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Am J Physiol Regul Integr Comp Physiol 279: R448-R454, 2000;
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Vol. 279, Issue 2, R448-R454, August 2000

Role of potassium channels in catecholamine secretion in the rat adrenal gland

Takahiro Nagayama1, Yasuo Fukushima1, Makoto Yoshida1, Mizue Suzuki-Kusaba1, Hiroaki Hisa1, Tomohiko Kimura2, and Susumu Satoh1

1 Laboratory of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Sendai 980-8578; and 2 Department of Dental Pharmacology, The Nippon Dental University School of Dentistry at Niigata, Hamaura-cho, Niigata 951-8580, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We elucidated the functional contribution of K+ channels to cholinergic control of catecholamine secretion in the perfused rat adrenal gland. The small-conductance Ca2+-activated K+ (SKCa)-channel blocker apamin (10-100 nM) enhanced the transmural electrical stimulation (ES; 1-10 Hz)- and 1,1-dimethyl-4-phenyl-piperazinium (DMPP; 5-40 µM)-induced increases in norepinephrine (NE) output, whereas it did not affect the epinephrine (Epi) responses. Apamin enhanced the catecholamine responses induced by acetylcholine (6-200 µM) and methacholine (10-300 µM). The putative large-conductance Ca2+-activated K+ channel blocker charybdotoxin (10-100 nM) enhanced the catecholamine responses induced by ES, but not the responses induced by cholinergic agonists. Neither the KA channel blocker mast cell degranulating peptide (100-1000 nM) nor the KV channel blocker margatoxin (10-100 nM) affected the catecholamine responses. These results suggest that SKCa channels play an inhibitory role in adrenal catecholamine secretion mediated by muscarinic receptors and also in the nicotinic receptor-mediated secretion of NE, but not of Epi. Charybdotoxin-sensitive Ca2+-activated K+ channels may control the secretion at the presynaptic site.

small-conductance Ca2+-activated K+; large-conductance Ca2+-activated K+; KA channel; KV channel; apamin; charybdotoxin; mast cell degranulating peptide; margatoxin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CATECHOLAMINE SECRETION FROM the adrenal medulla is controlled by nicotinic and muscarinic receptors. The elevation of intracellular free Ca2+ acts as an essential mediator in the exocytotic secretion of catecholamines (6, 20). Stimulation of nicotinic receptors opens voltage-dependent Ca2+ channels (5, 7) by depolarizing the membrane of chromaffin cells (4, 9, 19). Stimulation of muscarinic receptors triggers release of Ca2+ from intracellular storage sites by initiating phosphatidylinositol turnover (11, 25, 35). The elevation of intracellular free Ca2+ is considered to be counteracted by activation of K+ channels. The membrane depolarization may activate voltage-dependent K+ channels leading to the facilitation of repolarization, and the elevation of intracellular free Ca2+ may activate Ca2+-activated K+ (KCa) channels leading to hyperpolarization, either of which could inhibit further influx of Ca2+.

KCa channels, such as small (SKCa)- and large (BKCa)-conductance KCa channels, are present on adrenal chromaffin cells (26), but the role of each type in the catecholamine secretion is not fully understood. SKCa channels are characterized by indirect regulation of Ca2+ movement and catecholamine secretion in bovine (23, 39) and cat (27, 37, 38) chromaffin cells. Recently, Nagayama and colleagues (28, 29) suggested that SKCa channels play an inhibitory role in adrenal catecholamine secretion in the dog. On the other hand, the contribution of BKCa channels to catecholamine secretion remains controversial. Blockade of BKCa channels enhances the catecholamine secretion induced by carbachol, a nicotinic agonist, in bovine chromaffin cells (39), but it does not affect the transmural electrical stimulation (ES)-induced catecholamine secretion in adrenal gland of the cat (27) and the dog (28). Rat chromaffin cells possess SKCa and BKCa channels (31), but there has been no evidence for participation of these KCa channels in catecholamine secretion. Voltage-dependent K+ channels, such as KA and KV channels, are present on sympathetic neurons in the rat (2, 3, 13). Although blockade of KA channels does not affect Ca2+ influx in bovine chromaffin cells (39), we have observed that a KA-channel blocker enhanced the adrenal secretion of catecholamines induced by splanchnic nerve stimulation, but not by ACh in the dog (29). However, the physiological role of KV channels in adrenal catecholamine secretion has not been examined.

The aim of this study is to elucidate the functional role of K+ channels in controlling the adrenal catecholamine secretion. We examined the effects of apamin, an SKCa-channel blocker (17), charybdotoxin, a BKCa-channel blocker (15), mast cell degranulating (MCD) peptide, a KA-channel blocker (34), and margatoxin, a KV-channel blocker (1), on the secretion of epinephrine (Epi) and norepinephrine (NE) from the isolated perfused rat adrenal gland in response to ES, the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP), ACh, and the muscarinic agonist methacholine.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 230-300 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 (30). 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 on a bipolar platinum electrode used for ES. The adrenal gland, together with an electrode, was placed in a water-jacketed chamber, the temperature of which 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 (in mM): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 2.6 CaCl2, 1.2 KH2PO4, 24.9 NaHCO3, and 11.1 glucose. Krebs-Henseleit solution was maintained at 37°C by the thermostat bath and bubbled with a mixture of 95% O2-5% CO2. Perfusate samples were collected in chilled tubes containing 50 µl of 0.1 M perchloric acid to prevent oxidation of catecholamines. Before the start of an experiment, the adrenal gland was initially perfused for 60 min with Krebs-Henseleit solution.

ES. ES (duration 1 ms; supramaximal voltage 50 V) was applied by a bipolar platinum electrode with an electronic stimulator (SEN-3301, Nihon Kohden, Tokyo, Japan) and an isolation unit (SS-302J, Nihon Kohden). Stimulus frequency was raised stepwise from 1 to 2, 5, and 10 Hz at 5-min intervals, stimulation at each frequency being applied for 40 s.

Administration of cholinergic agonists. Different concentrations of DMPP, ACh, and 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 16 groups. In group 1a (n = 10), the effects of apamin on the ES-induced increases in catecholamine (Epi and NE) output were examined. The first set of ES (1, 2, 5, and 10 Hz) was regarded as a control (1st trial). Perfusion with apamin (10 and 100 nM)-containing Krebs-Henseleit solution was started 10 min before the start of the second and third trials, respectively. In group 1b (n = 11), the effects of apamin on the DMPP-induced increases in catecholamine output were examined. The first set of DMPP infusion (5, 10, 20, and 40 µM) was regarded as a control (1st trial). Perfusion with apamin-containing Krebs-Henseleit solution was started 10 min before the start of the second and third trials, respectively. In groups 1c (n = 7) and 1d (n = 10), the effects of apamin on the ACh (6, 20, 60, and 200 µM)- and methacholine (10, 30, 100, and 300 µM)-induced increases in catecholamine output were examined, respectively, with the same protocol as used in group 1b. The effects of charybdotoxin (10 and 100 nM) on increases in catecholamine output induced by ES (group 2a; n = 7), DMPP (group 2b; n = 8), ACh (group 2c; n = 7), and methacholine (group 2d; n = 13) were examined with the same protocol described above. The effects of MCD peptide (100 and 1,000 nM) on increases in catecholamine output induced by ES (group 3a; n = 7), DMPP (group 3b; n = 7), ACh (group 3c; n = 8), and methacholine (group 3d; n = 8) were examined with the same protocol. The effects of margatoxin (10 and 100 nM) on increases in catecholamine output induced by ES (group 4a; n = 8), DMPP (group 4b; n = 8), ACh (group 4c; n = 7), and methacholine (group 4d; n = 8) were also examined.

Previously, we demonstrated that the ES-, DMPP-, ACh-, and methacholine-induced increases in Epi and NE output were reproducible during repeated application of ES, DMPP, ACh, and methacholine (30).

Perfusate sampling. Perfusate was sampled before and during ES or infusion of the cholinergic agonists 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 stimuli-induced catecholamine responses returned to prestimulation level within ~20 s after stopping the ES or the agonist infusion. Thus the sampling during ES at each frequency or infusion of the cholinergic agonist at each concentration was 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, West Lafayette, IN), as described previously (21). 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 ES and infusion of the cholinergic agonists. The stimuli-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 DMPP iodide, ACh chloride, acetyl-beta -methylcholine (methacholine) chloride (Sigma Chemical, St. Louis, MO), apamin, charybdotoxin, MCD peptide, and margatoxin (Peptide Institute, Osaka, Japan). All drugs were dissolved in Krebs-Henseleit solution.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Increases in catecholamine output in response to ES, DMPP, ACh, and methacholine. Basal Epi and NE output from the adrenal gland at 60 min after initial perfusion was 14.5 ± 1.4 (n = 134) and 3.2 ± 0.2 ng/min (n = 134), respectively, in all groups. There were no differences in these basal values among the experimental groups. ES (1-10 Hz) or infusion of DMPP (5-40 µM), ACh (6-200 µM), and methacholine (10-300 µM) into the adrenal gland produced frequency- or concentration-dependent increases in Epi and NE output (groups 1-4, Figs. 1-5).


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  		Fig. 1.   Effects of apamin (Apa) on epinephrine (Epi) and norepinephrine (NE) output from the perfused rat adrenal glands in response to transmural electrical stimulation (ES; A, group 1a) and 1,1-dimethyl-4-phenyl-piperazinium (DMPP; B, group 1b) infused into the perfusate. ES or DMPP infusion was applied to the adrenal gland in the absence or presence of Apa (10 and 100 nM). Symbols and vertical bars represent means ± SE. * P < 0.05, ** P < 0.01 compared with corresponding control responses obtained before the Apa treatment.



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  		Fig. 2.   Effects of Apa on Epi and NE output from the perfused rat adrenal glands in response to ACh (A, group 1c) and methacholine (MCh; B, group 1d) infused into the perfusate. DMPP or MCh infusion was applied to the adrenal gland in the absence or presence of Apa (10 and 100 nM). Symbols and vertical bars represent means ± SE. * P < 0.05, ** P < 0.01 compared with corresponding control responses obtained before the Apa treatment.



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  		Fig. 3.   Effects of charybdotoxin (ChTX) on Epi and NE output from the perfused rat adrenal glands in response to ES (A, group 2a) and ACh (B, group 2c) infused into the perfusate. ES or ACh infusion was applied to the adrenal gland in the absence or presence of ChTX (10 and 100 nM). Symbols and vertical bars represent means ± SE. * P < 0.05, ** P < 0.01 compared with corresponding control responses obtained before the ChTX treatment.



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  		Fig. 4.   Effects of mast cell degranulating (MCD) peptide on Epi and NE output from the perfused rat adrenal glands in response to ES (A, group 3a) and ACh (B, group 3c) infused into the perfusate. ES or ACh infusion was applied to the adrenal gland in the absence or presence of MCD peptide (100 and 1,000 nM). Symbols and vertical bars represent means ± SE. There were no significant differences (P > 0.05) in catecholamine responses before (control) and during the MCD peptide treatment.



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  		Fig. 5.   Effects of margatoxin (MgTX) on Epi and NE output from the perfused rat adrenal glands in response to ES (A, group 4a) and ACh (B, group 4c) infused into the perfusate. ES or ACh infusion was applied to the adrenal gland in the absence or presence of MgTX (10 and 100 nM). Symbols and vertical bars represent means ± SE. There were no significant differences (P > 0.05) in catecholamine responses before (control) and during the MgTX treatment.

Effects of apamin on the ES-, DMPP-, ACh-, and methacholine-induced increases in catecholamine output. Apamin (10 and 100 nM) enhanced the ES- and DMPP-induced increases in NE output, but it did not affect the increases in Epi output (group 1a, Fig. 1A; group 1b, Fig. 1B). The enhancements by apamin of the ES- and DMPP-induced increases in NE output were small, but they were statistically significant. Percentages of enhancement by the highest concentration (100 nM) of apamin of increases in NE output induced by 10 Hz ES and 40 µM DMPP were 30 ± 8 and 21 ± 9%, respectively. The ACh- and methacholine-induced increases in Epi and NE output were enhanced by apamin (group 1c, Fig. 2A; group 1d, Fig. 2B). Percentages of enhancement by apamin of increases in catecholamine output induced by 200 µM ACh and 300 µM methacholine were 25 ± 6 and 63 ± 15% in Epi and 41 ± 9 and 108 ± 22% in NE, respectively. Basal Epi and NE output were not affected by apamin (data not shown).

Effects of charybdotoxin. Charybdotoxin (10 and 100 nM) enhanced increases in Epi and NE output induced by ES (group 2a, Fig. 3A), but not the increases induced by ACh (group 2c, Fig. 3B). The DMPP- and methacholine-induced increases in Epi and NE output were not affected even by the highest concentration (100 nM) of charybdotoxin (groups 2b and 2d). The 40 µM DMPP- and 300 µM methacholine-induced increases in Epi output were 215 ± 19 (n = 8) and 131 ± 15 (n = 13) during control period and 179 ± 16 and 119 ± 12 ng/min during treatment with 100 nM charybdotoxin, respectively, and the increases in NE output were 54 ± 6 (n = 8) and 10 ± 1 (n = 13) during control period and 40 ± 5 and 11 ± 1 ng/min during treatment with 100 nM charybdotoxin, respectively. Basal Epi and NE output were not affected by charybdotoxin (data not shown).

Effects of MCD peptide. MCD peptide (100 and 1,000 nM) did not affect increases in Epi and NE output induced by ES (group 3a, Fig. 4A) and ACh (group 3c, Fig. 4B). The DMPP- and methacholine-induced increases in Epi and NE output were not affected even by the highest concentration (1,000 nM) of MCD peptide (groups 3b and 3d). The 40 µM DMPP- and 300 µM methacholine-induced increases in Epi output were 215 ± 30 (n = 7) and 126 ± 22 (n = 8) during control period and 186 ± 23 and 120 ± 17 ng/min during treatment with 1,000 nM MCD peptide, respectively, and the increases in NE output were 66 ± 7 (n = 7) and 11 ± 2 (n = 8) during control period and 54 ± 6 and 10 ± 2 ng/min during treatment with 1,000 nM MCD peptide, respectively. Basal Epi and NE output were not affected by MCD peptide (data not shown).

Effects of margatoxin. Margatoxin (10 and 100 nM) did not affect increases in Epi and NE output induced by ES (group 4a, Fig. 5A) and ACh (group 4c, Fig. 5B). The DMPP- and methacholine-induced increases in Epi and NE output were not affected even by the highest concentration (100 nM) of margatoxin (groups 4b and 4d). The 40 µM DMPP- and 300 µM methacholine-induced increases in Epi output were 214 ± 39 (n = 8) and 131 ± 19 (n = 8) during control period and 198 ± 37 and 124 ± 18 ng/min during treatment with 100 nM margatoxin, respectively, and the increases in NE output were 56 ± 11 (n = 8) and 12 ± 1 (n = 8) during control period and 53 ± 11 and 12 ± 2 ng/min during treatment with 100 nM margatoxin, respectively. Basal Epi and NE output were not affected by margatoxin (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apamin, an SKCa-channel blocker (17), has been reported to enhance the secretion of catecholamines induced by DMPP in bovine chromaffin cells (23) and the secretion induced by methacholine (37, 38), ES, and ACh (27) in the perfused cat adrenal gland. Epi and NE are stored in and secreted from distinct chromaffin cells (8, 10, 16). However, the role of SKCa channels in the secretion of each catecholamine from these cells has not been explored. The present study is the first work in which the contribution of SKCa channels to the secretion of Epi or NE was demonstrated.

Apamin enhanced increases in NE output induced by ES and the nicotinic agonist DMPP. Previously, we demonstrated under the same experimental conditions as in this study that the ES-induced secretion of Epi and NE is mainly mediated by nicotinic receptors (30). Therefore, the results of this study suggest that SKCa channels play an inhibitory role in the adrenal secretion of NE mediated by nicotinic receptors from NE-containing cells. On the other hand, apamin did not affect increases in Epi output induced by ES and DMPP. Therefore, SKCa channels may not contribute to the nicotinic receptor-mediated secretion of Epi from Epi-containing cells. The adrenal secretion of each catecholamine is known to be controlled by the central nervous system. For example, hemorrhagic shock and hypoglycemia preferentially cause the secretion of NE and Epi, respectively (12, 18). Adding to the central mechanisms that mediate these reflex catecholamine secretions, the present study demonstrates the peripheral mechanisms that also participate in the control of differential secretion of Epi and NE through SKCa channels in response to nicotinic stimulation.

Apamin enhanced increases in Epi and NE output induced by ACh and the muscarinic agonist methacholine. The facilitatory effect of apamin on the methacholine-induced secretion suggests that SKCa channels play an inhibitory role in the adrenal secretion of Epi and NE mediated by muscarinic receptors. These results indicate that there is a difference in the contribution of SKCa channels to Epi secretion between nicotinic and muscarinic stimulation, although physiological relevance for these differences remains to be resolved. We demonstrated that ACh stimulates the secretion of Epi and NE by activating both nicotinic and muscarinic receptors (30). On the basis of these findings, the facilitatory effect of apamin on the ACh-induced secretion of Epi and NE is explained by its facilitatory action on the pathway mediated by muscarinic receptors and by both nicotinic and muscarinic receptors, respectively. Apamin did not affect the basal Epi and NE output. This indicates that apamin influences the secretion process, but the toxin does not stimulate the secretion process by itself.

Charybdotoxin, a BKCa-channel blocker (15), enhanced increases in Epi and NE output induced by ES, but not the increases induced by ACh, DMPP, and methacholine. These results are not consistent with the observation that charybdotoxin does not affect the ES-induced catecholamine secretion in the perfused cat adrenal gland (27). The authors applied charybdotoxin at the concentration of 10 nM. In the present study, 10 nM charybdotoxin did not affect the ES-induced secretion of catecholamines, but 100 nM charybdotoxin significantly enhanced the secretion. Therefore, this difference may be due to the differential concentrations of charybdotoxin used. Recently, charybdotoxin was reported to block KCa current (IKCa) channels, another type of KCa channels, as well as BKCa channels in vascular smooth muscle cells (32). Taken together, the facilitatory effect of 100 nM charybdotoxin on the ES-induced secretion of catecholamines may result from its blocking action on these charybdotoxin-sensitive KCa channels (BKCa and/or IKCa channels). The differential effects of charybdotoxin on the secretion between endogenous and exogenous ACh may be explained by assuming that distribution of charybdotoxin-sensitive KCa channels is limited at presynaptic nerve endings. The elevation of intracellular free Ca2+ induced by ES at the nerve endings may activate charybdotoxin-sensitive KCa channels, and the activated KCa channels may attenuate the release of ACh by diminishing the depolarizing phase. Charybdotoxin might produce the facilitation of the ACh release by blocking charybdotoxin-sensitive KCa channel-mediated negative control. Consequently, the secretion of catecholamines in response to ES, but not to the cholinergic agonists, may be facilitated. It was reported that BKCa channels are highly concentrated in terminal areas of prominent fiber tracts in rat brain (22) and that BKCa channels play an important role in transmitter release at a cholinergic presynaptic nerve terminal in the chick ciliary ganglion (36). These findings might support our hypothesis, although no report is available suggesting presynaptic localization of charybdotoxin-sensitive KCa channels in the adrenal gland.

MCD peptide, a KA-channel blocker (34), did not affect increases in Epi and NE output in response to ES, ACh, DMPP, and methacholine. The failure of MCD peptide to affect the catecholamine secretion responses may not be due to its poor tissue penetration ability or insufficient concentrations used, because submicromolar concentration (100 nM) of MCD peptide has been reported to block KA channels in rat sensory ganglion cells (34) and because MCD peptide has a smaller molecular size than charybdotoxin. In the present study, we used MCD peptide at a range of 100-1,000 nM. Our results suggest that KA channels have no role in the secretion of catecholamines from rat adrenal gland. Previously, we demonstrated that MCD peptide enhances the adrenal secretion of catecholamines induced by splanchnic nerve stimulation in the dog (29). Therefore, the contribution of KA channels to the secretion of catecholamines in response to sympathetic stimulation may differ from species to species.

Margatoxin has been shown to block KV channels in human T lymphocytes (1, 14). It has been reported that margatoxin increases the spontaneous and the electrically evoked [3H]dopamine release in rat striatum (33). However, the contribution of KV channels to the adrenal secretion of catecholamines is not known. In this study, margatoxin did not affect increases in Epi and NE output in response to ES, ACh, DMPP, and methacholine, and it did not affect the basal catecholamine secretion. The concentrations (10-100 nM) of margatoxin used in this study are known to block KV channels in human T lymphocytes (14, 24). Therefore, KV channels may have no role in the secretion of catecholamines from rat adrenal gland.

In conclusion, our results suggest that SKCa channels located on rat adrenal medullary cells play an inhibitory role in the secretion of Epi and NE mediated by muscarinic receptors and that they play the same role in the nicotinic receptor-mediated secretion of NE, but not of Epi. Charybdotoxin-sensitive KCa channels may control the secretion at presynaptic sites. KA and KV channels may not contribute to the secretion.

Perspectives

The results of this study suggest that SKCa channels functionally play an inhibitory role in adrenal secretion of catecholamines mediated by muscarinic receptors and also in the nicotinic receptor-mediated secretion of NE, but not of Epi. To strengthen these findings obtained in the isolated perfused rat adrenal gland preparation, it will be necessary to perform biochemical studies to clarify that SKCa channels are present in the rat adrenal medulla. Moreover, the results of this study suggest that charybdotoxin-sensitive KCa channels may play an inhibitory role in adrenal secretion of catecholamines through a presynaptic inhibition of ACh release. For confirmation of this hypothesis, it will be necessary to clarify the localization of charybdotoxin-sensitive KCa channels in splanchnic nerve endings by means of electrophysiological and histochemical studies and the effect of charybdotoxin on ACh release from the nerve endings.


    ACKNOWLEDGEMENTS

This work was supported in part by research fellowships of the Japan Society for the Promotion of Science for Young Scientists and by Grant 10877371 for scientific research from The Ministry of Education, Science, and Culture, Japan.


    FOOTNOTES

Address for reprint requests and other correspondence: H. Hisa, Laboratory of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku Univ., Aobayama, Sendai, 980-8578, Japan (E-mail: hhisa{at}mail.pharm.tohoku.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. §1734 solely to indicate this fact.

Received 29 October 1999; accepted in final form 15 March 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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