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Ca2+ inactivation of voltage-dependent Na+ channels in cultured bovine adrenal chromaffin cells: further studies on inhibition of veratridine-induced catecholamine secretion by external Ca2+.

The stimulatory actions of veratridine (VTD) on catecholamine secretion and Na+ influx in cultured bovine adrenal chromaffin cells were studied in the presence of high concentrations of Ca2+ in the incubation mixture. Catecholamine secretion evoked by VTD was reduced by elevating the external Ca2+ concentration to higher than 2 mM. Under the same conditions, VTD-stimulated 22Na+ uptake into the cells was also reduced by elevating the extracellular Ca2+ concentration. In contrast, the secretory action of VTD was not significantly suppressed by preloading Ca2+ to the cells. Furthermore, the effects of extracellular Ca2+ on the sensitivities of the cells to VTD and tetrodotoxin (TTX) were examined, and neither their sensitivities to VTD nor those to TTX were shown to be significantly altered by elevating the Ca2+ concentration in the incubation mixture. These results seem to indicate that the elevation of extracellular Ca2+ concentration may cause the inhibition of VTD-induced catecholamine secretion as a consequence of the inactivation of voltage-dependent Na+ channels, and suggest that Ca2+ may directly act on the cell surface, and the site of Ca2+ action is presumably distinct from the sites of both VTD and TTX actions in the plasma membranes of adrenal chromaffin cells.

ABSTRACT-The stimulatory actions of veratridine (VTD) on catecholamine secretion and Na+ influx in cultured bovine adrenal chromaffin cells were studied in the presence of high concentrations of Ca" in the incubation mixture. Catecholamine secretion evoked by VTD was reduced by elevating the external Ca" concentration to higher than 2 mM. Under the same conditions, VTD-stimulated 22Na+ uptake into the cells was also reduced by elevating the extracellular Ca 21 concentration. In contrast, the secretory action of VTD was not significantly suppressed by preloading Ca 21 to the cells. Furthermore, the effects of extracellular Ca 21 on the sensitivities of the cells to VTD and tetrodotoxin (TTX) were examined, and neither their sen sitivities to VTD nor those to TTX were shown to be significantly altered by elevating the Ca 2+ concentra tion in the incubation mixture. These results seem to indicate that the elevation of extracellular Ca2+ concen tration may cause the inhibition of VTD-induced catecholamine secretion as a consequence of the inactiva tion of voltage-dependent Na+ channels, and suggest that Ca 2+ may directly act on the cell surface, and the site of Ca 2+ action is presumably distinct from the sites of both VTD and TTX actions in the plasma mem branes of adrenal chromaffin cells.
Keywords: Veratridine, Extracellular Ca 2+, Na+ channel (voltage-dependent), Catecholamine secretion, Adrenal chromaffin cell Exocytosis is commonly appreciated as the mechanism for the release of neurotransmitters and hormones in vari ous cells and tissues, and it has been established that a series of intracellular events in exocytosis is initiated by a rise in the intracellular concentration of free Ca 2+, result ing from an increase in the influx of extracellular Ca 21 into the cell. In the adrenal chromaffin cell, the influx of extracellular Ca 2+ has been reported to be mediated by two different-types of Ca 21 channels, such as receptor operated and voltage-dependent Ca 21 channels (1 3). The stimulation of acetylcholine receptors on the cell sur face has been well established to stimulate the influx of both Na+ and Ca 21 through receptor-linked cation chan nels. On the other hand, various neurotoxins, such as veratridine (VTD), aconitine, batrachotoxin and scorpion venom, have been shown to stimulate Ca 21 influx through voltage-dependent Ca 21 channels as a result of the stimulation of tetrodotoxin (TTX)-sensitive Na+ chan nels (2,4). Thus, it seems reasonable to consider that Na+ channels may be functionally coupled with Ca 21 channels in the plasma membranes and probably play an important role in the regulation of stimulus-secretion coupling.
According to the general concept that an increase in the intracellular Ca2+ concentration may be responsible for the activation of the intracellular exocytotic process, it seems quite reasonable to consider that the secretory response of the adrenal chromaffin cell may directly be influenced by the mechanism(s) modulating the Ca 21 influx into the cell. In fact, the rate of catecholamine secre tion has previously been reported to be declining during prolonged stimulation of the cell with cholinergic agonists and depolarizing agents, and this decline has also been proposed to be attributable to a decrease in the influx of Ca 21 into the cell (2, 3, 5 7), resulting from the inactivation of Ca2+ channels by Ca 21 (3,8,9). Further more, since Na+ channels have been well known to be cou pled with Ca" channels, it also seems possible to assume that Na+ channels as well as Ca" channels may be inacti vated by Ca" itself. The earlier studies have shown that the electrical properties of excitable membranes can be modulated by elevating the external Ca" concentration, suggesting the possibility that the permeability of Na+ through the plasma membranes is presumably regulated by Ca" in squid axons (10) and mammalian skeletal mus cles (11). On the other hand, the stimulatory action of VTD on catecholamine secretion has previously been reported to be suppressed by elevating the external Ca" concentration in cultured bovine adrenal chromaffin cells (12). These findings suggest that Na+ channels as well as Ca 21 channels may be inactivated by Ca 21 itself in excit able cells and tissues, but little is known about the detailed mechanism underlying the inactivation of Na+ channels by external Ca 21 in the plasma membranes of excitable cells and tissues.
To characterize the pharmacological effect of Ca 21 on voltage-dependent Na+ channels in the adrenal chro maffin cell, the stimulatory actions of VTD on both cate cholamine secretion and Na+ influx were examined in the presence of relatively high concentrations of extracellular Ca t+, and confirmed that catecholamine secretion evoked by VTD was reduced by elevating the Ca 2+ concentration in the incubation mixture. This reduction was also shown to be accompanied by the inhibition of Na+ influx into the cells. Further studies on the mechanism of this Ca 2+ inhibition suggested the possiblity that the site(s) of Ca 21 action might be different from the sites of both VTD and TTX actions in the plasma membranes of adrenal chro maffin cells.

Cell preparation and culture
Chromaffin cells were enzymatically prepared from fresh bovine adrenal medulla according to the previously reported method (13). Cells were plated on a 24-well plastic cluster plate at a density of 5 x 105 cells/well and maintained as a monolayer culture in 1.5 ml of Eagle's minimum essential medium containing 5 % heat-inactivat ed newborn calf serum, 2 mM glutamine, 100 units/ml of penicillin, 100 pg/ml streptomycin, 50 pg/ml of gentamy cin, 2 pg/ml of fungizone and 10 pM cytosine arabino side. For the experiments, cells cultured for 3 or 4 days were usually employed.

Determination of catecholamine secretion
Cultured cells were washed with 1.0 ml of balanced salt solution consisting of 135 mM NaCI, 5.6 mM KCI, 1.2 mM MgSO4i 2 mM CaC12, 10 mM glucose and 20 mM 4-(2 hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.4, and then stimulated by secretagogues at 371C for 10 min in 250 , tl of balanced salt solution containing different concentrations of CaC12. At the end of the incu bation period, the medium covering the cells was withdrawn, and the cells were lysed by adding 250 p1 of 10°70 acetic acid followed by a freeze-thaw cycle. Both the medium and the lysates were centrifuged at the maximum speed for 2 min in an Eppendorf microcentrifuge, and the amounts of catecholamines in the supernatants were then determined by the previously reported method (14). Catecholamine secretion was expressed as the percentage of total cellular content secreted into the medium during the incubation period.
Measurement of 22Na+ uptake Cells were washed with balanced salt solution as described above and then incubated with VTD at 37V for 10 min in 250 pl of balanced salt solution containing various concentrations of CaC12 with 22NaC1 (0.5 pCi/ well). At the end of the incubation period, the medium was removed by aspiration, and the cells were washed 4 times with 1.0 ml of ice-cold balanced salt solution. The cells were solubilized by adding 200 p1 of 1 % Triton X 100, and the wells were washed twice with 200 pl of dis tilled water. The lysates and the washings were combined together, and the radioactivities in these solutions were de termined by a liquid scintillation spectrometer. The up take of Na+ into the cells was calculated on the basis of the initial specific activity of 22NaC1 in the incubation mix ture and expressed as nmoles Na+/well.
The concentration of NaCI in high K+ medium (56 mM KCI) was reduced from 135 mM to 84.6 mM to keep the solution isotonic. Standard deviation (S.D.) of the differ ence between two groups was calculated as (S.D.,2+ S.D.22)"2, and Student's t-test was employed to determine the statistical significance. and an approximately 50°7o inhibition of the secretory response was obtained at 8 mM. In contrast, catechol amine secretion evoked by either carbamylcholine or high K+ was substantially enhanced by elevating the external Ca 21 concentration to a level as high as that producing the marked reduction of VTD-induced secretion (Fig. 2). These results seemed to indicate that the VTD-stimulated step in the secretory process might be specifically influenced by elevating the extracellular Ca 21 concentra tion, thus suggesting the possibility that VTD-stimulated, voltage-dependent Na+ influx into the cells might be tight ly connected with the reduction of VTD-induced catechol amine secretion caused by high concentrations of extracel lular Ca2+.

Effect of external Ca" concentration on Na+ influx
To solve the question of whether the reduction of VTD induced catecholamine secretion caused by high concen trations of external Ca 21 might be attributed to an altera tion in the activity of VTD-stimulated, voltage-depend ent Na+ channels, the stimulatory action of VTD on 22Na+ uptake into the cells was examined in the presence of high concentrations of Ca 21 in the incubation mixture. As shown in Fig. 3, VTD-stimulated 22Na+ uptake into chromaffin cells was markedly reduced by elevating the extracellular Ca 2+ concentration, and this reduction of 22Na+ uptake was more pronouncedly observed in compari son with that of the VTD-induced secretion. These results seemed to provide evidence supporting the possibility that the reduction of VTD-induced catecholamine secretion caused by elevating the external Ca" concentration might be attributed to the reduction of Na+ influx through voltage-dependent Na+ channels.

Properties of Ca' action on voltage-dependent Na+ channels
To further elucidate the properties of Ca2+ action on VTD-induced catecholamine secretion, the cells were first incubated with the Ca 2+-ionophore A23187 in the me dium containing high concentration of Ca 21 (8 mM), and the secretory actions of various secretagogues on the Ca 2+ preloaded cells were examined in the presence of a normal concentration of Ca 2+ (2 mM). As shown in Fig. 4, the pretreatment of the cells with A23187 caused the enhance ment rather than the reduction of catecholamine secretion evoked by VTD as well as high K+, while the carbamyl choline-induced secretion was slightly reduced by pretreat ment of the cells with A23187 under the same conditions. These results seemed to indicate that the intracellular Ca 2+ was probably not related directly to the reduction of VTD-induced catecholamine secretion caused by elevat ing the Ca 21 concentration in the incubation mixture, thus suggesting strongly the possibility that the elevation of extracellular Ca 21 concentration might be able to cause the suppression of the secretory responses to VTD as a consequence of direct action of Ca 2+ on the cell surface.
To characterize the site of Ca 2+ action on the cell surface, the effect of extracellular Ca 2+ on the sensitivities of chro maffin cells to VTD were investigated by determining the secretory action of VTD in the presence of normal and high concentrations of Ca 2+. As shown in Fig. 5, catechol amine secretion was markedly enhanced by VTD in the presence of 2 mM Ca 2+, and this stimulatory action was observed in a concentration-dependent manner. In con trast, the VTD action on the secretion was markedly sup pressed by the presence of 8 mM Ca 2+, and this suppres sion was still observed when the cells were stimulated by high concentrations of VTD (50 and 100 1M). If the Lineweaver-Burk analysis was forcedly applied to these data obtained here, the apparent Km value of the cells for VTD and the Vmax of catecholamine secretion were esti mated as 57.5 iiM and 49.8% of the cellular catechol amines released/10 min in the presence of 2 mM Ca2+, and 50.9 pM and 20.1 % of the cellular content re leased/10 min in the presence of 8 mM Ca 2+. Thus, the elevation of extracellular Ca 21 concentration was thought to cause a marked decrease in the Vmax without any sig nificant alteration in the apparent Km value for VTD. It therefore seemed conceivable that the sensitivities of the cells to VTD might not be altered by elevating the Ca 21 concentration in the incubation mixture.
The inhibitory action of TTX, an inhibitor of voltage dependent Na+ channels, on VTD-induced catechol amine secretion was furtheremore examined in the incuba tion mixture containing 2 mM or 8 mM Ca 2+. As shown in Fig. 6, the inhibitory action of TTX on the VTD-in duced secretion was observed in a concentration-depend ent manner. The maximum inhibition of the secretory

DISCUSSION
The present study indicated that the stimulatory action of VTD on catecholamine secretion from cultured bovine adrenal chromaffin cells was markedly suppressed by elevating the extracellular concentration of Ca 21 from 2 mM to 8 mM (Fig. 1), thus confirming the previous find ing that VTD-induced catecholamine secretion is reduced by high concentrations of extracellular Ca 21 (12). However, the mechanism underlying this Ca2+ action on VTD-induced catecholamine secretion has not yet been elucidated. On the other hand, the secretory actions of carbamylcholine and high K+ were shown to be enhanced rather than suppressed by elevating the Ca 21 concentra tion to the level causing the suppression of VTD action (Fig. 2). Both carbamylcholine and high K+ are well known to stimulate the Ca 21 influx into the cells, and their stimulatory actions are also known to be mediated by mechanisms different from that underlying the stimula tory action of VTD on the Ca2+ influx. Namely, carbamyl choline can stimulate the influx of Ca 21 through acetyl choline receptor-mediated Ca 21 channels, and high K+ causes the stimulation of voltage-dependent Ca 21 influx without accompanying the Na+ influx into the cells. In view of these facts, it seems unlikely that the suppression of VTD action observed here may simply be due to the direct inactivation of Ca 21 channels in the plasma mem branes of adrenal chromaffin cells.
In view of the previous findings that VTD can stimulate voltage-dependent Na+ channels linked with voltage-de pendent Ca 21 channels, leading to the stimulation of cate cholamine secretion from adrenal medullary cells and tissues (1, 2,4,15,16), it seemed reasonable to assume that the reduction of VTD-induced catecholamine secre tion by elevating the external Ca 21 concentration is prob ably mediated by the inhibitory action of Ca 21 on VTD stimulated, voltage-dependent Na+ channels. In fact, the stimulatory action of VTD on Na+ influx into the cells as well as that on catecholamine secretion was shown to be markedly suppressed by elevating the Ca2+ concentration in the reaction mixture (Fig. 3). It therefore seems reason able to conclude that the elevation of external Ca 21 con centration may cause the reduction of VTD-induced cate cholamine secretion as a consequence of inhibition of Na+ influx through voltage-dependent Na+ channels in cultured bovine adrenal chromaffin cells. Furthermore, the suppression of VTD-induced catecholamine secretion was less pronouncedly observed as compared with that of VTD-stimulated Na+ influx under the experimental condi tions employed here. In view of the fact that catechol amine secretion is supported by the intracellular Ca 21 con centration which is determined by different mechanisms, such as the influx of extracellular Ca 21 through different type Ca 21 channels and the mobilization of intracellular Ca2+, it also seems possible to consider that the Ca2+ influx coupled with voltage-dependent Na+ influx may partly contribute to the secretory response of the chro maffin cell. The stimulatory action of VTD on catecholamine secre tion was shown to be suppressed by elevating the external Ca2+ concentration, and this suppression of the VTD ac tion has also been suggested to be the result of the inacti vation of voltage-dependent Na+ channels by Ca' itself. However, the question of whether the intracellular Ca 2+ level might be responsible for the inactivation of voltage dependent Na+ channels still remained to be elucidated.
To address this critical question, the secretory action of VTD was examined using Ca"-preloaded cells, which were obtained by preincubating the cells with the Ca2+ ionophore A23187 in the presence of a high concentration of extracellular Ca2+. Consequently, the enhancement rather than the suppression of the secretory action of VTD was observed in these Ca 2+-preloaded cells, although the Ca 21 level within these cells was presumed to be higher than the physiological level of Ca 21 concen tration in the adrenal chromaffin cell (Fig. 4). It therefore seems possible to exclude the possibility that the elevation of extracellular Ca 21 concentration may cause the inacti vation of voltage-dependent Na+ channels presumably through an alteration in the intracellular Ca 21 level. Fur thermore, the present study indicated that the secretory ac tion of carbamylcholine was not significantly suppressed by preincubation of the cells with the Ca 2+-ionophore A23187 in the presence of a high concentration of Ca2+, thus suggesting that the function of the stimulus-secretion coupling system is still kept intact during the Ca 21 preloading. In contrast, the secretory action of high K+ was shown to be enhanced by pretreatment of the cells with A23187 under the same conditions. In view of the previous finding that catecholamine secretion evoked by high K+ is markedly reduced by pretreatment of the cells with A23187 (9), the unexpected result described here seems to raise another critical question of whether the intracellular Ca 21 level in the A23187-pretreated cells may be maintained as high as that required to cause the inacti vation of voltage-dependent cation channels.
Voltage-dependent Na+ channels in the excitable mem branes have been well established to be stimulated by VTD and inhibited by TTX in various cells and tissues, and the binding site of VTD has been suggested to be distinct from that of TTX on the Na+ channel (17). Although the present results suggested that voltage-de pendent, TTX-sensitive Na+ channels might be inactivated by high concentrations of extracellular Ca2+, the question of whether the site affected by Ca 21 might be the same as or different from the binding site of VTD or that of TTX still remained to be solved. Further studies showed that the elevation of extracellular Ca 2+ concentration failed to cause any notable alteration in the potencies of both the secretory action of VTD (Fig. 5) and the inhibitory action of TTX on the VTD-induced secretion (Fig. 6), thus sug gesting the possibility that the sensitivities of Na+ chan nels to both VTD and TTX were probably not affected by elevating the external Ca 21 concentration. It therefore seems possible to consider that the site involved in the in activation of VTD-stimulated Na+ channels by high con centration of extracellular Ca 2+ may be different from both the binding site of VTD and that of TTX in the plas ma membranes of adrenal chromaffin cells. As one of pos sible mechanisms, it seems conceivable that external Ca 2+ may cause a non-specific stabilizing action on the plasma membranes, thus resulting in an alteration in the mem brane functions. Further studies on the characteristics of Na+ channels are still required to elucidate a possible mechanism(s) for the Ca 21 inactivation of Na+ channels as well as a possible site(s) of Ca 2+ action in the adrenal chromaffin cell.