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Effects of Ca²⁺ channel antagonists on nerve stimulation-induced and ischemia-induced myocardial interstitial acetylcholine release in cats

¹Department of Cardiovascular Dynamics, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute and ²Department of Cardiac Physiology, National Cardiovascular Center Research Institute, Osaka, Japan

Submitted 17 February 2006 ; accepted in final form 7 June 2006

	ABSTRACT

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Although an axoplasmic Ca²⁺ increase is associated with an exocytotic acetylcholine (ACh) release from the parasympathetic postganglionic nerve endings, the role of voltage-dependent Ca²⁺ channels in ACh release in the mammalian cardiac parasympathetic nerve is not clearly understood. Using a cardiac microdialysis technique, we examined the effects of Ca²⁺ channel antagonists on vagal nerve stimulation- and ischemia-induced myocardial interstitial ACh releases in anesthetized cats. The vagal stimulation-induced ACh release [22.4 nM (SD 10.6), n = 7] was significantly attenuated by local administration of an N-type Ca²⁺ channel antagonist -conotoxin GVIA [11.7 nM (SD 5.8), n = 7, P = 0.0054], or a P/Q-type Ca²⁺ channel antagonist -conotoxin MVIIC [3.8 nM (SD 2.3), n = 6, P = 0.0002] but not by local administration of an L-type Ca²⁺ channel antagonist verapamil [23.5 nM (SD 6.0), n = 5, P = 0.758]. The ischemia-induced myocardial interstitial ACh release [15.0 nM (SD 8.3), n = 8] was not attenuated by local administration of the L-, N-, or P/Q-type Ca²⁺ channel antagonists, by inhibition of Na⁺/Ca²⁺ exchange, or by blockade of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] receptor but was significantly suppressed by local administration of gadolinium [2.8 nM (SD 2.6), n = 6, P = 0.0283]. In conclusion, stimulation-induced ACh release from the cardiac postganglionic nerves depends on the N- and P/Q-type Ca²⁺ channels (with a dominance of P/Q-type) but probably not on the L-type Ca²⁺ channels in cats. In contrast, ischemia-induced ACh release depends on nonselective cation channels or cation-selective stretch activated channels but not on L-, N-, or P/Q type Ca²⁺ channels, Na⁺/Ca²⁺ exchange, or Ins(1,4,5)P₃ receptor-mediated pathway.

cardiac microdialysis; -conotoxin GVIA; -conotoxin MVIIC; KB-R7943; verapamil; vagal stimulation

Aside from the important role of the normal physiological regulation of the heart, the vagal nerve can be a therapeutic target for certain cardiovascular diseases (2, 3, 13, 22, 27). In previous studies, we have shown that acute myocardial ischemia causes myocardial interstitial ACh release in the ischemic region independently of efferent vagal nerve activity (12, 14). The comparison of the effects of Ca²⁺ channel antagonists on the ACh releases induced by vagal nerve stimulation and by acute myocardial ischemia may deepen our understanding about the ischemia-induced myocardial interstitial ACh release.

A cardiac microdialysis technique offers detailed analyses of in vivo myocardial interstitial ACh release (1, 15). Because the local administration of pharmacological agents through a dialysis probe can modulate ACh release without significantly affecting systemic hemodynamics, a combination of cardiac microdialysis with local pharmacological interventions is useful for analyzing the mechanisms of ACh release in vivo. In the present study, we examined the effects of Ca²⁺ channel antagonists on nerve stimulation- and ischemia-induced ACh releases in anesthetized cats. The results indicate that stimulation-induced ACh release from the cardiac parasympathetic postganglionic nerves depends on the N- and P/Q-type Ca²⁺ channels but probably not on the L-type Ca²⁺ channels. In contrast, ischemia-induced myocardial interstitial ACh release is resistant to the inhibition of L-, N-, and P/Q-type Ca²⁺ channels. In addition, the ischemia-induced myocardial ACh release is resistant to the inhibition of Na⁺/Ca²⁺ exchanger and the blockade of inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃] receptor but is suppressed by gadolinium, suggesting that nonselective cation channels or cation-selective stretch-activated channels are involved.

	MATERIALS AND METHODS

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Common Preparation

Animal care was provided in accordance with the Guiding Principles for the Care and Use of Animals in the Field of Physiological Sciences approved by the Physiological Society of Japan. All protocols were approved by the Animal Subjects Committee of the National Cardiovascular Center. Adult cats weighing from 2.2 to 4.2 kg were anesthetized via an intraperitoneal injection of pentobarbital sodium (30–35 mg/kg) and ventilated mechanically with room air mixed with oxygen. The depth of anesthesia was maintained with a continuous intravenous infusion of pentobarbital sodium (1–2 mg·kg^–1·h^–1) through a catheter inserted from the right femoral vein. Systemic arterial pressure was monitored from a catheter inserted from the right femoral artery. The vagi were sectioned bilaterally at the neck. The esophageal temperature of the animal, which was measured by a thermometer (CTM-303, TERUMO, Japan), was maintained at around 37°C using a heated pad and a lamp.

With the animal in the lateral position, the left fifth and sixth ribs were resected to expose the heart. A dialysis probe was implanted transversely, using a fine guiding needle, into the anterolateral free wall of the left ventricle perfused by the left anterior descending coronary artery (LAD). Heparin sodium (100 U/kg) was administered intravenously to prevent blood coagulation. At the end of the experiment, the experimental animals were killed with an overdose of pentobarbital sodium. Postmortem examination confirmed that the dialysis probe had been threaded in the middle layer of the left ventricular myocardium. The thickness of the left ventricular free wall was 7–8 mm, and the semipermeable membrane of the dialysis probe was positioned 3–4 mm from the epicardial surface.

Dialysis Technique

The materials and properties of the dialysis probe have been described previously (1). Briefly, we designed a transverse dialysis probe. A dialysis fiber of semipermeable membrane (13 mm length, 310 µm OD, 200 µm ID; PAN-1200, 50,000 molecular weight cutoff, Asahi Chemical, Japan) was glued at both ends to polyethylene tubes (25 cm length, 500 µm OD, 200 µm ID). The dialysis probe was perfused at a rate of 2 µl/min with Ringer solution containing a cholinesterase inhibitor eserine (physostigmine, 100 µM). Experimental protocols were started 2 h after the dialysis probe was implanted when the ACh concentration in the dialysate reached a steady state. The ACh concentration in the dialysate was measured by high-performance liquid chromatography with electrochemical detection (Eicom, Kyoto, Japan).

Local administration of a pharmacological agent was carried out through a dialysis probe. That is to say, we added the pharmacological agent to the perfusate and allowed 1 h for a settling time. The pharmacological agent should spread around the semipermeable membrane, thereby affecting the neurotransmitter release in the vicinity of the semipermeable membrane. Because the distribution across the semipermeable membrane is required, based on previous results (33, 34), we used the pharmacological agent at the concentration 10–100 times higher than that required for complete channel blockade in experimental settings in vitro.

Specific Preparation and Protocols

Protocol 1. Bipolar platinum electrodes were attached bilaterally to the cardiac ends of the sectioned vagi at the neck. The nerves and electrodes were covered with warmed mineral oil for insulation. The vagal nerves were stimulated for 15 min (20 Hz, 1 ms, 10 V). We measured the stimulation-induced ACh release in the absence of Ca²⁺ channel blockade (control, n = 7) and examined the effects of an L-type Ca²⁺ channel antagonist verapamil (100 µM, n = 5), an N-type Ca²⁺ channel antagonist -conotoxin GVIA (10 µM, n = 7), a P/Q-type Ca²⁺ channel antagonist -conotoxin MVIIC (10 µM, n = 6), and combined administration of -conotoxin GVIA and -conotoxin MVIIC (10 µM each, n = 6).

Protocol 2. Because a preliminary result from protocol 1 suggested that local administration of verapamil was ineffective in suppressing stimulation-induced ACh release, we examined the effects of the intravenous administration of verapamil (300 µg/kg, n = 6) on stimulation-induced ACh release in vagotomized animals as a supplemental experiment.

Protocol 3. A 60-min LAD occlusion was performed by using a 3-0 silk suture passed around the LAD just distal to the first diagonal branch. We measured the ACh levels during 45–60 min of ischemia in the absence of Ca²⁺ channel blockade (control, n = 8) and examined the effects of verapamil (100 µM, n = 5), -conotoxin GVIA (10 µM, n = 5), and -conotoxin MVIIC (10 µM, n = 5). A previous result indicated that the ischemia-induced ACh release reached the steady state during 45–60 min of ischemia (14). We also examined the effects of three additional agents, a Na⁺/Ca²⁺ exchange inhibitor KB-R7943 (10 µM, n = 5) (9, 10), an Ins(1,4,5)P₃ receptor blocker xestospongin C (500 µM, n = 6) (25), and a nonselective cation channel blocker or a cation-selective stretch activated channel blocker gadolinium (1 mM) (5, 17), on the ischemia-induced ACh release.

Statistical Analysis

All data are presented as mean (SD) values. In protocol 1, we compared stimulation-induced ACh release among the five groups using one-way analysis of variance followed by the Student-Neuman-Keuls test (6). In protocol 2, we used an unpaired-t test (two-sided) to examine the effect of intravenous verapamil administration on stimulation-induced ACh release. In protocol 3, we compared ischemia-induced ACh release among the seven groups using one-way analysis of variance followed by the Dunnett' test against the control. For all analyses, differences were considered significant when P < 0.05.

	RESULTS

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In protocol 1, the ACh level during electrical vagal stimulation was 22.4 nM (SD 10.6). Local administration of verapamil did not affect stimulation-induced ACh release (Fig. 1). In contrast, local administration of -conotoxin GVIA or -conotoxin MVIIC suppressed stimulation-induced ACh release. The extent of suppression was greater in the latter. The ACh level was significantly lower in the simultaneous administration group (-conotoxin GVIA + -conotoxin MVIIC) than that in the -conotoxin GVIA group but was not different from the -conotoxin MVIIC group.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Effects of local administration of verapamil, -conotoxin GVIA, -conotoxin MVIIC, or -conotoxin GVIA plus -conotoxin MVIIC on vagal nerve stimulation-induced myocardial interstitial ACh release. Both -conotoxin GVIA and -conotoxin MVIIC, but not verapamil, suppressed stimulation-induced ACh release. Data are mean (SD) values. *P < 0.01, P < 0.05. The exact P values are presented.

In protocol 3, the ACh level in the ischemic region was 14.9 nM (SD 8.3) during 45–60 min of acute myocardial ischemia. Inhibition of voltage-dependent Ca²⁺ channels by local administration of verapamil, -conotoxin GVIA, or -conotoxin MVIIC did not affect ischemia-induced ACh release (Fig. 2). Inhibition of the reverse mode action of Na⁺/Ca²⁺ exchange by local administration of KB-R7943 appeared to have augmented rather than suppressed ischemia-induced ACh release, though there was no statistically significant difference from the control. Blockade of the Ins(1,4,5)P₃ receptor by local administration of xestospongin C did not affect the ischemia-induced ACh release. In contrast, blockade of nonselective cation channels or cation-selective stretch-activated channels by local administration of gadolinium suppressed the ischemia-induced ACh release.

View larger version (9K): [in this window] [in a new window]   Fig. 2. Effects of local administration of verapamil, -conotoxin GVIA, -conotoxin MVIIC, KB-R7943, xestospongin C, or gadolinium on acute myocardial ischemia-induced myocardial interstitial ACh release in the ischemic region. Gadolinium alone suppressed the ischemia-induced ACh release. Data are mean (SD) values. P < 0.05. The exact P values are presented.

	DISCUSSION

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Although neurotransmitter release at mammalian sympathetic neuroeffector junctions predominantly depends on Ca²⁺ influx through N-type Ca²⁺ channels (23, 33, 34), the type(s) of Ca²⁺ channels involved in ACh release from cardiac parasympathetic neuroeffector junctions show diversity among reports (8, 28). One possible factor hampering investigations into parasympathetic postganglionic neurotransmitter release in response to vagal nerve stimulation in vivo is that the parasympathetic ganglia are usually situated in the vicinity of the effector organs, thereby making it difficult to separately assess ACh release from preganglionic and postganglionic nerves. In the previous study from our laboratory, intravenous administration, but not local administration of a ganglionic blocker, hexamethonium reduced vagal stimulation-induced ACh release assessed by cardiac microdialysis (1). The negligible effect of local hexamethonium administration on stimulation-induced ACh release suggests the lack of parasympathetic ganglia around the dialysis probe. In support of our speculation, a recent neuroanatomical finding indicates that three ganglia, away from the left anterior free wall targeted by the dialysis probe, provide the major source for left ventricular postganglionic innervation in cats: a cranioventricular ganglion, a left ventricular ganglion 2 (so designated), and an interventriculo-septal ganglion (11). Therefore, ACh, as measured by cardiac microdialysis, is considered to predominantly reflect ACh release from parasympathetic postganglionic nerves.

Local (protocol 1) or intravenous (protocol 2) administration of verapamil did not affect stimulation-induced ACh release. In contrast, vagal stimulation-induced ACh release was reduced in both the -conotoxin GVIA and -conotoxin MVIIC groups but to a greater extent in the latter (Fig. 1). Therefore, both N- and P/Q-type, but probably not L-type, Ca²⁺ channels are involved in stimulation-induced ACh release from the cardiac parasympathetic postganglionic nerves in cats. The contribution of P/Q type Ca²⁺ channels to ACh release might be greater than that of N-type Ca²⁺ channels. Hong and Chang (8) reported that the negative inotropic response to field stimulation depends predominantly on the P/Q-type Ca²⁺ channels in isolated guinea pig atria, whereas Serone et al. (28) reported the predominance of N-type Ca²⁺ channels. In those studies, the field stimulation employed differed from ordinary activation of the postganglonic nerves by nerve discharge and, in addition, ACh release was not directly measured. The present study directly demonstrated the involvement of P/Q- and N-type Ca²⁺ channels in the stimulation-induced ACh release in the cardiac parasympathetic postganglionic nerves. These results support the concept that multiple subtypes of the voltage-gated Ca²⁺ channel mediate transmitter release from the same population of parasympathetic neurons (31).

Stimulation-induced ACh release was suppressed by 50% in the -conotoxin GVIA group and by 80% in the -conotoxin MVIIC group. The algebraic summation of the extent of suppression exceeded 100%. The phenomenon may be in part due to the nonlinear dose-response relationship between Ca²⁺ influx and transmitter release (32). The supra-additive phenomenon may be also due to the affinity of -conotoxin MVIIC to N-type Ca²⁺ channels (8, 26, 36). Combined local administration of -conotoxin GVIA and -conotoxin MVIIC almost completely suppressed stimulation-induced ACh release to a level similar to that achieved by the Na⁺ channel inhibitor tetrodotoxin (15). Therefore, involvement of another untested type of Ca²⁺ channel(s) is unlikely in the stimulation-induced ACh release from the cardiac parasympathetic postganglionic nerves in cats.

Ca²⁺ Channels and Ischemia-Induced ACh Release

In a previous study, we showed that acute myocardial ischemia evokes myocardial interstitial ACh release in the ischemic region via a local mechanism independent of efferent vagal nerve activity (14). In that study, the inhibition of intracellular Ca²⁺ mobilization by local administration of 3,4,5-trimethoxybenzoic acid 8-(diethyl amino)-octyl ester (TMB-8) suppressed ischemia-induced ACh release, suggesting that an axoplasmic Ca²⁺ elevation is essential for the ischemia-induced ACh release. Because tissue K⁺ concentration increases in the ischemic region (7, 18), high K⁺-induced depolarization could activate voltage-dependent Ca²⁺ channels even in the absence of efferent vagal nerve activity. However, ischemia-induced ACh release was not suppressed by local administration of verapamil, -conotoxin GVIA, or -conotoxin MVIIC (Fig. 2). Therefore, Ca²⁺ entry through the voltage-dependent Ca²⁺ channels is unlikely a mechanism for the ischemia-induced myocardial interstitial ACh release.

Acute myocardial ischemia causes energy depletion in the ischemic region, which impairs Na⁺-K⁺-ATPase activity. Ischemia also causes acidosis in the ischemic region, which promotes Na⁺/H⁺ exchange. As a result, ischemia causes intracellular Na⁺ accumulation. The decrease in the Na⁺ gradient across the plasma membrane may then cause the Na⁺/Ca²⁺ exchanger to operate in the reverse mode, facilitating intracellular Ca²⁺ overload. KB-R7943 can inhibit the reverse mode of Na⁺/Ca²⁺ exchange (9, 10) and its potential to protect against ischemia-reperfusion injury has been reported (21). In the present study, however, local administration of KB-R7943 failed to suppress and rather increased ACh release during ischemia as opposed to our expectation. It is plausible that the inhibition of reverse mode of Na⁺/Ca²⁺ may have facilitated the accumulation of intracellular Na⁺ and induced adverse effects that cancelled the possible beneficial effects derived from the inhibition of Ca²⁺ entry through the Na⁺/Ca²⁺ exchanger itself. In addition, KB-R7943 could inhibit the forward mode of Na⁺/Ca²⁺ exchange and reduce Ca²⁺ efflux (16), contributing to the intracellular Ca²⁺ accumulation and ACh release. In the present study, we observed the effects of KB-R7943 only during the ischemic period. However, accumulation of intracellular Na⁺ through Na⁺/H⁺ exchange is enhanced on reperfusion due to the washout of extracellular H⁺ (20). The inhibition of Na⁺/Ca²⁺ exchange to suppress Ca²⁺ overload might become more important during the reperfusion phase. For instance, the percent segment shortening of the left ventricle was improved by KB-R7943 during reperfusion but not during ischemia (35).

As already mentioned, the ischemia-induced ACh release can be blocked by TMB-8 and thus the intracellular Ca²⁺ mobilization is required for the ischemia-induced ACh release (14). Besides the Ca²⁺ entries through voltage-dependent Ca²⁺ channels and via the reverse mode of Na⁺/Ca²⁺ exchanger, Ca²⁺ may be mobilized from the endoplasmic reticulum via pathological pathways. As an example, the mitochondrial permeability transition pore triggered in pathological conditions is linked to cytochrome c release. Cytochrome c can bind to the endoplasmic reticulum Ins(1,4,5)P₃ receptor, rendering the channel insensitive to autoinhibition by high cytosolic Ca²⁺ concentration and resulting in enhanced endoplasmic reticulum Ca²⁺ release (4, 30). In the present study, however, blockade of Ins(1,4,5)P₃ receptor by xestospongin C failed to suppress the ischemia-induced ACh release. In contrast, local administration of gadolinium significantly suppressed the ischemia-induced ACh release. Therefore, nonselective cation channels or cation-selective stretch-activated channels contribute to the ischemia-induced ACh release. During myocardial ischemia, the ischemic region can be subjected to paradoxical systolic bulging. Such bulging likely opens stretch-activated channels and causes myocardial interstitial ACh release, possibly leading to cardioprotection by ACh against ischemic injury (2).

Limitations

First, the experiment was performed under anesthetic conditions, which may have influenced basal autonomic activity. However, because we sectioned the vagi at the neck, basal autonomic activity may have had only a minor effect on ACh release during the vagal stimulation and during acute myocardial ischemia. Second, we added eserine to the perfusate to inhibit immediate degradation of ACh (24), which may have increased the ACh level in the synaptic cleft and activated regulatory pathways such as autoinhibition of ACh release via muscarinic receptors (24). However, the myocardial interstitial ACh level measured under this condition could reflect changes induced by Na⁺ channel inhibitor, choline uptake inhibitor, and vesicular ACh transport inhibitor as described in a previous study (15). Therefore, we think that the interpretation of the present results is reasonable. Third, tissue and species differences should be taken into account when extrapolating the present findings, because significant heterogeneity in the Ca²⁺ channels involved in the mammalian parasympathetic system may exist. Finally, we used verapamil to test the involvement of L-type Ca²⁺ channels in the ACh release. There are three major types of L-type Ca²⁺ channel antagonists with different binding domains (verapamil, nifedipine, and diltiazem) (19). Whether the effects on the ACh release are common among the three types of L-type Ca²⁺ channel antagonists remains unanswered.

In conclusion, the N- and P/Q-type Ca²⁺ channels (with the P/Q-type dominant), but probably not the L-type Ca²⁺ channels, are involved in vagal stimulation-induced ACh release from the cardiac parasympathetic postganglionic nerves in cats. In contrast, myocardial interstitial ACh release in the ischemic myocardium is resistant to the blockade of L-, N-, and P/Q-type Ca²⁺ channels. In addition, the ischemia-induced myocardial ACh release is resistant to the inhibition of Na⁺/Ca²⁺ exchanger and the blockade of Ins(1,4,5)P₃ receptor but is suppressed by gadolinium, suggesting that nonselective cation channels or cation-selective stretch-activated channels are involved.

	GRANTS

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This study was supported by Health and Labour Sciences Research Grant for Research on Advanced Medical Technology from the Ministry of Health, Labour and Welfare of Japan, Health and Labour Sciences Research Grant for Research on Medical Devices for Analyzing, Supporting and Substituting the Function of Human Body from the Ministry of Health, Labour and Welfare of Japan, Health and Labour Sciences Research Grant H18-Iryo-Ippan-023 from the Ministry of Health, Labour and Welfare of Japan, Program for Promotion of Fundamental Studies in Health Science from the National Institute of Biomedical Innovation, a Grant provided by the Ichiro Kanehara Foundation, Ground-based Research Announcement for Space Utilization promoted by Japan Space Forum, and Industrial Technology Research Grant Program in 03A47075 from New Energy and Industrial Technology Development Organization of Japan.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Kawada, Dept. of Cardiovascular Dynamics, Advanced Medical Engineering Center, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan (e-mail: torukawa{at}res.ncvc.go.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.

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