SELECTIVE TRANSLOCATION OF NONCONVENTIONAL PROTEIN-KINASE-C ISOENZYMES BY GONADOTROPIN-RELEASING-HORMONE (GNRH) IN THE GONADOTROPE-DERIVED ALPHA-T3-1 CELL-LINE

Gonadotropin-releasing hormone acts via G-protein coupled receptors to stimulate polyphosphoinositide-specific phospholipase C (PIG) with co nsequent elevation of cytosolic Ca2+ and activation of protein kinase C (PKC). Whereas Ca2+ is known to mediate stimulation of exocytotic go nadotropin release by GnRH, the identity of the PKC isoenzymes activat ed by;GnRH and their physiological role in gonadotropes are poorly und erstood. In many systems translocation of. PKC (from cytosolic to part iculate fractions of cellular homogenates) has been taken as evidence of hormonal activation of PKC and down regulation of PKC (by prolonged treatment with PKC-activating phorbol esters) has been used extensive ly to investigate the role of PKC in hormone action. Here we have asse ssed the influence of GnRH and phorbol esters on translocation and dow n regulation of PKC isoenzymes identified by Western blotting with iso enzyme-specific antibodies in alpha T3-1 cells (a gonadotrope-derived cell line). These cells were found to possess PKCs alpha, epsilon and zeta but not beta, delta (present in rat pituitaries) ol (present in r at brains). In short-term stimulations (10 min), the PKC-activating ph orbol esters, PMA and PDBu, caused concentration-dependent increases L n the proportion of PKC alpha and PKC epsilon recovered from the parti culate fraction of alpha T3-1 cells, but did not induce measurable tra nslocation of PKC zeta. The inactive phorbol ester 4 alpha PDBu did no t cause translocation of any of these isoenzymes. GnRH treatment induc ed a concentration-dependent increase in the proportion of particulate PKC epsilon and PKC zeta but had no measurable effect on PKC alpha tr anslocation. In longer incubations (6-48 h) GnRH failed to cause measu rable down-regulation of these isoenzymes whereas PMA treatment led to a clear down regulation of PKCs alpha and epsilon (albeit with differ ent kinetics). The data demonstrate the differential activation and do wn regulation of PKC isoenzymes by GnRH versus pertinent to the design of experiments intended to address the role of such isoenzymes in GnR H action. Moreover, they provide the first demonstration of hormonal r egulation of an atypical PKC isoenzyme (PKC zeta) in pituitary cells.