Desensitization of gonadotropin-releasing hormone action in the gonadotrope-derived alpha T3-1 cell line.

Abstract

Sustained exposure of gonadotropes to GnRH causes a pronounced desensitization of GnRH-stimulated gonadotropin release, but the mechanisms involved are poorly understood. Recent studies have suggested, however, that GnRH-stimulated phosphoinositidase C (PIC) activity does not undergo rapid ( < 5 min) homologous desensitization in alpha T3-1 cells, and we have, therefore, used this cell line to address the question of whether desensitization occurs distal to PIC activity and/or in an intermediate time frame. We show that GnRH stimulates a rapid increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P3; maximum at 10-20 sec with a modest reduction thereafter] and that the GnRH-stimulated accumulation of [3H]IPs (in cells stimulated in the presence of LiCl) increases linearly over 5-300 sec. This clearly indicates that desensitization of PIC does not occur within this period and that the dramatic reduction in the rate of Ins(1,4,5)P3 accumulation (10-30 sec) is due to its metabolism, rather than to a reduction in Ins(1,4,5)P3 generation. Pretreatment for 60 min with 10(-7)M GnRH reduced cell surface GnRH receptor number by 48% (without measurably altering Kd). The pretreatment also reduced maximal GnRH-stimulated [3H]IP accumulation (to 66% of the control) and increased the EC50 for GnRH-stimulated [3H]IP accumulation approximately 3-fold, demonstrating that desensitization of GnRH-stimulated [3H]IP accumulation can, indeed, occur within 60 min, but that this may be attributable to receptor loss (without appreciable uncoupling of residual receptors from their immediate effector system). Pretreatment for 60 min with GnRH also caused a dose-dependent reduction in both spike and plateau phases of the GnRH effect on cytosolic Ca2+. This effect could not be overcome by stimulation with high concentrations of GnRH and appears, therefore, to reflect not only receptor loss, but, also, an additional inability of agonist-occupied GnRH receptors to elevate cytosolic Ca2+. The effect of KCl on cytosolic Ca2+ was similarly reduced by GnRH pretreatment, suggesting that desensitization of voltage-operated Ca2+ channels mediates desensitization of the plateau phase Ca2+ response to GnRH. Such a mechanism could not, however, explain desensitization of the spike phase of the Ca2+ response to GnRH seen in normal or Ca2+ -free medium. Accordingly, the data reveal a novel mechanism for homologous desensitization to GnRH in which agonist-occupied GnRH receptors are rendered unable to mobilize intracellular Ca2+ and imply that desensitization of GnRH-stimulated Ins(1,4,5)P3 production and/or action occurs.

Rapid Down-regulation of the Type I Inositol 1,4,5-Trisphosphate Receptor and Desensitization of Gonadotropin-releasing Hormone-mediated Ca2+ Responses in αT3–1 Gonadotropes

“…The recent discovery that mammalian GnRH receptors do not show rapid homologous desensitization (3)(4)(5)(6)(7)(8) reveals that desensitization of GnRH-stimulated gonadotropin secretion must reflect changes distal to the receptor, as implied by earlier work showing that GnRH receptor regulation does not explain desensitization of gonadotropin secretion (5). Our work has revealed that pretreatment of ␣T3-1 cells with GnRH causes a pronounced desensitization of GnRHstimulated mobilization of Ca 2ϩ from intracellular stores (4, 5), which is of particular interest in the light of the established importance of Ca 2ϩ mobilization in mediation of GnRH-stimulated gonadotropin secretion.…”

“…Determination of Ins (1,4,5) Ϫ5 M unlabeled Ins(1,4,5)P 3 . The incubations were terminated by centrifugation and removal of supernatants by aspiration.…”

“…In contrast to nearly all other known PLC-coupled G-proteincoupled receptors (GPCRs), the GnRH receptor does not undergo rapid (seconds to minutes) desensitization following exposure to agonist (3)(4)(5)(6)(7)(8). Recent evidence has suggested that this lack of acute regulation is related to the lack of a C-terminal tail and the absence, therefore, of appropriate regulatory phospho-acceptor sites (8,9).…”

“…Despite no evidence for rapid desensitization of the GnRH receptor in these cells, pre-exposure to GnRH can cause a marked suppression of subsequent GnRH-mediated elevations of [Ca 2ϩ ] i . Both the spike phase of the response (which reflects Ins(1,4,5)P 3 -dependent mobilization of intracellular Ca 2ϩ ) and the sustained phase of the response (which is dependent upon Ca 2ϩ entry across the plasma membrane through voltage-operated Ca 2ϩ channels) were attenuated by GnRH pretreatment (4,5).…”

Lack of a C-terminal Tail in the Mammalian Gonadotropin-releasing Hormone Receptor Confers Resistance to Agonist-dependent Phosphorylation and Rapid Desensitization

“…This is true of the GnRH-R expressed either as an endogenous protein in an immortalized mouse pituitary cell line (␣T3-1) or as a recombinant protein in a number of cell types (2)(3)(4)(5). Furthermore, we have recently demonstrated that, although GnRH-Rs of ␣T3-1 cells are not acutely regulated, muscarinic M 3 receptors co-expressed as recombinant proteins in these cells are rapidly desensitized (6).…”