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 by measuring the incorporation of gamma-32P from [gamma-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. Ca2+ (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 Ca2+ to induce translocation of PKC, while being ineffective under Ca(2+)-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 (EC50) = 1 nM) translocation of the alpha, beta and delta species, while substance P stimulated time- and dose-dependent (EC50 = 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 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 alpha and beta.
Mau
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SE
Department of Physiology, Panum Institute, University of Copenhagen, Denmark.
Saermark
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Vilhardt
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D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000502:physiology; D004075:Diglycerides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D010741:Phospholipases A / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013373:Substance P / Q000494:pharmacology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D004075:Diglycerides; D007527:Isoenzymes; C044295:1,2-dioctanoylglycerol; D016718:Arachidonic Acid; D013373:Substance P; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D010741:Phospholipases A; D010727:Phosphoric Diester Hydrolases; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium