Prostacyclin: A potent stimulator of adrenal steroidogenesis

PROSTAGLANDINS PROSTACYCLIN: A POTENT STIMULATOR OF A D R E N A L STEROIDOGENESIS E.F.

Ellis,

J.C.

Shen, M.P. Schrey, and R.P. Rubin

R.A. Carchman

Department of Pharmacology, Medical College of Virginia Richmond, Virginia 23298

ABSTRACT The relative potencies of various prostaglandins were investigated in trypsin-dispersed cat adrenocortical cells. Prostacyclin proved to be the most potent steroidogenic prostaglandin, being 100-1000 times more potent than PGE2. This stimulant effect of prostacyclin was only partially dependent upon the presence of extracellular calcium and was associated with increased levels of cyclic AMP. These data suggest a possible role for prostacyclin in corticosteroidogenesis. INTRODUCTION Adrenal corticosteroid synthesis and release is one of the many biological functions in which prostaglandins (PGs) may be an essential component, since a number of laboratories, including our own, have shown that PGs are capable of enhancing steroidogenesis (1-4). Moreover, recent studies from our laboratory have also demonstrated that ACTH stimulates membranous phospholipase activity and PGE 2 and PGF2~ production in isolated cat adrenocortical cells (5,6). Recently, prostacyclin (PGI2), which is formed from the PG cyclic endoperoxides G 2 and H 2 (PGG 2 and PGH2), has been identified in various tissues (7-9). This labile PG has vasodilator properties (I0) and also inhibits platelet aggregation (ii) presumably by augmenting cyclic AMP levels (12). We report in this paper the potent stimulating effects of prostacyclin on corticosteroid production and release by isolated adrenocortical cells. METHODS The experiments were conducted on trypsin-dispersed cat adrenocortical cells incubated with Minimal Eagle Medium as previously described (13). In the experiments involving calcium deprivation, a special solution was prepared which was identical to MEM. When required, calcium was excluded from this solution and EGTA (0.4mM) added

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PROSTAGLANDINS to chelate residual calcium. After incubations of 1 hour the suspension (cells plus medium) was analyzed for corticosteroids by p r o t e i n - b i n d i n g assay (14)~ Cortical cells to be analyzed for cyclic AMP were incubated for 5 min in the p r e s e n c e or absence of prostacyclin. The beaker contents were d e c a n t e d into chilled glass test tubes and c e n t r i f u g e d at high speed for 1 min. The supernatant was discarded and 1 ml of cold t r i c h l o r o a c e t i c acid was added to the pellet. The m i x t u r e was then frozen for subsequent analysis by r a d i o i m m u n o a s s a y (15). P r o s t a c y c l i n was d i s s o l v e d in a m i x t u r e of ethanoltris buffer (9:1) (pH 9.3). The final c o n c e n t r a t i o n of the vehicle in the cell suspension, which never exceeded 0.35%, had no effect on basal or A C T H - s t i m u l a t e d steroid release. RESULTS Effect of p r o s t a c y c l i n

on s t e r o i d o g e n e s i s

A 60 min exposure to p r o s t a c y c l i n p r o d u c e d a dosedependent e n h a n c e m e n t of steroid release (Fig. i). Prostacyclin (10-9M) increased steroid release from a basal value of 59(±9) to 104(±50) ng/ml. Due to v a r i a b i l i t y in responsiveness from one p r e p a r a t i o n to another, this increase was not significant (p>0.5); however, the mean value for steroid release o b t a i n e d with 10-8M p r o s t a c y c l i n (178 ± 47 ng/ml) was s i g n i f i c a n t l y d i f f e r e n t from basal levels (p>0.01). M a x i m u m s t e r o i d o g e n i c responses were demonstrable with 10-5M p r o s t a c y c l i n (Fig. I) w h i c h were comparable in m a g n i t u d e to 200 ~U ACTH, a maximal stimulating c o n c e n t r a t i o n (i). Thus, mean values for steroid release induced by a 60 min exposure to p r o s t a c y c l i n (10-5M) and A C T H (200 ~U) were 371(±62) and 314(±58) ng/ml, respectively (N=4-6). Effect

of other PGs on s t e r o i d o g e n e s i s

Since the biologic activity of p r o s t a c y c l i n disappears w i t h i n i0 min at 37°C (7), and is readily converted to 6keto-PGFl , it was necessary to ascertain the relative p o t e n c y o~ this metabolite. A l t h o u g h 6-keto-PGFl~ was capable of enhancing steroid release, it was 100-1000 times less potent than p r o s t a c y c l i n (Fig. i). Similarly PGE~ and PGH 2 augmented steroid release, but they also were much less potent than prostacyclin, requiring threshold concentrations 100-1000 times greater than prostacyclin. At the highest c o n c e n t r a t i o n of PGH 2 tested (10-4M), the steroidogenic response d e c l i n e d from peak levels (Fig. i). The effects of PGH 2 may be the result of its conversion to some other active PG - possibly p r o s t a c y c l i n - since the stable m e t h y l e n e analogue of PGH~ (U-46619) p r o d u c e d i n c o n s i s t e n t and weak s t e r o i d o g e n i c activity at doses of 2 x 10-5 to 9 x 10-5M.

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PROSTAGLANDINS

Time course of prostac~clin action PGI 2 and ACTH augmented steroid release from a basal level of 37(±5) to 136(±42) and 117(±25) ng/ml, 500-

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PG conc.,M. Fig. i. Dose-response curves for PG-induced steroid release by isolated feline adrenocortical cells. Following dispersion by trypsin, adrenocortical cells (approximately 2.5 x 10~/ml) were incubated for 60 min at 37°C in 1.0 ml of Modified Eagle medium containing 0.2% bovine serum albumin plus 0.04% trypsin inhibitor. Stimulating agents were added as specified. Following incubation, the suspension was centrifuged and the corticosteroids were extracted from the supernatant with methylene chloride and assayed by competitive protein binding. Each point represents mean steroid release (±S.E.) derived from 3 or more different preparations using the following stimuli: prostacyclin • ; 6-ket°-PGFl~ A ; P G H 2 0 ; PGE2 /%,respectively within the first 15 min of incubation (Fig. 2) (p<0.05). However, the steroidogenic response to 10-6M prostacyclin was linear only up to 30 min, in contrast to the response to ACTH which was linear up to 90 min (Fig. 2). The loss of linearity

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PROSTAGLANDINS

with prostacyclin was probably related to its rapid degradation, as evidenced by maintained linearity of the response for up to 90 min with 10-4M prostacyclin (data not shown).

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Fi~. 2. Time course of steroid release evoked by prostacyclin and ACTH. Cells were incubated with and without prostacyclin (10-6M) or ACTH (50 ~U) for varying time intervals; the cells were centrifuged and the supernatant assayed for corticoids. The mean values (±S.E.) for basal [] and prostacyclin • and ACTH B stimulated release were derived from 4 different preparations. Effect of calcium deprivation on prostacyclin induced steroido~enesis Previous studies from our laboratory have demon-

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PROSTAGLANDINS s t r a t e d that the s t e r o i d o g e n i c a c t i o n of PGE~ is o n l y p a r t i a l l y d e p e n d e n t upon e x t r a c e l l u l a r c a l c i u m (i). Similarly, w h e n the i n c u b a t i o n m e d i u m was d e v o i d of c a l c i u m (Table i), the s t e r o i d o g e n i c r e s p o n s e to p r o s t a c y c l i n w a s d e p r e s s e d to 43% of control, in c o n t r a s t to the c o m p l e t e o b l i t e r a t i o n of the r e s p o n s e to an equip o t e n t c o n c e n t r a t i o n of A C T H (Table i). It s h o u l d be n o t e d that w i t h the p r e p a r e d m e d i u m u s e d in these e x p e r i m e n t s (see Methods) p r o s t a c y c l i n (see Fig. i) and A C T H w e r e i n e x p l i c a b l y less potent. Table

1

C o m p a r a t i v e E f f e c t s of C a l c i u m D e p r i v a t i o n on the S t e r o i d o g e n i c R e s p o n s e to P r o s t a c y c l i n and A C T H

ng/2.5 Expt Prostac~clin (10-~M)

Steroid x 105 cells % of C o n t r o l

+Ca +2

-Ca +2

Mean

1 2 3 ± SEM

125 43 84 84 ± 24

Mean

1 2 3 ± SEM

83 90 i01 91 ± 5

ACTH (5 ~U)

49 19 39 36 ± 9*

39 44 46 43 ± 2

7 5 3 5 ± i*

8 5 3 5 ± 1

C o r t i c a l cells w e r e i n c u b a t e d for 60 m i n in n o r m a l or c a l c i u m free m e d i u m in the p r e s e n c e or a b s e n c e of the a p p r o p r i a t e stimulus. Data are e x p r e s s e d as n g / 2 . 5 x 105 cells. The v a l u e s for s t i m u l a t e d cells in the a b s e n c e of c a l c i u m are also r e p r e s e n t e d as a p e r c e n t of v a l u e s d e r i v e d from c o r r e s p o n d i n g cells i n c u b a t e d w i t h calcium. A v e r a g e basal v a l u e s in the p r e s e n c e and a b s e n c e of c a l c i u m w e r e 8(±4) and 6(±5) ng, r e s p e c t i v e l y . * S i g n i f i c a n t l y d i f f e r e n t from c o r r e s p o n d i n g controls (p<0.05) as d e t e r m i n e d by o n e - t a i l t-test. E f f e c t of p r o s t a c y c l i n

on cyclic AMP

levels

Since p r o s t a c y c l i n is a p o t e n t s t i m u l a t o r of p l a t e let c y c l i c AMP (12) some p r e l i m i n a r y e x p e r i m e n t s w e r e c a r r i e d out on the e f f e c t of p r o s t a c y c l i n on cyclic AMP levels in c o r t i c a l cells. Cyclic AMP a c c u m u l a t i o n i n c r e a s e d m o r e than 3 . 5 - f o l d after 5 min in r e s p o n s e to p r o s t a c y c l i n (10-6M) (Table 2). Accurate measurement of cyclic AMP levels at e a r l i e r time p o i n t s was not

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PROSTAGLANDINS possible due to the experimental conditions employed. Table 2 Effect of Prostacyclin on Cortical Cyclic AMP Levels Cyclic AMP Content pmols/105 cells Control Prostacyclin

(10-6M)

0.52 ± 0.12 1.46 ± 0.ii

% Control 355 ± 122"

Cells were incubated with and without prostacyclin for 5 min and analyzed for cyclic AMP. The mean values (±SEM) represent cyclic AMP content of pellets obtained from 4 different preparations. *Average relative increase in cyclic AMP formation induced by prostacyclin. Paired t test analysis gives p<0.02 for differences between cyclic AMP levels in prostacyclin-treated and in unstimulated cells. DISCUSSION Previous reports from our laboratory have provided evidence that certain effects of ACTH may be expressed through the activation of phospholipase and the resulting enhancement of PG synthesis (17). The present experiments have shown that the more labile prostacyclin is by far the most potent steroidogenic PG so far encountered, possessing steroid-releasing activity comparable to ACTH; in fact, the lability of prostacyclin may be masking an even greater potency. In view of its potent steroidogenic capacity, it seems reasonable to speculate that prostacyclin may be a mediator of steroid synthesis and release. It is relevant to note here that prostacyclin synthetase activity has recently been demonstrated in bovine and monkey adrenal glands, as detected by the formation of its stable metabolite 6-keto-PGFl~ from radiolabeled PGH 2 (18). However, more quantitative information relating to prostacyclin concentrations in cortical cells is required to compare endogenous levels with concentrations of exogenous prostacyclin required to elicit steroid release. The potential significance of prostacyclin as a mediator of steroidogenesis as suggested by the present investigation may have important implications for characterizing the nature of this process, especially with regard to the critical roles of calcium and cyclic nucleotides (19,20). Some insight into this problem may

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PROSTAGLANDINS be gleaned from the finding that the steroidogenic action of prostacylin was only partially dependent upon extracellular calcium, in contrast to the absolute calcium-dependency of the steroidogenic response to ACTH. These data may be interpreted to mean that prostacyclin circumvents one or more calcium-dependent events which take place at the cell membrane, as for example the calcium-dependent activation of phospholipase A by ACTH (5,17). It should also be noted, however, that prostacyclin was capable of producing an early activation of the adenylate cyclase-cAMP system in isolated cortical cells, suggesting that expression of at least certain effects of prostacyclin is mediated through cyclic AMP. Moreover, since calcium appears to exert a primary action on adenylate cyclase (21), at least a portion of the inhibitory effect of calciumdeprivation on evoked steroidogenesis may be a consequence of impaired cyclic AMP formation. Extrapolation of the potent effects of prostacyclin on steroid production and release to other secretory organs appears tentatively justified on the basis of a recent publication reporting the potent stimulant action of prostacyclin on renin secretion (22). We anticipate further studies on other secretory systems testing the hypothesis that prostacyclin is a critical mediator of events associated with the secretory process. ACKNOWLEDGEMENTS We thank Drs. J.E. Pike and G.L. Bundy of the Upjohn Company for generously supplying the various PGs used in this study. Synthetic ACTH (8 1-24) (Synacthen) was contributed by Ciba Pharmaceuticals. This work was supported by the USPHS (Grant AM 18066). REFERENCES 1. 2. 3. 4.

5.

Warner, W. & Rubin, R.P. Evidence for a possible prostaglandin link in ACTH-induced steroidogenesis. Prostaglandins 9:83-95, 1975. Flack J.D., Jessup, R. & Ramwell, P.W. Prostaglandin stimulation of rat corticosteroidogenesis. Science 163:691, 1969. Saruta, T. & Kaplan, N.M. Adrenocortical steroidogenesis: The effects of prostaglandins. J. Clin. Invest. 51:2246-2251, 1972. Honn, W. & Chavin, W. Effects of A and B series prostaglandins on cAMP, cortisol and aldosterone production by the human adrenal. Biochem. Biophys. Res. Commun. 76:977-982, 1977. Laychock, S.G~, Franson, R.C., Weglicki, W.B. & Rubin, R.P. Identification and partial characterization of phospholipases in isolated adreno-

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490

cortical cells: The effects of ACTH and calcium. Biochem. J. 164:753-756, 1977. Laychock, S.G., Warner, W. & Rubin, R.P. Further studies on the mechanisms controlling prostaglandin biosynthesis in the cat adrenal cortex: the role of calcium and cyclic AMP. Endocrinology 100:7481, 1977. Gryglewski, R.J., Bunting, S., Moncada, S., Flower, R.J. & Vane, J.R. Arterial walls are protected against deposition of platelet thrombi by a substance (prostaglandin X) which they make from prostaglandin endoperoxides. Prostaglandins 12: 685-713, 1976. Villa, S. & deGaetano, G. Prostacyclin-like activity in rat vascular tissues. Fast, long-lasting inhibition by treatment with lysine acetylsalicylate. Prostaglandins 14:1117-1124, 1977. Pace-Asciak, C.R. & N ~ h a t , M. Mechanistic studies on the biosynthesis of 6-ketoprostaglandin Flu. Biochim. Biophys. Acta 487:495-507, 1977. Dusting, G.J., Moncada, S. & Vane, J.R. Prostacyclin (PGX) is the endogenous metabolite responsible for relaxation of coronary arteries induced by arachidonic acid. Prostaglandins 13:3-15, 1977. Moncada, S., Gryglewski, R., Bunding, S. & Vane, J.R. An enzyme isolated from arteries transforms prostaglandin endoperoxides to an unstable substance that inhibits platelet aggregation. Nature 263:663-665, 1976. Gorman, R.R., Bunting, S. & Miller, O.V° Modulation of human platelet adenylate cyolase by prostacyclin (PGX). Prostaglandins 13:377-388, 1977. Rubin, R.P. & Warner, W. Nicotine-induced stimulation of steroidogenesis in adrenocortical cells of the cat. Brit. J. Pharmaccl. 53:357-362, 1975. Jaanus, S.D., Carchman, R.A. & Ru~-fn, R.P. Further studies on the relationship between cyclic AMP levels and adrenocortical activity. Endocrinology 91:8870895, 1972. ~ b i n , R.P., Laychock, S.G. & End, D. On the role of cyclic AMP and cyclic GMP in steroid production by bovine cortical cells. Biochim. Biophys. Acta 496:329-338, 1977. Johnson, R.A., Morton, D.R., Kinner, J.H., Gorman, R.P., McGuire, J.C. & Sun, F.F. The chemical structure of prostaglandin X (prostacyclin). Prostaglandins 12:915-928, 1978. Rubin, R.P. & L ~ c h o c k , S.G. Prostaglandins and calcium-membrane interactions in secretory glands. Ann. N.Y. Acad. Sci. 307:377-390, 1978. Sun, F.F., Chapman, J.P. & McGuire, J.C. Metabolism of prostaglandin endoperoxide in animal tissues. Prostaglandins 14:1055-1074, 1977. Rubin, R.P. Calcium and t-he Secretory Process. Plenum Press, 1974. Kuehl, F.A. Prostaglandins, cyclic nucleotides and cell function. Prostaglandins 5:325-340, 1974. Rubin, R.P., Jaanus, S.D. & Car~hman, R.A. The role of calcium and adenosine cyclic 3'5' phosphate in the action of adrenocorticotropin. Nature 240:150-152, 1972. Whorton~--A.R., Misono, K., Hollifield, J.C., Frolich, J.C., Inagami, T. & Oates, J.A. Prostaglandins and renin release: I. Stimulation of renin release from rabbit renal cortical slices by PGI 2. Prostaglandins 14:1095-1104, 1977.

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