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Vol. 282, Issue 3, 1337-1344, 1997
Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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The primary goal of this study was to determine the extent that
selective muscarinic receptor antagonists could discriminate between
the chronotropic and coronary vasoconstrictor responses to
acetylcholine in isolated rat hearts perfused at constant flow rate.
Bolus injections of acetylcholine caused dose-dependent decreases in
heart rate and increases in perfusion pressure. The ED50
(95% confidence) of acetylcholine for decreasing rate was 0.463 (0.336-0.640) nmol and the dose that increased perfusion pressure by
30 mm Hg (ED30 mmHg
) was 3.19 (2.00-5.08)
nmol. The M2 selective antagonist methoctramine (3.16 µM)
produced a 307-fold increase in the ED50 for bradycardia
but had no significant effect on the pressor response to acetylcholine.
In marked contrast, the M3 antagonist
hexahydrosiladifenidol displayed a distinct preference for inhibiting
coronary vasoconstrictor responses to acetylcholine. When present at
316 nM, this drug produced a 66-fold increase in the ED30
mmHg but only a 6-fold increase in the ED50
for bradycardia. The M1 selective antagonist pirenzepine (316 nM) produced a 5- to 7-fold increase in both parameters. Pretreatment with pertussis toxin (25 µg/kg, i.p.) essentially eliminated acetylcholine-evoked bradycardia although pressor responses persisted with some reduction. These observations demonstrate that
cardiac and coronary vascular effects of acetylcholine can be clearly
discriminated with specific muscarinic antagonists. Furthermore, they
provide evidence that the M3 receptor subtype mediates the
vasoconstrictor effect of acetylcholine on resistance vessels in rat
heart.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Muscarinic
receptors are found throughout the heart (Hancock et al.,
1987
) and have a major role in regulating cardiac function (Löffelholz and Pappano, 1985; Levy and Warner, 1994). Activation of these receptors, by muscarinic agonists or endogenous ACh released from postganglionic vagal nerve fibers, causes prominent decreases in
heart rate, atrioventricular conduction velocity and myocardial contractility (Löffelholz and Pappano, 1985; Levy and Warner, 1994). Most of these responses can be attributed entirely to the activation of receptors localized to cardiac myocytes. However, the
effect of vagal nerve stimulation to decrease ventricular contractility
also appears to involve the stimulation of inhibitory prejunctional
muscarinic receptors on sympathetic nerve fibers (Levy and Warner,
1994).
The coronary vasculature also contains muscarinic receptors, but the
nature of its response to cholinergic agonists depends on species
(Kalsner, 1989; Feigl, 1994) and can be affected by pathology (Ludmer
et al., 1986; Treasure et al., 1992; Egashira et al., 1995). Vasodilation is the exclusive response evoked
from dog epicardial coronary arteries (Kalsner, 1989; Feigl, 1994), although muscarinic agonists produce coronary vasoconstriction in pigs
(Gräser et al., 1986; Kalsner, 1989; Kawamura et
al., 1989). The effect of ACh on human epicardial arteries that
were judged to be normal has varied between studies, with some
investigators observing vasodilation (Ludmer et al., 1986;
Bossaller et al., 1987; Egashira et al., 1995)
and others finding only vasoconstriction (Kalsner, 1989; Toda and
Okamura, 1989). However, vasoconstriction is either the sole or
dominant response of epicardial arteries in patients with
atherosclerosis or hypertension (Ludmer et al., 1986;
Hodgson and Marshall, 1989; Treasure et al., 1992; Egashira et al., 1995). Muscarinic agonists produce coronary
vasoconstriction directly by stimulating receptors localized to
vascular smooth muscle cells, although vasodilation is an indirect
effect that results from the stimulation of muscarinic receptors on
endothelial cells and is mediated by endothelium-derived relaxing
factor (Kalsner, 1989). Accordingly, the nature of the response to
muscarinic agonists is presumed to depend on the relative abundance of
receptors at these sites. Human epicardial arteries appear to have
muscarinic receptors in both endothelial and smooth muscle layers, and
the endothelial receptors may predominate in the absence of pathology. Vasoconstriction becomes the dominant effect in patients with atherosclerosis or hypertension due to reduction or loss of
endothelium-dependent vasodilation. The physiological significance of
muscarinic receptors in the coronary vasculature is currently unknown,
but there is clear evidence from animal studies that these receptors
can be activated upon vagal stimulation (van Charldorp et
al., 1987; Feigl, 1994).
Four subtypes of the muscarinic receptor (i.e.,
M1 through M4) can be
distinguished through the use of various antagonists in functional and
radioligand binding studies (Mei et al., 1989; Hulme
et al., 1990; Lazareno et al., 1990), although
five subtypes (i.e., m1 through m5) have been identified in
molecular cloning studies (Mei et al., 1989; Hulme et
al., 1990). Most evidence suggests that subtypes identified
through pharmacological methods correspond to the like-numbered
molecular form (e.g., M3 = m3). The
M2 muscarinic receptor was first identified in
the heart, and the gene that encodes this receptor was subsequently
cloned using cardiac tissue (Mei et al., 1989; Hulme
et al., 1990). Pharmacological and molecular studies have
provided evidence that the m2 subtype is the major muscarinic receptor
expressed by mammalian cardiac myocytes (Mei et al., 1989;
Li et al., 1991; Hoover et al., 1994) and
mediates inhibitory effects of ACh on cardiac function. Recent evidence
also suggests that cardiomyocytes contain a smaller population of m1
receptors that could mediate stimulatory responses reported to occur
with high concentrations of some muscarinic agonists (Gallo et
al., 1993; Sharma et al., 1996). The receptor subtype present in the coronary vasculature has been evaluated for several species in functional experiments with muscarinic antagonists (van
Charldorp and van Zwieten, 1989; Duckles, 1990; Bognar et al., 1990; Ren et al., 1993). The results from these
studies are consistent with the conclusion that
M3 receptors mediate both vasodilation and
vasoconstriction of coronary arteries.
It has been proposed that vagally induced coronary vasoconstriction may
be involved in the pathogenesis of coronary vasospasm in patients with
variant angina (Yasue et al., 1986; Kalsner, 1989). The
observation that different muscarinic receptor subtypes mediate cardiac
and coronary vascular responses to ACh suggests that it may be feasible
to block undesirable coronary vasoconstrictor responses while retaining
the ability of ACh to regulate cardiac function. However, the
muscarinic receptor antagonists that are available currently do not
have complete selectivity for a single receptor subtype. Accordingly,
the primary aim of our study was to determine the extent to which
cardiac and coronary vasoconstrictor responses of isolated rat hearts
to ACh could be discriminated by specific antagonists. Further
characterization of the muscarinic receptor subtype mediating coronary
vasoconstriction in rats was also achieved. A preliminary report of the
results was presented at the Sixth International Symposium on Subtypes
of Muscarinic Receptors (Hoover and Neely, 1995).
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
References
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Isolated heart preparation.
Male Sprague Dawley rats
(350-550 g) were pretreated with heparin (500 U/100 g) approximately
20 min before undergoing decapitation while anesthetized with sodium
pentobarbital (75 mg/kg, i.p.). The heart was rapidly removed and
placed into a Petri dish containing ice-cold perfusion buffer to enable
cannulation of the ascending aorta. After flushing the heart with 5 ml
cold buffer, it was transferred to an isolated heart apparatus for
perfusion by a modification of the Langendorff technique (Broadley,
1979). The perfusion solution was a modified Krebs-Ringer bicarbonate
buffer (pH 7.35-7.4) of the following composition (millimolar): NaCl, 127.2; KCl, 4.7; CaCl2, 2.5;
KH2PO4, 1.2;
NaHCO3, 24.9; MgSO4, 1.2;
sodium pyruvate, 2.0; dextrose, 5.5; and 0.1% bovine serum albumin. A
Masterflex peristaltic pump (Cole Parmer, St. Louis, MO) was used to
perfuse hearts at a constant rate of 8 ml/min. Buffer in the reservoir
was continuously gassed with 95% O2-5% CO2. The temperature of the buffer was maintained
at 37°C by passage through a heated, glass coil, and the heart was
surrounded by a glass jacket kept at the same temperature. Cardiac
contractions were measured by attaching one end of a piece of silk
suture to the apex of the heart and the other end to an isometric force transducer. Diastolic tension was adjusted to approximately 1 g.
Output from the force transducer was sent to a Gould Universal amplifier and a Gould Biotach amplifier to monitor contractions and
ventricular rate, respectively, with a Gould 2400 recorder. Perfusion
pressure was monitored using a transducer that was attached to the
sidearm of a 3-way stopcock located at the proximal end of the aortic
cannula. Experiments were started after a 30- to 40-min stabilization
period.
Administration of ACh. ACh chloride was dissolved in saline and administered by bolus injection through a short length of polyethylene 20 tubing (Clay Adams, Parsippany, NJ) that emptied into the perfusion buffer at a point near the 3-way stopcock attached to the aortic cannula. A volume of 100 µl was given over approximately 3 sec. Injections of saline caused a small injection artifact in the perfusion pressure recording that was easily distinguished from pressor responses to ACh. Serial injections of ACh were made in order of increasing dose with effective doses separated by at least five min. Heart rates returned to baseline after doses of ACh that produced a submaximal rate response, but hearts frequently remained quiescent after the first or second dose of ACh that caused cardiac arrest. Accordingly, it was necessary to collect a portion of the dose-response data for the pressor effect of ACh from non-beating hearts. Only a single set of dose-response data could be collected from each heart.
Protocol for evaluation of muscarinic receptor antagonists.
Three selective muscarinic receptor antagonists and atropine were
evaluated to determine their effects on negative chronotropic and
pressor responses to ACh. Methoctramine and HHSiD were chosen because
of their relative selectivity for M2 (Giraldo
et al., 1988; Buckley et al., 1989) and
M3 (Waelbroeck et al., 1987; Buckley et al., 1989) receptors, respectively. Effects of
pirenzepine were evaluated because of its high affinity for
M1 muscarinic receptors (Hammer et
al., 1980; Buckley et al., 1989). Each antagonist was
evaluated at two or three concentrations in separate hearts. Antagonists were present in the perfusion buffer for the entire duration of the experiment, so hearts had equilibrated with these drugs
for well over 30 min before the first administration of ACh.
Pertussis toxin pretreatment.
Even-numbered muscarinic
receptors are know to couple preferentially with PTX-sensitive
G-proteins for signal transduction while the odd-numbered subtypes do
not (Mei et al., 1989; Hulme et al., 1990;
Felder, 1995). Therefore, pretreatment with PTX was used to determine
which of these groups contains the muscarinic receptor subtype that
mediates coronary pressor responses to ACh. The PTX was administered at
a dose of 25 µg/kg (i.p.) 48 hr before the experiment (Lasley and
Mentzer, 1993). Control animals were pretreated with vehicle consisting
of 50% glycerol, 50 mM Tris, 10 mM glycine and 0.5 M NaCl (pH 7.5).
Data analysis.
Decreases in heart rate and increases in
diastolic perfusion pressure produced by ACh were determined relative
to base-line values immediately before each injection. Changes in heart
rate were expressed as a percentage of base-line for subsequent
analysis. Dose-response values of each heart were fit with a sigmoidal
curve in order to obtain estimates of ED50, a
slope coefficient and maximum pressor response (GraphPad Prism version
2.0, GraphPad Software, San Diego, CA). In several cases, the estimated
maximum perfusion pressure response exceeded the highest recorded value by 50% or more. For this reason, computer-generated estimates from
perfusion pressure data are not presented. However, curve fitting was
still used to construct most of the dose-response curves for perfusion
pressure shown in the figures. To estimate the relative potency of
antagonists in affecting pressor responses to ACh, we determined the
dose of ACh required to increase perfusion pressure by 30 mmHg
(i.e., ED30 mmHg) by linear
interpolation from doses evoking responses immediately above and below
this value.
)
using SYSTAT version 4.0 (SYSTAT, Inc., Evanston, IL). One-factor ANOVA
was used to compare ED50, slope (heart rate data) or ED30 mmHg from the control group and groups
with different concentrations of a single antagonist. Post
hoc comparisons were made using the Newman-Keuls procedure. Values
for ED50 and ED30
mmHg were log-transformed for analysis. Two-factor ANOVA
with repeated measures on the ACh dose factor was used where indicated
in the text for evaluation of baseline data and dose-response data. The arithmetic mean (± S.E.) or geometric mean (95% confidence interval) were used to summarize group data. A probability level of 0.05 or
smaller was used to indicate statistical significance.
Drugs used. ACh chloride, atropine sulfate, pirenzepine and PTX were purchased from Sigma (St. Louis, MO). Methoctramine and HHSiD were purchased from Research Biochemicals International (Natick, MA).
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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A summary of baseline values of heart rate and perfusion pressure from experiments with selective muscarinic antagonists is given in table 1. Although means varied somewhat between groups, the magnitudes of differences were relatively small. All but one of the group means was within a standard deviation of the mean determined for all similar base-line values (e.g., base-line heart rate before first dose of ACh). Statistical evaluation of these data, by two-factor ANOVA with repeated measures, demonstrated significant, overall increases in base-line heart rates (F1,47 = 20.0, P < .001) and perfusion pressures (F1,49 = 93.1, P < .001) over the time of experiments.
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Control responses to ACh.
Bolus injections of ACh caused
dose-dependent decreases in heart rate and increases in perfusion
pressure (fig. 1). Dose-response curves
for bradycardia were steeper than those for the pressor response to
ACh. Cardiac arrest occurred at doses of ACh that were submaximal for
increasing perfusion pressure (fig. 1). A majority of the hearts failed
to resume spontaneous beating after the first or second dose of ACh
that caused cardiac arrest. Pressor responses to ACh could still be
evoked in these arrested hearts.
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Effect of methoctramine on responses to ACh. Methoctramine had a prominent effect to inhibit negative chronotropic responses to ACh but had no significant influence on pressor responses (fig. 1; table 2). Antagonism of the rate response was already evident in the presence of 31.6 nM methoctramine and displayed concentration dependence. The highest concentration of methoctramine increased the ED50 for the negative chronotropic response by 307-fold (F3,24 = 134.8, P < .001).
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Effect of HHSiD on responses to ACh.
HHSiD, in marked contrast
to methoctramine, showed a distinct selectively for antagonizing
pressor responses to ACh (fig. 2; table
2). The lowest concentration of HHSiD increased the ED30
mmHg for the vascular response by about 22-fold although having only a slight effect on the ED50 for
bradycardia. A 10-fold greater concentration of HHSiD caused a 66-fold
increase in the ED30 mmHg but only a 6-fold
increase in the ED50 for bradycardia compared
with the control group.
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Effect of pirenzepine on responses to ACh.
Pirenzepine had
weak activity in antagonizing rate responses to ACh (fig.
3A; table 2). A 1 µM concentration of
this antagonist increased the ED50 of ACh for
evoking bradycardia by only 7-fold compared with control. Pressor
responses to ACh were unaffected by 100 nM pirenzepine (fig. 3B; table
2), but were reduced at higher concentrations of this blocker. The
ED30 mmHg was increased 10-fold in the
presence of 316 nM pirenzepine compared with the control group. The
dose-response curve for the pressor response to ACh was very shallow
with 1 µM pirenzepine (fig. 3B), and ED30
mmHg was not achieved in half of these hearts
(i.e., value > 1 µmol ACh).
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Effect of PTX pretreatment on responses to ACh.
Pretreatment
with PTX had no effect on base-line values for heart rate or perfusion
pressure. Negative chronotropic responses to ACh were almost eliminated
after pretreatment with PTX (figs. 4 and
5A). The highest dose of ACh
(i.e., 1 µmol) evoked a prominent bradycardia in two of
the four hearts obtained from PTX-treated rats, but lower doses had no
effect. Pressor responses to ACh were reduced significantly by PTX but
affected much less than negative chronotropic responses (figs. 4 and
5B).
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Effect of atropine on responses to ACh.
The nonselective
muscarinic receptor antagonist, atropine, was a potent inhibitor of
negative chronotropic and pressor responses to ACh (fig.
6). The ED50 for
bradycardia was increase 561-fold in the presence of 1 µM atropine
and an additional 10-fold with 10 µM atropine. Mean values of
ED50 for all groups differed significantly (F2,12 = 574, P < .001). Atropine produced
comparable shifts in the pressor response to ACh based on the dose of
ACh required to increase perfusion pressure by 15 mmHg. The geometric
mean (95% C.I.) of this value was 1.49 nmol (0.545-4.09) for control, 3.06 µmol (2.37-3.96) in the presence of 1 µM atropine and 34.8 µmol (21.0-57.7) with 10 µM atropine. These means were all
significantly different (F2,11 = 270, P < .001). The pressor response to 10 µmol ACh (i.e., highest
dose used in experiments with selective antagonists) was virtually
eliminated in the presence of 10 µM atropine.
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Our results demonstrate that specific muscarinic receptor antagonists selectively inhibit either negative chronotropic or coronary vasoconstrictor responses to ACh in a preparation with both parameters recorded simultaneously. They also show that vascular and cardiac responses to ACh involve different G-proteins. These findings provide strong evidence that different subtypes of the muscarinic receptor mediate pressor and negative chronotropic responses to ACh in the isolated perfused rat heart. Based on the rank order of potency for selective antagonists and the effects of PTX pretreatment, it is concluded that M2 receptors mediate bradycardia and M3 receptors mediate coronary vasoconstriction.
An exclusive role of M2 muscarinic receptors in
mediating direct, inhibitory cardiac responses to ACh in mammals has
been established by numerous functional and biochemical studies (Mei et al., 1989; Hulme et al., 1990). This knowledge
provided an important internal reference point for evaluating the
muscarinic receptor subtype in the coronary vasculature. Experiments
with cloned muscarinic receptors have established that methoctramine binds with highest affinity to m2 muscarinic receptors (Buckley et al., 1989), and this antagonist was the most effective at
blocking rate responses in our study. Pirenzepine and HHSiD, agents
with highest affinity for cloned m1 and m3 receptors, respectively (Buckley et al., 1989), displayed only weak activity against
negative chronotropic responses to ACh. In the presence of 316 nM
methoctramine, the ED50 for bradycardia was
increased 31-fold compared with control, although the same
concentration of pirenzepine and HHSiD produced only a 5- to 6-fold
increase. Accordingly, our results with antagonists support the
conclusion that M2 muscarinic receptors mediate
bradycardia evoked by ACh in the isolated rat heart. The observation
that negative chronotropic responses to ACh are abolished by
pretreatment with PTX is consistent with this conclusion since
M2 muscarinic receptors are known to couple
preferentially to PTX-sensitive G-proteins (Mei et al.,
1989; Hulme et al., 1990; Felder, 1995).
Perfusion pressure and chronotropic responses to ACh were
differentially affected by several of the agents studied but this was
most striking in the case of PTX. Pretreatment with this agent essentially eliminated acetylcholine-evoked bradycardia although pressor responses persisted, albeit with some reduction. This finding
suggests that the pressor response could be mediated by M1, M3 or M
5 muscarinic receptors because these subtypes
exhibit preferential coupling to PTX-insensitive G-proteins (Mei
et al., 1989; Hulme et al., 1990; Felder, 1995).
HHSiD had the highest potency for antagonizing pressor responses to
ACh, and also provided a clear discrimination between the receptors
mediating pressor and negative chronotropic responses. The lowest
concentration of this M3 selective antagonist
caused a 22-fold increase in the ED30 mmHg
for the pressor response while having only a slight effect on the
negative chronotropic response to ACh. In marked contrast, the highest
concentration of methoctramine (i.e., 3.16 µM) and 100 nM
pirenzepine had no significant effect on the pressor response. This
rank order of potency for antagonists (i.e., HHSiD > pirenzepine 
methoctramine) suggests that pressor responses to
ACh in the isolated perfused rat heart are mediated by
M3 muscarinic receptors. Other investigators have
previously reported that HHSiD and M2-selective
antagonists (i.e., AF-D × 116 and methoctramine)
differ in ability to antagonize vagally-induced pressor responses in
isolated rat hearts (Bognar et al., 1990), but the ability
of these antagonists to discriminate between negative chronotropic and
pressor responses was not studied. Rather, they made simultaneous
measurements of perfusion pressure and estimates of ACh release. They
found that vagally induced pressor responses were antagonized by HHSiD
but enhanced by the M2 blockers. The enhanced
pressor response was attributed to selective blockade of prejunctional
M2 receptors that function to inhibit release of
ACh, although M3 or M1
receptors were considered possible targets for mediating the inhibitory
effect of HHSiD. In view of the present findings, it is probable that
HHSiD antagonized vagally induced pressor responses through blockade of
M3 receptors.
In our study, the highest concentration of pirenzepine
(i.e., 1 µM) inhibited pressor responses to ACh by a much
larger magnitude than predicted based on the effect obtained with a
half-log unit lower concentration of this antagonist. We are unable to
offer a definitive explanation for this phenomenon, however, it may be
due to a combination of brief exposures to agonist and relatively slow
dissociation of pirenzepine from the M3
muscarinic receptor in the coronary vasculature. ACh was given by bolus
injection, and perfusion buffer was not recirculated. Therefore,
unbound agonist would be cleared from the system rapidly. The
dissociation of [3H]pirenzepine from muscarinic
receptors in rat cortical membranes occurs with a
T1/2 of about 5 min (Luthin and Wolfe,
1984). Therefore, relatively few M3 receptors may
have been available to be activated by ACh in the presence of 1 µM
pirenzepine. At lower concentrations of pirenzepine, the impact of
these factors on the response to ACh could have been minimized by the
presence of spare receptors. Pirenzepine has a lower affinity for the
M2 receptors that mediate negative chronotropic
responses to ACh, and the dose-response curve for this effect was not
shifted to the right by an inordinate amount in the presence of 1 µM
pirenzepine.
The first observation that ACh causes coronary vasoconstriction in rats
was made in experiments with isolated, donor-perfused hearts (Sakai,
1980). In this preparation, low doses of ACh decreased perfusion
pressure without affecting cardiac function although higher doses
increased perfusion pressure and decreased heart rate and left
ventricular contractile function. Comparable pressor responses occurred
in paced and spontaneously beating hearts. Experiments with
donor-perfused hearts also established that the coronary
vasoconstrictor action of ACh was not an indirect effect mediated by
norepinephrine, was unaffected by nicotinic receptor blockade but was
eliminated by treatment with atropine to block muscarinic receptors.
Therefore, it was concluded that pressor responses to ACh occurred
through stimulation of muscarinic receptors located in the coronary
vasculature (Sakai, 1980). Our observations with atropine support the
conclusion that ACh causes coronary vasoconstriction in rats through
stimulation of muscarinic receptors. Vasodilator responses to ACh were
not detected in our study and have been absent or minor at basal
conditions in other studies with isolated, buffer-perfused rat hearts
(Yang et al., 1993; Weselcouch et al., 1995). The
absence of this effect has been attributed to a low coronary vascular
tone since nitric oxide-mediated vasodilation can be demonstrated after
baseline tone (perfusion pressure) has been increased by including the
thromboxane mimetic, U-46619, in the perfusion buffer (Weselcouch
et al., 1995).
It is known that ACh can produce vasoconstriction directly by
stimulating muscarinic receptors on vascular smooth muscle (Kalsner, 1989) and indirectly through muscarinic receptors located on the vascular endothelium (Lüscher et al., 1992).
Thromboxane A2, cyclic endoperoxides, endothelin
and superoxide have been proposed as mediators of endothelium-dependent
vasoconstriction (Lüscher et al., 1992). Recent work
has shown that ACh can evoke endothelium-dependent and -independent
contractions of rabbit left coronary artery (Jino et al.,
1996). At least a portion of the pressor response to ACh in isolated
rat hearts could occur by an endothelium-dependent mechanism since it
has been reported that the magnitude of this effect is reduced in the
presence of a thromboxane A2 antagonist (Yang
et al., 1993).
Studies evaluating muscarinic receptor subtype in the coronary
vasculature of other species have focused on epicardial arteries. Coronary arteries from pigs have muscarinic receptors localized to the
media and contract in response to muscarinic agonists. Evidence from
functional studies and radioligand binding experiments suggests that
M3 receptors mediate coronary vasoconstriction in this species (Rinner et al., 1988; van Charldorp and van
Zwieten, 1989). Bovine and simian epicardial coronary arteries can be
relaxed through stimulation of muscarinic receptors on endothelial
cells and contracted by activation of muscarinic receptors localized to
smooth muscle cells. Evidence from pharmacological studies indicates
M3 receptors mediate both relaxation and
contraction of coronary vessels from these species (Duckles, 1990;
Brunner et al., 1991; Ren et al., 1993).
Accordingly, M3 muscarinic receptors are commonly
present in the coronary vasculature where they mediate relaxation and
contraction. We are not aware of any functional studies in which human
coronary arteries have been evaluated for muscarinic receptor subtypes.
However, it was recently proposed that M3
receptors mediate the vasodilation of human forearm resistance vessels
evoked by infusion of muscarinic receptor agonists (Bruning et
al., 1994).
In summary, our experiments with the isolated perfused rat heart preparation, have demonstrated that selective inhibition of negative chronotropic and coronary vasoconstrictor responses to ACh can be achieved with methoctramine and HHSiD, respectively. We have also provided new evidence in support of the conclusion that M3 muscarinic receptors mediate the coronary vasoconstrictor response to ACh in the rat. These observations suggest that HHSiD or another M3 selective antagonist could be useful for evaluating the proposed involvement of vagal nerve activity in the pathogenesis of coronary vasospasm in patients with variant angina.
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Acknowledgments |
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The authors are grateful to Dr. Peter Rice for information he provided during the preparation of this manuscript and to Dr. John Kalbfleisch for consultation on statistical analysis.
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Footnotes |
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Accepted for publication May 9, 1997.
Received for publication January 8, 1997.
1 This study was supported by a Grant-in-Aid from the American Heart Association, Tennessee Affiliate, Inc.
Send reprint requests to: Dr. Donald B. Hoover, Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614.
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Abbreviations |
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ACh, acetylcholine; HHSiD, hexahydrosiladifenidol; PTX, pertussis toxin; ANOVA, analysis of variance.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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-monophosphate formation in atherosclerotic human coronary artery and rabbit aorta.
J. Clin. Invest.
79: 170-174, 1987.
reappraisal of its role as a research and teaching model for coronary vasoactive drugs.
J. Pharmacol. Methods
2: 143-156, 1979.This article has been cited by other articles:
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Y. Zhang and D. B. Hoover Signaling Mechanisms for Muscarinic Receptor-Mediated Coronary Vasoconstriction in Isolated Rat Hearts J. Pharmacol. Exp. Ther., April 1, 2000; 293(1): 96 - 106. [Abstract] [Full Text]
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