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Vol. 293, Issue 1, 96-106, April 2000
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
Materials and Methods
Results
Discussion
References
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Signaling mechanisms for muscarinic receptor-mediated vasoconstriction in coronary resistance arteries were studied in potassium-arrested isolated rat hearts perfused at a constant flow rate. The cholinergic agonist bethanechol was given by bolus injection or constant infusion. Perfusion pressure was monitored as an indicator of coronary vascular resistance. Bolus injection of bethanechol evoked a phasic vasoconstriction in a dose-dependent manner, whereas infusion of bethanechol evoked a tonic vasoconstriction without producing tachyphylaxis. Bethanechol-induced phasic vasoconstriction was eliminated by perfusion with a Ca2+-free buffer. The L-type voltage-operated Ca2+ channel blocker nifedipine decreased the maximal constrictor response to bethanechol by 59 ± 2% (n = 4, P < .001), whereas the putative receptor-operated Ca2+ channel blocker SK&F 96365 converted this vasoconstriction into vasodilation that was not mediated by nitric oxide. The protein kinase C inhibitor chelerythrine reduced the maximal phasic vasoconstrictor response to bethanechol by 78 ± 2% (n = 6, P < .001) Bethanechol-induced tonic vasoconstriction was rapidly converted to a sustained vasodilation during infusion of SK&F 96365 or nifedipine, whereas infusion of chelerythrine gradually attenuated the tonic response to bethanechol. Results from other experiments do not support a role for phospholipase A2-dependent mediators in generating coronary vasoconstrictor responses to bethanechol. It is concluded that voltage-independent receptor-operated Ca2+ channels, voltage-operated Ca2+ channels, and protein kinase C are major signaling components for muscarinic receptor-mediated contraction of rat coronary resistance arteries.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Muscarinic
receptors are present in many vascular beds and commonly produce
endothelium-dependent vasodilation when activated. The coronary
vasculature also contains muscarinic receptors, but the response evoked
by stimulation of these receptors varies according to species and can
be affected by coronary artery diseases (Kalsner, 1989
; Treasure et
al., 1992; Egashira et al., 1995). Acetylcholine (ACh) and other
muscarinic receptor agonists cause endothelium-dependent relaxation of
canine coronary arteries (Kalsner, 1989; Feigl, 1998), whereas only
vasoconstriction occurs with porcine coronary arteries (Kawamura et
al., 1989). Bovine coronary arteries fall between these extremes and
can exhibit relaxation or contraction depending on the level of
existing tone and the concentration of agonist (Brunner et al., 1991).
It is most likely that such differences in response can be attributed
to variations among species in the relative abundance of muscarinic
receptors on endothelial and smooth muscle cells of the coronary
arteries. Activation of the endothelial receptors produces
vasodilation, mainly mediated by endothelium-derived relaxing factor
(EDRF), whereas stimulation of muscarinic receptors on coronary smooth
muscle cells evokes vasoconstriction. It is also possible that the
endothelium could contribute to coronary constriction by releasing
endothelium-derived contracting factors (Furchgott and Vanhoutte,
1989). Relaxation and contraction of isolated human coronary arteries
have been reported to occur with muscarinic receptor stimulation, but
vasoconstrictor responses become predominant with the progression of
coronary artery diseases (Hodgson and Marshall, 1989; Treasure et al., 1992; Egashira et al., 1995). Because there is anatomical and functional evidence that the coronary vasculature is innervated by
cholinergic neurons (Van Charldorp et al., 1987; Young et al., 1988;
Kalsner, 1989; Feigl, 1998), it is possible that the parasympathetic nervous system could play a role in coronary vasospasm.
Previous pharmacological studies have provided evidence that the m3
subtype of the muscarinic receptor mediates both coronary vasoconstrictor and vasodilator responses (Van Charldorp and Van Zwieten, 1989; Brunner et al., 1991; Boulanger et al., 1994; Hoover and
Neely, 1997). Experiments with cells expressing cloned m3 receptors
have demonstrated that activation of this receptor subtype can
stimulate several intracellular signaling pathways by coupling through
G-proteins (Felder, 1995). This mechanism can lead to activation of
phospholipase C and generation of diacylglycerol and inositol
1,4,5-trisphosphate, as well as stimulation of phospholipase A2 (PLA2) and generation of
arachidonic acid and several vasoactive eicosanoids. Voltage- and
store-operated calcium channels (VOCCs and SOCCs) can be opened as an
indirect response to m3 receptor stimulation (Felder, 1995), and recent
evidence suggests that m3 receptors can directly activate a novel class
of voltage-independent calcium channel called the receptor-operated
calcium channel (ROCC) (Murray et al., 1993; Felder, 1995). Coupling of
m3 receptors to ROCCs does not require diffusible second messengers and
may entail a direct interaction between the receptor and channel
(Felder, 1995).
It is well known that muscarinic receptor agonists and vagal
stimulation cause constriction of coronary resistance vessels in the
rat (Van Charldorp et al., 1987; Kalsner, 1989). Our previous experiments with spontaneously beating isolated rat hearts established that coronary vasoconstrictor responses to ACh were resistant to
pretreatment with pertussis toxin and inhibited by the m3 antagonist hexahydrosiladifenidol (Hoover and Neely, 1997). The goal of the present study was to elucidate signaling mechanisms that contribute to
muscarinic receptor-evoked constriction of rat coronary resistance vessels. Potassium-arrested hearts were used to eliminate mechanical and metabolic influences on the coronary system that might occur in
beating hearts as a consequence of drug effects on cardiac function.
The effects of specific enzyme inhibitors, channel blockers, and
receptor antagonists on phasic and tonic vasoconstrictor responses to
bethanechol were determined. Our observations suggest that ROCCs have a
significant role in generating phasic coronary vasoconstrictor responses, whereas VOCCs and protein kinase C (PKC) are important for
producing tonic and phasic responses.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Isolated Heart Preparation
Male Sprague-Dawley rats (250-350 g) were pretreated with
heparin (500 U/100 g b.wt., i.p.) approximately 20 min before
undergoing decapitation while deeply anesthetized with sodium
pentobarbital (68 mg/kg b.wt., 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
of ice-cold buffer, it was immediately transferred to an isolated heart
apparatus for retrograde perfusion by a modified Langendorff technique
(Broadley, 1979). The nonrecirculating perfusion solution was a
Krebs-Ringer-bicarbonate buffer containing (in mM): 127.2 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 KH2PO4, 24.9 NaHCO3, 1.2 MgSO4, 5.5 dextrose, 2.0 sodium pyruvate, and 0.1% BSA. Perfusion buffer in the
reservoir was continuously gassed with 95% O2,
5% CO2 (pH 7.35-7.4). A peristaltic pump
(Masterflex; Cole Parmer, St. Louis, MO) was used to perfuse hearts at
a constant flow rate of 8 ml/min. Because flow through the coronary
vasculature was held constant, changes in perfusion pressure directly
reflected alterations in coronary vascular resistance, an increase
signifying vasoconstriction and a decrease vasodilation. To maintain
the temperature at 37°C, the buffer passed through a water-heated glass coil and the heart was surrounded by a water-heated glass jacket.
Perfusion pressure was measured by a Statham-Gould P23ID pressure
transducer (Gould, Cleveland, OH) that was attached to the sidearm of a
three-way stopcock located at the proximal end of the aortic cannula.
Output from the pressure transducer was sent to a recorder (Gould
2400S) for monitoring perfusion pressure.
Hearts were initially perfused with a normal Krebs-Ringer-bicarbonate buffer for a 25-min equilibration period. Perfusions were subsequently switched to a modified buffer containing 20 mM KCl for the remainder of the experiment. The concentration of NaCl in this buffer was reduced and replaced with an equimolar concentration of KCl to maintain isotonicity. Perfusion with high K+ buffer caused cardiac arrest and a gradual increase in perfusion pressure over the experimental period (Table 1). Administration of bethanechol was started after perfusion of hearts with the high K+ buffer for 30 min.
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Administration of Bethanechol
The selective muscarinic receptor agonist bethanechol was administrated by bolus injections (100 µl) to produce phasic pressor responses or by constant infusion (100 µl/min) to cause a tonic increase in perfusion pressure. Injections and infusions were made through a short length of polyethylene 20 tubing (Clay Adams, Parsippany, NJ) that emptied into the perfusion buffer at a point close to the three-way stopcock attached to the aortic cannula. Bolus injections were given over approximately 2 to 3 s. A Harvard syringe infusion pump (Harvard Apparatus, South Natick, MA) was used to infuse bethanechol.
Experimental Design
Evaluation of Phasic Vasoconstrictor Responses to Bethanechol. Dose-response studies. Bolus injections of bethanechol were given in order of ascending dose with each injection made immediately after recovery from the response to the preceding dose. Two sets of dose-response data (i.e., trials 1 and 2) were collected from each heart preparation, and an interval of 30 min was allotted between trials. A control group of hearts was used to compare dose-response data obtained while perfusing with drug-free buffer during trials 1 and 2. For other groups, a specific blocker or enzyme inhibitor was present during one trial to determine its effect on the dose-response curve for bethanechol. The main protocol for these experiments was to use trial 1 as a control and trial 2 for treatments. In this case, we either switched to perfusion buffer containing the drug of interest or began infusing it after completion of trial 1. Trial 2 was started 30 min later with the inhibitor or blocker present in the buffer. Thus, each inhibitor had a 30-min period for its concentration to reach a steady state in the tissue before injection of bethanechol. This sequence was reversed in several experiments to determine whether the effects of treatment drugs on the vasoconstrictor response to bethanechol were reversible or dependent on the time of administration. For these experiments, treatment drugs were present from the start of perfusion with high K+ buffer through trial 1. Hearts were perfused with drug-free buffer for the remainder of these experiments.
A similar protocol was used to determine the effect of Ca2+-free buffer on vasoconstrictor responses to bethanechol. After obtaining control dose-response data during trial 1, the perfusion was switched to a modified Krebs-Ringer buffer prepared by omission of CaCl2 and addition of EGTA (1 mM). The effect of EGTA is mainly to chelate extracellular Ca2+ but not intracellular Ca2+ (Katsuyama et al., 1991). Trial 2 was
started after 30 min of perfusion with Ca2+-free buffer.
Dose dependence of inhibition by calcium channel blockers. To determine whether the concentrations of nifedipine and SK&F 96365 used in the preceding experiments were sufficient to cause maximum inhibition of the vasoconstrictor response to bethanechol, we measured the change in perfusion pressure caused by a bolus injection of 32 nmol of bethanechol (~ED50) during perfusion with drug-free buffer and again after stepwise infusion of increasing concentrations of blocker. Each blocker was studied in separate preparations. Bethanechol injections were given 4 min after starting infusion at each concentration of blocker.
Evaluation of Tonic Vasoconstrictor Responses to Bethanechol. Two concentrations of bethanechol (32 and 100 nmol/100 µl/min) were studied in one group of hearts, whereas other groups were used to determine the effect of treatment drugs on the tonic vasoconstrictor response to the lower concentration of bethanechol. Bethanechol was infused two times per heart for periods of 10 min. The control response to bethanechol infusion was recorded first, and the effect of vehicle or drug coinfusion was evaluated second. Coinfusions were of 3-min duration and began 3 min after starting the bethanechol infusion.
Drugs and Sources
The following drugs were used: acetylcholine chloride,
anthracene-9-carboxylic acid (A-9-C), bethanechol chloride,
NG-nitro-L-arginine
methyl ester hydrochloride (L-NAME), and
2,4'-dibromoacetophenone (p-BPB) (Sigma Chemical Co., St. Louis, MO);
chelerythrine chloride, indomethacin, nifedipine, and
[1S-[1
,2(Z),3,4]]-7-[3- [[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic
acid (SQ-29548) (Research Biochemicals International, Natick, MA);
esculetin, leukotriene D4
(LTD4), 1-[
-[3-(4-methoxyphenyl)proxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SK&F 96365) and 9,11-dideoxy-9,11-methanoepoxy prostaglandin F2
(U-46619) (Biomol Research
Laboratories, Inc., Plymouth Meeting, PA); L-660711 (a generous gift
from Merck Frosst Canada Inc., Pointe Claire-Dorval, Quebec, Canada).
Preparation and Storage of Drug Solutions
ACh and bethanechol were dissolved and diluted in saline.
Chelerythrine, L-NAME, L-660711, and SK&F 96365 were
dissolved in distilled water. A-9-C, LTD4, and
U-46619 were dissolved in ethanol (final ethanol concentration <1%).
Esculetin, indomethacin, nifedipine, p-BPB, and SQ-29548 were dissolved
in dimethyl sulfoxide (DMSO; final concentration 
0.1%). All stock
solutions, except LTD4 (10 µM in ethanol),
SQ-29548 (10 mM in DMSO), and U-46619 (29 mM in ethanol) were prepared
freshly for daily use. Aliquots of LTD4, SQ-29548, and U-46619 stock solutions were stored at 
80°C. Saline was used to make serial dilutions. The specific inhibitors, blockers, and agonists are summarized in Table 2
with their function and the amounts applied in this study.
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Statistical Analysis
The change in perfusion pressure evoked by a bolus injection or an infusion of bethanechol was calculated relative to the baseline value immediately before administration of the muscarinic agonist. Dose-response values of each heart were fit with a sigmoidal curve to obtain estimates of ED50 and the slope coefficient using GraphPad Prism version 2.01 (GraphPad Software, San Diego, CA). Data were analyzed by Microsoft Excel version 7.0a (Microsoft Excel software, Redmond, WA), and the values obtained were expressed as the arithmetic mean ± S.E. or the geometric mean with a 95% confidence interval. Values for ED50 were log-transformed for statistical analysis. The maximal vasoconstrictor responses and the estimated parameters for trials 1 and 2 dose-response curves for bethanechol in each heart preparation were compared by paired t test in each group. Two-factor ANOVA with repeated measures was used for evaluation of baseline perfusion pressure data. One-factor ANOVA was used to assess the baseline values in each column in Table 1. Dose-dependent vasoconstrictor responses to bethanechol were evaluated by three-factor ANOVA with multiple measures to assess the difference between the dose-response curves for control and treatment. A probability level of .05 or smaller was considered as significantly different.
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Results |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Baseline perfusion pressure data from control dose-response experiments with bolus injections of bethanechol and those in which effects of specific enzyme inhibitors, channel blockers, or receptor antagonists were evaluated are summarized in Table 1. Statistical evaluation of these data by two-factor ANOVA with repeated measures demonstrated significant increases in baseline perfusion pressure over the time of experiments (F1,84 = 454.5, P < .001 for the first dose-response curve; F1,84 = 449.3, P < .001 for the second dose-response curve). Although means for baseline perfusion pressure varied somewhat between groups, the differences were relatively small. All group means for columns in Table 1 are within one standard deviation of the corresponding column mean. Comparison of baseline data for the third column by one-factor ANOVA demonstrated that treatment of hearts with the indicated drugs, except L-NAME, had no significant effect on baseline perfusion pressure over the period in which dose-response data for bethanechol were collected.
Control Responses to Bethanechol.
Bolus injections of saline
produced a small injection artifact in the perfusion pressure recorder
tracing (Fig. 1A, left panel). In contrast, injections
of 32 nmol of bethanechol evoked a larger phasic increase in perfusion
pressure (maximum of 42 ± 1 mm Hg, n = 80) that
was easily distinguished from the injection artifact (Fig. 1A, right
panel). Serial bolus injections of bethanechol produced dose-dependent
vasoconstrictor responses with an ED50 of 26.9 nmol (95% CI of 12.9-56.1 nmol) and a maximal increase in perfusion
pressure of 67 ± 3 mm Hg (n = 6). Two sets of
dose-response data obtained from the same heart demonstrated good
replication (i.e., trials 1 and 2 in Fig.
2), providing a basis for the paired design used in subsequent dose-response studies with bolus injections of bethanechol.
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Effect of Extracellular Ca2+ on Responses to Bolus Injections of Bethanechol. Baseline perfusion pressure was decreased by 14 ± 4 mm Hg (n = 6) when perfusion was changed to Ca2+-free buffer for trial 2 and remained at that level. Bethanechol did not affect perfusion pressure under this condition (not shown).
Effect of A-9-C on Responses to Bolus Injections of
Bethanechol.
During treatment with the
Ca2+-dependent Cl
channel
blocker A-9-C (1 mM), baseline perfusion pressure was initially reduced by 5 ± 1 mm Hg (n = 3) but gradually returned to
the previous level. The maximum increase in perfusion pressure evoked
by bethanechol was reduced by 31 ± 1% (n = 3) in
the presence of A-9-C, but the ED50 level for
bethanechol was unaffected (Fig. 3).
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Effect of Nifedipine on Responses to Bethanechol.
The role of
L-type VOCCs in producing coronary vasoconstrictor responses to
bethanechol was evaluated in a series of experiments. We initially
examined the effect of 10 µM nifedipine on phasic responses to
bethanechol. Treatment with this concentration of nifedipine caused a
rapid decrease in baseline perfusion pressure (13 ± 2 mm Hg,
n = 4), which gradually returned to the initial value
over a 20-min period. When 10 µM nifedipine was present during trial
2 for bolus injections of bethanechol, the maximal vasoconstriction was
reduced by 59 ± 2%, and the ED50 was
increased by 3-fold (Fig. 4A). Comparable
suppression of responses to bethanechol was observed with 10 µM
nifedipine present during trial 1, and this inhibitory effect of
nifedipine was not reversed by perfusion with drug-free buffer (Fig.
4B).
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Effect of SK&F 96365 on Responses to Bethanechol.
Phasic
coronary vasoconstrictor responses to bethanechol were replaced by
vasodilation when 10 µM SK&F 96365 was present in the perfusion
buffer during trial 2 (Fig. 7A). The
maximal response to bethanechol was changed from an increase of 65 ± 2 mm Hg in perfusion pressure during trial 1 to a decrease of
15 ± 4 mm Hg in trial 2 (paired t test,
t = 11.74, P < .001). When 10 µM
SK&F 96365 was present in trial 1, bolus injections of bethanechol caused vasodilation, decreasing perfusion pressure by a maximum of
10 ± 2 mm Hg (Fig. 7B). This effect of SK&F 96365 could be reversed because bolus injections of bethanechol during subsequent perfusion with drug-free buffer (i.e., trial 2 in Fig. 7B) caused dose-dependent vasoconstrictor responses.
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Effect of Chelerythrine on Responses to Bethanechol.
During
treatment with 5 µM chelerythrine, baseline perfusion pressure was
initially decreased by 5 ± 0.3 mm Hg and gradually recovered.
Phasic responses to bethanechol were reduced significantly by 78 ± 2% (paired t test, P < .0001) compared
with control when chelerythrine was present during trial 2 (Fig.
8A). When chelerythrine was present
during trial 1, bolus injection of bethanechol increased perfusion
pressure by only 11 ± 2 mm Hg (Fig. 8B). This inhibitory effect
of chelerythrine on bethanechol-evoked vasoconstriction could not be
reversed on washout.
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Effects of p-BPB, Indomethacin, and Esculetin on Responses to Bolus Injections of Bethanechol. When present during trial 2, p-BPB (5 µM) decreased the maximum vasoconstrictor response to bethanechol by 43 ± 3%, whereas indomethacin (10 µM) and esculetin (10 µM) caused reductions of 31 ± 2 and 30 ± 1%, respectively (Table 4). Treatment with a combination of indomethacin and esculetin did not produce additional inhibition (n = 3, not shown, paired t test for maximal vasoconstriction, t = 0.01, P = .25). When indomethacin or esculetin was present in trial 1, no inhibitory effect on the vasoconstrictor response to bethanechol was observed (n = 3 each, paired t tests for maximal vasoconstriction: P = .87 for indomethacin and P = .33 for esculetin).
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Effect of SQ-29548 on Responses to Bethanechol.
The
thromboxane A2 (TxA2) analog
U-46619 produced a dose-dependent constriction of rat coronary
resistance vessels (Fig. 10A). Responses to U-46619 were eliminated in the presence of 1 µM SQ-29548 (n = 3, not shown). The maximum response to bolus
injection of bethanechol was reduced by 16 ± 1% when 1 µM
SQ-29548 was present during trial 2 (Table 4) but unaffected by its
presence during trial 1 (n = 3, paired t
test for maximal vasoconstriction, t = 0.01, P = .09).
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Effect of L-660711 on Responses to Bethanechol. LTD4 exhibited a high potency for constricting rat coronary resistance vessels when given by bolus injection (Fig. 10B). These responses were abolished during treatment with the selective LTD4 receptor antagonist L-660711 (10 µM, n = 3, not shown). The maximal vasoconstrictor response to bolus injections of bethanechol was reduced by 20 ± 4% when L-660711 was present in trial 2 (Table 4) but unaffected by the presence of LTD4 receptor antagonist during trial 1 (n = 3, not shown, paired t test for maximal vasoconstriction, t = 0.01, P = .46).
Coinfusion of L-660711 (10 µM final concentration) did not affect the tonic vasoconstriction produced by infusion of bethanechol at 32 nmol/100 µl/min (n = 3, not shown).| |
Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Muscarinic receptor-mediated coronary vasoconstriction is
important because of its potential role in the pathophysiology of vasospasm and its value as a model for evaluating mechanisms regulating contraction of coronary vascular smooth muscle. This study used potassium-arrested hearts to analyze some major signaling mechanisms that could function in linking muscarinic receptor activation to
contraction of smooth muscle in coronary resistance vessels. Our
observations demonstrate that bethanechol has a direct vasoconstrictor effect on rat coronary resistance vessels. Bolus injection of bethanechol dose-dependently evokes a phasic contraction as observed by
Sakai (1980), whereas infusion of bethanechol produces a tonic vasoconstriction without tachyphylaxis. Our results highlight the
importance of extracellular Ca2+ and
Ca2+ channels for both phasic and tonic
vasoconstrictor responses to bethanechol. Experiments with SK&F 96365 have provided the first evidence that ROCC could have a signaling
function in these vessels. Additionally, the effect of chelerythrine to
attenuate phasic and tonic responses to bethanechol suggests that PKC
activity is important for coronary vasoconstriction evoked by
muscarinic receptor activation. Lastly, our results indicate that
mediators downstream of PLA2 are not required for
the coronary vasoconstrictor effect of bethanechol in
potassium-arrested rat hearts.
Although K+ concentration in the perfusion buffer
was increased in our experiments to arrest the hearts, it is recognized
that this procedure also affected coronary vessels. Elevation of
extracellular K+ causes a concentration-dependent
depolarization of smooth muscle cells, Ca2+
influx through VOCCs, and smooth muscle contraction (Shimamoto et al.,
1993). These factors presumably contribute to the gradual increase in
baseline perfusion pressure that occurred over the experimental period.
We have also noted that the vasoconstrictor potency of bethanechol is
increased in potassium-arrested hearts compared with spontaneously
beating hearts (Y.Z. and D.B.H., unpublished observation). Both of
these changes presumably reflect an increase in coronary vascular tone
in the presence of 20 mM KCl. Despite these changes, the preparations
exhibited very consistent vasoconstrictor responses to bethanechol.
Entry of extracellular Ca2+ is required to refill
cytosolic Ca2+ stores, activate enzymes, and
regulate smooth muscle contractile elements directly (Berridge, 1997).
In the present study, bethanechol-evoked phasic vasoconstriction was
abolished by perfusion with Ca2+-free buffer.
Similar results were observed in spontaneously beating rat hearts
(Nuutinen et al., 1985). Therefore, influx of extracellular Ca2+ is essential for muscarinic
receptor-mediated constriction of rat coronary resistance vessels. This
finding concurs with evidence that resistance arteries have a greater
dependence on extracellular Ca2+ than elastic
capacitance vessels (Bulbring and Tomita, 1987) and that extracellular
Ca2+ influx becomes more dominating for
contraction as arteries get smaller (Low et al., 1996).
It is thought that VOCCs are the predominant Ca2+
entry pathway for vascular smooth muscle contraction (Malarkey et al.,
1996). Others have implicated VOCCs in muscarinic receptor-mediated
vasoconstriction produced by carbachol in potassium-arrested rat hearts
(Nuutinen et al., 1985). However, these investigators did not study
phasic responses to bolus injections of carbachol. We found that tonic and phasic vasoconstrictor responses to bethanechol differ in susceptibility to the VOCC blocker nifedipine. Tonic increases in
perfusion pressure were completely reversed by 10 µM nifedipine, whereas 40% of the maximum phasic response to bethanechol was resistant to blockade by the same concentration of VOCC blocker. Ca2+ influx through VOCCs has been suggested to
contribute predominantly to the maintenance of muscarinic
receptor-mediated vasoconstriction (Felder, 1995). Our results
demonstrate that VOCCs are heavily involved in phasic and tonic
contractions of rat coronary resistance vessels, although they play a
greater role in tonic responses.
Opening of VOCCs in smooth muscle by muscarinic agonists might be a
result of Ca2+-activated
Cl current-induced depolarization (Wayman et
al., 1997). In this regard, we observed that the
Ca2+-activated Cl current
inhibitor A-9-C caused a 31% decrease in the maximum pressor response
evoked by bolus injections of bethanechol. This finding suggests that
Ca2+-activated Cl
channels contribute to activation of VOCCs by bethanechol. However, additional mechanisms must be involved because the magnitude of inhibition produced by A-9-C was only half that of nifedipine.
It is well established that SK&F 96365 can produce reversible blockade
of voltage-independent ROCCs in excitable and nonexcitable cells
(Merritt et al., 1990; Okada et al., 1998). Nevertheless, this compound
also exhibits a similar potency for inhibiting VOCCs and SOCCs (Merritt
et al., 1990; Wayman et al., 1998), so these actions need to be
considered when evaluating results with SK&F 96365. Calcium entry
through SOCCs occurs as a consequence of Ca2+
depletion from intracellular stores and is required for sustaining contraction of some smooth muscles (Gibson et al., 1998). However, our
observation that tonic responses to bethanechol were completely reversed by nifedipine argues against a significant contribution from
SOCCs in rat coronary resistance vessels. Reversal of tonic vasoconstrictor responses by SK&F 96365 might be attributed to blockade
of VOCCs by this agent. In contrast, a significant portion of the
phasic response to bethanechol was insensitive to maximally effective
concentrations of nifedipine, whereas SK&F 96365 abolished phasic
vasoconstrictor responses. These findings suggest that ROCCs
mediate the nifedipine-insensitive component of phasic responses to bethanechol.
We speculate that an endothelium-dependent mechanism is responsible for
bethanechol-evoked vasodilation in the presence of SK&F 96365. This
response may also be dependent on Ca2+ influx via
VOCCs, because it was not detected during treatment with a combination
of SK&F 96365 and nifedipine. Our experiments using L-NAME
indicate the vasodilator response to bethanechol is not mediated by
EDRF. However, they did provide evidence for a high level of basal EDRF
release in potassium-arrested rat heart. Treatment with
L-NAME increased perfusion pressure by 81% in our study,
whereas a much smaller increase occurs in spontaneously beating rat
hearts (Yang et al., 1993; Y.Z. and D.B.H., unpublished observations). These observations suggest that high
K+ increases basal release of EDRF.
The involvement of PKC in ACh-induced coronary vasoconstriction has
been suggested by measuring PKC translocation and using phorbol esters
(Itoh et al., 1988; Haller et al., 1990). The functional role of PKC in
muscarinic receptor-mediated vasoconstriction in coronary resistance
vessels was first evaluated in our study. Chelerythrine reduced
bethanechol-evoked phasic vasoconstriction by a maximum of 79%. Tonic
vasoconstrictor responses to bethanechol were also partially reversed
by chelerythrine. The onset of response to infused chelerythrine was
gradual instead of rapid as observed with nifedipine and SK&F 96365. This difference is presumably related to the localization of specific
target proteins and biochemical mechanisms underlying effects of PKC
inhibition. Nifedipine and SK&F 96365 produce rapid effects because
they directly block channels in the cell membrane. For effects of
chelerythrine to be manifest, the drug must enter the cell to interact
with PKC and phosphatases must act to reverse the protein
phosphorylation produced by prior PKC activity. According to the
two-phase model for smooth muscle contraction, PKC plays a major role
in tonic contractions but not in phasic responses (Rasmussen et al.,
1987). Our results suggest that PKC activity is important for rat
coronary resistance vessels to generate both phasic and tonic responses
to bethanechol. Evidence from work with other models indicates that PKC
may promote vasoconstriction by inhibiting myosin light chain
phosphatase, by phosphorylating calponin and caldesmon, and by
enhancing the activity of VOCCs (Malarkey et al., 1996). The
later mechanism may be especially relevant in rat coronary resistance
vessels because VOCCs have a major role in muscarinic receptor-mediated vasoconstriction. Because Ca2+ is required at the
cell membrane for activation of phospholipase C and some isoforms of
PKC, it is possible that muscarinic receptor-mediated opening of ROCCs
could be important for activation of this pathway as well.
Results of the present study do not provide strong evidence that
muscarinic receptor-mediated coronary vasoconstriction involves signaling through the PLA2, COX, or lipoxygenase
pathways in potassium-arrested rat hearts. Although specific inhibitors
or blockers for this system reduced the maximum phasic response to
bethanechol when added during trial 2, treatment with several of these
agents during trial 1 had no effect. The reason for this trial
dependence is unclear, because the time for equilibration with the
tissue before challenge with bethanechol was the same regardless of the
trial used for treatment. It is possible that these pathways are
activated only after prolonged exposure to a high
K+ buffer and may not be a normal signaling
component for the response to bethanechol. Alternatively, addition of
specific inhibitors of these pathways during trial 2 may have a general
effect to reduce smooth muscle contraction. We also observed that tonic vasoconstrictor responses to bethanechol were unaffected by coinfusion of SQ-29548 or L-660711. Accordingly, neither
TxA2 nor LTD4 appears to
have a role in tonic vasoconstrictor responses to bethanechol in
potassium-arrested hearts. Other investigators have reported that
inhibition of COX reduces the maximum coronary vasoconstriction evoked
by ACh in isolated rat hearts (Yang et al., 1993; Nasa et al., 1997).
One of these groups also reported that SQ-29548 reduced the pressor
response to 1 µM ACh by 27% in beating hearts. We have no
explanation for the discrepancy between these reports and ours.
However, the effectiveness of SQ-29548 was established in our
experiments by verifying that it blocked coronary vasoconstrictor responses to the TxA2 analog U-46619.
In conclusion, the present study has provided evidence that multiple signaling mechanisms contribute to muscarinic receptor-mediated contraction of coronary resistance arteries in rat hearts. Voltage-independent ROCCs, VOCCs, and PKC have been identified as major components in generating coronary vasoconstrictor responses to bethanechol. Activation of ROCCs through stimulation of muscarinic receptors may play an important role in the subsequent activation of PKC and opening of VOCCs.
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Footnotes |
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Accepted for publication December 21, 1999.
Received for publication August 26, 1999.
1 This work was supported by National Institutes of Health Grant HL-54633. Preliminary reports of the results were presented at the 8th International Symposium on Subtypes of Muscarinic Receptors and the 28th Annual Meeting of the Society for Neuroscience.
Send reprint requests to: Dr. Donald B. Hoover, Department of Pharmacology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614-0577. E-mail: hoover{at}etsu.edu
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Abbreviations |
|---|
ACh, acetylcholine;
A-9-C, anthracene-9-carboxylic acid;
COX, cyclooxygenase;
p-BPB, 2,4'-dibromoacetophenone;
ROCC, receptor-operated calcium channel;
SK&F 96365, 1-[-[3-(4-methoxyphenyl)proxy]-4-methoxyphenethyl]-1H-imidazole
hydrochloride;
SQ-29548, [1S-[1,2(Z),3,4]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic
acid;
U-46619, 9,11-dideoxy-9,11-methanoepoxy prostaglandin
F2;
VOCC, voltage-operated calcium channel;
EDRF, endothelium-derived relaxing factor;
PLA2, phospholipase
A2;
PKC, protein kinase C;
L-NAME, NG-nitro-L-arginine methyl ester
hydrochloride;
LTD4, leukotriene D4;
DMSO, dimethyl sulfoxide.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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