Published Online
on January 14, 2008

A more recent version of this article appeared on February 1, 2008

This Article Full Text (PDF) Data Supplement All Versions of this Article: 51/2/481    most recent HYPERTENSIONAHA.107.103275v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Han, W. Articles by Yu, P. Search for Related Content PubMed PubMed Citation Articles by Han, W. Articles by Yu, P. Pubmed/NCBI databases Compound via MeSH Substance via MeSH Hazardous Substances DB CHOLESTEROL Related Collections Hypertension - basic studies Oxidant stress

Submitted on October 14, 2007
Revised on November 5, 2007

Lipid Rafts Keep NADPH Oxidase in the Inactive State in Human Renal Proximal Tubule Cells

Weixing Han; Hewang Li; Van Anthony M. Villar; Annabelle M. Pascua; Mustafa I. Dajani; Xiaoyang Wang; Aruna Natarajan; Mark T. Quinn; Robin A. Felder; Pedro A. Jose; and Peiying Yu^*

From the Department of Pediatrics (W.H., H.L., V.A.M.V., A.M.P., M.I.D., X.W., A.N., P.A.J., P.Y.), Georgetown University School of Medicine, Washington, DC; the Department of Veterinary Molecular Biology (M.Q.), Montana State University, Bozeman, Mont; and the Department of Pathology (R.A.F.), University of Virginia, Charlottesville, Va.

^* To whom correspondence should be addressed. E-mail: yup{at}georgetown.edu.

Abstract—Recent studies have indicated the importance of cholesterol-rich membrane lipid rafts (LRs) in oxidative stress-induced signal transduction. Reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidases, the major sources of reactive oxygen species, are implicated in cardiovascular diseases, including hypertension. We tested the hypothesis that NADPH oxidase subunits and activity are regulated by LRs in human renal proximal tubule cells. We report that a high proportion of p22^phox and the small GTPase Rac1 are expressed in LRs in human renal proximal tubule cells. The D₁-like receptor agonist, fenoldopam (1 µmol/L per 20 minutes) dispersed Nox subunits within LRs and non-LRs and decreased oxidase activity (30.7±3.3%). In contrast, cholesterol depletion (2% methyl--cyclodextrin [CD]) translocated NADPH oxidase subunits out of LRs and increased oxidase activity (154.0±10.5% versus control, 103.1±3.4%), which was reversed by cholesterol repletion (118.9±9.9%). Moreover, NADPH oxidase activation by CD (145.5±9.0%; control: 98.6±1.6%) was also abrogated by the NADPH oxidase inhibitors apocynin (100.4±3.2%) and diphenylene iodonium (9.5±3.3%). Furthermore, CD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0±5.1% versus CD: 162.0±2.0%) or Nox4 (108.0±10.8% versus CD: 152.0±9.8%). We have demonstrated for the first time that disruption of LRs results in NADPH oxidase activation that is abolished by antioxidants and silencing of Nox2 or Nox4. Therefore, in human renal proximal tubule cells, LRs maintain NADPH oxidase in an inactive state.

Key words: NADPH oxidase • ROS • dopamine receptors • lipid rafts

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