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 by measuring the incorporation of γ-32P from [γ-32P]ATP into a synthetic peptide substrate, MBP4–14, and by immunoblotting of PKC isozymes. Using cells permeabilized with digitonin the effects of PKC cofactors on the distribution of the enzyme were studied. Ca+ (50 nm) and dioctanoyl-sn-glycerol had no effect when tested alone, but in combination they caused a redistribution of PKC from the soluble to the particulate fraction. Arachidonic acid needed Ca+ 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 α, β, δ, and ζ 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 (EC50)=1 nm) translocation of the α, β and δ species, while substance P stimulated time- and dose-dependent (EC50=1 nm) redistribution of the α and β isozymes. ζ subtype immunoreactivity could not be translocated by either agonist; neither could the immunoreactivity of ζ be down-regulated by long-term treatment (24 h) with TPA.
The results indicate that simultaneous activation of phospholipases C and A2 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 α and β.
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