Cytosolic free calcium levels in cultured pituitary cells separated by centrifugal elutriation: Effect of gonadotropin-releasing hormone

The cytosolic concentration of free Ca²⁺ ([Ca²⁺]i) in normal rat pituitary cells separated by centrifugal elutriation was monitored with the fluorescent Ca²⁺ indicator Quin 2. GnRH (10^-7 M) induced a rapid rise (6–8 sec) in the gonadotroph's [Ca²⁺]i, followed by a plateau phase of prolonged elevated [Ca²⁺]i which lasted about 15 min. The stimulatory effect of GnRH was dose dependent, with an ED50 of 10^-9 M, and was blocked by the potent antagonist [Dp-Glu¹.pclPhe².DTrp^3,6]GnRH. GnRH elevated [Ca²⁺]iOnly in gonadotroph- enriched cell fractions, whereas TRH and GH-releasing factor (GRF) elevated [Ca²⁺]i in mammotroph- and somatotroph- enriched cells fractions, respectively. A rapid increase (first phase) in [Ca²⁺]i induced by GnRH was observed in Ca²⁺-free medium containing EGTA, but this rapid phase was terminated within 2 min. Readdition of Ca²⁺ to the medium induced a second slower rise in [Ca²⁺]i (plateau phase). Addition of K⁺ caused a rapid rise in [Ca²⁺]i, which was dependent on extracellular Ca²⁺, but was not affected by prior stimulation with GnRH. On the other hand, stimulation of gonadotroph's [Ca²⁺]i response by GnRH desensitized the cells to a subsequent GnRH challenge within the time frame studied. These findings indicate an elevation of [Ca²⁺]i induced by GnRH, TRH, and GRF in their respective separated target cells in the rat pituitary. The rise in [Ca²⁺]i in GnRH-stimulated gonadotrophs originates partly from intracellular Ca²⁺ pools and partly from influx of Ca²⁺ across the cell membrane.