dopamine receptor regulation of reactive oxygen species production, NADPH oxidase, and blood pressure. Am J Physiol Regul Integr Comp Physiol 290: R96 -R104, 2006; doi:10.1152/ajpregu.00434.2005.-Activation of D1-like receptors (D 1 and/or D5) induces antioxidant responses; however, the mechanism(s) involved in their antioxidant actions are not known. We hypothesized that stimulation of the D 5 receptor inhibits NADPH oxidase activity, and thus the production of reactive oxygen species (ROS). We investigated this issue in D 5 receptor-deficient (D5Ϫ/Ϫ) and wild-type (D 5ϩ/ϩ) mice. NADPH oxidase protein expression (gp91 phox , p47 phox , and Nox 4) and activity in kidney and brain, as well as plasma thiobarbituric acid-reactive substances (TBARS) were higher in D 5Ϫ/Ϫ than in D5ϩ/ϩ mice. Furthermore, apocynin, an NADPH oxidase inhibitor, normalized blood pressure, renal NADPH oxidase activity, and plasma TBARS in D 5Ϫ/Ϫ mice. In HEK-293 cells that heterologously expressed human D 5 receptor, its agonist fenoldopam decreased NADPH oxidase activity, expression of one of its subunits (gp91 phox ), and ROS production. The inhibitory effect of the D 5 receptor activation on NADPH oxidase activity was independent of cAMP/PKA but was partially dependent on phospholipase D2. The ability of D5 receptor stimulation to decrease ROS production may explain, in part, the antihypertensive action of D 5 receptor activation. hypertension DURING THE PAST DECADE, DOPAMINE has been shown as an important regulator of blood pressure, sodium balance, and renal and adrenal function through an independent peripheral dopaminergic system (32). Dopamine exerts its actions via two families of cell surface receptors that belong to the superfamily of G protein-coupled receptors. D 1 -like receptors (D 1 and D 5 ) stimulate adenylyl cyclases, while D 2 -like receptors (D 2 , D 3 , and D 4 ) inhibit adenylyl cyclases (32,45). Abnormal signaling of D 1 -like receptors has been shown to be involved in rodent models of genetic hypertension and in humans with essential hypertension (7,10,17,28,32,55). However, the precise D 1 -like receptor involved remains to be determined. There is an abnormal renal D 1 function in hypertension, which is caused by activated variants of the G protein-coupled receptor kinase type 4 (16, 32). The D 5 locus is not linked to hypertension in Dahl salt-sensitive rats (22), and mutations of the D 5 are not found in spontaneously hypertensive rats (3). However, the locus of the human D 5 (hD 5 ), 4p15.1-16

Abstract-Dysfunction of D 2 -like receptors has been reported in essential hypertension. Disruption of D 2 R in mice (D 2 Ϫ/Ϫ ) results in high blood pressure, and several D 2 R polymorphisms are associated with decreased D 2 R expression. Because D 2 R agonists have antioxidant activity, we hypothesized that increased blood pressure in D 2 Ϫ/Ϫ is related to increased oxidative stress. D 2 Ϫ/Ϫ mice had increased urinary excretion of 8-isoprostane, a parameter of oxidative stress; increased activity of reduced nicotinamide-adenine dinucleotide phosphate oxidase in renal cortex; increased expression of the reduced nicotinamide-adenine dinucleotide phosphate oxidase subunits Nox1, Nox2, and Nox4; and decreased expression of the antioxidant enzyme heme-oxygenase-2 in the kidneys, suggesting that regulation of reactive oxygen species (ROS) production by D 2 R involves both pro-oxidant and antioxidant systems. Apocynin, a reduced nicotinamide-adenine dinucleotide phosphate oxidase inhibitor, or hemin, an inducer of heme oxigenase-1, normalized the blood pressure in D 2 Ϫ/Ϫ mice. Because D 2 Rs in the adrenal gland are implicated in aldosterone regulation, we evaluated whether alterations in aldosterone secretion contribute to ROS production in this model. Urinary aldosterone was increased in D 2 Ϫ/Ϫ mice and its response to a high-sodium diet was impaired. Spirolactone normalized the blood pressure in D 2 Ϫ/Ϫ mice and the renal expression of Nox1 and Nox4, indicating that the increased blood pressure and ROS production are, in part, mediated by impaired aldosterone regulation. However, spironolactone did not normalize the excretion of 8-isoprostane and had no effect on expression of Nox2 or heme-oxygenase-2. Our results show that the D 2 R is involved in the regulation of ROS production and that, by direct and indirect mechanisms, altered D 2 R function may result in ROS-dependent hypertension. [1][2][3] There is abundant evidence that an intact dopaminergic system is necessary to maintain normal blood pressure and that genetic hypertension is associated with alterations in dopamine production and receptor function. [1][2][3][4] In humans and rodents, some dopamine receptor genes and their regulators are in loci linked to hypertension. 3,5 The natriuretic effect of D 1 -like agonists is impaired in genetically hypertensive rats 3,4 and in human essential hypertension. 3,4 Alterations in D 2 -like receptor function have also been reported in hypertension. 1,2 Loci in chromosome 11, where the D 2 R gene is located, are linked to hypertension. 5,6 A polymorphism in exon 6 of the D 2 R gene is associated with elevated blood pressure, 7 and a TaqI polymorphism is associated with human essential hypertension. 8 Several D 2 R polymorphisms are associated with decreased D 2 R expression 9,10 and affect D 2 R mRNA stability and synthesis of the receptor. 11 The disruption of any of the dopamine receptor genes in mice produces dopamine receptor subtype-specific hypertension. 3,[12][13][14] Specifically, disruption of the D 2 R...

The D2 dopamine receptor (D2R) is important in the pathogenesis of essential hypertension. We have already reported that systemic deletion of the D2R gene in mice results in reactive oxygen species (ROS)-dependent hypertension, suggesting that the D2R has antioxidant effect. However, the mechanism of this effect is unknown. DJ-1 is a protein which has antioxidant properties. D2R and DJ-1 are expressed in the mouse kidney and colocalize and co-imunoprecipitate in mouse renal proximal tubule cells. We hypothesized that D2Rs regulate renal ROS production in the kidney through regulation of DJ-1 expression or function. Heterozygous D2+/− mice have increased blood pressure, urinary 8-isoprostanes, and renal Nox 4 expression, but decreased renal DJ-1 expression. Silencing D2R expression in mouse renal proximal tubule cells increases ROS production and decreases the expression of DJ-1. Conversely, treatment of these cells with a D2R agonist increases DJ-1 expression and decreases Nox 4 expression and NADPH oxidase activity, effects that are partially blocked by a D2R antagonist. Silencing DJ-1 expression in mouse renal proximal tubule cells increases ROS production and Nox 4 expression. Selective renal DJ-1 silencing by the subcapsular infusion of DJ-1 siRNA in mice increases blood pressure, and renal Nox 4 expression and NADPH oxidase activity. These results suggest that the inhibitory effects of D2R on renal ROS production are at least, in part, mediated by a positive regulation of DJ-1 expression/function and that DJ-1 may have a role in the prevention of hypertension associated with increased ROS production.

The dopamine D2 receptor (D2R) regulates renal reactive oxygen species (ROS) production and impaired D2R function results in ROS-dependent hypertension. Paraoxonase 2 (PON2), which belongs to the paraoxonase gene family, is expressed in various tissues, acting to protect against cellular oxidative stress. We hypothesized that PON2 may be involved in preventing excessive renal ROS production and thus may contribute to maintenance of normal blood pressure. Moreover, the D2R may decrease ROS production, in part, through regulation of PON2. D2R co-localized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33%±6%) in D2-/- relative to D2+/+ mice. The renal subcapsular infusion of PON2 siRNA decreased PON2 protein expression (-55%), increased renal oxidative stress (2.2-fold), associated with increased renal NADPH oxidase expression (Nox1: 1.9-fold; Nox2: 2.9-fold; and Nox4: 1.6-fold) and activity (1.9-fold), and elevated arterial blood pressure (systolic: 134±5 vs. 93±6 mmHg; diastolic: 97±4 vs. 65±7 mmHg; mean: 113±4 vs. 75±7 mmHg). To determine the relevance of the PON2 and D2R interaction in humans, we studied human renal proximal tubule cells. Both D2R and PON2 were found in non-lipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D2R/D3R agonist quinpirole (1μM, 24h) decreased ROS production (-35%±6%), associated with decreased NADPH oxidase activity (-32%±3%) and expression of Nox2 (-41%±7%) and Nox4 (-47%±8%) protein, and increased expression of PON2 mRNA (2.1-fold) and protein (1.6-fold) at 24h. Silencing PON2 (siRNA, 10nM, 48 h) not only partially prevented the quinpiroleinduced decrease in ROS production by 36%, but also increased basal ROS production (1.3-fold) which was associated with an increase in NADPH oxidase activity (1.4-fold) and expression of Nox2 (2.1-fold) and Nox4 (1.8-fold) protein. Inhibition of NADPH oxidase with diphenylene iodonium (10 μM/30 min) inhibited the increase in ROS production caused by PON2 silencing. Our results suggest that renal PON2 is involved in the inhibition of renal NADPH oxidase activity and ROS production and contributes to the maintenance of normal blood pressure. PON2 is positively regulated by D2R and may, in part, mediate the inhibitory effect of renal D2R on NADPH oxidase activity and ROS production.