© 1995 American Heart Association, Inc.
Articles |
Calcitonin Gene-Related Peptide Mediates Acetylcholine-Induced Endothelium-Independent Vasodilation in Mesenteric Resistance Blood Vessels of the Rat
From the First Department of Internal Medicine (M.T., T.E.) and the Department of Pharmacology (A.W.), Miyazaki (Japan) Medical College, and the Department of Hospital Pharmacy (H.K.), Okayama (Japan) University Medical School.
Correspondence to Hiromu Kawasaki, PhD, Department of Hospital Pharmacy, Okayama University Medical School, 2-5-1 Shikata-Cho, Okayama 700, Japan.
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Abstract |
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Abstract The role of calcitonin gene-related peptide (CGRP)–containing vasodilator nerves in acetylcholine chloride (ACh)–induced vasodilation was studied in the perfused mesenteric vascular bed isolated from the rat. Bolus infusions of ACh at smaller doses (0.1 and 1 nmol) produced rapid and short-lived vasodilation. However, larger doses (10 and 100 nmol) of ACh caused a rapid and subsequent long-lasting vasodilator response in which the duration of vasodilation was prolonged in a concentration-dependent manner. Pretreatment with capsaicin (1 µmol/L for 20 minutes) significantly shortened the duration of vasodilator response to ACh but did not affect the initial rapid phase of ACh-induced vasodilation. Chemical removal of the vascular endothelium by perfusion with sodium deoxycholate (1.75 to 1.80 mg/mL) for 30 seconds and subsequent treatment with N
-nitro-L-arginine
(100 µmol/L) to inhibit nitric oxide synthesis abolished the initial
rapid vasodilator action of ACh at any given concentration. However, in
the same preparation, increasing concentrations (from 1 to 1000 nmol)
of ACh produced only the long-lasting vasodilator responses in a
concentration-dependent manner. This long-lasting vasodilator response
to ACh infusion was abolished by capsaicin pretreatment (1 µmol/L),
human CGRP[8-37] (CGRP receptor antagonist, 1 µmol/L), and atropine
(muscarinic ACh receptor antagonist, 1, 10, and 100 nmol/L) but not by
hexamethonium (nicotinic ACh receptor antagonist, 1 and 10 µmol/L).
In the preparations without endothelium, the bolus infusion of ACh (300
nmol for 30 seconds) evoked a long-lasting vasodilation and release of
CGRP-like immunoreactivities into the perfusate. These results suggest
that the ACh-induced vasorelaxation consists of two elements: an
initial transient endothelium-dependent component and a
secondary long-lasting endothelium-independent
component. Moreover, ACh activates muscarinic receptors located on
CGRP-containing neurons to release CGRP, which then acts at CGRP
receptors on vascular smooth muscles to cause the
endothelium-independent vasodilation.
Key Words: acetylcholine • endothelium-independent vasodilation • calcitonin gene-related peptide • muscarinic receptor • rat mesenteric vascular bed
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Since the initial work by Furchgott and Zawadzki,1 it is widely accepted that acetylcholine chloride (ACh)–induced vasodilation is absolutely dependent on an intact endothelium and is mediated by endothelium-derived relaxing factors (EDRF). Evidence has been accumulated that EDRF is nitric oxide (NO) or an NO-containing compound.2 However, subsequent studies have shown that ACh-induced vasodilation is not fully antagonized by the NO synthase inhibitor3 N
-nitro-L-arginine (L-NA), the
potent inactivator of EDRF methylene blue,4 or mechanical
removal of vascular endothelium from ring segments.5 6 In
addition, in in vivo3 and in vitro7 8
studies, the presence of a capsaicin (a toxin for peptidergic and
sensory neurons)–sensitive vasodilator component of the ACh-induced
vasodilation has been reported. However, mechanisms underlying
ACh-induced endothelium-independent vasodilation and
its mediator and the possible role of the capsaicin-sensitive component
in the ACh-induced vasodilation remain unknown. Although the tone of the peripheral blood vessels is mainly regulated by vascular adrenergic nerves, recent studies have demonstrated that many blood vessels have nonadrenergic noncholinergic (NANC) nerves or capsaicin-sensitive sensory nerves. Previously, we showed that the mesenteric resistance blood vessels of the rat are innervated by NANC vasodilator nerves, which are sensitive to tetrodotoxin (a neurotoxin) and capsaicin. Calcitonin gene-related peptide (CGRP), a potent vasodilator neuropeptide,9 10 acts as a neurotransmitter in NANC vasodilator nerves10 11 and is widely distributed in perivascular nerves throughout the vascular system.12 13 14 Thus, we proposed that the tone of the peripheral resistance vascular bed is controlled not only by sympathetic adrenergic nerves but also by CGRP-containing vasodilator nerves.10 13 15 Therefore, the present study was undertaken to investigate the role of CGRP-containing nerves in ACh-induced vasodilation. For this purpose, we examined the effects of treatment with capsaicin, chemical denudation with sodium deoxycholate (SD) and L-NA (an antagonist of NO synthase), and treatment with human CGRP[8-37] (a CGRP receptor antagonist) on the response to bolus infusion of ACh in the mesenteric resistance blood vessels of the rat.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals
Male Wistar rats, weighing 350 to 450 g, were used in the present study. Animals were given food and water ad libitum. They were housed in the experimental animal center of Miyazaki Medical College at a controlled ambient temperature of 22°C with 50±10% relative humidity and with a 12-hour light/12-hour dark cycle (light on at 7:30 AM).
Perfusion of the Mesenteric Vascular Bed
While the rats were under pentobarbital anesthesia, the
mesenteric vascular bed was isolated and prepared for perfusion as
described previously.10 The superior mesenteric artery was
cannulated and gently flushed with a modified Krebs' solution
containing (mmol/L) NaCl 120.0, KCl 5.0, CaCl2 2.4,
MgSO4 1.2, NaHCO3 25.0, 2NaEDTA 0.027, and
dextrose 11.0 (pH 7.4) to eliminate blood from the vascular bed. The
mesenteric vascular bed was separated from the intestine by cutting
close to the intestinal wall. Only four main arterial branches from the
superior mesenteric trunk running to the terminal ileum were perfused.
All other branches of the superior mesenteric artery were tied off. The
preparation was perfused with Krebs' solution at a constant flow rate
of 5 mL/min with a peristaltic pump (model SJ-1215, ATTO Co) and
superfused with the same solution at a rate of 0.5 mL/min to prevent
drying. The Krebs' solution was bubbled with a mixture of 95%
O2/5% CO2 before passage through a
warming coil maintained at 37°C. Changes in the perfusion pressure
measured with a pressure transducer (model MPU-0.5A, Nihon Kohden) were
recorded on a polygraph (model RM-25, Nihon Kohden).
Bolus Infusion of Drugs and Periarterial Nerve Stimulation
After equilibration for 30 minutes, the preparation was perfused
with Krebs' solution containing guanethidine (5 µmol/L) to block
adrenergic neurotransmission and methoxamine (5 to 7 µmol/L) to
induce submaximal contraction. After stabilization of the elevated
perfusion pressure, the mesenteric vascular bed was subjected to bolus
infusion of drugs and periarterial nerve stimulation (PNS).
Drugs were infused directly into the perfusate proximal to the arterial cannula with an infusion pump (model 975, Harvard Apparatus). The volumes of infusion were 100 µL for 10 seconds.
PNS was applied for 30 seconds by using bipolar platinum ring electrodes placed around the superior mesenteric artery. Rectangular pulses of 1 millisecond and supramaximal voltage (60 V) were applied at 1 and 4 Hz by using an electronic stimulator (model SEN 3301, Nihon Kohden).
Chemical Denudation of Vascular Endothelium
To remove the vascular endothelium, preparations with resting
tone were perfused with 1.75 to 1.80 mg/mL of SD in saline for 30
seconds.16 17 This caused a transient increase (10 to 20
mm Hg) in perfusion pressure. The preparation was rinsed with SD-free
Krebs' solution for 10 minutes and then perfused with Krebs' solution
containing 100 µmol/L L-NA for 30 minutes. After the preparation was
contracted by perfusion with Krebs' solution containing methoxamine,
guanethidine, and L-NA, chemical removal of the endothelium was
assessed by the lack of relaxant effect of bolus infusion of 1 nmol ACh
and by histological examination following staining with toluidine blue
in some preparations.
Experimental Protocols
The duration of the vasodilator response to bolus infusion of
ACh (0.1, 1, 10, and 100 nmol) was measured in preparations with intact
endothelium and precontracted with methoxamine in the presence of
guanethidine.
In another series of experiments, preparations were perfused with Krebs' solution containing vehicle (0.4 µg/mL ethanol in Krebs' solution) or 1 µmol/L capsaicin for the first 20 minutes, rinsed with capsaicin-free Krebs' solution for 90 minutes, and contracted by perfusion of Krebs' solution containing guanethidine and methoxamine. Thereafter, the duration and pattern of vascular response to bolus infusion of ACh (0.1, 1, 10, and 100 nmol) were examined.
In the endothelium removal experiment, the active tone of preparations with intact endothelium was produced by methoxamine (7 µmol/L) and guanethidine (5 µmol/L), and then PNS (1 Hz) and bolus infusions of isoproterenol (1 nmol) and ACh (1 nmol) were performed. Subsequently, the Krebs' solution was switched to methoxamine-free Krebs' solution, and the preparation was rinsed for 30 to 40 minutes until the perfusion pressure returned to the control level. Then, chemical denudation with SD and then with the addition of L-NA was carried out. After chemical denudation, the active tone of the preparation was reproduced by perfusion with Krebs' solution containing guanethidine (5 µmol/L), methoxamine (5 µmol/L), and L-NA (100 µmol/L). After the elevated perfusion pressure was allowed to stabilize, PNS (1 Hz) and a bolus infusion of isoproterenol (1 nmol) were performed to determine the presence of CGRP-containing nerves10 11 and the occurrence of vasodilation mediated via ß-adrenoceptors, which causes an increase in cAMP, respectively. Subsequently, successful removal of vascular endothelium was confirmed by the lack of relaxant effect of 1 nmol ACh infusion, and vascular responses to bolus infusions of ACh at concentrations of 3, 10, 30, 100, 300, and 1000 nmol were examined.
In another series of experiments, chemical denudation was performed in capsaicin-treated preparations. After the preparation was contracted with Krebs' solution containing guanethidine (5 µmol/L), methoxamine (5 µmol/L), and L-NA (100 µmol/L), vascular responses to bolus infusions of ACh (1 to 1000 nmol) were examined. PNS (1 and 4 Hz) and a bolus infusion of CGRP (30 pmol) were also performed to determine the denervation of CGRP-containing nerves by capsaicin and the presence of postsynaptic CGRP receptors, respectively.
The effects of human CGRP[8-37], atropine, and hexamethonium on the vasodilator response to ACh (100 nmol) were examined in preparations without endothelium. After control responses to the first (I1) and second (I2) bolus infusions of ACh were obtained, perfusion of CGRP[8-37] (1 µmol/L), atropine (1, 10, and 100 nmol/L), or hexamethonium (1 and 10 µmol/L) was begun 10 minutes before and throughout the third (I3) bolus infusion of ACh. PNS (1 Hz) was also performed during perfusion of CGRP[8-37]. The effects of CGRP[8-37], atropine, and hexamethonium were expressed as the ratio between the vasodilator response induced by I3 and I2 bolus infusions of 100 nmol ACh or as the percentage of vasorelaxation during and after perfusion of the antagonist.
At the end of each experiment, the preparation was perfused with 100 µmol/L papaverine, a vasodilator that causes an increase in cAMP, to induce complete relaxation.
Release of CGRP-Like Immunoreactivity
In preparations without endothelium, the perfusate was collected
for 5 minutes before and after bolus infusion of ACh (300 nmol/30 s).
Each sample was applied to a Sep-Pak C18 cartridge (Waters
Associates), and the adsorbed peptide was eluted with 3 mL of 60%
acetonitrile in 0.1% trifluoroacetic acid. The eluate was evaporated
under vacuum and subjected to radioimmunoassay (RIA) for CGRP as
described by Fujimori et al.11 The incubation buffer for
the RIA was 50 mmol/L sodium phosphate buffer (pH 7.4) containing 0.1%
bovine serum albumin, 0.1% Triton X-100, 80 mmol/L NaCl, 25 mmol/L
2NaEDTA, 0.05% NaN3, and 500 KIU/mL
aprotinin.18 [125I]Tyr0 rat CGRP
was prepared by the lactoperoxidase method,18 and the
monoiodinated form was separated from the other
iodinated species by reverse-phase high-performance liquid
chromatography. The samples were preincubated with rabbit anti-human
CGRP-II serum (Peninsula Laboratories, Inc) at 4°C for 12 hours.
Then, the reaction mixture was incubated with
[125I]Tyr0 rat CGRP for an additional 24 to
36 hours at 4°C. The antibody-bound antigen was separated from free
antigens by double-antibody precipitation. The lower detection limit
was 1 fmol per tube for CGRP-like immunoreactivity (CGRP-LI).
Statistical Analysis
Results, expressed as mean±SEM, were analyzed statistically by
Student's paired or unpaired t test and one-way ANOVA
followed by Dunnett's test. A value of P<.05 was
considered statistically significant.
Drugs
The following drugs were used: ACh (Daiichi Pharmaceutica Co),
atropine sulfate (Sigma Chemical Co), capsaicin (Sigma), guanethidine
sulfate (Tokyo Kasei), hexamethonium bromide (Sigma), human
CGRP[8-37] (Peptide Institute), L-isoproterenol
hydrochloride (Nikken Chemical Co), methoxamine hydrochloride (Nihon
Shinyaku), L-NA (Sigma), papaverine hydrochloride (Dainihon Seiyaku),
rat CGRP (Peptide Institute), and SD (Sigma). All drugs, except
capsaicin and SD, were dissolved in distilled water and diluted with
Krebs' solution containing 5 to 7 µmol/L methoxamine, 5 µmol/L
guanethidine, and 100 µmol/L L-NA. Capsaicin was dissolved in 50%
ethanol and diluted with Krebs' solution (final alcohol concentration,
0.4 µg/mL). SD was dissolved in 0.9% saline. ACh, when infused in
the preparation with intact endothelium, was diluted with Krebs'
solution containing 7 µmol/L methoxamine and 5 µmol/L
guanethidine.
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Results |
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Vascular Responses to Bolus Infusion of ACh
In mesenteric vascular beds contracted by 7 µmol/L methoxamine in the presence of 5 µmol/L guanethidine, the mean perfusion pressure was 81.3±7.8 mm Hg (n=6). As shown in Fig 1A
, a bolus
infusion of ACh produced a rapid drop in perfusion pressure due to
vasodilation, which occurred 1 to 2 seconds after ACh infusion. The
vasodilator responses to ACh at low doses were characterized by a rapid
and short-lived drop in perfusion pressure, fast in both onset
(appearing 1 to 2 seconds after the start of infusion) and decay
(returning to preinjection level within 2 to 5 minutes). However,
higher doses of ACh (10 and 100 nmol) caused a rapid drop in perfusion
pressure, followed by long-lasting vasodilation in which the decay was
prolonged in a concentration-dependent manner; the perfusion pressure
gradually returned to the preinjection level within 15 to 22 minutes
(Figs 1A and 2A). The maximum decrease in perfusion
pressure, which was reached shortly after infusion of ACh, became
greater with large doses of ACh (Fig 2B).
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and
indicate bolus infusions of ACh and PNS, respectively; PPV,
papaverine perfusion.
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Effect of Capsaicin Treatment on Vasodilator Responses to Bolus
Infusion of ACh
There was no significant difference in the mean perfusion pressure
between contracted preparations treated and untreated with capsaicin
(73.2±12.2 mm Hg [n=6] versus 81.3±7.8 mm Hg [n=6]). In
mesenteric vascular beds treated with 1 µmol/L capsaicin, the PNS (1
Hz) did not cause a vasodilator response (Figs 1B
and 2B). In this
preparation, capsaicin treatment did not affect the maximum decrease in
perfusion pressure induced by bolus infusion of ACh at any doses (Figs 1B and 2B). However, this treatment significantly shortened the
duration of the vasodilator response to ACh infusion; the time
(minutes) for recovery of vasodilation to preinjection levels of
perfusion pressure was significantly shorter in treated preparations
than in untreated preparations (Figs 1B and 2A).
Effect of Chemical Denudation of Vascular Endothelium on
Vasodilator Response to Bolus Infusion of ACh
Since chemical denudation of vascular endothelium increased the
vasoconstriction induced by methoxamine, the concentration of
methoxamine required to raise the tone was reduced to 5 µmol/L after
denudation. The mean perfusion pressures of preparations contracted
with methoxamine before and after chemical denudation by perfusion with
SD were 60.6±6.5 mm Hg (n=6) and 79.0±10.3 mm Hg (n=6),
respectively.
Chemical denudation abolished the initial rapid component of the
vasodilator response to bolus infusion of 1 nmol ACh, indicating that
the endothelium was successfully removed (Fig 3A and 3B). However, PNS (1 Hz) and a bolus infusion of
isoproterenol (1 nmol) still induced vasodilator responses (Fig 3A and 3B).
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, and 
indicate bolus infusions of ACh, ISO, and CGRP,
respectively;
In the preparation without endothelium, as shown in Fig 3B, the initial
rapid vasodilator component of ACh that was observed with intact
endothelium (Figs 1A and 3A) disappeared at all concentrations of ACh
tested, and increasing concentrations (3 to 1000 nmol) of ACh produced
long-lasting vasodilator responses in a concentration-dependent manner,
appearing 10 to 20 seconds after the infusion and returning to
preinfusion level within 10 to 15 minutes.
Effect of Capsaicin Treatment on Vasodilator Response to Bolus
Infusion of ACh in Mesenteric Vascular Bed Without Endothelium
As shown in Figs 3C and 4, capsaicin treatment in
the endothelium-removed preparation abolished the
long-lasting vasodilation induced by ACh infusion (1 to 1000 nmol). In
the same preparation, the vasodilator responses to PNS (1 and 4 Hz)
were also markedly attenuated (Figs 3C and 4). However, bolus infusion
of CGRP (30 pmol) produced a vasodilator response in the same
preparation, indicating that CGRP receptors were left intact.
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Effects of Various Drugs on Vasodilator Response to Bolus Infusion
of ACh in Mesenteric Vascular Beds Without Endothelium
To examine the effects of human CGRP[8-37], atropine, and
hexamethonium, a dose of 100 nmol ACh, which produced 
50%
vasodilation in response to bolus infusion of 1000 nmol ACh, was used.
Repeated infusions of 100 nmol ACh caused reproducible and long-lasting
vasodilator responses in the endothelium-removed
preparations.
As shown in Figs 5A and 6, perfusion of
Krebs' solution containing 1 µmol/L CGRP[8-37] resulted in a
transient increase in mean perfusion pressure (7.8±1.9 mm Hg, n=5)
and markedly attenuated long-lasting vasodilator responses both to
bolus infusion of ACh (100 nmol) and to PNS (1 Hz). After rinsing with
CGRP[8-37]–free Krebs' solution, the long-lasting vasodilator
responses to bolus infusion of ACh and PNS were reproduced (Fig 5A).
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There was no significant difference in mean perfusion pressure before
and during perfusion of Krebs' solution containing atropine or
hexamethonium. The long-lasting vasodilator response to bolus infusion
of ACh (100 nmol) was attenuated by atropine (1, 10, and 100 nmol/L) in
a concentration-dependent manner (Figs 5B and 7A) but
was not affected by hexamethonium (1 and 10 µmol/L) (Figs 5C and 7B).
Atropine at a concentration of 10 nmol/L almost abolished the
vasodilator response to bolus infusion of ACh, and the calculated
ED50 was 3 nmol/L.
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Release of CGRP-LI Induced by ACh in Mesenteric Vascular Beds
Without Endothelium
In the noncontracted preparation without endothelium, basal
overflow of CGRP-LI in the perfusate was 28.2±2.7 fmol/5 min
(n=6).
Bolus infusion of ACh (300 nmol/30 s) produced long-lasting
vasodilation in endothelium-removed mesenteric vascular
beds precontracted with 5 µmol/L methoxamine plus 5 µmol/L
guanethidine and 100 µmol/L L-NA. As shown in Fig 8,
ACh infusion significantly increased overflow of CGRP-LI in the
perfusate of the mesenteric vascular bed from 66.7±9.4 to 94.1±13.1
fmol/5 min (P<.01, n=6) within the first 5 minutes after
the start of ACh infusion.
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) and during (
) ACh infusion. *P<.01 compared
with the release before ACh infusion by Student's paired t
test. Data indicate mean±SEM of six experiments. SD indicates sodium
deoxycholate; L-NA,
N |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The present study demonstrated that in the perfused mesenteric artery with intact endothelium and with active tone produced by methoxamine in the presence of guanethidine, bolus infusion of ACh induced a vasodilator response consisting of two elements: an initial rapid component and a subsequent long-lasting component. The secondary long-lasting vasodilation in response to ACh infusion was sensitive to capsaicin, a toxin for peptidergic neurons, which shortened the duration of vasodilation without affecting the initial rapid response. These findings indicate that capsaicin-sensitive neurons are involved in the ACh-induced vasodilation as reported previously in both in vivo3 and in vitro7 8 studies.
In the present study, to investigate the influence of endothelium removal on the vasodilator response to ACh, the endothelium was chemically removed from the mesenteric vascular preparation by perfusion with the detergent SD.16 17 To exclude the influence of NO synthesis in not only the vascular endothelium but also the vascular smooth muscle layer, L-NA, a potent inhibitor of NO synthase,19 20 which has no antagonistic effect on muscarinic receptors,21 was also added into the perfusate. In these preparations, PNS still induced the vasodilator response, and the short-lived vasodilator response to bolus infusion of 1 nmol isoproterenol, a ß-adrenoceptor agonist, was not suppressed. These results suggest that the functions of perivascular nerves and vascular smooth muscles are maintained after chemical denudation of endothelium with SD.
Chemical removal of the endothelium in the mesenteric artery abolished the initial rapid component of the vasodilator response to ACh infusion at all concentrations tested. However, increasing concentrations of ACh produced only long-lasting vasodilator responses in a concentration-dependent manner. These results suggest that in the mesenteric resistance artery of the rat, the initial rapid phase of ACh-induced vasodilation is endothelium dependent, but its long-lasting phase is independent on function(s) of the endothelium.
In preparations with intact endothelium, capsaicin treatment did not alter the initial rapid phase of ACh-induced vasodilation but significantly shortened the duration of the vasodilator response. Moreover, simultaneous endothelium removal and treatment with capsaicin abolished the long-lasting endothelium-independent vasodilation caused by ACh. Capsaicin has been shown to deplete neuropeptides including tachykinins (substance P, neurokinin A, and neurokinin B) and CGRP10 22 but not vasoactive intestinal polypeptide23 24 from sensory neurons. Previous studies of the mesenteric vascular bed demonstrated that the neurogenic vasodilator response to PNS is mediated via CGRP-containing vasodilator nerves, because the neurogenic vasodilation is markedly attenuated by capsaicin treatment10 and by human CGRP[8-37], an antagonist of CGRP receptors.15 25 Furthermore, PNS of the perfused mesenteric vascular bed elicits increased release of CGRP-LI into the perfusate, which was inhibited by capsaicin treatment.26 In the present study, human CGRP[8-37] abolished the endothelium-independent vasodilation in response to ACh infusion as well as the PNS-induced vasodilation. Moreover, exposure of ACh to these preparations increased overflow of CGRP-LI in the perfusate. Taken together, these findings strongly suggest that the long-lasting endothelium-independent component of ACh-induced vasodilation is elicited by activation of CGRP-containing nerves. This activation releases CGRP from perivascular CGRP-containing nerves; released CGRP then acts on CGRP receptors in vascular smooth muscle to cause long-lasting vasodilation. This may be supported by the findings that the ACh- or methacholine-induced vasodilation was attenuated by local anesthetics or in surgically denervated experimental models.27 28 CGRP has been shown to be released by some exogenous or endogenous substances, including capsaicin,14 29 30 31 32 bradykinin,14 29 30 31 32 nicotine,30 32 histamine,32 ouabain,30 arachidonic acid,29 and prostaglandins,29 and by conditions such as low-pH medium,33 high-K+ medium,31 vagal stimulation,32 and ischemia30 in various experimental models. The present study is the first report that ACh has the ability to cause release of CGRP from CGRP-containing vasodilator nerves.
In the present study, atropine, a muscarinic ACh receptor antagonist, inhibited the long-lasting endothelium-independent component of ACh-induced vasodilation in a concentration-dependent manner, whereas hexamethonium, a nicotinic ACh receptor antagonist, had no effect. Therefore, it is more likely that muscarinic ACh receptors, presumably located on CGRP-containing vasodilator nerves, are responsible for the long-lasting endothelium-independent vasodilator response to ACh infusion.
Recent studies have demonstrated that many blood vessels are innervated by NANC nerves or capsaicin-sensitive sensory nerves. We have shown previously that the mesenteric resistance blood vessels of the rat are innervated by NANC vasodilator nerves, in which CGRP acts as a vasodilator neurotransmitter.10 11 Because CGRP-containing vasodilator nerves are widely distributed throughout the vascular system12 13 14 and CGRP is a potent vasodilator,9 10 we have proposed that the tone of the peripheral resistance vascular bed is controlled not only by sympathetic adrenergic nerves but also by CGRP-containing vasodilator nerves.10 13 15 Although the concentrations of endogenous ACh in tissues surrounding blood vessels have not been measured, cholinergic innervation in blood vessels, including the cephalic arteries,5 34 femoral artery,35 and coronary arteries,36 has been demonstrated. Thus, taken together, it can be inferred that endogenous CGRP released from CGRP-containing nerves by endogenous ACh may play an important role in the regulation of tone of peripheral resistance vessels.
In conclusion, the present study demonstrated that the ACh-induced vasodilation in the mesenteric resistance vessels of the rat consisted of two elements, an initial rapid endothelium-dependent component and a secondary long-lasting endothelium-independent component that is mediated by activation of perivascular CGRP-containing nerves and muscarinic ACh receptors. The present study is the first to report that ACh is capable of producing endothelium-independent vasodilation in the mesenteric resistance arteries of the rat, by activating the putative muscarinic receptors located on perivascular CGRP-containing nerves to elicit the release of CGRP and, in turn, acting at CGRP receptors on vascular smooth muscle to cause vasorelaxation. These results suggest that endogenous ACh contributes to the control of vascular tone through CGRP-containing vasodilator nerves.
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Acknowledgments |
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We are grateful to Dr Atsushi Kisanuki for the histological examination, Keizo Masumoto for technical assistance, and Keiko Kawabata for secretarial assistance.
Received January 18, 1995; accepted March 15, 1995.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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