CA2-T3-1 CELLS - DOES STORE-DEPENDENT CA2+ INFLUX MEDIATE GONADOTROPIN-RELEASING-HORMONE ACTION( ENTRY IN GONADOTROPHS AND ALPHA)

In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) inc reases in cytosolic Ca2+ ([Ca2+](i)) and gonadotrophin release. The sp ike phases reflect mobilization of stored Ca2+ and the plateau respons es are attributed, in part, to Ca2+ influx via voltage-sensitive Ca2channels. In recent years, store-dependent Ca2+ influx (SDCI), in whic h depletion of the intracellular inositol 1,4,5-trisphosphate-mobiliza ble pool stimulates Ca2+ influx, has emerged as a major form of Ca2+ e ntry activated by phosphoinositidase C-coupled receptors in non-excita ble cells. More recent evidence also indicates a role for SDCI in exci table cells. We have used dynamic video imaging of [Ca2+](i) in alpha T3-1 cells (a gonadotroph-derived cell line) and manipulation of the f illing state of the GnRH-mobilizable Ca2+ pool to test the possible ro le of SDCI in GnRH action. In Ca2+-containing medium, GnRH caused a bi phasic increase in [Ca2+](i) whereas in Ca2+-free medium only a transi ent 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+-sequesterin g ATPase of the endoplasmic reticulum) also transiently increased [Ca2 +](i) in Ca2+-free medium and depleted the GnRH-mobilizable pod as ind icated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca2+-containing medium becaus e 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 o r 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 hi gher than that seen in control cells) indicating the activation of SDC I, whereas pool depletion by GnRH pretreatment had no such effect. To ensure maintained pool depletion after Ca2+ re-addition, similar studi es were performed in which the thapsigargin and GnRH treatments were n ot washed off, but were retained through the period of return to Ca2+- containing medium. Return of GnRH-treated cells to Ca2+-containing med ium 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 i ncreased by GnRH and thapsigargin, indicating that both stimulate Ca2 influx via Mn2+ permeant channels. The GnRH effect was abolished by n icardipine 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 tha t (a) thapsigargin stimulates SDCI in alpha T3-1 cells via nicardipine -insensitive Ca2+ channels, ro) in spite of the fact that GnRH deplete s the hormone-mobilizable Ca2+ pool, it fails to stimulate SDCI, (c) G nRH stimulates Ca2+ entry predominantly via nicardipine-sensitive chan nels, a route not activated by SDCI and (d) in rat gonadotrophs, GnRH- stimulated LH release is not mediated by SDCI.