During conditions of moderate sodium excess, the dopaminergic system sits at the fulcrum of homeostatic control of water and electrolyte balance and blood pressure (1, 2). Dopamine promotes natriuresis by inhibiting sodium chloride reabsorption in specific segments of the nephron. Dopamine exerts its action on dopamine receptors, which belong to the family of G protein-coupled receptors (GPCRs). The signal transduction that follows ligand occupation of a GPCR is tightly regulated to limit the specificity and extent of cellular response. GPCR-mediated signal transduction is rapidly dampened via receptor desensitization or the waning of the responsiveness of the receptor to agonist with time. Desensitization involves receptor phosphorylation and is carried out by either GPCR kinases (GRKs) or second messenger-activated kinases such as protein kinase A and protein kinase C. Homologous desensitization involves GRKs that selectively phosphorylate only agonist-activated receptors, whereas heterologous desensitization is carried out by second messenger-dependent kinases that indiscriminately phosphorylate agonist-activated receptors and those that have not been exposed to the agonist (7).The GRKs are serine/threonine protein kinases comprising seven isoforms that are grouped into three subfamilies. GRK1 and GRK7 belong to the rhodopsin kinase subfamily and are expressed exclusively in the retina (8 -10). GRK2 and GRK3 phosphorylate the ␤-adrenergic receptor and belong to the ␤-adrenergic receptor kinase subfamily (11), and GRK4, GRK5, and GRK6 belong to the GRK4 subfamily. GRK4 is highly enriched in the testis and, to a lesser degree, in the kidneys (12, 13). Four splice variants of human GRK4 result from the alternative splicing of exons 2 and 15 (11). GRK4-␣ is considered the full-length version, whereas GRK4-␤, -␥, and -␦ are shortened versions of . The coding region of the GRK4 gene, whose 4p16.3 locus has been linked to essential hypertension (15, 16), contains several single nucleotide polymorphisms, including R65L, A142V, and A486V, which have been linked to * This work was supported, in whole or in part, by National Institutes of Health Grants HL023081, HL074940, DK039308, HL092196, and HL068686.

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...

Angiotensin II can cause hypertension through enhanced vasoconstriction of renal vasculature. One proposed mechanism for reduction of angiotensin II-induced hypertension is through inhibition of the mitogen-activated protein kinase kinase (MEK)/ extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase cascade. MEK/ERK has been shown to phosphorylate the regulatory subunit of myosin light chain at identical positions as myosin light chain kinase. There are multiple mechanisms proposed regarding angiotensin II-mediated ERK activation. We hypothesized that renal microvascular smooth muscle cells (RVSMCs) signal through a unique pathway compared with thoracic aorta smooth muscle cells (TASMCs), which is involved in blood pressure regulation. Use of epidermal growth factor (EGF) and platelet derived growth factor (PDGF) receptor-specific inhibitors 4-(3-chloroanilino)-6,7-dimethoxyquinazoline (AG1478) and 6,7-dimethoxy-3-phenylquinoxaline (AG1296), respectively, demonstrates that angiotensin II activates ERK in TASMCs, but not RVSMCs, through transactivation of EGF and PDGF receptors. In addition, inhibition of Src with its specific inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo [3,4-d]pyrimidine (PP2) abolishes angiotensin II-, but not EGF-or PDGF-, mediated phosphorylation of ERK in RVSMCs, yet it has no effect in TASMCs. The physiological significance of transactivation was examined in vivo using anesthetized Wistar-Kyoto rats with 15 mg/kg 2Ј-amino-3Ј-methoxyflavone (PD98059), an MEK inhibitor, as well as 20 mg/kg AG1478 and 1.5 mg/kg AG1296 in an acute model of angiotensin II-mediated increase in blood pressure. None of the inhibitors had an effect on basal blood pressure, and only PD98059 reduced angiotensin II-mediated increase in blood pressure. Moreover, in RVSMCs, but not TASMCs, angiotensin II localizes phosphorylated ERK to actin filaments. In conclusion, angiotensin II signals through a unique mechanism in the renal vascular bed that may contribute to hypertension.Extracellular signal-regulated kinases ERK1 and ERK2 (herein referred to as ERK) are involved in smooth muscle cell contraction (Touyz et al., 1999), attenuation of vascular relaxation (Touyz et al., 2002a), and blood pressure control (Muthalif et al., 2000a,b;Hu et al., 2007); however, this is not a universal mechanism (Watts et al., 1998;Touyz et al., 2002a). In addition, the molecular mechanism underlying the role of ERK in control of vascular contraction is not completely understood; currently there are two proposed mechanisms. One pathway is an ERK-mediated phosphorylation of the 20-kDa myosin light chain regulatory subunit (MLC20) at the same position as myosin light chain kinase (D'Angelo and Adam, 2002;Roberts, 2004). A second pathway involves ERK-mediated phosphorylation of caldesmon (Adam and Hathaway, 1993;D'Angelo et al., 1999). Phosphorylation of This work was supported by National Institutes of Health Grants HL074940 (Georgetown University; to Dr. Pedro A. Jose) and Grant DK52612 (to B.T.A.) a...