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,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 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.
D000634:Aminoquinolines; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002469:Cell Separation / Q000379:methods; D002498:Centrifugation / Q000379:methods; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes; D007987:Gonadotropin-Releasing Hormone / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D000634:Aminoquinolines; D005456:Fluorescent Dyes; D007987:Gonadotropin-Releasing Hormone; C035739:Quin2; D002118:Calcium