Downregulation of protein kinase C levels leads to inhibition of GnRH-stimulated gonadotropin secretion from dispersed pituitary cells of goldfish.

Abstract

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 cells greatly reduced both the peak and plateau phases of sGnRH- and cGnRHII-stimulated GtH secretion;TPA-induced GtH release is also greatly attenuated.(ABSTRACT TRUNCATED AT 250 WORDS)