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RC Fowkes, W Forrest-Owen and CA McArdle

C-type natriuretic peptide (CNP), the third member of the natriuretic peptide family, has been found at its highest tissue concentrations in the anterior pituitary, where it is localised in gonadotrophs. Its specific guanylyl cyclase-containing receptor, GC-B, is also expressed on several anterior pituitary cell types, and CNP potently stimulates cGMP accumulation in rat pituitary cell cultures and pituitary cell lines. The mouse gonadotroph-derived alpha T3-1 cell line has been shown to express CNP as well as GC-B (but not GC-A) receptors, suggesting that CNP may well be an autocrine regulator of gonadotrophs. Comparing effects of three natriuretic peptides (atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP) and CNP) on cGMP accumulation in four pituitary cell lines (alpha T3-1, TtT-GF, AtT-20 and GH(3)) we find that CNP is most potent and effective in alpha T3-1 cells. In these cells, CNP-stimulated cGMP accumulation was found to desensitise during a 30 min exposure to CNP. Pretreatment with CNP for up to 6 h also caused a significant reduction in the ability of CNP to subsequently stimulate cGMP accumulation. This effect was receptor specific, because pretreatment with sodium nitroprusside (an activator of nitric oxide-sensitive guanylyl cyclase), or with ANP or BNP, did not cause desensitisation of CNP-stimulated cGMP accumulation. Protein kinase C activation with phorbol esters also inhibited CNP-stimulated cGMP accumulation and such inhibition was also seen in cells desensitised by pretreatment with CNP. Thus it appears that the endogenous GC-B receptors of alpha T3-1 cells are subject to both homologous and heterologous desensitisation, that the mechanisms underlying these forms of desensitisation are distinct, and that cGMP elevation alone is insufficient to desensitise GC-B receptors.

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C A McArdle, W Forrest-Owen, J S Davidson, R Fowkes, R Bunting, W T Mason, A Poch and M Kratzmeier

Abstract

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 αT3–1 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 pool 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 αT3–1 cells via nicardipine-insensitive Ca2+ channels, (b) 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.

Journai of Endocrinology (1996) 149, 155–169