In:
Journal of Endocrinology, Bioscientifica, Vol. 149, No. 1 ( 1996-04), p. 155-169
Abstract:
In pituitary gonadotrophs GnRH causes biphasic (spike and plateau) increases in cytosolic Ca 2+ ([Ca 2+ ] i ) and gonadotrophin release. The spike phases reflect mobilization of stored Ca 2+ and the plateau responses are attributed, in part, to Ca 2+ influx via voltage-sensitive Ca 2+ channels. In recent years, store-dependent Ca 2+ influx (SDCI), in which depletion of the intracellular inositol 1,4,5-trisphosphate-mobilizable pool stimulates Ca 2+ influx, has emerged as a major form of Ca 2+ 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 [Ca 2+ ] i , in αT3–1 cells (a gonadotroph-derived cell line) and manipulation of the filling state of the GnRH-mobilizable Ca 2+ pool to test the possible role of SDCI in GnRH action. In Ca 2+ -containing medium, GnRH caused a biphasic increase in [Ca 2+ ] i whereas in Ca 2+ -free medium only a transient increase occurred. The response to a second stimulation with GnRH in Ca 2+ -free medium was reduced by 〉 95% (demonstrating that Ca 2+ pool depletion had occurred) and was recovered after brief exposure to Ca 2+ -containing medium (which enables refilling of the pool). Ionomycin (a Ca 2+ ionophore) and thapsigargin (which inhibits the Ca 2+ -sequestering ATPase of the endoplasmic reticulum) also transiently increased [Ca 2+ ] i , in Ca 2+ -free medium and depleted the GnRH-mobilizable pool as indicated by greatly reduced subsequent responses to GnRH. Pool depletion also occurs on stimulation with GnRH in Ca 2+ -containing medium because addition of ionomycin and Ca 2+ -free medium during the plateau phase of the GnRH response caused only a reduction in [Ca 2+ ] i rather than the transient increase seen without GnRH. To deplete intracellular Ca 2+ pools, cells were pretreated in Ca 2+ -free medium with thapsigargin or GnRH and then, after extensive washing, returned to Ca 2+ -containing medium. Pretreatment with thapsigargin augmented the increase in [Ca 2+ ] i seen on return to Ca 2+ -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 Ca 2+ 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 Ca 2+ -containing medium. Return of GnRH-treated cells to Ca 2+ -containing medium caused an increase in [Ca 2+ ] i which was inhibited by nicardipine, whereas the increase seen on return of thapsigargin-treated cells to Ca 2+ -containing medium was not reduced by nicardipine. The quench of fura-2 fluorescence by MnCl 2 (used as a reporter of Ca 2+ influx) was increased by GnRH and thapsigargin, indicating that both stimulate Ca 2+ influx via Mn 2+ permeant channels. The GnRH effect was abolished by nicardipine whereas that of thapsigargin was not. Finally, depletion of intracellular Ca 2+ pools by pretreatment of superfused rat pituitary cells with GnRH or thapsigargin in Ca 2+ -free medium did not enhance LH release on return to Ca 2+ -containing medium. The results indicate that (a) thapsigargin stimulates SDCI in αT3–1 cells via nicardipine-insensitive Ca 2+ channels, (b) in spite of the fact that GnRH depletes the hormone-mobilizable Ca 2+ pool, it fails to stimulate SDCI, (c) GnRH stimulates Ca 2+ 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. Journai of Endocrinology (1996) 149, 155–169
Type of Medium:
Online Resource
ISSN:
0022-0795
,
1479-6805
DOI:
10.1677/joe.0.1490155
Language:
Unknown
Publisher:
Bioscientifica
Publication Date:
1996
detail.hit.zdb_id:
1474892-7
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