D ₁ -Like Receptors Regulate NADPH Oxidase Activity and Subunit Expression in Lipid Raft Microdomains of Renal Proximal Tubule Cells

Abstract

Abstract-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 D 1 -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 (58Ϯ3.3%) than in hypertensive rats (31Ϯ5.2%; PϽ0.05; nϭ6 per group). Fenoldopam decreased the amount of Nox2 that coimmunoprecipitated with p67 phox in cells from normotensive rats. D 1 -like receptors may decrease oxidase activity by disrupting the distribution and assembly of oxidase subunits in cell membrane microdomains. The cholesterol-depleting reagent methyl-␤-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 Ⅲ dopamine receptor Ⅲ reactive oxygen species Ⅲ lipid rafts T he NADPH oxidase (Nox) enzyme family is a major source of reactive oxygen species (ROS), eg, superoxide anion, hydrogen peroxide, and the hydroxyl radical. 1,2 Noxdependent ROS regulate diverse cellular processes, including angiotensin II-mediated renal growth and cardiovascular remodeling. [3][4][5][6] The increased generation of ROS by Nox contributes to human 5-7 and animal hypertension. 8 -15 Nox activity and superoxide formation are increased in vascular smooth muscle, 5,13 endothelial, 4,14 and neural cells 15 in genetic and acquired hypertension. The renal contribution to the pathogenesis of hypertension has also been ascribed to increased ROS production. [7][8][9][10]12,14 Lipid rafts (LRs) are membrane microdomains composed of glycosylphosphatidylinositol-linked proteins, glycosphingolipids, and cholesterol. 16 -19 Caveolae and LRs have been implicated in protein trafficking and signal transduction. 16 -19 They also serve as compartments for the recruitment of cell signaling components and enzymes to increase the efficient and rapid coupling of receptors to Ͼ1 effector system. G protein-coupled receptors, including dopamine receptors, are associated with caveolar and noncaveolar LRs. 16 -20 We and others have reported the presence of noncaveolar LRs in immortal...

Regulation of NADPH oxidase (Nox2) by lipid rafts in breast carcinoma cells

“…Under basal conditions, both gp91 phox and p22 phox are located in LRs. Disruption of lipid rafts with MßCD allowed the NADPH oxidase subunits (gp91 phox and p22 phox ) to move from raft to non-raft positions in the membrane; this finding is similar to previous reports (51,52). These results suggest that LRs play a pivotal role in the regulation of NADPH oxidase.…”

NADPH Oxidase-derived Reactive Oxygen Species Increases Expression of Monocyte Chemotactic Factor Genes in Cultured Adipocytes

“…In the resting state, NOX2 and p22 phox are closely associated with each other in non-LR parts of the cell. Upon activation, they move into LRs and assemble together with the cytosolic activators, p47 phox and p67 phox , thereby forming the active NOX2 enzyme complex that generates superoxide using NADPH (36). Little information was available as to whether NOX4 also moves from the cytoplasm into LRs and, if so, whether this translocation is required for NOX4 to increase ROS generation in adipocytes.…”

“…Some proteins are reported to become activated when they move into LRs (36). To determine whether NOX4 is translocated to LRs following exposure of adipocytes to high glucose or palmitate, we isolated LRs by ultracentrifugation.…”

Vascular Functions of NADPH Oxidases

“…20 In cells of the proximal tubule, the NADPH oxidase components Nox2, Nox4, and Rac1 are expressed and even further induced by hypertension. 21 Interestingly, a role for lipid rafts in controlling NADPH oxidase activity in the proximal tubule has been suggested through an action on the oxidase activator Rac1: under nonstimulated conditions, membrane-bound Rac1 appears to be associated with lipid rafts. Destruction of rafts by cholesterol depletion activates the ROS formation from Nox2 and Nox4 NADPH oxidases in human proximal tubule cells.…”

“…22 Importantly, it was observed that dopamine D1 receptor agonists also release Rac1 from lipid rafts and thereby induce an agonist-stimulated ROS production in the kidney. 21 However, it should be mentioned that overexpression studies failed to link Rac1 with Nox4 activation. 23,24 Under the latter condition, Nox4 is, however, also predominantly observed in the endoplasmic reticulum, 25 and it is currently unknown whether interacting proteins as are present in smooth muscle cells 26 associate Nox4 with lipid rafts in the kidney.…”