Calcium-Dependent Regulation of Progesterone Production by Isolated Rat Granulosa Cells: Effects of the Calcium lonophore A23187, Prostaglandin E2, dl-Isoproterenol and Cholera Toxin 1

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Abstract
The role of Ca in the regulation of ovarian steroidogenesis was investigated in granulosa cells from estradiol-treated immature rats. Incubation of granulosa cells with various Ca channel blockers (verapamil, Co or Mn) and a Ca chelator (EGTA [ethylene glycol bis(.beta.-aminoethyl ether)-N,N,N'',N''-tetraacetic acid]) resulted in marked decreases in progesterone production in response to FSH, cholera toxin, prostaglandin E2, dl-isoproterenol and dibutyryl cAMP (Bt2 cAMP). cAMP production was unaffected by treatment with EGTA and verapamil at concentrations which attenuated steroidogensis (0.1-1.0 mM and 125 .mu.M, respectively). Two inhibitors of the Ca-dependent regulatory protein, calmodulin [trifluoperazine, 40 .mu.M and 1[bis-(p-chlorophenyl)methyl] 3-[2,4-dichloro-.beta.-(2,4-dichlorobenzyloxy)-phenethyl] imidazolium chloride (R24571) 20 .mu.M] significantly inhibited cAMP and progesterone production elicited by these stimulatory agents. Over the concentration range of 62.5 ng/ml to 1.0 .mu.g/ml, the Ca ionophore A23187 increased basal progesterone production in a dose-dependent manner, with half-maximal stimulation at .apprx. 0.14 .mu.g/ml. Maximal steroidogenic response to the Ca ionophore (1 .mu.g/ml) however, was only 50% of that evoked by FSH (0.33 .mu.gml). A23187 (0.5 .mu.g/ml) significantly enhanced progesterone production stimulated by a low concentration of FSH (0.025 .mu.g/ml) but failed to potentiate the maximally stimulatory action of the gonadotropin (0.33 .mu.g/ml). The Ca-calmodulin system evidently plays a central role in the gonadotropic regulation of ovarian steroidogenesis. A transmembrane flux of extracellular Ca may be an important and common step in the mechanism of stimulation of granulosa cell progesterone production.