TRANSLOCATION OF PROTEIN-KINASE-C ISOZYMES IN LACTOTROPH-ENRICHED RAT ANTERIOR-PITUITARY CELL-CULTURES - DIFFERENTIAL-EFFECTS OF SUBSTANCE-P AND PHORBOL ESTER

Translocation of protein kinase C (PKC) from the cytosol to the plasma membranes is believed to reflect activation of the enzyme. We have studied translocation of PKC in lactotroph-enriched anterior pituitary cell cultures bg measuring the incorporation of gamma-P-32 from [gamma-P-32]ATP into a synthetic peptide substrate, MBP(4-14), and by immunoblotting of PKC isozymes. Using cells permeabilized with digitonin the effects of PKC cofactors on the distribution of the enzyme were studied. Ca2+ (50 nM) and dioctanoyl-sn-glycerol had no effect when tested alone, but in combination they caused a redistribution of PKC from the acid needed Ca2+ to induce translocation of PKC, while being ineffective under Ca2+-free conditions. Western blot analysis of partly purified PKC from lactotroph-enriched pituitary cells revealed the presence of the alpha, beta, delta and zeta isozymes. 12-O-Tetradecanoylphorbol 13-acetate (TPA) and substance P displayed different patterns of redistribution of PKC isozyme immunoreactivity from soluble to membrane-attached forms. Thus, TPA induced time- and dose-dependent (mean effective concentration (EC(50))=1 nM) translocation of the alpha, beta and delta species, while substance P stimulated time- and dose-dependent (EC(50)=1 nM) redistribution of the alpha and beta isozymes. zeta subtype immunoreactivity could not be translocated by either agonist; neither could the immunoreactivity of zeta be down-regulated by long-term treatment (24 h) with TPA. The results indicate that simultaneous activation of phospholipases C and A(2) induces a synergistic activation of PKC. Finally it is suggested that substance P may exert some of its effects in lactotrophs by translocation of PKC isozymes alpha and beta.