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 stimula te gonadotropin (GtH) secretion and elevate intracellular Ca2+ levels are mimicked by the protein kinase C (PKC) stimulators, 1,2-dioctanoyl glycerol (DiC8) and 12-O-tetradecanoyl phorbol-13-acetate (TPA). The a bility of PKC inhibitors to attenuate GnRH-stimulated GtH secretion wa s also demonstrated. In the present study, the involvement of PKC was examined through the reduction of cellular PKC levels by prolonged pre incubation of the cells with TPA (TPA desensitization). TPA pretreatme nt reduced the levels of PKC in fish pituitary cells as measured by im munoblotting (Western blot). Pretreatment of dispersed goldfish pituit ary cells in static culture with TPA abolished the GtH responses to sG nRH, cGnRH II and ionomycin, and drastically reduced TPA-and DiC8-stim ulated GtH release, but had no major effect on forskolin-induced GtH r elease. TPA pretreatment also reduces the cell content of GtH in goldf ish pituitary cells. Interestingly, treatment with all of the pharmaco logical secretagogues tested led to a decrease in cellular contents of GtH, however, the two native GnRHs had no such effect. In rapid colum n perifusion experiments (1-min fractions), the GtH responses induced by both native GnRHs were characterized by an initial acute increase i n 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 gre atly attenuated. In contrast, forskolin-stimulated GtH release was enh anced by TPA desensitization. The ability of forskolin to stimulate Gt H release at similar (in static culture) or enhanced (in column perifu sion) levels following TPA desensitization, despite a reduction in cel l content of GtH, strongly indicates that the reduced GtH response to sGnRH and cGnRH II following TPA pretreatment is not due to a simple r eduction in the availability of releasable GtH. Additionally, the magn itude of decreases in cellularcontents of GtH was not sufficient to to tally account for the inhibition of GnRH action observed in the PKC-de pleted cells. These results strongly suggest that PKC plays an importa nt role in both acute and prolonged GnRH-stimulated GtH secretion in t he goldfish.