Hypotension and Reduced Catecholamines in
Neuropeptide Y Transgenic Rats
Mieczyslaw Michalkiewicz, Kriss M. Knestaut, Elena Yu. Bytchkova, Teresa Michalkiewicz
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Abstract—The neurons that control blood pressure express neuropeptide Y. Administered centrally, this neuropeptide
reduces blood pressure and anxiety, together with lowering sympathetic outflow. The generation of neuropeptide Y
transgenic rats overexpressing this peptide, under its natural promoter, has allowed us to examine the role of endogenous
neuropeptide Y in the long-term control of blood pressure by the sympathetic nervous system. This study tested a
hypothesis that endogenous neuropeptide Y acts to reduce blood pressure and catecholamine release. Blood pressure was
measured by radiotelemetry in conscious male transgenic and nontransgenic littermates (control). Novel cage with cold
water and forced swimming were used as stressors. Catecholamines were determined in 24-hour urine (baseline) and
plasma (cold water stress) by a radioenzymatic assay. Blood pressures in baseline and during the stresses were
significantly reduced in the transgenic rats. The lower blood pressure was associated with reduced catecholamines, lower
decrease in pressure after autonomic ganglionic blockade, and increased longevity. Data obtained through the use of this
transgenic rat model support and extend the evidence for the previously postulated sympatholytic and hypotensive
effects of neuropeptide Y and provide novel evidence for an important physiological role of endogenous peptide in blood
pressure regulation. As indicated by the increased longevity of these rats, in long-term regulation, these buffering actions
of neuropeptide Y may have important cardiovascular protective effects against sympathetic hyperexcitation.
(Hypertension. 2003;41:●●●-●●●.)
Key Words: hypotension 䡲 nervous system, sympathetic renal 䡲 stress 䡲 sympatholytics
䡲 catecholamines 䡲 rats, transgenic
T
he neurons involved in blood pressure regulation express
high quantities of neuropeptide Y (NPY). In the central
nervous system, a high degree of NPY expression is present,
particularly in the paraventricular nucleus of the hypothalamus, ventrolateral medulla, nucleus of tractus solitari, and in
the bulbospinal neurons.1–5 In the peripheral organs, NPY is
expressed in sympathetic neurons innervating arteries and
veins.6,7 A remarkable feature of this peptide is its close
coexistence and corelease with norepinephrine.5– 8 Exogenous
NPY exerts either a hypertensive or a hypotensive effect,
depending on the site of its administration. Administered
centrally, the peptide has potent sympatholytic, hypotensive,
and anxiolytic effects.9 –13 In contrast, acute administration of
NPY into the systemic circulation increases blood pressure.6
Altogether, the accumulated data indicate that endogenous
NPY is an important transmitter of the sympathetic nervous
system involved in regulation of blood pressure. However,
because of its broad distribution in the nervous system, the
complexity of signaling (which involves at least 5 different
receptors), the peptide interaction with other neurotransmitters, and the lack of specific antagonists, it is difficult to
understand the factual physiological role of endogenous NPY
in the regulation of blood pressure.
The present study aimed to elucidate the role of endogenous NPY in the long-term regulation of blood pressure by
the sympathetic nervous system. In the light of the reported
hypotensive, anxiolytic, and sympatholytic effects of synthetic NPY, our hypothesis was that endogenous NPY acts to
reduce the activity of the sympathetic nervous system. Hence,
we reasoned that genetic NPY upregulation will inhibit
norepinephrine release and reduce blood pressure.
For this study, we have generated transgenic rats that
overexpress rat NPY under the control of its natural regulatory sequences.14 –17 The expression of this transgene may
thus be regulated in a manner similar to that of endogenous
NPY.16 To date, the cardiovascular role of NPY has been
mostly studied in the rat. Thus, the generation of NPY
transgenic rat has provided a suitable model for studies of the
role of endogenous NPY because it allows a direct comparison with the previously described effects of exogenous NPY
in the rat model.
Our previous blood pressure studies with these transgenic
rats were based on short-term blood pressure recordings with
a tethered catheter or a tail-cuff method.16 Those methods
showed either no change in baseline blood pressure or
increased tail pressure, respectively. In view of the deficien-
Received October 24, 2002; first decision November 19, 2002; revision accepted March 4, 2003.
From the Department of Physiology, Medical College of Wisconsin, Milwaukee.
Correspondence to M. Michalkiewicz, DVM, PhD, Department of Physiology, Medical College of Wisconsin, 8701 Watertown Plank Road, PO Box
26509, Milwaukee, WI 53226-0509. E-mail [email protected]
© 2003 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
DOI: 10.1161/01.HYP.0000066623.64368.4E
1
2
Hypertension
May 2003
cies of short-term blood pressure measurements with the use
of catheters or the tail-cuff,18 it is important to repeat these
hemodynamic studies using radiotelemetry to more accurately measure the long-term effect of NPY on resting blood
pressure. This method allows the continuous collection of
data from animals residing in their home cages for days
without the stress from human contact.
In this study, we demonstrate in conscious rats that
endogenous NPY is a potent neurotransmitter that lowers
catecholamine release and blood pressure in baseline and
during stress.
Methods
The study was reviewed and approved by the Medical College of
Wisconsin Committee on Animal Care and Use.
Animals
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
We used the NPY transgenic (NPY-tg) Sprague-Dawley rats and
nontransgenic littermates (control).14 –17 Four- to 5-month-old NPY
transgenic hemizygote male rats carrying 5 copies of the transgene
(line No. 400) were used. The rats were housed in a temperaturecontrolled (21° to 22°C) and light-dark cycle– controlled (lights on
6:00 AM to 6:00 PM) room and were provided 5001 Purina Rodent
chow and water ad libitum.
Radiotelemetry
The Dataquest A.R.T. 2.2 (Data Sciences International) system was
used for radiotelemetric acquisition and analysis of blood pressure,
heart rate, locomotor activity, and body temperature. Under anesthesia (Nembutal, 50 mg/kg IP), a transducer (TL11 mol/L2-C50-PXT)
was placed into the abdominal cavity and its catheter threaded
through the abdominal wall and inserted into the femoral artery.
After 10 days’ recovery, when all animals had regained their
circadian hemodynamic rhythms and weights, baseline data were
recorded for 10 seconds, every minute, continuously for 7 days.
Signals were recorded from several animals simultaneously. The
data were averaged over 1-hour intervals and divided into 2 time
periods: with the lights on and off. Calibration of each transmitter
was verified before implantation and at the end of recording after
explantation.
Blood Pressure During “Novelty” and Forced
Swimming Stresses
After 1-hour baseline recording, rats were transferred either into a
new cage containing 2 cm of iced water,19 with blood pressure
recorded for 20 minutes (“novelty”), or into a glass cylinder (44 cm
high⫻30cm in diameter) filled with water at a temperature of 25°C
(30 cm high) for 5 minutes (forced swimming).20
Pressor Response to Ganglionic Blockade
The fall in blood pressure to ganglionic blockade with hexamethonium (20 mg/kg IV through chronic femoral vein catheter) was
measured in conscious, freely moving rats through the use of a
chronic femoral artery tethered catheter and a cardiovascular monitoring system (BPA-100, Micro-Med), as previously described in
detail.16 Ganglionic blockade response was determined at the nadir
of the pressure response.
Urine and Blood Sample Collection for
Catecholamine Assay
For 24-hour urine collection, rats were housed in Nalgene metabolic
cages. After a 24-hour adaptation period, 2 consecutive 12-hour
urine collections were drawn into tubes containing 20 ␮L of 2.5
mol/L HCl. Subsequently, aliquots were stored below ⫺78°C in
tubes containing glutathione (1.2 mg/mL) and EGTA (1.8 mg/mL).
Chronic femoral artery catheters were used for blood sample
collection16,21 during the cold water stress. After surgery, the rats
were brought to the laboratory every day for 3 to 4 days and the
catheters were flushed with heparinized saline solution. On the day
of the experiment, 2 hours before blood sampling, the catheters were
connected to tubing extensions that permitted the blood sample
withdrawal without disturbing the rat. After the baseline sample was
collected (into a tube containing 1.2 mg/mL glutathione and 1.8
mg/mL EGTA and stored below ⫺78°C), the rats were transferred to
a novel cage containing ice water and another blood sample was
withdrawn 20 minutes later.
Catecholamine Assay
Norepinephrine and epinephrine were measured in urine and plasma
with the use of a (3H)radioenzymatic assay kit from Amersham.21
Food Intake and Central NPY Concentrations
Food intake was recorded daily for 14 days. NPY concentrations
were measured in extracts from micropunches of selected brain areas
with the use of a method and a specific radioimmunoassay as
previously described.15,21,22
Longevity
Pairs of male rats of the same strain were housed in plastic cages.
The animals were inspected daily for spontaneously dead or terminally ailing animals. The terminally ill rats with visible signs of
ailing, for example, immobility or impaired respiration, were euthanized and were included in the analysis. No evidence of infectious
disease was observed in these rats.
Data Analysis
Data are presented as mean⫾SEM. The statistical methods used are
provided in each figure legend. A probability value of ⬍0.05 was
considered statistically significant.
Results
Baseline Blood Pressure
In the transgenic and nontransgenic strains, the mean arterial
blood pressure showed very regular diurnal changes with
lower levels during the day and higher during the nighttime
(Figure 1A, left panel). As compared with the nontransgenic
siblings, the blood pressure of the NPY-tg rats was significantly lower during both periods (P⬍0.05, Figure 1A, right
panel). There was no significant interaction between the
effects of genotype and time, indicating that the time of day
had a similar effect on blood pressure in the 2 genotypes.
Heart rates underwent a similar diurnal oscillation in both
strains, without any significant difference between them
(Figure 1B). A comparable reduction in basal blood pressure
(by 5.2 mm Hg, P⬍0.05, n⫽5 to 6) was also observed in
another NPY-transgenic line (No. 438) tested.
Food Intake, Body Weight, Locomotor Activity,
and Body Temperature
The 2 strains did not differ regarding body weight before
implantation of the transmitters (440.4⫾32.7 versus
448.8⫾32.3 g), and at the end of the recording, the weight
gain was similar (21.3⫾7.6 versus 17.2⫾7.7 g) in wild-type
versus NPY-tg, respectively. Food intake was also similar in
both groups (24.3⫾0.6 versus 23.7⫾0.4 g/d, NPY-tg versus
control, respectively). Similar to the hemodynamic variables,
locomotor activity and body temperature had consistent
diurnal variations, however, without any significant difference between the strains (data not shown).
Michalkiewicz et al
Hypotension in Neuropeptide Y Transgenic Rats
3
Figure 1. Telemetric measurements of blood
pressure (A) and heart rate (B) in conscious
NPY-tg and nontransgenic littermates (wildtype) rats. Left panels represent 7-day profiles
of radiotelemetry data averaged over 1-hour
intervals. Bars on right panels represent average of 7 days of recording data divided into
light and dark periods. Two-way repeatedmeasures ANOVA (1-factor repetition) with
Tukey post hoc test was used to determine the
effect of genotype on blood pressure for each
period. *,#Statistically significant difference
between periods within strain or between
strains within the period, respectively; n⫽16.
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Urine Concentrations of Catecholamines
As Figure 2A demonstrates, levels of norepinephrine and
epinephrine in urine were significantly (P⬍0.05) reduced in
NPY-tg rats. Urine volume was similar in both groups
(22.9⫾2.5 versus 22.8⫾2.3 mL/d, control versus transgenic
rats, respectively).
Pressor Response to Ganglionic Blockade
To determine sympathetic drive to the blood vessels, we used
ganglionic blockade with hexamethonium. The blood pressure fall in response to ganglionic blockade was significantly
(P⬍0.05) attenuated in the transgenic rats (Figure 2B). This
suggests that the sympathetic drive to the blood vessels was
reduced in the NPY-tg rats.
Blood Pressure and Plasma Catecholamines
During Novelty Stress
The strains responded to the stress with a significant increase
in blood pressure (Figure 3A). However, the blood pressure
of the NPY-tg rats during the stress was 6.2⫾1.2 mm Hg
lower than their nontransgenic siblings, showing significant
effect of the genotype (P⬍0.05). Heart rates increased in both
groups during the stress without any significant difference
between them (Figure 3B). Baseline circulating levels of
catecholamines were not different between the strains (Figures 3C and 3D). During the stress, highly significant
increases in plasma norepinephrine were observed in nontransgenic littermates but not in the NPY-tg group (P⬍0.05).
Circulating epinephrine increased significantly in both groups
of animals; however, its levels in the NPY-tg rats during the
stress were significantly lower than those in the wild type
(P⬍0.05).
Blood Pressure During Forced Swimming
Swimming increased blood pressure in both groups; however,
during this stress the pressure in the transgenic rats was
9.1⫾1.3 mm Hg lower than that of the control rats (Figure
4A). Similarly, in the NPY-tg rats, blood pressure increased
by a smaller amount than in the control (25.5⫾1.6 versus
31.4⫾1.8 mm Hg, P⬍0.05 by t test). A significant overall
effect of the genotype was observed (F⫽7.65; P⫽0.01), and
a highly significant swimming⫻genotype interaction term
(F⫽5.64; P⫽0.024) indicated that the stress differentially
affected the 2 genotypes. The stress-induced heart rate increases were not different between the strains (Figure 4B).
Longevity
The survival curve of the transgenic strain was shifted to the
right (P⫽0.059), suggesting an increased longevity in this
strain (Figure 5A). The mean longevity was 633.1⫾24.6
versus 697.5⫾20.1 days (Figure 5B), and the 50th percent
survival estimates were 638 (95% CI, 556 to 718) versus 698
(95% CI, 638 to 761) days in wild-type versus NPY-tg rats,
respectively (P⫽0.05).
Figure 2. Urine excretion of catecholamines (A) and arterial
pressure response to hexamethonium (B) in NPY-tg and wildtype rats. *Significant difference between strains (P⬍0.05 by
unpaired Student t test); n⫽14 to 16 or n⫽8 in A and B,
respectively.
Central Concentrations of NPY
NPY concentrations were significantly higher in the paraventricular, suprachiasmatic, and supraoptic nuclei and tended to
be increased in the arcuate nucleus (Table).
4
Hypertension
May 2003
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Figure 4. Mean blood pressure (A) and heart rate (B) in the 2
strains during forced swimming. Statistical analysis and symbols
are as described in the legend of Figure 3; n⫽15 to 16.
this approach allowed for the most accurate determination of
the factual role of endogenous NPY in long-term regulation
of blood pressure by the sympathetic nervous system.
Influence of NPY Upregulation on Blood Pressure
Figure 3. Mean blood pressure (A), heart rate (B), and plasma
levels of norepinephrine (C) and epinephrine (D) in the 2 strains
during novelty stress. Two-way repeated-measures ANOVA with
the Tukey post hoc test was used to determine the effect of genotype on blood pressure, heart rate, and plasma levels of catecholamines in baseline and during stress. *,#Statistically significant difference between treatments within the strain or between
strains within the treatment, respectively; n⫽11 to 14.
The hypotensive effect of NPY upregulation is consistent
with the anatomic findings that NPY is abundantly expressed
in the neurons of the hypothalamus and the brain stem, which
control sympathetic outflow to the preganglionic sympathetic
neurons involved in blood pressure regulation.1–5,23 The
present findings also corroborate the functional observations
demonstrating that this peptide decreases neuronal excitabil-
Discussion
In this study, overexpression of endogenous NPY in transgenic rats was associated with lower blood pressure in
baseline and during stress. The lower pressure occurred
together with reduced levels of catecholamines and lower
sympathetic drive to the periphery, thus indicating that the
hypotensive effect of NPY upregulation was due to a reduced
adrenergic signaling. The hypotension appeared to be specifically related to the increased NPY signaling in the cardiovascular regulatory system because it was observed in the
absence of the genotype effect on appetite, body weight,
locomotor activity, or body temperature. It was also observed
in an additional transgenic line tested. Based on this finding,
we suggest that endogenous NPY is a potent hypotensive and
sympatholytic neurotransmitter involved in the long-term
regulation of blood pressure by the sympathetic nervous
system.
In the current experiment, genetic NPY upregulation was
combined with telemetric blood pressure measurement. Thus,
Figure 5. Survival curves (A) and longevity (B) for NPY-tg and
nontransgenic littermates. Kaplan-Meier method estimates were
used to calculate survival probabilities.43 Survival curves were
compared by means of a Wilcoxon test (P⫽0.059) and longevity
by t test (*P⫽0.05) (n⫽20).
Michalkiewicz et al
NPY Concentrations in Selected Brain Areas of Nontransgenic
Littermates (Wild Type) and NPY-tg Rats
Area
Paraventricular nucleus
Suprachiasmatic nucleus
Wild Type (n)
9.7⫾0.4 (12)
27.0⫾3.0 (9)
NPY-Tg (n)
P
13.2⫾1.3 (13)
⬍0.05
50.9⫾10.1 (6)
⬍0.05
12.0⫾3.8 (9)
⬍0.05
Supraoptic nucleus
3.7⫾1.2 (11)
Posterior pituitary*
0.42⫾0.03 (10)
0.54⫾0.02 (10)
⬍0.05
Arcuate nucleus
14.6⫾1.1 (11)
16.7⫾1.2 (11)
NS
Medial preoptic nucleus
8.1⫾1.2 (10)
8.5⫾1.6 (8)
NS
Ventromedial hypothalamus
6.5⫾2.6 (10)
8.2⫾1.2 (14)
Dorsomedial hypothalamus
11.0⫾2.0 (12)
10.4⫾1.6 (9)
NS
NS
Values are mean⫾SE. Tissue concentrations of neuropeptide Y (NPY) are
expressed in ng/mg of total protein, except for the posterior pituitary (*), where
the peptide concentrations are expressed in ng/gland. Unpaired Student t test
was used to estimate statistical significance of the difference between the
genotypes. NS indicates not significant; NPY-tg, neuropeptide Y-transgenic
rats.
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
ity, reduces the releases of other neurotransmitters,24 –26 and
lowers sympathetic outflow and arterial pressure when microinjected into selected areas of the hypothalamus or the
medulla oblongata of conscious rats.9 –12
Similarly to endogenous NPY, the mechanism of transgenic NPY action to reduce blood pressure could involve
both central and peripheral levels, and the present study does
not distinguish between the two. Acting centrally within the
nuclei of the hypothalamus and the “vasomotor center” of the
medulla oblongata NPY could reduce sympathetic outflow to
the peripheral vasculature. This suggestion is strongly supported by the present observation of the attenuated pressor
response to hexamethonium in the NPY-overexpressing rats,
which is compatible with lower sympathetic outflow and
lower adrenergic contribution to blood pressure. Moreover, in
these transgenic rats, we have demonstrated a significant
reduction of the susceptibility to stress and epileptogenesis,
together with inhibition of memory acquisition, and related
these effects to a modulatory action of NPY on GABA-ergic
and glutamatergic transmissions in the central nervous system.15,17 Concurrently, the mechanism of hypotension could
involve peripheral presynaptic action of excess NPY to
decrease norepinephrine release from the vascular nerve
terminals27–29 akin to action of an ␣2-adrenergic agonist.12 In
this study, we found reduced urine and plasma levels of
catecholamines in this strain (discussed below). Our earlier
observation of an increased sensitivity of the vascular adrenoceptors to norepinephrine in this strain also indicated a
reduced norepinephrine release at the neurovascular
synapse.16
The blood pressures in transgenic rats were also lower
during an acute novelty stress and during forced swimming.
These results indicate important cardiovascular protective
function of NPY in long-term regulation and may explain the
increased life span in this strain. By maintaining lower blood
pressure in baseline and in stress during the entire life span,
the NPY gene product may protect cardiovascular and other
end organs from adverse effects of increased pressure. The
present observations corroborate the behavioral insensitivity
to restraint stress in this transgenic strain15 and give further
Hypotension in Neuropeptide Y Transgenic Rats
5
support to the suggestion that the hypotensive effect of NPY
upregulation was of a central origin. Behavioral studies in the
rat have demonstrated that endogenous NPY reduces anxiety
and may serve as a physiological stabilizer of neural activity
in circuits involved in arousal and anxiety— behavioral states
associated with blood pressure increase.13,15,26,30 It is not clear
why during the “novelty” stress, despite the blunted increase
in catecholamines, the change in blood pressure was similar
in both groups. It may be that during this stress, the
NPY-induced blood pressure reduction was overwhelmed by
the release of some other vasoconstrictors or by increased
responsiveness of vascular adrenoceptors in this strain. In
fact, we have reported an increased pressor response to
exogenous norepinephrine in these transgenic rats.16
Heart rate was not decreased in the NPY-tg rats, as it could
be expected from the lower catecholamines and blood pressure or from the reported transient bradycardia caused by
2-week cerebral delivery of NPY.31 It is possible that a
reduction of heart rate in these transgenic rats was prevented
by some compensatory mechanism, including baroreceptor
reflex. In fact, the reported bradycardiac effect of synthetic
NPY also disappeared in the second week of treatment.31
NPY is expressed in the groups of neurons of the baroreflex
arc,1–5 and treatment with NPY increased the sensitivity of
the aortic baroreceptor reflex in the rat.32 In addition, it could
be that NPY signaling is more involved in the sympathetic
pathways controlling blood pressure than in those controlling
heart rate. There are data demonstrating that the central
pathways mediating the increased sympathetic vasomotor
activity could, at least in part, be separated from the pathways
mediating the tachycardia.33
Influence of NPY Upregulation on
Catecholamine Release
The lower baseline urine and stress-induced circulating catecholamines in the NPY-tg rats indicate that the increased
NPY signaling brought about a reduced sympathetic signaling in baseline and diminished the immediate sympathetic
response to stress. Therefore, we suggest that endogenous
NPY acts to buffer the activity of the sympathetic nervous
system.
The limitation of this study was that plasma and urinary
norepinephrine are indirect markers of sympathetic nervous
system activity. Plasma concentrations, and ultimately urine
concentrations of norepinephrine depend not only on the rates
of the neurotransmitter release but also on the rate of its
neuronal reuptake and clearance from plasma.34 Nonetheless,
the existence of proportionality between plasma or urine
concentrations of norepinephrine and the activity of the
sympathetic nervous system has been well documented in
human and in animal models.35,36
The mechanism of reduced catecholamines in the NPY-tg
rats may involve an inhibitory interaction of transgenic NPY
with the group of neurons of the hypothalamus and the brain
stem responsible for sympathetic outflow, as discussed earlier. The present observation of reduced sympathetic drive to
the blood vessels in the NPY-overexpressing rats supports
this suggestion. Moreover, intracerebroventricular injection
of synthetic NPY also reduced sympathetic nerve activity to
6
Hypertension
May 2003
the kidney or brown adipose tissue.9,10 In addition to the
central influence, at the same time, a direct inhibition at the
presynaptic membranes of the peripheral sympathetic nerves
could also contribute to the reduced norepinephrine in the
NPY-tg rats. The peptide inhibits norepinephrine release from
the sympathetic nerves of the mesenteric, femoral, portal,
renal, or atrial arteries.27–29
Influence of NPY Upregulation on Appetite, Body
Weight, and Longevity
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Central administration of synthetic NPY has been consistently shown to increase appetite and weight.31 Therefore, it
was surprising that food intake and body weight were not
changed in the NPY-tg rats. It may be that the effect of NPY
upregulation in the transgenic rats was counterbalanced by
increased expression of anorexigenic factors, for example,
melanocortins or cocaine/amphetamine-regulated transcript
(CART).37 It is also possible that NPY overexpressions in
areas affecting appetite were lower than in those affecting
sympathetic activity or that lower amounts of NPY may
affect sympathetic activity without altering appetite. However, the observations in this transgenic rat completely agree
with the reported NPY knockout experiments in mouse, in
which body weight and appetite were also not changed.38,39
Our transgenic data clearly demonstrate that hypotensive
effects of NPY can be separated from its metabolic influences. This is somewhat in contrast with the recent report
demonstrating that 2-week cerebral delivery of synthetic
NPY in the rat increased food intake and body weights
without altering arterial pressure.31 Different sites of action or
opposing pressor effects of obesity-induced sympathoactivation and NPY-induced sympathoinhibition could account for
these divergences.
The sympatholytic and hypotensive effects of NPY overexpression appear to be functional, as they were associated
with increased longevity of this strain. We have no explanation for the direct mechanism of the increased longevity of
the NPY transgenic rats. Nonetheless, the present observation
is in agreement with the known cardiovascular protective and
life span–increasing effects of reduced sympathetic activity
and lower blood pressure.40,41 Interestingly, both the hypotensive and sympatholytic effects of central NPY are significantly diminished in the SHR.24,42
In conclusion, genetic upregulation of NPY in transgenic
rats led to reduced catecholamine release and to lower blood
pressure in baseline and during stress. At the same time, these
effects were associated with increased longevity of these rats.
Thus, we suggest that endogenous NPY buffers adrenergic
signaling to blood vessels. In long-term regulation, such
antiadrenergic action of NPY within the sympathetic nervous
system may protect the cardiovascular system from excessive
adrenergic excitations and sudden blood pressure bursts and
consequently may increase longevity.
Perspectives
It has been difficult to determine the role of endogenous NPY
in long-term blood pressure regulation because in the cardiovascular system, NPY is expressed in the circuits consisting
of multiple synaptic pathways and multiple neurotransmitters.
The type of the transgene used in this model most likely
ensured that excess NPY was released in the appropriate sites
and at the required times. Therefore, this transgenic rat
provides a unique model for study of the physiological
importance of NPY in the sympathetic nervous system,
particularly its functional interaction with norepinephrine.
The present results suggest that endogenous NPY is a
potent sympathoinhibitory neurotransmitter, acting centrally
and peripherally. Its central action within the hypothalamus
and the brain stem may inhibit sympathetic outflow. While in
the peripheral postganglionic adrenergic varicosities, by the
virtue of close coexistence with norepinephrine, NPY may
directly inhibit the neurotransmitter release, or even its
synthesis. Such close interaction may provide a basis for an
ultra–short-loop homeostatic mechanism, ensuring steady and
balanced sympathetic tone to the blood vessels. In this way,
NPY may serve as an important buffering system, preventing
desensitization of the adrenergic receptor or protecting endorgans from adverse effects of excess catecholamines and
blood pressure bursts during stress.
In combination with other techniques, for example, microinjections of NPY antagonists into the specific nuclei of the
brain and recording nerve activity, this genetic model could
be used to identify the synaptic sites of NPY action in the
sympathetic pathways. In addition, these sympatholytic rats
are useful to determine the sympathetic component in the rat
models of hypertension or heart failure, for example Goldblatt, L-NAME, or DOCA.
Acknowledgments
This study was supported by National Institutes of Health grant
HL-57921. We thank Dr Hyun Ja Yun and Dan Eastwood, MS, for
help with statistical analysis, Mae J. Racadio for technical support,
and Janet Steward for editing the manuscript.
References
1. Dumont Y, Martel JC, Fournier A, St-Pierre S, Quirion R. Neuropeptide
Y and neuropeptide Y receptor subtypes in brain and peripheral tissues.
Prog Neurobiol. 1992;38:125–167.
2. Glass MJ, Chan J, Pickel VM. Ultrastructural localization of neuropeptide
Y Y1 receptors in the rat medial nucleus tractus solitarius: relationships
with neuropeptide Y or catecholamine neurons. J Neurosci Res. 2002;67:
753–765.
3. Stornetta RL, Akey PJ, Guyenet PG. Location and electrophysiological
characterization of rostral medullary adrenergic neurons that contain
neuropeptide Y mRNA in rat medulla. J Comp Neurol. 1999;415:
482–500.
4. Minson JB, Llewellyn-Smith IJ, Pilowsky PM, Chalmers JP. Bulbospinal
neuropeptide Y-immunoreactive neurons in the rat: comparison with
adrenaline-synthesising neurons. J Auton Nerv Syst. 1994;47:233–243.
5. Tseng CJ, Lin HC, Wang SD, Tung CS. Immunohistochemical study of
catecholamine enzymes and neuropeptide Y in the rostral ventrolateral
medulla and bulbospinal projection. J Comp Neurol. 1993;334:294 –303.
6. Zukowska-Grojec Z, Wahlestedt C. Origin and actions of NPY in the
cardiovascular system. In: Colmers WF, Wahlestedt C, eds. The Biology
of Neuropeptide Y and Related Peptides. Clifton, NJ: Humana Press;
1993:315–389.
7. Fried G, Terenius L, Dockray G, Fahrenkrug J, Goldstein M, Hokfelt T.
Neuropeptide Y and noradrenaline coexist in sympathetic neurons innervating the bovine spleen. Cell Tissue Res. 1986;243:495–508.
8. Lundberg JM, Fried G, Pernow J, Theodorsson-Norheim E. Co-release of
neuropeptide Y and catecholamines upon adrenal activation in the cat.
Acta Physiol Scand. 1986;126:231–238.
9. Egawa M, Yoshimatsu H, Bray GA. Neuropeptide Y suppresses sympathetic activity to interscapular brown adipose tissue in rats. Am J Physiol.
1991;260:R328 –R334.
Michalkiewicz et al
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
10. Matsumura K, Tsuchihashi T, Abe I. Central cardiovascular action of
neuropeptide Y in conscious rabbits. Hypertension. 2000;36:1040 –1044.
11. Tseng CJ, Mosqueda-Garcia R, Appalsamy M, Robertson D. Cardiovascular effects of neuropeptide Y in rat brainstem nuclei. Circ Res. 1989;
64:55– 61.
12. McAuley MA, Macrae IM, Reid JL. The cardiovascular actions of
clonidine and neuropeptide-Y in the ventrolateral medulla of the rat. Br J
Pharmacol. 1989;97:1067–1074.
13. Wahlestedt C, Pich EM, Koob GF, Yee F, Heilig M. Modulation of
anxiety and neuropeptide Y-Y1 receptors by antisense oligodeoxynucleotides. Science. 1993;259:528 –531.
14. Michalkiewicz M, Michalkiewicz T. Developing transgenic neuropeptide
Y rats. In: Balasubramaniam A, ed. Methods in Molecular Biology:
Neuropeptide Y Protocols. Totowa, NJ: Humana Press; 2000:73– 89.
15. Thorsell A, Michalkiewicz M, Dumont Y, Quirion R, Caberlotto L,
Rimondini R, Mathé A, Heilig M. Behavioral insensitivity to restraint
stress, absent fear suppression of behavior and impaired spatial learning
in NPY transgenic rats. Proc Natl Acad Sci U S A. 2000;97:12852–12857.
16. Michalkiewicz M, Michalkiewicz T, Kreulen D, McDougall S. Increased
vascular responses in neuropeptide Y transgenic rats. Am J Physiol.
2001;281:R417-R426.
17. Vezzani A, Michalkiewicz M, Michalkiewicz T, Moneta D, Ravizza T,
Richichi C, Aliprandi M, Mulé F, Gobbi M, Schwarzer C, Sperk G.
Seizure susceptibility and epileptogenesis are decreased in transgenic rats
overexpressing neuropeptide Y. Neuroscience. 2002;110:237–243.
18. Ferrari AU, Daffonchio S, Albergati F, Bertoli P, Mancia G. Intra-arterial
pressure alterations during tail-cuff blood pressure measurements in normotensive and hypertensive rats. J Hypertens. 1990;8:909 –911.
19. Zukowska-Grojec Z, Shen GH, Capraro PA, Vaz CA. Cardiovascular,
neuropeptide Y, and adrenergic responses in stress are sexually differentiated. Physiol Behav. 1991;49:771–777.
20. Garcia-Lecumberri C, Ambrosio E. Differential effect of low doses of
intracerebroventricular corticotropin-releasing factor in forced swimming
test. Pharmacol Biochem Behav. 2000;67:519 –525.
21. Michalkiewicz M, Dey M, Huffman LJ, Hedge GA. The neuropeptides,
VIP and NPY, that are present in the thyroid nerves are not released into
the thyroid vein. Thyroid. 1998;8:1073–1079.
22. Michalkiewicz M, Huffman LJ, Hedge GA. Endogenous neuropeptide Y
regulates thyroid blood flow. Am J Physiol. 1993;264:E699 –E705.
23. Schreihofer AM, Stornetta RL, Guyenet PG. Regulation of sympathetic
tone and arterial pressure by rostral ventrolateral medulla after depletion
of C1 cells in rat. Physiology. 2000;529:221–236.
24. Tsuda K, Tsuda S, Masuyama Y, Goldstein M. Norepinephrine release
and neuropeptide Y in medulla oblongata of spontaneously hypertensive
rats. Hypertension. 1990;15:784 –790.
25. Klapstein GJ, Colmers WF. On the sites of presynaptic inhibition by
neuropeptide Y in rat hippocampus in vitro. Hippocampus. 1993;3:
103–111.
26. Bacci A, Huguenard JR, Prince DA. Differential modulation of synaptic
transmission by neuropeptide Y in rat neocortical neurons. Proc Natl
Acad Sci U S A. 2002;99:17125–17130.
Hypotension in Neuropeptide Y Transgenic Rats
7
27. Westfall TC, Carpentier S, Chen X, Beinfeld MC, Naes L, Meldrum MJ.
Prejunctional and postjunctional effects of neuropeptide Y at the noradrenergic neuroeffector junction of the perfused mesenteric arterial bed of
the rat. J Cardiovasc Pharmacol. 1987;10:716 –722.
28. Lundberg JM, Pernow J, Tatemoto K, Dahlof C. Pre- and postjunctional
effects of NPY on sympathetic control of rat femoral artery. Acta Physiol
Scand. 1985;123:511–513.
29. Malmstrom RE, Lundberg JN, Weitzberg E. Effects of the neuropeptide
Y Y2 receptor antagonist BIIE0246 on sympathetic transmitter release in
the pig in vivo. Naunyn Schmiedebergs Arch Pharmacol. 2002;365:
106 –111.
30. Kask A, Harro J, von Horsten S, Redrobe JP, Dumont Y, Quirion R. The
neurocircuitry and receptor subtypes mediating anxiolytic-like effects of
neuropeptide Y. Neurosci Biobehav Rev. 2002;26:259 –283.
31. Correia ML, Morgan DA, Sivitz WI, Mark AL, Haynes WG. Hemodynamic consequences of neuropeptide Y-induced obesity. Am J Hypertens.
2002;15:137–142.
32. Minson RB, McRitchie RJ, Chalmers JP. Effects of neuropeptide Y on
baroreflex control of heart rate and myocardial contractility in conscious
rabbits. Clin Exp Pharmacol Physiol. 1990;17:39 – 49.
33. Fontes MA, Tagawa T, Polson JW, Cavanagh SJ, Dampney RA.
Descending pathways mediating cardiovascular response from dorsomedial hypothalamic nucleus. Am J Physiol. 2001;280:H2891–H2901.
34. Esler M, Jennings G, Lambert G, Meredith I, Horne M, Eisenhofer G.
Overflow of catecholamine neurotransmitters to the circulation: source,
fate, and functions. Physiol Rev. 1990;70:963–985.
35. Pierdomenico SD, Bucci A, Costantini F, Lapenna D, Cuccurullo F,
Mezzetti A. Twenty-four-hour autonomic nervous function in sustained
and “white coat” hypertension. Am Heart J. 2000;140:672– 677.
36. Hansell P, Isaksson B, Sjoquist M, Sjoquist M. Renal dopamine and
noradrenaline excretion during CNS-induced natriuresis in spontaneously
hypertensive rats: influence of dietary sodium. Acta Physiol Scand. 2000;
168:257–266.
37. Rahmouni K, Haynes WG. Leptin signaling pathways in the central
nervous system: interactions between neuropeptide Y and melanocortins.
Bioessays. 2001;23:1095–1099.
38. Erickson JC, Clegg KE, Palmiter RD. Sensitivity to leptin and susceptibility to seizures of mice lacking neuropeptide Y. Nature. 1996;381:
415– 421.
39. Marsh DJ, Hollopeter G, Kafer KE, Palmiter RD. Role of the Y5 neuropeptide Y receptor in feeding and obesity. Nat Med. 1998;4:718 –721.
40. Julius S, Valentini M. Consequences of the increased autonomic nervous
drive in hypertension, heart failure and diabetes. Blood Press. 1998;(suppl
3):5–13.
41. Mancia G, Cattaneo BM, Grassi G. Clinical benefits of a consistent
reduction in blood pressure. J Hypertens Suppl. 1998;16:S35–S39.
42. Westfall TC, Han SP, Knuepfer M, Martin J, Chen X, Del Valle K, Naes
L. Neuropeptides in hypertension: role of neuropeptide Y and calcitonin
gene related peptide. Br J Clin Pharmacol. 1990;30:75S-82S.
43. Gross AJ, Clark VA. Survival Distributions. New York, NY: Wiley;
1975.
Hypotension and Reduced Catecholamines in Neuropeptide Y Transgenic Rats
Mieczyslaw Michalkiewicz, Kriss M. Knestaut, Elena Yu. Bytchkova and Teresa Michalkiewicz
Hypertension. published online March 31, 2003;
Downloaded from http://hyper.ahajournals.org/ by guest on June 16, 2017
Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2003 American Heart Association, Inc. All rights reserved.
Print ISSN: 0194-911X. Online ISSN: 1524-4563
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://hyper.ahajournals.org/content/early/2003/03/31/01.HYP.0000066623.64368.4E.citation
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial
Office. Once the online version of the published article for which permission is being requested is located,
click Request Permissions in the middle column of the Web page under Services. Further information about
this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Hypertension is online at:
http://hyper.ahajournals.org//subscriptions/