MECHANISMS OF ACTION FOR AN ANDROGEN-MEDIATED AUTOREGULATORY PROCESS IN RAT THECAL-INTERSTITIAL CELLS

In rat thecal-interstitial cells (TIC), treatment with the synthetic a ndrogen mibolerone has led to the documentation of an autoregulatory p rocess for androgen production. In the present study, accumulated evid ence h2s provided insight into the mechanisms of mibolerone action tha t control this process. Investigations using the nonsteroidal antiandr ogen hydroxyflutamide were conducted to characterize mibolerone's mode of action. Hydroxyflutamide had differential effects on hCG action, t he 1-muM dose stimulating hCG-induced androsterone synthesis by 27% an d the 10-muM concentration decreasing the androgen levels by 84%. In a ddition, treatment with 1 muM hydroxyflutamide was effective in partia lly reversing the inhibitory action of mibolerone on hCG-stimulated an drosterone production. Thus, the data indicated that mibolerone's mode of action may be mediated, at least in part, via dic androgen recepto r. The possibility that mibolerone had multiple sites of action prompt ed studies on the effectiveness of this androgen to alter various sign aling pathways. Treatment with increasing concentrations (0.01-100 nM) of the phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), whic h activates protein kinase C, resulted in a 75% decrease in hCG-stimul ated androgen production at a dose of 100 nM TPA. Treatment with mibol erone (100 nM) was unable to alter the action of TPA on androgen synth esis when doses of 1 and 10 nM TPA were employed. It was also found th at Ca2+ can serve as a mediator of mibolerone action. Treatment with a 0.01-muM dose of A23187, a Ca2+ ionophore known to increase intracell ular Ca2+, was ineffective in altering hCG-stimulated androsterone syn thesis. The concurrent treatment of mibolerone (100 nM) and A23187 (0. 01 muM) resulted in the potentiation of mibolerorie's inhibitory effec ts on hCG-stimulated androgen production, thereby suggesting that mibo lerone can stimulate Ca2+ influx. Additional studies revealed that the administration of a 1-muM dose of the L-type Ca2+ channel blocker ver apamil to TIC cultures was able to partially block the inhibitory effe ct of mibolerone on androgen synthesis. Evidence for an additional sit e of mibolerone action was revealed through an analysis of the mRNA le vels of P450(src) and P450(17alpha) enzymes. Although hCG and insulin- like growth factor I treatment resulted in 20- and 32-fold increases i n the amount of P450scc and P450(17alpha) mRNA, respectively, the addi tion of mibolerone (100 nM) reduced only P450(17alpha) mRNA levels by 91%. Overall, the evidence indicates that mibolerone has multiple site s of action in exerting its regulatory effect on androgen synthesis.