Effect of protein kinase inhibitors on ACTH-stimulated aldosterone production in rat zona glomerulosa cells

Life Sciences, Vol. Printed in the USA

51, pp.

i157-i163

Pergamon

Press

EFFECT OF PROTEIN KINASE INHIBITORS ON ACTH-STIMULATED ALDOSTERONE PRODUCTION IN RAT ZONA GLOMERULOSA CELLS Doris Kurscheid-Reich*, Lutz Hegemann#, and Stefan Wohlfeil** * Yale University, School of Medicine, Dept. Internal Medicine, New Haven, USA # Thomas Jefferson University, Dept. Dermatology, Philadelphia, USA **Bayer AG, Pharma Research Center, Institute for Biochemistry,Wuppertal, Germany (Received

in final

form July 27,

1992)

Summarv In order to obtain further evidence for the involvement of protein kinases in the short-term ACTH-stimulated aldosterone synthesis in rat zona glomerulosa cells, the effects of three different compounds with protein kinase inhibitory properties were investigated. Staurosporine, H-7 and trifluoperazine inhibited ACTH-stimulated aldosterone release in a dosedependent manner. While the inhibitory effect of H-7 was reversible upon washing of the cells with inhibitor-free medium, the inhibition was maintained in cells treated with staurosporine or trifluoperazine. In contrast to the stimulated production, basal release of aldosterone even at the highest drug concentrations tested was not completely inhibited. We thus conclude that protein kinases may play a crucial role in short-term ACTH-stimulated aldosterone production in rat glomerulosa cells. It is well accepted that binding of adrenocorticotropin (ACTH) to its receptor in zona glomerulosa cells leads to an intracellular increase in cyclic adenosine 3', 5' -monophosphate (cAMP) (1,2, 3). Recent investigations have shown that also other second messengers such as free cytosolic calcium (4, 5) and diacylglycerol (4, 6) are involved in the signal transduction process of ACTH. Calcium ions are known to activate different protein kinases, such as Ca2+/calmodulin-dependent protein kinase (7) and, together with diacylglycerol, Ca2+/phospholipid-dependent protein kinase (PKC) (8). Since ACTH has been demonstrated to induce protein phosphorylation in zona glomerulosa cells (9, 10, 11, 12), a crucial role for protein kinases in the regulation of aldosterone secretion is likely. The stimulatory activity of ACTH on steroidogenesis can be distinguished by two phases. In its short-term action it activates the conversion of cholesterol to pregnenolone by supplying cholesterol to the precursor pool available for steroidogenesis (13, 14, 15). Its long-term effect is to stimulate biosynthesis of the enzymes involved in steroidogenesis by increasing their mRNA levels (16, 17, 18). Increased mRNA levels could be detected not earlier than four hours after the onset of Corresponding Author: Dr. Doris Kurscheid-Reich, Yale University, School of Medicine, Department of Internal Medicine, P.O. Box 3333, Fitkin 134, New Haven, CT 065108O56, USA Copyright

0024-3205/92 $5.00 + .00 © 1992 Pergamon Press Ltd All rights

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ACTH treatment (17). The mode by which ACTH regulates the short-term action on steroidogenesis is still uncertain. In contrast to its long-term action, the role of protein kinases in the activation of the early phase has not been positively identified (3). The present study was designed in order to obtain further evidence for the involvement of protein kinases in the short term ACTH-induced aldosterone production in zona glomerulosa cells. Therefore, the effects of three different, chemically unrelated compounds were investigated. Staurosporine and 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H-7) are known to inhibit protein kinase activity in general (for review, see 19). Since the PKC activating diacylglycerol has been demonstrated to be released during ACTH-induced transmembrane signaling (4, 5) we also included trifluoperazine in our study, which has been shown to act at the regulatory domain of PKC (20). Materials and Methods Animals and Chemicals: Female, adult Wistar rats (250-300 g) were obtained from Winkelmann, Borchem, Germany. The following chemicals were purchased from the companies indicated. ACTH (1-24), 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine (H-7) from Sigma Chemical Co., St. Louis, MO, USA; staurosporine, Dulbecco's modified eagle medium (DMEM), L-Glutamin from Boehringer Mannheim, Mannheim,Germany; trifluoperazine from Smith, Kline and French Laboratories, Philadelphia, PA, USA; Hepes-buffer from GIBCO-Europe GmbH, Karlsruhe, Germany; penicillin/streptomycinsolution from Flow Lab., Scotland; collagenase "Worthington", Type CLS II (173 U/mg) from Biochrom KG, Berlin, Germany. Preoaration of zona olomerulosa cells from the rat: Zona glomerulosa cells were prepared according to the method of Tait et al. (21). Shortly, rats were killed in CO 2saturated atmosphere and both adrenals were taken out. The medulla and the two inner zones of the cortex were removed by extrusion. To isolate the cells, the capsular portion with the attached zona glomerulosa were incubated with collagenase (80 mg/ 20 ml incubation medium) at 37 oC for 1,5 h with shaking. The remaining tissue in the suspension was removed by filtering through nylon gauze. After centrifugation (200 xg, 15 min) the cells were suspended in fresh incubation medium (DMEM with 1% Hepesbuffer, 1% L-Glutamin, 1% penicillin/streptomycin-solution, 0.2% bovine serum albumin). Contamination with other than zona glomerulosa cells (zona fasciculata cells, red and white blood cells) made up ca. 10%. Viability of the cells were examined by dye exclusion with trypan blue and made up more than 95%. Incv~ation of zona olomerulosa cells: The cell suspension was dispensed into Eppendorf - 2.2 ml - plastic tubes at a level of 2-3 x 105 cells/ml. Staurosporine (10 -9 10-5 M), H-7 (10 `7 - 10-4 M) and trifluoperazine (10 -7 - 10-4 M) were added in triplicate for each dosage. After 1 h-incubation (95% 0 2, 7% CO 2, 37 oc) ACTH (5 x 10 -9 M) was added and incubation was continued for 2 h. For investigating the effects of the inhibitors on basal aldosterone production, the cell suspension was first preincubated for 2 h in incubation medium. Then, cells were centrifuged, resuspended in fresh incubation medium, and dispensed into plastic tubes. Staurosporine, H-7 and trifluoperazine were added and incubation was continued for 2 h. For investigating the reversibility of the inhibition of aldosterone release, zona glomerulosa cells were preincubated with different concentrations of the appropriate inhibitor for 1 h, then washed twice with medium and resuspended in fresh incubation medium without inhibitor. ACTH (5 x 10 -9 M) was added to the tubes and incubation was continued for 2 h. Viability of the cells after each incubation was determined using trypan blue.

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Extraction of aldosterone from the incubation medium and radioimmunoassav of aldosterone: Aldosterone was extracted from the incubation medium using S-EP-PAK C18-cartridges (Millipore, Milford, USA). The cartridges were prewetted with 2 ml of acetonitril 100% and flushed with 10 ml aq. dest., before 800 t~1 of the sample (incubation medium with produced aldosterone) were applied. After washing the cartridges with 5 ml of aq. dest. and 2 ml of acetonitril 20%, aldosterone was eluted with 3 x 1 ml of acetonitril 90%. Recovery of the extraction was 90%, as judged from extracted radioactive aldosterone. The eluates were lyophilized and resolved in human, aldosterone-free plasma. The aldosterone content of the samples was determined by a radioimmunoassay-Kit (Aldosterone-MAIA), obtained from Serono, Freiburg, Germany.

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Fig. 1 Inhibition of short-term ACTH (5 x 10 -9 M)-induced increase of aldosterone production by staurosporine, H-7 and trifluoperazine. Data represent the means + SE of triplicate values from one out of three independent experiments. The absence of error bars indicates a SE smaller than the size of the symbol. Basal aldosterone production was 0.75 + 0.25 ng/2-3 x 105 cells, ACTH-stimulated aldosterone release was 7.1+ 2.3 ng/2-3 x 105 cells. Results and Discussion In the present study, all three compounds, staurosporine, H-7 and trifluoperazine, dose-dependently inhibited ACTH-stimulated aldosterone production from rat zona glomerulosa cells (Fig. 1). Staurosporine was the most potent inhibitor, which is consistent with other investigations on cellular systems as well as with isolated PKC. It acted on the ACTH-induced aldosterone release with an IC50-value of 350 + 120 nM. This is in agreement with reports, where staurosporine inhibited specifically protein kinase-mediated effects in other cell systems with IC50-values that varied between 20 nM and 10 I~M (for review: 19). The ACTH-induced cellular response was less potently inhibited by H-7 and trifluoperazine, with IC50-values of 28 + 8 I~M and 30 + 12 I~M, respectively. Both compounds are reported to act in vivo and in vitro with IC50-values between 10 and 100 I~M (19). As examined by the trypan blue exclusion method none

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of the compounds even at the highest concentrations tested, showed reduced cell viability. The inhibition of hormone secretion observed with the different drugs are thus not thougt to be related to cytotoxicity. The inhibitory effects of staurosporine and H-7, which are thought to be kinase inhibitors in general (for review: 19), strongly indicate that protein kinases play a regulatory role in short-term ACTH-induced aldosterone production. Since trifluoperazine is known as a dual PKC/calmodulin inhibitor (22, 23), whereas staurosporine does not antagonize calmodulin function (Hegemann, unpublished data), the present data point to a regulatory role of PKC and/or calcium/calmodulindependent protein kinases in this cellular response.

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Fig. 2 Inhibition of basal aldosterone production by staurosporine (10 -6 M), trifluoperazine (10 -4 M) and H-7 (10 -4 M). Data represent the mean + SE of triplicate values from one out of three independent experiments. In order to determine whether the compounds tested specifically interfered with the ACTH-stimulated aldosterone production or with the cellular response in general, the drugs were also tested on basal aldosterone release from zona glomerulosa cells. Cells first passed a preincubation period of two hours in order to assure that cells were in unstimulated, basal conditions before the study was carried out. In contrast to our findings after stimulation with ACTH, none of the drugs tested even in high concentrations (10 -6 M for staurosporine, 10 -4 M for H-7 and trifluoperazine) was able to completely inhibit basal aldosterone release; staurosporine, H-7 and trifluoperazine maximally decreased zona glomerulosa hormone secretion by 52, 62 and 68 %, respectively (Fig. 2) Therefore, the present data indicate that different mechanisms might underly the regulation of basal aldosterone release and the release after stimulation with ACTH. In further experiments, reversibility of the inhibitor action on ACTH-induced aldosterone production was examined. Although the cells were thoroughly washed after preincubation, the inhibition of staurosporine and trifluoperazine on aldosterone release persisted, while the action of H-7 was completely abolished (Fig. 3 a-c). After preincubation with H-7 at the maximum concentration tested (10 -4 M), the cells showed normal stimulation with ACTH.

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a)

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Fig. 3 a-c Reversibility of the inhibitory action of staurosporine, H-7 and trifluoperazine on the ACTH (5 x 10-9 M)-induced increase of aldosterone production. In each figure data represent the means + SE of triplicate values from one out of three independent experiments.

In contrast to our finding, staurosporine-induced inhibition of luteinizing hormone secretion from rat anterior pituitary cells has been described as being reversible after washing (24), whereas, in a different study (25), inhibition of PKC-mediated protein phosphorylation by staurosporine was found to be sustained after washing inside-out-

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vesicles from erythrocyte membranes with inhibitor-free buffer. However, staurosporine (26), as well as trifluoperazine (22, 23) have been shown to bind to protein kinases and/or calmodulin in a reversible manner. Therefore, it seems most likely that different physico-chemical properties of the various cells with respect to their membrane permeability for the specific drug or different washing and incubation conditions account for these conflicting results. We thus conclude that protein kinases are involved in the early phase of ACTH-induced aldosterone production from rat zona glomerulosa cells. It seems most likely that not only cAMP-dependent protein kinase (27) but also protein kinase C and/or calcium/calmodulin-dependent protein kinase are involved in the regulation of the early phase of steroidogenesis. The aldosterone-stimulating activity of phorbol esters, known activators of protein kinase C, in adrenal glomerulosa cells show the existence of PKC in this cell system (28). However, further studies are necessary to elaborate the exact role of the different kinases. References 1. R.C. HAYNES, S.B. KORITZ and F.G. PERON, J. Biol. Chem. 234 1421-1423 (1959), 2. D.G. GRAHAM-SMITH, R.W. BUTCHER, R.L. NEY and E.W. SUTHERLAND, J. Biol. Chem. 242 5535-5541 (1967). 3. P.J. HORNSBY, Hormones and their Actions. part II, B.A. Cooke et al. (eds.), 193210, Elsevier Science Publishers, (1988). 4. R.B. FARESE, L.F. FANJUL, C.M DE RUIZ GALARRETA, J.S. DAVIS and D.R. COOPER, Life Sci. 41 2631-2632 (1987). 5. H. RASMUSSEN and B.Q. BARRETT, Hormones and their Actions, part II, B.A. Cooke et al. (eds.), 93-111, Elsevier Science Publishers, (1988). 6. E.N. COZZA, M. DEL CARMEN VILA, M. ACEVEDO-DUNCAN, C.E. GOMEZSANCHEZ and R.V. FARESE, J. Steroid Biochem. 35 343-351 (1990). 7. A.M. EDELMAN, D.K. BLUMENTHAL and E.C. KREBS, Ann. Rev. Biochem. 56 567613 (1987). 8. Y. NISHIZUKA, Sciene 233 305-312 (1986). 9. T.M. KOROSCIL and S. GALLANT, J. Biol. Chem. 225 6276-6283 (1980). 10. A.F. BRISTOW, D. SCHULSTER and R. RODNIGHT, Biochim. Biophys. Acta 67,5 24-28 (1981). 11. K. MIKAMI and C.A. STROTT, Biochem. Biophys. Res. Commun. 138 895-901 (1986). 12. M.E. ELLIOTT, Life Sci. 46 1479-1488 (1990). 13. P.T. KOVANEN, J.R. FAUST, M.S. BROWN and J.L. GOLDSTEIN, Endocrinology 104 599-609 (1979). 14. G.V. VAHOUNY, R. CHANDERBHAN, B.J. NOLAND and T.J. SCALLEN, Endocrine Res. 10 473-505 (1984/85). 15. G.S. BOYD, B. MCNAMARA, K.E. SUCKLING and D.R. TOCHER, J. Steroid Biochem. 1~).1017-1027 (1983). 16. M.R. WATERMAN and E.R. SIMPSON, Mol. Cell. Endocrinol. 3981-89 (1985). 17. E.R. SIMPSON and M.R. WATERMAN, Ann. Rev. Phys. 50427-440 (1988). 18. W.L. MILLER, Endocrine Res. 15 1611-1618 (1989). 19. U.T. RUEGG and G.M. BURGESS, Trends in Pharmacol. Sci. 10218-220 (1989). 20. S. PONTREMOLI, E. MELLONI, M. MICHETTI, O. SACCO, F. SALAMINO, B. SPARATORE and B.L. HORECKER, J. Biol. Chem. 261 8309-8313 (1986). 21. J.F. TAIT, S.A.S. TAIT, R.P. GOULD and M.S.R. MEE, Proc. R. Soc. Lond. B 18,5 375-385 (1974). 22. B.C. WISE and J.F. KUO, Biochem. Pharmacol. 32 1259-1265 (1983).

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23. L. MASSOM, H. LEE and H.W. JARRETT, Biochemistry 29671-681 (1990). 24. H. DAN-COHEN and Z. NAOR, Mol. Cell. Endocrinol. 69 135-144 (1990). 25. M. WOLF and M. BAGGIOLINI, Biochem. Biophys. Res. Commun. 155 643-648 (1988). 26. J.M. HERBERT, E. SEBAN and J.P. MAFFRAND, Biochem. Biophys. Res. Commun. 171 189-195 (1990). 27. L.D. GARREN, G.N. GILL, H. MASIN and G.M WALTON, Recent Prog. Horm. Res. 27 433-439 (1971). 28. D. LANGLOIS, J.-M. SAEZ, M. BEGEOT, Biochem. Biophys. Res. Commun. 146 517-523 (1987)