Reduced renal responses to nitric oxide synthase inhibition in mice lacking the gene for gp91phox subunit of NAD(P)H oxidase

Both short-term and long-term nitric oxide (NO) blockade were shown to cause an increase in O2− activity. To assess the contribution of such enhanced O2− activity in the kidney, responses to administration of the NO synthase inhibitor nitro-l-arginine methyl ester (l-NAME; 200 μg·min−1·kg body wt−1) were assessed in knockout mice the lacking NAD(P)H oxidase subunit gp91 phox (KO; n = 10) and in wild-type (WT; n = 10) mice. Renal blood flow (RBF) and glomerular filtration rate (GFR) were determined by PAH and inulin clearances, respectively. Baseline RBF was higher in KO compared with WT mice (5.8 ± 0.5 vs. 4.5 ± 0.3 ml·min−1·g−1; P < 0.04) without significant differences in GFR (0.62 ± 0.04 vs. 0.73 ± 0.05 ml·min−1·g−1) and in mean arterial pressure (MAP; 91 ± 6 vs. 88 ± 4 mmHg). l-NAME infusion for 60 min caused similar increases in MAP (114 ± 6 vs. 113 ± 3 mmHg) in both groups but resulted in a lesser degree of reduction in RBF in KO compared with WT mice (−7 ± 3 vs. −17 ± 3%; P < 0.02), although GFR remained unchanged in both groups. The natriuretic response to systemic l-NAME infusion was attenuated in KO compared with WT mice (Δ: 3.1 ± 0.7 vs. 5.2 ± 0.6 μmol·min−1·g−1). l-NAME increased urinary 8-isoprostane excretion rate in WT (5.9 ± 1 to 7.7 ± 1 pg·min−1·g−1; P < 0.02) but not in KO mice (5.6 ± 1 to 4.9 ± 0.3 pg·min−1·g−1). In contrast, responses to another vasoconstrictor, norepinephrine, were similar in both strains of mice. These data indicate that activation of NAD(P)H oxidase results in the enhancement of O2− activity that influences renal hemodynamics and excretory function in the condition of NO deficiency.

Absence of Proton Channels in COS-7 Cells Expressing Functional NADPH Oxidase Components

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O2−) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H+ efflux was thought to be contained within the gp91phox subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063–36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COSphox). The 7D5 antibody, which detects an extracellular epitope of the gp91phox protein, labeled 96–98% of COSphox cells. NADPH oxidase was functional because COSphox (but not COSWT) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COSWT) or COSphox cells studied at pHo 7.0 and pHi 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H+ current in COSWT or COSphox cells. Therefore, gp91phox does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

NADPH oxidase of chondrocytes contains an isoform of the gp91phox subunit

Previously it has been reported that chondrocytic cells produce oxygen free radicals and express the cytosolic components of NADPH oxidase. Here we report the expression of large subunit of the flavocytochrome of NADPH oxidase in chondrocytes and, further, show that the cDNA sequence contains three single base pair differences compared with the phagocyte gp91phox gene sequence. These base-pair differences may account for the different activity profiles reported between phagocytic and non-phagocytic cells.

Cloning of murine gp91phox cDNA and functional expression in a human X- linked chronic granulomatous disease cell line

The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.

Cloning of murine gp91phox cDNA and functional expression in a human X- linked chronic granulomatous disease cell line

The phagocyte cytochrome b558, a heterodimer comprised of gp91phox and p22phox, is a flavocytochrome that mediates the transfer of electrons from NADPH to molecular oxygen in the respiratory burst oxidase. The human gene encoding the glycosylated gp91phox subunit is the site of mutations in X-linked chronic granulomatous disease (CGD). Reverse transcriptase-polymerase chain reaction was used to obtain a full- length clone for the murine gp91phox cDNA, which was 87% identical to the human gp91phox cDNA. The encoded murine protein had 39 amino acids out of 570 that differed from the human, many of which were conservative substitutions. Nonconservative replacements occurred in hydrophilic regions outside of domains previously implicated in binding to NADPH, flavin, and the cytosolic oxidase subunit p47phox. Some substitutions altered potential N-glycosylation sites, which is likely to explain why the glycosylated murine protein migrates with an apparent molecular mass of 58 kD instead of 91 kD as seen for the human protein. Expression of murine gp91phox in a human myeloid cell line with a null gp91phox allele using a mammalian expression plasmid or a retroviral vector rescued stable expression of the p22phox subunit and fully reconstituted respiratory burst activity. This suggests that the murine gp91phox subunit forms a functional cytochrome b558 heterodimer with human oxidase subunits, consistent with the high degree of identity between the mouse and human proteins in domains implicated in cytochrome function.