Role of Protein Kinase C in Catecholamine Secretion from Digitonin-permeabilized Bovine Adrenal Medullary Cells*

The effects of staurosporine and K-252a, potent in- hibitors of protein kinases, and 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on catecholamine secretion and protein phosphorylation in digitonin-permeabil-ized bovine adrenal medullary cells were investigated. Staurosporine and K-252a (0.01-10 p ~ ) did not cause large changes in catecholamine secretion evoked by Caz+ in digitonin-permeabilized cells whereas these compounds strongly prevented TPA-induced enhance- ment of catecholamine secretion in a concentration-dependent manner. Incubation of digitonin-permeabil-ized cells with [y3’P]ATP resulted in 32Pi incorpora- tion into a large number of proteins, detected as several major bands and darkened background in autoradi- ograms. Ca2+ and TPA increased phosphorylation of these proteins. Staurosporine and K-252a markedly inhibited Ca2+-induced and TPA-induced increases in protein phosphorylation as well as basal (0 Ca2+) pro- tein phosphorylation in digitonin-permeabilized cells. Long term treatment (24 h) of adrenal medullary cells with 1 PM TPA markedly decreased total cellular protein kinase C activity to about 5.3% of control. Pre- treatment of the cells with 1 p~ TPA strongly inhibited the TPA-induced enhancement scintillation and 10 PM K-252a). Incubations were stopped by replacing the medium with SDS buffer. Samples were subjected to SDS-polyacryl-amide (7515% gradient) gel electrophoresis and autoradiography as described under “Experimental Procedures.” by a suppression of protein phosphorylation.

Role of Protein Kinase C in Catecholamine Secretion from Digitoninpermeabilized Bovine Adrenal Medullary Cells* (Received for publication, April 15, 1991) Minoru IsosakiS, Toshikatsu Nakashima, and Yutaka Kurogochi From the Department of Pharmacology, Nara Medical Uniuersity, Kashihara 634, Japan The effects of staurosporine and K-252a, potent inhibitors of protein kinases, and 12-0-tetradecanoylphorbol-13-acetate (TPA) on catecholamine secretion and protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells were investigated. Staurosporine and K-252a (0.01-10 p~) did not cause large changes in catecholamine secretion evoked by Caz+ in digitonin-permeabilized cells whereas these compounds strongly prevented TPA-induced enhancement of catecholamine secretion in a concentrationdependent manner. Incubation of digitonin-permeabilized cells with [y3'P]ATP resulted in 32Pi incorporation into a large number of proteins, detected as several major bands and darkened background in autoradiograms. Ca2+ and TPA increased phosphorylation of these proteins. Staurosporine and K-252a markedly inhibited Ca2+-induced and TPA-induced increases in protein phosphorylation as well as basal (0 Ca2+) protein phosphorylation in digitonin-permeabilized cells. Long term treatment (24 h) of adrenal medullary cells with 1 PM TPA markedly decreased total cellular protein kinase C activity to about 5.3% of control. Pretreatment of the cells with 1 p~ TPA strongly inhibited the TPA-induced enhancement of catecholamine secretion whereas it did not cause large changes in total cellular catecholamine amounts, Caz'-induced catecholamine secretion, and CAMP-induced enhancement of catecholamine secretion from digitonin-permeabilized cells. From these results we conclude that protein kinase C plays a modulatory role in catecholamine secretion rather than being essential for initiating catecholamine secretion.
Stimulation of nicotinic acetylcholine receptors in bovine adrenal medullary cells causes an influx of extracellular Caz+ into the cells and a rise in cytosolic Ca2+ concentration, and it results in exocytotic catecholamine secretion (1,2). However, little is known about the details of the biochemical mechanisms by which intracellular Ca2+ causes exocytotic catecholamine secretion. An increase in cytosolic free Ca2+ concentration leads to phosphorylation of many proteins in adrenal medullary cells (3, 4). Because protein phosphorylation seems to occur prior to catecholamine secretion it has been proposed that protein phosphorylation may be a prerequisite for catecholamine secretion. Furthermore, several lines of evidence indicate that Ca2+-activated, phospholipid-dependent protein kinase C is intimately involved in the process * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be sent: Dept. of Pharmacology, Nara Medical University, Kashihara 634, Japan. of exocytotic catecholamine secretion (4)(5)(6)(7)(8)(9)(10)(11). Lee and Holz examined extensively the effects of various phorbol ester analogs on protein phosphorylation and catecholamine secretion, and they reported that certain phorbol esters, which could substitute for diacylglycerol and similarly increased the affinity of protein kinase C for Ca2+ i n vitro, also enhanced protein phosphorylation and Ca2+-induced catecholamine secretion in adrenal medullary cells (4). However, it is still controversial whether protein kinase C is essential for initiating exocytotic catecholamine secretion (8)(9)(10)(11). Trypsin inhibits a phorbol ester-induced increase in catecholamine secretion without any effect on primary secretion (10). On the other hand, various protein kinase C inhibitors depress both primary secretion and a phorbol ester-induced increase in secretion (8).
Staurosporine and K-252a' have been recently reported to be novel and potent inhibitors of protein kinases (12,13). These compounds nonselectively inhibit protein kinase C, myosin light chain kinase, CAMP-dependent protein kinase, and cGMP-dependent protein kinase at nanomolar concentrations i n vitro. In the present study we examined the effects of staurosporine, K-252a, and TPA on catecholamine secretion and protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells to elucidate the role of protein kinase C in catecholamine secretion.

EXPERIMENTAL PROCEDURES
Culture ofAdrenal Medullary Cells-Bovine adrenal medullary cells were isolated by sequential digestion of adrenal medullary slices with collagenase as reported previously (14), purified by differential plating (15), and maintained as monolayer cultures in Eagle's minimal essential medium containing 10% heat-inactivated calf serum, 100 units/ ml penicillin, 100 pg/ml streptomycin, 0.3 pg/ml amphotericin B, and 10 p~ cytosine arabinoside. The cells were plated a t a density of 5 X lo5 cells/well in 24-well cluster plates and 1.5 X IO5 cells/well in 96well cluster plates. For the experiments of down-regulation of protein kinase C 3-day-cultured cells were incubated for 24 h in the presence of TPA or vehicle (Me,SO).
Catecholamine Secretion from Permeabilized Cells-The culture cells were washed with 0.5 ml of permeabilizing medium consisting of 150 mM monosodium glutamate and 10 mM PIPES (pH 7.0) and then permeabilized for 6 min with 0.5 ml of permeabilizing medium containing 10 p~ digitonin and 1 mM EGTA. The permeabilized cells were then stimulated for the indicated time in 0.5 ml of permeabilizing medium containing 2 mM MgATP, 5 mM EGTA, various amounts of CaCI,, and vehicle (Me,SO) or test compounds. Free Ca2+ concentrations in the medium were measured with a Ca'+-sensitive electrode constructed using the neutral ligand ETH 1001 (16). In some cases, with 0.5 ml of permeabilizing medium containing Me2S0 or test compounds and then stimulated for 10 min with 0.5 ml of permeabilizing medium containing 10 p~ digitonin, 2 mM MgATP, various concentrations of free Ca2+, and Me,SO or test compounds. This medium was then removed and centrifuged at 700 X g for 5 min and the supernatant assayed for catecholamines. Catecholamines were determined by the ethylenediamine condensation method (17). The experiments were performed at room temperature, between 23 and 28 "C. Protein .5 mM Tris-HC1 (pH 6.81, and 10 pg/ml bromphenol blue. Samples were incubated at 100 "C for 5 min and then analyzed for phosphoprotein by SDSpolyacrylamide (7.5-15% gradient) slab gel electrophoresis in the buffer system of Laemmli (18). Gels were stained with Coomassie Brilliant Blue R, dried, and exposed to Kodak X-OMAT AR-2 film preexposed to Ahd0 = about 0.1 above the absorbance of unexposed film. The relative intensity of each band was quantitated by densitometric tracing of the autoradiograms at 430 nm using a Shimazu chromatogram scanner model CS-930. The molecular marker proteins used were a-lactoalhumin (14,400), soybean trypsin inhibitor (20,100), carbonic anhydrase (30,000), ovalbumin (43,000), bovine serum albumin (67,000), and phosphorylase b (94,000).
Protein mM EGTA, or 5 mM EGTA-calcium, 2 mM dithiothreitol, 0.02% Triton X-100, and 10 pg/ml leupeptin. After a 10-min incubation at 37 "C the reaction was stopped by the addition of 10 pl of 10 mM ATP and 100 mM EDTA. 50-p1 aliquots were then dropped onto 2.2 X 1.2-cm phosphocellulose paper (Whatman P81), which was washed five times with water. The radioactivity on each piece of paper was determined by scintillation counting. The reaction was linear with respect to time and enzyme concentration under the conditions used. Because protein kinase C activity was maximal with about 10 nM TPA and did not largely change at around 100 nM TPA used in assay, protein kinase C activity in the samples is not dependent on TPA treatment of the cells.
Materials-Staurosporine and K-252a were purchased from Kyowa Medex Co. ETH 1001 was from Fluka Chemie AG. Other chemicals and materials were obtained from commercial sources.

RESULTS
Effects of Staurosporine and K-252a on Catecholamine Secretion-TPA activates protein kinase C, which is the only known cellular receptor for this compound (19,20) and therefore has been used widely as a means of testing whether protein kinase C is involved in cellular events. TPA (1-1,000 nM) increased catecholamine secretion evoked by Ca2+ in digitonin-permeabilized bovine adrenal medullary cells in a concentration-dependent manner but had little effect on basal (0 Ca'+) catecholamine secretion. Enhancement of Ca2+-dependent catecholamine secretion was almost maximal with 100 nM TPA (data not shown). On the other hand, 4-aphorbol 12,13-didecanoate, inactive phorbol ester on protein kinase C, had little effect on catecholamine secretion. These results are in good agreement with the reports that protein kinase C is intimately involved in catecholamine secretion from digitonin-permeabilized cells (4,6,7,(9)(10)(11). Fig. 1 shows the effects of various concentrations of staurosporine and K-252a on Ca2+-and TPA-dependent catecholamine secretion from digitonin-permeabilized adrenal medullary cells. Staurosporine (0.01-10 PM) caused a little inhibition on catecholamine secretion evoked by 0.95 FM Ca2+ whereas this compound at the same range of concentrations strongly prevented TPA-induced enhancement of catecholamine secretion. Inhibition of TPA-induced enhancement of catecholamine secretion was detectable with as little as 48 nM staurosporine and was half-maximal with about 0.48 PM. Almost complete inhibition was observed with 2.2 PM staurosporine. K-252a had little effect on Ca2+-induced catecholamine secretion but inhibited TPA-induced enhancement of catecholamine secretion in a concentration-dependent manner (IC,,,, about 1 PM). Moreover, staurosporine and K-252a did not cause large changes in catecholamine secretion induced by 2.1 PM Ca2+, comparable to that by 0.95 PM Ca2+ plus 100 nM TPA (Fig.  1). Therefore, the possibility that staurosporine and K-252a solely inhibit more vigorous exocytosis is unlikely. Fig. 2A shows staurosporine on the Ca'+ dose response for catecholamine secretion. The preincubation of the cells with TPA resulted in a larger increase in Ca2+-dependent catecholamine secretion than was the case without the preincubation with TPA.
This result is consistent with the report that active phorbol esters inhibit the release of protein kinase C molecules from the digitonin-permeabilized cells by increasing the percentage of membrane-bound protein kinase C (21). On the other hand, there was virtually no difference in the effects of staurosporine and K-252a on Caz+-induced and TPA-induced catecholamine secretion from the digitonin-permeabilized cells with and without the preincubation with these compounds. Staurosporine (3 p~) had only a small inhibitory effect on the Ca2+ dose-response relation for catecholamine secretion. It is unclear whether the inhibitory effect of staurosporine on Ca2+induced catecholamine secretion reflects a small role for protein kinase C in Ca2+-induced catecholamine secretion in the absence of phorbol ester or whether the compound has a nonspecific deleterious effect on the cells. Staurosporine at the same concentration almost completely inhibited the TPAinduced enhancement of catecholamine secretion in response to various concentrations of Ca2+. Preincubation with 10 p~ K-252a following the protocol in Fig. 2C resulted in a small increase in Ca2+-evoked catecholamine secretion and a slight leftward shift of the Ca2+ dose-response curve (data not shown). This slight increase in catecholamine secretion by the preincubation with K-252a was not explored. K-252a in this case also inhibited the TPA-induced enhancement of catecholamine secretion.
Effects of Staurosporine and K-252a on Protein Phosphorylation-We examined the effects of staurosporine and K-252a on protein phosphorylation in digitonin-permeabilized bovine adrenal medullary cells to investigate whether the inhibitory effects of staurosporine and K-252a on TPA-induced enhancement of catecholamine secretion are caused by a suppression of protein phosphorylation. Incubation of digitonin-permeabilized cells with permeabilizing medium containing 2 mM [y-32P]ATP-magnesium, and 5 mM EGTA resulted in 32Pi incorporation into a large number of proteins, detected as several major bands of 100,89,59, 39,26, 18, and 16 kDa and darkened background in autoradiograms (Fig. 3). The intense band at 59 kDa has been characterized as tyrosine hydroxylase (3,7), the rate-limiting enzyme in catecholamine biosynthesis. An increase in the Ca2+ concentration resulted in enhanced phosphorylation of these proteins. Staurosporine (3 p~) and K-252a (10 p~) inhibited basal and Ca2+-induced phosphorylation of these proteins although the degree of inhibition depends on each protein. These results are in good agreement with the experiments that staurosporine and K-252a nonselectively inhibit various protein kinases in vitro (12,13).
TPA (100 nM) caused a small but significant increase in protein phosphorylation (Fig. 3). Preincubation of intact cells with 100 nM TPA for 15 min before permeabilization resulted in a marked increase in phosphorylation of many proteins (Fig. 4). Staurosporine (3 p~) and K-252a (10 p~) inhibited not only basal and Ca2+-induced protein phosphorylation but also TPA-induced protein phosphorylation. The relative intensity of the bands was quantitated by densitometric tracing of the autoradiograms at 430 nm using a Shimazu chromatogram scanner model CS-930. The TPA-induced increase in the intensity of the band at 59 kDa was 66 f 12%, 23 f 7%, and 5 f 4% (means +-S.E. of three experiments) at 0.1, 1.0, and 10 pM staurosporine, respectively, and 78 f ll%, 42 f 15%, and 8 f 5% at 0.1, 1.0, and 10 p~ K-252a, respectively. These dose-response curves are similar to the dose-response curves of these compounds on the TPA-induced increase in catecholamine secretion but not to those on Ca'+-induced catecholamine secretion in the absence of TPA (Fig. 1). The results suggest that selective inhibition of staurosporine and Incubations were stopped by replacing the medium with SDS buffer. Samples were subjected to SDS-polyacrylamide ( 7 5 1 5 % gradient) gel electrophoresis and autoradiography as described under "Experimental Procedures." K-252a on TPA-induced enhancement of catecholamine secretion is caused by a suppression of protein phosphorylation.
Effect of Long Term Treatment of TPA-In many cell types prolonged protein kinase C stimulation with active phorbol esters leads to down-regulation of protein kinase C with a loss of enzyme activity and the protein itself as detected by immunoprecipitation and binding of ['Hlphorbol 12,13-dibutyrate (9,22,23). However, it is still controversial whether downregulation of protein kinase C results in a decrease in primary secretion of catecholamine in bovine adrenal medullary cells (9,11). We also studied the effect of the long term treatment of TPA on catecholamine secretion and protein kinase C activity in bovine adrenal medullary cells (Fig. 5). 24-h pretreatment of the cells with 1 p~ TPA markedly decreased total cellular protein kinase C activity from a control level of cretion. Our experiments on the down-regulation of protein kinase C are in good agreement with the conclusion that protein kinase C is not essential for primary catecholamine secretion.

DISCUSSION
Effects of a variety of protein kinase C inhibitors on catecholamine secretion from electropermeabilized bovine adrenal medullary cells have already been reported (8).
All protein kinase C inhibitors tested inhibited Ca2+-evoked catecholamine secretion as well as TPA-induced enhancement of catecholamine secretion from the electropermeabilized cells. The discrepancy between the previous report and our results probably reflects the difference in specificity of the inhibitors tested. Staurosporine and K-252a are nonselective inhibitors of various protein kinases. However, staurosporine and K-252a inhibit protein kinase C with Ki values of as low as 3 and 25 nM, respectively, in vitro (12,13). These values are approximately 2 or 3 orders of magnitude lower than those of the protein kinase C inhibitors that were tested previously. From our results it seems likely that staurosporine and K-252a specifically inhibit TPA-dependent regulatory process(es) of the membrane events (membrane fusion and fission) in exocytosis but not primary membrane event(s) itself. The effects of staurosporine and K-252a on Ca2+-activated protein kinase C without TPA or diacylglycerol have not been reported. The possibility that Ca2+-activated protein kinase C without TPA might be less sensitive to drug inhibition than is the case with Ca2+-activated protein kinase C with TPA is possible. However, because protein kinase C cannot be activated without TPA or diacylglycerol at physiological concentrations of Ca'+ (24), such a possibility cannot account for our results.
Protein kinase C is a family of at least seven subspecies having Ca2+-dependent types (a, PI, f i 2 , and y) and Ca2+independent types (6, 6, and {) (25). At present, the distribution of these protein kinase C subspecies in bovine adrenal medullary cells and their relevance to catecholamine secretion have not been reported. However, Ca2+-independent protein kinase C subspecies should not be a "Ca2+ receptor" which is responsible for primary catecholamine secretion. Moreover, because TPA itself cannot induce Catecholamine secretion without Ca", Ca2+-independent subspecies are not putative intermediate between Ca2+ receptor and exocytosis. K-252a had little effect on Ca2+-evoked catecholamine secretion from digitonin-permeabilized adrenal medullary cells (Figs. 1 and 2). Staurosporine showed only a little inhibition of Ca2+-induced catecholamine secretion. However, these compounds markedly inhibited phosphorylation of many proteins (Figs. 3 and 4). It has been reported that an increase in protein phosphorylation may be essential for initiating cate-cholamine secretion (3). From our results it seems unlikely that Ca2+-induced phosphorylation of these proteins is a prerequisite for catecholamine secretion. However, further experiments are required to determine exactly the relationship between the initiation of catecholamine secretion and phosphorylation of each protein.
K-252a was reported to inhibit concomitantly serotonin release and phosphorylation of proteins of 20 and 40 kDa without a significant effect on the rise of intracellular free Ca2+ induced by platelet-activating factor, suggesting that phosphorylation of these proteins may be a prerequisite for serotonin release in platelets (26). In our work K-252a inhibited phosphorylation of many proteins in digitonin-permeabilized bovine adrenal medullary cells but did not inhibit catecholamine secretion evoked by Ca2+ (Figs. 1-4). Although we have no definitive explanation for this discrepancy at present it seems conceivable that it is because of a difference in the regulatory systems of exocytosis. One of the most striking differences of Ca2+-dependent exocytotic release between platelets and adrenal medullary cells is its sensitivity to phorbol ester. TPA has been shown to cause a much larger increase in the apparent affinity of exocytosis for Ca2+ in electropermeable platelets than that in electropermeable adrenal medullary cells (27).
We conclude that protein kinase C plays a modulatory role in catecholamine secretion rather than being essential for initiating catecholamine secretion in bovine adrenal medullary cells.