Dual intracellular pathways in gonadotropin releasing hormone (GNRH) induced desensitization of luteinizing hormone (LH) secretion.

The mechanisms of GnRH-induced desensitization of LH secretion are poorly understood. Protein kinase C (PKC) and protein kinase A (PKA) desensitize some receptors of the 7-membrane type, and the GnRH receptor has consensus phosphorylation sites for PKC in the first and third intracellular loops, and a site for PKA in the first intracellular loop. In the first set of experiments we determined whether synthetic peptides representing the three intracellular loops of the receptor could be phosphorylated in vitro by purified PKC and PKA. As compared with a model substrate peptide for PKC, the third intracellular loop was phosphorylated 74% and the first intracellular loop 21%; PKA-phosphorylated the first intracellular loop peptide 17% as well as a model peptide substrate. In the second set of experiments, we used phorbol 12-myristate 13 acetate (PMA), an established PKC stimulator, and cholera toxin (CTX), established to activate the Gs protein and presumed to activate PKA, to treat cultured rat pituitary cells followed by LH measurements. Treatment with both drugs severely impaired GnRH-stimulated LH secretion whereas neither drug alone reduced LH secretion. Dibutyryl cAMP did not duplicate the effects of cholera toxin suggesting that the CTX action could not be explained by an increase in cAMP. These results suggest that more than one intracellular signaling pathway requires activation in order to induce desensitization; one pathway involves PKC and the other involves a pathway stimulated by cholera toxin, presumably Gs protein, which does not involve PKA.

A temporally intermediate mode of gonadotropin releasing hormone-induced desensitization of luteinizing hormone secretion.

The classical mode of luteinizing hormone (LH) secretory desensitization in the rat appears after 3-6 h of continuous in vitro administration of gonadotropin (GnRH). A second mode has been reported to occur very rapidly (< 2 min) after the onset of GnRH administration, and to reverse within 3 min after its withdrawal. Here, the existence of a third mode of desensitization is reported. occurring at 40-50 min after initiation of continuous GnRH administration. Rat pituitary cells were perifused with 10(-8) M GnRH for 6 h: 10 min samples were collected for LH measurements by radioimmunoassay. As expected, the pattern of LH release was biphasic: LH levels peaked in the first phase at 30 min, decreased at 40-50 min, increased in the second phase to maximal levels at 90-110 min, and then decreased in the classical desensitization mode to near-baseline values by 300-360 min. Static incubations of pituitary cells in Petri dishes in the presence of high (10(-8) M) or submaximal (10(-9) M) GnRH concentrations confirmed the decrease in LH secretion at 40-50 min. Measurement of LH by reverse hemolytic plaque assay (RHPA) confirmed the existence of this new mode of desensitization; since 93% of all gonadotropes had become secretory at 40-50 min, the possibility of two subpopulations of gonadotropes accounting for the two phases of LH secretion appears to be ruled-out. GnRH receptor binding studies demonstrated a approximately 50% decrease in cell-surface binding in association with the desensitization at 40-50 min. These studies suggest the existence of a third mode of GnRH-induced LH secretory desensitization that is not due to gonadotrope subpopulations but may be causally associated with decreased GnRH receptor binding.

Gonadotropin-releasing hormone-induced desensitization may account for the decrease in pituitary responsiveness after the preovulatory luteinizing hormone surge.

The LH secretory response of gonadotropes to GnRH varies during the estrous cycle of the rat. The increased secretion of estrogens during the 24-48 h before the preovulatory surge of LH secretion and the enhanced quantities of progesterone secreted acutely during the surge elevate the responsiveness of hypophysial gonadotropes to GnRH. However, the cause of the massive decline in GnRH responsiveness that occurs during or after the surge remains unknown. In the present studies, we investigated the possibility that it is due to GnRH-induced desensitization of gonadotropes. Dispersed pituitary cells from proestrous and estrous rats were preincubated with GnRH (3 or 6 h, 10(- 10) or 10(-9) M), progesterone (13 h, 100 or 200 nM), GnRH plus progesterone, or medium alone. Then, the cells were retrypsinized to permit performance of the reverse hemolytic plaque assay for measurement of LH secretin, during which they were treated with GnRH(0,10(-11),10(-10), and 10(-8)M) for 2 h. The cells from estrous animals showed the large decline in GnRH responsiveness typical of that day of the cycle compared to those from proestrous animals (the total amount of LH secreted decreased by 50-70%). Preincubation of cells from proestrous rat pituitary glands with GnRH in concentrations and for durations that were designed to mimic the physiological situation induced a decline in GnRH responsiveness similar to that observed at estrus. Preincubation with progesterone also reduced the pituitary responsiveness to GnRH in a dose-dependent manner, but did not show additive effects with GnRH. Our results suggest that the major increase in GnRH secretion that induces the preovulatory surge of LH secretion may also participate in inducing the major decrease in pituitary responsiveness to GnRH that occurs from proestrus to estrus.