title: ET-1 receptors in C-6 cells: homologous down-regulation and modulation by protein kinase C.
abstract: Endothelin (ET-1) receptors were studied in the C-6 glia cell line. ET-1 binds to C-6 cells in a temperature- and time-dependent manner, with an apparent Kd of 1.16 +/- 0.07 10(-10) M and a Bmax of 96,500 +/- 6000 sites/cell (mean +/- SEM, n = 27). Stimulation of protein kinase C (PKC) with the diacylglycerol (DAG) analog phorbol 12-myristate 13-acetate (PMA) resulted in a decrease in the number of receptors in a dose-dependent manner. Inhibition of PKC with H-7 eliminated the effect of PMA on the reduction of binding sites. Treatment with exogenous 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 1,2-dioctanoyl-sn-glycerol (DOG), release of endogenous DAG with phospholipase C, and inhibition of the metabolism of DAG with the diacylglycerol kinase inhibitor R 59022 also resulted in a decrease in the number of receptors. The effect of these agents was inhibited by H-7. ET-1-mediated down-regulation of receptors was also demonstrated, but the down-regulation was not affected by H-7 or by depletion of cellular PKC with chronic, high dose of PMA. Internalization constants of ET-1-receptor complex was also measured according to the model of Wiley and Cunningham (Cell 25 (1981) 433). PMA- and ET-1-mediated down-regulation of receptors was associated with an increase in the endocytosis constant for the hormone-receptor complex and a decrease in the rate of insertion of receptor into the plasma membrane. PMA, but not ET-1, increased the rate of endocytosis of unoccupied receptors. Radioiodinated ET-1 was crosslinked to the receptor after binding, extracted and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A band at 66 kDa was obtained. These studies show that ET-1 and PKC activation produce down-regulation of ET-1 membrane receptors and that ET-1-mediated down-regulation probably does not involve the activation of PKC.
authors.affiliation: Department of Internal Medicine, College of Medicine, University of South Florida, Tampa.
authors.forename: E N
authors.initials: EN
authors.lastname: Cozza
authors.forename: M C
authors.initials: MC
authors.lastname: Vila
authors.forename: C E
authors.initials: CE
authors.lastname: Gomez-Sanchez
mesh_terms: D000818:Animals; D015536:Down-Regulation; D016232:Endothelins; D007700:Kinetics; D008970:Molecular Weight; D009457:Neuroglia / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000187:drug effects / Q000378:metabolism*; D017466:Receptors, Endothelin; D013696:Temperature; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
chemical_list: D016232:Endothelins; D010455:Peptides; D011956:Receptors, Cell Surface; D017466:Receptors, Endothelin; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate