Characterization of phorbol ester- and diacylglycerol-stimulated secretion in permeable GH3 pituitary cells....

Prolactin (PRL) release in permeable GH3 pituitary cells was stimulated by the protein kinase C activators 12-O-tetradecanoylphorbol 13-acetate (TPA) and 1-oleoyl-2-acetyl-sn-glycerol (OAG). Both agents stimulated secretion at 10 nM Ca2+, but higher [Ca2+] (greater than 0.1 microM) potentiated TPA and OAG action. Maximal potentiation occurred at 1 microM calculated free Ca2+, and a similar value was obtained when the cytoplasmic [Ca2+] was measured with the Ca2+-sensitive dye Quin 2. Release of a secretory sulfated proteoglycan was also stimulated by TPA and OAG in permeable GH3 cells, with characteristics similar to those for PRL release. Trifluoroperazine, polymyxin B, neomycin, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate all inhibited both TPA- and Ca2+-stimulated PRL release, but in each case the half-maximal inhibitory concentrations were approximately 2-fold higher for TPA-stimulated release compared to Ca2+-stimulated release. Thyrotropin-releasing hormone (TRH) and guanosine 5'-Q-thiotriphosphate, which stimulate polyphosphoinositide breakdown in permeable cells, were found to be only weak stimulators of PRL release, compared to TPA and exogenous diacylglycerol. However, a much stronger effect of TRH was seen if cells were briefly treated with TRH prior to permeabilization. PRL release from TRH-pretreated permeable cells resembled TPA- and OAG-stimulated secretion, with [Ca2+] greater than 0.1 microM potentiating the effect of TRH pretreatment. These studies support the hypothesis that PRL release in GH3 cells can be stimulated directly by a diacylglycerol-activated secretory mechanism whose activity is modulated by [Ca2+].