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NEUROHUMORAL CONTROL OF CIRCULATION AND HYPERTENSION

Effects of Ca²⁺ channel antagonists on acetylcholine and catecholamine releases in the in vivo rat adrenal medulla

¹Department of Cardiac Physiology and ²Department of Cardiovascular Dynamics, National Cardiovascular Center Research Institute, Suita, Osaka 565–8565, Japan

Submitted 20 October 2003 ; accepted in final form 13 March 2004

	ABSTRACT

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To elucidate the types of voltage-dependent Ca²⁺ channels controlling ACh and catecholamine releases in the in vivo adrenal medulla, we implanted microdialysis probes in the left adrenal medulla of anesthetized rats and investigated the effects of Ca²⁺ channel antagonists on ACh, norepinephrine, and epinephrine releases induced by nerve stimulation. The dialysis probes were perfused with Ringer solution containing a cholinesterase inhibitor, neostigmine. The left splanchnic nerves were electrically stimulated at 2 and 4 Hz before and after intravenous administration of Ca²⁺ channel antagonists. -Conotoxin GVIA (an N-type Ca²⁺ channel antagonist, 10 µg/kg) inhibited ACh release at 2 and 4 Hz by 40%, norepinephrine release at 4 Hz by 50%, and epinephrine release at 2 and 4 Hz by 45%. A fivefold higher dose of -conotoxin GVIA (50 µg/kg) did not further inhibit these releases. -Conotoxin MVIIC (a P/Q-type Ca²⁺ channel antagonist, 50 µg/kg) inhibited ACh and epinephrine releases at 4 Hz by 30%. Combined -conotoxin GVIA (50 µg/kg) and MVIIC (250 µg/kg) inhibited ACh release at 2 and 4 Hz by 70% and norepinephrine and epinephrine releases at 2 and 4 Hz by 80%. Nifedipine (an L-type Ca²⁺ channel antagonist, 300 and 900 µg/kg) did not change ACh release at 2 and 4 Hz; however, nifedipine (300 µg/kg) inhibited epinephrine release at 4 Hz by 20%, and nifedipine (900 µg/kg) inhibited norepinephrine and epinephrine releases at 4 Hz by 30%. In conclusion, both N- and P/Q-type Ca²⁺ channels control ACh release on preganglionic splanchnic nerve endings while L-type Ca²⁺ channels do not. L-type Ca²⁺ channels are involved in norepinephrine and epinephrine releases on chromaffin cells.

anesthetized rats; microdialysis; norepinephrine; epinephrine; preganglionic autonomic nerve endings

In the in vivo adrenal medulla, catecholamine release is controlled by central sympathetic neurons through preganglionic splanchnic nerves. Splanchnic nerve endings make synaptic-like contacts with chromaffin cells (9). ACh released from splanchnic nerve endings consequently evokes catecholamine release from chromaffin cells by activation of cholinergic receptors. Thus, in vivo catecholamine release requires Ca²⁺ influx through the voltage-dependent Ca²⁺ channels at two different sites in the adrenal medulla: splanchnic nerve endings and chromaffin cells. Numerous studies have investigated the nature of Ca²⁺ channels controlling transmitter release from postganglionic autonomic nerve endings (8, 11, 32, 33, 36, 37). Little information is, however, available on the type of Ca²⁺ channels controlling the ACh release from preganglionic autonomic nerve endings including splanchnic nerve endings. Moreover, although the types of Ca²⁺ channels controlling catecholamine release have been investigated using isolated chromaffin cells in various species (5, 6, 13, 16, 21, 23, 24), it remains unknown whether endogenous ACh induces Ca²⁺ influx through the same types of Ca²⁺ channels on chromaffin cells.

We have recently developed a dialysis technique to simultaneously monitor ACh and catecholamine releases in the in vivo adrenal medulla (2). This method makes it possible to characterize Ca²⁺ channels controlling ACh release from splanchnic nerve endings and catecholamine release from adrenal medulla in the in vivo state. In the present study, we applied the microdialysis technique to the adrenal medulla of anesthetized rats and investigated the effects of Ca²⁺ channel antagonists on dialysate ACh and catecholamine responses induced by the electrical stimulation of splanchnic nerves.

	MATERIALS AND METHODS

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Animal preparation. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85–23, revised 1996). Adult male Wistar rats weighing 380–450 g were anesthetized with pentobarbital sodium (50–55 mg/kg ip). A cervical midline incision was made to expose the trachea, which was then cannulated. The rats were ventilated with a constant-volume respirator using room air mixed with oxygen. The left femoral artery and vein were cannulated for monitoring arterial blood pressure and administration of anesthetic, respectively. The level of anesthesia was maintained with a continuous intravenous infusion of pentobarbital sodium (15–25 mg·kg^–1·h^–1 iv). Electrocardiogram was monitored for recording heart rate. A thermostatic heating pad was used to keep the esophageal temperature within a range of 37–38°C. With the animal in the lateral position, the left adrenal gland and left splanchnic nerve were exposed by a subcostal flank incision, and the left splanchnic nerve was transected. Shielded bipolar stainless steel electrodes were applied to the distal end of the nerve, which was then stimulated with a digital stimulator (SEN-7203, Nihon Kohden) with a rectangular pulse (10 V and 1 ms in duration).

Dialysis technique. The materials of the dialysis probe were the same as those used in our previous dialysis experiments (1, 2). Briefly, each end of the dialysis fiber (0.31 mm OD, and 0.20 mm ID; PAN-1200 50,000 mol wt cutoff, Asahi Chemical) was inserted into the polyethylene tube (25-cm length, 0.5 mm OD, and 0.2 mm ID; SP-8) and glued. The length of the dialysis fiber exposed was 3 mm.

The left adrenal gland was gently lifted, and the dialysis probe was implanted in the medulla of the left adrenal gland along the long axis by using a fine guiding needle. The dialysis probe was perfused with Ringer solution containing a cholinesterase inhibitor, neostigmine (10 µM), at a speed of 10 µl/min using a microinjection pump (CMA/100, Carnegie Medicin). Ringer solution with no buffer consisted of (in mM) 147.0 NaCl, 4.0 KCl, and 2.25 CaCl₂. One sampling period was 2 min (1 sample volume = 20 µl). We started the protocols followed by a stabilization period of 3–4 h and sampled dialysate taking the dead space volume into account.

Dialysate ACh, norepinephrine (NE), and epinephrine (Epi) concentrations were measured as indexes of ACh and catecholamine releases in the adrenal medulla. Half of the dialysate sample was used for the measurement of ACh, and the remaining half for the measurement of NE and Epi. ACh and catecholamine assays were separately conducted using each high-performance liquid chromatography with electrochemical detection as previously described (3, 4).

Experimental design. The experiment was performed based on the previous experiment showing that dialysate ACh and catecholamine responses were reproducible on repetition of stimulation (2). The left splanchnic nerves were electrically stimulated for 2 min at 30-min intervals. Three dialysate samples were continuously collected per electrical stimulation: one before, one during, and one after stimulation. Stimulations at two different frequencies (2 and 4 Hz) were performed before and after intravenous administration of Ca²⁺ channel antagonists.

We tested three types of Ca²⁺ channel antagonists (25): the N-type Ca²⁺ channel antagonist -conotoxin GVIA, the P/Q-type Ca²⁺ channel antagonist -conotoxin MVIIC, and the L-type Ca²⁺ channel antagonist nifedipine. We determined the first doses of Ca²⁺ channel antagonists based on the dose used in the earlier experiments (7, 14, 26, 29, 37) and tested -conotoxin GVIA (10 µg/kg) in six rats, -conotoxin MVIIC (50 µg/kg) in six rats, and nifedipine (300 µg/kg) in six rats. Second, we tested a fivefold higher dose of -conotoxin GVIA (50 µg/kg) in six rats, a combination of fivefold higher doses of -conotoxin GVIA (50 µg/kg) and MVIIC (250 µg/kg) in six rats, and a threefold higher dose of nifedipine (900 µg/kg) in six rats. We did not test a higher dose of -conotoxin MVIIC singly because a high dose of -conotoxin MVIIC loses its selectivity for P/Q-type and inhibits N-type Ca²⁺ channels (18).

Nifedipine was administered twice before 2- and 4-Hz stimulation, but -conotoxin GVIA and MVIIC were administered once before 2-Hz stimulation because the -conotoxin family has long-lasting blocking actions (8, 18, 36). We assessed the responses to nerve stimulation 30, 20, and 10 min after administration of -conotoxin GVIA, -conotoxin MVIIC, and nifedipine, respectively, when heart rate and arterial pressure had already been stabilized.

At the end of the experiment the rats were killed with pentobarbital sodium, and the implant sites were examined. The dialysis probes were confirmed to have been implanted in the adrenal medulla, and no bleeding or necrosis was found macroscopically.

Drugs. Drugs were mixed fresh for each experiment. Neostigmine methylsulfate (Shionogi), -conotoxin GVIA (Peptide Institute), and -conotoxin MVIIC (Peptide Institute) were dissolved and diluted in Ringer solution. Nifedipine (Sigma Chemical) was dissolved in ethanol and diluted in Ringer solution.

Statistical methods. To examine the effects of nerve stimulation and Ca²⁺ channel antagonists, we analyzed heart rate and mean arterial pressure and dialysate ACh, NE, and Epi responses by using one-way ANOVA with repeated measures. When statistical significance was detected, the Newman-Keuls test was applied (35). Statistical significance was defined as P < 0.05. Values are presented as means ± SE.

	RESULTS

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Effects of Ca²⁺ channel antagonists on heart rate and mean arterial pressure. -Conotoxin GVIA (10 µg/kg) decreased heart rate from 418 ± 9 to 328 ± 13 beats/min (P < 0.05) and mean arterial pressure from 115 ± 2 to 74 ± 2 mmHg (P < 0.05). -Conotoxin GVIA (50 µg/kg) did not further decrease heart rate and mean arterial pressure. -Conotoxin MVIIC decreased heart rate from 408 ± 3 to 390 ± 5 beats/min (P < 0.05) but did not change mean arterial pressure. Combined -conotoxin GVIA and MVIIC decreased heart rate from 415 ± 10 to 327 ± 4 beats/min (P < 0.05) and mean arterial pressure from 124 ± 2 to 57 ± 2 mmHg (P < 0.05). Nifedipine (300 µg/kg) decreased mean arterial pressure from 113 ± 4 to 86 ± 4 mmHg (P < 0.05) but did not change heart rate. Nifedipine (900 µg/kg) decreased mean arterial pressure from 124 ± 3 to 73 ± 2 mmHg (P < 0.05).

Effects of Ca²⁺ channel antagonists on ACh and catecholamine releases. ACh could not be detected in dialysate before or after stimulation. Thus we expressed dialysate ACh concentration during stimulation as an index of ACh release induced by stimulation. In contrast, substantial amounts of NE and Epi were observed in dialysate before stimulation. Intravenous administration of Ca²⁺ channel antagonists did not affect these basal NE and Epi releases (Table 1). Dialysate NE and Epi concentrations increased by nerve stimulation and rapidly declined after the stimulation. Thus we subtracted the dialysate NE and Epi contents before stimulation from those during stimulation and expressed these values as indexes of NE and Epi releases induced by stimulation.

Effects of -conotoxin GVIA.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Effects of -conotoxin GVIA on ACh, norepinephrine (NE), and epinephrine (Epi) releases. -Conotoxin GVIA (10 µg/kg) inhibited ACh release at 2 and 4 Hz, NE release at 4 Hz, and Epi release at 2 and 4 Hz (A). A 5-fold higher dose of -conotoxin GVIA (50 µg/kg) did not further inhibit these releases (B). Values are means ± SE from 6 rats. *P < 0.05 vs. ACh, NE, or Epi release at same frequency before administration.

Effects of -conotoxin MVIIC.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Effects of -conotoxin MVIIC on ACh, NE, and Epi releases. -Conotoxin MVIIC (50 µg/kg) inhibited ACh and Epi releases at 4 Hz. Values are means ± SE from 6 rats. *P < 0.05 vs. ACh, NE, or Epi release at same frequency before administration.

Effects of combined -conotoxin GVIA and MVIIC.

View larger version (20K): [in this window] [in a new window]   Fig. 3. Effects of combined -conotoxin GVIA and MVIIC on ACh, NE, and Epi releases. Combined -conotoxin GVIA (50 µg/kg) and MVIIC (250 µg/kg) inhibited ACh, NE, and Epi releases at 2 and 4 Hz. Values are means ± SE from 6 rats. *P < 0.05 vs. ACh, NE, or Epi release at same frequency before administration.

View larger version (29K): [in this window] [in a new window]   Fig. 4. Effects of nifedipine on ACh, NE, and Epi releases. Nifedipine (300 µg/kg) did not change ACh release at either frequency but inhibited Epi release at 4 Hz (A). Nifedipine (900 µg/kg) did not change ACh release at either frequency but inhibited NE and Epi releases at 4 Hz (B). Values are means ± SE from 6 rats. *P < 0.05 vs. ACh, NE, or Epi release at same frequency before administration.

	DISCUSSION

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In isolated rat adrenal glands, catecholamine release induced by field stimulation is sensitive to P/Q-type Ca²⁺ channel antagonist, whereas that induced by exogenous ACh is insensitive (27). This indirect study suggested the involvement of P/Q-type Ca²⁺ channels in ACh release but failed to show the involvement of N-type Ca²⁺ channels. In isolated bovine adrenal glands, a direct measurement study showed that a reduction of the extracellular Ca²⁺ concentration inhibits ³H-labeled ACh release induced by field stimulation, but N- and L-type Ca²⁺ channel antagonists do not (28). Thus our findings are in part consistent with these direct and indirect studies but inconsistent as to the involvement of N-type Ca²⁺ channels.

This discrepancy might be ascribed to the experimental method. The contribution of Ca²⁺ channels may vary with the type of method used to evoke ACh release. In these studies, ACh release was evoked by electrical field stimulation of isolated adrenal glands, which is known to induce ACh release but not direct depolarization of chromaffin cells (34). In the present study, ACh release was evoked in the in vivo state by electrical stimulation of splanchnic nerves. The type of Ca²⁺ channels involved in ACh release may vary with the frequency, amplitude, or time period of stimulation. Actually, in the present study, we observed the involvement of P/Q-type Ca²⁺ channels at only high-frequency stimulation, while it has been reported in perfused rat adrenal glands that N-type Ca²⁺ channels are involved in the maintenance of catecholamine release in response to long splanchnic nerve stimulation (31). The time period of 2 min in the present study seems to be longer than those in earlier studies but could be within the physiological range. Moreover, the blocking action of -conotoxin GVIA is time dependent as well as dose dependent and irreversible (8, 11, 32, 36). The maximum functional effect of -conotoxin GVIA has been observed to be at least 15 min after administration. We evaluated the effect of -conotoxin GVIA 30 min after intravenous administration, when heart rate and mean arterial pressure had already been stabilized. The evaluation early after administration might lead to underestimation of the inhibitory effects of -conotoxin GVIA.

There are many similarities between synaptic transmission from splanchnic nerves to chromaffin cells and sympathetic ganglionic transmission (17). In isolated guinea pig paravertebral ganglia, an electrophysiological study has shown that both N- and P-type Ca²⁺ channel antagonists reduce cholinergic synaptic conductance, whereas L-type Ca²⁺ channel antagonist does not (19). In isolated rat superior cervical ganglia, both N- and P-type Ca²⁺ channel antagonists inhibit the rise in Ca²⁺ concentration in the terminal boutons (22). Moreover, in isolated rat superior cervical ganglia, ³H-labeled ACh release induced by high K⁺ is inhibited by both N- and P-type Ca²⁺ channel antagonists but unaffected by L-type Ca²⁺ channel antagonist (15). Our findings are similar to these findings obtained from isolated sympathetic preganglionic nerves.

The inhibition by -conotoxin GVIA (50 µg/kg) was almost the same as that by -conotoxin GVIA (10 µg/kg). Moreover, the inhibition by combined -conotoxin GVIA (50 µg/kg) and MVIIC (250 µg/kg) was almost algebraically the sum of the individual inhibition by -conotoxin GVIA (10 µg/kg) and MVIIC (50 µg/kg). These results suggest that fivefold higher doses of -conotoxin GVIA and MVIIC are sufficient to cause inhibition of Ca²⁺ channels. However, 30% of ACh release was resistant to combined -conotoxin GVIA (50 µg/kg) and MVIIC (250 µg/kg). Other types of Ca²⁺ channels except for N- and P/Q-types may be involved in ACh release from splanchnic nerve endings. Further examination could be needed.

Effects of Ca²⁺ channel antagonists on catecholamine release from chromaffin cells. In the present study, nifedipine (300 µg/kg) did not change ACh release at 2 and 4 Hz but inhibited Epi release at 4 Hz by 20%. A threefold higher dose of nifedipine (900 µg/kg) did not change ACh release at 2 and 4 Hz but inhibited NE and Epi releases at 4 Hz by 30%. Adrenal chromaffin cells are divided into two populations: NE- and Epi-storing cells (10). L-type Ca²⁺ channels could be present on the surface of both NE- and Epi-storing cells and play a role in NE and Epi releases.

Approximately 70% of catecholamine release was resistant to nifedipine (900 µg/kg). This result suggests that other types of Ca²⁺ channels except for L-type are present on chromaffin cells and involved in NE and Epi releases, although we cannot exclude the possibility of incomplete inhibition of L-type Ca²⁺ channels. Species differences in the types of Ca²⁺ channels controlling Ca²⁺ influx and catecholamine release have been shown with rat, cat, and bovine chromaffin cells (5, 6, 13, 24). In patch-clamp studies of isolated rat chromaffin cells, Ca²⁺ inward current elicited by depolarization is sensitive to both L- and N-type Ca²⁺ channel antagonists (16, 21). The study measuring Ba²⁺ current by patch-clamp technique has shown the existence of L-, N-, and P/Q-type Ca²⁺ channels on rat chromaffin cells and the following distribution of Ca²⁺ channels in decreasing order: L-type > N-type > P/Q-type (13). In the present study, -conotoxin GVIA (10 and 50 µg/kg) inhibited NE release at 4 Hz and Epi release at 2 and 4 Hz by approximately 45–50%. -Conotoxin MVIIC (50 µg/kg) inhibited Epi release at 4 Hz by 30%. Combined -conotoxin GVIA (50 µg/kg) and MVIIC (250 µg/kg) inhibited NE and Epi releases at 2 and 4 Hz by approximately 75–85%. However, these Ca²⁺ channel antagonists simultaneously inhibited ACh release to almost the same extent. It is difficult to determine how much Ca²⁺ antagonists are acting on chromaffin cells when Ca²⁺ channel antagonists inhibit ACh release. Thus, although much of these inhibitions of catecholamine release may be considered to be consequences of the inhibition of ACh release, we cannot exclude the possibility that N- or P/Q-type Ca²⁺ channels may be involved in the in vivo catecholamine release on chromaffin cells.

The inhibition of NE release at 2 Hz by -conotoxin GVIA (10 and 50 µg/kg) and the inhibition of NE release at 4 Hz by -conotoxin MVIIC (50 µg/kg) were not statistically significant despite significant inhibitions of ACh and Epi releases. In the same preparation, we have shown that cholinergic antagonists almost inhibited NE and Epi releases induced by nerve stimulation (1, 2). However, the correlation between ACh and NE releases was poorer than that between ACh and Epi releases when stimulation frequency was raised stepwise (2). Insignificant inhibitions of NE release may be ascribed to this poor correlation.

In the present study, Ca²⁺ channel antagonists did not affect basal dialysate NE and Epi levels. In our previous study of the same preparation, these basal levels were not affected by neostigmine, hexamethonium, or atropine (1). We then concluded that these basal dialysate NE and Epi levels reflect noncholinergic catecholamine release. N-, P/Q-, and L-type Ca²⁺ channels may not play a major role in basal noncholinergic catecholamine release from adrenal medulla.

Methodological considerations. We administered neostigmine locally to adrenal medulla through a dialysis probe. Cholinesterase inhibitor was necessary to monitor endogenous ACh even during splanchnic nerve stimulation because released ACh is rapidly degraded by acetylcholinesterase before reaching the dialysis fiber. In the same preparation, local administration of neostigmine enhanced the dialysate catecholamine response to nerve stimulation by approximately threefold, but dialysate ACh and catecholamine responses are correlated with the stimulation frequency of splanchnic nerves in the presence of neostigmine (2). Thus dialysate ACh and catecholamine responses are likely to be correlated with the amount of Ca²⁺ influx from voltage-dependent Ca²⁺ channels even in the presence of neostigmine.

Intravenous administration of Ca²⁺ channel antagonists induced changes in heart rate or mean arterial pressure. These changes might affect ACh and catecholamine releases through a baroreflex mechanism. Moreover, these hemodynamic changes might decrease the spillover of ACh or catecholamine from adrenal medulla and affect the dialysate ACh or catecholamine concentrations (20). In our preparation, however, splanchnic nerves had been transected before control sampling, and basal dialysate catecholamine concentrations did not change before or after administration. Thus effects of these hemodynamic changes could be negligible when we considered the effects of Ca²⁺ channel antagonists on nerve stimulation-induced dialysate responses.

In conclusion, we applied dialysis technique to the adrenal medulla of anesthetized rats and investigated the effects of Ca²⁺ channel antagonists on ACh and catecholamine releases induced by electrical stimulation of splanchnic nerves. Both N- and P/Q-type Ca²⁺ channels control ACh release on preganglionic splanchnic nerve endings while L-type Ca²⁺ channels do not. L-type Ca²⁺ channels are involved in norepinephrine and epinephrine releases on chromaffin cells.

	GRANTS

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This study was supported by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (of Japan); by a Health Sciences Research Grant for Advanced Medical Technology from the Ministry of Health and Welfare of Japan; by a Ground-Based Research Grant for the Space Utilization promoted by the National Space Development Agency of Japan and Japan Space Forum; and by grants-in-aid for scientific research from the Ministry of Education, Science.

	FOOTNOTES

 

Address for reprint requests and other correspondence: T. Akiyama, Dept. of Cardiac Physiology, National Cardiovascular Center Research Institute, 5–7-1 Fujishiro-dai, Suita, Osaka, 565–8565 Japan (E-mail: takiyama{at}ri.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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