Ca2+ entry in gonadotrophs and alpha T3-1 cells: Does store-dependent Ca2+ influx mediate gonadotrophin-releasing hormone action?

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca2+ ([Ca2+](i)) and gonadotrophin release. The spike phases reflect mobilization of stored Ca2+ and the plateau responses are attributed, in part, to Ca2+ influx via voltage-sensitive Ca2+ channels. In recent years, store-dependent Ca2+ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca2+ influx, has emerged as a major form of Ca2+ entry activated by phosphoinositidase C-coupled receptors in non-excitable cells. More recent evidence also indicates a role for SDCI in excitable cells. We have used dynamic video imaging of [Ca2+](i) in alpha T3-1 cells (a gonadotroph-derived cell line) and manipulation of the filling state of the GnRH-mobilizable Ca2+ pool to test the possible role of SDCI in GnRH action. In Ca2+-containing medium, GnRH caused a biphasic increase in [Ca2+](i) whereas in Ca2+-free medium only a transient increase occurred. The response to a second stimulation with GnRH in Ca2+-free medium was reduced by >95% (demonstrating that Ca2+ pool depletion had occurred) and was recovered after brief exposure to Ca2+-containing medium (which enables refilling of the pool). Ionomycin (a Ca2+ ionophore) and thapsigargin (which inhibits the Ca2+-sequestering ATPase of the endoplasmic reticulum) also transiently increased [Ca2+](i) in Ca2+-free medium and depleted the GnRH-mobilizable pod as indicated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca2+-containing medium because addition of ionomycin and Ca2+-free medium during the plateau phase of the GnRH response caused only a reduction in [Ca2+](i) rather than the transient increase seen without GnRH. To deplete intracellular Ca2+ pools, cells were pretreated in Ca2+-free medium with thapsigargin or GnRH and then, after extensive washing, returned to Ca2+-containing medium. Pretreatment with thapsigargin augmented the increase in [Ca2+](i) seen on return to Ca2+-containing medium (to two- to threefold higher than that seen in control cells) indicating the activation of SDCI, whereas pool depletion by GnRH pretreatment had no such effect. To ensure maintained pool depletion after Ca2+ re-addition, similar studies were performed in which the thapsigargin and GnRH treatments were not washed off, but were retained through the period of return to Ca2+-containing medium. Return of GnRH-treated cells to Ca2+-containing medium caused an increase in [Ca2+](i) which was inhibited by nicardipine, whereas the increase seen on return of thapsigargin-treated cells to Ca2+-containing medium was not reduced by nicardipine. The quench of fura-2 fluorescence by MnCl2 (used as a reporter of Ca2+ influx) was increased by GnRH and thapsigargin, indicating that both stimulate Ca2+ influx via Mn2+ permeant channels. The GnRH effect was abolished by nicardipine whereas that of thapsigargin was not. Finally, depletion of intracellular Ca2+ pools by pretreatment of superfused rat pituitary cells with GnRH or thapsigargin in Ca2+ free medium did not enhance LH release on return to Ca2+-containing medium. The results indicate that (a) thapsigargin stimulates SDCI in alpha T3-1 cells via nicardipine-insensitive Ca2+ channels, ro) in spite of the fact that GnRH depletes the hormone-mobilizable Ca2+ pool, it fails to stimulate SDCI, (c) GnRH stimulates Ca2+ entry predominantly via nicardipine-sensitive channels, a route not activated by SDCI and (d) in rat gonadotrophs, GnRH-stimulated LH release is not mediated by SDCI.