GNRH SIGNALING PATHWAYS AND GNRH-INDUCED HOMOLOGOUS DESENSITIZATION IN A GONADOTROPE CELL-LINE (ALPHA-T3-1)

Exposure of the gonadotrope cells to gonadotropin-releasing hormone (G nRH) reduces their responsiveness to a new GnRH stimulation (homologou s desensitization). The time frame as well as the mechanisms underlyin g this phenomenon are yet unclear. We studied in a gonadotrope cell li ne (alpha T3-1) the effects of short as well as long term GnRH pretrea tments on the GnRH-induced phospholipases-C (PLC), -A(2) (PLA(2)) and -D (PLD) activities, by measuring the production of IP3, total inosito l phosphates (IPs), arachidonic acid (AA) and phosphatidylethanol (PEt ) respectively. We demonstrated that although rapid desensitization of GnRH-induced IF, formation did not occur in these cells, persistent s timulation of cells with GnRH or its analogue resulted in a time-depen dent attenuation of GnRH-elicited IPs formation. GnRH-induced IPs dese nsitization was potentiated after direct activation of PKC by the phor bol ester TPA, suggesting the involvement of distinct mechanisms in th e uncoupling exerted by either GnRH or TPA on GnRH-stimulated PI hydro lysis. The levels of individual phosphoinositides remained unchanged u nder any desensitization condition applied. Interestingly, while the G nRH-induced PLA(2) activity was rapidly desensitized (2.5 min) after G nRH pretreatments, the neuropeptide-evoked PLD activation was affected at later times, indicating an important time-dependent contribution o f these enzymatic activities in the sequential events underlying the G nRH-induced homologous desensitization processes in the gonadotropes. Under GnRH desensitization conditions, TPA was still able to induce PL D activation and to further potentiate the GnRH-evoked PLD activity. a lpha T3-1 cells possess several PKC isoforms which, except PKC zeta, w ere differentially down-regulated by TPA (PKC alpha, beta(II), delta, epsilon, eta) or GnRH (PKC beta(II), delta, epsilon, eta). In spite of the presence of PKC inhibitors or down-regulation of PKC isoforms by TPA, the desensitizing effect of the neuropeptide on GnRH-induced IPs, AB and PEt formation remained unchanged. In conclusion, in alpha T3-1 cells the GnRH-induced homologous desensitization affects the GnRH co upling with PLC, PLA, and PLD by mechanism(s) which do not implicate T PA-sensitive PKC isoforms, but likely reflect time-dependent modificat ion(s) on the activation processes of the enzymes. (C) 1998 Elsevier S cience Ireland Ltd. All rights reserved.