Cytosolic Free Calcium Levels in Cultured Pituitary Cells Separated by Centrifugal Elutriation: Effect of Gonadotropin-Releasing Hormone*

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Abstract
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 ] 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,pc1Phe2,DTrp3,6]GnRH. GnRH elevated [Ca2+]i only 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 with 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.