title: Dopamine D1 receptor-mediated inhibition of NADPH oxidase activity in human kidney cells occurs via protein kinase A-protein kinase C cross talk.
abstract: Dopamine cellular signaling via the D(1) receptor (D(1)R) involves both protein kinase A (PKA) and protein kinase C (PKC), but the PKC isoform involved has not been determined. Therefore, we tested the hypothesis that the D(1)R-mediated inhibition of NADPH oxidase activity involves cross talk between PKA and a specific PKC isoform(s). In HEK-293 cells heterologously expressing human D(1)R (HEK-hD(1)), fenoldopam, a D(1)R agonist, and phorbol 12-myristate 13-acetate (PMA), a PKC activator, inhibited oxidase activity in a time- and concentration-dependent manner. The D(1)R-mediated inhibition of oxidase activity (68.1±3.6%) was attenuated by two PKA inhibitors, H89 (10μmol/L; 88±8.1%) and Rp-cAMP (10μmol/L; 97.7±6.7%), and two PKC inhibitors, bisindolylmaleimide I (1μmol/L; 94±6%) and staurosporine (10nmol/L; 93±8%), which by themselves had no effect (n=4-8/group). The inhibitory effect of PMA (1μmol/L) on oxidase activity (73±3.2%) was blocked by H89 (100±7.8%; n=5 or 6/group). The PMA-mediated inhibition of NADPH oxidase activity was accompanied by an increase in PKCθ(S676), an effect that was also blocked by H89. Fenoldopam (1μmol/L) also increased PKCθ(S676) in HEK-hD(1) and human renal proximal tubule (RPT) cells. Knockdown of PKCθ with siRNA in RPT cells prevented the inhibitory effect of fenoldopam on NADPH oxidase activity. Our studies demonstrate for the first time that cross talk between PKA and PKCθ plays an important role in the D(1)R-mediated negative regulation of NADPH oxidase activity in human kidney cells.
authors.affiliation: Center for Molecular Physiology Research, Children's National Medical Center, Department of Pediatrics, George Washington University School of Medicine, Washington, DC 20010, USA. pyu@cnmc.org
authors.forename: Peiying
authors.initials: P
authors.lastname: Yu
authors.forename: Weixing
authors.initials: W
authors.lastname: Han
authors.forename: Van Anthony M
authors.initials: VA
authors.lastname: Villar
authors.forename: Hewang
authors.initials: H
authors.lastname: Li
authors.forename: Francis B
authors.initials: FB
authors.lastname: Arnaldo
authors.forename: Gisela P
authors.initials: GP
authors.lastname: Concepcion
authors.forename: Robin A
authors.initials: RA
authors.lastname: Felder
authors.forename: Mark T
authors.initials: MT
authors.lastname: Quinn
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Jose
mesh_terms: D002461:Cell Line, Transformed; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004298:Dopamine / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D018818:Fenoldopam / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D007668:Kidney / Q000187:drug effects / Q000201:enzymology; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D020033:Protein Isoforms / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000819:agonists* / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D019311:Staurosporine / Q000494:pharmacology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
chemical_list: D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D017447:Receptors, Dopamine D1; D019255:NADPH Oxidases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D019311:Staurosporine; D018818:Fenoldopam; D013755:Tetradecanoylphorbol Acetate; D004298:Dopamine