Gonadotropin-releasing hormone-induced Ca²⁺ transients in single identified gonadotropes require both intracellular Ca²⁺ mobilization and Ca²⁺ influx

We examined the effects of gonadotropin-releasing hormone (GnRH) on the intracellular free Ca²⁺ concentration ([Ca²⁺](i)) in single rat anterior pituitary gonadotropes identified by a reverse hemolytic plaque assay. Concentrations of GnRH >10 pM elicited increases in [Ca²⁺](i) in identified cells but not in others. In contrast, depolarization induced by 50 mM K⁺ increased [Ca²⁺](i) in all cells. Ca²⁺ transients induced by GnRH exhibited a complex time course. After an initial rapid rise, the [Ca²⁺](i) fell to near basal levels only to be followed by a secondary extended rise and fall. Analysis of the Ca²⁺ transients on a rapid time base revealed that responses frequently consisted of several rapid oscillations in [Ca²⁺](i). Removal of extracellular Ca²⁺ or addition of the dihydropyridine Ca²⁺-channel blocker nitrendipine completely blocked the secondary rise in [Ca²⁺](i) but had no effect whatsoever on the initial spike. Nitrendipine also blocked 50 mM K⁺-induced increases in [Ca²⁺](i) in identified gonadotropes. The secondary rise induced by GnRH could be enhanced by a phorbol ester in a nitrendipine-sensitive fashion. Multiple spike responses to GnRH stimulation of the same cell could only be obtained if subsequent Ca²⁺ influx was permitted either by allowing a secondary rise to occur or by producing a Ca²⁺ transient by depolarizing the cells with 50 mM K⁺. It therefore appears that the response to GnRH consists of an initial phase of Ca²⁺ mobilization, probably mediated by inositol trisphosphate, and a subsequent phase of Ca²⁺ influx through nitrendipine-sensitive Ca²⁺ channels that may be activated by protein kinase C. The relative roles of these phases in the control of gonadotropin secretion are discussed.