Stimulation of Luteinizing Hormone (LH) Release and Phospholipid Breakdown by Guanosine Triphosphate in Permeabilized Pituitary Gonadotropes: Antagonist Action Suggests Association of a G Protein and Gonadotropin-Releasing Hormone Receptor*

Abstract

The stimulation of gonadotropin release from pituitary cell cultures by GnRH has been linked to inositol phospholipid breakdown to diacylglycerols and subsequent activation of protein kinase C as well as Ca2+ mobilization. In order to examine the means of receptor coupling to a phospholipase C-type reaction, we evaluated the role of guanine nucleotides in inositol phospholipid breakdown. In these studies ATP (50 .mu.M) was used for cell permeabilization to allow guanine nucleotides access to the intracellular compartment. Under these conditions GTP and the GTP analog, guanylylimidodiphosphate (GMP-PNP), stimulated a time- and dose-dependent incrase in LH release and inositol phosphate accumulation. These actions of GTP and GMP-PNP were not observed unless ATP was included in the treatment media. Other closely related nucleotides and nucleoside alone, or in the presence of ATP, did not elevate LH release above basal levels. We also evaluated the actions of pertussis toxin and cholera toxin on mediating the effect of GTP, GMP-PNP, and GnRH on LH release and inositol phosphate accumulation. After treatment with these agents, no changes were observed in the ability of GnRH, GTP, or GMP-PNP to stimulate either LH release or inositol phosphate accumulation. The additional observation that GnRH-, GTP-, or GMP-PNP-stimulated LH release and inositol phosphate accumulation were blocked by a potent GnRH antagonist suggests that a G protein is functionally associated with the GnRH receptor recognition site.