Effect of a tumour-promoting phorbol ester on atrial peptide-induced testosterone production and cyclic GMP accumulation by isolated mouse Leydig cells

Abstract

The objective of this study was to investigate the effects of 4β-phorbol 12-myristate 13-acetate (4β-PMA) — a potent activator of protein kinase C — on the responsiveness of mouse Leydig cells to stimulation with rat atriopeptin II (rAP-II). We report that, in these cells, the stimulation of testosterone production by rAP-II could be inhibited in a dose-dependent manner by 4β-PMA (1–200 nM). In contrast, the basal steroidogenesis was stimulated 2-fold by 4β-PMA. There was no inhibition of testosterone production when the cells were stimulated with 8-bromo cyclic GMP (8Br-cGMP) in the presence of 4β-PMA. Furthermore, addition of 4β-PMA resulted in a marked reduction in the amount of cGMP accumulated in response to rAP-II stimulation. 4α-Phorbol 12-myristate 13-acetate (4α-PMA) was found to have no effect at all. The inhibitory effect of 4β-PMA on-steroidogenesis could be completely reversed by the addition of 0.25 mM 3-isobutyl 1-methylxanthine (IBMX), a phosphodiesterase inhibitor. Also, the 4β-PMA-induced lowering of cGMP content could be partially reversed by IBMX. Membrane fractions from cells treated with 4β-PMA or 4α-PMA did not differ in their contents of either basal or rAP-II-stimulated guanylate cyclase activities. We conclude that the 4β-PMA-mediated inhibition of testosterone production by Leydig cells stimulated with rAP-II results from an activation of a phosphodiesterase enzyme, hypothetically through an activated protein kinase C. This leads to a reduction in the cellular cGMP content through an increased metabolic removal of cGMP formed in response to rAP-II stimulation.