Alteration of the Intracellular Calcium Level Stimulates Gonadotropin Release from Cultured Rat Anterior Pituitary Cells*

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Abstract
We demonstrated that stimulation of LH release from pituitary cells by gonadotropin-releasing hormone (GnRH) requires extracellular Ca2+ (optimally, 1 mM). Mg2+ (up to 10 mM) did not substitute for this requirement. Stimulated, but not basal, LH release was blocked by La3+ which competes for cellular calcium-binding sites. Antagonists of Ca2+ flux (D-600, methoxyverapamil) and Ca2+ transport (Ruthenium red) also inhibited GnRH stimulation of LH release. In the present work, we incubated pituitary cells with compounds which alter the intracellular level of Ca2+ and examined the effect on LH release. In separate studies, cultured cells were incubated with ionophores A23187 (Lilly) or X537A (Roche) or with Ca2+- and Mg2+-bearing lipid vesicles (liposomes). Ionophore A23187, which inserts Ca2+ channels into the plasma membrane, stimulated LH release from the cells in the presence of extracellular Ca2+. When A23187 and GnRH were both present in saturating amounts, the cells did not release more LH than with either secretogogue alone. Ionophore X537A, a less specific ionophore which may enter cells, did not show a marked dependency on either extracellular Ca2+ or Mg2+ for stimulation of LH release. Thus, intracellular ion pools appeared sufficient to support maximal stimulation of LH release even though extracellular Ca2+ was required for GnRH stimulation. Ion-bearing (Ca2+ or Mg2+) liposomes were used to insert particular ions into the gonadotropes in order to establish the specific ion(s) which stimulated LH release. Introduction of Ca2+-bearing, but not Mg2+-bearing or control, liposomes stimulated LH release above the basal level. The data in the present studies show that artificial introduction of Ca2+ into cultured pituitary cells resulted in the release of LH from the gonadotrope in the absence of GnRH. By sequential and concomitant GnRH and ionophore incubations, these secretogogues appeared to act on the same releasable pool of LH. The data provide additional support for a role of Ca2+ in LH release from cultured rat pituitary cells.