D1 Dopamine Receptor Down‐regulates NADPH Oxidase Activity via a Protein Kinase C and Protein kinase A Cross‐talk in Human Kidney Cells

Abstract

D1‐like dopamine receptors inhibit NADPH oxidase activity via stimulation of protein kinase A (PKA) and inhibition of phospholipase D. We tested the hypothesis that a cross‐talk between PKA and protein kinase C (PKC) is involved in the regulation of NADPH oxidase activity in HEK‐293 cells heterologously expressing human D1 dopamine receptor (hD1R) (n=6/group). The D1‐like receptor agonist, fenoldopam (Fen., 1 μM), decreased NADPH oxidase activity (63±5.0% vs. vehicle=101.6±6.2%) that was completely blocked by the D1‐like receptor antagonist, Sch23390 (5μM) (84±6.2%), which, by itself, had no effect. The PKA inhibitor, H89 (10 μM), by itself, had no effect on NADPH oxidase activity but prevented (85.6±5.0%) the inhibitory effect of fenoldopam (P<0.01, vs. others, ANOVA Newman‐Keuls test). The D1R‐mediated decrease in oxidase activity (65±3.0%) was also completely reversed by PKC inhibitors, e.g., bisinolylmaleimide I (1 μM)(94±6.0%) and staurosporine (10 nM) (93.4±7.8%), which, by themselves, had no effect (P<0.001). Moreover, the PKC activator, phobol‐12‐myristate‐13‐acetate (PMA,1 μM), inhibited oxidase activity (72.7±8.1%), that was accompanied by increased PKCθS647 phosphorylation, effects that were also abolished by H89 (103±7.4%), which, by itself, had no effect (P<0.05). We conclude that D1R inhibition of NADPH oxidase activity is dependent on both PKA and PKC (PKCθS647) signaling. Our studies demonstrate for the first time that a cross‐talk between PKA and PKC signaling pathways plays an important role in the D1R negative regulation of NADPH oxidase activity in human kidney cells.