INHIBITION OF NAD(P)H OXIDASE REDUCES FIBRONECTIN EXPRESSION IN STROKE‐PRONE RENOVASCULAR HYPERTENSIVE RAT BRAIN
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SUMMARY The aim of the present study was to test the hypothesis that in vivo chronic inhibition of NAD(P)H oxidase reduces cerebrovascular fibronectin expression in stroke‐prone renovascular hypertensive rats (RHRSP). The RHRSP model was induced by two clips and NAD(P)H oxidase was inhibited with apocynin. The mRNA and protein expression of NAD(P)H oxidase subunit p22 phox in brains of RHRSP and Sprague‐Dawley (control) rats was determined using real‐time reverse transcription–polymerase chain reaction, western blot and immunohistochemistry. The expression of fibronectin protein was localized immunohistochemically in cerebral vessels and then quantified by western blot. Cerebrovascular fibronectin levels in RHRSP ( n = 6) were significantly higher than control ( n = 5) levels 8 weeks after operation (1.29 ± 0.04 vs 1.15 ± 0.02, respectively; P = 0.007). The p22 phox immunopositive reactivity was localized in the cerebral vasculature of control rats and RHRSP. Furthermore, chronic treatment of RHRSP with apocynin, a selective NAD(P)H oxidase inhibitor, in the drinking water for 4 weeks (1.5 mmol/L, 5 weeks after operation) resulted in a significant decrease in the expression of p22 phox protein (0.85 ± 0.01 vs 0.93 ± 0.01 in non‐treated RHRSP; n = 5; P = 0.002), with a concomitant reduction of fibronectin levels in the cerebral vasculature (1.31 ± 0.03 vs 1.56 ± 0.05 in non‐treated RHRSP; n = 5; P = 0.002). No significant differences were detected in the expression of p22 phox mRNA and protein between RHRSP (4 and 8 weeks after renal artery constriction) and the control group. These findings suggest that the chronic inhibition of NAD(P)H oxidase in vivo by apocynin reduces cerebrovascular fibronectin levels, which may lessen hypertensive cerebrovascular fibrosis.Keywords:
Renovascular Hypertension
Stroke
NAD(P)H oxidase
Accumulating evidence suggests a critical role of increased reactive oxygen species production for left ventricular (LV) remodeling and dysfunction after myocardial infarction (MI). An increased myocardial activity of the NAD(P)H oxidase, a major oxidant enzyme system, has been observed in human heart failure; however, the role of the NAD(P)H oxidase for LV remodeling and dysfunction after MI remains to be determined. MI was induced in wild-type (WT) mice (n=46) and mice lacking the cytosolic NAD(P)H oxidase component p47 phox (p47 phox −/− mice) (n=32). Infarct size was similar among the groups. NAD(P)H oxidase activity was markedly increased in remote LV myocardium of WT mice after MI as compared with sham-operated mice (83±8 versus 16.7±3.5 nmol of O 2 − · μg −1 ·min −1 ; P <0.01) but not in p47 phox −/− mice after MI (13.5±3.6 versus 15.5±3.5 nmol of O 2 − · μg −1 ·min −1 ), as assessed by electron-spin resonance spectroscopy using the spin probe CP-H. Furthermore, increased myocardial xanthine oxidase activity was observed in WT, but not in p47 phox −/− mice after MI, suggesting NAD(P)H oxidase–dependent xanthine oxidase activation. Myocardial reactive oxygen species production was increased in WT mice, but not in p47 phox −/− mice, after MI. LV cavity dilatation and dysfunction 4 weeks after MI were markedly attenuated in p47 phox −/− mice as compared with WT mice, as assessed by echocardiography (LV end-diastolic diameter: 4.5±0.2 versus 6.3±0.3 mm, P <0.01; LV ejection fraction, 35.8±2.5 versus 22.6±4.4%, P <0.05). Furthermore, cardiomyocyte hypertrophy, apoptosis, and interstitial fibrosis were substantially reduced in p47 phox −/− mice as compared with WT mice. Importantly, the survival rate was markedly higher in p47 phox −/− mice as compared with WT mice after MI (72% versus 48%; P <0.05). These results suggest a pivotal role of NAD(P)H oxidase activation and its subunit p47 phox for LV remodeling/dysfunction and survival after MI. The NAD(P)H oxidase system represents therefore a potential novel therapeutic target to prevent cardiac failure after MI.
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Phorbol myristate acetate activated in normal human neutrophils a single enzymatic entity that was dormant in unstimulated cells, optimally active at pH 7.0, and capable of oxidizing either NADH or NADPH, producing NAD(P)+ and superoxide (O27). Comparative fluorometric and spectrophotometric measurements supported the stoichiometry NAD(P)H + 20(2) leads to NAD(P)+ + 20(27) + H+. the seemingly considerable NAD(P)+ production at pH 5.5 and 6.0 was due largely to nonenzymatic oxidation of NAD(P)H by chain reactions initiated by HO27 (perhydroxyl radical), the conjugate acid of O27. This artifact, responsible for earlier erroneous assignments of an acid pH optimum for NAD(P)H oxidase, was prevented by including superoxide dismutase in fluorometric assays. NAD(P)H oxidase was more active towards NADPH (Km = 0.15 +/- 0.03 mM) than NADH (Km = 0.68 +/- 0.2 mM). No suggestion that oxidase activity was allosterically regulated by NAD(P)H was seen. Phorbol myristate acetate-induced O27 production was noted to be modulated by pH in intact neutrophils, suggesting that NAD(P)H oxidase is localized in the plasma membrane where its activity may be subject to (auto) regulation by local H+ concentrations.
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Oxidative stress has long been identified as one if the mechanism in glycerol-induced acute renal failure (ARF), a disease model of human rhabdomyolysis. Besides NAD(P)H Oxidase, cyclooxygenase (COX) and xanthine oxidase (XO) are important oxygenase system that contributes in free radical generation in the biological system. In this study we explore involvement of these oxygenase system as the source of free radical in ARF and investigate the relationship between NAD(P)H oxidase, COX and XO in this process. Renal failure was induced in male Sprague Dawley rats by injecting glycerol (8 ml / kg; 50% v/v; i.m) with or without pretreatment of apocynin (Apo: mg/kg in drinking water, 7 days) a NAD(P)H Oxidase inhibitor. Rats were sacrificed 24 hrs after inducing ARF and kidney tissues were analyzed for biochemical assays. Glycerol increased free radical production by 39% as evident by elevated 8-isoprostane. Inhibition of NAD(P)H oxidase by apocynin reduced free radicals by 46%. In ARF rats, NAD(P)H oxidase, COX and XO activities were elevated by 106%, 70%, and 208%, respectively. Apocynin prevented this attenuation of NAD(P)H oxidase (54%; p<0.05) and COX (62%; p<0.05) but did not alter XO activity. These data suggests that NAD(P)H oxidase, COX, and XO are involved in generating oxidative stress in ARF. We also propose that COX-mediated free radical generation requires a functional NAD(P)H oxidase.
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β-NAD+e (extracellular β-NAD+), present at nanomolar levels in human plasma, has been implicated in the regulation of [Ca2+]i (the intracellular calcium concentration) in various cell types, including blood cells, by means of different mechanisms. Here, we demonstrate that micromolar NAD+e (both the α and the β extracellular NAD+ forms) induces a sustained [Ca2+]i increase in human granulocytes by triggering the following cascade of causally related events: (i) activation of adenylate cyclase and overproduction of cAMP; (ii) activation of protein kinase A; (iii) stimulation of ADP-ribosyl cyclase activity and consequent overproduction of cADP-ribose, a universal Ca2+ mobilizer; and (iv) influx of extracellular Ca2+. The NAD+e-triggered [Ca2+]i elevation translates into granulocyte activation, i.e. superoxide and nitric oxide generation, and enhanced chemotaxis in response to 0.1–10 μM NAD+e. Thus extracellular β-NAD+e behaves as a novel pro-inflammatory cytokine, stimulating human granulocytes and potentially recruiting them at sites of inflammation.
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Accumulating evidence has suggested that NAD (including NAD+ and NADH) and NADP (including NADP+ and NADPH) could belong to the fundamental common mediators of various biological processes, including energy metabolism, mitochondrial functions, calcium homeostasis, antioxidation/generation of oxidative stress, gene expression, immunological functions, aging, and cell death: First, it is established that NAD mediates energy metabolism and mitochondrial functions; second, NADPH is a key component in cellular antioxidation systems; and NADH-dependent reactive oxygen species (ROS) generation from mitochondria and NADPH oxidase-dependent ROS generation are two critical mechanisms of ROS generation; third, cyclic ADP-ribose and several other molecules that are generated from NAD and NADP could mediate calcium homeostasis; fourth, NAD and NADP modulate multiple key factors in cell death, such as mitochondrial permeability transition, energy state, poly(ADP-ribose) polymerase-1, and apoptosis-inducing factor; and fifth, NAD and NADP profoundly affect aging-influencing factors such as oxidative stress and mitochondrial activities, and NAD-dependent sirtuins also mediate the aging process. Moreover, many recent studies have suggested novel paradigms of NAD and NADP metabolism. Future investigation into the metabolism and biological functions of NAD and NADP may expose fundamental properties of life, and suggest new strategies for treating diseases and slowing the aging process.
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Nicotinamide adenine dinucleotide (NAD) is a critical regulator of metabolic networks, and declining levels of its oxidized form, NAD+, are closely associated with numerous diseases. While supplementing cells with precursors needed for NAD+ synthesis has shown poor efficacy in combatting NAD+ decline, an alternative strategy is the development of synthetic materials that catalyze the oxidation of NADH into NAD+, thereby taking over the natural role of the NADH oxidase (NOX) present in bacteria. Herein, we discovered that metal-nitrogen-doped graphene (MNGR) materials can catalyze the oxidation of NADH into NAD+. Among MNGR materials with different transition metals, Fe-, Co-, and Cu-NGR displayed strong catalytic activity combined with >80% conversion of NADH into NAD+, similar specificity to NOX for abstracting hydrogen from the pyridine ring of nicotinamide, and higher selectivity than 51 other nanomaterials. The NOX-like activity of FeNGR functioned well in diverse cell lines. As a proof of concept of the in vivo application, we showed that FeNGR could specifically target the liver and remedy the metabolic flux anomaly in obesity mice with NAD+-deficient cells. Overall, our study provides a distinct insight for exploration of drug candidates by design of synthetic materials to mimic the functions of unique enzymes (e.g., NOX) in bacteria.
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