The dependence of LH responses to GnRH on extracellular calcium was investigated in cultured rat pituitary cells exposed to GnRH for 3 h in static culture or for 2 min during column perifusion. During static culture in normal medium, LH release was stimulated by GnRH with an ED50 of 0.3 nM and by K+ with an ED50 of 32 mM. Incubation in Ca2+-deficient (no added Ca2+) or Ca2+-free medium (containing 100 microM EGTA) substantially decreased, but did not abolish, the LH responses to 10 and 100 nM GnRH, whereas K+-induced LH release was almost completely abolished in Ca2+-deficient medium. The Ca2+ channel agonist (BK 8644) and antagonists (nifedipine, nicardipine, verapamil, and Co2+) respectively enhanced or reduced the LH responses to both GnRH and K+. However, the calcium antagonists completely abolished the LH response to depolarization by K+, but only partially inhibited the LH response to GnRH, confirming the existence of a significant component of GnRH action that is not dependent on extracellular Ca2+. In perifused pituitary cells, exposure to Ca2+-deficient medium or normal medium containing 5 mM EGTA or 5 mM EDTA, reduced the initial rapid LH response to 2-min pulses of 10 nM GnRH and abolished the second phase of LH release. Reintroduction of Ca2+-containing medium at the end of the GnRH pulse caused recovery of the second phase of LH secretion, demonstrating that influx of extracellular Ca2+ is not required for the early phase of the LH response to GnRH but, rather, appears to be essential for its prolongation. The release of LH in response to arachidonic acid, which has been implicated in the mechanism of the secretory action of GnRH, was completely independent of extracellular Ca2+ and unaffected by addition of 10 nM BK 8644. These observations indicate that the initiation of the secretory response to GnRH is largely independent of calcium entry, whereas the prolongation of gonadotropin secretion is maintained by calcium influx, in part through voltage-sensitive calcium channels. The role of arachidonic acid metabolites in GnRH action is probably related to the calcium-independent component of GnRH-induced LH secretion. Since GnRH is secreted episodically and for short periods, much of its physiological action on pulsatile gonadotropin release could be independent of calcium influx from the extracellular fluid.

The relative contributions of arachidonic acid (AA)- and protein kinase C-dependent pathways during the immediate LH response to short term stimulation by GnRH were analyzed in perifused anterior pituitary cells cultured on Cytodex beads. The LH response to a 2-min pulse of 10 nM GnRH was biphasic, with a rapid increase to an initial peak, followed by a second peak or shoulder before the gradual return to baseline release. Retinal, which inhibits activation of protein kinase C, reduced the total LH response to GnRH by 35-40% and advanced the termination of the response, but did not alter the height, position, or rate of onset of the initial LH peak. In contrast, pretreatment with the lipoxygenase inhibitor nordihydroguaiaretic acid decreased the total LH response to GnRH by 60%, reduced the magnitude and latency of the first LH peak, and shortened the duration of the response. Pretreatment with both retinal and nordihydroguaiaretic acid abolished the GnRH-induced LH release. Addition of 2-min pulses of AA induced LH responses of short duration that coincided with the first phase of GnRH-stimulated LH release. Application of 2-min pulses of either tetradecanoyl phorbol 13-acetate (TPA) or dioctanoyl glycerol generated LH responses with delayed onsets that corresponded to the second phase of the GnRH-induced response. The LH response to the combined action of AA and TPA approximated that induced by GnRH. These results suggest that mobilization and metabolism of AA are important in the rapid initial phase of the LH response to GnRH, and that activation of protein kinase C-dependent mechanisms participates in the maintenance of the LH response. During continuous perifusion with 10 nM GnRH, addition of 2-min pulses of 100 nM GnRH and 100 microM AA, but not 100 nM TPA, stimulated further increases in LH release. This suggests that during prolonged GnRH action, LH release is primarily maintained by protein kinase C-dependent mechanisms. The results of this study indicate that GnRH stimulation of LH release requires the coordinated actions of at least two major interrelated mechanisms, namely those activated by AA and/or its metabolites and those maintained by protein kinase C-dependent pathways.

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