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

Department of Hormone Research (R.L., Z.N.), The Weizmann Institute of Science Rehouot, Israel 76100
Timsit Institute of Reproductive Endocrinology (R.L., D.A.), Tel-Aviv University School of Medicine Tel-Aviv, Israel
Division of Endocrinology (A.M.C.), University Hospital CH-1211 Geneva 4, Switzerland
Department of Anatomy (G.V.C.), University of Texas Medical Branch Galveston, Texas 77550

Address requests for reprints and all correspondence to: Dr. Zvi Naor, Department of Hormone Research, the Weizmann Institute of Science, Rehovot 76100 Israel.

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 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. (Endocrinology 120: 497–503, 1987)

Footnotes

^* Supported by NIH Grant HD-16279 (to Z.N.); NIH Grants HD-15472 and RCDA HD-19930 (to G.C.); the United States-Israel Binational Science Foundation (to Z.N.); a grant from EMBO (to A.M.C.); and by Grant 3.914-0.83 from the Swiss National Foundation (to A.M.C.).

Received June 26, 1986.

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