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 muM 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.