Mechanism of action of GnRH: The participation of calcium mobilization and activation of protein kinase C in gonadotropin secretion

Abstract

The role of protein kinase C in luteinizing hormone (LH) release was analyzed in studies on the secretory responses to gonadotropin releasing hormone (GnRH) and phorbol esters in pituitary cell cultures. 12-O-tetradecanoyl-phorbol 13-acetate (TPA), 4β-phorbol 12,13-bibenzoate, and 4β -phorbol 12,13-diacetate stimulated LH release with ED 50s of 5, 10 and 1000 nM, respectively, and with about 70% of the efficacy of GnRH. Phorbol ester-stimulated LH secretion was decreased but not abolished by progressive reduction of [Ca 2+] in the incubation medium, and the residual response was identical with that of GnRH in Ca 2+-defficient medium. TPA increased [Ca 2+i to a peak after 30 s in normal medium but not in the absence of extracellular Ca 2+, indicating that protein kinase C promotes calcium entry but can also mediate secretory responses without changes in calcium influx and [Ca 2+]i. The extracellular Ca 2+-dependent action of TPA on LH release was blocked by CoCl 2 but not by nifedipine. The secretory actions of TPA and GnRH were additive at low doses and converged to a common maximum LH response at high concentrations of the agonists. TPA caused rapid translocation of cytosolic protein kinase C to the particulate fraction, followed by a progressive decrease in total enzyme activity to < 10% after 6 h. Partial recovery of the cytosolic enzyme (to 20%) occurred after washing and reincubation for 15h. Such kinase C-depleted cells showed prominent dose-dependent reductions in the actions of both GnRH and TPA on LH release in normal and Ca 2+-deficient media. These observations show that the actions of kinase C on LH release include extracellular Ca 2+-dependent and independent components, and support the hypothesis that protein kinase C participates in the LH secretory response to GnRH in pituitary gonadotrophs.