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Department of Pharmacology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The possible role of type II
(cGMP-stimulated cAMP hydrolysis) phosphodiesterase (PDE) in the
accentuated antagonism of muscarinic effects on heart rate during
-stimulation via endogenous nitric oxide (NO) was evaluated. The
canine isolated sinoatrial node preparation was cross circulated with
arterial blood of a support dog. The sinoatrial rate of the preparation
was 96 ± 5 beats/min (n = 16) at control.
Methacholine (MCh; 0.01-1 µg) injected into the right coronary
artery in a bolus fashion caused dose-dependent decreases in sinoatrial
rate. Under an intra-arterial infusion of isoproterenol (1 µM),
resulting in ~50% increase in sinoatrial rate, MCh-induced decreases
were markedly augmented from 
18 ± 3% to 44 ± 4% at
0.3 mg of MCh. When
NG-nitro-L-arginine methyl ester
(100 µM) or
NG-monomethyl-L-arginine (100 µM)
were continuously infused, the augmented MCh-induced decreases in
sinoatrial rate were significantly suppressed (29 ± 3% or
25 ± 3%, respectively, P < 0.01).
Pretreatment with either 3-isobutyl-1-methylxanthine (IBMX; 20 µM), a
non-selective PDE inhibitor, or amrinone (20 µM), a selective type
III (cGMP inhibited cAMP hydrolysis) PDE inhibitor, doubled the
isoproterenol-induced increase in the sinoatrial rate. However, the
augmented MCh-induced decreases in sinoatrial rate were significantly
depressed by IBMX (from 23 ± 5% to 14 ± 1%,
P < 0.01) but not by amrinone (to 20 ± 3%).
These results suggest that MCh-induced accentuated antagonism in the
sinoatrial node pacemaker activity can be modulated by endogenous NO
via an activation of the type II cyclic GMP-stimulated cAMP PDE.
negative chronotropic effect; NG-nitro-L-arginine methyl ester; NG-monomethyl-L-arginine; 3-isobutyl-1-methylxanthine; amrinone; nitric oxide; phosphodiesterase; methacholine
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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NITRIC OXIDE (NO) is known to play an important role as a modulator of many cellular processes in various tissues (23). In particular, NO has been found to play physiological and pathophysiological roles in regulating cardiac functions, whereas various types of NO synthase (NOS) have been identified in cardiac myocytes, endocardium, intracardiac nerves, macrophages, and vascular endothelium (15). The NADPH diaphorase technique and immunohistological approaches have demonstrated NOS-positive neurons in the atrial myocardium of guinea pig, in particular the sinoatrial (SA) node and atrioventricular (AV) node (17, 27); these latter tissues are generally considered to be densely innervated by postganglionic parasympathetic neurons.
Recently, it was reported that NO-mediated intracellular
signaling pathways are involved in muscarinic cholinergic inhibition of
heart rate and cardiac contraction (15). In particular,
Han and colleagues (9-11) substantially demonstrated
in mammalian SA and AV node cells that NO modulated muscarinic
cholinergic regulation of L-type Ca current
(ICa,L) when intracellular cAMP was
increased by -adrenoceptor stimulation. This has been known as
"accentuated antagonism," a term first introduced by Levy
(20) for the sympathetic-parasympathetic interactions in
the heart. In addition, Han et al. (10) also suggested
that NO-mediated muscarinic cholinergic modulation of
ICa,L might be in part due to an activation of
the type II cGMP-stimulated cAMP phosphodiesterase (PDE).
On the other hand, muscarinic inhibition of the SA node pacemaker
activity results not only from inhibition of
ICa,L but also from activation of atrial
muscarinic-activated K+ current
(IK,ACh) and an inhibition of
hyperpolarization-activated inward pacemaker current
(If) carried by Na+
(14). The latter could predominate in the absence of
-adrenoceptor stimulation. In the absence of -adrenoceptor
stimulation, the physiological roles of endogenous NO, which is
synthesized by cNOS, in muscarinic cholinergic nerves have been
controversial (15, 29). NOS inhibitors had little effect
on ICa,L, even in the presence of a muscarinic
cholinergic agonist when intracellular cAMP was not elevated (1,
11). Furthermore, NOS inhibitors also did not affect muscarinic
stimulation of IK,ACh, which is known to be
mediated by a pertussis toxin-sensitive G protein subunit
(18). Thus the effect of endogenous NO on
IK,ACh also needs to be further assessed.
In addition to the pacemaker activity, it was demonstrated that NO also
suppressed -adrenoceptor-mediated increases in
ICa,L and contraction of ventricular myocytes,
which are postulated to be mediated in large part by increases in
intracellular cGMP (22). However, the roles of NO in
muscarinic inhibitions of ventricular functions have remained
controversial even under -adrenoceptor stimulation (1, 3, 12,
29, 30).
In the present study, we attempted to clarify the following issues:
1) the possible roles of NO in the accentuated antagonism observed in muscarinic interventions under -adrenoceptor
stimulation, 2) possible roles of type II PDE, and
3) potential differences between chronotropism and
inotropism in relation to the interaction between NO regulation and
parasympathetic innervation to the cardiac tissues. For these purposes,
we used canine isolated blood-perfused SA node and papillary muscle
(PM) preparations, in which drugs were administered directly into each
nutrient coronary artery. The negative chronotropic and inotropic
effects of methacholine (MCh), a long-acting analog of ACh, were
compared under similar physiological conditions.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Isolated, Blood-Perfused SA Node and PM Preparations
Experiments were carried out on the canine isolated SA node (19), and PM (8) preparations were cross circulated with heparinized arterial blood of a support dog (24, 25). The hearts were obtained from mongrel dogs of either sex weighing 8-12 kg. The animals were anesthetized with pentobarbital sodium (30 mg/kg iv), given heparin calcium (500 U/kg iv), and exsanguinated. The heart was rapidly excised and plunged into cold Tyrode solution kept at ~4°C.The SA node preparation consists of the entire right atrium in which the sinus node artery was cannulated through the right coronary artery. The SA rate of the spontaneous beating preparation was measured with a cardiotachograph (1321; NEC San-ei Instruments, Tokyo, Japan) triggered by bipolar atrial electrograms recorded from the atrial epicardium close to the SA node.
The PM preparation consists of the anterior papillary muscle of the right ventricle attached to the interventricular septum. The cannulated anterior septal artery supplying to the PM was perfused by the support dog. The PM preparation was electrically driven at a fixed basal cycle length of 500 ms by a stimulator (DHM-226-3; Dia Medical, Tokyo, Japan) and an isolation unit (DSP-110; Dia Medical) with rectangular pulses of 1-3 V (~20% above the threshold voltage) and 5-ms duration through bipolar stimulating electrodes sutured onto the endocardium of the ventricular septum close to the base of the PM. Developed tension of the PM preloaded with a 2-g weight was measured isometrically using a force-displacement transducer (DRM-T200; Dia Medical) and an amplifier (DRM-T20; Dia Medical).
Cross Circulation
The SA node and PM preparations were each placed in double-wall glass jackets maintained at 38°C by circulating warm water. Both preparations were simultaneously cross circulated with the heparinized arterial blood of a support dog through the cannulated arteries at a constant perfusion pressure of 120 mmHg with a Harvard peristaltic pump and a Starling pneumatic resistance placed parallel to the perfusion system. Venous blood from each preparation and excess blood passing through the pneumatic resistance were collected in the blood reservoir and returned to the support dog through the jugular vein. The coronary blood flow rate in each cannulated artery was measured using an electromagnetic flowmeter (MFV-1100, Nihon Kohden, Tokyo, Japan) and 2-mm cannulating flow probes.Adult mongrel dogs of either sex, weighing 12-18 kg, used as
support dogs, were anesthetized with an initial intravenous dose of 30 mg/kg of pentobarbital sodium and maintained with a continuous intravenous infusion of 5 mg · kg
1 · h1 at a rate of
10 ml/h by using an infusion pump (model STC-521, Terumo, Tokyo,
Japan). The animals received an initial dose of 500 U/kg heparin
calcium, followed by 200 U/kg every hour. Respiration was controlled
using an animal respirator (SN-3041, Shinano Instruments, Tokyo,
Japan). Systemic blood pressure at the femoral artery and heart rate
triggered by the R wave of the limb II lead of the electrocardiogram
were monitored continuously with a polygraph (361-6, NEC San-ei
Instruments). Blood pressure and heart rate of the support dog were
kept constant during experiments. Mean blood pressure and heart rate
were 97 ± 6 mmHg and 117 ± 8 beats/min at the beginning of
the protocol and 95 ± 5 mmHg and 109 ± 7 beats/min 2 h
after the protocol, respectively.
Experimental Protocol
Protocol 1. To confirm that endothelium-dependent coronary vasodilatation was intact, the effects of ACh (0.01-1µg), which is known as an endothelium-dependent vasodilator, and MCh (0.01-1 µg), which is a long-acting analog of ACh, on the coronary blood flow, SA rate, and developed tension were compared by injecting them into each nutrient artery of the preparation with a microsyringe (Terumo, Tokyo, Japan). Peak changes in each parameter were measured and expressed as percentages of corresponding basal predrug values. Dose-response curves for negative chronotropic, negative inotropic, and coronary vasodilator effects of MCh and ACh were obtained, respectively.
Protocol 2.
For -adrenoceptor stimulation, isoproterenol (Iso) was continuously
infused into each nutrient artery to produce a steady-state concentration of 1 µM in the arterial blood, which produced an ~50% increase in SA rate and a 100% increase in developed tension, respectively. The blood concentration of Iso at 1 µM was calculated by measuring the rate of coronary blood flow and adjusting the concentration of Iso in the syringe for a continuous infusion at a rate
of 0.1 ml/min into the nutrient artery. During the infusion, the
coronary blood flow was essentially unchanged. As a result, the
infusion rate to achieve a target concentration for Iso of 1 µM in
the arterial blood could be reliably calculated.
-adrenoceptor stimulation, MCh (0.01-1 µg)
was administrated into each nutrient artery of the preparations, and
dose-response curves for negative chronotropic and negative inotropic
effects were obtained as percentages of the corresponding pre-MCh
values that had been enhanced by Iso.
After the parameters of the preparations returned to stable states,
NG-nitro-L-arginine methyl ester
(L-NAME), a nonselective NOS inhibitor, was infused to give
a concentration of 100 µM in arterial blood, calculated and adjusted
according to the coronary blood flow rate and drug concentration in the
syringe. Iso (1 µM in arterial blood) was then infused again and
subsequently MCh was injected again, and dose-response curves for
negative chronotropic and negative inotropic effects were obtained.
Similarly, NG-monomethyl-L-arginine
(L-NMMA), another nonselective NOS inhibitor, was infused
into the right coronary artery (100 µM in arterial blood), and then
the negative chronotropic effects of MCh were evaluated during Iso (1 µM) infusion in the SA node preparation.
Protocol 3.
Likewise, MCh (0.01-1µg) was administrated into the right
coronary artery of the SA node preparation in the presence of
-adrenoceptor stimulation with 1 µM of Iso, and the dose-response
curve for negative chronotropic effects of MCh was obtained again.
After the SA rate and coronary blood flow of the SA node preparation returned to stable baseline states, 3-isobutyl-1-methylxanthine (IBMX),
which is a nonspecific PDE inhibitor, or amrinone, which is a selective
PDE III inhibitor, were infused to give a concentration of 20 µM,
calculated and adjusted according to the coronary blood flow rate and
drug concentration in the syringe. Iso (1 µM in arterial blood) was
then infused, subsequently MCh was injected again, and dose-response
curves for negative chronotropic effects of MCh were obtained, respectively.
Protocol 4.
In contrast to protocol 2, influences of L-NAME
(100 µM in arterial blood) or L-arginine (100 µM in
arterial blood), a substrate for NOS, on the negative chronotropic and
inotropic effects of MCh were determined in the absence of
-adrenoceptor stimulation, respectively. Thereafter,
L-arginine was infused after an infusion of
L-NAME, and the dose-response curves for negative
chronotropic and negative inotropic effects of MCh were obtained again.
Drugs
Drugs used were MCh chloride (Sigma, St. Louis, MO), Iso hydrochloride (Nikken Kayaku, Tokyo, Japan), L-NAME (Wako, Osaka, Japan), IBMX (Wako, Osaka, Japan), amrinone (Yamanouchi, Tokyo, Japan), L-NMMA (Wako), and L-arginine (Wako).Statistics
All values in text are arithmetic means ± SE. Statistical analyses were performed using ANOVA. When F statistics were significantly larger than the appropriate critical values, post hoc t-tests were performed. Simultaneous multiple comparisons were then done by the Bonferroni methods (28). When P values were <0.05, the difference was taken to be significant.Ethics
The experiments were performed in accordance with guidelines for animal experimentation of Hirosaki University.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Comparison of the Effects of MCh and ACh in the SA Node and PM Preparations
In the SA node preparations, the basal SA rate was 96 ± 8 beats/min (n = 8), and the basal coronary blood flow through the right coronary artery was 3.0 ± 0.3 ml/min. MCh or ACh in a dose range of 0.01-1 µg injected into the right coronary artery in a bolus fashion produced dose-dependent decreases in SA rate (negative chronotropic effects). The maximal negative chronotropic effect of MCh was comparable with that of ACh. The doses that decreased the SA rate by 20% were 0.30 ± 0.10 µg for MCh and 0.47 ± 0.15 µg for ACh (n = 8), respectively.In the PM preparations, after ventricular fibrillation was terminated ~1 h after the start of cross circulation, the basal developed tension was 6.5 ± 0.2 g, and the basal coronary blood flow through the anterior septal artery was 5.2 ± 0.3 ml/min (n = 8). When MCh or ACh in a dose range of 0.01-1 µg was injected into the anterior septal artery, dose-dependent decreases of developed tension (negative inotropic effects) and increases in coronary blood flow rate through the anterior septal artery (coronary vasodilator effects) were produced. The maximal negative inotropic effect and coronary vasodilator effect of MCh were comparable with those of ACh. The doses that decreased the developed tension by 20% were 0.75 ± 0.20 µg for MCh and 0.54 ± 0.15 µg for ACh (n = 8), and the doses that increased the coronary blood flow rate through the anterior septal artery by 20% were 0.022 ± 0.009 µg for MCh and 0.011 ± 0.005 µg for ACh (n = 8), respectively. There were no significant differences between the doses of MCh and ACh.
Accentuated Antagonisms of MCh-Induced Negative Chronotropic Effects in the SA Node and Negative Inotropic Effects in the PM Preparations
When Iso was continuously infused into the nutrient coronary artery to give a calculated blood concentration of 1 µM (see METHODS), the SA rate was increased from 92 ± 3 beats/min to 158 ± 6 beats/min (n = 8). As shown in Fig. 1, A and B, when MCh was injected into the right coronary artery in the presence of Iso, the SA rate was slowed markedly more compared with the bradycardia observed in the absence of Iso. In addition to the augmented decreases in SA rate, the duration of the negative chronotropic effect was prolonged in the case of administration of a larger dose of MCh in the presence of Iso. Similarly, as shown in Fig. 2, A and B, decreases in the developed tension produced by MCh were augmented in the presence of Iso compared with those in the absence of Iso.
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In Fig. 3A, the averaged
dose-response curves are shown for the negative chronotropic effects of
MCh in the absence and presence of Iso. Quantitatively, for example,
the decrease in SA rate, which was 18 ± 3% at 0.3 µg of MCh in
the absence of Iso, was significantly augmented to 44 ± 4% at
the same dose of MCh in the presence of Iso (n = 8, P < 0.01). The averaged dose-response curves for the
negative inotropic effect of MCh are shown in Fig. 3B. At
control, the basal developed tension was 7.0 ± 0.3 g
(n = 8), and the decrease in developed tension was
15 ± 1% at 0.3 µg of MCh. In the presence of Iso (1 µM), the
pre-MCh developed tension was increased to 17.0 ± 0.5 g
(n = 8), and the decrease in developed tension
was significantly enhanced to 31 ± 4% at the same dose of MCh
(n = 8, P < 0.01).
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These phenomena have been termed "accentuated antagonism" by Levy (20). In both preparations, however, MCh-induced increases in coronary blood flow were not enhanced in the presence of Iso. The increases were 18 ± 2% (n = 8) in the SA node and 28 ± 3% (n = 8) in the PM preparations at 0.3 µg of MCh in the absence of Iso, whereas increases were 17 ± 3 (n = 8) and 30 ± 5% (n = 8) in the presence of Iso, respectively.
Effects of NOS Inhibition on Accentuated Antagonisms of MCh-Induced Negative Chronotropic and Inotropic Effects
In the SA node preparations, when the SA rate returned to the basal level, at least 1 h after cessation of Iso infusion, a continuous intracoronary infusion of L-NAME was started, calculated as 100 µM in blood. After an infusion of L-NAME alone, the SA rate was slightly reduced from 92 ± 2 to 90 ± 2 beats/min (n = 8, P < 0.05), and the coronary blood flow was similarly reduced from 3.1 ± 0.4 to 2.9 ± 0.4 ml/min (n = 8, P < 0.05). After treatment with L-NAME, the augmented negative chronotropic effect of MCh in the presence of Iso (Fig. 1B) was markedly suppressed, as shown in Fig. 1C. Quantitatively, as shown in Fig. 3A, the MCh-induced (0.3 µg) decrease in SA rate in the presence of Iso was significantly suppressed from 44 ± 4 (Iso alone) to 29 ± 3% after pretreatment of L-NAME (100 µM) (n = 8, P < 0.01). The increases in coronary blood flow induced by MCh were not significantly affected.By contrast, in the PM preparation, the magnitude of the MCh-induced negative inotropic effects in the presence of Iso were not affected by L-NAME (Fig. 2C). As shown in Fig. 3B, the averaged decreases in developed tension in the presence of Iso (1 µM) were 31 ± 4 and 32 ± 4% before and after pretreatment of L-NAME (100 µM), respectively. There was no significant difference between them. Although the coronary blood flow through the anterior septal artery of the PM preparation was also slightly reduced from 5.6 ± 0.8 to 4.8 ± 0.8 ml/min (n = 8, P < 0.01) after L-NAME (100 µM), the increases in coronary blood flow induced by MCh were not significantly changed.
Identical results were obtained when L-NMMA was used
instead of L-NAME. Augmented negative chronotropic effects
of MCh in the presence of Iso were similarly suppressed after an
infusion of L-NMMA. Figure 4
shows averaged dose-response curves for the negative chronotropic
effects of MCh in the absence and presence of Iso and for Iso + L-NMMA. At control, the basal SA rate was 94 ± 6 beats/min (n = 6), and 0.3 µg of MCh decreased the SA
rate 19 ± 3%. In the presence of Iso (1 µM), the pre-MCh SA
rate increased to 150 ± 9 beats/min (n = 6), and the
decrease in SA rate was significantly augmented to 42 ± 5% at
the same dose of MCh. After treatment of L-NMMA
(100 µM), the augmented decrease in SA rate was significantly
suppressed to 25 ± 3% at 0.3 µg of MCh in the presence of
Iso (n = 6, P < 0.01).
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Effects of PDE Inhibition on Accentuated Antagonisms of MCh-Induced Negative Chronotropic Effect
In the SA node preparation, after confirmation of the accentuated antagonism and the SA rate had returned to the basal level, at least 1 h after cessation of Iso infusion, a continuous intracoronary infusion of IBMX or amrinone (20 µM in arterial blood) was started. Although an infusion of either IBMX or amrinone did not significantly affect the SA rate, the SA rate was almost doubled by a subsequent infusion of Iso from 95 ± 3 to 175 ± 8 beats/min. As shown in Fig. 5, the augmented negative chronotropic effects of MCh in the presence of Iso were significantly suppressed by IBMX, but not affected by amrinone, even in the presence of Iso. For example, at 0.3 µg of MCh, decreases in SA rate were 17 ± 4% at control, 23 ± 5% in the presence of Iso, 14 ± 1% in the presence of Iso + IBMX, and 20 ± 3% in the presence of Iso + amrinone, respectively.
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Effects of NOS Inhibition on the Negative Chronotropic and
Inotropic Effects of MCh in the Absence of -Adrenoceptor
Stimulation
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The averaged dose-response curves for negative chronotropic and
inotropic effects of MCh in the absence of Iso are shown in Fig.
8. In both preparations,
L-NAME alone or excess L-arginine per se did
not affect the MCh-induced negative chronotropic and inotropic effects
in the absence of Iso (Fig. 8, A and B). However, the negative chronotropic effects induced by larger doses of MCh were
suppressed by excess L-arginine infused subsequently to
L-NAME (Fig. 8A). On the other hand, the
MCh-induced negative inotropic effects were not affected by
L-arginine + L-NAME (Fig. 8B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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In the present study, applying the canine isolated, blood-perfused
SA node and PM preparations, we demonstrated the following findings:
1) MCh-induced muscarinic cholinergic interventions in the
SA node pacemaker activity can be modulated by endogenous NO at least
in the presence of -adrenoceptor stimulation; 2) under an
increased adrenergic tone, accentuated antagonism of MCh-induced
negative chronotropic effect can be partially modulated by endogenous
NO via an activation of the type II cGMP-stimulated cAMP PDE;
3) however, in the absence of -adrenoceptor stimulation, NO does not play an important role in MCh-induced muscarinic
cholinergic interventions in heart rate; and 4) on the other
hand, muscarinic cholinergic interventions in ventricular contractility
cannot be modulated by endogenous NO, even in the presence of
-adrenoceptor stimulation.
In the present experiments, L-NAME was mainly used for inhibition of NO synthase. Certainly, L-NAME maneuver has a wide variety of other actions as reviewed by Campbell and Harder (4). In addition, as reported by Buxton et al. (5), L-NAME has an antimuscarinic action, which could have an influence on the effects of MCh. According to Buxton et al. (5), however, L-NMMA does not possess an atropine-like action. Therefore, the experiment was repeated using L-NMMA instead of L-NAME, and almost identical qualitative results were obtained, as shown in Fig. 4. Thus the results obtained by using L-NAME as well as L-NMMA were equally reliable under the conditions of the present study.
In the present study, the magnitude of accentuated antagonism was
expressed as a percentage of pre-MCh values. This means that pre-MCh
values had been increased by -adrenoceptor stimulation with or
without PDE inhibition and before or after NOS inhibition. Thus the
larger percentage decrease in SA rate in the presence of
-adrenoceptor stimulation means a further large decrease in actual
SA rate, that is, accentuated antagonism. Even in the actual values,
however, L-NAME did not affect the accentuated antagonism of MCh-induced negative inotropic effect. On the other hand, the duration of the negative chronotropic effects in accentuated antagonism at larger doses of MCh might be due to the shift of the predominant pacemaker within the SA node, because the negative chronotropic effect
abruptly terminated and then gradually returned to the basal value.
Such hypothetical changes would likely be similar to the observations
for the predominant pacemaker shift in the AV junctional area
(26).
The SA node is usually under the tonic influence of both divisions of
the autonomic nervous system in the mammalian heart. Ordinarily,
parasympathetic tone predominates over sympathetic tone at the SA node,
but changes in heart rate are evoked by the interaction of both
divisions. Muscarinic inhibition of SA nodal automaticity primarily
results from inhibition of If and
ICa,L and an activation of
IK,ACh (14). Muscarinic receptors,
when stimulated, are coupled with the pertussis toxin-sensitive G
proteins; the latter are heterotrimers consisting of 
-, -, and
-subunits and are postulated to be the critical components for
activating ICa and
IK,ACh. Under an increased adrenergic tone, it
has been demonstrated that ACh can antagonize the activation of
adenylyl cyclase via mechanisms involving G proteins (probably
Gi and/or Go) and that it can inhibit Ca
channel phosphorylation, thereby decreasing ICa
(13). In this case, an inhibitory effect of ACh on
-adrenoceptor activation is mainly modulated by interactions between
the -subunit of Gi (Gi) and adenylyl
cyclase, resulting in a decrease in cAMP level (4).
It has been suggested in various species that NO donors release NO and increase the intracellular cGMP levels by enhancing guanylyl cyclase activity in cardiac myocytes, and thereby modulate the activity of ICa,L. For example, in frog and guinea pig ventricular cells, sodium nitroprusside (in µM concentration), similar to cGMP, had little effect in altering the amplitude of ICa,L in the absence of cAMP elevation (22). When the intracellular cAMP concentration was elevated, however, sodium nitroprusside administration inhibited ICa,L, which was attributed to cGMP-stimulated PDE in the frog and to cGMP-activation of cGMP-dependent protein kinase in the guinea pig (22). In mammalian hearts, it was demonstrated that type II (cGMP stimulated) and type III (cGMP inhibited) PDE are primarily localized in cardiac tissue (2). In this connection, in the present study, we demonstrated that IBMX, a nonspecific PDE inhibitor, suppressed the accentuated antagonism of MCh-induced negative chronotropic effect, but amrinone, a selective PDE III inhibitor, did not. Therefore, our present results indirectly, but strongly, suggested that the accentuated antagonism of MCh-induced negative chronotropic effect could be modulated by type II PDE, which is stimulated by cGMP produced by endogenous NO via activation of cGMP-dependent protein kinase.
On the other hand, in the absence of -adrenoceptor stimulation, the
physiological role of the NO-mediated signaling pathway in the SA node
has been controversial. At a baseline stable state, NO did not play a
significant role in parasympathetic modulation of even the SA node,
because L-NAME did not affect heart rate at all. However,
when excess L-arginine was infused, this intracellular signaling pathway began to operate in parasympathetic modulation of the
SA node, and thereafter L-NAME began to inhibit MCh-induced negative chronotropic effects. Hence, even in the absence of
-adrenoceptor stimulation, excess L-arginine may play a
key role in modulation of NO-mediated muscarinic cholinergic transmission.
As discussed above, the muscarinic receptors are also coupled with the
ACh-regulated K+ channels by the - and -subunit
(G) of pertussis toxin-sensitive G protein and do not require
intracellular buildup of second messengers, mainly cAMP, to exert
beat-by-beat control of heart rate (21). Hence, if
endogenous NO modulates heart rate through the NO-mediated muscarinic
cholinergic signaling pathway even in a basal condition, it should
affect not only ICa,L but also
IK,G, i.e., IK,ACh. In the present study, however, we demonstrated that even the MCh-induced negative chronotropic effect was not influenced significantly by
pharmacological interventions to NO synthesis in the absence of
-adrenoceptor stimulation. These results may imply that endogenous NO-mediated intracellular signaling does not intervene to G, which mediates the activity of ACh-regulated K+ channels.
Furthermore, it has remained controversial whether NO donors affect the
IK,ACh that directly binds G. Recently, we
investigated the effects of an NO donor, nitroglycerin, on MCh-induced
negative chronotropic and inotropic effects, but we could not
demonstrate any direct interactions between the NO donor and muscarinic
cholinergic interventions in the absence of -adrenoceptor stimulation. These results indicate that endogenous NO may have different actions than exogenous NO in the muscarinic cholinergic modulation of the SA node pacemaker activity. Thus, in the heart, cNOS
may exert different actions from inducible NOS for pacemaker activity.
In the present study, augmented MCh-induced negative chronotropic
effect was suppressed substantially by NOS inhibition with L-NAME, but the enhanced MCh-induced negative inotropic
effect was not affected. These results have convinced us of the
existence of an NO-mediated signaling pathway at least in the SA node,
as already discussed, and suggest that this pathway may have tissue specificity. Roles of NO in muscarinic inhibition of ventricular functions have been controversial even in the presence of
-adrenoceptor stimulation. It has been reported in an in vivo study
(12) that NO mediates vagal inhibition of the inotropic
response to -adrenergic stimulation. However, NO did not affect the
muscarinic inhibition of cAMP-regulated ion (Cl) channels
in ventricular myocytes (30) and did not elicit an acute
negative inotropic effect in unstimulated ventricular muscle (29). This tissue specificity of the action might be
partly due to the difference of distribution of M2-receptors between the atrium and the ventricle. Parasympathetic nerves were shown to
densely innervate the atria, in particular the SA node in dogs, whereas
the ventricles are sparsely innervated by the vagi, although species
differences do exist in the distribution of M2-receptors (7,
16). This difference of parasympathetic innervation could be
attributed to the heterogeneity in the distribution of M2-receptors in
the heart.
In conclusion, NO-mediated modulation of muscarinic cholinergic
transmission was clearly demonstrated only in the case of augmented
MCh-induced negative chronotropic effect under an increased adrenergic
tone, when ICa, ICa,L, or
T-type Ca current could be enhanced by -adrenoceptor
stimulation. These modulations were mediated via an activation of the
type II cGMP-stimulated cAMP PDE. In the basal condition, however, the
MCh-induced negative chronotropic effect, which could be mainly
mediated by activation of IK,G, i.e.,
IK,ACh, might not be modulated by endogenous NO. Consequently, expression of endogenous NO-mediated actions might be
largely dependent on both the presence of -adrenoceptor stimulation and the magnitude of parasympathetic cholinergic innervation, which
predominates in the SA node.
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ACKNOWLEDGEMENTS |
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The authors thank I. Miki, K. Komune, T. Kuramae, and K. Inoue for technical assistance.
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FOOTNOTES |
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This work was supported in part by Grants-In-Aid for Scientific Research (07672454, 09670086) from the Minister of Education, Science, and Culture, Japan, and the Karoji Memorial Fund for Medical Research in Hirosaki University, Hirosaki, Japan.
Address for reprint requests and other correspondence: S. Motomura, Dept. of Pharmacology, Hirosaki University School of Medicine, 5 Zaifu-cho, Hirosaki 036-8562, Japan (E-mail: moto{at}cc.hirosaki-u.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 24 September 1999; accepted in final form 22 June 2000.
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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