The transport activity of the human cationic amino acid transporter hCAT-1 is downregulated by activation of protein kinase C.
1. The human cationic amino acid transporter hCAT-1 contains several consensus sequences for phosphorylation by protein kinase C (PKC). This study investigates the effect of PKC activation on hCAT-1-mediated transport. 2. When expressed in Xenopus laevis oocytes, hCAT-1-mediated L-arginine transport was reduced to 44+/-3% after a 30 min treatment of the oocytes with 100 nM phorbol-12-myristate-13-acetate (PMA). 4 alpha-phorbol-12,13-didecanoate (4 alpha-PDD, 100 nM) had no effect. 3. In EA.hy926 endothelial cells, maximal inhibition of hCAT-1-mediated L-arginine transport (to 3 -- 11% of control) was observed after treatment of the cells with 100 nM PMA for 4 h. A 20 -- 30 h exposure of the cells to 100 nM PMA led to the recovery of the L-arginine uptake rate that was now resistant to a second application of PMA. Phorbol-12,13-dibutyrate had similar effects as PMA, whereas 4 alpha-PDD had no effect. One microM bisindolylmaleimide I reduced the PMA effect significantly. 4. Interestingly, a 4 h treatment with 100 nM PMA increased the expression of hCAT-1 mRNA 3 -- 5 fold. hCAT-1 protein levels were unchanged for up to 4 h after PMA treatment and then increased slightly between 8 -- 28 h. 5. It is concluded that PMA downregulates the intrinsic activity of hCAT-1 by a pathway involving protein kinase C.
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Department of Pharmacology, Johannes Gutenberg University, Obere Zahlbacher Str. 67, 55101 Mainz, Germany.
Förstermann
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Closs
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D026923:Amino Acid Transport Systems, Basic; D000818:Animals; D001120:Arginine / Q000378:metabolism; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D015536:Down-Regulation; D004789:Enzyme Activation; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection; D014982:Xenopus laevis
D026923:Amino Acid Transport Systems, Basic; D002352:Carrier Proteins; D008565:Membrane Proteins; D001120:Arginine; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate