Co-regulation of constitutive nitric oxide synthases and NADPH oxidase by the small GTPase Rac

Nitric oxide (NO), generated by NO synthases (NOSs), has multifarious roles in signal transduction. Reactive oxygen species (ROS), generated by ubiquitous NADPH oxidases (NOXs), also participate in cellular signaling. However, the coordination of signals conveyed by NO and ROS is poorly understood. We show that the small GTPase Rac, a component of some NOXs, also interacts with and regulates the constitutively-expressed NOSs. Cellular NO and O(2)(-) production increase or decrease together following activation or inhibition of Rac, and Rac inhibition reveals transduction mechanisms that depend upon NO (vasodilation), ROS (actin polymerization) or both (cytoskeletal organization). Thus, signaling by NO and ROS may be coordinated through a common control element.     

Production of nitric oxide under Ultraviolet-B irradiation is mediated by hydrogen peroxide through activation of nitric oxide synthase

Excised leaves of kidney bean (Phaseolus vulgaris) were used to investigate the mechanism of NO generation under UV-B stress. We showed that two signaling molecules, NO and H₂O₂, were produced in the irradiated leaves. NO release was blocked by LNNA, an inhibitor of NOS. Application of CAT (EC 1.11.1.6) not only effectively eliminated H₂O₂ in the leaves, but also inhibited the activity of NOS and the emission of NO. In contrast, treatment with exogenous H₂O₂ increased both of those events. Therefore, we suggest that, under UV-B stress, NO production is mediated by H₂O₂ through greater NOS activity.     

Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation

When activated by proinflammatory stimuli, microglia release substantial levels of glutamate, and mounting evidence suggests this contributes to neuronal damage during neuroinflammation. Prior studies indicated a role for the Xc exchange system, an amino acid transporter that antiports glutamate for cystine. Because cystine is used for synthesis of glutathione (GSH) synthesis, we hypothesized that glutamate release is an indirect consequence of GSH depletion by the respiratory burst, which produces superoxide from NADPH oxidase. Microglial glutamate release triggered by lipopolysaccharide was blocked by diphenylene iodonium chloride and apocynin, inhibitors of NADPH oxidase. This glutamate release was also blocked by vitamin E and elicited by lipid peroxidation products 4-hydroxynonenal and acrolein, suggesting that lipid peroxidation makes crucial demands on GSH. Although NADPH oxidase inhibitors also suppressed nitrite accumulation, vitamin E did not; moreover, glutamate release was largely unaffected by nitric oxide donors, inhibitors of nitric oxide synthase, or changes in gene expression. These findings indicate that a considerable degree of the neurodegenerative consequences of neuroinflammation may result from conversion of oxidative stress to excitotoxic stress. This phenomenon entails a biochemical chain of events initiated by a programmed oxidative stress and resultant mass-action amino acid transport. Indeed, some of the neuroprotective effects of antioxidants may be due to interference with these events rather than direct protection against neuronal oxidation.     

The role of nitric oxide and reactive oxygen species in the positive inotropic response to mechanical stretch in the mammalian myocardium

The endothelial nitric oxide synthase (eNOS) has been implicated in the rapid (Frank-Starling) and slow (Anrep) cardiac response to stretch. Our work and that of others have demonstrated that a neuronal nitric oxide synthase (nNOS) localized to the myocardium plays an important role in the regulation of cardiac function and calcium handling. However, the effect of nNOS on the myocardial response to stretch has yet to be investigated. Recent evidence suggests that the stretch-induced release of angiotensin II (Ang II) and endothelin 1 (ET-1) stimulates myocardial superoxide production from NADPH oxidases which, in turn, contributes to the Anrep effect. nNOS has also been shown to regulate the production of myocardial superoxide, suggesting that this isoform may influence the cardiac response to stretch or ET-1 by altering the NO-redox balance in the myocardium. Here we show that the increase in left ventricular (LV) myocyte shortening in response to the application of ET-1 (10 nM, 5 min) did not differ between nNOS^−/− mice and their wild type littermates (nNOS^+/+). Pre-incubating LV myocytes with the NADPH oxidase inhibitor, apocynin (100 μM, 30 min), reduced cell shortening in nNOS^−/− myocytes only but prevented the positive inotropic effects of ET-1 in both groups. Superoxide production (O₂⁻) was enhanced in nNOS^−/− myocytes compared to nNOS^+/+; however, this difference was abolished by pre-incubation with apocynin. There was no detectable increase in O₂⁻ production in ET-1 pre-treated LV myocytes. Inhibition of protein kinase C (chelerythrine, 1 μM) did not affect cell shortening in either group, however, protein kinase A inhibitor, PKI (2 μM), significantly reduced the positive inotropic effects of ET-1 in both nNOS^+/+ and nNOS^−/− myocytes. Taken together, our findings show that the positive inotropic effect of ET-1 in murine LV myocytes is independent of nNOS but requires NADPH oxidases and protein kinase A (PKA)-dependent signaling. These results may further our understanding of the signaling pathways involved in the myocardial inotropic response to stretch.     

NADPH氧化酶抑制剂apocynin对力竭运动大鼠运动性蛋白尿的影响

目的：研究NADPH氧化酶抑制剂apocynin对力竭运动大鼠运动性蛋白尿产生的影响及其机制。方法：32只SD雄性大鼠随机分为安静对照组（C组）、对照+药物组（CA组）、力竭运动组（E组）、力竭运动+药物组（EA组）。药物注射按10 mg/kg体重,每天一次,连续3 d,并在末次药物注射1 h后进行一次性跑台力竭运动。测定运动后尿UP、血液BUN水平、肾脏ROS浓度、NOS活性、NOS与3-NT蛋白含量。结果：结果显示,E组UP、肾脏ROS、iNOS含量及活性、3-NT明显升高,而EA组的这些指标与C组相比无显著性差异。结论：力竭运动可明显增加肾组织NADPH氧化酶活性,从而产生大量的ROS,后者可迅速地与由肾脏iNOS催化生成的NO反应,产生过量的ONOO^-,诱发运动性蛋白尿的生成。     

Catalase activity prevents exercise-induced up-regulation of vasoprotective proteins in venous tissue

Physical activity induces favourable changes of arterial gene expression and protein activity, although little is known about its effect in venous tissue. Although our understanding of the initiating molecular signals is still incomplete, increased expression of endothelial nitric oxide synthase (eNOS) is considered a key event. This study sought to investigate the effects of two different training protocols on the expression of eNOS and extracellular superoxide dismutase (ecSOD) in venous and lung tissue and to evaluate the underlying molecular mechanisms. C57Bl/6 mice underwent voluntary exercise or forced physical activity. Changes of vascular mRNA and protein levels and activity of eNOS, ecSOD and catalase were determined in aorta, heart, lung and vena cava. Both training protocols similarly increased relative heart weight and resulted in up-regulation of aortic and myocardial eNOS. In striking contrast, eNOS expression in vena cava and lung remained unchanged. Likewise, exercise up-regulated ecSOD in the aorta and in left ventricular tissue but remained unchanged in lung tissue. Catalase expression in lung tissue and vena cava of exercised mice exceeded that in aorta by 6.9- and 10-fold, respectively, suggesting a lack of stimulatory effects of hydrogen peroxide. In accordance, treatment of mice with the catalase inhibitor aminotriazole for 6 weeks resulted in significant up-regulation of eNOS and ecSOD in vena cava. These data suggest that physiological venous catalase activity prevents exercise-induced up-regulation of eNOS and ecSOD. Furthermore, therapeutic inhibition of vascular catalase might improve pulmonary rehabilitation.     

Glycolate oxidase is an alternative source for H2O2 production during plant defense responses and functions independently from NADPH oxidase

The photorespiratory enzyme glycolate oxidase (GOX) was found to be involved in nonhost resistance by regulating plant defense responses through the production of H₂O₂. Silencing of a gene encoding NADPH oxidase (AtRBOHD) in the gox mutants did not further increase susceptibility to a nonhost pathogen, P. syringae pv tabaci, although it caused an increase in bacterial growth in the Atgox1 and Atgox3 mutant backgrounds. In order to confirm this finding, we created double homozygous knockouts AtrbohD x Atgox1 and AtrbohD x Atgox3 to evaluate symptom development and bacterial growth. Here we show that there is no additive effect of disease symptoms or bacterial growth in the AtrbohD x Atgox1 and AtrbohD x Atgox3 double mutants when compared with individual mutants. Slight additive effect observed previously upon silencing of AtRBOHD in Atgox1 and Atgox3 mutants was most likely due to cross-silencing of AtRBOHF. These results further prove that GOX plays a role in nonhost resistance independent of NADPH oxidase.     

Partial Purification and Some Properties of ADP-glucose Phosphorylase from Potato Tubers

ADP-glucose phosphorylase [adenosine diphosphate glucose: orthophosphate adenyl- yltransferase; Dankert et ah, Biochim. Biophys. Acta, 81, 78 (1964)] was found to be widely distributed in plant tissues. The enzyme was purified 570-fold in a 24% yield from cell- free extract of growing tubers of potato (Solanum tuberosum L.). The following reaction catalyzed by the purified enzyme was found to proceed stoichiometrically. ADP-glucose +P₁→ADP+glucose-1-P

Maximal activity was observed at pH 8. The enzyme was the most stable at pH 7, showing 50% loss of its original activity after 50 min heating at 57°C. The following kinetic parameters were obtained: activation energy, 11.1 kcal/mole; Km (P₁), 2.5 mm; Km (ADP-glucose), 0.05 mm. The enzyme did not act on GDP-mannose, GDP-glucose and UDP-glucose. Neither activator nor inhibitor was found among various phosphorylated metabolites tested. The enzyme was inhibited by metal-binding reagents, EDTA and o-phenanthroline. None of the metal ions tested was found to recover the activity of chelator-treated enzyme.     

NADPH oxidase- and mitochondrion-derived superoxide at rostral ventrolateral medulla in endotoxin-induced cardiovascular depression

We evaluated the contribution of superoxide anion (O2*-) generated by NADPH oxidase or mitochondria in the rostral ventrolateral medulla (RVLM), where sympathetic premotor neurons for arterial pressure maintenance are located, on cardiovascular depression induced by inducible nitric oxide synthase-derived NO after Escherichia coli lipopolysaccharide (LPS) treatment. In Sprague-Dawley rats maintained under propofol anesthesia, microinjection of LPS bilaterally into the RVLM induced progressive hypotension, bradycardia, and reduction in sympathetic vasomotor outflow over our 240-min observation period. This was accompanied by an increase in O2*- production (60-240 min) in the RVLM, alongside phosphorylation of p47(phox) or p67(phox), upregulation of gp91(phox) or p47(phox) protein, and increase in Rac-1 or NADPH oxidase activity (60-120 min), and a depression of mitochondrial respiratory enzyme activity (120-240 min). Whereas inhibition of NADPH oxidase or knockdown of the gp91(phox) or p47(phox) gene blunted the early phase (60-150 min), coenzyme Q10 or mitochondrial K(ATP) channel inhibitor antagonized the delayed phase (120-240 min) of LPS-induced increase in O2*- production in RVLM and cardiovascular depression. We conclude that, whereas NADPH oxidase-derived O2*- in RVLM participates predominantly in the early phase, O2*- generated by depression in mitochondrial respiratory enzyme activity or opening of mitoK(ATP) channels mediates the delayed phase of LPS-induced cardiovascular depression.     

Iron, Nitric Oxide, and Myeloperoxidase in Asthmatic Patients

Plasma nitric oxide (NO), myeloperoxidase (MPO), and iron (Fe) levels were determined in bronchial asthma. The relations among these parameters in different stages of asthma were interpreted. Their association with airway inflammation observed in patients with bronchial asthma as well as the roles and the contributions to the pathological processes were evaluated. A total of 62 individuals, 32 asthmatics and 30 controls, were included into the scope of this study. Plasma nitric oxide metabolites (NOx) and MPO and Fe levels were determined by the Griess reaction, ELISA, and the automated TPTZ (2,4,6-tri[2-pyridyl]-5-triazine) method, respectively. In the asthmatic individuals, plasma NOx, MPO, and Fe concentrations were 133 +/- 13 microM, 95 +/- 20 ng/ml, and 159 +/- 20 microg/dl, respectively; in the control group these values were 82 +/- 11 microM, 62 +/- 11 ng/ml, and 96 +/- 9 microg/dl. Increased values were detected for plasma MPO (p > 0.05), NOx (p < 0.01), and Fe (p < 0.01) concentrations in asthmatic individuals. Considering the facts that NO modulates the catalytic activity of MPO and induces the expression of heme oxygenase as important contributors to the mechanisms causing free Fe release, it is concluded that elevated NOx, MPO, and Fe levels observed in the asthmatic group act in a concerted manner and appear to be involved in the pathogenesis of asthma.