DESENSITIZATION OF GONADOTROPIN-RELEASING-HORMONE ACTION IN THE GONADOTROPE-DERIVED ALPHA-T3-1 CELL-LINE

Sustained exposure of gonadotropes to GnRH causes a pronounced desensi tization of GnRH-stimulated gonadotropin release, but the mechanisms i nvolved are poorly understood. Recent studies have suggested, however, that GnRH-stimulated phosphoinositidase C (PIC) activity does not und ergo rapid (<5 min) homologous desensitization in alpha T3-1 cells, an d we have, therefore, used this cell Line to address the question of w hether desensitization occurs distal to PIC activity and/or in an inte rmediate time frame. We show that GnRH stimulates a rapid increase in inositol 1,4,5-trisphosphate [Ins(1,4,5)P-3; maximum at 10-20 sec with a modest reduction thereafter] and that the GnRH-stimulated accumulat ion of [H-3]IPs (in cells stimulated in the presence of LiCl) increase s Linearly over 5-300 sec. This clearly indicates that desensitization of PIC does not occur within this period and that the dramatic reduct ion in the rate of Ins(1,4,5)P-3 accumulation (10-30 sec) is due to it s metabolism, rather than to a reduction in Ins(1,4,5)P-3 generation. Pretreatment for 60 min with 10(-7) M GnRH reduced cell surface GnRH r eceptor number by 48% (without measurably altering K-d). The pretreatm ent also reduced maximal GnRH-stimulated [H-3]IP-accumulation (to 66% of the control) and increased the EC(50) for GnRH-stimulated [H-3]IP a ccumulation approximately 3-fold, demonstrating that desensitization o f GnRH-stimulated [H-3]IP accumulation can, indeed, occur within 60 mi n, but that this may be attributable to receptor loss (without appreci able uncoupling of residual receptors from their immediate effector sy stem). Pretreatment for 60 min with GnRH also caused a dose-dependent reduction in both spike and plateau phases of the GnRH effect on cytos olic Ca2+. This effect could not be overcome by stimulation with high concentrations of GnRH and appears, therefore, to reflect not only rec eptor loss, but, also, an additional inability of agonist-occupied GnR H receptors to elevate cytosolic Ca2+. The effect of KCl on cytosolic Ca2+ was similarly reduced by GnRH pretreatment, suggesting that desen sitization 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+ resp onse to GnRH seen in normal or Ca2+-free medium. Accordingly, the data reveal a novel mechanism for homologous desensitization to GnRH in wh ich agonist-occupied GnRH receptors are rendered unable to mobilize in tracellular Ca2+ and imply that desensitization of GnRH-stimulated Ins (1,4,5)P-3 production and/or action occurs.