Gonadotropin-Releasing Hormone-Induced Rise in Cytosolic Free Ca²⁺ Levels: Mobilization of Cellular and Extracellular Ca²⁺ Pools and Relationship to Gonadotropin Secretion

Addition of GnRH to pituitary gonadotrophs preloaded with Quin 2 resulted in a rapid (~8 s) mobilization of an ionomycin-sensitive intracellular Ca²⁺ pool. A second component of Ca²⁺ entry via voltage dependent channels contributed about 45% of the peak cytosolic free Ca²⁺ concentration ([Ca²⁺],). Thereafter, influx of Ca²⁺ via voltage-sensitive and -insensitive channels is responsible for maintenance of elevated [Ca²⁺], during the second phase of GnRH action. Addition of inositol 1, 4, 5-trisphosphate (IP₃) to permeabilized pituitary cells resulted in a Ca²⁺ transient, released from a nonmitochondrial pool, which maintained ambient free Ca²⁺ concentration around 170 mu in an ATP-dependent mechanism. Successive stimulations of the cells with IP₃ produced an attenuated response. Elevation of the gonadotroph [Ca²⁺]_i by ionomycin, to levels equivalent to that induced by GnRH, resulted in LH release amounting to only 45% of the response to the neurohormone. Activation of the voltage-dependent Ca²⁺ channels by the dihydropyridine Ca²⁺- agonist [methyl 1, 4-dihydro-2, 6-dimethyl-3-nitro-4-(2-tri-fluoromethylphenyl)-pyridine-5-carboxylate (BAYK8644)] stimulated LH release, 36% of the GnRH (100 nm) response being reached by 10^-8 m of the drug, both [Ca²⁺], elevation and GnRH-induced LH released were inhibited similarly (40-50%) by the dihydropyridine Ca²⁺- antagonist nifedipine. The results indicate that peak [Ca²⁺]_i induced by GnRH in pituitary gonadotrophs is derived mainly from ionomycin-sensitive cellular stores most likely via IP₃ formation. L type voltage-sensitive Ca²⁺ channels contribute partially to the peak [Ca²⁺]_i, and may participate with other types of Ca²⁺ channels (i.e. transient T channels and voltageinsensitive channels) in the secondary elevation of [Ca²⁺]_i and gonadotropin secretion. Influx of Ca²⁺ is necessary but not sufficient to mediate GnRH-stimulation of gonadotropin secretion.