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(Hypertension. 2009;53:1054.)
© 2009 American Heart Association, Inc.

Original Articles

D₁-Like Receptors Regulate NADPH Oxidase Activity and Subunit Expression in Lipid Raft Microdomains of Renal Proximal Tubule Cells

Hewang Li; Weixing Han; Van Anthony M. Villar; Lindsay B. Keever; Quansheng Lu; Ulrich Hopfer; Mark T. Quinn; Robin A. Felder; Pedro A. Jose; Peiying Yu

From the Center for Molecular Physiology Research (H.L., V.A.M.V., L.B.K., Q.L., P.A.J., P.Y.), Children’s National Medical Center, Washington, DC; Department of Pediatrics (H.L., V.A.M.V., L.B.K., Q.L., P.A.J., P.Y.), George Washington University School of Medicine, Washington, DC; Department of Cardiovascular Medicine (W.H.), Anhui Medical University, Hefei, Anhui, People’s Republic of China; Department of Physiology and Biophysics (U.H.), Case Western Reserve School of Medicine, Cleveland, Ohio; Department of Veterinary Molecular Biology (M.T.Q.), Montana State University, Bozeman; and the Department of Pathology (R.A.F.), University of Virginia Health Sciences Center, Charlottesville.

Correspondence to Peiying Yu, Center for Molecular Physiology Research, Children’s National Medical Center, 111 Michigan Ave, NW, Washington, DC 20010. E-mail pyu{at}cnmc.org

NADPH oxidase (Nox)–dependent reactive oxygen species production is implicated in the pathogenesis of cardiovascular diseases, including hypertension. We tested the hypothesis that oxidase subunits are differentially regulated in renal proximal tubules from normotensive and spontaneously hypertensive rats. Basal Nox2 and Nox4, but not Rac1, in immortalized renal proximal tubule cells and brush border membranes were greater in hypertensive than in normotensive rats. However, more Rac1 was expressed in lipid rafts in cells from hypertensive rats than in cells from normotensive rats; the converse was observed with Nox4, whereas Nox2 expression was similar. The D₁-like receptor agonist fenoldopam decreased Nox2 and Rac1 protein in lipid rafts to a greater extent in hypertensive than in normotensive rats. Basal oxidase activity was 3-fold higher in hypertensive than in normotensive rats but was inhibited to a greater extent by fenoldopam in normotensive (58±3.3%) than in hypertensive rats (31±5.2%; P<0.05; n=6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67^phox in cells from normotensive rats. D₁-like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl–β-cyclodextrin decreased oxidase activity and cholesterol content to a greater extent in hypertensive than in normotensive rats. The greater basal levels of Nox2 and Nox4 in cell membranes and Nox2 and Rac1 in lipid rafts in hypertensive rats than in normotensive rats may explain the increased basal oxidase activity in hypertensive rats.

Key Words: NADPH oxidase • dopamine receptor • reactive oxygen species • lipid rafts