In rat thecal-interstitial cells (
TIC), treatment with the
synthetic androgen mibolerone has led to the documentation of an autoregulatory process for
androgen production. In the present study, accumulated evidence has provided insight into the mechanisms of
mibolerone action that control this process. Investigations using the nonsteroidal
antiandrogen hydroxyflutamide were conducted to characterize
mibolerone's mode of action.
Hydroxyflutamide had differential effects on hCG action, the 1-microM dose stimulating hCG-induced
androsterone synthesis by 27% and the 10-microM concentration decreasing the
androgen levels by 84%. In addition, treatment with 1 microM
hydroxyflutamide was effective in partially reversing the inhibitory action of
mibolerone on hCG-stimulated
androsterone production. Thus, the data indicated that
mibolerone's mode of action may be mediated, at least in part, via the
androgen receptor. The possibility that
mibolerone had multiple sites of action prompted studies on the effectiveness of this
androgen to alter various signaling pathways. Treatment with increasing concentrations (0.01-100 nM) of the
phorbol ester 12-0-tetradecanoylphorbol 13-acetate (TPA), which activates
protein kinase C, resulted in a 75% decrease in hCG-stimulated
androgen production at a dose of 100 nM TPA. Treatment with
mibolerone (100 nM) was unable to alter the action of TPA on
androgen synthesis when doses of 1 and 10 nM TPA were employed. It was also found that Ca2+ can serve as a mediator of
mibolerone action. Treatment with a 0.01-microM dose of
A23187, a Ca2+
ionophore known to increase intracellular Ca2+, was ineffective in altering hCG-stimulated
androsterone synthesis. The concurrent treatment of
mibolerone (100 nM) and
A23187 (0.01 microM) resulted in the potentiation of
mibolerone's inhibitory effects on hCG-stimulated
androgen production, thereby suggesting that
mibolerone can stimulate Ca2+ influx. Additional studies revealed that the administration of a 1-microM dose of the L-type Ca2+ channel blocker
verapamil to
TIC cultures was able to partially block the inhibitory effect of
mibolerone on
androgen synthesis. Evidence for an additional site of
mibolerone action was revealed through an analysis of the
mRNA levels of P450scc and P450(17) alpha
enzymes. Although hCG and
insulin-like growth factor I treatment resulted in 20- and 32-fold increases in the amount of P450scc and P450(17) alpha
mRNA, respectively, the addition of
mibolerone (100 nM) reduced only P450(17) alpha
mRNA levels by 91%. Overall, the evidence indicates that
mibolerone has multiple sites of action in exerting its regulatory effect on
androgen synthesis.