title: Lipid rafts keep NADPH oxidase in the inactive state in human renal proximal tubule cells.
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 D(1)-like receptor agonist, fenoldopam (1 micromol/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-beta-cyclodextrin [beta 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 beta 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, beta CD-induced reactive oxygen species production was reversed by knocking down either Nox2 (81.0+/-5.1% versus beta CD: 162.0+/-2.0%) or Nox4 (108.0+/-10.8% versus beta 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.
authors.affiliation: Department of Pediatrics, Georgetown University School of Medicine, Washington, DC, USA.
authors.forename: Weixing
authors.initials: W
authors.lastname: Han
authors.forename: Hewang
authors.initials: H
authors.lastname: Li
authors.forename: Van Anthony M
authors.initials: VA
authors.lastname: Villar
authors.forename: Annabelle M
authors.initials: AM
authors.lastname: Pascua
authors.forename: Mustafa I
authors.initials: MI
authors.lastname: Dajani
authors.forename: Xiaoyang
authors.initials: X
authors.lastname: Wang
authors.forename: Aruna
authors.initials: A
authors.lastname: Natarajan
authors.forename: Mark T
authors.initials: MT
authors.lastname: Quinn
authors.forename: Robin A
authors.initials: RA
authors.lastname: Felder
authors.forename: Pedro A
authors.initials: PA
authors.lastname: Jose
authors.forename: Peiying
authors.initials: P
authors.lastname: Yu
mesh_terms: D000098:Acetophenones / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D002784:Cholesterol / Q000378:metabolism; D018491:Dopamine Agonists / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018818:Fenoldopam / Q000494:pharmacology; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000235:genetics; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics; D021962:Membrane Microdomains / Q000502:physiology*; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009861:Onium Compounds / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D017447:Receptors, Dopamine D1 / Q000819:agonists; D047392:beta-Cyclodextrins / Q000494:pharmacology
chemical_list: D000098:Acetophenones; D018491:Dopamine Agonists; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008562:Membrane Glycoproteins; D009861:Onium Compounds; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D017447:Receptors, Dopamine D1; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; C007517:diphenyleneiodonium; D002784:Cholesterol; C056165:acetovanillone; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; D018818:Fenoldopam