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D5 dopamine receptor decreases NADPH oxidase, reactive oxygen species and blood pressure via heme oxygenase-1.

ABSTRACT: D5 dopamine receptor (D5R) knock-out mice (D5(-/-)) have a higher blood pressure (BP) and higher reactive oxygen species (ROS) production than their D5R wild-type littermates (D5(+/+)). We tested the hypothesis that the high BP and increased ROS production in D5(-/-) mice may be caused by decreased heme oxygenase-1 (HO-1) expression and activity. We found that renal HO-1 protein expression and HO enzyme activity were decreased (65 and 50%, respectively) in D5(-/-) relative to D5(+/+) mice. A 24?h of administration of hemin, an HO-1 inducer, increased HO-1 expression and HO activity (6.8- and 1.9-fold, respectively) and normalized the increased ROS production and BP in D5(-/-) mice. Expression of HO-1 protein and HO activity were increased (2.3- and 1.5-fold, respectively) in HEK cells that heterologously expressed human wild-type D5R (HEK-hD5R), but not the empty vector-transfected HEK-293 cells. Fenoldopam (Fen), a D5R agonist, increased HO activity (3?h), HO-1 protein expression, HO-1 and D5R colocalization and co-immunoprecipitation in HEK-hD5R cells. Cellular NADPH oxidase activity was decreased by 35% in HEK-hD5R that was abrogated with silencing of the heme oxygenase 1 gene (HMOX1). HMOX1 siRNA also impaired the ability of Fen to decrease NADPH oxidase activity in HEK-hD5R cells. In summary, the D5R positively regulates HO-1 through direct protein/protein interaction in the short-term and by increasing HO-1 protein expression in the long-term. The impaired D5R regulation of HO-1 and ROS production contributes to the pathogenesis of hypertension in D5(-/-) mice.

SUBMITTER: Lu Q

PROVIDER: S-EPMC4061571 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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D5 dopamine receptor decreases NADPH oxidase, reactive oxygen species and blood pressure via heme oxygenase-1.

Lu Quansheng Q Yang Yu Y Villar Van Anthony VA Asico Laureano L Jones John E JE Yu Peiying P Li Hewang H Weinman Edward J EJ Eisner Gilbert M GM Jose Pedro A PA

Hypertension research : official journal of the Japanese Society of Hypertension 20130221 8

D5 dopamine receptor (D5R) knock-out mice (D5(-/-)) have a higher blood pressure (BP) and higher reactive oxygen species (ROS) production than their D5R wild-type littermates (D5(+/+)). We tested the hypothesis that the high BP and increased ROS production in D5(-/-) mice may be caused by decreased heme oxygenase-1 (HO-1) expression and activity. We found that renal HO-1 protein expression and HO enzyme activity were decreased (65 and 50%, respectively) in D5(-/-) relative to D5(+/+) mice. A 24 ...[more]

PMID: 23425954

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Project description:The dopamine D(2) receptor (D(2)R) regulates renal reactive oxygen species (ROS) production, and impaired D(2)R 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, D(2)R may decrease ROS production, in part, through regulation of PON2. D(2)R colocalized with PON2 in the brush border of mouse renal proximal tubules. Renal PON2 protein was decreased (-33±6%) in D(2)(-/-) relative to D(2)(+/+) mice. 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±6mmHg; diastolic, 97±4 vs 65±7mmHg; mean 113±4 vs 75±7mmHg). To determine the relevance of the PON2 and D(2)R interaction in humans, we studied human renal proximal tubule cells. Both D(2)R and PON2 were found in nonlipid and lipid rafts and physically interacted with each other. Treatment of these cells with the D(2)R/D(3)R agonist quinpirole (1?M, 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, 48h) not only partially prevented the quinpirole-induced 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?M/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 D(2)R and may, in part, mediate the inhibitory effect of renal D(2)R on NADPH oxidase activity and ROS production.

Project description:BackgroundNADPH-oxidases (Nox) and the related Dual oxidases (Duox) play varied biological and pathological roles via regulated generation of reactive oxygen species (ROS). Members of the Nox/Duox family have been identified in a wide variety of organisms, including mammals, nematodes, fruit fly, green plants, fungi, and slime molds; however, little is known about the molecular evolutionary history of these enzymes.ResultsWe assembled and analyzed the deduced amino acid sequences of 101 Nox/Duox orthologs from 25 species, including vertebrates, urochordates, echinoderms, insects, nematodes, fungi, slime mold amoeba, alga and plants. In contrast to ROS defense enzymes, such as superoxide dismutase and catalase that are present in prokaryotes, ROS-generating Nox/Duox orthologs only appeared later in evolution. Molecular taxonomy revealed seven distinct subfamilies of Noxes and Duoxes. The calcium-regulated orthologs representing 4 subfamilies diverged early and are the most widely distributed in biology. Subunit-regulated Noxes represent a second major subdivision, and appeared first in fungi and amoeba. Nox5 was lost in rodents, and Nox3, which functions in the inner ear in gravity perception, emerged the most recently, corresponding to full-time adaptation of vertebrates to land. The sea urchin Strongylocentrotus purpuratus possesses the earliest Nox2 co-ortholog of vertebrate Nox1, 2, and 3, while Nox4 first appeared somewhat later in urochordates. Comparison of evolutionary substitution rates demonstrates that Nox2, the regulatory subunits p47phox and p67phox, and Duox are more stringently conserved in vertebrates than other Noxes and Nox regulatory subunits. Amino acid sequence comparisons identified key catalytic or regulatory regions, as 68 residues were highly conserved among all Nox/Duox orthologs, and 14 of these were identical with those mutated in Nox2 in variants of X-linked chronic granulomatous disease. In addition to canonical motifs, the B-loop, TM6-FAD, VXGPFG-motif, and extreme C-terminal regions were identified as important for Nox activity, as verified by mutational analysis. The presence of these non-canonical, but highly conserved regions suggests that all Nox/Duox may possess a common biological function remained in a long history of Nox/Duox evolution.ConclusionThis report provides the first comprehensive analysis of the evolution and conserved functions of Nox and Duox family members, including identification of conserved amino acid residues. These results provide a guide for future structure-function studies and for understanding the evolution of biological functions of these enzymes.

Project description:BackgroundActivation of microglia causes the production of proinflammatory factors and upregulation of NADPH oxidase (NOX) that form reactive oxygen species (ROS) that lead to neurodegeneration. Previously, we reported that 10 daily doses of ethanol treatment induced innate immune genes in brain. In the present study, we investigate the effects of chronic ethanol on activation of NOX and release of ROS, and their contribution to ethanol neurotoxicity.MethodsMale C57BL/6 and NF-κB enhanced GFP mice were treated intragastrically with water or ethanol (5 g/kg, i.g., 25% ethanol w/v) daily for 10 days. The effects of chronic ethanol on cell death markers (activated caspase-3 and Fluoro-Jade B), microglial morphology, NOX, ROS and NF-κB were examined using real-time PCR, immunohistochemistry and hydroethidine histochemistry. Also, Fluoro-Jade B staining and NOX gp91phox immunohistochemistry were performed in the orbitofrontal cortex (OFC) of human postmortem alcoholic brain and human moderate drinking control brain.ResultsEthanol treatment of C57BL/6 mice showed increased markers of neuronal death: activated caspase-3 and Fluoro-Jade B positive staining with Neu-N (a neuronal marker) labeling in cortex and dentate gyrus. The OFC of human post-mortem alcoholic brain also showed significantly more Fluoro-Jade B positive cells colocalized with Neu-N, a neuronal marker, compared to the OFC of human moderate drinking control brain, suggesting increased neuronal death in the OFC of human alcoholic brain. Iba1 and GFAP immunohistochemistry showed activated morphology of microglia and astrocytes in ethanol-treated mouse brain. Ethanol treatment increased NF-κB transcription and increased NOX gp91phox at 24 hr after the last ethanol treatment that remained elevated at 1 week. The OFC of human postmortem alcoholic brain also had significant increases in the number of gp91phox + immunoreactive (IR) cells that are colocalized with neuronal, microglial and astrocyte markers. In mouse brain ethanol increased gp91phox expression coincided with increased production of O2- and O2- - derived oxidants. Diphenyleneiodonium (DPI), a NOX inhibitor, reduced markers of neurodegeneration, ROS and microglial activation.ConclusionsEthanol activation of microglia and astrocytes, induction of NOX and production of ROS contribute to chronic ethanol-induced neurotoxicity. NOX-ROS and NF-κB signaling pathways play important roles in chronic ethanol-induced neuroinflammation and neurodegeneration.

Dataset Information

D5 dopamine receptor decreases NADPH oxidase, reactive oxygen species and blood pressure via heme oxygenase-1.

Publications

D5 dopamine receptor decreases NADPH oxidase, reactive oxygen species and blood pressure via heme oxygenase-1.

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