Effects of a phorbol ester, a calcium ionophore, and 3',5'-adenosine monophosphate production on hen granulosa cell plasminogen activator activity

Recent studies conducted in our laboratory have demonstrated that plasminogen activator (PA) is present in granulosa cells collected from the largest preovulatory follicle in the ovary of the domestic hen, and that its activity can be modulated by a variety of hormones in vitro. The present study was conducted to evaluate the intracellular mechanisms involved in the control of hen granulosa cell PA activity through the use of physiological and pharmacological agents. Treatment of granulosa cells with increasing doses (1, 10, and 50 ng/tube) of ovine LH resulted in a significant reduction of PA activity, which was accompanied by an increase in intracellular levels of cAMP. Furthermore, the effects of LH were potentiated by cotreatment with the phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (0.1 mM). Exposure of cells to increasing concentrations of the adenylyl cyclase activator forskolin (0.005, 0.01, 0.05, and 0.1 mM) resulted in a significant reduction in PA activity at all doses given. Similarly, the presence of the cAMP analog 8- bromo-cAMP (0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, and 10 mM) caused a dose-dependent inhibition of PA activity from 0.005 to 1.0 mM, further suggesting the involvement of cAMP in the inhibitory regulation of hen granulosa cell PA activity. The induction of intracellular calcium mobilization through the use of the calcium ionophore A23187 (0.1, 0.5, 1, and 2 μM) resulted in a dose-dependent suppression of PA activity. By contrast, treatment of granulosa cells with the tumor-promoting phorbol ester phorbol 12-myristate 13-acetate (PMA; 0.5, 5, 10, 25, and 50 Mg/tube), a compound that activates protein kinase- C, stimulated PA activity in a dose-dependent fashion; a nontumor- promoting phorbol ester (phorbol 13-monoacetate; 0.5, 10, and 50 ng/tube) was without effect. Coincubation of granulosa cells with a submaximal dose of PMA (5 ng/tube) and low concentrations of A23187 (0.001, 0.005, 0.01, and 0.05 nM) could not significantly enhance the stimulatory effects of PMA on PA activity; however, higher concentrations of the ionophore (0.1, 0.5, and 1.0 μM) completely abolished PMA-stimulated PA activity. The stimulatory effects of PMA could also be eliminated by cotreatment with a protein kinase-C inhibitor (H-7; 100 μM), a mRNA transcription blocker (actinomycin-D; 5 Mg/tube), or a protein synthesis inhibitor (cycloheximide; 50 μg/tube). Taken collectively, the data presented suggest that several intracellular mechanisms exist for the control of hen granulosa cell PA activity in vitro: 1) the adenylyl cyclase-cAMP second messenger system, which appears to regulate the suppression of PA activity; 2) intracellular calcium mobilization, also apparently responsible for the inhibitory regulation of PA activity; and 3) the diacylglycerol-protein kinase-C pathway, which appears to mediate the induction of PA activity by mechanisms requiring mRNA transcription and protein synthesis.