0013-7227/90/1261-0125$02.00/0 Endocrinology Copyright© 1990 by The Endocrine Society
Vol. 126, No. 1 Printed in U.S.A.
Role of Protein Kinase C on the Steroidogenic Effect of Angiotensin II in the Rat Adrenal Glomerulosa Cell SHIGERU NAKANO, PILAR CARVALLO, STEFANO ROCCO, AND GRETI AGUILERA Section on Endocrine Physiology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT. The role of protein kinase C (PKC) in the steroidogenic action of angiotensin II (All) was investigated by depletion of endogenous PKC using prolonged incubation with phorbol ester and direct measurement of PKC in isolated rat adrenal glomerulosa cells. PKC activity was measured by incorporation of 32P from [732P]ATP into histone in the presence of cytosolic and detergent-solubilized membrane fractions purified by diethylaminoethyl cellulose chromatography. Basal PKC activity was higher in cytosol than in membranes (1,000 ± 57 and 413 ± 14 pmol P incorporated/mg-min, respectively). After incubation of the cells with All for 5, 15, 30, and 60 min, PKC activity in the cytosol decreased by 5, 18, 25, and 27%, respectively, while in the membrane there was a transient increase of 15% at 15 min returning to basal by 60 min. Incubation of the cells with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in transient translocation of PKC activity to the membrane (15 min) which was followed by a 64% decrease in total cellular enzyme activity after 3 h. In PKC-depleted cells, the aldosterone response to ACTH was increased by 25% but Allstimulated steroidogenesis was unchanged. In contrast, in cells in which PKC was translocated to the membrane by a 15 min preincubation with TPA, aldosterone response to All was enhanced by 40%, while the response to ACTH was reduced by
30%; under these conditions membrane PKC levels rapidly returned to basal. However, the changes in aldosterone response were still evident when addition of All or ACTH was delayed for up to 30 min after removal of TPA, indicating a persistent modification in the cell membrane secondary to PKC activation. Aldosterone responses to potassium were not altered by preincubation of the cells with TPA. The inactive phorbol ester analog, 4a-hydroxyphorbol-12,13-dibutyrate, had no effect on the steroid responses to either stimulus. The small but significant translocation of PKC activity from cytosol to membrane after treatment of rat adrenal glomerulosa cells with All suggests that All activates PKC. However, the fact that aldosterone responses to All are potentiated during TPA-induced PKC translocation to the membrane suggests that All and phorbol esters do not share the same mechanism of action in the regulation of steroidogenesis. In addition, the full aldosterone response to All despite marked cellular PKC depletion after prolonged preincubation with TPA argues against the involvement of PKC in the stimulation of aldosterone production by AIL We propose that PKC activation by All may mediate effects other than steroidogenesis, such as trophic maintenance of the adrenal glomerulosa or adrenal growth. (Endocrinology 126: 125-133, 1990)
T
HE INTERACTION of angiotensin II (All) with its plasma membrane receptor in a number of target tissues, including liver, smooth muscle, and the adrenal glomerulosa, is associated with calcium mobilization and phospholipid turnover (1-3). In the adrenal glomerulosa All is known to stimulate phospholipase C with formation of diacylglycerol and inositol phosphates, and to induce calcium mobilization with the consequent increase in cytosolic calcium (1-5). Evidence for the role of calcium in the regulation of steroidogenesis by All has been shown by its dependence on extracellular calcium and the inhibitory effect of calmodulin antagonists and voltage-dependent calcium channel blockers (6-8). Calcium is involved in the regulation of a number of enzymes including protein kinase C (PKC) and calcium protein
kinases (9-11). PKC is present in the adrenal cortex (12, 13), and the factors necessary for its activation, diacylglycerol and calcium mobilization, are stimulated by All (1, 3). However, the participation of PKC in the mechanism of action of All is controversial. Although All has been shown to cause translocation (14, 15), and presumably activation of PKC in bovine adrenal glomerulosa cells, it is not clear whether activation of the enzyme is required for aldosterone secretion. Whereas in many systems phorbol esters mimic the effect of peptide hormones which act through PKC (16-18), this is not the case for All stimulation of aldosterone secretion (3). Since combined perifusion of bovine adrenal glomerulosa cells with phorbol esters and a calcium ionophore has been shown to cause stimulation of aldosterone production similar to that of All, it has been suggested that there is an interdependence between the PKC and calcium-calmodulin dependent pathways (12). However, this effect has not
Received September 11,1989. Address requests for reprints to: Dr. Greti Aguilera, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
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126
PROTEIN KINASE C ROLE IN All STEROIDOGENESIS
Endo • 1990 Vol 126* No 1
Histone type III-S, EGTA, leupeptin, pepstatin A, dithiothreitol, diethylaminoethyl (DEAE)-cellulose, sodium pyrophosphate, isobutylmethylxantine, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma, (St. Louis, MO); phosphatidylserine and 1,2-dioleoylglycerol from Avanti Polar Lipids, Inc. (Birmingham, AL); synthetic [He5] All and ACTH 1-24 from Peninsula Laboratories (Belmont, CA); [ 7 32 P]ATP and [3H]aldosterone from Dupont New England Nuclear (Wilmington, DE); and medium 199 containing 4.5 mM potassium from the NIH Media Supply Unit.
cells were incubated for 15 min with 0.1 nM [3H]PMA in 1 ml medium 199 containing 0.1% BSA at 37 C. To mimic the experimental conditions used for aldosterone secretion, triplicate samples of the cells were either immediately filtered, through glass fiber filters, or washed twice with 10 ml medium 199 and filtered or washed twice with medium 199 at room temperature, resuspended in 5 ml of media incubated for 30 min at 37 C, and then filtered (Table 1). To study the effect of more prolonged pretreatment with phorbol ester, dispersed glomerulosa cells, 10 million in 20 ml medium 199 containing 100 U/ml penicillin, 100 fig/ml streptomycin, 0.1% BSA, 5 Mg/ml insulin, 5 fig/ml transferrin, 0.2 mM ascorbic acid, 5 ^M metyrapone, and 100 mM dimethyl sulfoxide (DMSO) were cultured at 37 C under air/5% CO2, in two 50 ml plastic tubes, one of which contained 0.1 juM TPA. In these experimental conditions steroid responses to stimulation with All and ACTH were maintained for 18 to 24 h after cell dispersion. The use of antioxidants and blockade of glucocorticoid production by metyrapone have been shown to preserve the activity of cytochrome P-450-dependent enzymes of the biosynthetic pathway (21). After 6 h incubation, cells were centrifuged, washed twice with medium 199 at room temperature, resuspended in 20 ml fresh culture media, and culture continued overnight in the absence of TPA. Cells were then pelleted by centrifugation, washed twice in medium 199 containing 0.1% BSA, and used for stimulation with hormones and PKC assay.
Preparation and treatment of adrenal glomerulosa cells
Protein kinase C determination
been reproduced in other experimental conditions in bovine or rat adrenal glomerulosa cells (Ref. 3 and Aguilera G., unpublished). To more directly evaluate the role of PKC in the mechanism of action of All, we have studied the effect of changes in PKC distribution and activity on the ability of All to stimulate aldosterone secretion in rat adrenal glomerulosa cells. The results indicate that although All causes cellular redistribution of PKC activity in the adrenal glomerulosa and PKC can modulate the cell responsiveness to All, PKC activation is not required for steroidogenic effects of the peptide. Materials and Methods Materials
Isolated adrenal glomerulosa cells were prepared from 200250 g female Sprague-Dawley rats (Charles River, Wilmington, MA) by collagenase digestion as previously described (19). Cells were resuspended in medium 199 containing 0.1% BSA at a concentration of 1.5 to 2 million/ml. Two milliliter aliquots were incubated in 20 ml plastic scintillation vials with and without TPA for the time periods indicated, at 37 C under 95% O2/5% CO2. TPA was added after 20 min preincubation, and incubation of all vials was stopped at the same time. For PKC determination, vials were placed on ice, 10 ml ice-cold PBS were added, and the total volume was transferred to 15 ml conical tubes and centrifuged for 10 min at 100 X g to obtain the cell pellet. For measurement of aldosterone production, cells were washed twice with medium 199 at room temperature and resuspended in fresh media at a concentration of 105 cells/ml. It was necessary to wash the cells with medium at room temperature to preserve responsiveness of the cells, and preliminary experiments showed that this step did not influence PKC activity in membrane or cytosol compared with cells in which TPA preincubation was stopped on ice. One milliliter aliquots were incubated for 2 h in 12 X 75 mm glass tubes at 37 C, under 95% O2/5% CO2 in the presence of All, ACTH, and potassium. At the end of the incubation, tubes were centrifuged at 1,500 X g for 10 min and the supernatant decanted and stored at -20 C until aldosterone was determinated by direct RIA (20). The proportion of phorbol ester that remained in the cells after the wash and incubation was determined by [3H]phorbol-12-myristate-13-acetate ([3H]PMA) uptake. Nine aliquots of 0.5 million
Cell pellets were resuspended in 300 /A 20 mM Tris/HCl buffer (pH 7.5) containing 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 10 MM leupeptin, and 10 fiM pepstatin A (buffer A), and sonicated twice for 2 sec. The homogenate was centrifuged at 105,000 X g for 30 min at 4 C, and the supernatant was used as the cytosol fraction. The pellet was resonicated in buffer A containing 0.1% Nonidet P-40 and centrifuged at 105,000 X g for 30 min, and the supernatant was used as the membrane fraction. To partially purify PKC, 2-3 mg cytosol or solubilized membrane protein were applied to a 1.2 cm diameter X 1.5 cm height DEAE-cellulose column equilibrated with buffer A at 4 C. After washing with 10 ml buffer A, protein kinases were eluted with 1. Phorbol ester washout after preincubation of the cells with [3H]PMA
TABLE
Treatment (after incubation with [3H]PMA)
Cell associated phorbol ester
pmol cpm No wash 29,670 ± 89 1.3 2 washes in M199 1,250 ± 128 0.05 2 washes in M199, dilution, and 30 184 ± 15 0.01 min incubation After 15 min preincubation with [3H]PMA, cells were treated as described in the treatment column and filtered through glass fiber filters (Whatman GF/C). Filters were washed twice with 4 ml ice-cold PBS, and the radioactivity was counted in a liquid scintillation counter. Values are the mean ± SE of triplicate incubations. M199, Medium 199.
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PROTEIN KINASE C ROLE IN All STEROIDOGENESIS a 10 ml linear gradient (0-0.3 M) of NaCl in the same buffer. Fractions of 0.5 ml were collected and assayed for PKC activity. A peak of PKC activity was eluted with 90 mM NaCl (Fig. 1). Therefore, in all subsequent experiments the PKC fraction was obtained by elution of the column with 100 mM NaCl. A small peak of calcium phospholipid independent activity, presumably protein kinase M (85 ± 7.9 and 79 ± 6.2 pmol/mg-min, for cytosol and membrane, respectively) was observed in the fractions eluted with NaCl 150-170, in two of three experiments. In subsequent experiments protein kinase M (PKM) was measured in the 90-200 step elution fraction. PKC activity was assayed by measuring the incorporation of 32 P from [T- 3 2 P] ATP into histone III-S by a modified procedure from that described by Huang et al. (22). The reaction mixture contained 20 mM Tris-HCl (pH 7.5), 5 mM Mg acetate, 0.6 mM CaCl2, 40 MM [T- 3 2 P]ATP (specific activity, 400-600 cpm/
pmol), 1 mg/ml histone III-S, 40 ixg/ml phosphatidylserine, 8 Mg/ml l-2,dioleoylglycerol, and the sample protein in a final volume of 50 n\. The calcium and phospholipid-independent activity was measured under the same conditions without calcium and phospholipids and containing 1 mM EGTA. To examine the contribution of calcium-dependent protein kinase, activity was measured in the presence of 0.6 mM CaCl2. Reactions were initiated by addition of [7-32P]ATP and incubated for 15 min at 37 C, and terminated by adding 3 ml ice-cold 20% trichloroacetic acid containing 1 mM sodium pyrophosphate.
0.10
0.05
0.05
10 15 20 FRACTION NUMBER
-JO
FIG. 1. Anion exchange chromatography of adrenal glomerulosa PKC. Cytosol- or detergent-solubilized membranes from 20 million adrenal glomerulosa cells were applied to a column (1.2 x 1.5 cm) of DEAEcellulose equilibrated with buffer A. After sample addition the column was washed with buffer A and eluted with a linear (0-0.3 M) NaCl gradient in 0.5 ml fractions. PKC was determined in the presence (•— —•) and in the absence (O O) of Ca, phosphatidylserine, and diolein as described in Materials and Methods. Protein concentration is shown by the dotted line. Data are the mean of triplicate determinations.
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Preliminary experiments showed that the time course of PKC activity in cytosol and membrane fractions was linear up to 20 min. Precipitates were collected on filter paper (Whatman, GF/ C), washed twice with 5 ml 5% trichloroacetic acid containing 1 mM sodium pyrophosphate, and dried, and the radioactivity incorporated into protein was counted in a liquid scintillation counter. Determinations were performed using duplicate aliquots from each sample.
Results PKC activity in cytosol and membrane fractions from adrenal glomerulosa cells after DEAE-cellulose chromatography is shown in Fig. 1. Consistent with the recognized elution pattern of PKC, the peak of activity was obtained with 90 mM NaCl. In three experiments, DEAEcellulose chromatography resulted in more than a 10fold enrichment of PKC activity compared with crude fractions, from 77.3 ± 7.6 to 1,020 ± 57.9 pmol/mg-min in cytosol and from 39.3 ± 2.4 to 413.3 ± 14.4 pmol/mgmin in membrane. As shown in Table 2, PKC activity in the 100 mM NaCl elution fractions in the presence of EGTA or calcium alone was markedly reduced compared with activity in the presence of calcium, phosphatidylserine, and diacylglycerol. Therefore, all subsequent experiments were performed using PKC purified on DEAEcellulose. To determine whether All has an effect on PKC in rat adrenal glomerulosa cells, the enzyme activity was measured in cytosol and membrane fractions after incubation of the cells with All (Fig. 2). The time course of the effect of All on PKC activity, in two experiments, shows a small decrease in the cytosol to 94% the basal values at 5 min, followed by a further reduction to 82 ± 6.4%, 75 ± 9.2%, and 74 ± 6.4% at 15, 30, and 60 min, respectively. The converse changes were observed in the membrane fraction with an increase to 106 ± 4.2% and 133 ± 5.7% at 5 and 15 min, respectively, followed by a decrease to 113 ± 7.1% and 99 ± 7.5% at 30 and 60 min, respectively (mean ± SD). NO increase in PKM was observed after incubation of the cells with All up to 60 min. The ability of All to cause PKC translocation suggests that All can activate PKC in the rat adrenal TABLE 2. Effect of calcium, phosphatidylserine (PS), and diacylglycerol (DG) on phosphate incorporation into Histone III-S by DEAEcellulose-purified PKC in cytosol and membrane from adrenal glomerulosa cells
Incubation conditions EGTA Ca PS/DG/Ca
P incorporation (pmol/mg/min) Cytosol
Membrane
84.5 ± 4.6 91.8 ± 6.2 1128.0 ± 30.2
113.3 ± 14.2 145.6 ± 17.8 573.1 ± 59.7
Values are the mean ± SE from four experiments.
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128
PROTEIN KINASE C ROLE IN All STEROIDOGENESIS
15
30 45 TIME (min)
60
FIG. 2. Effect of All treatment on cytosolic and membrane-associated PKC in isolated adrenal glomerulosa cells. Aliquots containing 6 million cells were incubated with 10 nM All for the periods of time indicated and processed for PKC determination as described in Materials and Methods. Data are the mean of triplicate determinations in one of 2 similar experiments.
Endo • 1990 Voll26-Nol
(up to 6 h). In three to seven experiments the mean PKC values decreased from 1235 ± 209 pmol/mg-min to 827 ± 141, 519 ± 135, 341 ± 114, 389 ± 97, 320 ± 130, and 292 ± 111 pmol/mg-min at 5, 15, 30, 60, 180, and 360 min, respectively. The decrease in activity in the cytosol fraction was accompanied by a transient increase in PKC in the membrane, reaching a maximum at 15 min followed by a rapid decline over the next 15 min with a further decrease to basal levels for up to 6 h. The mean values for all experiments were 353 ± 101 for basal, 705 ± 189, 1036 ± 122, 655 ± 143, 436 ± 131, 265 ± 178, and 230 ± 149 pmol/mg-min at 5, 15, 30, 60, 180, and 360 min, respectively. In order to determine whether the decrease in PKC was a consequence of degradation with conversion to PKM, we measured kinase activity in the presence and in the absence of calcium and phospholipids in all fractions eluted from the DEAE-cellulose column or the 200 mM step gradient fractions. Pretreatment of the cells with TPA for 15 min (n = 2), 30 min (n = 4), or 180 min (n = 1) had no effect on Ca/phospholipid independent activity in these fractions, indicating the absence of PKM formation. The aldosterone response of cells preincubated with 0.1 (xM TPA for 15 min and 3 h is shown in Fig. 4. In five experiments after 3 h preincubation with TPA when PKC content of the cells was maximally reduced, aldosterone response to All was identical to that in control cells. In contrast, aldosterone production stimulated by maximal and submaximal concentrations of ACTH was enhanced by 110%, 229%, 78%, and 25% with 1,10, 100, and 1,000 pM, respectively. To study the possibility that residual PKC after the 3 h TPA treatment may be activated by All and account for the steroidogenic effect of the peptide, cells were •n. i.o
30
180 60 TIME (min)
360
FIG. 3. Effect of treatment with TPA on PKC activity isolated from adrenal glomerulosa cells. Cells (6 million) were incubated with 0.1 /*M TPA for the periods of time indicated. Cytosolic and membrane fractions were assayed for PKC activity as described in Materials and Methods. Total PKC activity in the cell was reduced by 70%, from 1652 to 505 pmol/mg-min after 3 h incubation with TPA. Data are the mean ± SE of the values of 3 to 7 experiments.
glomerulosa cell. To determine the role of PKC on the steroidogenic activity of All, the effect of aldosterone stimulants was evaluated in cells in which PKC activity was modified by preincubation with phorbol esters. As illustrated in Fig. 3, treatment of the cells with 0.1 /xM TPA resulted in a marked decrease in PKC activity in the cytosol, reaching about 25% of the basal activity at 3 h, and these levels were maintained with longer preincubation times
0
-11 -10-9 -8 Log All (M)
-12-11-10-9 Log ACTH (M)
6
8 10 12
K+(mM)
FIG. 4. Effect of 15 min (A A) and 3 h (4 \) pretreatment with TPA on the responsiveness of isolated rat adrenal glomerulosa cells to All, ACTH, and potassium. Control cells (O O) were preincubated for 3 h or 15 min in the absence of TPA. After preincubation with the phorbol ester, cells were washed and incubated for 2 h with increasing concentrations of the various stimuli. Data points are the mean ± SE of duplicate incubations in one of five similar experiments.
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PROTEIN KINASE C ROLE IN All STEROIDOGENESIS
preincubated with TPA for 3 h, washed, and then treated with TPA or All for 15 min to assess their ability to induce further PKC translocation. As shown in Table 3, treatment of control cells with TPA or All for 15 min caused the expected changes in cytosolic and membrane PKC. In contrast, when PKC levels were reduced by 3 h preincubation with the phorbol ester, no further changes in PKC activity were observed after 15 min incubation with TPA or AIL The results obtained after 3 h preincubation with TPA were confirmed in two experiments in which cells cultured overnight were preincubated with TPA for 6 h to achieve a more marked reduction in cellular PKC. In these experiments, total PKC was reduced by 91% (from 1187 ± 102 to 95 ± 11 pmol/mgmin in the cytosol and from 338 ± 45 to 36 ± 9 in the membrane). Similar to the results in cells preincubated with TPA for 3 h, aldosterone responses to All were unaffected despite the marked reduction in cellular PKC content (Fig. 5A). Also, consistent with the experiments above, the aldosterone response to ACTH was enhanced in the cells preincubated with TPA for 6 h (Fig. 5B). After 15 min preincubation with TPA which induces maximum PKC translocation to the membrane, aldosteTABLE 3. Effect of 15 min incubation with TPA or All in adrenal glomerulosa cells preincubated with and without 0.1 nM TPA for 3 h Protein Kinase C activity {% of basal) Incubation conditions BASAL TPA All
Control
TPA preincubation
Cytosol
Membrane
Cytosol
Membrane
100 40.7 ± 2.8° 79.3 ± 3.7°
100 289 ±10.1° 135 ± 6.2°
100 99.7 ± 1.2 98.8 ± 0.88
100 101.4 ± 1.8 98.5 ± 1.6
Basal PKC values were 1016 ± 126 and 376 ± 14.8 pmol/mg-min P incorporation for cytosol and membrane, respectively, in the control cells, and 306 ± 30.8 and 336 ± 20 pmol/mg min 32P incorporation for cytosol and membrane, respectively, in TPA-pretreated cells. P < 0.01 vs. basal (n = 3). 32
129
rone responses to AH and ACTH were affected in the opposite manner. While All-stimulated aldosterone production was enhanced by 28%, 65%, 102%, and 171% with 0.01,0.1. .1 and 10 nM All, respectively, the response to ACTH was reduced by 29%, 24%, 19%, and 28% with 1,10,100, and 1,000 pM ACTH, respectively. Potassiumstimulated aldosterone production was completely unaffected by preincubation with TPA for 15 min or 3 h. Preincubation of the cells with the inactive phorbol ester, 4a-hydroxyphorbol 12,13 dibutyrate, had no significant effect on the aldosterone response to subsequent stimulation with other stimulants (not shown). In the experiments described above in which cells were preincubated with TPA for 15 min, the time of maximum PKC membrane translocation, measurement of aldosterone production required a further 2 h incubation period with the stimulators. There is, thus the important question concerning the extent to which PKC remains translocated during the 2 h incubation with the stimulants. In order to evaluate this question, measurement of PKC activity and addition of stimuli were performed at increasing time intervals after the 15 min treatment with TPA. As shown in Fig. 6, PKC activity in the cytosol decreased by 70% after 15 min incubation with TPA and remained at this level for up to 2 h after removal of TPA. In the membrane the 100% increase in activity observed at the end of the TPA preincubation rapidly decreased to basal values at 3 min, and declined to less than basal values 10 min after removal of TPA. To determine whether the changes in aldosterone responses after 15 min TPA preincubation were correlated with the membrane location of PKC activity, All or ACTH was added 2 and 30 min after removal of TPA, when membrane PKC activity was still increased by 50% or was less than
• Cytoeol O Membrane
•n LOG A D (M)
LOG ACTH (M)
FIG. 5. Effect of prolonged incubation with TPA on the responsiveness of isolated rat adrenal glomerulosa cells to All and ACTH. Cells were preincubated for 6 h with 0.1 fiM TPA, washed, and cultured for additional 10 h as described in Materials and Methods. Cells were then washed, resuspended in medium 199/0.1% BSA and incubated for 2 h with ACTH or AIL Data points are the mean ± SE of duplicate incubations in one of two experiments.
-15
0
15
30
120
TIME (min)
FIG. 6. Time course of the changes in PKC activity in cytosol and membrane fractions after preincubation of adrenal glomerulosa cells with TPA for 15 min. After preincubation, cells were washed and incubated in fresh medium 199 for the time periods indicated before PKC determination. Points are the mean of triplicate determinations in two or three experiments.
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PROTEIN KINASE C ROLE IN All STEROIDOGENESIS
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the basal value, respectively. As observed in the previous experiments All-stimulated aldosterone production was increased by 30% when All was added immediately after the 15 min preincubation with TPA (Fig. 7, time 0). In these experiments the level of potentiation was identical when the addition of All was delayed to 2 or 30 min after the removal of TPA. Similarly ACTH-stimulated aldosterone secretion was inhibited by 30%, irrespective of the time of addition of ACTH, with no delay, 2 or 30 min after removal of TPA. In contrast with the effects of TPA preincubation on stimulated steroidogenesis, direct treatment of the cells for 2 h with TPA had no significant effect on aldosterone production. In six experiments, aldosterone production was unchanged, remaining at 1.0 ± 0.05 times the basal values after incubation with 0.1 M TPA, while with maximum stimulatory concentrations of All, ACTH, and potassium it was increased by 6.8 ± 0.62, 22.3 ± 1.7, and 23.3 ± 3.3 fold, respectively. In two experiments, simultaneous incubation of the cells with 10 nM All and increasing TPA concentrations inhibited rather than potentiating aldosterone production. A small decrease of 14.4 ± 0.84% (mean ± SD) was observed with 0.1 nM TPA. Larger concentrations of 1 juM caused a marked but nonspecific inhibition, since the effects of all stimulators, including that of 8-Br-cAMP were equally decreased. Discussion The purpose of these experiments was to investigate the role of PKC in the steroidogenic action of All in rat
1
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Vol. 126, No. 1 Printed in U.S.A.
Role of Protein Kinase C on the Steroidogenic Effect of Angiotensin II in the Rat Adrenal Glomerulosa Cell SHIGERU NAKANO, PILAR CARVALLO, STEFANO ROCCO, AND GRETI AGUILERA Section on Endocrine Physiology, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892
ABSTRACT. The role of protein kinase C (PKC) in the steroidogenic action of angiotensin II (All) was investigated by depletion of endogenous PKC using prolonged incubation with phorbol ester and direct measurement of PKC in isolated rat adrenal glomerulosa cells. PKC activity was measured by incorporation of 32P from [732P]ATP into histone in the presence of cytosolic and detergent-solubilized membrane fractions purified by diethylaminoethyl cellulose chromatography. Basal PKC activity was higher in cytosol than in membranes (1,000 ± 57 and 413 ± 14 pmol P incorporated/mg-min, respectively). After incubation of the cells with All for 5, 15, 30, and 60 min, PKC activity in the cytosol decreased by 5, 18, 25, and 27%, respectively, while in the membrane there was a transient increase of 15% at 15 min returning to basal by 60 min. Incubation of the cells with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) resulted in transient translocation of PKC activity to the membrane (15 min) which was followed by a 64% decrease in total cellular enzyme activity after 3 h. In PKC-depleted cells, the aldosterone response to ACTH was increased by 25% but Allstimulated steroidogenesis was unchanged. In contrast, in cells in which PKC was translocated to the membrane by a 15 min preincubation with TPA, aldosterone response to All was enhanced by 40%, while the response to ACTH was reduced by
30%; under these conditions membrane PKC levels rapidly returned to basal. However, the changes in aldosterone response were still evident when addition of All or ACTH was delayed for up to 30 min after removal of TPA, indicating a persistent modification in the cell membrane secondary to PKC activation. Aldosterone responses to potassium were not altered by preincubation of the cells with TPA. The inactive phorbol ester analog, 4a-hydroxyphorbol-12,13-dibutyrate, had no effect on the steroid responses to either stimulus. The small but significant translocation of PKC activity from cytosol to membrane after treatment of rat adrenal glomerulosa cells with All suggests that All activates PKC. However, the fact that aldosterone responses to All are potentiated during TPA-induced PKC translocation to the membrane suggests that All and phorbol esters do not share the same mechanism of action in the regulation of steroidogenesis. In addition, the full aldosterone response to All despite marked cellular PKC depletion after prolonged preincubation with TPA argues against the involvement of PKC in the stimulation of aldosterone production by AIL We propose that PKC activation by All may mediate effects other than steroidogenesis, such as trophic maintenance of the adrenal glomerulosa or adrenal growth. (Endocrinology 126: 125-133, 1990)
T
HE INTERACTION of angiotensin II (All) with its plasma membrane receptor in a number of target tissues, including liver, smooth muscle, and the adrenal glomerulosa, is associated with calcium mobilization and phospholipid turnover (1-3). In the adrenal glomerulosa All is known to stimulate phospholipase C with formation of diacylglycerol and inositol phosphates, and to induce calcium mobilization with the consequent increase in cytosolic calcium (1-5). Evidence for the role of calcium in the regulation of steroidogenesis by All has been shown by its dependence on extracellular calcium and the inhibitory effect of calmodulin antagonists and voltage-dependent calcium channel blockers (6-8). Calcium is involved in the regulation of a number of enzymes including protein kinase C (PKC) and calcium protein
kinases (9-11). PKC is present in the adrenal cortex (12, 13), and the factors necessary for its activation, diacylglycerol and calcium mobilization, are stimulated by All (1, 3). However, the participation of PKC in the mechanism of action of All is controversial. Although All has been shown to cause translocation (14, 15), and presumably activation of PKC in bovine adrenal glomerulosa cells, it is not clear whether activation of the enzyme is required for aldosterone secretion. Whereas in many systems phorbol esters mimic the effect of peptide hormones which act through PKC (16-18), this is not the case for All stimulation of aldosterone secretion (3). Since combined perifusion of bovine adrenal glomerulosa cells with phorbol esters and a calcium ionophore has been shown to cause stimulation of aldosterone production similar to that of All, it has been suggested that there is an interdependence between the PKC and calcium-calmodulin dependent pathways (12). However, this effect has not
Received September 11,1989. Address requests for reprints to: Dr. Greti Aguilera, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892.
125
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126
PROTEIN KINASE C ROLE IN All STEROIDOGENESIS
Endo • 1990 Vol 126* No 1
Histone type III-S, EGTA, leupeptin, pepstatin A, dithiothreitol, diethylaminoethyl (DEAE)-cellulose, sodium pyrophosphate, isobutylmethylxantine, and 12-O-tetradecanoylphorbol-13-acetate (TPA) were obtained from Sigma, (St. Louis, MO); phosphatidylserine and 1,2-dioleoylglycerol from Avanti Polar Lipids, Inc. (Birmingham, AL); synthetic [He5] All and ACTH 1-24 from Peninsula Laboratories (Belmont, CA); [ 7 32 P]ATP and [3H]aldosterone from Dupont New England Nuclear (Wilmington, DE); and medium 199 containing 4.5 mM potassium from the NIH Media Supply Unit.
cells were incubated for 15 min with 0.1 nM [3H]PMA in 1 ml medium 199 containing 0.1% BSA at 37 C. To mimic the experimental conditions used for aldosterone secretion, triplicate samples of the cells were either immediately filtered, through glass fiber filters, or washed twice with 10 ml medium 199 and filtered or washed twice with medium 199 at room temperature, resuspended in 5 ml of media incubated for 30 min at 37 C, and then filtered (Table 1). To study the effect of more prolonged pretreatment with phorbol ester, dispersed glomerulosa cells, 10 million in 20 ml medium 199 containing 100 U/ml penicillin, 100 fig/ml streptomycin, 0.1% BSA, 5 Mg/ml insulin, 5 fig/ml transferrin, 0.2 mM ascorbic acid, 5 ^M metyrapone, and 100 mM dimethyl sulfoxide (DMSO) were cultured at 37 C under air/5% CO2, in two 50 ml plastic tubes, one of which contained 0.1 juM TPA. In these experimental conditions steroid responses to stimulation with All and ACTH were maintained for 18 to 24 h after cell dispersion. The use of antioxidants and blockade of glucocorticoid production by metyrapone have been shown to preserve the activity of cytochrome P-450-dependent enzymes of the biosynthetic pathway (21). After 6 h incubation, cells were centrifuged, washed twice with medium 199 at room temperature, resuspended in 20 ml fresh culture media, and culture continued overnight in the absence of TPA. Cells were then pelleted by centrifugation, washed twice in medium 199 containing 0.1% BSA, and used for stimulation with hormones and PKC assay.
Preparation and treatment of adrenal glomerulosa cells
Protein kinase C determination
been reproduced in other experimental conditions in bovine or rat adrenal glomerulosa cells (Ref. 3 and Aguilera G., unpublished). To more directly evaluate the role of PKC in the mechanism of action of All, we have studied the effect of changes in PKC distribution and activity on the ability of All to stimulate aldosterone secretion in rat adrenal glomerulosa cells. The results indicate that although All causes cellular redistribution of PKC activity in the adrenal glomerulosa and PKC can modulate the cell responsiveness to All, PKC activation is not required for steroidogenic effects of the peptide. Materials and Methods Materials
Isolated adrenal glomerulosa cells were prepared from 200250 g female Sprague-Dawley rats (Charles River, Wilmington, MA) by collagenase digestion as previously described (19). Cells were resuspended in medium 199 containing 0.1% BSA at a concentration of 1.5 to 2 million/ml. Two milliliter aliquots were incubated in 20 ml plastic scintillation vials with and without TPA for the time periods indicated, at 37 C under 95% O2/5% CO2. TPA was added after 20 min preincubation, and incubation of all vials was stopped at the same time. For PKC determination, vials were placed on ice, 10 ml ice-cold PBS were added, and the total volume was transferred to 15 ml conical tubes and centrifuged for 10 min at 100 X g to obtain the cell pellet. For measurement of aldosterone production, cells were washed twice with medium 199 at room temperature and resuspended in fresh media at a concentration of 105 cells/ml. It was necessary to wash the cells with medium at room temperature to preserve responsiveness of the cells, and preliminary experiments showed that this step did not influence PKC activity in membrane or cytosol compared with cells in which TPA preincubation was stopped on ice. One milliliter aliquots were incubated for 2 h in 12 X 75 mm glass tubes at 37 C, under 95% O2/5% CO2 in the presence of All, ACTH, and potassium. At the end of the incubation, tubes were centrifuged at 1,500 X g for 10 min and the supernatant decanted and stored at -20 C until aldosterone was determinated by direct RIA (20). The proportion of phorbol ester that remained in the cells after the wash and incubation was determined by [3H]phorbol-12-myristate-13-acetate ([3H]PMA) uptake. Nine aliquots of 0.5 million
Cell pellets were resuspended in 300 /A 20 mM Tris/HCl buffer (pH 7.5) containing 0.5 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 10 MM leupeptin, and 10 fiM pepstatin A (buffer A), and sonicated twice for 2 sec. The homogenate was centrifuged at 105,000 X g for 30 min at 4 C, and the supernatant was used as the cytosol fraction. The pellet was resonicated in buffer A containing 0.1% Nonidet P-40 and centrifuged at 105,000 X g for 30 min, and the supernatant was used as the membrane fraction. To partially purify PKC, 2-3 mg cytosol or solubilized membrane protein were applied to a 1.2 cm diameter X 1.5 cm height DEAE-cellulose column equilibrated with buffer A at 4 C. After washing with 10 ml buffer A, protein kinases were eluted with 1. Phorbol ester washout after preincubation of the cells with [3H]PMA
TABLE
Treatment (after incubation with [3H]PMA)
Cell associated phorbol ester
pmol cpm No wash 29,670 ± 89 1.3 2 washes in M199 1,250 ± 128 0.05 2 washes in M199, dilution, and 30 184 ± 15 0.01 min incubation After 15 min preincubation with [3H]PMA, cells were treated as described in the treatment column and filtered through glass fiber filters (Whatman GF/C). Filters were washed twice with 4 ml ice-cold PBS, and the radioactivity was counted in a liquid scintillation counter. Values are the mean ± SE of triplicate incubations. M199, Medium 199.
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PROTEIN KINASE C ROLE IN All STEROIDOGENESIS a 10 ml linear gradient (0-0.3 M) of NaCl in the same buffer. Fractions of 0.5 ml were collected and assayed for PKC activity. A peak of PKC activity was eluted with 90 mM NaCl (Fig. 1). Therefore, in all subsequent experiments the PKC fraction was obtained by elution of the column with 100 mM NaCl. A small peak of calcium phospholipid independent activity, presumably protein kinase M (85 ± 7.9 and 79 ± 6.2 pmol/mg-min, for cytosol and membrane, respectively) was observed in the fractions eluted with NaCl 150-170, in two of three experiments. In subsequent experiments protein kinase M (PKM) was measured in the 90-200 step elution fraction. PKC activity was assayed by measuring the incorporation of 32 P from [T- 3 2 P] ATP into histone III-S by a modified procedure from that described by Huang et al. (22). The reaction mixture contained 20 mM Tris-HCl (pH 7.5), 5 mM Mg acetate, 0.6 mM CaCl2, 40 MM [T- 3 2 P]ATP (specific activity, 400-600 cpm/
pmol), 1 mg/ml histone III-S, 40 ixg/ml phosphatidylserine, 8 Mg/ml l-2,dioleoylglycerol, and the sample protein in a final volume of 50 n\. The calcium and phospholipid-independent activity was measured under the same conditions without calcium and phospholipids and containing 1 mM EGTA. To examine the contribution of calcium-dependent protein kinase, activity was measured in the presence of 0.6 mM CaCl2. Reactions were initiated by addition of [7-32P]ATP and incubated for 15 min at 37 C, and terminated by adding 3 ml ice-cold 20% trichloroacetic acid containing 1 mM sodium pyrophosphate.
0.10
0.05
0.05
10 15 20 FRACTION NUMBER
-JO
FIG. 1. Anion exchange chromatography of adrenal glomerulosa PKC. Cytosol- or detergent-solubilized membranes from 20 million adrenal glomerulosa cells were applied to a column (1.2 x 1.5 cm) of DEAEcellulose equilibrated with buffer A. After sample addition the column was washed with buffer A and eluted with a linear (0-0.3 M) NaCl gradient in 0.5 ml fractions. PKC was determined in the presence (•— —•) and in the absence (O O) of Ca, phosphatidylserine, and diolein as described in Materials and Methods. Protein concentration is shown by the dotted line. Data are the mean of triplicate determinations.
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Preliminary experiments showed that the time course of PKC activity in cytosol and membrane fractions was linear up to 20 min. Precipitates were collected on filter paper (Whatman, GF/ C), washed twice with 5 ml 5% trichloroacetic acid containing 1 mM sodium pyrophosphate, and dried, and the radioactivity incorporated into protein was counted in a liquid scintillation counter. Determinations were performed using duplicate aliquots from each sample.
Results PKC activity in cytosol and membrane fractions from adrenal glomerulosa cells after DEAE-cellulose chromatography is shown in Fig. 1. Consistent with the recognized elution pattern of PKC, the peak of activity was obtained with 90 mM NaCl. In three experiments, DEAEcellulose chromatography resulted in more than a 10fold enrichment of PKC activity compared with crude fractions, from 77.3 ± 7.6 to 1,020 ± 57.9 pmol/mg-min in cytosol and from 39.3 ± 2.4 to 413.3 ± 14.4 pmol/mgmin in membrane. As shown in Table 2, PKC activity in the 100 mM NaCl elution fractions in the presence of EGTA or calcium alone was markedly reduced compared with activity in the presence of calcium, phosphatidylserine, and diacylglycerol. Therefore, all subsequent experiments were performed using PKC purified on DEAEcellulose. To determine whether All has an effect on PKC in rat adrenal glomerulosa cells, the enzyme activity was measured in cytosol and membrane fractions after incubation of the cells with All (Fig. 2). The time course of the effect of All on PKC activity, in two experiments, shows a small decrease in the cytosol to 94% the basal values at 5 min, followed by a further reduction to 82 ± 6.4%, 75 ± 9.2%, and 74 ± 6.4% at 15, 30, and 60 min, respectively. The converse changes were observed in the membrane fraction with an increase to 106 ± 4.2% and 133 ± 5.7% at 5 and 15 min, respectively, followed by a decrease to 113 ± 7.1% and 99 ± 7.5% at 30 and 60 min, respectively (mean ± SD). NO increase in PKM was observed after incubation of the cells with All up to 60 min. The ability of All to cause PKC translocation suggests that All can activate PKC in the rat adrenal TABLE 2. Effect of calcium, phosphatidylserine (PS), and diacylglycerol (DG) on phosphate incorporation into Histone III-S by DEAEcellulose-purified PKC in cytosol and membrane from adrenal glomerulosa cells
Incubation conditions EGTA Ca PS/DG/Ca
P incorporation (pmol/mg/min) Cytosol
Membrane
84.5 ± 4.6 91.8 ± 6.2 1128.0 ± 30.2
113.3 ± 14.2 145.6 ± 17.8 573.1 ± 59.7
Values are the mean ± SE from four experiments.
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128
PROTEIN KINASE C ROLE IN All STEROIDOGENESIS
15
30 45 TIME (min)
60
FIG. 2. Effect of All treatment on cytosolic and membrane-associated PKC in isolated adrenal glomerulosa cells. Aliquots containing 6 million cells were incubated with 10 nM All for the periods of time indicated and processed for PKC determination as described in Materials and Methods. Data are the mean of triplicate determinations in one of 2 similar experiments.
Endo • 1990 Voll26-Nol
(up to 6 h). In three to seven experiments the mean PKC values decreased from 1235 ± 209 pmol/mg-min to 827 ± 141, 519 ± 135, 341 ± 114, 389 ± 97, 320 ± 130, and 292 ± 111 pmol/mg-min at 5, 15, 30, 60, 180, and 360 min, respectively. The decrease in activity in the cytosol fraction was accompanied by a transient increase in PKC in the membrane, reaching a maximum at 15 min followed by a rapid decline over the next 15 min with a further decrease to basal levels for up to 6 h. The mean values for all experiments were 353 ± 101 for basal, 705 ± 189, 1036 ± 122, 655 ± 143, 436 ± 131, 265 ± 178, and 230 ± 149 pmol/mg-min at 5, 15, 30, 60, 180, and 360 min, respectively. In order to determine whether the decrease in PKC was a consequence of degradation with conversion to PKM, we measured kinase activity in the presence and in the absence of calcium and phospholipids in all fractions eluted from the DEAE-cellulose column or the 200 mM step gradient fractions. Pretreatment of the cells with TPA for 15 min (n = 2), 30 min (n = 4), or 180 min (n = 1) had no effect on Ca/phospholipid independent activity in these fractions, indicating the absence of PKM formation. The aldosterone response of cells preincubated with 0.1 (xM TPA for 15 min and 3 h is shown in Fig. 4. In five experiments after 3 h preincubation with TPA when PKC content of the cells was maximally reduced, aldosterone response to All was identical to that in control cells. In contrast, aldosterone production stimulated by maximal and submaximal concentrations of ACTH was enhanced by 110%, 229%, 78%, and 25% with 1,10, 100, and 1,000 pM, respectively. To study the possibility that residual PKC after the 3 h TPA treatment may be activated by All and account for the steroidogenic effect of the peptide, cells were •n. i.o
30
180 60 TIME (min)
360
FIG. 3. Effect of treatment with TPA on PKC activity isolated from adrenal glomerulosa cells. Cells (6 million) were incubated with 0.1 /*M TPA for the periods of time indicated. Cytosolic and membrane fractions were assayed for PKC activity as described in Materials and Methods. Total PKC activity in the cell was reduced by 70%, from 1652 to 505 pmol/mg-min after 3 h incubation with TPA. Data are the mean ± SE of the values of 3 to 7 experiments.
glomerulosa cell. To determine the role of PKC on the steroidogenic activity of All, the effect of aldosterone stimulants was evaluated in cells in which PKC activity was modified by preincubation with phorbol esters. As illustrated in Fig. 3, treatment of the cells with 0.1 /xM TPA resulted in a marked decrease in PKC activity in the cytosol, reaching about 25% of the basal activity at 3 h, and these levels were maintained with longer preincubation times
0
-11 -10-9 -8 Log All (M)
-12-11-10-9 Log ACTH (M)
6
8 10 12
K+(mM)
FIG. 4. Effect of 15 min (A A) and 3 h (4 \) pretreatment with TPA on the responsiveness of isolated rat adrenal glomerulosa cells to All, ACTH, and potassium. Control cells (O O) were preincubated for 3 h or 15 min in the absence of TPA. After preincubation with the phorbol ester, cells were washed and incubated for 2 h with increasing concentrations of the various stimuli. Data points are the mean ± SE of duplicate incubations in one of five similar experiments.
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PROTEIN KINASE C ROLE IN All STEROIDOGENESIS
preincubated with TPA for 3 h, washed, and then treated with TPA or All for 15 min to assess their ability to induce further PKC translocation. As shown in Table 3, treatment of control cells with TPA or All for 15 min caused the expected changes in cytosolic and membrane PKC. In contrast, when PKC levels were reduced by 3 h preincubation with the phorbol ester, no further changes in PKC activity were observed after 15 min incubation with TPA or AIL The results obtained after 3 h preincubation with TPA were confirmed in two experiments in which cells cultured overnight were preincubated with TPA for 6 h to achieve a more marked reduction in cellular PKC. In these experiments, total PKC was reduced by 91% (from 1187 ± 102 to 95 ± 11 pmol/mgmin in the cytosol and from 338 ± 45 to 36 ± 9 in the membrane). Similar to the results in cells preincubated with TPA for 3 h, aldosterone responses to All were unaffected despite the marked reduction in cellular PKC content (Fig. 5A). Also, consistent with the experiments above, the aldosterone response to ACTH was enhanced in the cells preincubated with TPA for 6 h (Fig. 5B). After 15 min preincubation with TPA which induces maximum PKC translocation to the membrane, aldosteTABLE 3. Effect of 15 min incubation with TPA or All in adrenal glomerulosa cells preincubated with and without 0.1 nM TPA for 3 h Protein Kinase C activity {% of basal) Incubation conditions BASAL TPA All
Control
TPA preincubation
Cytosol
Membrane
Cytosol
Membrane
100 40.7 ± 2.8° 79.3 ± 3.7°
100 289 ±10.1° 135 ± 6.2°
100 99.7 ± 1.2 98.8 ± 0.88
100 101.4 ± 1.8 98.5 ± 1.6
Basal PKC values were 1016 ± 126 and 376 ± 14.8 pmol/mg-min P incorporation for cytosol and membrane, respectively, in the control cells, and 306 ± 30.8 and 336 ± 20 pmol/mg min 32P incorporation for cytosol and membrane, respectively, in TPA-pretreated cells. P < 0.01 vs. basal (n = 3). 32
129
rone responses to AH and ACTH were affected in the opposite manner. While All-stimulated aldosterone production was enhanced by 28%, 65%, 102%, and 171% with 0.01,0.1. .1 and 10 nM All, respectively, the response to ACTH was reduced by 29%, 24%, 19%, and 28% with 1,10,100, and 1,000 pM ACTH, respectively. Potassiumstimulated aldosterone production was completely unaffected by preincubation with TPA for 15 min or 3 h. Preincubation of the cells with the inactive phorbol ester, 4a-hydroxyphorbol 12,13 dibutyrate, had no significant effect on the aldosterone response to subsequent stimulation with other stimulants (not shown). In the experiments described above in which cells were preincubated with TPA for 15 min, the time of maximum PKC membrane translocation, measurement of aldosterone production required a further 2 h incubation period with the stimulators. There is, thus the important question concerning the extent to which PKC remains translocated during the 2 h incubation with the stimulants. In order to evaluate this question, measurement of PKC activity and addition of stimuli were performed at increasing time intervals after the 15 min treatment with TPA. As shown in Fig. 6, PKC activity in the cytosol decreased by 70% after 15 min incubation with TPA and remained at this level for up to 2 h after removal of TPA. In the membrane the 100% increase in activity observed at the end of the TPA preincubation rapidly decreased to basal values at 3 min, and declined to less than basal values 10 min after removal of TPA. To determine whether the changes in aldosterone responses after 15 min TPA preincubation were correlated with the membrane location of PKC activity, All or ACTH was added 2 and 30 min after removal of TPA, when membrane PKC activity was still increased by 50% or was less than
• Cytoeol O Membrane
•n LOG A D (M)
LOG ACTH (M)
FIG. 5. Effect of prolonged incubation with TPA on the responsiveness of isolated rat adrenal glomerulosa cells to All and ACTH. Cells were preincubated for 6 h with 0.1 fiM TPA, washed, and cultured for additional 10 h as described in Materials and Methods. Cells were then washed, resuspended in medium 199/0.1% BSA and incubated for 2 h with ACTH or AIL Data points are the mean ± SE of duplicate incubations in one of two experiments.
-15
0
15
30
120
TIME (min)
FIG. 6. Time course of the changes in PKC activity in cytosol and membrane fractions after preincubation of adrenal glomerulosa cells with TPA for 15 min. After preincubation, cells were washed and incubated in fresh medium 199 for the time periods indicated before PKC determination. Points are the mean of triplicate determinations in two or three experiments.
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PROTEIN KINASE C ROLE IN All STEROIDOGENESIS
130
the basal value, respectively. As observed in the previous experiments All-stimulated aldosterone production was increased by 30% when All was added immediately after the 15 min preincubation with TPA (Fig. 7, time 0). In these experiments the level of potentiation was identical when the addition of All was delayed to 2 or 30 min after the removal of TPA. Similarly ACTH-stimulated aldosterone secretion was inhibited by 30%, irrespective of the time of addition of ACTH, with no delay, 2 or 30 min after removal of TPA. In contrast with the effects of TPA preincubation on stimulated steroidogenesis, direct treatment of the cells for 2 h with TPA had no significant effect on aldosterone production. In six experiments, aldosterone production was unchanged, remaining at 1.0 ± 0.05 times the basal values after incubation with 0.1 M TPA, while with maximum stimulatory concentrations of All, ACTH, and potassium it was increased by 6.8 ± 0.62, 22.3 ± 1.7, and 23.3 ± 3.3 fold, respectively. In two experiments, simultaneous incubation of the cells with 10 nM All and increasing TPA concentrations inhibited rather than potentiating aldosterone production. A small decrease of 14.4 ± 0.84% (mean ± SD) was observed with 0.1 nM TPA. Larger concentrations of 1 juM caused a marked but nonspecific inhibition, since the effects of all stimulators, including that of 8-Br-cAMP were equally decreased. Discussion The purpose of these experiments was to investigate the role of PKC in the steroidogenic action of All in rat
1
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