INTRODUCTION

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CATECHOLAMINE SECRETION from the adrenal medulla is controlled by nicotinic and muscarinic receptors. Stimulation of muscarinic receptors causes G protein-mediated activation of phospholipase C, and produced inositol trisphosphate triggers release of Ca²⁺ from intracellular storage sites (3, 4, 7, 16). The elevation of intracellular free Ca²⁺ induces the secretion of catecholamines (4, 8, 12) and simultaneously activates SK_Ca channels (13, 16).

Apamin, a selective small-conductance Ca²⁺-activated K⁺ (SK_Ca) channel blocker (2, 6), has been reported to enhance the secretion of catecholamines induced by muscarine in the dog adrenal gland in vivo (9) and by the muscarinic agonist methacholine in the isolated perfused cat adrenal gland (17, 18). These studies suggest that SK_Ca channels play an inhibitory role in the secretion of catecholamines mediated by muscarinic receptors. We observed that apamin facilitated catecholamine secretion induced by methacholine and that the facilitation disappeared during treatment with Ca²⁺-free solution in the rat adrenal gland. These results indicate that the facilitatory effect of apamin depends on extracellular Ca²⁺. It is well established that catecholamine secretion is caused by the influx of extracellular Ca²⁺ through L-type Ca²⁺ channels. Although the muscarinic receptor-mediated secretion of catecholamines is considered to result from Ca²⁺ release from intracellular storage sites, we could hypothesize that the Ca²⁺ influx through L-type Ca²⁺ channels might contribute to the facilitatory effect of apamin on the muscarinic receptor-mediated secretion.

The aim of this study was to clarify the interaction of SK_Ca channels and L-type Ca²⁺ channels in muscarinic receptor-mediated control of adrenal catecholamine secretion. First, we examined the effect of apamin, the L-type Ca²⁺ channel blocker nifedipine, the L-type Ca²⁺ channel activator Bay k 8644, and Ca²⁺-free solution on the secretion of epinephrine (Epi) and norepinephrine (NE) induced by methacholine from the isolated perfused rat adrenal gland. Then the effects of apamin during treatment with nifedipine or Ca²⁺-free solution on catecholamine secretion induced by methacholine were examined. Finally, the effects of nifedipine or Bay k 8644 during treatment with apamin on catecholamine secretion induced by methacholine were examined.

	MATERIALS AND METHODS

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Animal preparation. All procedures for handling animals were approved by the Animal Experimentation Committee of Tohoku University Graduate School of Pharmaceutical Sciences. Male Wistar rats, weighing 250-350 g, were housed at 21-24°C and maintained on a standard diet and water ad libitum. Rats were anesthetized with pentobarbital sodium (50 mg/kg ip). The surgical procedure used in the present study was described previously (11). A polyethylene cannula, used for perfusion of the adrenal gland, was inserted into the adrenal vein through the renal vein. Then the adrenal gland was removed from the animal, and a small slit was made into the adrenal cortex just opposite the entrance of the adrenal vein. Perfusion of the adrenal gland was started to ensure that no leak was present and the perfusate escaped only from the slit of the adrenal gland. The adrenal gland was placed in a water-jacketed chamber in which the temperature was maintained at 37°C with thermostatically controlled water circulator (NTT-1200, EYELA, Tokyo, Japan). After extraction of the adrenal gland, the animal was killed by exsanguination.

Perfusion of the adrenal gland. The adrenal gland was perfused by means of a peristaltic pump (MP-3A, EYELA) at a rate of 0.2 ml/min. The perfusion was carried out with Krebs-Henseleit solution of the following composition (mM): 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 2.6 CaCl₂, 1.2 KH₂PO₄, 24.9 NaHCO₃, and 11.1 glucose. Krebs solution was maintained at 37°C by the thermostat bath and bubbled with a mixture of 95% O₂ and 5% CO₂. Perfusate samples were collected in chilled tubes containing 50 µl of 0.1 M perchloric acid to prevent oxidation of catecholamines. Before an experiment was started, the adrenal gland was initially perfused for 60 min with Krebs solution.

Administration of methacholine. Different concentrations of methacholine solution were administered into the perfusion stream through a branching polyethylene catheter. These drugs were infused by using a microsyringe pump (CMA/200, Bioanalytical Systems, West Lafayette, IN). Stimulus concentration was raised stepwise at 5-min intervals, stimulation at each concentration being applied for 40 s.

Experimental protocol. The rats were divided into eight groups. In group 1 (n = 8), the effects of apamin (1 µM) on the methacholine-induced increases in catecholamine (Epi and NE) output were examined. The first set of methacholine (10, 30, 100, and 300 µM) infusion was regarded as a control. Perfusion with apamin-containing Krebs-Henseleit solution was started 10 min before the start of the second trial. In groups 2 (n = 8), 3 (n = 8), and 4 (n = 9), the effects of nifedipine (3 µM), Bay k 8644 (1 µM), and perfusion with Ca²⁺-free solution on increases in catecholamine output induced by methacholine were examined, respectively, with the same protocol as used in group 1. In group 5 (n = 8), the effects of apamin (1 µM) during treatment with nifedipine (3 µM) on increases in catecholamine output induced by methacholine were examined. Perfusion with nifedipine-containing Krebs-Henseleit solution was started 10 min before the start of the first set of methacholine infusions. After the first trial, perfusion with apamin- and nifedipine-containing Krebs-Henseleit solution was started 10 min before the start of the second trial. In group 6 (n = 9), the effects of apamin (1 µM) during treatment with Ca²⁺-free solution on increases in catecholamine output induced by methacholine were examined with the same protocol as used in group 5. In groups 7 (n = 9) and 8 (n = 9), the effects of nifedipine (3 µM) and Bay k 8644 (1 µM) during treatment with apamin (1 µM) on increases in catecholamine output induced by methacholine were examined with the same protocol described above.

Perfusate sampling. Perfusate was sampled before and during infusion of methacholine to determine catecholamine output. The sampling during the basal state was performed for 60 s just before the stimulation. In preliminary experiments, it was found that the methacholine-induced catecholamine responses returned to prestimulation level within ~20 s after stopping the methacholine infusion. Therefore, the samplings during infusion of methacholine at each concentration were performed for 60 s.

Determination of adrenal catecholamine output. Catecholamines in perfusate sample were measured by high-performance liquid chromatography with electrochemical detection (LC-4C, Bioanalytical Systems), as described previously (5). Epi and NE output (ng/min) were calculated by multiplying perfusate catecholamine concentration (ng/ml) by perfusion rate (0.2 ml/min). The basal catecholamine output was determined from sample collected just before infusion of methacholine. The methacholine-induced increases in catecholamine output were calculated by subtracting basal catecholamine output from that obtained during the stimulus state.

Analysis of data. The results are expressed as means ± SE. Two-factor ANOVA with Dunnett's test was used for statistical analysis of data. P values <0.05 were considered to be statistically significant.

Drugs. The drugs used were nifedipine, S-[]-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridine carboxylic acid methyl ester (Bay k 8644), acetyl--methylcholine (methacholine) chloride (Sigma Chemical, St. Louis, MO), and apamin (Peptide Institute, Osaka, Japan). Nifedipine and Bay k 8644 were dissolved in ethanol and diluted to the required concentrations with Krebs-Henseleit solution under dim light immediately before use. Other drugs were dissolved in Krebs-Henseleit solution.

	RESULTS

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Increases in catecholamine output in response to methacholine. Basal Epi and NE output from the adrenal gland at 60 min after initial perfusion were 20.4 ± 1.6 (n = 68) and 4.2 ± 0.4 ng/min (n = 68), respectively, in all groups. There were no significant differences in these basal values among the experimental groups. Infusion of methacholine (10, 30, 100, and 300 µM) into the adrenal gland produced concentration-dependent increases in Epi and NE output in first experimental period of each experimental group (groups 1-8, Figs. 1-4).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effects of apamin (A; group 1) and nifedipine (B; group 2) on the methacholine (MCh)-induced increases in epinephrine (Epi) and norepinephrine (NE) output from the perfused rat adrenal glands. MCh infusion was applied to the adrenal gland in the absence or presence of apamin (1 µM) or nifedipine (3 µM). Symbols and vertical bars represent means ± SE of changes in Epi and NE output from basal output levels. **P < 0.01 compared with corresponding control response obtained before apamin treatment. There were no significant differences (P > 0.05) in catecholamine responses before and during nifedipine treatment.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effects of Bay k 8644 (A; group 3) and perfusion with Ca2+-free solution (B; group 4) on the MCh-induced increases in Epi and NE output from the perfused rat adrenal glands. MCh infusion was applied to the adrenal gland in the absence or presence of Bay k 8644 (1 µM) or Ca2+-free solution. Symbols and vertical bars represent means ± SE of changes in Epi and NE output from basal output levels. **P < 0.01 compared with corresponding control response obtained before Bay k 8644 or Ca2+-free solution treatment.

View larger version (24K): [in this window] [in a new window]   Fig. 3.   Effects of apamin during treatment with nifedipine (A; group 5) and Ca2+-free perfusion solution (B; group 6) on the MCh-induced increases in Epi and NE output from the perfused rat adrenal glands. The adrenal gland was perfused with nifedipine (3 µM)-containing or Ca2+-free Krebs-Henseleit solution. MCh infusion was applied to the adrenal gland in the absence or presence of apamin (1 µM). Symbols and vertical bars represent means ± SE of changes in Epi and NE output from basal output levels. There were no significant differences (P > 0.05) in catecholamine responses before and during apamin treatment.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Effects of nifedipine (A; group 7) and Bay k 8644 (B; group 8) during treatment with apamin on the MCh-induced increases in Epi and NE output from the perfused rat adrenal glands. The adrenal gland was perfused with apamin (1 µM)-containing Krebs-Henseleit solution. MCh infusion was applied to the adrenal gland in the absence or presence of nifedipine (3 µM) or Bay k 8644 (1 µM). Symbols and vertical bars represent means ± SE of changes in Epi and NE output from basal output levels. *P < 0.05, **P < 0.01 compared with corresponding control response obtained with apamin alone.

Effects of apamin, nifedipine, Bay k 8644, and perfusion with Ca²⁺-free solution on the methacholine-induced increases in catecholamine output. Apamin (1 µM) significantly enhanced the methacholine-induced increases in Epi and NE output (group 1, Fig. 1A). Percentages of enhancement by apamin in catecholamine output induced by 100 and 300 µM methacholine were 45 ± 11 and 73 ± 16% in Epi and 46 ± 12 and 128 ± 20% in NE, respectively. Nifedipine (3 µM) did not affect increases in Epi and NE output induced by methacholine (group 2, Fig. 1B). Bay k 8644 (1 µM) significantly enhanced the methacholine-induced increases in Epi and NE output (group 3, Fig. 2A). Percentages of enhancement by Bay k 8644 of increases in catecholamine output induced by 100 and 300 µM methacholine were 24 ± 12 and 57 ± 30% in Epi and 104 ± 23 and 157 ± 61% in NE, respectively. Perfusion with Ca²⁺-free solution significantly inhibited the methacholine-induced increases in Epi and NE, but the inhibition was not complete (group 4, Fig. 2B). Percentages of inhibition by Ca²⁺-free solution of increases in catecholamine output induced by 100 and 300 µM methacholine were 50 ± 8 and 50 ± 6% in Epi and 76 ± 8 and 65 ± 5% in NE, respectively. Neither apamin, nifedipine, Bay k 8644, nor Ca²⁺-free solution affected the basal catecholamine output. Values of basal Epi output before and during drug treatment were 20.5 ± 6.6 and 25.1 ± 6.7 ng/min with apamin, 25.4 ± 8.4 and 20.7 ± 3.0 ng/min with nifedipine, 17.4 ± 2.1 and 21.0 ± 1.1 ng/min with Bay k 8644, and 16.3 ± 2.9 and 22.1 ± 4.7 ng/min with Ca²⁺-free solution, respectively. Values of basal NE output before and during drug treatment were 3.3 ± 0.7 and 2.8 ± 0.8 ng/min with apamin, 3.1 ± 1.8 and 2.7 ± 1.3 ng/min with nifedipine, 4.8 ± 0.5 and 6.6 ± 0.7 ng/min with Bay k 8644, and 4.2 ± 0.8 and 5.7 ± 1.2 ng/min with Ca²⁺-free solution, respectively.

Effects of apamin during treatment with nifedipine or Ca²⁺-free perfusion solution. Apamin (1 µM) did not affect the methacholine-induced increases in Epi and NE output during treatment with nifedipine (3 µM; group 5, Fig. 3A) or Ca²⁺-free solution (group 6, Fig. 3B). Apamin did not affect the basal catecholamine output during treatment with nifedipine or Ca²⁺-free solution. Values of basal Epi output before and during apamin were 16.0 ± 3.7 and 17.4 ± 3.1 ng/min in the presence of nifedipine and 22.8 ± 4.0 and 21.3 ± 3.3 ng/min in the presence of Ca²⁺-free solution, respectively. Values of basal NE output before and during apamin were 4.3 ± 0.7 and 4.9 ± 0.8 ng/min in the presence of nifedipine and 3.6 ± 0.5 and 3.6 ± 0.4 ng/min in the presence of Ca²⁺-free solution, respectively.

Effects of nifedipine and Bay k 8644 during treatment with apamin. Nifedipine (3 µM) significantly inhibited increases in Epi and NE output induced by methacholine during treatment with apamin (1 µM; group 7, Fig. 4A). Percentages of inhibition by nifedipine during treatment with apamin in catecholamine output induced by 100 and 300 µM methacholine were 45 ± 4 and 42 ± 3% in Epi and 64 ± 3 and 66 ± 2% in NE, respectively. Bay k 8644 (1 µM) did not affect the methacholine-induced increase in Epi output during treatment with apamin (1 µM; group 8, Fig. 4B). The methacholine-induced increase in NE output was significantly enhanced by Bay k 8644 (1 µM) during treatment with apamin (1 µM), but the enhancing effect of Bay k 8644 in the presence of apamin was smaller than that in the absence of apamin. Percentages of enhancement by Bay k 8644 during treatment with apamin in catecholamine output induced by 100 and 300 µM methacholine were 16 ± 7 and 6 ± 4% for Epi and 63 ± 14 and 66 ± 12% for NE, respectively. Nifedipine and Bay k 8644 did not affect the basal catecholamine output during treatment with apamin. Values of basal Epi output before and during nifedipine treatment in the presence of apamin were 20.2 ± 2.9 and 15.6 ± 2.6 ng/min. Before and after Bay k 8644 treatment, Epi output was 23.1 ± 3.1 and 35.0 ± 4.3 ng/min, respectively. Values of basal NE output before and during drug treatment in the presence of apamin were 4.5 ± 0.6 and 4.9 ± 0.6 ng/min with nifedipine and 5.0 ± 0.8 and 6.9 ± 1.0 ng/min with Bay k 8644, respectively.

	DISCUSSION

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Apamin enhanced increases in Epi and NE output induced by methacholine without affecting the basal Epi and NE output. This indicates that apamin enhances the secretion by affecting the process mediated by muscarinic receptors but that the toxin does not stimulate the secretion process by itself. This result suggests that SK_Ca channels play an inhibitory role in the secretion of Epi and NE mediated by muscarinic receptors in the rat adrenal gland. This finding is consistent with the previous observations in the isolated perfused cat adrenal gland (17, 18) and the dog adrenal gland in vivo (9, 10).

The methacholine-induced increases in Epi and NE output, although they were blunted, still remained during treatment with Ca²⁺-free solution. This result indicates that the muscarinic receptor-mediated secretion of catecholamines can occur in the absence of extracellular Ca²⁺. The blunted catecholamine output responses observed may be due to reduction of Ca²⁺ in intracellular storage sites resulting from long exposure of chromaffin cells to Ca²⁺-free solution, as suggested by Harish et al. (4). Previously we demonstrated under the same experimental conditions as in this study that the influx of extracellular Ca²⁺ through L-, N-, or P/Q-type Ca²⁺ channels did not contribute to the muscarinic receptor-mediated secretion of catecholamines (11). Therefore, the elevation of intracellular free Ca²⁺ mobilized from intracellular storage sites may mainly contribute to the muscarinic receptor-mediated secretion of catecholamines. Electrophysiological studies have suggested that muscarine-triggered release of Ca²⁺ from intracellular storage sites activates SK_Ca channels on the surface of guinea pig (15) and rat chromaffin cells (13). On the basis of these findings, SK_Ca channels might be activated by Ca²⁺ mobilized from intracellular storage sites during muscarinic receptor stimulation and thereby inhibit the secretion of catecholamines in the rat adrenal gland. Apamin may enhance the muscarinic receptor-mediated secretion of catecholamines by blocking the inhibitory effect of SK_Ca channels.

In the absence of extracellular Ca²⁺, apamin did not affect the methacholine-induced increases in Epi and NE output. This result indicates that extracellular Ca²⁺ is essential to the facilitatory effect of apamin on the methacholine-induced secretion. Here, the question arises as to how extracellular Ca²⁺ contributes to the facilitatory effect of apamin on the muscarinic receptor-mediated secretion of catecholamines. Previously we demonstrated that voltage-dependent Ca²⁺ channels were not involved in the muscarinic receptor-mediated secretion of catecholamines, whereas L- but not N- or P/Q-type Ca²⁺ channels contribute to the nicotinic receptor-mediated secretion in the rat adrenal gland (11). This finding indicates that rat chromaffin cells possess L-type Ca²⁺ channels that participate in the secretion of catecholamines. Therefore, the influx of extracellular Ca²⁺ through L-type Ca²⁺ channels might contribute to the facilitatory effect of apamin on the muscarinic receptor-mediated secretion. To clarify this hypothesis, we examined the effects of nifedipine or Bay k 8644 on the facilitation by apamin of the methacholine-induced increases in catecholamine output in the absence or presence of apamin.

Nifedipine, an L-type Ca²⁺ channel blocker, did not affect increases in Epi and NE output induced by methacholine in the absence of apamin, suggesting that L-type Ca²⁺ channels do not contribute to the muscarinic receptor-mediated secretion of catecholamines. On the other hand, nifedipine inhibited the methacholine-induced catecholamine output responses in the presence of apamin. Moreover, apamin did not affect the catecholamine output responses induced by methacholine in the presence of nifedipine. These results indicate that the facilitatory effect of apamin appears only when L-type Ca²⁺ channels are intact and that the facilitation is blocked by nifedipine. Therefore, it is suggested that the facilitation is caused by the influx of extracellular Ca²⁺ through L-type Ca²⁺ channels. Bay k 8644, an L-type Ca²⁺ channel activator, enhanced increases in Epi and NE output induced by methacholine without affecting the basal Epi and NE output in the absence of apamin. This result indicates that the activation of L-type Ca²⁺ channels by Bay k 8644 can enhance the methacholine-induced secretion of catecholamines, although L-type Ca²⁺ channels normally play no role in the methacholine-induced secretion. The failure of Bay k 8644 to increase the basal catecholamine output suggests that Bay k 8644 itself may not sufficiently elevate intracellular free Ca²⁺ over the threshold level for catecholamine secretion. Bay k 8644 did not affect the methacholine-induced increase in Epi output in the presence of apamin, indicating that apamin blocks the enhancing effect of Bay k 8644. The increase in NE output induced by methacholine was enhanced by Bay k 8644 in the presence of apamin, but the enhancing effect of Bay k 8644 was smaller than that observed in the absence of apamin. These results indicate that apamin interferes with the facilitatory effect of Bay k 8644, suggesting that blockade of SK_Ca channels activates L-type Ca²⁺ channels. Therefore, the activation of SK_Ca channels might inhibit the activation of L-type Ca²⁺ channels in the muscarinic receptor-mediated secretion of catecholamines.

The sequence of events underlying the facilitatory effect of apamin could be explained as follows. Muscarinic receptor stimulation produces the release of Ca²⁺ from intracellular storage sites (3, 4, 7, 16). The elevation of intracellular free Ca²⁺ triggers the secretion of catecholamines (4, 8, 12) and simultaneously activates SK_Ca channels (13, 15). Activation of SK_Ca channels causes the efflux of K⁺ from the cell, which changes the membrane potential in a hyperpolarizing direction. The resulting hyperpolarization may inhibit the opening of L-type Ca²⁺ channels located on chromaffin cells (see Fig. 5). Therefore, it is probable that blockade of SK_Ca channels by apamin leads to depolarization by preventing K⁺ efflux and thereby induces the influx of extracellular Ca²⁺ through L-type Ca²⁺ channels in the rat adrenal gland.

View larger version (21K): [in this window] [in a new window]   Fig. 5.   Schematic diagram demonstrating possible role of small-conductance Ca2+-activated K+ (SKCa) channels and L-type Ca2+ channels (L-type) in catecholamine (CA) secretion mediated by muscarinic receptors (Mus) in the rat adrenal gland. Mus receptor stimulation produces the release of Ca2+ from intracellular storage sites, and the released Ca2+ activates SKCa channels. Activation of SKCa channels causes the efflux of K+ from the cell, which changes the membrane potential in a hyperpolarizing direction. The resulting hyperpolarization inhibits the influx of extracellular Ca2+ through L-type Ca2+ channels. [Ca2+]i, intracellular Ca2+ concentration.

In conclusion, this study demonstrated that apamin enhanced the secretion of Epi and NE induced by methacholine in the isolated perfused rat adrenal gland and that the facilitatory effect of apamin on the methacholine-induced secretion of catecholamines was prevented by nifedipine or Ca²⁺-free solution. We also found that nifedipine did not affect the methacholine-induced catecholamine responses but inhibited the responses in the presence of apamin. Bay k 8644 enhanced the methacholine-induced catecholamine responses, whereas the enhancement of the methacholine-induced Epi and NE responses were prevented and attenuated by apamin, respectively. These results suggest that SK_Ca channels are activated by muscarinic receptor stimulation, which inhibits the opening of L-type Ca²⁺ channels and thereby attenuates adrenal catecholamine secretion in the rat adrenal gland.