PMA-sensitive protein kinase C is not necessary in TRH-stimulated prolactin release from female rat primary pituitary cells

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In GH 3 cells and other clonal rat pituitary tumor cells, TRH has been shown to mediate its effects on prolactin release via a rise of cytosolic Ca 2+ and activation of protein kinase C. In this study, we examined the role of protein kinase C in TRH- stimulated prolactin release from female rat primary pituitary cell culture. Both TRH and PMA stimulated prolactin release in a dose-dependent manner. When present together at maximal concentrations, TRH and PMA produced an effect which was slightly less than additive. Pretreatment of rat pituitary cells with 10 −6 M PMA for 24 hrs completely down-regulated protein kinase C, since such PMA-pretreated cells did not release prolactin in response to a second dose of PMA. Interestingly, protein kinase C down-regulation had no effect on TRH-induced prolactin release from rat pituitary cells. In contrast, PMA-pretreated GH 3 cells did not respond to a subsequent stimulation by either PMA or TRH. Pretreatment of rat pituitary cells with TRH (10 −7 M, 24 hrs) inhibited the subsequent response to TRH, but not PMA. Forskolin, an adenylate cyclase activator, stimulated prolactin release by itself and in a synergistic manner when incubated together with TRH or PMA. The synergistic effects of forskolin on prolactin release was greater in the presence of PMA than TRH. Down-regulation of protein kinase C by PMA pretreatment abolished the synergistic effect produced by PMA and forskolin but had no effect on those generated by TRH and forskolin. sn-1,2-Dioctanylglycerol (DOG) pretreatment attenuated the subsequent responses to DOG and PMA but not TRH. The effect of TRH, but not PMA, on prolactin release required the presence of extracellular Ca 2+. In conclusion, the mechanism by which TRH causes prolactin release from rat primary pituitary cells is different from that of GH 3 cells; the former is a protein kinase C-independent process whereas the latter is at least partially dependent upon the activation of protein kinase C.