DOWN-REGULATION OF PROTEIN-KINASE-C LEVELS LEADS TO INHIBITION OF GNRH-STIMULATED GONADOTROPIN-SECRETION FROM DISPERSED PITUITARY-CELLS OF GOLDFISH

We have previously shown that the abilities of the two native goldfish GnRHs, salmon GnRH (sGnRH) and chicken GnRH II (cGnRH II), to stimulate gonadotropin (GtH) secretion and elevate intracellular Ca2+ levels are mimicked by the protein kinase C (PKC) stimulators, 1,2-dioctanoylglycerol (DiC8) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). The ability of PKC inhibitors to attenuate GnRH-stimulated GtH secretion was also demonstrated. In the present study, the involvement of PKC was examined through the reduction of cellular PKC levels by prolonged preincubation of the cells with TPA (TPA desensitization). TPA pretreatment reduced the levels of PKC in fish pituitary cells as measured by immunoblotting (Western blot). Pretreatment of dispersed goldfish pituitary cells in static culture with TPA abolished the GtH responses to sGnRH, cGnRH II and ionomycin, and drastically reduced TPA-and DiC8-stimulated GtH release, but had no major effect on forskolin-induced GtH release. TPA pretreatment also reduces the cell content of GtH in goldfish pituitary cells. Interestingly, treatment with all of the pharmacological secretagogues tested led to a decrease in cellular contents of GtH, however, the two native GnRHs had no such effect. In rapid column perifusion experiments (1-min fractions), the GtH responses induced by both native GnRHs were characterized by an initial acute increase in hormone secretion followed by a 'plateau' phase which is smaller in magnitude relative to the initial phase. TPA pretreatment of perifused celts greatly reduced both the peak and plateau phases of sGnRH- and cGnRH II-stimulated GtH secretion; TPA-induced GtH release is also greatly attenuated. In contrast, forskolin-stimulated GtH release was enhanced by TPA desensitization. The ability of forskolin to stimulate GtH release at similar (in static culture) or enhanced (in column perifusion) levels following TPA desensitization, despite a reduction in cell content of GtH, strongly indicates that the reduced GtH response to sGnRH and cGnRH II following TPA pretreatment is not due to a simple reduction in the availability of releasable GtH. Additionally, the magnitude of decreases in cellularcontents of GtH was not sufficient to totally account for the inhibition of GnRH action observed in the PKC-depleted cells. These results strongly suggest that PKC plays an important role in both acute and prolonged GnRH-stimulated GtH secretion in the goldfish.