Calcium-dependent actions of gonadotropin-releasing hormone agonist and luteinizing hormone upon cyclic AMP and progesterone production in rat ovarian granulosa cells

The Ca²⁺ dependency of the direct stimulatory effect of the gonadotropin-releasing hormone (GnRH) agonist analog [d-Ser(t-Bu)⁶]des-Gly¹⁰-GnRH-N-ethylamide (GnRHa) on progesterone production was investigated and compared to that of luteinizing hormone (LH) in rat granulosa cells from preovulatory follicles. Removal of extracellular Ca²⁺ by EGTA, or the use of the Ca²⁺ channel blockers verapamil and La³⁺, resulted in complete inhibition of GnRHa-induced progesterone production and a partial inhibition of LH-stimulated progesterone production (80, 80 and 50% inhibition respectively for EGTA, verapamil and La³⁺). Removal of extracellular Ca²⁺ increased the ED₅₀ for LH-induced cAMP production by four-fold (from 80 to 330 ng/ml) and decreased maximal nucleotide formation by 44%. LH-induced cAMP production was also inhibited partially by verapamil (35%) at 10^-4 M drug concentration. GnRHa had no effect on cAMP production in the presence or absence of Ca²⁺, GnRHa and LH were found to have maximal effects on progesterone production at about 0.5 mM of Ca²⁺ in the incubation medium. On the other hand the stimulatory effect of dibutyryl cAMP ((Bu)₂cAMP) on progesterone production showed little dependency on extracellular Ca²⁺. The calmodulin antagonist trifluoperazine (TFP) caused concentration-dependent inhibition of the stimulatory action of GnRHa and LH on progesterone production with IC₅₀ values of 3 and 8 μM, respectively. The stimulatory effect of (Bu)₂cAMP on progesterone synthesis was attenuated by verapamil and TFP. These results indicate that the direct stimulatory effect of GnRH on ovarian progesterone production is absolutely dependent on Ca²⁺. About 60% of LH-induced cAMP production, 20% and 45% of LH and cAMP-induced progesterone production, respectively, are Ca²⁺ independent. At least two sites of Ca²⁺ requirements are involved in LH action. The first is involved in LH-induced cAMP production, and the second is involved in a later step in the steroidogenic response.