on the range of contexts, including central and peripheral as well
as in vitro and in vivo settings, that these interactions have been previously evaluated.
Although our present studies do not directly investigate potential AT1-CB1 signaling
interactions, our data showing enhanced baroreflex sensitivity and reductions in blood
pressure by CB1 receptor blockade in conditions associated with hypertension are
consistent with disruption of enhanced Ang II-mediated signaling through interactions
between CB1 and AT1 receptors.
Future studies will establish whether interactions
between the endocannabinoid system and brain RAS contribute to impaired baroreflex
function, elevated blood pressure or insulin resistance and obesity during Ang IIdependent hypertension or normal aging. However, certain properties of the transgenic
and aged animals utilized in our studies require us to consider additional, as-yet
unexamined interactions between the endocannabinoid system and brain RAS.
As described above, exogenous Ang-(1-7) in the NTS restores baroreflex
sensitivity in both young adult (mRen2)27 and aged Sprague-Dawley rats,42, 44 but not
blockade of AT1 receptors,43 implicating an absence of tonic Ang-(1-7) input rather than
upregulated Ang II signaling in the NTS underlying impaired baroreflex function in these
198
animals. This further implies a potential role for Ang-(1-7) or mas receptor signaling in
the modulation of endocannabinoid production, perhaps functioning to oppose the
stimulating effect of Ang II on 2-AG production.
Interactions between the
endocannabinoid system and Ang-(1-7) are unexplored to date, but would certainly be
worth investigating as a potential mechanism underlying increased 2-AG production in
the dorsal medulla of (mRen2)27 and aged Sprague-Dawley rats relative to ASrAOGEN
and younger Sprague-Dawley rats, respectively. The CB1 receptor is reported to interact
with a wide variety of G protein-coupled receptors in the brain,112, 113 so the discovery of
an additional interaction with the mas receptor would be an important result.
Another potential interaction between the CB1 receptor and brain RAS during
central blood pressure regulation that has not yet been investigated concerns modulation
of shared downstream intracellular signaling pathways that contribute to baroreflex
function. For example, activation of both CB1 and AT1 receptors is positively linked to
both MAPK- and PI3 kinase-mediated signaling,114-116 which is upregulated in brainstem
nuclei during hypertension117 and contributes to elevated blood pressure and suppressed
baroreflex sensitivity in both SHR118 and (mRen2)27 rats.119
Therefore, future
experiments will determine whether CB1 receptor blockade alters activity of shared
downstream signaling targets of CB1 and AT1 receptors as a potential mechanism of
action underlying modulation of baroreflex sensitivity by the endocannabinoid system.
199
7. Viability of CB1 Receptor Blockade as a Therapeutic Strategy
The results contained in the present studies support CB1 receptor antagonists as having
efficacy in conditions associated with RAS-dependent hypertension, with concomitant
beneficial effects on metabolic profile and autonomic blood pressure regulation. An
entire body of literature suggests that systemic CB1 receptor blockade is an effective
therapy against many disorders, including metabolic syndrome, obesity-related
hypertension, cardiovascular diseases, and various drug dependence syndromes. 120
Indeed, SR141716A was once approved by the European Medicines Agency as an
obesity treatment and was the subject of several Phase III clinical trials in the U.S.
However, it induces significant psychiatric side effects, primarily depression and anxiety,
and was withdrawn from the market in October 2008.121
Despite challenges associated with the endocannabinoid system as a therapeutic
target, interest persists in developing novel agents that block CB1 receptor actions.
Pharmacological treatment options for obesity are limited, while antihypertensive
medications lose their effectiveness in elderly and obese patients and have negative side
effects of their own. Thus, there is an enormous unmet need for drugs that reduce blood
pressure, body weight and associated health problems. New proposals aim to circumvent
or mitigate the negative psychiatric symptoms of central CB1 receptor blockade. Current
strategies include the development of peripherally restricted CB1 antagonists, and a
personalized medicine approach to identify patients with low risk to the adverse effects of
the treatment.122, 123
Several CB1 receptor antagonists that poorly penetrate the blood-brain barrier
show promising results for the improvement of cardiometabolic risk factors.
200
For
example, AM6545, a peripherally restricted analogue of SR141716A with high affinity
and selectivity for the CB1 receptor, improves glucose homeostasis and plasma lipid
profile in mice with diet-induced obesity, without eliciting the behavioral responses of
SR141716A.124, 125 However, functional data from these drugs in humans are lacking,
and effects on cardiovascular functions are not yet known. Furthermore, questions about
the efficacy of peripherally restricted CB1 antagonists are raised by studies showing that
the anti-obesity effects of these drugs are markedly reduced compared to SR141716A.103,
126
Therefore, while blockade of CB1 receptors in the periphery may improve certain
cardiometabolic risk factors without central side effects, it may not be sufficient for a
complete antiobesity or cardioprotective effect.
For patients with unsatisfactory therapeutic responses to peripherally restricted
CB1 antagonists, a brain-penetrating agent may be needed. Another strategy targeting the
endocannabinoid system utilizes techniques from the field of personalized medicine to
identify genetic markers in patients that indicate if they are susceptible to the adverse
psychiatric effects of CB1 receptor antagonists. A meta-analysis of the clinical trials
studying SR141716A in the treatment of cardiometabolic risk found that 26% of
participants reported mood symptoms compared to 13% taking placebo. 127 Therefore,
treatment with SR141716A may be safe and tolerable for a considerable portion of
patients, making it crucial to obtain tools that can identify patients who are at a low risk
of developing negative side effects during CB1 receptor antagonist therapy.
One potential tool to assess patient risk to this treatment is the screening for genes
or gene polymorphisms that are involved in mediating the depression-like and anxiogenic
effects of CB1 receptor antagonists. This requires a more complete understanding of
201
brain endocannabinoid signaling pathways that promote depression and anxiety,
however, several close associations between the CB1 receptor and serotonergic or
metabolic enzyme genes have emerged that may predict susceptibility to anxiety or
depression.122 An additional clinical observation is that adverse effects of SR141716A
treatment are more likely to manifest in patients with a history of depression, which
suggests CB1 receptor blockade is more likely to exacerbate already existing anxiety and
depressive states rather than generate new cases.122, 128 Therefore, a thorough screening
process, including relevant genetic markers and history of psychiatric symptoms, may
identify patients who will benefit most from CB1 receptor antagonist therapy with the
lowest risk of adverse side effects.
An additional strategy for pursuing blockade of the endocannabinoid system as a
pharmacotherapy involves combination treatment with agents that target systems known
to interact with the endocannabinoid system.
For example, SR141716A produces
synergistic effects with an insulin sensitizing agent to improve insulin signaling in
diabetic rats.129
A similar therapeutic effect to either agent alone is found during
combination treatment with lower doses, thus inviting the possibility of reduced side
effects.
Based on data from the present studies, we propose a similar additive or
synergistic effect may exist between CB1 receptor and RAS blockade during treatment of
certain forms of hypertension. During combination treatment with a RAS inhibitor, the
risk of psychiatric side effects of a CB1 receptor antagonist may be further reduced due to
both a lower efficacious dose required as well as the anxiolytic and antidepressant
behavioral profile of Ang II blockade.130,
131
202
Further study of the endocannabinoid
system-RAS interaction during hypertension will be necessary, but we conclude that the
viability of CB1 receptor blockade as a therapeutic strategy once again holds promise.
203
8. General Limitations of Studies
The present studies provide novel insights into the maintenance of baroreflex function
and blood pressure regulation during conditions associated with hypertension. However,
they do not conclusively identify molecular mechanisms underlying central blood
pressure regulation by the endocannabinoid system.
Thus, while these studies
demonstrate a role for the endocannabinoid system in the neural control of the circulation
during conditions associated with hypertension and support our primary hypotheses, they
also generate new hypotheses described throughout this chapter that will drive future
research. Additional limitations that should be considered while interpreting the results
of our present studies include the use of anesthesia, microinjection procedures, and the
transgenic animal models we utilize to investigate our hypotheses.
Anesthesia
All baroreflex measurements were obtained while animals were either under the
influence of urethane-chloralose anesthesia or while conscious after recovering from
surgery performed 48 hours previous under isoflurane anesthesia.
Microinjection studies are performed in the conscious state in many brain
areas,132-134 however, a fixed micropipette and movement of the neck can damage
medullary tissue and eliminate baroreflex responses.
Therefore, the majority of
microinjection experiments at the level of the NTS require the use of anesthesia.
Anesthesia of any kind can significantly influence autonomic nervous system function
and suppress baroreflex sensitivity for control of heart rate.135 Combination urethanechloralose anesthesia is widely used to study neural control of the circulation because it
preserves autonomic function to a greater extent relative to other anesthetics.135
204
Furthermore, there is no overall effect of urethane-chloralose anesthesia on blood
pressure in Sprague-Dawley rats, as demonstrated in the present as well as previous
studies from our laboratory.44,
108
However, under urethane-chloralose anesthesia,
(mRen2)27 and ASrAOGEN rats exhibit altered blood pressure that suggests differential
anesthesia-induced effects on the sympathetic nervous system in these animals. Blood
pressure and heart rate are paradoxically increased in ASrAOGEN rats under urethanechloralose anesthesia, suggesting activation of the sympathetic nervous system by this
anesthetic.15 In contrast, under urethane-chloralose anesthesia, (mRen2)27 rats exhibit
lower blood pressure and heart rate.42
sympathetic nervous system,135,
136
Since urethane inhibits overactivity of the
this observation provides further evidence for
enhanced sympathetic activity in (mRen2)27 rats.
Anesthetics also produce
cardiorespiratory depression,137 so anesthetized animals used during microinjection
experiments were supplemented with a mixture of room air and oxygen to stabilize
ventilation. The same percentage and flow rate of oxygen was used in all microinjection
studies to control for respiratory variability among animals.
Baroreflex sensitivity for control of heart rate is reduced in anesthetized relative
to conscious rats,135 although as illustrated by data from Chapters Two and Three, the
relative level of baroreflex function under anesthesia parallels the trend observed in
conscious animals across strains or age, regardless of anesthesia-induced changes in
blood pressure. For instance, relative to young adult Sprague-Dawley rats, conscious
ASrAOGEN rats have higher, and aged Sprague-Dawley and (mRen2)27 rats have lower
resting baroreflex sensitivity, respectively.18
This pattern of baseline baroreflex
sensitivity is maintained in rats under the influence of urethane-chloralose anesthesia.15,
205
42, 44
Moreover, the relative contributions of Ang II and Ang-(1-7) to baroreflex function
at the level of the NTS are maintained in conscious relative to anesthetized SpragueDawley rats.132 These data, together with the observation that (mRen2)27 rats still
exhibit suppressed baroreflex function despite blood pressure-lowering effects of
anesthesia, confirm that baroreflex sensitivity is modulated independently of changes in
arterial pressure. However, urethane-chloralose anesthesia induces 5-fold increases in
plasma renin activity and circulation of Ang II levels, which may affect autonomic
nervous system activity and cardiovascular function.135
Isoflurane anesthesia was used during catheter surgery for the analysis of
conscious baroreflex function in (mRen2)27 rats in Chapter Four. Similar to combination
urethane-chloralose anesthesia, isoflurane also dampens baroreflex function in rats and
humans,138,
139
and increases circulating Ang II and vasopressin levels in patients.140
However, in humans under isoflurane anesthesia, baroreflex sensitivity for control of
heart rate recovered to a level statistically similar to conscious baseline within 25 minutes
of recovery.139 The recovery time of a minimum of 48 hours allowed to animals prior to
the start of testing is consistent with previous studies in our laboratory.141, 142
Microinjection Procedures
A heterogeneous population of neurons exists within the NTS, including afferent and
efferent neurons and local interneurons.143 Determining which neurons are accessed by
the spread of injectate is a consistent problem for microinjection studies. Since injections
may spread to neuronal cell bodies and presynaptic vagal afferent fibers converging on
the NTS, both of which express CB1 receptors,26 it is unclear which elements mediate
baroreflex responses to the CB1 receptor antagonist used in our studies. The components
206
mediating baroreflex function in response to CB1 receptor activation are currently
unknown.
The spread of the injectate may also access other nuclei in close association with
the NTS including the dmnX and area postrema. Several studies address the spread of
drug microinjections within the NTS. For instance, microinjection of 100 nL of a typical
drug spans a 300 µm radius within the NTS with a concentration gradient radiating from
the pipette tip.143 In addition, the spread of 100 nL of [125I]Sar-Thr-Ang II is limited to
an area mostly confined to within the NTS.144 Even at small volumes it is possible that
the injection may diffuse beyond a single subnucleus within the NTS, potentially
reaching the dmnX. However, functional assessments show that a 50 nL injection of an
AT1 receptor antagonist into the dmnX does not alter baroreflex responses within the
NTS.145 The microinjections performed in our present studies used a volume of 120 nL
and thus should be confined within the NTS.
Furthermore, our demonstrations of
unaltered chemoreflex responses following NTS microinjections of SR141716A indicate
selective actions of the drug on baroreflex modulation.
Transgenic Animal Models
Transgenic rodents with differential expression of RAS components either systemically
or in specific tissue systems are useful models to investigate the role of the RAS to
physiology and pathophysiology. The (mRen2)27 rat is a model used to mimic essential
hypertension in humans as well as being a practical model of the effects of high Ang II
hypertension on end-organ damage. Likewise, the ASrAOGEN rat is a model used to
evaluate the contribution of the glial RAS to various physiological processes. However,
it is generally unknown which systems are affected by the deletion or addition of a
207
transgene over lifespan or during embryonic development. Therefore, the (mRen2)27
and ASrAOGEN transgenic rat models cannot be used to explore the etiology of human
hypertension, and the ability to directly translate our findings to other animal models or
humans may be limited.
The strength of these transgenic models is the ability to determine interactions
between the singular genetic events that initiated under- or overexpression of RAS
components in ASrAOGEN or (mRen2)27 rats, respectively, and the genetic background
against which these interactions occur.111 This is in contrast to other animal models of
hypertension, such as the SHR, in which the undefined genetic origin of hypertension and
the difficulty of comparing these rats to outbred genetic controls results in a broad variety
of effects described in this hypertensive strain. An additional difficulty in the study of
the causes of human hypertension is to distinguish between effects of elevated blood
pressure itself and effects that are secondary to the development of hypertension. The
use of transgenic animals offers the possibility to circumvent this challenge in part,
because the phenotype of the (mRen2)27 and ASrAOGEN rats must originate with the
single genetic change that was introduced.111
Plasma levels of insulin, leptin, IGF-1 and RAS peptides are similar at 16 weeks
of age among Sprague-Dawley, (mRen2)27 and ASrAOGEN rats.
In addition, the
phenotype of ASrAOGEN rats resembles the phenotype of animals treated chronically
with RAS inhibitors beginning at 6 months of age.146 Together, these data suggest that
transgene insertion does not result in developmental abnormalities in (mRen2)27 or
ASrAOGEN rats with respect to the variables studied in our experiments, and that the
brain RAS is responsible for many of the phenotypic qualities of these animals.
208
9. Concluding Remarks
In summary, we demonstrate the following:
1.
Dorsal medullary endocannabinoid production is increased in conditions associated
with hypertension, including imbalances in the brain RAS and during normal aging.
2.
Blockade of CB1 receptors in the NTS improves the parasympathetic baroreflex
sensitivity for control of heart rate in young adult hypertensive rats with an
overactive brain RAS, as well as in older Sprague-Dawley rats that exhibit impaired
baseline baroreflex function.
3.
Acute and chronic systemic blockade of CB1 receptors significantly lowers blood
pressure in hypertensive rats with an overactive brain RAS.
4.
Chronic systemic blockade of CB1 receptors significantly improves indices of
spontaneous autonomic control of blood pressure in RAS-dependent hypertensive
rats.
5.
Chronic systemic blockade of CB1 receptors significantly improves the metabolic
profile of RAS-dependent hypertensive rats.
Restoration of baroreflex sensitivity and blockade of the endocannabinoid system
are novel targets for the treatment of cardiovascular disease and associated risk factors,
including hypertension, aging and obesity.5, 120 Despite controversy regarding the precise
mechanisms underlying the autonomic imbalance observed in conditions associated with
hypertension, the studies in this volume each provide evidence for the participation of the
209
endocannabinoid system in the central dysregulation of blood pressure associated with an
activated Ang II versus Ang-(1-7) system. Furthermore, these studies are consistent with
the novel hypothesis that interactions between the endocannabinoid system and Ang IIAT1 signaling contribute to the pathogenesis of hypertension and its related pathologies.
Therefore, blockade of endocannabinoid signaling through CB1 receptors may be
protective against cardiovascular diseases through a variety of sites and mechanisms.
Whether a direct link can be established among upregulated CB1 receptor and RAS tone,
baroreflex impairment, hypertension and metabolic syndrome is a subject of continued
investigation.
The figure below represents our laboratory’s updated working hypothesis for
modulation of sympathovagal balance at the level of the NTS during conditions
associated with hypertension:
210
To conclude, we propose the following experiments for the future:
1.
Determine if excitatory or inhibitory neurotransmitter content in the NTS is
altered following microinjection of CB1 receptor ligands in rats with altered brain
RAS expression.
2.
Determine if CB1 and AT1 or mas receptors in the NTS share a common signaling
pathway to modulate baroreflex sensitivity.
3.
Determine if chronic systemic blockade of CB1 receptors alters expression of
RAS components in brain nuclei associated with neural control of the circulation.
4.
Determine if combination systemic treatment with a RAS inhibitor and CB1
receptor antagonist produces additive or synergistic effects on blood pressure,
autonomic regulation and metabolic profile in hypertensive (mRen2)27 rats.
5.
Determine if long-term systemic CB1 receptor blockade extends the lifespan of
(mRen2)27 rats.
211
References
1.
Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, Jr.,
Jones DW, Materson BJ, Oparil S, Wright JT, Jr., Roccella EJ. Seventh report of
the Joint National Committee on Prevention, Detection, Evaluation, and
Treatment of High Blood Pressure. Hypertension. 2003;42:1206-1252.
2.
Go AS, Mozaffarian D, Roger VL, et. a. Heart disease and stroke statistics--2014
update: a report from the american heart association. Circulation. 2014;129:e28e292.
3.
Cutler JA, Sorlie PD, Wolz M, Thom T, Fields LE, Roccella EJ. Trends in
hypertension prevalence, awareness, treatment, and control rates in United States
adults between 1988-1994 and 1999-2004. Hypertension. 2008;52:818-827.
4.
Thayer JF, Yamamoto SS, Brosschot JF. The relationship of autonomic
imbalance, heart rate variability and cardiovascular disease risk factors. Int J
Cardiol. 2010;141:122-131.
5.
Thayer JF, Lane RD. The role of vagal function in the risk for cardiovascular
disease and mortality. Biol Psychol. 2007;74:224-242.
6.
Rodriguez de Fonseca F, Del Arco I, Bermudez-Silva FJ, Bilbao A, Cippitelli A,
Navarro M. The endocannabinoid system: physiology and pharmacology. Alcohol
Alcohol. 2005;40:2-14.
7.
Cote M, Matias I, Lemieux I, Petrosino S, Almeras N, Despres JP, Di Marzo V.
Circulating endocannabinoid levels, abdominal adiposity and related
cardiometabolic risk factors in obese men. Int J Obes (Lond). 2007;31:692-699.
8.
Pacher P, Mukhopadhyay P, Mohanraj R, Godlewski G, Batkai S, Kunos G.
Modulation of the endocannabinoid system in cardiovascular disease: therapeutic
potential and limitations. Hypertension. 2008;52:601-607.
9.
Turu G, Simon A, Gyombolai P, Szidonya L, Bagdy G, Lenkei Z, Hunyady L.
The role of diacylglycerol lipase in constitutive and angiotensin AT1 receptorstimulated cannabinoid CB1 receptor activity. J Biol Chem. 2007;282:7753-7757.
10.
Turu G, Varnai P, Gyombolai P, Szidonya L, Offertaler L, Bagdy G, Kunos G,
Hunyady L. Paracrine transactivation of the CB1 cannabinoid receptor by AT1
angiotensin and other Gq/11 protein-coupled receptors. J Biol Chem.
2009;284:16914-16921.
11.
Tiyerili V, Zimmer S, Jung S, Wassmann K, Naehle CP, Lutjohann D, Zimmer A,
Nickenig G, Wassmann S. CB1 receptor inhibition leads to decreased vascular
AT1 receptor expression, inhibition of oxidative stress and improved endothelial
function. Basic Res Cardiol. 2010;105:465-477.
212
12.
Rozenfeld R, Gupta A, Gagnidze K, Lim MP, Gomes I, Lee-Ramos D, Nieto N,
Devi LA. AT1R-CB(1)R heteromerization reveals a new mechanism for the
pathogenic properties of angiotensin II. EMBO J. 2011;30:2350-2363.
13.
Szekeres M, Nadasy GL, Turu G, Soltesz-Katona E, Toth ZE, Balla A, Catt KJ,
Hunyady L. Angiotensin II Induces Vascular Endocannabinoid Release, Which
Attenuates Its Vasoconstrictor Effect via CB1 Cannabinoid Receptors. J Biol
Chem. 2012;287:31540-31550.
14.
Borgonio A, Pummer S, Witte K, Lemmer B. Reduced baroreflex sensitivity and
blunted endogenous nitric oxide synthesis precede the development of
hypertension in TGR(mREN2)27 rats. Chronobiol Int. 2001;18:215-226.
15.
Sakima A, Averill DB, Kasper SO, Jackson L, Ganten D, Ferrario CM, Gallagher
PE, Diz DI. Baroreceptor reflex regulation in anesthetized transgenic rats with
low glia-derived angiotensinogen. Am J Physiol Heart Circ Physiol.
2007;292:H1412-1419.
16.
Pinto E. Blood pressure and ageing. Postgrad Med J. 2007;83:109-114.
17.
Monahan KD. Effect of aging on baroreflex function in humans. Am J Physiol
Regul Integr Comp Physiol. 2007;293:R3-R12.
18.
Diz DI, Varagic J, Groban L. Aging and the brain renin-angiotensin system:
relevance to age-related decline in cardiac function. Future Cardiol. 2008;4:237245.
19.
Arnold AC, Gallagher PE, Diz DI. Brain renin-angiotensin system in the nexus of
hypertension and aging. Hypertens Res. 2013;36:5-13.
20.
Lee MA, Bohm M, Paul M, Bader M, Ganten U, Ganten D. Physiological
characterization of the hypertensive transgenic rat TGR(mREN2)27. Am J
Physiol. 1996;270:E919-929.
21.
Liu AJ, Ma XJ, Shen FM, Liu JG, Chen H, Su DF. Arterial baroreflex: a novel
target for preventing stroke in rat hypertension. Stroke. 2007;38:1916-1923.
22.
Benowitz NL, Jones RT. Cardiovascular effects of prolonged delta-9tetrahydrocannabinol ingestion. Clin Pharmacol Ther. 1975;18:287-297.
23.
Schmid K, Schonlebe J, Drexler H, Mueck-Weymann M. The effects of cannabis
on heart rate variability and well-being in young men. Pharmacopsychiatry.
2010;43:147-150.
24.
Mailleux P, Vanderhaeghen JJ. Distribution of neuronal cannabinoid receptor in
the adult rat brain: a comparative receptor binding radioautography and in situ
hybridization histochemistry. Neuroscience. 1992;48:655-668.
213
25.
Glass M, Dragunow M, Faull RL. Cannabinoid receptors in the human brain: a
detailed anatomical and quantitative autoradiographic study in the fetal, neonatal
and adult human brain. Neuroscience. 1997;77:299-318.
26.
Burdyga G, Varro A, Dimaline R, Thompson DG, Dockray GJ. Expression of
cannabinoid CB1 receptors by vagal afferent neurons: kinetics and role in
influencing neurochemical phenotype. Am J Physiol Gastrointest Liver Physiol.
2010;299:G63-69.
27.
Himmi T, Perrin J, El Ouazzani T, Orsini JC. Neuronal responses to cannabinoid
receptor ligands in the solitary tract nucleus. Eur J Pharmacol. 1998;359:49-54.
28.
Rademacher DJ, Patel S, Hopp FA, Dean C, Hillard CJ, Seagard JL.
Microinjection of a cannabinoid receptor antagonist into the NTS increases
baroreflex duration in dogs. Am J Physiol Heart Circ Physiol. 2003;284:H15701576.
29.
Seagard JL, Dean C, Patel S, Rademacher DJ, Hopp FA, Schmeling WT, Hillard
CJ. Anandamide content and interaction of endocannabinoid/GABA modulatory
effects in the NTS on baroreflex-evoked sympathoinhibition. Am J Physiol Heart
Circ Physiol. 2004;286:H992-1000.
30.
Brozoski DT, Dean C, Hopp FA, Seagard JL. Uptake blockade of
endocannabinoids in the NTS modulates baroreflex-evoked sympathoinhibition.
Brain Res. 2005;1059:197-202.
31.
Brozoski DT, Dean C, Hopp FA, Hillard CJ, Seagard JL. Differential
endocannabinoid regulation of baroreflex-evoked sympathoinhibition in
normotensive versus hypertensive rats. Auton Neurosci. 2009;150:82-93.
32.
Schaich CL, Howlett AC, Diz DI. Dose-Related Biphasic Modulation of
Baroreflex Sensitivity by the CB1 Receptor in Rat Solitary Tract Nucleus.
Hypertension. 2011;58:e105 (Abstract).
33.
Mechoulam R, Parker LA. The endocannabinoid system and the brain. Annu Rev
Psychol. 2013;64:21-47.
34.
Durakoglugil MS, Orer HS. Cannabinoid receptor activation in the nucleus tractus
solitaries produces baroreflex-like responses in the rat. Int J Biomed Sci.
2008;4:229-237.
35.
Batkai S, Pacher P, Osei-Hyiaman D, Radaeva S, Liu J, Harvey-White J,
Offertaler L, Mackie K, Rudd MA, Bukoski RD, Kunos G. Endocannabinoids
acting at cannabinoid-1 receptors regulate cardiovascular function in
hypertension. Circulation. 2004;110:1996-2002.
36.
Sugamura K, Sugiyama S, Nozaki T, Matsuzawa Y, Izumiya Y, Miyata K,
Nakayama M, Kaikita K, Obata T, Takeya M, Ogawa H. Activated
214
endocannabinoid system in coronary artery disease and antiinflammatory effects
of cannabinoid 1 receptor blockade on macrophages. Circulation. 2009;119:2836.
37.
Naito Y, Tsujino T, Akahori H, Ohyanagi M, Mitsuno M, Miyamoto Y,
Masuyama T. Augmented cannabinoid receptors expression in human aortic valve
stenosis. Int J Cardiol. 2010;145:535-537.
38.
Aileru AA, Logan E, Callahan M, Ferrario CM, Ganten D, Diz DI. Alterations in
sympathetic ganglionic transmission in response to angiotensin II in (mRen2)27
transgenic rats. Hypertension. 2004;43:270-275.
39.
Ribeiro JM, Santos RA, Pesquero JB, Bader M, Krieger EM. Autonomic control
in rats with overactivity of tissue renin-angiotensin or kallikrein-kinin system.
Regul Pept. 2005;129:155-159.
40.
Mullins JJ, Peters J, Ganten D. Fulminant hypertension in transgenic rats
harbouring the mouse Ren-2 gene. Nature. 1990;344:541-544.
41.
Hotta H, Uchida S. Aging of the autonomic nervous system and possible
improvements in autonomic activity using somatic afferent stimulation. Geriatr
Gerontol Int. 2010;10 Suppl 1:S127-136.
42.
Diz DI, Garcia-Espinosa MA, Gallagher PE, Ganten D, Ferrario CM, Averill DB.
Angiotensin-(1-7) and baroreflex function in nucleus tractus solitarii of
(mRen2)27 transgenic rats. J Cardiovasc Pharmacol. 2008;51:542-548.
43.
Isa K, Arnold AC, Westwood BM, Chappell MC, Diz DI. Angiotensin-converting
enzyme inhibition, but not AT(1) receptor blockade, in the solitary tract nucleus
improves baroreflex sensitivity in anesthetized transgenic hypertensive
(mRen2)27 rats. Hypertens Res. 2011;34:1257-1262.
44.
Sakima A, Averill DB, Gallagher PE, Kasper SO, Tommasi EN, Ferrario CM, Diz
DI. Impaired heart rate baroreflex in older rats: role of endogenous angiotensin(1-7) at the nucleus tractus solitarii. Hypertension. 2005;46:333-340.
45.
Arnold AC, Sakima A, Ganten D, Ferrario CM, Diz DI. Modulation of reflex
function by endogenous angiotensins in older transgenic rats with low glial
angiotensinogen. Hypertension. 2008;51:1326-1331.
46.
Wheal AJ, Bennett T, Randall MD, Gardiner SM. Cardiovascular effects of
cannabinoids in conscious spontaneously hypertensive rats. Br J Pharmacol.
2007;152:717-724.
47.
Arnold AC, Shaltout HA, Gilliam-Davis S, Kock ND, Diz DI. Autonomic control
of the heart is altered in Sprague-Dawley rats with spontaneous hydronephrosis.
Am J Physiol Heart Circ Physiol. 2011;300:H2206-2213.
215
48.
Kano M, Ohno-Shosaku T, Hashimotodani Y, Uchigashima M, Watanabe M.
Endocannabinoid-mediated control of synaptic transmission. Physiol Rev.
2009;89:309-380.
49.
Colombari E, Bonagamba LG, Machado BH. Mechanisms of pressor and
bradycardic responses to L-glutamate microinjected into the NTS of conscious
rats. Am J Physiol. 1994;266:R730-738.
50.
Ohta H, Talman WT. Both NMDA and non-NMDA receptors in the NTS
participate in the baroreceptor reflex in rats. Am J Physiol. 1994;267:R1065-1070.
51.
Chen CY, Bonham AC. Non-NMDA and NMDA receptors transmit area
postrema input to aortic baroreceptor neurons in NTS. Am J Physiol.
1998;275:H1695-1706.
52.
Aylwin ML, Horowitz JM, Bonham AC. Non-NMDA and NMDA receptors in
the synaptic pathway between area postrema and nucleus tractus solitarius. Am J
Physiol. 1998;275:H1236-1246.
53.
Bonham AC, Chen CY. Glutamatergic neural transmission in the nucleus tractus
solitarius: N-methyl-D-aspartate receptors. Clin Exp Pharmacol Physiol.
2002;29:497-502.
54.
Weston M, Wang H, Stornetta RL, Sevigny CP, Guyenet PG. Fos expression by
glutamatergic neurons of the solitary tract nucleus after phenylephrine-induced
hypertension in rats. J Comp Neurol. 2003;460:525-541.
55.
Chen CY, Bonham AC. Glutamate suppresses GABA release via presynaptic
metabotropic glutamate receptors at baroreceptor neurones in rats. J Physiol.
2005;562:535-551.
56.
Sartor DM, Verberne AJ. The role of NMDA and non-NMDA receptors in the
NTS in mediating three distinct sympathoinhibitory reflexes. Naunyn
Schmiedebergs Arch Pharmacol. 2007;376:241-252.
57.
Hildreth CM, Goodchild AK. Role of ionotropic GABA, glutamate and glycine
receptors in the tonic and reflex control of cardiac vagal outflow in the rat. BMC
Neurosci. 2010;11:128.
58.
Len WB, Chan JY. Rostral ventrolateral medulla suppresses reflex bradycardia by
the release of gamma-aminobutyric acid in nucleus tractus solitarii of the rat.
Synapse. 2001;39:23-31.
59.
Zubcevic J, Potts JT. Role of GABAergic neurones in the nucleus tractus solitarii
in modulation of cardiovascular activity. Exp Physiol. 2010;95:909-918.
216
60.
Callera JC, Bonagamba LG, Nosjean A, Laguzzi R, Machado BH. Activation of
GABA receptors in the NTS of awake rats reduces the gain of baroreflex
bradycardia. Auton Neurosci. 2000;84:58-67.
61.
Mei L, Zhang J, Mifflin S. Hypertension alters GABA receptor-mediated
inhibition of neurons in the nucleus of the solitary tract. Am J Physiol Regul
Integr Comp Physiol. 2003;285:R1276-1286.
62.
Catelli JM, Sved AF. Enhanced pressor response to GABA in the nucleus tractus
solitarii of the spontaneously hypertensive rat. Eur J Pharmacol. 1988;151:243248.
63.
Moreira TS, Takakura AC, Colombari E. Important GABAergic mechanism
within the NTS and the control of sympathetic baroreflex in SHR. Auton
Neurosci. 2011;159:62-70.
64.
Yao F, Sumners C, O'Rourke ST, Sun C. Angiotensin II increases GABAB
receptor expression in nucleus tractus solitarii of rats. Am J Physiol Heart Circ
Physiol. 2008;294:H2712-2720.
65.
Zhang Q, Yao F, O'Rourke ST, Qian SY, Sun C. Angiotensin II enhances
GABA(B) receptor-mediated responses and expression in nucleus tractus solitarii
of rats. Am J Physiol Heart Circ Physiol. 2009;297:H1837-1844.
66.
Garcia-Espinosa MA, Shaltout HA, Olson J, Westwood BM, Robbins ME, Link
K, Diz DI. Proton magnetic resonance spectroscopy detection of
neurotransmitters in dorsomedial medulla correlate with spontaneous baroreceptor
reflex function. Hypertension. 2010;55:487-493.
67.
Matsuo I, Hirooka Y, Hironaga K, Eshima K, Shigematsu H, Shihara M, Sakai K,
Takeshita A. Glutamate release via NO production evoked by NMDA in the NTS
enhances hypotension and bradycardia in vivo. Am J Physiol Regul Integr Comp
Physiol. 2001;280:R1285-1291.
68.
Gomes da Silva AQ, Xavier CH, Campagnole-Santos MJ, Caligiorne SM, Baltatu
OC, Bader M, Santos RA, Fontes MA. Cardiovascular responses evoked by
activation or blockade of GABA(A) receptors in the hypothalamic PVN are
attenuated in transgenic rats with low brain angiotensinogen. Brain Res.
2012;1448:101-110.
69.
Chen CY, Bonham AC, Dean C, Hopp FA, Hillard CJ, Seagard JL. Retrograde
release of endocannabinoids inhibits presynaptic GABA release to second-order
baroreceptive neurons in NTS. Auton Neurosci. 2010;158:44-50.
70.
Derbenev AV, Stuart TC, Smith BN. Cannabinoids suppress synaptic input to
neurones of the rat dorsal motor nucleus of the vagus nerve. J Physiol.
2004;559:923-938.
217
71.
Zygmunt PM, Petersson J, Andersson DA, Chuang H, Sorgard M, Di Marzo V,
Julius D, Hogestatt ED. Vanilloid receptors on sensory nerves mediate the
vasodilator action of anandamide. Nature. 1999;400:452-457.
72.
Zsombok A, Gao H, Miyata K, Issa A, Derbenev AV. Immunohistochemical
localization of transient receptor potential vanilloid type 1 and insulin receptor
substrate 2 and their co-localization with liver-related neurons in the
hypothalamus and brainstem. Brain Res. 2011;1398:30-39.
73.
Peters JH, McDougall SJ, Fawley JA, Smith SM, Andresen MC. Primary afferent
activation of thermosensitive TRPV1 triggers asynchronous glutamate release at
central neurons. Neuron. 2010;65:657-669.
74.
Shoudai K, Peters JH, McDougall SJ, Fawley JA, Andresen MC. Thermally
active TRPV1 tonically drives central spontaneous glutamate release. J Neurosci.
2010;30:14470-14475.
75.
Andresen MC, Fawley JA, Hofmann ME. Peptide and lipid modulation of
glutamatergic afferent synaptic transmission in the solitary tract nucleus. Front
Neurosci. 2012;6:191.
76.
Derbenev AV, Monroe MJ, Glatzer NR, Smith BN. Vanilloid-mediated
heterosynaptic facilitation of inhibitory synaptic input to neurons of the rat dorsal
motor nucleus of the vagus. J Neurosci. 2006;26:9666-9672.
77.
Anwar IJ, Derbenev AV. TRPV1-dependent regulation of synaptic activity in the
mouse dorsal motor nucleus of the vagus nerve. Front Neurosci. 2013;7:238.
78.
Wang W, Cao X, Liu C, Liu L. Cannabinoid WIN 55,212-2 inhibits TRPV1 in
trigeminal ganglion neurons via PKA and PKC pathways. Neurol Sci.
2012;33:79-85.
79.
Rinaldi-Carmona M, Barth F, Heaulme M, Shire D, Calandra B, Congy C,
Martinez S, Maruani J, Neliat G, Caput D, et al. SR141716A, a potent and
selective antagonist of the brain cannabinoid receptor. FEBS Lett. 1994;350:240244.
80.
Rinaldi-Carmona M, Barth F, Heaulme M, Alonso R, Shire D, Congy C, Soubrie
P, Breliere JC, Le Fur G. Biochemical and pharmacological characterisation of
SR141716A, the first potent and selective brain cannabinoid receptor antagonist.
Life Sci. 1995;56:1941-1947.
81.
Varga K, Lake K, Martin BR, Kunos G. Novel antagonist implicates the CB1
cannabinoid receptor in the hypotensive action of anandamide. Eur J Pharmacol.
1995;278:279-283.
82.
Wagner JA, Varga K, Jarai Z, Kunos G. Mesenteric vasodilation mediated by
endothelial anandamide receptors. Hypertension. 1999;33:429-434.
218
83.
Lake KD, Martin BR, Kunos G, Varga K. Cardiovascular effects of anandamide
in anesthetized and conscious normotensive and hypertensive rats. Hypertension.
1997;29:1204-1210.
84.
Ho WS, Gardiner SM. Acute hypertension reveals depressor and vasodilator
effects of cannabinoids in conscious rats. Br J Pharmacol. 2009;156:94-104.
85.
Szczepanska-Sadowska E, Paczwa P, Lon S, Ganten D. Increased pressor
function of central vasopressinergic system in hypertensive renin transgenic rats.
J Hypertens. 1998;16:1505-1514.
86.
Kasper SO, Carter CS, Ferrario CM, Ganten D, Ferder LF, Sonntag WE,
Gallagher PE, Diz DI. Growth, metabolism, and blood pressure disturbances
during aging in transgenic rats with altered brain renin-angiotensin systems.
Physiol Genomics. 2005;23:311-317.
87.
Putnam K, Shoemaker R, Yiannikouris F, Cassis LA. The renin-angiotensin
system: a target of and contributor to dyslipidemias, altered glucose homeostasis,
and hypertension of the metabolic syndrome. Am J Physiol Heart Circ Physiol.
2012;302:H1219-1230.
88.
Bramlage P, Schonrock E, Odoj P. Metabolic effects of an AT1-receptor blockade
combined with HCTZ in cardiac risk patients: a non interventional study in
primary care. BMC Cardiovasc Disord. 2008;8:30.
89.
Janiak P, Bidouard JP, Cadrouvele C, Poirier B, Gouraud L, Grataloup Y, Pierre
F, Bruneval P, O'Connor SE, Herbert JM. Long-term blockade of angiotensin
AT1 receptors increases survival of obese Zucker rats. Eur J Pharmacol.
2006;534:271-279.
90.
Sloniger JA, Saengsirisuwan V, Diehl CJ, Kim JS, Henriksen EJ. Selective
angiotensin II receptor antagonism enhances whole-body insulin sensitivity and
muscle glucose transport in hypertensive TG(mREN2)27 rats. Metabolism.
2005;54:1659-1668.
91.
Engeli S, Bohnke J, Feldpausch M, Gorzelniak K, Janke J, Batkai S, Pacher P,
Harvey-White J, Luft FC, Sharma AM, Jordan J. Activation of the peripheral
endocannabinoid system in human obesity. Diabetes. 2005;54:2838-2843.
92.
Kunos G, Osei-Hyiaman D, Liu J, Godlewski G, Batkai S. Endocannabinoids and
the control of energy homeostasis. J Biol Chem. 2008;283:33021-33025.
93.
Cota D, Marsicano G, Tschop M, Grubler Y, Flachskamm C, Schubert M, Auer
D, Yassouridis A, Thone-Reineke C, Ortmann S, Tomassoni F, Cervino C, Nisoli
E, Linthorst AC, Pasquali R, Lutz B, Stalla GK, Pagotto U. The endogenous
cannabinoid system affects energy balance via central orexigenic drive and
peripheral lipogenesis. J Clin Invest. 2003;112:423-431.
219
94.
Vickers SP, Webster LJ, Wyatt A, Dourish CT, Kennett GA. Preferential effects
of the cannabinoid CB1 receptor antagonist, SR 141716, on food intake and body
weight gain of obese (fa/fa) compared to lean Zucker rats. Psychopharmacology
(Berl). 2003;167:103-111.
95.
Janiak P, Poirier B, Bidouard JP, Cadrouvele C, Pierre F, Gouraud L, Barbosa I,
Dedio J, Maffrand JP, Le Fur G, O'Connor S, Herbert JM. Blockade of
cannabinoid CB1 receptors improves renal function, metabolic profile, and
increased survival of obese Zucker rats. Kidney Int. 2007;72:1345-1357.
96.
Poirier B, Bidouard JP, Cadrouvele C, Marniquet X, Staels B, O'Connor SE,
Janiak P, Herbert JM. The anti-obesity effect of rimonabant is associated with an
improved serum lipid profile. Diabetes Obes Metab. 2005;7:65-72.
97.
Scheen AJ. CB1 receptor blockade and its impact on cardiometabolic risk factors:
overview of the RIO programme with rimonabant. J Neuroendocrinol. 2008;20
Suppl 1:139-146.
98.
Osei-Hyiaman D, Liu J, Zhou L, Godlewski G, Harvey-White J, Jeong WI, Batkai
S, Marsicano G, Lutz B, Buettner C, Kunos G. Hepatic CB1 receptor is required
for development of diet-induced steatosis, dyslipidemia, and insulin and leptin
resistance in mice. J Clin Invest. 2008;118:3160-3169.
99.
Bensaid M, Gary-Bobo M, Esclangon A, Maffrand JP, Le Fur G, Oury-Donat F,
Soubrie P. The cannabinoid CB1 receptor antagonist SR141716 increases Acrp30
mRNA expression in adipose tissue of obese fa/fa rats and in cultured adipocyte
cells. Mol Pharmacol. 2003;63:908-914.
100.
Despres JP, Golay A, Sjostrom L. Effects of rimonabant on metabolic risk factors
in overweight patients with dyslipidemia. N Engl J Med. 2005;353:2121-2134.
101.
Di Marzo V, Goparaju SK, Wang L, Liu J, Batkai S, Jarai Z, Fezza F, Miura GI,
Palmiter RD, Sugiura T, Kunos G. Leptin-regulated endocannabinoids are
involved in maintaining food intake. Nature. 2001;410:822-825.
102.
Nedvidkova J, Smitka K, Kopsky V, Hainer V. Adiponectin, an adipocyte-derived
protein. Physiol Res. 2005;54:133-140.
103.
Pavon FJ, Serrano A, Perez-Valero V, Jagerovic N, Hernandez-Folgado L,
Bermudez-Silva FJ, Macias M, Goya P, de Fonseca FR. Central versus peripheral
antagonism of cannabinoid CB1 receptor in obesity: effects of LH-21, a
peripherally acting neutral cannabinoid receptor antagonist, in Zucker rats. J
Neuroendocrinol. 2008;20 Suppl 1:116-123.
104.
Ruiz P, Basso N, Cannata MA, Taquini AC. The renin-angiotensin system in
different stages of spontaneous hypertension in the rat (S H R). Clin Exp
Hypertens A. 1990;12:63-81.
220
105.
de Kloet AD, Pioquinto DJ, Nguyen D, Wang L, Smith JA, Hiller H, Sumners C.
Obesity induces neuroinflammation mediated by altered expression of the reninangiotensin system in mouse forebrain nuclei. Physiol Behav. 2014
106.
Arnold AC, Okamoto LE, Gamboa A, Shibao C, Raj SR, Robertson D, Biaggioni
I. Angiotensin II, independent of plasma renin activity, contributes to the
hypertension of autonomic failure. Hypertension. 2013;61:701-706.
107.
Ryan JP, Sheu LK, Verstynen TD, Onyewuenyi IC, Gianaros PJ. Cerebral blood
flow links insulin resistance and baroreflex sensitivity. PLoS One. 2013;8:e83288.
108.
Arnold AC, Shaltout HA, Gallagher PE, Diz DI. Leptin impairs cardiovagal
baroreflex function at the level of the solitary tract nucleus. Hypertension.
2009;54:1001-1008.
109.
Gyombolai P, Pap D, Turu G, Catt KJ, Bagdy G, Hunyady L. Regulation of
endocannabinoid release by G proteins: a paracrine mechanism of G proteincoupled receptor action. Mol Cell Endocrinol. 2012;353:29-36.
110.
Gyires K, Ronai AZ, Zadori ZS, Toth VE, Nemeth J, Szekeres M, Hunyady L.
Angiotensin II-induced activation of central AT1 receptors exerts
endocannabinoid-mediated gastroprotective effect in rats. Mol Cell Endocrinol.
2014;382:971-978.
111.
Engler S, Paul M, Pinto YM. The TGR(mRen2)27 transgenic rat model of
hypertension. Regul Pept. 1998;77:3-8.
112.
Wager-Miller J, Westenbroek R, Mackie K. Dimerization of G protein-coupled
receptors: CB1 cannabinoid receptors as an example. Chem Phys Lipids.
2002;121:83-89.
113.
Mackie K. Cannabinoid receptor homo- and heterodimerization. Life Sci.
2005;77:1667-1673.
114.
Kanagasundaram V, Jaworowski A, Hamilton JA. Association between
phosphatidylinositol-3 kinase, Cbl and other tyrosine phosphorylated proteins in
colony-stimulating factor-1-stimulated macrophages. Biochem J. 1996;320 ( Pt
1):69-77.
115.
Saward L, Zahradka P. Angiotensin II activates phosphatidylinositol 3-kinase in
vascular smooth muscle cells. Circ Res. 1997;81:249-257.
116.
Gomez del Pulgar T, Velasco G, Guzman M. The CB1 cannabinoid receptor is
coupled to the activation of protein kinase B/Akt. Biochem J. 2000;347:369-373.
117.
Reja V, Goodchild AK, Phillips JK, Pilowsky PM. Upregulation of angiotensin
AT1 receptor and intracellular kinase gene expression in hypertensive rats. Clin
Exp Pharmacol Physiol. 2006;33:690-695.
221
118.
Sun C, Zubcevic J, Polson JW, Potts JT, Diez-Freire C, Zhang Q, Paton JF,
Raizada MK. Shift to an involvement of phosphatidylinositol 3-kinase in
angiotensin II actions on nucleus tractus solitarii neurons of the spontaneously
hypertensive rat. Circ Res. 2009;105:1248-1255.
119.
Logan EM, Aileru AA, Shaltout HA, Averill DB, Diz DI. The functional role of
PI3K in maintenance of blood pressure and baroreflex suppression in (mRen2)27
and mRen2.Lewis rat. J Cardiovasc Pharmacol. 2011;58:367-373.
120.
Pacher P, Batkai S, Kunos G. The endocannabinoid system as an emerging target
of pharmacotherapy. Pharmacol Rev. 2006;58:389-462.
121.
Sam AH, Salem V, Ghatei MA. Rimonabant: From RIO to Ban. J Obes.
2011;2011:432607.
122.
Lazary J, Juhasz G, Hunyady L, Bagdy G. Personalized medicine can pave the
way for the safe use of CB(1) receptor antagonists. Trends Pharmacol Sci.
2011;32:270-280.
123.
Kirilly E, Gonda X, Bagdy G. CB1 receptor antagonists: new discoveries leading
to new perspectives. Acta Physiol (Oxf). 2012;205:41-60.
124.
Tam J, Vemuri VK, Liu J, Batkai S, Mukhopadhyay B, Godlewski G, OseiHyiaman D, Ohnuma S, Ambudkar SV, Pickel J, Makriyannis A, Kunos G.
Peripheral CB1 cannabinoid receptor blockade improves cardiometabolic risk in
mouse models of obesity. J Clin Invest. 2010;120:2953-2966.
125.
Cluny NL, Vemuri VK, Chambers AP, Limebeer CL, Bedard H, Wood JT, Lutz
B, Zimmer A, Parker LA, Makriyannis A, Sharkey KA. A novel peripherally
restricted cannabinoid receptor antagonist, AM6545, reduces food intake and
body weight, but does not cause malaise, in rodents. Br J Pharmacol.
2010;161:629-642.
126.
Son MH, Kim HD, Chae YN, Kim MK, Shin CY, Ahn GJ, Choi SH, Yang EK,
Park KJ, Chae HW, Moon HS, Kim SH, Shin YG, Yoon SH. Peripherally acting
CB1-receptor antagonist: the relative importance of central and peripheral CB1
receptors in adiposity control. Int J Obes (Lond). 2010;34:547-556.
127.
Mitchell PB, Morris MJ. Depression and anxiety with rimonabant. Lancet.
2007;370:1671-1672.
128.
Moreira FA, Crippa JA. The psychiatric side-effects of rimonabant. Rev Bras
Psiquiatr. 2009;31:145-153.
129.
Literati-Nagy Z, Tory K, Literati-Nagy B, Bajza A, Vigh L, Jr., Vigh L, Mandl J,
Szilvassy Z. Synergic insulin sensitizing effect of rimonabant and BGP-15 in
Zucker-obese rats. Pathol Oncol Res. 2013;19:571-575.
222
130.
Pavel J, Benicky J, Murakami Y, Sanchez-Lemus E, Saavedra JM. Peripherally
administered angiotensin II AT1 receptor antagonists are anti-stress compounds in
vivo. Ann N Y Acad Sci. 2008;1148:360-366.
131.
Kangussu LM, Almeida-Santos AF, Bader M, Alenina N, Fontes MA, Santos RA,
Aguiar DC, Campagnole-Santos MJ. Angiotensin-(1-7) attenuates the anxiety and
depression-like behaviors in transgenic rats with low brain angiotensinogen.
Behav Brain Res. 2013;257:25-30.
132.
Michelini LC, Bonagamba LG. Angiotensin II as a modulator of baroreceptor
reflexes in the brainstem of conscious rats. Hypertension. 1990;15:I45-50.
133.
Mayorov DN, Head GA. Influence of rostral ventrolateral medulla on renal
sympathetic baroreflex in conscious rabbits. Am J Physiol Regul Integr Comp
Physiol. 2001;280:R577-587.
134.
Shaltout HA, Abdel-Rahman AA. Mechanism of fatty acids induced suppression
of cardiovascular reflexes in rats. J Pharmacol Exp Ther. 2005;314:1328-1337.
135.
Shimokawa A, Kunitake T, Takasaki M, Kannan H. Differential effects of
anesthetics on sympathetic nerve activity and arterial baroreceptor reflex in
chronically instrumented rats. J Auton Nerv Syst. 1998;72:46-54.
136.
Shimokawa A, Jin QH, Ishizuka Y, Kunitake T, Takasaki M, Kannan H. Effects
of anesthetics on norepinephrine release in the hypothalamic paraventricular
nucleus region of awake rats. Neurosci Lett. 1998;244:21-24.
137.
Sear JW. Practical treatment recommendations for the safe use of anaesthetics.
Drugs. 1992;43:54-68.
138.
Seagard JL, Elegbe EO, Hopp FA, Bosnjak ZJ, von Colditz JH, Kalbfleisch JH,
Kampine JP. Effects of isoflurane on the baroreceptor reflex. Anesthesiology.
1983;59:511-520.
139.
Takeshima R, Dohi S. Comparison of arterial baroreflex function in humans
anesthetized with enflurane or isoflurane. Anesth Analg. 1989;69:284-290.
140.
Goldmann A, Hoehne C, Fritz GA, Unger J, Ahlers O, Nachtigall I, Boemke W.
Combined vs. Isoflurane/Fentanyl anesthesia for major abdominal surgery:
Effects on hormones and hemodynamics. Med Sci Monit. 2008;14:CR445-452.
141.
Nautiyal M, Katakam PV, Busija DW, Gallagher PE, Tallant EA, Chappell MC,
Diz DI. Differences in oxidative stress status and expression of MKP-1 in dorsal
medulla of transgenic rats with altered brain renin-angiotensin system. Am J
Physiol Regul Integr Comp Physiol. 2012;303:R799-806.
223
142.
Nautiyal M, Shaltout HA, de Lima DC, do Nascimento K, Chappell MC, Diz DI.
Central angiotensin-(1-7) improves vagal function independent of blood pressure
in hypertensive (mRen2)27 rats. Hypertension. 2012;60:1257-1265.
143.
Andresen MC, Kunze DL. Nucleus tractus solitarius--gateway to neural
circulatory control. Annu Rev Physiol. 1994;56:93-116.
144.
Campagnole-Santos MJ, Diz DI, Ferrario CM. Baroreceptor reflex modulation by
angiotensin II at the nucleus tractus solitarii. Hypertension. 1988;11:I167-171.
145.
Fow JE, Averill DB, Barnes KL. Mechanisms of angiotensin-induced hypotension
and bradycardia in the medial solitary tract nucleus. Am J Physiol.
1994;267:H259-266.
146.
Gilliam-Davis S, Payne VS, Kasper SO, Tommasi EN, Robbins ME, Diz DI.
Long-term AT1 receptor blockade improves metabolic function and provides
renoprotection in Fischer-344 rats. Am J Physiol Heart Circ Physiol.
2007;293:H1327-1333.
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SCHOLASTIC VITA
CHRISTOPHER LEE SCHAICH
EDUCATION
Ph.D., Integrative Physiology and Pharmacology (2014)
Certificate in College Level Teaching
Wake Forest University, Winston-Salem, North Carolina, USA
Advisor:
Dissertation title:
Debra I. Diz, Ph.D.
Blood Pressure Regulation by the Endocannabinoid System in
Conditions Associated with Hypertension
B.A., Psychology, Magna Cum Laude, With Distinction (2006)
University of Connecticut, Storrs, CT
Minors in Neuroscience and Ecology & Evolutionary Biology
PROFESSIONAL EXPERIENCE
2009 – Present: Wake Forest School of Medicine, Winston-Salem, NC
Graduate Student; Predoctoral Fellow
Department of Physiology and Pharmacology; and Hypertension & Vascular
Research Center
2007 – 2009: Lundbeck Research USA, Inc., Paramus, NJ
Neuroscience Department – Functional Neuroanatomy group
Research Associate
2006 – 2007: PsychoGenics, Inc., Tarrytown, NY
Spinal Muscular Atrophy (SMA) project
Research Associate
2004 – 2006: Neurobiology of Learning and Memory Lab, Storrs, CT
Research Assistant
University of Connecticut, Department of Psychology
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Summer 2005: Behavioral Healthcare Options, Inc., Las Vegas, NV
Behavioral Health and Detoxification Clinic
Clinical Intern, Longford Medical Center
MANUSCRIPTS
1.
Schaich CL, Grabenauer M, Thomas BF, Gallagher PE, Howlett AC, Diz DI.
Medullary Endocannabinoids Contribute to the Differential Resting
Baroreflex Sensitivity in Rats with Altered Brain Renin-Angiotensin System
Expression. In revision to Hypertension.
2.
Schaich CL, Shaltout HA, Brosnihan KB, Howlett AC, Diz DI. Acute and
Chronic Systemic CB1 Cannabinoid Receptor Blockade Improves Blood
Pressure Regulation and Metabolic Profile in Hypertensive (mRen2)27 Rats.
Submitted to Journal of Pharmacology and Experimental Therapeutics. (Under
review).
3.
Schaich CL, Grabenauer M, Thomas BF, Gallagher PE, Howlett AC, Diz DI.
Alterations in the Medullary Endocannabinoid System Contribute to AgeRelated Impairment of Baroreflex Sensitivity. Submitted to Hypertension.
(Under review).
4.
Schaich CL, Howlett AC, Diz DI. Integrative Modulation of Blood Pressure
Regulation and Metabolic Profile by the Endocannabinoid System. Review in
preparation; expected submission April 2014.
PUBLISHED ABSTRACTS
1.
Chris L. Schaich, Brian F. Thomas, Megan A. Grabenauer, Allyn C. Howlett,
Debra I. Diz; Blockade of CB1 Receptors Reveals that Elevated Medullary
Endocannabinoids Contribute to Age-Related Impairment of Baroreflex
Sensitivity in Sprague-Dawley Rats. The FASEB Journal 28:681.4, 2014.
2.
Chris L. Schaich, Allyn C. Howlett, K. Bridget Brosnihan, Debra I. Diz;
Differential Blood Pressure Response in Normotensive Rats and Rats with
Angiotensin II-Dependent Hypertension Following Systemic CB1
Cannabinoid Receptor Blockade. The FASEB Journal 28:1140.11, 2014.
3.
Chris L. Schaich, Allyn C. Howlett, Brian F. Thomas, Megan A. Grabenauer,
Debra I. Diz; Medullary Endocannabinoid Content Contributes to the
Differential Resting Baroreflex Sensitivity in Rats with Altered Brain ReninAngiotensin System Expression. Hypertension 62:A182, 2013.
226
4.
Chris L. Schaich, Hossam A. Shaltout, Allyn C. Howlett, Debra I. Diz; Chronic
Systemic CB1 Receptor Blockade Reduces Blood Pressure and Weight Gain
and Improves Baroreflex Function and Heart Rate Variability in
Hypertensive (mRen2)27 Rats. Hypertension 62:A183, 2013.
5.
Chris L. Schaich, Allyn C. Howlett, Mark C. Chappell, Manisha M. Nautiyal,
Debra I. Diz; Enhanced CB1 Cannabinoid Receptor Tone Contributes to
Impaired Baroreflex Sensitivity in Hypertensive (mRen2)27 Transgenic Rats.
The FASEB Journal 27:926.13, 2013.
6.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz; Contribution of
Endocannabinoids via CB1 Receptors to the Impaired Baroreflex Sensitivity
in (mRen2)27 Hypertensive Rats. Hypertension 60:A117, 2012.
7.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz; Dose-Related Biphasic
Modulation of Baroreflex Sensitivity by the CB1 Receptor in Rat Solitary
Tract Nucleus. Hypertension 58:e105, 2011.
8.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz. Attenuation of Baroreflex
Sensitivity by Cannabinoid Receptor Activation in Rat Solitary Tract
Nucleus. Hypertension 56:e106, 2010.
SPEAKING INVITATIONS AND ORAL PRESENTATIONS
1.
Chris L. Schaich, Brian F. Thomas, Megan A. Grabenauer, Allyn C. Howlett,
Debra I. Diz; Blockade of CB1 Receptors Reveals that Elevated Medullary
Endocannabinoids Contribute to Age-Related Impairment of Baroreflex
Sensitivity in Sprague-Dawley Rats. American Physiological Society Data
NCARnation, Experimental Biology 2014. San Diego, CA. April 26, 2014.
2.
Chris L. Schaich; Chronic Systemic CB1 Cannabinoid Receptor Blockade
Reduces Blood Pressure and Improves Baroreflex Function in Angiotensin
II-Dependent Hypertension. Department of Physiology and Pharmacology
Seminar Series, Wake Forest University School of Medicine. Winston-Salem,
NC. October 14, 2013.
3.
Chris L. Schaich, Allyn C. Howlett, Brian F. Thomas, Megan A. Grabenauer,
Debra I. Diz; Chronic Systemic CB1 Receptor Blockade Reduces Weight Gain
and Lowers Blood Pressure in Hypertensive (mRen2)27 Rats. 23rd Annual
Symposium on the Cannabinoids, International Cannabinoid Research Society,
Abstract #44. Vancouver, BC. June 22, 2013.
4.
Chris L. Schaich; Endocannabinoid Modulation of Baroreflex Sensitivity and
Blood Pressure in Renin-Angiotensin System-Dependent Hypertension.
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Department of Physiology and Pharmacology Seminar Series, Wake Forest
University School of Medicine. Winston-Salem, NC. March 11, 2013.
5.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz; Contribution of
Endocannabinoids to the Impaired Baroreflex Sensitivity in (mRen2)27
Hypertensive Rats. Presented at Carolina Cannabinoid Collaborative. Greenville,
NC. October 20, 2012.
6.
Chris L. Schaich; Modulation of Baroreflex Sensitivity by the CB1
Cannabinoid Receptor in Normotensive and Hypertensive Rats. Department
of Physiology and Pharmacology Seminar Series, Wake Forest University School
of Medicine. Winston-Salem, NC. December 12, 2011.
7.
Chris L. Schaich; Modulation of Baroreflex Sensitivity by Cannabinoid
Receptor Activation in Rat Solitary Tract Nucleus. Department of Physiology
and Pharmacology Seminar Series, Wake Forest University School of Medicine.
Winston-Salem, NC. February 7, 2011.
CONFERENCE PROCEEDINGS
1.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz; Blockade of CB1 Cannabinoid
Receptors in the Solitary Tract Nucleus Improves Baroreflex Sensitivity in
Hypertensive Rats. Eleventh Annual Charlotte Life Sciences Conference.
Charlotte Research Institute, Charlotte, NC. October 25, 2012.
2.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz; Modulation of Baroreflex
Sensitivity by the CB1 Cannabinoid Receptor in Normotensive and
Hypertensive Rats. Twelfth Annual Graduate Student and Postdoctoral Fellow
Research Day. Wake Forest University, Winston-Salem, NC. March 29, 2012.
3.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz; Differential Modulation of
Baroreflex Sensitivity by Solitary Tract Nucleus CB1 Cannabinoid Receptors
in Normotensive and Hypertensive Rats. Nineteenth Annual Residents’ and
Fellows’ Research Day. Division of Surgical Sciences, Wake Forest University,
Winston-Salem, NC. November 17, 2011.
4.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz; Dose-Related Biphasic
Modulation of Baroreflex Sensitivity by the CB1 Receptor in Rat Solitary
Tract Nucleus. 21st Annual Symposium on the Cannabinoids, International
Cannabinoid Research Society. St. Charles, IL. July 5 – 10, 2011.
5.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz. Modulation of Baroreflex
Sensitivity by Cannabinoid Receptor Activation in Rat Solitary Tract
Nucleus. Vasoactive Peptides VIII International Symposium. Ouro Preto, Minas
Gerais, Brazil. April 28 – 30, 2011.
228
6.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz. Modulation of Baroreflex
Sensitivity by Cannabinoid Receptor Activation in Rat Solitary Tract
Nucleus. Eleventh Annual Graduate Student and Postdoctoral Fellow Research
Day. Wake Forest University, Winston-Salem, NC. March 22, 2011.
7.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz. Attenuation of Baroreflex
Sensitivity by Cannabinoid Receptor Activation in Rat Solitary Tract
Nucleus. Eighteenth Annual Residents’ and Fellows’ Research Day. Division of
Surgical Sciences, Wake Forest University, Winston-Salem, NC. November 11,
2010.
8.
Chris L. Schaich, Allyn C. Howlett, Debra I. Diz. Attenuation of Baroreflex
Sensitivity by Cannabinoid Receptor Activation in Rat Solitary Tract
Nucleus. Presented at the Carolina Cannabinoid Collaborative. Bridgewater, VA.
October 23, 2010.
9.
C. Schaich, M. Dovlatyan, Y. Jiang, S. Miller, M. Packiarajan, S. Hong, G. Ma,
C. Giuseppina-Mazza, M. Sabio, A. White. The Use of Ex Vivo Occupancy as
the Primary Screen in the MCH1 Drug Discovery Project. Presented at Annual
Project Review, H. Lundbeck A/S. Copenhagen, Denmark. September 22 – 24,
2008.
10.
Schaich C.L. The Effects of Oxotremorine-M on Medial Septum Circuits
Regulating Memory. Presented at University of Connecticut Frontiers in
Undergraduate Research Symposium. Storrs, CT. April 21 – 22, 2006.
11.
C. Schaich, N. Ron, C. Hamelin. Episodic Memory in the Rat: A DelayedMatch-to-Sample Radial Water Maze Task. Presented at University of
Connecticut Frontiers in Undergraduate Research Symposium. Storrs, CT. April
23 – 24, 2005.
TEACHING EXPERIENCE
Spring 2014:
DPT-6206 Pharmacology, Winston-Salem State University (NC), Department of
Physical Therapy, under the direction of Allyn C. Howlett, Ph.D.
 Autonomic Nervous System Drugs (2 lecture hour)
 Cardiovascular Pharmacology (2 lecture hours)
o Cardiac Arrhythmias
o Treatment of Angina
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Fall 2013:
BIO-2311 Anatomy and Physiology I, Winston-Salem State University,
Department of Biology, under the direction of Allyn C. Howlett, Ph.D.
 Brain/nervous system block (4 lecture hours)
BIO-2312 Anatomy and Physiology II, Winston-Salem State University,
Department of Biology
 Endocrinology block (3 lecture hours)
Summer 2013:
DPT-6403 Applied Physiology, Winston-Salem State University, Department of
Physical Therapy, under the direction of Allyn C. Howlett, Ph.D.
 Autonomic Nervous System (3 lecture hours)
HONORS AND AWARDS
Finalist, 3 Minute Thesis (3MT) competition, Wake Forest University (2014)
ASPET Graduate Student Travel Award (2014)
 To attend Experimental Biology 2014, San Diego, CA, April 26 – 30, 2014.
Onsite Poster Competition Award: Council for High Blood Pressure Research (2013)
 High Blood Pressure Research Conference, New Orleans, LA (September 11,
2013)
ICRS Travel Award (2013)
 To attend the 23rd Annual Symposium of the International Cannabinoid Research
Society, Vancouver, BC, June 21 – 26, 2013.
Finalist, Nature Careers Columnist Competition (2013)
Mary A. Bell Award, Systems Neuroscience. Western North Carolina Society for
Neuroscience Research Day (December 6, 2012)
Finalist, Sixth Annual Graduate Student Poster Competition, Charlotte Life Sciences
Conference (October 25, 2012)
Runner-Up Award, Integrative Sciences. Twelfth Annual Graduate Student and
Postdoctoral Fellow Research Day. Wake Forest University (March 29, 2012)
230
Wake Forest University Alumni Travel Award (2011)
 To attend the 21st Annual Symposium of the International Cannabinoid Research
Society conference, St. Charles, IL, July 5 - 10, 2011.
Wake Forest University Alumni Travel Award (2010)
 To attend the High Blood Pressure Research Council 2010 Scientific Sessions,
Washington, DC, October 13 - 16, 2010.
Lundbeck 3R Award (2008)
 For contributions to the reduction, refinement, and replacement of wasteful
resource and animal use.
PROFESSIONAL MEMBERSHIP
American Society for Pharmacology and Experimental Therapeutics (2012 – present)
Society for Neuroscience – Western North Carolina Chapter (2011 – present)
International Cannabinoid Research Society (2011 – present)
American Heart Association (2010 – present)
American Physiological Society (2010 – present)
NEWSLETTER ARTICLES
1. Chris Schaich. Insulin May Be Key to New Treatments for Alzheimer’s
Disease. The Neurotransmitter: Newsletter of the Western North Carolina
Chapter of the Society for Neuroscience – Issue #16 (November 2013).
2. Chris Schaich. Popular Science Book Review: Hallucinations by Oliver Sacks.
The Neurotransmitter: Newsletter of the Western North Carolina Chapter of the
Society for Neuroscience – Issue #15 (August 2013).
3. Chris Schaich. Popular Science Book Review: Consciousness: Confessions of a
Romantic Reductionist by Christof Koch. The Neurotransmitter: Newsletter of
the Western North Carolina Chapter of the Society for Neuroscience – Issue #14
(March 2013).
4. Chris Schaich. Neuroscience in the News: PET Scanning Reveals
Neurodegenerative Condition in Retired NFL Players. The Neurotransmitter:
Newsletter of the Western North Carolina Chapter of the Society for
Neuroscience – Issue #14 (March 2013).
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