Brain Research, 571 (1992) 154-158 © 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

154 BRES 25014

A mu intc reoeptor allonist mobilla Ca inoe4tol-1,4,5-- . • Ca 2+ chromafftn cells

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Masaru Sorimachi, Kazuhiko Yamagami and Shigeto Nishimura Department of Physiology, Kagoshima University, School of Medicine, Kagoshima (Japan) (Accepted 15 October 1991)

Key words: Cytosolic-free calcium; Fura-2 microfluorimetry; Caffeine; Inositol-l,4,5-trisphosphate; Adrenal chromaffin cell; Cat

To gain some understanding of the characteristics of intraceUular C a 2+ stores of cat adrenal chromaffm cells, we investigated the effects of ryanodine, a blocker of Ca2+-induced Ca2+ release channels in muscle, on both cytosolic Ca2+ concentration and catecholamine secretion induced by caffeine or methacholine. The results suggest that Ca2+ stores consist of at least two compartments, one which is sensitive to both caffeine and inositol-l,4,5-trisphosphate (IP3), and the other which is sensitive to IP3 alone. Depolarizing stimuli such as nicotine, high K+,Na ÷ channel activators, etc., evoke catecholamine (CA) secretion from adrenal chromaffin cells by increasing the cytosolic free calcium concentration ([Ca]in) that results from Ca 2+ influx. In contrast, the inositol-l,4,5-tdsphosphate (IPa)-mobilizing agonists were found to cause littie, if any, secretion in bovine chromaffin cells, possibly because IP3-sensitive Ca 2+ stores 12 and the intracellular Ca 2+ signal 3 were spatially restricted. Recent findings 2 have shown that in addition to possessing IP3-sensitive Ca 2+ stores, which are likely a portion of endoplasmic reticulum, these ceils also possess caffeine-sensitive stores, which are likely the calciosome. Results of another study 13, in which Ca 2÷ release from permeabilized bovine cells was measured, suggested that these Ca 2+ stores did not overlap, since the effect of caffeine but not of IP3 was blocked by ryanodine, a blocker of Ca 2+induced Ca 2÷ release (CICR) on skeletal 6'14 and smooth muscle s. On the other hand, in bovine cells, the caffeineinduced rise in [Ca]i n became smaller after stimulation with MCh, and MCh failed to increase [Ca]in in the caffeine-treated cells 1°, suggesting the overlapping of these Ca 2÷ stores. Also, the [Ca]in transients caused by IP 3 mobilizing agonists in rat ceils pretreated with ryanodine became smaller than those in controls 11. Thus, whether caffeine- and IPa-sensitive stores reside in two separate organeUes is still unclear. We therefore investigated the relationship between two Ca 2+ stores. We took advantage of cat chromaffln cells, since we previously found that the MCh-induced [Ca]in rise and CA secre-

tion, which are due to intracellular Ca 2÷ mobilization, were much larger and more consistently obtained in cat cells that those in bovine cells 16. Our results indicate that ryanodine added with caffeine, but not with MCh, not only abolished the subsequent response to caffeine but also strongly reduced the response to MCh due to intracellular Ca 2+ mobilization. Isolated cat chromaffin cells were prepared by digestion with collagenase (Worthington, type 4) and were maintained in culture for 1-5 days on collagen-coated glass coverslips, as described previously 16. The cells were loaded with 1.5/zM fura-2 acetoxymethyl ester (fura-2/ AM) for 60 rain at 37°C and [Ca]i n was measured by dual excitation (340/380 nm) microfluorimetry, using a CAM200 spectrofluorimeter (Jasco, Japan) 15. Cells on glass coverslips were continuously superfused at 0.5-1 ml/min with a standard or test solution at room temperature (25-27°C). The standard solution was composed of NaC1 150 mM, KC1 5 mM, CaC12 2 mM, MgC12 1 raM, HEPES 10 mM, and glucose 5.5 mM (pH was adjusted to 7.4 by adding Tris base). [Ca]in was calculated by using a formula described previously 7. For measurements of CA secretion, cat adrenals were retrogradely perfused through the adrenal vein s. Since collagenase treatment of cat adrenals for 50 min had little effect on secretory responses to nicotine, MCh, high K +, and caffeine, enzyme treatment was omitted in the secretion studies. CA in the effluents was determined by the trihydroxyindole method 1, without the intermediate alumina absorption procedure, and total CA was obtained by reading fluo-

Correspondence: M. Sorimachi, Department of Physiology, Kagoshima University, School of Medicine, Kagoshima 890, Japan.

155 rescence intensity at 4001495 nm (excitation/emission), a condition under which equimolar concentrations of adrenaline and noradrenaline produced the same intensity. Caffeine (10-40 mM) caused a transient rise in [Ca]i n in the presence and absence of external Ca 2+. Although the peak value of the [Ca]in transient was comparable under the two conditions in many cells, [Ca]in returned to its basal value somewhat more slowly in the presence of Ca 2+ than in its absence. The application of caffeine (20 mM) in the presence of ryanodine (10/zM) caused a [Ca]i n transient, but then the basal [Ca]in reached a state higher than its initial value (Fig. 1, upper and lower panels). Ryanodine-treated cells became completely unresponsive to subsequent caffeine stimulation. These resuits are consistent with previous observations that, in skeletal muscle sarcoplasmic reticulum, ryanodine locks CICR channels 6'14 open, and also suggest an important role of this Ca 2+ stores in buffering [Ca]in4. The MCh (50 #M)-induced rise in [Ca]i n in the absence of Ca 2+ was also markedly reduced in ten cells (Fig. 1, lower panel), while substantial increases in [Call n were still observed in four other cells (Fig. 1, upper panel). The persistence of the MCh-induced response, even though it was reduced, may rule out the possibility that the caffeine-induced [Call n rise indirectly activated IP3-sensitive Ca 2+ stores via the activation of phospholipase C. In contrast, the

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MCh-induced [Ca]in rise in the presence of Ca 2+ in these cells was little affected by ryanodine treatment (Fig. 1, upper and lower panels; n = 23). CA secretion induced by MCh at 2 mM Ca 2+ was similarly unaffected by ryanodine treatment; 0.65 + 0.25 /~g/5 rain (mean + S.E.M.; n = 5) of CA was released with 2-rain MCh stimulation after ryanodine treatment, as compared to 0.83 + 0.27/zg/5 rain with the first control stimulation. These results suggest that MCh-induced CA secretion in the presence of Ca 2+ is mainly due to increased Ca 2+ influx. To further assess the inhibitory effect of ryanodine plus caffeine on the MCh-induced Ca 2+ release, we next investigated the effects of this agent on the [Ca]in rise and CA secretion induced by MCh during prolonged high K + depolarization. We have previously shown that both responses to MCh during depolarization result mainly from intracellular Ca 2+ mobilization 16. Application of caffeine or MCh, in each case repeated at least three times during depolarization, consistently caused transient rises in [Ca]in as well as in CA secretion. It is of interest to note that the secretory response to caffeine during depolarization was much larger than that under the non-depolarized conditions, supporting a previous suggestion that intracellular Ca 2+ mobilization becomes a strong stimulus for increasing secretion when the [Ca]in elevated above the basal level was maintained by con-

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Fig. 1. Effects of ryanodine (ryano) added with caffeine (Car) on the [Ca]in rise induced by subsequent appfication of caffeine or MCh in the presence and absence of external Ca2+. In the presence of Ca 2+, ryanodine completely abolished the response to caffeine, without affecting the response to MCh. Two representative responses to MCh in the absence of external Ca 2+ are shown in the upper and lower panels. Note that after ryanodine treatment, the basal [Ca]i. reached a new state which is higher than its initial level.

156 tinuous Ca 2+ influx 16. Application of caffeine together with ryanodine during depolarization completely abolished the [Ca]i . rise (Fig. 2, upper panel; n = 20) and C A secretion due to subsequent addition of caffeine (Fig. 2, lower panel; n = 4). The MCh-induced rise in [Ca]i . was similarly abolished in 15 cells, but in 5 other cells small responses were still observed (Fig. 2, upper panel); the peak rise above the prestimulation value was 27 + 12 nM (n = 20) after ryanodine treatment, as compared to

346 + 59 nM in control response. Consistent with these results, MCh-induced C A secretion from the depolarized gland was markedly reduced, but a small increase was still observed (Fig. 2, lower panel). These results raise two possibilities. One is that a major part of the Ca 2+ stores is sensitive to both caffeine and IP3, while a smaller part is sensitive to only IP 3. The other is that the caffeine- and IP3-sensitive stores are non-overlapping, but the MCh-induced response was me-

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