0013-7227/92/1304-2230$03.00/O Endocrinology Copyright 0 1992 by The Endocrine
Vol. 130, No. 4 Society
Printed
The Role of Protein Kinase-C in Control Production by Rat Adrenal Glomerulosa Activation of Protein Kinase-C by Stimulation with Potassium* GYORGY ANDRAS
HAJNOCZKY, SPAT
PfiTER
VARNAI,
LliSZL6
BUDAY,
ANNA
in U.S.A.
of Aldosterone Cells:
FARAGO,
AND
Department of Physiology and First Department of Biochemistry (L.B., A.F.), SemmelweisUniversity Medical School,Budapest,Hungary
ABSTRACT. The role of protein kinase-C (PKC) in control of the function of rat adrenal glomerulosa cells was studied. Phorbol 12-mvristate ll-acetate (PMA). an activator of PKC. inhibited the itimulation of aldosmrone ‘production induced by K+ (6.4 mM) or ACTH (5 PM) in a dose-dependent manner. Phorbol 12,13dibutyrate, another phorbol ester that activates PKC, also exerted an inhibitory effect, whiie the inactive 4~ phorbol 12,13didecanoate failed to affect aldosterone production. The inhibitory effect of PMA (5 nM) was reversed by preincubation of the cells with staurosporine (ST, 50 nM), an inhibitor of PKC. These data suggest that pharmacological activation of PKC initiates an inhibitory mechanism in rat glomendosa cells. To elucidate whether PKC is activated by physiological stimuli. the effects of ST and down-reeulation of PKC bv nroloneed pretreatment with PMA on stimulation of aldosterone prod&tion were studied. The effects of angiotensin-II (AK) and K+, but not that of ACTH, were enhanced by ST pretreatment. This potentiation was prompt and transient in the case of AI1 (2.5 nM), while it developed gradually when the cells were stimulated
T
with K+ (5.4 or 18 mM). Long term pretreatment (6 h) of glomerulosa cells with PMA also enhanced the stimulatory effect of AI1 (396 PM) and K+ (5.4 mM). These data together suggest that the actions of AI1 and K+ on aldosterone production involve a PKC-mediated inhibition. Activation of PKC by AI1 is probably due to formation of diacylglycerol via receptor-mediated activation of phosphoinositide-specific phospholipase-C. Stimulation with K+ caused a moderate accumulation of [3H]inositol phosphate in a concentration-dependent manner. Since this effect was abolished by nifedipine, activation of phospholipase-C may have been secondary to Cal+ entry. The concomitant formation of diacylglycerol may contribute to activation of PKC in K+ stimulated cells. In conclusion, our data support the view that PKC participates in the physiological control of aldosterone production by rat adrenal domerulosa cells. In addition to AII. K+ mav activate PKC. Rega&lless of whether the enzyme is activated by phorbol esters or physiological stimuli, it exerts an inhibitory, rather than stimulatory, action on steroid production. (Endocrinology 130: 2230-2236,1992)
HE ENZYMES of the protein kinase-C (PKC) family are well known to participate in the control of several cellular processes (see references in Refs. l-4). Data reported on the role of PKC in adrenal glomerulosa cells are controversial. In bovine and human cells, pharmacological activation of PKC by phorbol esters induced a slowly developing increase in aldosterone production (5-7), which was potentiated by coadministration of Ca2+ ionophores ($6). In isolated rat glomerulosa cells, phorbol esters evoked the translocation of PKC (8, 9), yet phorbol 12-myristate 13-acetate (PMA) failed to stimu-
late aldosterone production (9-11). Nevertheless, phorbol esters modified hormone production induced by angiotensin-II (AII), ACTH (9), or vasopressin (12), suggesting that PKC may be involved in the control of aldosterone production. The aim of the present study was to clarify the effect of PKC activation on the secretory response to the major physiological stimuli (K+, AII, and ACTH). We also wanted to elucidate whether stimuli other than AI1 can also activate PKC in rat glomerulosa cells.
Received October 15, 1991. Address all correspondence and requests for reprints to: Dr. And& Spat, Department of Physiology, Semmelweis University Medical School, H-1444, P.O. Box 259, Budapest 8, Hungary. *This work was supported by Grant 1111 from the Hungarian National Science Foundation and Grant T-176/1990 from the Hungarian Council of Medical Sciences.
Materials
Materials
and Methods
AI1 (Ile5-AII) was obtained from Serva (Heidelberg, FRG), ACTH-( l-24) (Synacthen) from Ciba-Geigy (Basel, Switzerland), phorbol estersfrom Sigma (St. Louis, MO), staurosporine (ST) from Calbiochem (La Jolla, CA), proteaseinhibitors
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ACTIVATION
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from Sigma, myo-[2-3H]inositol (16.6 Ci/mmol) from DuPontNew England Nuclear (Boston, MA), [32P]ATP (10 Ci/mmol) from Izinta (Budapest, Hungary), and medium 199 (with Earle’s salts) from Gibco (Grand Island, NY). Thapsigargin was kindly provided by S. B. Christensen (Copenhagen, Denmark) All other chemicals were of analytical grade. Cell isolation and aklosterone experiments Adrenal glomerulosa cells were prepared from capsular tissues of male Wistar rats (200-300 g) using collagenase and mechanical dispersion, as described previously (13). The viability of the cells was examined by a trypan blue exclusion test; usually less than 5% of the cells were trypan blue positive. Cells were preincubated for 3 h in a mixture of modified KrebsRinger bicarbonate-glucose solution and medium 199 (2:1, vol/ vob final concentrations: Na, 145 mM; K, 3.6 mM; Ca, 1.2 mM; and Mg, 0.5 mM) supplemented with human serum albumin (fraction V; 2 g/liter) at 37 C under a mixture of 95% OS-5% CO* (pH 7.4). When cells were pretreated for 6 h with phorbol esters, the medium also contained gentamycin (100 mg/liter) and cefalexin (50 mg/liter). Then, the cells were washed and incubated (-1.5 x 10’ cells/500 ~1) for 60 or 120 min under identical conditions. The aldosterone content of the cell suspension was measured by RIA (14). Assays were performed in triplicate. Mean @EM) basal aldosterone production was 17.1 f 3.2 pmol/lO’ cells-h (n = 3). Cell superfusion was carried out as previously described (15). Briefly, isolated cells (-lo6 cells/column) were mixed with 600 ~1 preswollen Bio-Gel P2 resin and loaded onto small columns prepared from 2.0-ml syringes. Cells on the column were superfused with a flow rate of 8.0-10.0 ml/h using the medium detailed above. Effluent fractions were collected on ice, and their aldosterone concentration was measured by RIA (14). Measurement of form&on
of inositol phosphates
Experiments on the formation of inositol phosphates were performed as described previously (16). The labeling period (3 h in the presence of 15 &i/ml [3H]inositol) was followed by a 15-min stimulation in the presence of LiCl (10 mM). Inositol phosphates were separated by anion exchange chromatography (17). Assays were performed in duplicate. The control values were 10,619 f 2,815,822 + 272, and 183 + 70 cpm/106 cells for inositol monophosphates, bisphosphates, and trisphosphates, respectively (mean f SEM; n = 3) Measurement of PKC activity Cells (-1 million/500 ~1) were incubated in the above-described medium supplemented with gentamycin and cefalexin in the presence or absence of PMA at 37 C. After incubation, the cells were centrifuged at 100 x g, then suspended in 1 ml of a medium (lysing medium) containing 250 mM sucrose, 5 mM EGTA, 2 mM EDTA, 0.002% leupeptin, 1 mM benzamidine, 2 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, and 20 mM Tris-HCl (pH 7.4). The suspension was sonicated in an ice bath and centrifuged at 100,000 x g for 30 min. The supernatant was referred to as the cytosolic fraction. The pellet was resuspended in 1 ml lysing medium containing 0.02% Triton X-100. The suspension was incubated at 0 C for 60 min
2231
and then centrifuged at 40,000 x g for 15 min. The supernatant obtained by this procedure was referred to as the membrane fraction. Estimation of the activity of PKC was based on [32P]phosphate incorporation into the nonapeptide substrate Ala-AlaAla-Ser-Phe-Lys-Ala-Lys-Lys-amide, as previously described (8). Briefly, the assay was carried out in a mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgC12, 1.2 mM EGTA, 0.01 mM [32P]ATP (-lo6 cpm/400 rl), and the synthetic nonapeptide (0.75 mM). Activation of PKC was induced by phosphatidylserine (20 pg/ml), CaCl, (2.5 mM), and PMA (40 nM). The reaction (10 min; 37 C) was terminated by adding glacial acetic acid, and the peptide was separated on a phosphocellulose column (8). All assays were performed in triplicate. PKC activity was calculated as the difference in the 32P activity of matched samples with and without the nonapeptide and activators (phosphatydilserine, PMA, and Ca”). Peptide kinase activity was 26.3 f 5.4 pmol phosphate/min. lo6 cells in the cytosolic fraction and 9.7 f 3.4 pmol/min- lo6 cells in the particulate fraction (mean f SEM; n = 3) Statistics The mean + SEM are shown in the aldosterone and inositol phosphate experiments. The effects of PMA and ST on aldosterone production were tested with paired sample t test. The significance of the interaction between the effects of stimuli and down-regulation of PKC on aldosterone production was estimated by three-way analysis of variance.
Results Effect of short term incubation aldosterone production
with phorbol esters on
First, the interaction of PMA with three physiological stimuli of glomerulosa cells was examined. The tested stimuli have different modes of action; potassium ions induce membrane depolarization with consecutive Ca*+ entry, the effect of AI1 is mediated by Ca*+ mobilization and Ca*’ entry, while CAMP is the second messenger responsible for the effect of ACTH. PMA (50 nM) did not significantly affect basal aldosterone production (lo), but inhibited the aldosterone response to K+ (elevated from 3.6 to 5.4 mM; Fig. 1, left panel; P < 0.01; n = 4) and ACTH (5 pM; Fig. 1, middle panel; P < 0.01; n = 5) in a cell superfusion system. The ratio of aldosterone output in the presence vs. the absence of PMA (upper panels) shows the sustained duration of inhibition. A similarly sustained inhibition by PMA was observed when aldosterone production was stimulated by the microsomal Ca*+-ATPase inhibitor thapsigargin (0.6 PM), known to evoke a cytoplasmic Ca*+ signal similar to that of AI1 (16) (data not shown). Yet, the aldosterone-stimulating action of AI1 (300 PM) was only slightly and transiently decreased by PMA (for the 15- to 30-min period of stimulation, P < 0.1; n = 4), and the later period of stimulation was not at all affected by the drug
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F’IG. 1. Effect of PMA on aldosterone production stimulated by K+, ACTH, and AII. Aldosterone production by superfused glomeruloaa cells was followed. After a 2-h control period, 50 nM PMA (closed symbols on lower pan&) or solvent (open symbols on lower panels) was added for 15 min. Then, agonists were added, PMA or its solvent was also present throughout the 2-h stimulation. Stimuli were K+ (5.4 mM; left panel; n = 4), ACTH (5 PM; middle panel; n = 5), and AI1 (300 PM; right panel; n = 4). The lower panels show aldosterone production in picomoles per ml for l@ cells. Ratios of agonist-induced aldosterone production in the presence us. the absence of PMA are shown on the upper panels (mean + SEM).
(Fig. 1, r&tpaneZ). The dose-response curve of inhibition of the K+ (5.4 mM)-induced aldosterone production by PMA was determined in static incubations (Fig. 2). Half-maximal inhibition was attained at about 5 nM, and the highest PMA concentration applied (500 nM) completely inhibited the effect of K+. The specificity of PMA’s effects was first examined by the application of other phorbol esters. Phorbol 12,13dibutyrate (PDBu; 50 nM), another activator of PKC, exerted a similar inhibitory effect on K+-stimulated aldosterone production (Fig. 3, upper panel; P c 0.05; n = 4). In contrast to this, ricu-phorbol 12,13didecanoate (4aPDD; 50 nM), which is not able to stimulate PKC, did not influence the effect of K+ (Fig. 3, lower panel). Effect of ST on aldosterone production PMA (5 nM) inhibited K+-induced aldosterone production when it was added subsequently to K+ (5.4 mM; Fig. 4). Preincubation of the cells with ST, an inhibitor of PKC (18), prevented the effect of PMA (Fig. 4). Figure 4 also shows that the stimulatory action of 5.4 mM K+ was enhanced by coadministration of ST (for the second hour of stimulation, P < 0.01; n = 5).
Possible modification of the effects of K+, ACTH, and AI1 by ST was also tested using supramaximal concentrations of these agonists. The effect of 18 mM K+ was enhanced by ST (Fig. 5,leftpanel; P < 0.05; n = 4). The stimulatory effect of ST developed gradually at both K+ concentrations (5.4 and 18 mM), and it was higher for 18 InM K+. ACTH (5 n&&induced aldosterone output was not significantly affected by ST (Fig. 5, middle panel), and the effect of AI1 (2.5 nM) was only transiently enhanced by ST (Fig. 5, right panel; for the first 30 min of stimulation, P < 0.02; n = 6). Effect of long term incubation with PMA on PKC activity and aldosterone production Fifteen-minute treatment of glomerulosa cells with PMA (50 nM) reduced the activity of PKC in the cytosol fraction and induced a parallel increase in the membrane fraction (Fig. 6). After 6 h of incubation with PMA, both the cytosolic and membrane fractions of PKC activity were below the control level, and no further decrease was found after 24 h of PMA administration (Fig. 6). Data obtained with ST raised the possibility that PKC was activated by both K+ and AIL To elucidate the physiological significance of such an activation, we ex-
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ACTIVATION
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n
PYA 1
0.1
100 ;PYA]
1000
';nM)
2. Inhibition of potassium-stimulated aldosterone production by PMA. Cells were stimulated with K+ (5.4 mM) in the presence of different concentrations of PMA in static incubation for 1 h. Aldosterone production was expressed as a percentage of the control stimulation, i.e. in the absence of phorbol ester (mean f SEM; n = 3). Aldosterone production by unstimulated cells (3.6 mM K+) was 17.1 f 3.2 pmol/lO’ cells and that by K+-stimulated cells (5.4 mM K+) was 33.1 f 2.3 pmol/lOs cells (n = 3). hG.
*
uj
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K+6.4
'54 >
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TJ
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3. Effect of PDBu and 4aPDD on K+-stimulated. aldosterone production. Cells were preincubated with 50 nM PDBu (upper panel) or 50 nM 4aPDD (lowerpanel) for 15 min, and then the K+ concentration was increased from 3.6 to 5.4 mM in the superfusion medium. The aldosterone concentration of the effluent was measured as described in Materials and Methods, and ratios of K+-induced aldosterone production in the presence us. the absence of the phorbol ester are shown. The mean f SEM are shown for four (upper panel) or three (lower panel) separate experiments. FIG.
0
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FIG. 4. Effect of ST on PMA inhibition of aldosterone production stimulated by K+. After the 120-min control period, cells were superfused with either 50 nM ST (closed symbols) or control medium (open sytikr) for 60 min. Then, the K+ concentration was increased from 3.6 to 5.4 mM, and after 120-min stimulation, PMA (5 nM) was also added. Aldosterone production was normalized for 10’ cells (mean f SEM; n = 3). ST per se caused no changes in aldosterone production between -60 and 0 min; thus, in this period, the closed symbols are valid for both groups.
amined the effect of physiological stimuli in PKC-depleted cells. Basal aldosterone production by cells preincubated for 6 h with the inactive phorbol ester 4aPDD (50 nM) was 14.7 + 4.4 pmol/106 cel1s.h; preincubation with 50 nM PMA had no significant effect (13.1 -I- 2.8 pmol/106 cells. h; n = 6). Acute exposure to PMA (5 nM) inhibited the response to K+ (5.4 mM) by 47.8 + 6.0% in control cells (i.e. without prior exposure to PMA) and by only 10.4 f 12.3% after the cells became depleted of PKC (by long term preincubation with PMA; n = 4). The increase in l-h hormone production induced by AI1 (300 PM) was significantly higher in PKC-depleted than in control cells (11.4 f 2.0 us. 7.0 + 1.2 pmol/106 cells; for the interaction between the effects of AI1 and PKC depletion, P < 0.05; n = 6), and similarly, an increase was found in the effect of 5.4 mM K+ (24.1 + 3.7 us. 16.9 + 4.2 pmol/106 cells; for the interaction between the effects of K+ and PKC depletion, P < 0.05; n = 6). Considering the slow onset of enhancement of the effect of K+ by ST, 2-h hormone production was also measured for K+. In fact, the increase was more pronounced at 120 min than at 60 min of incubation (Fig. 7). In view of these observations, we examined whether potassium affects the activity of phospholipase-C. As an indicator of enzyme activity, the accumulation of 3Hlabeled inositol phosphates was measured at different concentrations of K+. Compared with control (3.6 InM) conditions, the formation of inositol trisphosphates in-
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FIG. 5. Effect of ST on aldosterone responses to K+, ACTH, and AK. After the control period, cells were preincubated with 50 nM ST (closed symbols) or solvent (open symbols) for 1 h. Then, stimuli were applied: K+ (18 mM; left panel; n = 4), ACTH (5 nM; middle panel; n = 3), and AI1 (2.5 nM; rig& panel; n = 6). In the lower pan.& aldosterone production is expressed as picomoles per ml for 10’ cells. In the upper panels ratios are shown that ware calculated by dividing the respective aldosterone concentrations obtained in response to stimuli in the presence vs. the absence of ST. The mean f SEM are shown.
creased by 13 + lo%, 37 + ll%, and 70 + 5% in the presence of 5.4, 8.4, and 18 mM K+, respectively (n = 3). The labeling of the inositol bisphosphate fraction increased to a similar extent, while that of the monophosphate fraction was twice as high. A possible physiological significance of these observations is shown by the significant accumulation of inositol phosphates (mono-, bis-, and tris-) in the presence of 5.4 mM K+ (122 + 5% compared to 3.6 mM; t = 4.26; n = 3) The stimulatory action of 18 mM K+ was completely abolished by nifedipine (1 PM), indicating that activation of phospholipaseC was secondary to influx of Ca2+ through voltage-dependent channels. This indirect means of activation of the enzyme may account for the small effect of K+ on the formation of inositol phosphates (mono-, bis-, and tris-) compared to that of AI1 (18 mM K+, 2.1 + O.O-fold increase; 25 nM AII, 10.8 + 1.9-fold increase; n = 3). Discussion The purpose of the present study was to elucidate the role of the enzyme family PKC in the physiological control of aldosterone production by rat adrenal glomerulosa cells. PKC enzymes are activated by diacylglycerol (DAG) in the presence of phospholipids and Ca2+ (for
review, see Refs. 1 and 19). Certain phorbol esters (PMA and PDBu) may substitute DAG in the activation of PKC (1). Considering that in bovine glomerulosa cells simultaneous administration of a Ca2+ ionophore and PMA induces aldosterone production resembling the effect of AII, it was postulated that Ca2+-mobilizing stimuli exert their effect through elevation of the cytoplasmic Ca2+ concentration and activation of PKC (20). However, PMA has no stimulatory effect on aldosterone production by isolated rat glomerulosa cells (9-11) and decreases, rather than increases, the effect of the Ca2+ ionophore ionomycin (Hajnoczky, Gy., and A. Spat, unpublished). Furthermore, the present study revealed that PMA diminished the aldosterone response to thapsigargin. Thapsigargin brings about a cytoplasmic Ca2+ signal in glomerulosa cells through mobilization of Ca2+ from intracellular stores (16) and, thus, mimics the Ca2+ branch of the AII-activated messenger mechanisms. Therefore, in contrast to bovine glomerulosa cells, in rat cells PKC does not complete the effect of Ca2+ in inducing the secretory response. However, the possible contribution of PKC to regulation of steroid production is suggested by two recent papers (9, 12). In the present study we analyzed the interaction of PKC with physiological stimuli, primarily K+, since activation of the cells
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ACTIVATION
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FIG. 6. Time-course relationship of PKC activity in glomerulosa cells treated with PMA. PKC activity was measured in the cytosol and membrane fractions of glomerulosa cells, as described in M&TF~ULS and Methods. Cells were treated with PMA (50 nM) for 15 min, 6 h, and 24 h. Results were expressed as the percentage of PKC activity in the cytosol and membrane fractions of control cells, respectively. The two symbols represent the data from two separate experiments.
PDD pretreated PYA pretreated
K+ 5.4mY:
60 min
120 min
7. Comparison of the effect of K+ on aldosterone production by glomerulosa cells after prolonged treatment with PMA and 4aPDD. Cells were preincubated with ~(YPDD (50 nM; 0) or PMA (50 nM; W) for 6 h. Then, the cells were washed, and aldosterone production was measured under static incubation conditions for 60 or 120 min. The aldosterone content of the cell suspension was normalized for lo6 cells (60 and 120 min aldosterone production at 3.6 mM K+ were 11.9 f: 2.6 and 12.4 f 3.0 pmol/lO’ cells after 4aPDD pretreatment and 11.6 f 2.2 and 13.3 f 2.1 pmol/106cells after PMA pretreatment, respectively). The mean f SEM are shown for four separate experiments. FIG.
with K+ provides the simplest model of stimulation of glomerulosa cells through the Ca2+ signal. PMA exerted a marked inhibitory effect on aldosterone production induced by a physiological elevation of the K+ concentration (from 3.6 to 5.4 IIIM). The ACTH-
2235
induced hormone response was similarly sensitive to PMA, while the response to AI1 was diminished to a small extent. The mediatory role of PKC in the effect of PMA is indicated by the following results. 1) Halfmaximal inhibition was attained at 5 nM PMA, a concentration reported to activate PKC in several cell types (3). 2) PDBu, another pharmacological activator of PKC, also inhibited K+-stimulated hormone production, while the inactive phorbol analog (~cxPDD) was ineffective. 3) ST, an inhibitor of PKC, reversed the effect of PMA. 4) Down-regulation of PKC activity in glomerulosa cells reduced the inhibitory action of PMA. These data suggest that in rat glomerulosa cells PKC may inhibit the aldosterone secretory response to the major physiological stimuli. In static incubation, 15min preincubation with PMA (100 nM) markedly inhibited the secretory response to ACTH, but not to K+ and AI1 in the experiments of Nakano et al. (9). Apart from sexual and some technical differences, the explanation for the partial discrepancy between the results of the two studies is unknown. What is the mode of action of PKC? Since PKC phosphorylates cytochrome P450, in bovine cells (21), and PMA affects the expression of several types of cytochrome P450 in human adrenocortical cells (22), the site of the action of PKC might be on the final common pathway of steroidogenesis. However, formation of CAMP by ACTH (23) and accumulation of inositol phosphates by vasopressin (12) are also sensitive to phorbol esters in rat glomerulosa cells. Inhibition of AII-induced inositol phosphate formation (24, 25) and modulation of voltage-operated Ca2+ channels by PMA (for review, see Ref. 3) were observed in several other cell types. Thus, PKC may also influence the specific signal-transducing mechanisms of the three stimuli studied. Do the physiological stimuli of glomerulosa cells activate PKC? Enhancement of AII-induced aldosterone production by inhibition or down-regulation of PKC is compatible with the view that AI1 activates PKC (9,26). On the other hand, ST did not influence the ACTHelicited steroid response; thus, we have no reason to suppose the involvement of PKC in the action of ACTH. Unexpectedly, the effect of K+ was potentiated by ST and also by depletion of PKC activity. These data suggest that PKC may be activated by K+ in rat glomerulosa cells. Since this potentiation took place when K+ was increased from 3.6 to only 5.4 mM, the effect of K+ on PKC may have physiological significance. The mode of action of K+ is stimulation of Ca2+ entry via voltage-operated Ca2+ channels. The rise in cytoplasmic Ca2+ facilitates the interaction of PKC with lipid activators (27); thus, the cytoplasmic Ca2+ signal may contribute to the activation of PKC. Phospholipase-C is also sensitive to Ca2+; thus, Ca2+ entry may enhance the generation of DAG. In fact, we observed slight but sig-
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nificant stimulation of inositol phosphate formation by K+; this phenomenon was inhibited by nifedipine. Therefore, we attribute the K+-induced activation of PKC to elevation of cytoplasmic Ca2+ and a secondary moderate enhancement of DAG generation. This explanation is in agreement with the recently proposed mechanism of activation of PKC by stimuli that act through stimulation of receptor or voltage-operated Ca2+ entry (4, 27). As an additional effect, Ca’+-induced formation of DAG from phosphatidyl choline should be considered (28). Activation of PKC by K+ in rat glomerulosa cells may also contribute to the well documented bell-shaped doseresponse curve for K+-induced aldosterone production (see references in Ref. 29). Above 8-10 InM K+, the secretory response is increasingly more short-lived (29). In the present study we found that ST prevents the decline in the aldosterone response to 18 mM K+, which suggests that PKC contributes to the transient nature of the secretory response at high K+. In accordance with this assumption are the observations that ST stimulated aldosterone production more potently at 18 than at 5.4 mM K+, and stimulated the disappearance of the aldosterone inhibitory action of PMA after 2-h stimulation more potently with 18 mM [K+] (Hajnoczky, Gy., and A. Spat, unpublished) than with 5.4 mM. In conclusion, our data suggest that PKC exerts an inhibitory role in the control of aldosterone secretion by rat glomerulosa cells. This inhibitory effect may have significance during stimulation with AI1 and potassium ions. Acknowledgments We wish to thank medical students Mr. Attila Bago and Mr. Gyiirgy Csordasfor their enthusiastic work during the experiments. The excellent technical help of Miss Erika Kovacs and Mrs. Agnes Ribir is greatly appreciated. Aldosterone antibody was a gift from the NIAMD (Bethesda,MD), and Synacthen was a gift from Ciba-Geigy (Basel, Switzerland).
References 1. Nishizuka Y 1989 The family of protein kinase C for signal transduction. JAMA 2621826-1833 2. Huang KP 1989 The mechanism of protein kinase C activation. Trends Neurosci 12:425-432 3. Shearman MS, Sekiguchi K, Nishizuka Y 1989 Modulation of ion channel activitv: a kev function of the nrotein kinase C enzvme family. Pharmacol Rev 41:211-237 4. Faragi, A, Nishizuka Y 1990 Protein kinase C in transmembrane signalling. FEBS Lett 268:350-354 5. Koiima I. Linnes H. Koiima K. Rasmussen H 1983 Aldosterone sec”retionI effect of phorbol ester and A23 187. Biochem Biophys Res Commun 116:555-562 6. Kojima I, Kojima K, Kreutter D, Rasmussen H 1984 The temporal integration of aldosterone secretory response to angiotensin II occurs via two intracellular pathways. J Biol Chem 260:4248-4256 7. Laird SN, Hinson JP, Vinson GP, Mallick N, Kapas S, Teja R 1990 Control of steroidogenesis by the calcium messenger system in human adrenocortical cells. J Mol Endocrinol6:45-51 8. Farago A, Sepriidi J, Split A 1988 Subcellular distribution of protein
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kinase C in rat adrenal glomerulosa cells. Biochem Biophys Res Commun X6:62&633 9. Nakano S, Carvallo P, Rocco S, Aguilera G 1990 Role of protein kinase C on the steroidogenic effect of angiotensin II in the rat adrenal glomerulosa cell. Endocrinology 126125-133 10. Spat A 1988 Stimulus-secretion coupling in angiotensin-stimulated adrenal glomerulosa cells. J Steroid Biochem 29443453 11. Vinson GP, Laird SM. Whitehouse BJ, Hinson JP 1989 Snecific effects of agonists of the calcium messenger system on secretion of “late-pathway” steroid products by intact tissue and dispersed cells of the rat adrenal glomerulosa. J Mol Endocrinol2:157-165 12. Gallo-Payet N, Chouinard L, Balestre MN. Guillon G 1991 Involvement of protein kinase C in the coupling between the Vi vasopressin recentor and Dhosnholinase C in rat elomerulosa cells: effects on aldo&.erone secretion. Endocrinology i29:623-634 13. Spat A, Balla I, Balla T, Cragoe Jr EJ, Hajnoczky Gy, Hunyady L 1989 Angiotensin II and potassium activate different Ca*+ entry mechanisms in rat adrenal glomerulosacells. J Endocrinol122:361370 -._ 14. Enyedi P, Spat A 1981 Effect of reduced extracellular sodium concentration on the function of adrenal glomerulosa cells: studies on isolated glomerulosa cells from rat. J Endocrinol89417-421 15. Enyedi P, Szab6 B, Spat A 1985 Reduced resnonsiveness of elomerulosa cells aft&prolonged stimulation with‘angiotensin II. Am J Physiol248:E209-214 16. Hajnoczky Gy, V&nai P, Ho116 Zs, Christensen SB, Balla T, Enyedi P, Spat A 1991 Thapsigargin-induced increase in cytoplasmic Ca*+ concentration and aldosterone production in rat adrenal glomerulosa cells: interaction with potassium and anaiotensin-II. Endocrinology 1282639-2644 17. Berridge MJ, Dawson RMC, Downes CP, Heslop JP, Irvine RF 1983 Changes in the levels of inositol phosphates after agonistdependent hydrolysis of membrane phosphoinositides. Biochem J 212473-482 18. Tamaoki T, Nomoto H, Takahashi I, Kato Y, Morimoto M, Tomita F 1986 Staurosporine a potent inhibitor of phospholipid/Ca*+ denendent protein kinase. Biochem Bionhvs ” I Res Commun 135:397-40219. Bell RM. Burns DJ 1991 Linid activation of nrotein kinase C. J Biol Chem 2664661-4664 20. Rasmussen H, Barrett PQ 1984 Calcium messenger system: an integrated view. Physiol Rev 64:938-984 21. Vilerain I. Defave G. Chambaz EM 1984 Adrenocortical cvtochrome P:450 responsible for cholesterol side chain cleavage”(P450,) is phosphorylated by the calcium-activated, phospholipidsensitive protein kinase (protein kinase C). Biochem Biophys Res Commun 125554-561 22. Ilvesmiiki V, Voutilainen R 1991 Interaction of phorbol ester and adrenocorticotropin in the regulation of steroidogenic P450 genes in human fetal and adult adrenal cell cultures. Endocrinoloav“1281450-1458 23. Rocco S, Nakano S, Aguilera G, Mechanism of the modulatory effect of phorbol esters on ACTH-stimulated aldosterone production in rat adrenal glomerulosa cells. 72nd Annual Meeting of The Endocrine Society, Atlanta GA, 1990 (Abstract 1529) 24. Kojima I, Shibata H, Ogata E 1986 Phorbol ester inhibits angiotensin-induced activation of phospholipase C in adrenal glomerulosa cells. Biochem J 232253-258 25. Pfeilschifter J, Ochsner M, Whitebread S, Gasparo M 1989 Downregulation of protein kinase C potentiates angiotensin II-stimulated polyphosphoinositide hydrolysis in vascular smooth-muscle cells. Biochem J 262:285-291 26. Lang U, Vallotton MB 1987 Angiotensin II but potassium induces subcellular redistribution of protein kinase C in bovine adrenal glomerulosa cells. J Biol Chem 262:8047-8050 27. Wollheim CB, Regazzi R 1990 Protein kinase C in insulin releasing cells. FEBS Lett 268376-380 28. Augert G, Bocckino SB, Blackmore PF, Exton JH 1989 Hormonal stimulation of diacylglycerol formation in hepatocytes. Evidence for phosphatidylcholine breakdown. J Biol Chem 26421689-21698 29. Balla T, VCnai P, Ho116 Zs, Spat A 1990 Effects of high potassium concentration and dihydropyridine calcium channel agonists on cytoplasmic Ca*+ and aldosterone production in rat adrenal glomerulosa cells. Endocrinology 127:815-822
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Vol. 130, No. 4 Society
Printed
The Role of Protein Kinase-C in Control Production by Rat Adrenal Glomerulosa Activation of Protein Kinase-C by Stimulation with Potassium* GYORGY ANDRAS
HAJNOCZKY, SPAT
PfiTER
VARNAI,
LliSZL6
BUDAY,
ANNA
in U.S.A.
of Aldosterone Cells:
FARAGO,
AND
Department of Physiology and First Department of Biochemistry (L.B., A.F.), SemmelweisUniversity Medical School,Budapest,Hungary
ABSTRACT. The role of protein kinase-C (PKC) in control of the function of rat adrenal glomerulosa cells was studied. Phorbol 12-mvristate ll-acetate (PMA). an activator of PKC. inhibited the itimulation of aldosmrone ‘production induced by K+ (6.4 mM) or ACTH (5 PM) in a dose-dependent manner. Phorbol 12,13dibutyrate, another phorbol ester that activates PKC, also exerted an inhibitory effect, whiie the inactive 4~ phorbol 12,13didecanoate failed to affect aldosterone production. The inhibitory effect of PMA (5 nM) was reversed by preincubation of the cells with staurosporine (ST, 50 nM), an inhibitor of PKC. These data suggest that pharmacological activation of PKC initiates an inhibitory mechanism in rat glomendosa cells. To elucidate whether PKC is activated by physiological stimuli. the effects of ST and down-reeulation of PKC bv nroloneed pretreatment with PMA on stimulation of aldosterone prod&tion were studied. The effects of angiotensin-II (AK) and K+, but not that of ACTH, were enhanced by ST pretreatment. This potentiation was prompt and transient in the case of AI1 (2.5 nM), while it developed gradually when the cells were stimulated
T
with K+ (5.4 or 18 mM). Long term pretreatment (6 h) of glomerulosa cells with PMA also enhanced the stimulatory effect of AI1 (396 PM) and K+ (5.4 mM). These data together suggest that the actions of AI1 and K+ on aldosterone production involve a PKC-mediated inhibition. Activation of PKC by AI1 is probably due to formation of diacylglycerol via receptor-mediated activation of phosphoinositide-specific phospholipase-C. Stimulation with K+ caused a moderate accumulation of [3H]inositol phosphate in a concentration-dependent manner. Since this effect was abolished by nifedipine, activation of phospholipase-C may have been secondary to Cal+ entry. The concomitant formation of diacylglycerol may contribute to activation of PKC in K+ stimulated cells. In conclusion, our data support the view that PKC participates in the physiological control of aldosterone production by rat adrenal domerulosa cells. In addition to AII. K+ mav activate PKC. Rega&lless of whether the enzyme is activated by phorbol esters or physiological stimuli, it exerts an inhibitory, rather than stimulatory, action on steroid production. (Endocrinology 130: 2230-2236,1992)
HE ENZYMES of the protein kinase-C (PKC) family are well known to participate in the control of several cellular processes (see references in Refs. l-4). Data reported on the role of PKC in adrenal glomerulosa cells are controversial. In bovine and human cells, pharmacological activation of PKC by phorbol esters induced a slowly developing increase in aldosterone production (5-7), which was potentiated by coadministration of Ca2+ ionophores ($6). In isolated rat glomerulosa cells, phorbol esters evoked the translocation of PKC (8, 9), yet phorbol 12-myristate 13-acetate (PMA) failed to stimu-
late aldosterone production (9-11). Nevertheless, phorbol esters modified hormone production induced by angiotensin-II (AII), ACTH (9), or vasopressin (12), suggesting that PKC may be involved in the control of aldosterone production. The aim of the present study was to clarify the effect of PKC activation on the secretory response to the major physiological stimuli (K+, AII, and ACTH). We also wanted to elucidate whether stimuli other than AI1 can also activate PKC in rat glomerulosa cells.
Received October 15, 1991. Address all correspondence and requests for reprints to: Dr. And& Spat, Department of Physiology, Semmelweis University Medical School, H-1444, P.O. Box 259, Budapest 8, Hungary. *This work was supported by Grant 1111 from the Hungarian National Science Foundation and Grant T-176/1990 from the Hungarian Council of Medical Sciences.
Materials
Materials
and Methods
AI1 (Ile5-AII) was obtained from Serva (Heidelberg, FRG), ACTH-( l-24) (Synacthen) from Ciba-Geigy (Basel, Switzerland), phorbol estersfrom Sigma (St. Louis, MO), staurosporine (ST) from Calbiochem (La Jolla, CA), proteaseinhibitors
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ACTIVATION
OF PKC BY K+ STIMULATION
from Sigma, myo-[2-3H]inositol (16.6 Ci/mmol) from DuPontNew England Nuclear (Boston, MA), [32P]ATP (10 Ci/mmol) from Izinta (Budapest, Hungary), and medium 199 (with Earle’s salts) from Gibco (Grand Island, NY). Thapsigargin was kindly provided by S. B. Christensen (Copenhagen, Denmark) All other chemicals were of analytical grade. Cell isolation and aklosterone experiments Adrenal glomerulosa cells were prepared from capsular tissues of male Wistar rats (200-300 g) using collagenase and mechanical dispersion, as described previously (13). The viability of the cells was examined by a trypan blue exclusion test; usually less than 5% of the cells were trypan blue positive. Cells were preincubated for 3 h in a mixture of modified KrebsRinger bicarbonate-glucose solution and medium 199 (2:1, vol/ vob final concentrations: Na, 145 mM; K, 3.6 mM; Ca, 1.2 mM; and Mg, 0.5 mM) supplemented with human serum albumin (fraction V; 2 g/liter) at 37 C under a mixture of 95% OS-5% CO* (pH 7.4). When cells were pretreated for 6 h with phorbol esters, the medium also contained gentamycin (100 mg/liter) and cefalexin (50 mg/liter). Then, the cells were washed and incubated (-1.5 x 10’ cells/500 ~1) for 60 or 120 min under identical conditions. The aldosterone content of the cell suspension was measured by RIA (14). Assays were performed in triplicate. Mean @EM) basal aldosterone production was 17.1 f 3.2 pmol/lO’ cells-h (n = 3). Cell superfusion was carried out as previously described (15). Briefly, isolated cells (-lo6 cells/column) were mixed with 600 ~1 preswollen Bio-Gel P2 resin and loaded onto small columns prepared from 2.0-ml syringes. Cells on the column were superfused with a flow rate of 8.0-10.0 ml/h using the medium detailed above. Effluent fractions were collected on ice, and their aldosterone concentration was measured by RIA (14). Measurement of form&on
of inositol phosphates
Experiments on the formation of inositol phosphates were performed as described previously (16). The labeling period (3 h in the presence of 15 &i/ml [3H]inositol) was followed by a 15-min stimulation in the presence of LiCl (10 mM). Inositol phosphates were separated by anion exchange chromatography (17). Assays were performed in duplicate. The control values were 10,619 f 2,815,822 + 272, and 183 + 70 cpm/106 cells for inositol monophosphates, bisphosphates, and trisphosphates, respectively (mean f SEM; n = 3) Measurement of PKC activity Cells (-1 million/500 ~1) were incubated in the above-described medium supplemented with gentamycin and cefalexin in the presence or absence of PMA at 37 C. After incubation, the cells were centrifuged at 100 x g, then suspended in 1 ml of a medium (lysing medium) containing 250 mM sucrose, 5 mM EGTA, 2 mM EDTA, 0.002% leupeptin, 1 mM benzamidine, 2 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, and 20 mM Tris-HCl (pH 7.4). The suspension was sonicated in an ice bath and centrifuged at 100,000 x g for 30 min. The supernatant was referred to as the cytosolic fraction. The pellet was resuspended in 1 ml lysing medium containing 0.02% Triton X-100. The suspension was incubated at 0 C for 60 min
2231
and then centrifuged at 40,000 x g for 15 min. The supernatant obtained by this procedure was referred to as the membrane fraction. Estimation of the activity of PKC was based on [32P]phosphate incorporation into the nonapeptide substrate Ala-AlaAla-Ser-Phe-Lys-Ala-Lys-Lys-amide, as previously described (8). Briefly, the assay was carried out in a mixture containing 50 mM Tris-HCl (pH 7.5), 10 mM MgC12, 1.2 mM EGTA, 0.01 mM [32P]ATP (-lo6 cpm/400 rl), and the synthetic nonapeptide (0.75 mM). Activation of PKC was induced by phosphatidylserine (20 pg/ml), CaCl, (2.5 mM), and PMA (40 nM). The reaction (10 min; 37 C) was terminated by adding glacial acetic acid, and the peptide was separated on a phosphocellulose column (8). All assays were performed in triplicate. PKC activity was calculated as the difference in the 32P activity of matched samples with and without the nonapeptide and activators (phosphatydilserine, PMA, and Ca”). Peptide kinase activity was 26.3 f 5.4 pmol phosphate/min. lo6 cells in the cytosolic fraction and 9.7 f 3.4 pmol/min- lo6 cells in the particulate fraction (mean f SEM; n = 3) Statistics The mean + SEM are shown in the aldosterone and inositol phosphate experiments. The effects of PMA and ST on aldosterone production were tested with paired sample t test. The significance of the interaction between the effects of stimuli and down-regulation of PKC on aldosterone production was estimated by three-way analysis of variance.
Results Effect of short term incubation aldosterone production
with phorbol esters on
First, the interaction of PMA with three physiological stimuli of glomerulosa cells was examined. The tested stimuli have different modes of action; potassium ions induce membrane depolarization with consecutive Ca*+ entry, the effect of AI1 is mediated by Ca*+ mobilization and Ca*’ entry, while CAMP is the second messenger responsible for the effect of ACTH. PMA (50 nM) did not significantly affect basal aldosterone production (lo), but inhibited the aldosterone response to K+ (elevated from 3.6 to 5.4 mM; Fig. 1, left panel; P < 0.01; n = 4) and ACTH (5 pM; Fig. 1, middle panel; P < 0.01; n = 5) in a cell superfusion system. The ratio of aldosterone output in the presence vs. the absence of PMA (upper panels) shows the sustained duration of inhibition. A similarly sustained inhibition by PMA was observed when aldosterone production was stimulated by the microsomal Ca*+-ATPase inhibitor thapsigargin (0.6 PM), known to evoke a cytoplasmic Ca*+ signal similar to that of AI1 (16) (data not shown). Yet, the aldosterone-stimulating action of AI1 (300 PM) was only slightly and transiently decreased by PMA (for the 15- to 30-min period of stimulation, P < 0.1; n = 4), and the later period of stimulation was not at all affected by the drug
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ACTIVATION
2232
OF PKC BY K+ STIMULATION
Endo Voll30
l l
1992 No 4
PMA 1
0.0 -30
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-30
(min)
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120
0
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F’IG. 1. Effect of PMA on aldosterone production stimulated by K+, ACTH, and AII. Aldosterone production by superfused glomeruloaa cells was followed. After a 2-h control period, 50 nM PMA (closed symbols on lower pan&) or solvent (open symbols on lower panels) was added for 15 min. Then, agonists were added, PMA or its solvent was also present throughout the 2-h stimulation. Stimuli were K+ (5.4 mM; left panel; n = 4), ACTH (5 PM; middle panel; n = 5), and AI1 (300 PM; right panel; n = 4). The lower panels show aldosterone production in picomoles per ml for l@ cells. Ratios of agonist-induced aldosterone production in the presence us. the absence of PMA are shown on the upper panels (mean + SEM).
(Fig. 1, r&tpaneZ). The dose-response curve of inhibition of the K+ (5.4 mM)-induced aldosterone production by PMA was determined in static incubations (Fig. 2). Half-maximal inhibition was attained at about 5 nM, and the highest PMA concentration applied (500 nM) completely inhibited the effect of K+. The specificity of PMA’s effects was first examined by the application of other phorbol esters. Phorbol 12,13dibutyrate (PDBu; 50 nM), another activator of PKC, exerted a similar inhibitory effect on K+-stimulated aldosterone production (Fig. 3, upper panel; P c 0.05; n = 4). In contrast to this, ricu-phorbol 12,13didecanoate (4aPDD; 50 nM), which is not able to stimulate PKC, did not influence the effect of K+ (Fig. 3, lower panel). Effect of ST on aldosterone production PMA (5 nM) inhibited K+-induced aldosterone production when it was added subsequently to K+ (5.4 mM; Fig. 4). Preincubation of the cells with ST, an inhibitor of PKC (18), prevented the effect of PMA (Fig. 4). Figure 4 also shows that the stimulatory action of 5.4 mM K+ was enhanced by coadministration of ST (for the second hour of stimulation, P < 0.01; n = 5).
Possible modification of the effects of K+, ACTH, and AI1 by ST was also tested using supramaximal concentrations of these agonists. The effect of 18 mM K+ was enhanced by ST (Fig. 5,leftpanel; P < 0.05; n = 4). The stimulatory effect of ST developed gradually at both K+ concentrations (5.4 and 18 mM), and it was higher for 18 InM K+. ACTH (5 n&&induced aldosterone output was not significantly affected by ST (Fig. 5, middle panel), and the effect of AI1 (2.5 nM) was only transiently enhanced by ST (Fig. 5, right panel; for the first 30 min of stimulation, P < 0.02; n = 6). Effect of long term incubation with PMA on PKC activity and aldosterone production Fifteen-minute treatment of glomerulosa cells with PMA (50 nM) reduced the activity of PKC in the cytosol fraction and induced a parallel increase in the membrane fraction (Fig. 6). After 6 h of incubation with PMA, both the cytosolic and membrane fractions of PKC activity were below the control level, and no further decrease was found after 24 h of PMA administration (Fig. 6). Data obtained with ST raised the possibility that PKC was activated by both K+ and AIL To elucidate the physiological significance of such an activation, we ex-
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ACTIVATION
OF PKC BY K+ STIMULATION
2233
n
PYA 1
0.1
100 ;PYA]
1000
';nM)
2. Inhibition of potassium-stimulated aldosterone production by PMA. Cells were stimulated with K+ (5.4 mM) in the presence of different concentrations of PMA in static incubation for 1 h. Aldosterone production was expressed as a percentage of the control stimulation, i.e. in the absence of phorbol ester (mean f SEM; n = 3). Aldosterone production by unstimulated cells (3.6 mM K+) was 17.1 f 3.2 pmol/lO’ cells and that by K+-stimulated cells (5.4 mM K+) was 33.1 f 2.3 pmol/lOs cells (n = 3). hG.
*
uj
1.3
K+6.4
'54 >
;
H + -$
1.0
0.7
L H
TJ
'24
-50
0
30
80
90
120
-30
0
30
60
90
120
Time
(min)
3. Effect of PDBu and 4aPDD on K+-stimulated. aldosterone production. Cells were preincubated with 50 nM PDBu (upper panel) or 50 nM 4aPDD (lowerpanel) for 15 min, and then the K+ concentration was increased from 3.6 to 5.4 mM in the superfusion medium. The aldosterone concentration of the effluent was measured as described in Materials and Methods, and ratios of K+-induced aldosterone production in the presence us. the absence of the phorbol ester are shown. The mean f SEM are shown for four (upper panel) or three (lower panel) separate experiments. FIG.
0
P -60
=
t 0
Time
I
I
60
120
160
(min)
FIG. 4. Effect of ST on PMA inhibition of aldosterone production stimulated by K+. After the 120-min control period, cells were superfused with either 50 nM ST (closed symbols) or control medium (open sytikr) for 60 min. Then, the K+ concentration was increased from 3.6 to 5.4 mM, and after 120-min stimulation, PMA (5 nM) was also added. Aldosterone production was normalized for 10’ cells (mean f SEM; n = 3). ST per se caused no changes in aldosterone production between -60 and 0 min; thus, in this period, the closed symbols are valid for both groups.
amined the effect of physiological stimuli in PKC-depleted cells. Basal aldosterone production by cells preincubated for 6 h with the inactive phorbol ester 4aPDD (50 nM) was 14.7 + 4.4 pmol/106 cel1s.h; preincubation with 50 nM PMA had no significant effect (13.1 -I- 2.8 pmol/106 cells. h; n = 6). Acute exposure to PMA (5 nM) inhibited the response to K+ (5.4 mM) by 47.8 + 6.0% in control cells (i.e. without prior exposure to PMA) and by only 10.4 f 12.3% after the cells became depleted of PKC (by long term preincubation with PMA; n = 4). The increase in l-h hormone production induced by AI1 (300 PM) was significantly higher in PKC-depleted than in control cells (11.4 f 2.0 us. 7.0 + 1.2 pmol/106 cells; for the interaction between the effects of AI1 and PKC depletion, P < 0.05; n = 6), and similarly, an increase was found in the effect of 5.4 mM K+ (24.1 + 3.7 us. 16.9 + 4.2 pmol/106 cells; for the interaction between the effects of K+ and PKC depletion, P < 0.05; n = 6). Considering the slow onset of enhancement of the effect of K+ by ST, 2-h hormone production was also measured for K+. In fact, the increase was more pronounced at 120 min than at 60 min of incubation (Fig. 7). In view of these observations, we examined whether potassium affects the activity of phospholipase-C. As an indicator of enzyme activity, the accumulation of 3Hlabeled inositol phosphates was measured at different concentrations of K+. Compared with control (3.6 InM) conditions, the formation of inositol trisphosphates in-
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2234
ACTIVATION
-30
0
30
Time
60
90
120
OF PKC BY K+ STIMULATION
-30
0
(min)
30
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60
(min)
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120
Endo. 1992 Voll30. No 4
-30
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(min)
FIG. 5. Effect of ST on aldosterone responses to K+, ACTH, and AK. After the control period, cells were preincubated with 50 nM ST (closed symbols) or solvent (open symbols) for 1 h. Then, stimuli were applied: K+ (18 mM; left panel; n = 4), ACTH (5 nM; middle panel; n = 3), and AI1 (2.5 nM; rig& panel; n = 6). In the lower pan.& aldosterone production is expressed as picomoles per ml for 10’ cells. In the upper panels ratios are shown that ware calculated by dividing the respective aldosterone concentrations obtained in response to stimuli in the presence vs. the absence of ST. The mean f SEM are shown.
creased by 13 + lo%, 37 + ll%, and 70 + 5% in the presence of 5.4, 8.4, and 18 mM K+, respectively (n = 3). The labeling of the inositol bisphosphate fraction increased to a similar extent, while that of the monophosphate fraction was twice as high. A possible physiological significance of these observations is shown by the significant accumulation of inositol phosphates (mono-, bis-, and tris-) in the presence of 5.4 mM K+ (122 + 5% compared to 3.6 mM; t = 4.26; n = 3) The stimulatory action of 18 mM K+ was completely abolished by nifedipine (1 PM), indicating that activation of phospholipaseC was secondary to influx of Ca2+ through voltage-dependent channels. This indirect means of activation of the enzyme may account for the small effect of K+ on the formation of inositol phosphates (mono-, bis-, and tris-) compared to that of AI1 (18 mM K+, 2.1 + O.O-fold increase; 25 nM AII, 10.8 + 1.9-fold increase; n = 3). Discussion The purpose of the present study was to elucidate the role of the enzyme family PKC in the physiological control of aldosterone production by rat adrenal glomerulosa cells. PKC enzymes are activated by diacylglycerol (DAG) in the presence of phospholipids and Ca2+ (for
review, see Refs. 1 and 19). Certain phorbol esters (PMA and PDBu) may substitute DAG in the activation of PKC (1). Considering that in bovine glomerulosa cells simultaneous administration of a Ca2+ ionophore and PMA induces aldosterone production resembling the effect of AII, it was postulated that Ca2+-mobilizing stimuli exert their effect through elevation of the cytoplasmic Ca2+ concentration and activation of PKC (20). However, PMA has no stimulatory effect on aldosterone production by isolated rat glomerulosa cells (9-11) and decreases, rather than increases, the effect of the Ca2+ ionophore ionomycin (Hajnoczky, Gy., and A. Spat, unpublished). Furthermore, the present study revealed that PMA diminished the aldosterone response to thapsigargin. Thapsigargin brings about a cytoplasmic Ca2+ signal in glomerulosa cells through mobilization of Ca2+ from intracellular stores (16) and, thus, mimics the Ca2+ branch of the AII-activated messenger mechanisms. Therefore, in contrast to bovine glomerulosa cells, in rat cells PKC does not complete the effect of Ca2+ in inducing the secretory response. However, the possible contribution of PKC to regulation of steroid production is suggested by two recent papers (9, 12). In the present study we analyzed the interaction of PKC with physiological stimuli, primarily K+, since activation of the cells
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ACTIVATION
f
0
1
I
i
0
6
12
Time
(hours)
OF PKC BY K+ STIMULATION
membrane-
1
1
16
24
1
FIG. 6. Time-course relationship of PKC activity in glomerulosa cells treated with PMA. PKC activity was measured in the cytosol and membrane fractions of glomerulosa cells, as described in M&TF~ULS and Methods. Cells were treated with PMA (50 nM) for 15 min, 6 h, and 24 h. Results were expressed as the percentage of PKC activity in the cytosol and membrane fractions of control cells, respectively. The two symbols represent the data from two separate experiments.
PDD pretreated PYA pretreated
K+ 5.4mY:
60 min
120 min
7. Comparison of the effect of K+ on aldosterone production by glomerulosa cells after prolonged treatment with PMA and 4aPDD. Cells were preincubated with ~(YPDD (50 nM; 0) or PMA (50 nM; W) for 6 h. Then, the cells were washed, and aldosterone production was measured under static incubation conditions for 60 or 120 min. The aldosterone content of the cell suspension was normalized for lo6 cells (60 and 120 min aldosterone production at 3.6 mM K+ were 11.9 f: 2.6 and 12.4 f 3.0 pmol/lO’ cells after 4aPDD pretreatment and 11.6 f 2.2 and 13.3 f 2.1 pmol/106cells after PMA pretreatment, respectively). The mean f SEM are shown for four separate experiments. FIG.
with K+ provides the simplest model of stimulation of glomerulosa cells through the Ca2+ signal. PMA exerted a marked inhibitory effect on aldosterone production induced by a physiological elevation of the K+ concentration (from 3.6 to 5.4 IIIM). The ACTH-
2235
induced hormone response was similarly sensitive to PMA, while the response to AI1 was diminished to a small extent. The mediatory role of PKC in the effect of PMA is indicated by the following results. 1) Halfmaximal inhibition was attained at 5 nM PMA, a concentration reported to activate PKC in several cell types (3). 2) PDBu, another pharmacological activator of PKC, also inhibited K+-stimulated hormone production, while the inactive phorbol analog (~cxPDD) was ineffective. 3) ST, an inhibitor of PKC, reversed the effect of PMA. 4) Down-regulation of PKC activity in glomerulosa cells reduced the inhibitory action of PMA. These data suggest that in rat glomerulosa cells PKC may inhibit the aldosterone secretory response to the major physiological stimuli. In static incubation, 15min preincubation with PMA (100 nM) markedly inhibited the secretory response to ACTH, but not to K+ and AI1 in the experiments of Nakano et al. (9). Apart from sexual and some technical differences, the explanation for the partial discrepancy between the results of the two studies is unknown. What is the mode of action of PKC? Since PKC phosphorylates cytochrome P450, in bovine cells (21), and PMA affects the expression of several types of cytochrome P450 in human adrenocortical cells (22), the site of the action of PKC might be on the final common pathway of steroidogenesis. However, formation of CAMP by ACTH (23) and accumulation of inositol phosphates by vasopressin (12) are also sensitive to phorbol esters in rat glomerulosa cells. Inhibition of AII-induced inositol phosphate formation (24, 25) and modulation of voltage-operated Ca2+ channels by PMA (for review, see Ref. 3) were observed in several other cell types. Thus, PKC may also influence the specific signal-transducing mechanisms of the three stimuli studied. Do the physiological stimuli of glomerulosa cells activate PKC? Enhancement of AII-induced aldosterone production by inhibition or down-regulation of PKC is compatible with the view that AI1 activates PKC (9,26). On the other hand, ST did not influence the ACTHelicited steroid response; thus, we have no reason to suppose the involvement of PKC in the action of ACTH. Unexpectedly, the effect of K+ was potentiated by ST and also by depletion of PKC activity. These data suggest that PKC may be activated by K+ in rat glomerulosa cells. Since this potentiation took place when K+ was increased from 3.6 to only 5.4 mM, the effect of K+ on PKC may have physiological significance. The mode of action of K+ is stimulation of Ca2+ entry via voltage-operated Ca2+ channels. The rise in cytoplasmic Ca2+ facilitates the interaction of PKC with lipid activators (27); thus, the cytoplasmic Ca2+ signal may contribute to the activation of PKC. Phospholipase-C is also sensitive to Ca2+; thus, Ca2+ entry may enhance the generation of DAG. In fact, we observed slight but sig-
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2236
ACTIVATION
OF PKC BY K+ STIMULATION
nificant stimulation of inositol phosphate formation by K+; this phenomenon was inhibited by nifedipine. Therefore, we attribute the K+-induced activation of PKC to elevation of cytoplasmic Ca2+ and a secondary moderate enhancement of DAG generation. This explanation is in agreement with the recently proposed mechanism of activation of PKC by stimuli that act through stimulation of receptor or voltage-operated Ca2+ entry (4, 27). As an additional effect, Ca’+-induced formation of DAG from phosphatidyl choline should be considered (28). Activation of PKC by K+ in rat glomerulosa cells may also contribute to the well documented bell-shaped doseresponse curve for K+-induced aldosterone production (see references in Ref. 29). Above 8-10 InM K+, the secretory response is increasingly more short-lived (29). In the present study we found that ST prevents the decline in the aldosterone response to 18 mM K+, which suggests that PKC contributes to the transient nature of the secretory response at high K+. In accordance with this assumption are the observations that ST stimulated aldosterone production more potently at 18 than at 5.4 mM K+, and stimulated the disappearance of the aldosterone inhibitory action of PMA after 2-h stimulation more potently with 18 mM [K+] (Hajnoczky, Gy., and A. Spat, unpublished) than with 5.4 mM. In conclusion, our data suggest that PKC exerts an inhibitory role in the control of aldosterone secretion by rat glomerulosa cells. This inhibitory effect may have significance during stimulation with AI1 and potassium ions. Acknowledgments We wish to thank medical students Mr. Attila Bago and Mr. Gyiirgy Csordasfor their enthusiastic work during the experiments. The excellent technical help of Miss Erika Kovacs and Mrs. Agnes Ribir is greatly appreciated. Aldosterone antibody was a gift from the NIAMD (Bethesda,MD), and Synacthen was a gift from Ciba-Geigy (Basel, Switzerland).
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