Role of protein kinase C in the regulation of prolactin and growth hormone release

Protein kinase C has been implicated in the mechanism of action of some hormones. Phorbol esters are known to activate this enzyme, but although short-term exposure t o such compounds causes activation, prolonged exposure can reduce both the amount and the activity of the enzyme to low levels (Ballester & Rosen, 1985). A number of investigators have used such down-regulation of protein kinase C to investigate its role in the actions of hormones and regulation of hormone secretion (Hii et al., 1987; Smal & De Meyts, 1987; Ohmura et al., 1988). We have used down-regulation by the phorbol ester, tetradecanoyl phorbol acetate (TPA) to investigate the role of protein kinase C in the actions of growth hormone releasing hormone (GHRH), thyrotrophin releasing hormone (TRH), dopamine and somatostatin on growth hormone (GH) and prolactin secretion in cultured ovine anterior pituitary cells. Cells from ovine anterior pituitary glands were dispersed and placed in short-term culture for 3-4 days as described by Ray & Wallis (1982). Before use in experiments. they were washed with serum-free medium and then incubated in fresh serum-free medium containing test materials, as indicated, for 30 min. GH and prolactin secreted into the medium and intracellular cyclic AMP concentrations were measured by radioimmunoassay as described previously (Ray & Wallis, 1982). TPA (100 ng/ml) stimulated secretion of GH and prolactin from cultured ovine anterior pituitary cells, as shown previously (Ray et ul.. 1986). Pretreatment of the cells with TPA (0.1-1000 ng/ml) for 5 h resulted in a dose-dependent attenuation of this stimulatory effect on both prolactin and G H release. Pretreatment with 1 pg of TPA/ml completely abolished the stimulatory effect of 100 ng of TPA/ml on release of these hormones, indicating complete down-regulation of protein kinase C. Pretreatment of ovine anterior pituitary cells with 1 pug of TPA/ml for 5 h had no significant effect on the ability of TRH (100 nM) to stimulate prolactin release. The ability of dopamine (100 n M ) to inhibit TRH-stimulated prolactin release was also not significantly affected by the TPA pretreatment. TPA pretreatment ( 5 h) did attenuate the stimulatory effect of GHRH ( 1 nM) on GH release (44 k 5%). The ability of somatostatin (100 nM) to inhibit GHRH-stimulated GH release was also partially blocked by the TPA pretrcatment. The ability of 10 pM-forskohn to stimulate GH release was similarly blocked, as was the inhibitory effect of somatostatin on forskolin-stimulated GH release.