Hypertension
Adenosine Modulates Cardiovascular Functions Through
Activation of Extracellular Signal-Regulated Kinases
1 and 2 and Endothelial Nitric Oxide Synthase in
the Nucleus Tractus Solitarii of Rats
Wen-Yu Ho, MD; Pei-Jung Lu, PhD; Michael Hsiao, DVM, PhD; Hwong-Ru Hwang, MD;
Yu-Chou Tseng, BS; Mao-Hsiung Yen, PhD; Ching-Jiunn Tseng, MD, PhD
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Background—The nucleus tractus solitarius (NTS) is the primary integrative center for baroreflex. Adenosine has been
shown to play an important modulatory role in blood pressure control in the NTS. Our previous results demonstrated
that adenosine decreases blood pressure, heart rate, and renal sympathetic nerve activity and modulates baroreflex
responses in the NTS. We also demonstrated that a nitric oxide synthase (NOS) inhibitor may block the cardiovascular
effects of adenosine in the NTS, which suggests interaction between the adenosine receptor and NOS. However, the
signaling mechanisms of adenosine that induce nitric oxide release in the NTS remain uncertain. The aim of the present
study was to investigate the possible signal pathways involved in the cardiovascular regulation of adenosine in the NTS.
Methods and Results—Adenosine was microinjected into the NTS of urethane-anesthetized male Sprague-Dawley rats.
Blood pressure and heart rate decreased significantly after microinjection. The cardiovascular effects of adenosine were
attenuated by prior administration of the mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor
PD98059 (6 nmol/60 nL) or an endothelial NOS–selective inhibitor, L-NIO (6 nmol/60 nL); however, the neuronal
NOS–specific inhibitor vinyl-L-NIO (600 pmol/60 nL) did not attenuate the cardiovascular effects of adenosine. Western
blot and immunohistochemistry studies demonstrated that adenosine induced extracellular signal-regulated kinases 1
and 2 and endothelial NOS phosphorylation in the NTS. Pretreatment with PD98059 diminished the endothelial NOS
phosphorylation evoked by adenosine.
Conclusions—These results represent a novel finding that extracellular signal-regulated kinases 1 and 2 is involved in
cardiovascular regulation in the NTS. They also indicate that the cardiovascular modulatory effects of adenosine in the
NTS are accomplished by activation of mitogen-activated protein kinase/extracellular signal-regulated kinases 1 and 2
and then endothelial NOS. (Circulation. 2008;117:773-780.)
Key Words: adenosine 䡲 baroreceptors 䡲 signal transduction 䡲 nitric oxide 䡲 brain
T
he nucleus tractus solitarius (NTS) is the primary integrative center for cardiovascular control and other autonomic functions in the central nervous system. The NTS not
only integrates convergent information but itself is the site of
substantial modulation. Our previous studies demonstrated
that several neuromodulators are involved in cardiovascular
control in the NTS, including ATP,1 adenosine,1,2 angiotensin
II,3 angiotensin III,4 nitric oxide,5–7 carbon monoxide,8 –11 and
insulin.12 Among these neuromodulators, adenosine has been
shown to play an important role in central cardiovascular
control.1,2,13 Microinjection of adenosine into NTS produces
slowly developed, long-lasting (minutes), and dose-related
decreases in blood pressure (BP) and heart rate (HR).2,14
Clinical Perspective p 780
Adenosine acts as a neuromodulator at many levels of the
central nervous system, including the brainstem, which is the
central cardiovascular control center.15,16 The natural precursor
of neuromodulator adenosine in the central nervous system is
ATP, which is dephosphorylated and rapidly becomes adenosine
via the activity of ectonucleotidases on synaptic release.17
Among 4 adenosine receptor subtypes, the A1 and A2A receptor
Received April 18, 2007; accepted November 21, 2007.
From the Graduate Institute of Medical Sciences (W.-Y.H.) and Department of Pharmacology (M.-H.Y., C.-J.T.), National Defense Medical Center,
Taipei, Taiwan; Genomics Research Center (M.H.), Academia Sinica, Taipei, Taiwan; Graduate Institute of Clinical Medicine (P.-J.L.) and Department
of Pharmacology (Y.-C.T.), National Cheng Kung University, Tainan, Taiwan; Institute of Biomedical Sciences (C.-J.T.), National Sun Yat-sun
University, Kaohsiung, Taiwan; Institute of Clinical Medicine (C.-J.T.), National Yang-Ming University, Taipei, Taiwan; Department of Internal
Medicine (H.-R.H.) and Department of Medical Education and Research (C.-J.T.), Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan.
The online-only Data Supplement, consisting of figures, is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.
107.746032/DC1.
Correspondence to Ching-Jiunn Tseng, MD, PhD, Department of Medical Education and Research, Kaohsiung Veterans General Hospital, 386,
Ta-Chung 1st Rd, Kaohsiung, Taiwan 813, Taiwan, Republic of China. E-mail [email protected]
© 2008 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org
DOI: 10.1161/CIRCULATIONAHA.107.746032
773
774
Circulation
February 12, 2008
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
subtypes have been demonstrated to play neuromodulatory roles
in the NTS.15,18,19 The A1 receptor is expressed in the central
nervous system ubiquitously and has been shown to inhibit
neuronal activity, whereas the A2A receptor is expressed at high
levels in limited regions of the brain that are primarily linked to
adenylyl cyclase activation.17,20 In the NTS, activation of the A1
and A2A receptors has been demonstrated to elicit dose-related
increases and decreases in BP, respectively.14,15 Together, these
studies suggest that the cardiovascular modulatory effects of
adenosine in the NTS are mediated predominantly by the A2A
receptor.
Studies including ours have demonstrated that the depressor
and bradycardic effects of adenosine in the NTS were attenuated
by a nitric oxide synthase (NOS) inhibitor.7,21 In addition,
microinjection of nitric oxide (NO) donors into subpostremal NTS
evokes a pattern of regional sympathetic responses that increases
in preganglionic adrenal nerve activity and decreases in renal,
postganglionic sympathetic nerve activity. Interestingly, these
sympathetic responses are similar to those evoked by stimulation
of the adenosine A2A receptor in the NTS.13 All of these results
indicate that the cardiovascular modulatory effects of adenosine
in the NTS might be accomplished by NOS activation and
subsequent NO release; however, the underlying signal mechanism between adenosine receptor activation and NO production
in the NTS remains unclear. Recently, it has been demonstrated
that activation of the adenosine A2A receptor can cause NO
release in endothelial cells.22,23 In addition, it has been reported
that adenosine induces endothelial NOS (eNOS) activation in a
Ca2⫹-insensitive manner that involves extracellular signalregulated kinase (ERK) 1 and 2 phosphorylation in human
umbilical vein endothelial cells.24 This raises the possibility that
the cardiovascular modulatory effects of adenosine in the NTS
might be mediated through activation of ERK1/2 and eNOS.
In the present experiments, we tested whether adenosine
could induce ERK1/2 and eNOS activation and whether
specific ERK1/2 and eNOS inhibitors are able to inhibit the
cardiovascular effects of adenosine in the NTS. In addition,
we examined whether a specific ERK inhibitor could attenuate the eNOS activation induced by adenosine in the NTS.
Our results suggest that a possible adenosine-ERK-eNOS
signaling pathway is well regulated in the modulation of
cardiovascular responses to adenosine in the NTS.
Methods
Experimental Chemicals
All experimental chemicals were purchased from Sigma-Aldrich (St
Louis, Mo) except when otherwise noted. Chemicals microinjected
into the NTS (eg, adenosine, PD98059, L-NIO, and vinyl-L-NIO)
were dissolved in reduced-serum medium (Opti-MEM I; Invitrogen,
Carlsbad, Calif).
Animals
Male Sprague-Dawley rats (weight 250 to 350 g) were obtained from
the National Science Council Animal Facility (Taipei, Taiwan) and
housed in the animal room of Kaohsiung Veterans General Hospital
(Kaohsiung, Taiwan). The rats were kept in individual cages in a room
where lighting was controlled (12-hour light/12-hour dark cycle), and
temperature was maintained at 23°C to 24°C. The rats were given
normal rat chow (Purina; St. Louis, Mo) and tap water ad libitum. All
animal research protocols had been approved by the Research Animal
Facility Committee of Kaohsiung Veterans General Hospital.
Intra-NTS Microinjection and
Hemodynamic Measurements
Rats were anesthetized with urethane (1.0 g/kg IP and 0.3 g/kg IV if
necessary). The preparation of animals for intra-NTS microinjection
and the methods used to locate the NTS have been described
previously.5 Briefly, a polyethylene cannula was inserted into the
femoral vein for fluid supplementation, and BP was measured via a
femoral-artery cannula by pressure transducer and polygraph (Gould,
Cleveland, Ohio). HR was monitored by a tachograph preamplifier
(Gould). To verify that the needle tip of the glass electrode was
exactly in the NTS, L-glutamate (0.154 nmol/60 nL) was microinjected. This would induce a characteristically abrupt decrease in BP
(⌬BP ⱖ35 mm Hg) and HR (⌬HR ⱖ50 bpm) if the needle tip was
located precisely in the NTS.
To investigate whether ERK 1 and ERK 2 participate in cardiovascular responses to adenosine in the NTS, 2 groups of rats (6 rats
per group) received microinjection of adenosine (0.77 nmol/60 nL)
in the unilateral NTS, and changes in BP and HR were measured.
The rats were then allowed to rest for at least 30 minutes until BP and
HR returned to basal levels. An MEK inhibitor, PD98059 (6 pmol/60
nL), or vehicle was microinjected into the NTS, then the same dose
of adenosine (0.77 nmol) was microinjected into the NTS 10, 30, 60,
and 90 minutes after PD98059 or vehicle administration. Cardiovascular parameters were recorded.
To investigate whether eNOS or neuronal NOS (nNOS) also
participates in the cardiovascular responses to adenosine in the NTS,
similar experimental procedures were performed to study the effects
of pretreatment with a selective eNOS inhibitor, L-NIO (L-N5-(1iminoethyl)-ornithine; Calbiochem, San Diego, Calif; 6 nmol/60 nL),
and a specific nNOS inhibitor, vinyl-L-NIO (L-N5-(1-imino-3butenyl)-ornithine; Alexis Biochemicals, San Diego, Calif; 600
pmol/60 nL), on the cardiovascular effects of adenosine in the NTS.
Western Blot Analysis
Four groups of rats (6 rats per group) were enrolled in this test. Rats
received intra-NTS microinjection with vehicle, PD98059, or L-NIO,
respectively, 10 minutes before adenosine injection; another group of
rats received microinjection with the A2A receptor agonist CGS21680
(24 pmol/60 nL). Ten to 20 minutes after adenosine injection, rats were
euthanized, and brainstems were excised immediately. The NTS was
dissected by a micropunch (1-mm inner diameter) from a brainstem
slice 1 mm thick at the level of the obex under a microscope. Total
protein extract was prepared by homogenization of the NTS in lysis
buffer with protease inhibitor cocktail and phosphatase inhibitor cocktail
and was incubated for 1 hour at 4°C. Protein extracts (20 ␮g per sample
assessed by BCA protein assay; Pierce) were subjected to 7.5% to 10%
SDS-Tris glycerin gel electrophoresis and transferred to a polyvinylidene difluoride membrane (GE Healthcare, Buckingamshire, UK). The
membrane was blocked with 5% nonfat milk in TBS/Tween 20 buffer
(10 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween 20, pH 7.4),
incubated with anti-p-ERK1/2T202/Y204 antibody (Cell Signaling Technology, Danvers, Mass), anti-ERK1/2 antibody (Cell Signaling Technology), anti-p-eNOSS1177 antibody (Cell Signaling Technology), and antieNOS antibody (BD Biosciences, San Jose, Calif) at 1:1000 in PBST
with 5% BSA, and incubated at 4°C overnight. Peroxidase-conjugated
anti-mouse or anti-rabbit antibody (1:5000) was used as secondary
antibody. The membrane was detected with an ECL-Plus detection kit
(GE Healthcare) on film. The films were scanned by photo scanner
(4490, Epson, Long Beach, Calif) and were analyzed with NIH Image
densitometry analysis software (National Institutes of Health, Bethesda,
Md).
Immunohistochemistry Analysis
Adenosine (0.77 nmol/60 nL) was administered into the NTS with or
without PD98059 (6 nmol/60 nL) pretreatment. Ten to 20 minutes
after adenosine microinjection, brainstems were excised and fixed
with 4% formaldehyde. Paraffin-embedded serial sections were cut
at 5-␮m thickness. The sections were dewaxed, quenched in 3%
H2O2/methanol, antigen-retrieved in citric buffer (10 mmol/L, pH
6.0), blocked in 5% goat serum, and incubated with anti-phospho-
Ho et al
Adenosine Activates ERK1/2 and eNOS in the NTS
775
ERK1/2T202/Y204 antibody (1:100; Cell Signaling Technology) or
anti-phospho-eNOSS1177 antibody (1:50; Zymed Laboratories, South
San Francisco, Calif) at 4°C overnight. Afterward, sections were
incubated in biotinylated secondary antibody (1:200; Vector Laboratories, Burlingame, Calif) for 1 hour and in AB complex (1:100)
for 30 minutes at room temperature. Sections were visualized with a
DAB substrate kit (Vector Laboratories) and counterstained with
hematoxylin. The sections were then photographed with a microscope equipped with a charge-coupled device camera.
Statistical Analysis
Group data are expressed as mean⫾SEM. The paired Student t test
(for comparisons of cardiovascular parameters before and after
pretreatment), unpaired Student t test (for control and study group
comparisons), or 1-way ANOVA with Scheffé post hoc comparison
was applied to compare group differences. Differences with P⬍0.05
were considered significant.
The authors had full access to the data and take full responsibility
for its integrity. All authors have read and agree to the manuscript as
written.
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Results
Effects of MEK Inhibitor on Cardiovascular
Responses to Adenosine in the NTS
To assess whether ERK1 and ERK2 participates in the
cardiovascular modulatory effects of adenosine in the NTS,
we investigated the inhibitory effect of an MEK inhibitor,
PD98059, on the cardiovascular effects of adenosine in the
NTS. Microinjection of adenosine into unilateral NTS induced hypotension and bradycardia. The depressor effect of
single-dose adenosine lasted for more than 5 minutes, and the
bradycardic effect lasted for more than 15 minutes (Figure
1A). These results were consistent with our previous findings.7,8 Pretreatment with PD98059 did not elicit significant
cardiovascular responses (Figure 1A). Interestingly, the depressor and bradycardic responses of the same dose of
adenosine in the NTS were attenuated 10 minutes after
PD98059 treatment (⫺37.9⫾2.9 versus ⫺21.7⫾3.1 mm Hg
and ⫺78.6⫾13.2 versus ⫺52.9⫾12.0 bpm, respectively;
P⬍0.05, paired t test; n⫽6; Figure 1A and 1B). The cardiovascular effects of adenosine in the NTS recovered gradually
30 minutes after PD98059 treatment (Figure 1A). To clarify
the specificity of the inhibitory effects of PD98059 in the
NTS, we tested the effect of PD98059 on the cardiovascular
response of L-glutamate in the NTS. The results revealed that
pretreatment with PD98059 did not alter the depressor and
bradycardic effects of L-glutamate (Data Supplement Figure
I), which is the major neurotransmitter in the NTS.
Effects of eNOS Inhibitor and nNOS Inhibitor on
Cardiovascular Responses to Adenosine in the NTS
Our previous studies suggested that cardiovascular modulatory
responses to adenosine are accomplished through activation of
NOS in the NTS.7 To identify which constitutive NOS contributes to these cardiovascular responses to adenosine in the NTS,
we investigated the effects of a selective eNOS inhibitor, L-NIO,
and an nNOS-specific inhibitor, vinyl-L-NIO, on the cardiovascular responses induced by adenosine in the NTS. Ten minutes
after L-NIO (6 nmol/60 nL) pretreatment, hypotensive and
bradycardic responses to the same dose of adenosine were
attenuated significantly (⫺36.0⫾1.7 versus ⫺15.8⫾2.8 mm Hg
and ⫺79.3⫾7.7 versus ⫺29.3⫾8.5 bpm, respectively; P⬍0.05;
Figure 1. The cardiovascular effects of adenosine in the NTS
before and after administration of the MEK inhibitor PD98059. A,
Representative tracings demonstrate the cardiovascular effects of
adenosine (0.77 nmol) in unilateral NTS before and 10 minutes
after pretreatment with PD98059 (6 pmol) in anesthetized rats. The
depressor and bradycardic effects of adenosine were significantly
attenuated by PD98059. As PD98059 washed out, the cardiovascular effects of adenosine recovered. B, Graph reveals the effects
of pretreated PD98059 on mean BP (MBP) and HR after microinjection of adenosine into unilateral NTS. Both the MBP and HR
effects of adenosine were significantly attenuated by PD98059.
Adenosine was administered 10 minutes after vehicle or PD98059.
Values are shown as mean difference⫾SEM, n⫽6. *P⬍0.05 vs
control group. ADO indicates adenosine; PD, PD98059.
Figure 2A and 2B). As the effects of L-NIO washed out, the
hypotensive and bradycardic effects of adenosine recovered
(Figure 2A). The almost 60% inhibition in adenosine-mediated
hypotensive and bradycardic responses caused by the eNOS
inhibitor L-NIO indicated that eNOS activity plays a role in the
downstream signaling of adenosine. In contrast, pretreatment
with vinyl-L-NIO (600 pmol/60 nL) did not diminish the
depressor (⫺38.6⫾2.8 versus ⫺38.2⫾2.7 mm Hg, P⬎0.05,
n⫽6) and bradycardic effects (⫺84.6⫾12.4 versus ⫺94.4⫾9.3
bpm, P⬎0.05, n⫽6) of adenosine in the NTS (Data Supplement
Figure II). These results indicate that nNOS did not play a role
in adenosine-mediated hypotensive and bradycardic responses in
the NTS.
Adenosine Induced eNOS Phosphorylation in
the NTS
Using a specific pharmacological inhibitor, we demonstrated
that eNOS but not nNOS was involved in the adenosinemediated hypotensive and bradycardic responses in the NTS.
776
Circulation
February 12, 2008
ERK1/2 was determined by immunoblotting and immunohistochemistry staining with an antibody specifically against
phospho-ERK1/2T202/Y204. Immunoblotting analysis of protein
extract of adenosine-treated NTS revealed that ERK1/2 phosphorylation was increased compared with that of the control
group (1.8⫾0.1 versus 1.0⫾0.1 for ERK1 and 1.8⫾0.2
versus 1.0⫾0.1 for ERK2, P⬍0.05; n⫽6; Figure 4A and 4B).
To confirm that adenosine induces ERK1/2 phosphorylation
in situ, paraffin sections of the brainstem were subjected to
immunohistochemistry staining analysis with phosphoERK1/2T202/Y204 antibodies. Figure 4C shows that there were
more phospho-ERK1/2–positive neuronal cells in the NTS of
the adenosine-administered side than in the control side
(frame c versus frame d). The results of immunoblotting and
immunohistochemistry staining were consistent and indicated
that adenosine can induce ERK1/2 phosphorylation in situ.
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
MEK1/2 Inhibitor Attenuated Adenosine-Induced
eNOS Phosphorylation in the NTS
Figure 2. The cardiovascular effects of adenosine in the NTS
before and after administration of an eNOS selective inhibitor,
L-NIO. A, Representative tracings demonstrate cardiovascular
effects of microinjection of adenosine (0.77 nmol) into unilateral
NTS before and 10 minutes after pretreatment with L-NIO (6 nmol)
in anesthetized rats. The depressor and bradycardic effects of
adenosine were significantly attenuated by L-NIO. As L-NIO
washed out, the cardiovascular responses of adenosine recovered.
B, Graph reveals the effects of pretreated L-NIO on MBP and HR
after microinjection of adenosine into unilateral NTS. Both the MBP
and HR effects of adenosine were significantly attenuated by L-NIO
(*P⬍0.05 vs vehicle group, n⫽6). Values are shown mean
difference⫾SEM. ADO indicates adenosine.
We further investigated whether adenosine may induce eNOS
phosphorylation in the NTS. The phosphorylation of eNOS
was determined by immunoblotting and immunohistochemistry staining with antibodies specifically against phosphoeNOSS1177. The results of immunoblotting analysis demonstrated that adenosine could increase eNOS phosphorylation
in the NTS (1.9⫾0.2 versus 1.0⫾0.3, P⬍0.05, n⫽6; Figure
3A and 3B). To confirm that intra-NTS adenosine administration induces eNOS phosphorylation, paraffin sections of
the brainstem were subjected to immunohistochemistry staining. Immunohistochemistry stain against phospho-eNOSS1177
of the NTS revealed the adenosine-administered side had
more phospho-eNOS S1177–positive cells than the control side
(Figure 3C, frame c versus frame d). The immunohistochemistry results were consistent with those of immunoblotting.
Adenosine Induced ERK1/2 Phosphorylation in
the NTS
PD98059 inhibited the depressor and bradycardic effects of
adenosine in NTS, which suggested that ERK1 and ERK2
contribute to cardiovascular responses to adenosine in the
NTS. We further investigated whether adenosine can induce
ERK1/2 phosphorylation in the NTS. Phosphorylation of
To test whether ERK1/2 may induce eNOS phosphorylation,
we investigated the inhibitory effect of PD98059 on eNOS
phosphorylation in the NTS. The results from the immunoblotting analysis demonstrated that pretreatment with the
MEK inhibitor PD98059 could attenuate adenosine-induced
eNOS phosphorylation in the NTS (0.8⫾0.2 versus 1.9⫾0.2,
P⬍0.01; Figure 3A and 3B). These results suggested that
ERK-eNOS signaling served as the downstream molecules of
adenosine-mediated cardiovascular responses in the NTS.
This conclusion was further supported by the results of
immunohistochemistry staining against phospho-eNOSS1177,
which demonstrated that the number of phospho-eNOS–
positive cells on the PD98059-pretreated side was much
less than on the vehicle-pretreated side (Figure 3C, frame
g versus frame h).
Adenosine Activates ERK1/2 and eNOS via the
Adenosine A2A Receptor in the NTS
It was reported that the cardiovascular modulatory effects of
adenosine in the NTS are mediated predominantly through
the A2A receptor. The above results demonstrated that adenosine could induce ERK1/2 and eNOS phosphorylation in the
NTS. Therefore, we tested whether the induction of ERK1/2
and eNOS phosphorylation is via the adenosine A2A receptor
in the NTS. Immunoblotting of NTS treated with the A2A
agonist CGS21680 revealed increases in ERK1/2 phosphorylation (2.1⫾0.5 versus 1.0⫾0.4 for ERK1 and 2.0⫾0.1
versus 1.0⫾0.2 for ERK2, P⬍0.05; n⫽6; Figure 5A).
eNOSS1177 phosphorylation also increased in NTS that received CGS21680 microinjection (1.8⫾0.2 versus 1.0⫾0.2,
P⬍0.05; n⫽6; Figure 5B). Together, these results support the
idea that signal molecule activation by adenosine in the NTS
occurs predominantly through the A2A receptor.
Discussion
Adenosine has been demonstrated to play an important role in
cardiovascular modulation in the NTS.1 Our previous experiments demonstrated that the depressor and bradycardic
effects of adenosine in the NTS were blocked by the
nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester,
Ho et al
Adenosine Activates ERK1/2 and eNOS in the NTS
777
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Figure 3. Quantitative analysis of phospho-eNOS in the NTS after microinjection of adenosine with or without PD98059. A, Immunoblotting revealed that phosphorylated eNOSS1177 was increased in the NTS that received adenosine. Phosphorylation of eNOS was
blocked by pretreatment with PD98059 10 minutes before adenosine microinjection. B, Densitometric analysis of immunoblotting of
phospho-eNOSS1177 showed that adenosine induced an almost 2-fold increase in phospho-eNOSS1177 compared with the control group.
Phospho-eNOSS1177 was decreased by PD98059 pretreatment. Values are shown as mean⫾SEM, n⫽6. *P⬍0.05, control vs adenosine
group, #P⬍0.01, adenosine vs PD98059⫹adenosine group. C, Immunohistochemistry of the brainstem for phospho-eNOSS1177 demonstrated microinjection of adenosine into the NTS induced eNOSS1177 phosphorylation in situ (c vs d). Pretreatment with PD98059 significantly reduced eNOSS1177 phosphorylation in the NTS (g vs h). PD98059 or vehicle was administered 10 minutes before adenosine.
Scale bar⫽200 ␮m. ADO indicates adenosine.
which suggested that cardiovascular modulation by adenosine
was mediated by activation of NOS and subsequent NO
release.7 Results of the present study confirm and extend our
previous observations that eNOS but not nNOS participates
in the signal mechanism effect of adenosine on cardiovascular modulation in the NTS. The present data further indicate
that ERK1 and ERK2 are the intermediate signal molecules
that activate eNOS in the NTS. The present study demonstrated that adenosine-ERK-eNOS signaling exists and plays
an important role in modulation of cardiovascular responses
to adenosine in the NTS.
The ERK cascade is distinguished by a characteristic core
cascade of 3 kinases. The first kinase is a so-called MAP kinase
kinase kinase (MAPKKK; Raf-1 and B-Raf), which activates the
second kinase, a MAP kinase kinase (MAPKK; MEK), by
serine/threonine phosphorylation. MEK in turn activates
ERK1/2 by phosphorylation of both threonine and tyrosine
residues.25 The ERK cascade was originally discovered to be a
critical regulator of cell division and differentiation; however,
ERK1 and ERK2, as well as their upstream regulators and many
of their downstream targets, are highly expressed in mature
neurons.26 This raises the question of whether ERK1 and ERK2
not only regulate neuronal development but also play regulatory
roles in mature neurons in response to various physiological
stimuli. A number of studies have suggested the regulatory roles
of ERK1/2 in mature neurons. First, ERK1 and ERK2 have been
demonstrated to be phosphorylated and activated in neurons in
response to excitatory glutamatergic signaling, which controls
many forms of synaptic plasticity that are thought to underlie
higher brain processes such as learning and memory.26 Second,
activation of ERK1/2 has been identified in central neurons that
respond to specific stimuli, such as nociception, unfamiliar taste,
synaptic plasticity, and fear memory consolidation.27–29 Third,
peripheral administration of cholecystokinin and hydralazine can
induce ERK1/2 phosphorylation in the NTS.30,31 Finally, experiments on rat hippocampus brain slices revealed that a wide
variety of G-protein– coupled receptor neurotransmitters, including N-methyl-D-aspartate and adrenergic, dopamine, and muscarinic acetylcholine receptors, activate ERK1/2 by protein
kinase A or protein kinase C.32 All of this suggests the possibility
of a broad physiological role for the ERK cascade in the adult
central nervous system. Indeed, in the present study, we demonstrated that microinjection of adenosine into the NTS induced
ERK1/2 phosphorylation (Figure 4) and that blockade of
ERK1/2 activation by PD98059 attenuated the depressor and
bradycardic effects of adenosine (Figure 1). The present study
provides the first evidence to demonstrate the ERK1/2 activation
is involved in cardiovascular modulation in the NTS.
It would be interesting to know how adenosine activates
ERK1/2 in mature neurons. The activated adenosine A2A
receptor activates adenylyl cyclase, which in turn increases
cAMP.17 Interestingly, cAMP may either inhibit or activate
778
Circulation
February 12, 2008
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Figure 4. Quantitative analysis of phospho-ERK1/2 in the NTS with adenosine treatment. A, Phosphorylated ERK1/2T202/Y204 was increased in
the adenosine-microinjected group compared with the control group. B, Densitometric analysis of immunoblotting of phospho-ERK1/2T202/Y204
revealed adenosine induced an almost 2-fold increase in ERK1/2 phosphorylation. Values are mean⫾SEM, n⫽6. *P⬍0.05, control vs adenosine group. C, Immunohistochemistry stain of the brainstem for phospho-ERK1/2T202/Y204 demonstrated microinjection of adenosine into the
NTS induced ERK1/2T202/Y204 phosphorylation in situ (c vs d). Scale bar⫽200 ␮m. ADO indicates adenosine.
ERK1/2 in a cell-specific manner.33 Emerging new data
revealed that the determinant of whether cAMP activates
or inhibits the ERK cascade is B-Raf.34,35 In B-Raf–
expressing cells, cAMP may activate B-Raf by complex
mechanisms, and the activated B-Raf subsequently activates the MEK-ERK cascade.33 A convincing model of
how cAMP activates B-Raf is not currently available, but
the accumulated data suggest the involvement of RAS,
Rap1, Src, protein kinase A, and 14-3-3 proteins.36 Interestingly, B-Raf is the major RAF isoform expressed in
neurons.37 Therefore, we infer that adenosine activates
ERK1/2 in the NTS by activation of the A2A receptor, an
increase in cAMP, activation of B-Raf, and the ensuing
phosphorylation and activation of MEK.
Figure 5. Adenosine A2A receptor agonist, CGS21680, induced ERK1/2 and eNOS phosphorylation in the NTS. A, Immunoblotting revealed
there were at least 2-fold increases in phospho-ERK1 and phospho-ERK2 in the NTS that received CGS21680. B, CGS21680 also induced
an almost 2-fold increase in phospho-eNOS in the NTS. Values are mean⫾SEM, n⫽6. *P⬍0.05, control vs CGS21680 group.
Ho et al
Adenosine Activates ERK1/2 and eNOS in the NTS
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Signaling molecules that regulate eNOS activity include
PI3K/Akt, cyclic nucleotide-dependent kinases (protein kinase A and protein kinase G), protein kinase C, and ERKs.38
In the present study, we demonstrated that PD98059 inhibited
eNOSS1177 phosphorylation (Figure 3A and 3B). This result
suggests that adenosine may phosphorylate eNOS through
activation of MEK/ERK cascades in the NTS. This result is
further supported by reports that inhibition of ERK1/2 activation attenuated the eNOS phosphorylation that was induced
by estrogen and vascular endothelial growth factor.39 – 41
Although it has been reported for years that ERKs could
phosphorylate and activate eNOS,39 the exact signaling mechanisms that couple the activated ERK1/2 to eNOS activation
remained uncertain.
eNOS, originally identified in vascular endothelium, is
expressed in several nonendothelial cell types, including
neurons of various rat brain regions. Although the physiological functions of eNOS in cardiovascular modulation in the
NTS remain controversial, several studies, including the
present study, demonstrated that eNOS participates in cardiovascular modulation in the NTS. By reverse-transcription
polymerase chain reaction, Waki et al42 demonstrated that
endogenous eNOS mRNA is expressed in the NTS of adult
Wistar-Kyoto and spontaneously hypertensive rats. Suppression of the endogenous eNOS by overexpression of
dominant-negative eNOS in bilateral NTS increased spontaneous baroreceptor reflex gain in conscious Wistar rats and
decreased BP in mature spontaneously hypertensive rats.42,43
In the present study, our data revealed that eNOS not only
exists in the NTS (Figure 3A and 3C) but that it also mediates
cardiovascular modulation by adenosine (Figure 2). Microinjection of adenosine into the NTS induced eNOS phosphorylation in situ (Figure 3C).
Scislo et al21 reported the differential role of NO in regional
sympathetic responses to stimulation of NTS A2A adenosine
receptor. They demonstrated that the nNOS-selective antagonist
TRIM [1-(2-trifluoromethylphenyl) imidazole; 20 nmol/100 nL]
reversed normal depressor responses and significantly attenuated
the A2A receptor– elicited decreases in HR and renal sympathetic
nerve activity. They concluded that the hemodynamic and
sympathoinhibitory responses caused by stimulation of the NTS
A2A receptor were predominantly mediated by nNOS.21 Conversely, in the present study, only an eNOS-selective antagonist
attenuated the depressor and bradycardic effect of adenosine
(Figure 2B), whereas the nNOS-specific antagonist did not (Data
Supplement Figure II). TRIM is a potent inhibitor of nNOS (IC50
of 2.8 ␮mol/L) and a relatively weak inhibitor of eNOS (IC50
1057.5 ␮mol/L).44 The concentrations of TRIM used by Scislo
and coworkers21 (200 mmol/L) may have been too high, and
therefore, TRIM might have lost its nNOS selectivity and could
no longer discriminate which NOS mediated the cardiovascular
modulatory effects of adenosine in the NTS. In the present study,
we not only demonstrated the pharmacological effects of eNOS
inhibitors on cardiovascular parameters (Figure 2), we also
demonstrated eNOSS1177 phosphorylation in situ in the NTS by
immunohistochemistry staining (Figure 3C). These evidences
confirm that cardiovascular modulation by adenosine in the NTS
is predominantly through eNOS activation, with only a small
contribution, if any, of nNOS.
779
In conclusion, we present a novel adenosine-ERK-eNOS
signaling pathway in the NTS that allows us to dissect the
adenosine signaling pathway related to the production of NO
in the regulation of BP and HR. Our results suggest a possible
interaction between ERK1/2 and eNOS in the NTS. The
present data demonstrate that adenosine modulates central
cardiovascular control via activation of ERK1/2 and its
downstream eNOS.
Acknowledgments
The authors gratefully acknowledge the technical assistance of
Yi-Shan Wu and Ya-Chun Yang.
Sources of Funding
This work was supported by funding from the National Science
Council (NSC 94-2815-C-075B-001-B; NSC 95-2320-B-075– 002)
to Dr C.-J. Tseng; Kaohsiung Veterans General Hospital (VGHKS96-108) to Dr Hwang; and Zuoying Armed Forces General Hospital
(ZAFGH-9502) to Dr Ho.
Disclosures
None.
References
1. Tseng CJ, Biaggioni I, Appalsamy M, Robertson D. Purinergic receptors
in the brainstem mediate hypotension and bradycardia. Hypertension.
1988;11:191–197.
2. Tseng CJ, Ger LP, Lin HC, Tung CS. Attenuated cardiovascular response
to adenosine in the brain stem nuclei of spontaneously hypertensive rats.
Hypertension. 1995;25:278 –282.
3. Mosqueda-Garcia R, Tseng CJ, Appalsamy M, Robertson D. Cardiovascular effects of microinjection of angiotensin II in the brainstem of
renal hypertensive rats. J Pharmacol Exp Ther. 1990;255:374 –381.
4. Tseng CJ, Chou LL, Ger LP, Tung CS. Cardiovascular effects of angiotensin III in brainstem nuclei of normotensive and hypertensive rats.
J Pharmacol Exp Ther. 1994;268:558 –564.
5. Tseng CJ, Liu HY, Lin HC, Ger LP, Tung CS, Yen MH. Cardiovascular
effects of nitric oxide in the brain stem nuclei of rats. Hypertension.
1996;27:36 – 42.
6. Lo WJ, Liu HW, Lin HC, Ger LP, Tung CS, Tseng CJ. Modulatory
effects of nitric oxide on baroreflex activation in the brainstem nuclei of
rats. Chin J Physiol. 1996;39:57– 62.
7. Lo WC, Jan CR, Wu SN, Tseng CJ. Cardiovascular effects of nitric oxide
and adenosine in the nucleus tractus solitarii of rats. Hypertension. 1998;
32:1034 –1038.
8. Lin CH, Lo WC, Hsiao M, Tseng CJ. Interaction of carbon monoxide and
adenosine in the nucleus tractus solitarii of rats. Hypertension. 2003;42:
380 –385.
9. Lin CH, Lo WC, Hsiao M, Tung CS, Tseng CJ. Interactions of carbon
monoxide and metabotropic glutamate receptor groups in the nucleus
tractus solitarii of rats. J Pharmacol Exp Ther. 2004;308:1213–1218.
10. Lo WC, Hsiao M, Tung CS, Tseng CJ. The cardiovascular effects of nitric
oxide and carbon monoxide in the nucleus tractus solitarii of rats.
J Hypertens. 2004;22:1182–1190.
11. Lo WC, Lu PJ, Ho WY, Hsiao M, Tseng CJ. Induction of heme oxygenase-1 is involved in carbon monoxide-mediated central cardiovascular
regulation. J Pharmacol Exp Ther. 2006;318:8 –16.
12. Huang HN, Lu PJ, Lo WC, Lin CH, Hsiao M, Tseng CJ. In situ Akt
phosphorylation in the nucleus tractus solitarii is involved in central
control of blood pressure and heart rate. Circulation. 2004;110:
2476 –2483.
13. Scislo TJ, O’Leary DS. Purinergic mechanisms of the nucleus of the
solitary tract and neural cardiovascular control. Neurol Res. 2005;27:
182–194.
14. Phillis JW, Scislo TJ, O’Leary DS. Purines and the nucleus tractus
solitarius: effects on cardiovascular and respiratory function. Clin Exp
Pharmacol Physiol. 1997;24:738 –742.
15. Barraco RA, Phillis JW. Subtypes of adenosine receptors in the brainstem
mediate opposite blood pressure responses. Neuropharmacology. 1991;
30:403– 407.
780
Circulation
February 12, 2008
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
16. Mosqueda-Garcia R, Tseng CJ, Appalsamy M, Robertson D. Modulatory
effects of adenosine on baroreflex activation in the brainstem of normotensive rats. Eur J Pharmacol. 1989;174:119 –122.
17. Dunwiddie TV, Masino SA. The role and regulation of adenosine in the
central nervous system. Annu Rev Neurosci. 2001;24:31–55.
18. Scislo TJ, O’Leary DS. Activation of A2a adenosine receptors in the
nucleus tractus solitarius inhibits renal but not lumbar sympathetic nerve
activity. J Auton Nerv Syst. 1998;68:145–152.
19. Scislo TJ, O’Leary DS. Mechanisms mediating regional sympathoactivatory responses to stimulation of NTS A(1) adenosine receptors.
Am J Physiol. 2002;283:H1588 –H1599.
20. Ribeiro JA, Sebastiao AM, de Mendonca A. Adenosine receptors in the
nervous system: pathophysiological implications. Prog Neurobiol. 2002;
68:377–392.
21. Scislo TJ, Tan N, O’Leary DS. Differential role of nitric oxide in regional
sympathetic responses to stimulation of NTS A2a adenosine receptors.
Am J Physiol. 2005;288:H638 –H649.
22. Sobrevia L, Yudilevich DL, Mann GE. Activation of A2-purinoceptors by
adenosine stimulates L-arginine transport (system y⫹) and nitric oxide
synthesis in human fetal endothelial cells. J Physiol. 1997;499:135–140.
23. Li J, Fenton RA, Wheeler HB, Powell CC, Peyton BD, Cutler BS,
Dobson JG Jr. Adenosine A2a receptors increase arterial endothelial cell
nitric oxide. J Surg Res. 1998;80:357–364.
24. Wyatt AW, Steinert JR, Wheeler-Jones CP, Morgan AJ, Sugden D,
Pearson JD, Sobrevia L, Mann GE. Early activation of the p42/p44MAPK
pathway mediates adenosine-induced nitric oxide production in human
endothelial cells: a novel calcium-insensitive mechanism. FASEB J.
2002;16:1584 –1594.
25. Sweatt JD. The neuronal MAP kinase cascade: a biochemical signal integration system subserving synaptic plasticity and memory. J Neurochem.
2001;76:1–10.
26. Thomas GM, Huganir RL. MAPK cascade signalling and synaptic plasticity. Nat Rev. 2004;5:173–183.
27. Berman DE, Hazvi S, Neduva V, Dudai Y. The role of identified neurotransmitter systems in the response of insular cortex to unfamiliar taste:
activation of ERK1–2 and formation of a memory trace. J Neurosci.
2000;20:7017–7023.
28. Schafe GE, Atkins CM, Swank MW, Bauer EP, Sweatt JD, LeDoux JE.
Activation of ERK/MAP kinase in the amygdala is required for memory
consolidation of pavlovian fear conditioning. J Neurosci. 2000;20:
8177– 8187.
29. Dai Y, Iwata K, Fukuoka T, Kondo E, Tokunaga A, Yamanaka H,
Tachibana T, Liu Y, Noguchi K. Phosphorylation of extracellular signalregulated kinase in primary afferent neurons by noxious stimuli and its
involvement in peripheral sensitization. J Neurosci. 2002;22:7737–7745.
30. Sutton GM, Patterson LM, Berthoud HR. Extracellular signal-regulated
kinase 1/2 signaling pathway in solitary nucleus mediates
cholecystokinin-induced suppression of food intake in rats. J Neurosci.
2004;24:10240 –10247.
31. Springell DA, Costin NS, Pilowsky PM, Goodchild AK. Hypotension and
short-term anaesthesia induce ERK1/2 phosphorylation in autonomic
nuclei of the brainstem. Eur J Neurosci. 2005;22:2257–2270.
32. Roberson ED, English JD, Adams JP, Selcher JC, Kondratick C, Sweatt
JD. The mitogen-activated protein kinase cascade couples PKA and PKC
to cAMP response element binding protein phosphorylation in area CA1
of hippocampus. J Neurosci. 1999;19:4337– 4348.
33. Stork PJ, Schmitt JM. Crosstalk between cAMP and MAP kinase signaling in the regulation of cell proliferation. Trends Cell Biol. 2002;12:
258 –266.
34. Vossler MR, Yao H, York RD, Pan MG, Rim CS, Stork PJ. cAMP
activates MAP kinase and Elk-1 through a B-Raf- and Rap1-dependent
pathway. Cell. 1997;89:73– 82.
35. Dugan LL, Kim JS, Zhang Y, Bart RD, Sun Y, Holtzman DM, Gutmann
DH. Differential effects of cAMP in neurons and astrocytes: role of B-raf.
J Biol Chem. 1999;274:25842–25848.
36. Dumaz N, Marais R. Integrating signals between cAMP and the RAS/
RAF/MEK/ERK signalling pathways: based on the anniversary prize of
the Gesellschaft für Biochemie und Molekularbiologie Lecture delivered
on 5 July 2003 at the Special FEBS Meeting in Brussels. FEBS J.
2005;272:3491–3504.
37. Morice C, Nothias F, Konig S, Vernier P, Baccarini M, Vincent JD,
Barnier JV. Raf-1 and B-Raf proteins have similar regional distributions
but differential subcellular localization in adult rat brain. Eur J Neurosci.
1999;11:1995–2006.
38. Cale JM, Bird IM. Inhibition of MEK/ERK1/2 signalling alters endothelial nitric oxide synthase activity in an agonist-dependent manner.
Biochem J. 2006;398:279 –288.
39. Chen Z, Yuhanna IS, Galcheva-Gargova Z, Karas RH, Mendelsohn ME,
Shaul PW. Estrogen receptor alpha mediates the nongenomic activation
of endothelial nitric oxide synthase by estrogen. J Clin Invest. 1999;103:
401– 406.
40. Chen DB, Bird IM, Zheng J, Magness RR. Membrane estrogen receptordependent extracellular signal-regulated kinase pathway mediates acute
activation of endothelial nitric oxide synthase by estrogen in uterine
artery endothelial cells. Endocrinology. 2004;145:113–125.
41. Feliers D, Chen X, Akis N, Choudhury GG, Madaio M, Kasinath BS.
VEGF regulation of endothelial nitric oxide synthase in glomerular endothelial cells. Kidney Int. 2005;68:1648 –1659.
42. Waki H, Murphy D, Yao ST, Kasparov S, Paton JF. Endothelial NO
synthase activity in nucleus tractus solitarii contributes to hypertension in
spontaneously hypertensive rats. Hypertension. 2006;48:644 – 650.
43. Waki H, Kasparov S, Wong LF, Murphy D, Shimizu T, Paton JF. Chronic
inhibition of endothelial nitric oxide synthase activity in nucleus tractus
solitarii enhances baroreceptor reflex in conscious rats. J Physiol. 2003;
546:233–242.
44. Handy RL, Harb HL, Wallace P, Gaffen Z, Whitehead KJ, Moore PK.
Inhibition of nitric oxide synthase by 1-(2-trifluoromethylphenyl) imidazole (TRIM) in vitro: antinociceptive and cardiovascular effects.
Br J Pharmacol. 1996;119:423– 431.
CLINICAL PERSPECTIVE
The sympathetic nervous system has moved toward center stage in cardiovascular medicine. Studies have demonstrated
that sympathetic overactivity characterizes the hypertensive state and participates in the development, maintenance, and
progression of elevated blood pressure. The importance of sympathetic overactivity in heart failure progression and
mortality and in the generation of ventricular arrhythmias is now well established, and a central nervous system–mean
arterial pressure (CNS-MAP) set-point theory has recently been proposed. It has been hypothesized that hypertension
occurs as the result of a primary shift of the CNS-MAP set point to a higher operating pressure, which results in increased
sympathetic nerve activity. The nucleus tractus solitarius (NTS), located at the dorsal part of the brainstem, was discovered
to be an important sympathetic nervous system integral center in the central nervous system. Adenosine, a ubiquitously
distributed neuromodulator, was found to participate in sympathetic activity regulation in the NTS. In the present study,
we investigated the signaling mechanism of adenosine with regard to cardiovascular modulation in the NTS and found that
the MEK-ERK (mitogen-activated protein kinase– extracellular signal-regulated kinase) cascade, which was originally
discovered to be a critical regulator of cell division and differentiation, participates in adenosine-mediated central
cardiovascular control. In addition, we also demonstrated that endothelial nitric oxide synthase, which was originally
identified in the vascular endothelium, is present and participates in cardiovascular regulation in the NTS. Further
investigation of the molecular mechanisms involved in sympathetic nervous activity modulation might elucidate the
pathogenesis of the CNS-MAP set-point shift and sympathetic overactivity in essential hypertension.
Adenosine Modulates Cardiovascular Functions Through Activation of Extracellular
Signal-Regulated Kinases 1 and 2 and Endothelial Nitric Oxide Synthase in the Nucleus
Tractus Solitarii of Rats
Wen-Yu Ho, Pei-Jung Lu, Michael Hsiao, Hwong-Ru Hwang, Yu-Chou Tseng, Mao-Hsiung
Yen and Ching-Jiunn Tseng
Downloaded from http://circ.ahajournals.org/ by guest on June 16, 2017
Circulation. 2008;117:773-780; originally published online January 28, 2008;
doi: 10.1161/CIRCULATIONAHA.107.746032
Circulation is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2008 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7322. Online ISSN: 1524-4539
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circ.ahajournals.org/content/117/6/773
Data Supplement (unedited) at:
http://circ.ahajournals.org/content/suppl/2008/02/08/CIRCULATIONAHA.107.746032.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation 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 Circulation is online at:
http://circ.ahajournals.org//subscriptions/