Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.6.99.6 (NADPH oxidase)
 10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To understand the role of redox-sensitive mechanisms in vascular smooth muscle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase activation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-derived growth factor BB-induced ERK2 activation. In contrast, DPI only inhibited agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family along with the AP-1-dependent reporter gene revealed that AP-1 with c-Fos and JunB composition exhibited a higher transactivating activity than AP-1 with other compositions tested. All three agonists significantly stimulated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesis in VSMC. These results also suggest a role for NADH/NADPH oxidase activity in some subset of early signaling events such as p38 MAP kinase activation and c-Fos and JunB induction, which appear to be important in agonist-induced AP-1 activity and DNA synthesis in VSMC.
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PMID:JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells. 1002 27

The leukocyte NADPH oxidase catalyzes the one-electron reduction of oxygen to O2- at the expense of NADPH. It is a multicomponent enzyme comprising a membrane-bound flavocytochrome (cytochrome b558) and at least four cytosolic components: p47PHOX, p67PHOX, p40PHOX, and Rac, a small GTPase. All the oxidase components except p40PHOX are required for enzyme activity. Many aspects of their function, however, are unclear. Using the electron acceptor ferricyanide, we found that recombinant p67PHOX from baculovirus-infected Sf9 cells could mediate the dehydrogenation of NADPH. NADPH dehydrogenation was not dependent on FAD and was insensitive to superoxide dismutase. Several control experiments showed that NADPH dehydrogenation was accomplished by p67PHOX, not by a trace contaminant in the p67PHOX preparation. The NADPH dehydrogenase activity of p67PHOX was proportional to enzyme concentration, and showed saturation kinetics with NADPH (Km 92 +/- 5 microM), but was inhibited at high concentrations of ferricyanide. NADH was also used as a substrate by p67PHOX (Km 123 +/- 38 microM). Taken together, these results show that p67PHOX is able to mediate pyridine nucleotide dehydrogenation. These findings raise the possibility that p67PHOX might participate directly in electron transfer between NADPH and the oxidase flavin.
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PMID:NADPH dehydrogenase activity of p67PHOX, a cytosolic subunit of the leukocyte NADPH oxidase. 1023 25

Angiotensin (A) II is a potent constrictor as well as growth stimulant of vascular smooth muscle cell caused by activation of AT1 receptor signal transduction systems. There are two major signal systems of AT1 receptor: one leads to an increase in cytosolic free calcium levels causing smooth muscle contraction which may result in high blood pressure, and the other leads to smooth muscle proliferation and inflammation which may result in atherosclerosis. AT1 receptor activation induces phosphinositide hydrolysis by phospholipase C and creates an inositol phosphate, which release calcium from cytosolic calcium pools. Cytosolic calcium can also be elevated by activation of calcium channel via a link between AT1 receptor and a G protein. Protein phosphorylation triggered by AT1 receptor is important for cell growth, in which tyrosine kinase, serine/threonine kinase and protein kinase C are involved. Free radicals are generated by NADH/NADPH oxidase in response to AT1 receptor activation, causing expression of genes leading to atherosclerosis. On the other hand, activation of AT2 receptor is shown to play a role of lowering blood pressure. Some phosphatases and NO/cyclic GMP would be involved in the mechanism. In renal vasculature, endothelium dependent epoxygenase products are synthesized by AT2 receptor stimulation causing vasorelaxation. In summary, AT1 receptor signals are vasopressive and evoke atherosclerosis, whereas AT2 receptor signals may possibly be vasodilatory.
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PMID:[Signal transduction systems of angiotensin II receptors]. 1036 37

Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851, slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement an Escherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, and sll1484 have been designated ndbA, ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion of ndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.
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PMID:Type 2 NADH dehydrogenases in the cyanobacterium Synechocystis sp. strain PCC 6803 are involved in regulation rather than respiration. 1038 67

Angiotensin II and hypertension increase vascular oxidant stress. We examined how these might affect expression of the extracellular superoxide dismutase (ecSOD), a major form of vascular SOD. In mice, angiotensin II infusion (1.1 mg/kg for 7 days) increased systolic blood pressure from 107+/-3 to 152+/-9 mm Hg and caused a 3-fold increase in ecSOD, but there was no change in the cytosolic Cu/Zn SOD protein, as determined by Western blot analysis. This was associated with a similar increase in ecSOD mRNA as assessed by RNase protection assay and was prevented by losartan. Induction of ecSOD by angiotensin II was not due to hypertension alone, because hypertension caused by norepinephrine (5.6 mg. kg-1. d-1) had no effect on ecSOD. Similarly, exposure of mouse aortas to angiotensin II (100 nmol/L) in organoid culture increased ecSOD by approximately 2-fold. In the organoid culture, angiotensin II-induced upregulation of ecSOD was prevented by losartan (10 micromol/L) and PD985059 (30 micromol/L), a specific inhibitor of p42/44 MAP kinase kinase. Angiotensin II activates the NADH/NADPH oxidase; however, diphenyleneiodonium chloride (10 micromol/L), an inhibitor of this oxidase, did not prevent p42/44 MAP kinase phosphorylation or ecSOD induction by angiotensin II. Finally, in human aortic smooth muscle cells, angiotensin II moderately increased transcriptional rate (as assessed by nuclear run-on analysis) but markedly increased ecSOD mRNA stability. Thus, angiotensin II increases ecSOD expression independent of hypertension, and this increase involves both an increase in ecSOD transcription and stabilization of ecSOD mRNA. This effect of angiotensin II on ecSOD expression may modulate the oxidative state of the vessel wall in pathological processes in which the renin-angiotensin system is activated.
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PMID:Modulation of extracellular superoxide dismutase expression by angiotensin II and hypertension. 1040 Sep 7

An NAD(P)H-dependent H2O2 forming activity has been evidenced in thyroid tissue from patients with Grave's disease. Its biochemical properties were compared to those of the NADPH oxidase previously described in pig thyroid gland. Both were Ca2+-dependent and activated by inorganic phosphate anions in the same range of concentrations. Both are flavoproteins using FAD as cofactor, but the human enzyme was also able to utilize FMN. The apparent Km for NADPH of the human enzyme (100 microM) was 5-10 times higher than that of porcine enzyme. Vm was 3 to 10 times higher in pig (150 nmol x h(-1) x mg(-1)) than in man (14 to 45). Total content in human tissue was 7 to 9% of that in porcine tissue. An unidentified inhibitor has been detected in the 3000 g particulate fraction from most patients, which could account for this apparently low enzyme content. An NADH-dependent H2O2 production has also been observed in porcine and human thyroid tissues. This activity was only partly Ca2+-dependent (man, 50-70%; pig, 80-90%) and presented similar apparent Km values for NADH (man, 100 microM; pig, 200 microM). In pig thyrocytes, the expression of the Ca2+-dependent part of the NADH-oxidase activity was induced by TSH and down-regulated by TGFbeta, as was the NADPH oxidase activity. Furthermore, NADPH and NADH-dependent activities were not additive. We conclude that a single, inducible, NAD(P)H-oxidase can use NADPH or NADH as substrate to catalyse H2O2 formation, and that human and porcine NAD(P)H-oxidases are highly similar. Differences observed could be attributed to minor differences in enzyme structure and/or in membrane microenvironment. The NADH-dependent Ca2+-independent activity observed in human and porcine thyroid fractions could be attributed to a distinct and constitutive enzyme.
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PMID:Biochemical characterization of a Ca2+/NAD(P)H-dependent H2O2 generator in human thyroid tissue. 1040 72

Intracellular signaling events that mediate the long-term effects of Ang II in vascular smooth muscle cells are unclear, but oxidative stress may play an important role. This study examined the ability of Ang II to generate reactive oxygen species and investigated the putative role of phospholipase D (PLD)-dependent signaling pathways for its production in human vascular smooth muscle cells. In addition, we assessed whether redox-sensitive pathways influence Ang II-stimulated cell growth. Primary and low-passage cells (passages 1 to 4) derived from resistance arteries of subcutaneous gluteal biopsies from healthy subjects were studied. Oxidative stress was measured with the fluorescent probe 5-(and 6)-chloromethyl-2', 7'-dichlorodihydrofluorescein diacetate (CM-H(2)DCFDA) (8 micromol/L), and the role of PLD was assessed with the PLD inhibitor D-erythro-sphingosine, dihydro (sphinganine) (10 micromol/L). To determine whether NADH/NADPH oxidase contributes to production of reactive oxygen species, Ang II-stimulated cells were pretreated with the specific flavoprotein inhibitor diphenylene iodinium (DPI) (10 micromol/L). DNA and protein synthesis were determined by [(3)H]thymidine and [(3)H]leucine incorporation, respectively. Ang II increased CM-H(2)DCFDA fluorescence, and this was inhibited by catalase (350 U/mL), indicating that the fluorescence signal was derived predominantly from H(2)O(2). Ang II dose-dependently increased H(2)O(2) production (E(max)=57.6+/-1.7 nmol/L, pD(2)=7.7+/-0.06) and PLD activation (E(max)=207+/-3.3% of control, pD(2)=7.7+/-0.5). H(2)O(2) effects were evident within 1 hour, and maximal PLD activation occurred within 40 minutes after stimulation. DPI inhibited (P<0.01) Ang II-stimulated responses. PLD inhibition significantly attenuated (P<0.05) Ang II-elicited H(2)O(2) production (E(max)=29+/-5 nmol/L). DPI and sphinganine inhibited Ang II-induced DNA and protein synthesis. These data indicate that in vascular smooth muscle cells from human peripheral resistance arteries, Ang II increases H(2)O(2) generation via PLD-dependent, NADH/NADPH oxidase-sensitive pathways. These cascades may function as second messengers in long-term Ang II-mediated growth-signaling events.
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PMID:Ang II-stimulated superoxide production is mediated via phospholipase D in human vascular smooth muscle cells. 1052 94

Impairment of nitric oxide-dependent vascular relaxation is a characteristic feature of the insulin-resistant state. To understand those mechanisms, we examined imbalance of O2-/NO production in aortic endothelial cells obtained from high fructose-fed, exogenous hyperinsulinemic, and control rats. Aortic segments from both high fructose-fed and insulin-treated rats produced a 4-fold more O2- than control rats evaluated by a chemiluminescence method. The O2- production in the aortas of both high fructose-fed and insulin-treated rats was mediated through activation of NADH/NADPH oxidase. In isometric tension studies, high fructose vessels with endothelium elicited impaired relaxation in response to acetylcholine or a calcium ionophore A23187 when compared with control rats, whereas these impaired vascular responses were not found in insulin-treated rats. Furthermore, endothelial constitutive NO synthase activity was increased in vessels from insulin-treated rats, but decreased in vessels from high fructose-fed rats. These results indicate that relative excess of O2- production through activation of NADH/NADPH oxidase over NO generation in endothelial cells may contribute to impaired endothelial-dependent relaxation in insulin-resistant state.
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PMID:Free radical production in endothelial cells as a pathogenetic factor for vascular dysfunction in the insulin resistance state. 1058 73

Multiple enzymes may stimulate ROS production in VSMC and endothelial cells. These include NADH/NADPH oxidase, xanthine oxidase, lipoxygenases, cyclooxygenase, P-450 monooxygenases, and the enzymes of mitochondrial oxidative phosphorylation. In addition to generation of intracellular O2- by these enzymes, extracellular stimuli including lipophilic substrates, membrane permeant oxidants (e.g., H2O2), cytokines, and growth factors may modulate cellular redox state. Both intracellular and extracellular ROS act as second-messengers to activate tyrosine and serine-threonine kinases, such as the MAP kinase family. As discussed in the previous sections, regulation of the MAP kinases is one example of the complexity of ROS-dependent signal transduction. Although the complexity of ROS-mediated signal transduction is daunting, the diversity offers multiple therapeutic targets for pharmacologic intervention.
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PMID:Redox signals that regulate the vascular response to injury. 1060 87

Modular kinetic analysis was used to determine the sites in plant mitochondria where charge-screening stimulates the rate of electron transfer from external NAD(P)H to oxygen. In mitochondria isolated from potato (Solanum tuberosum L.) tuber callus, stimulation of the rate of oxygen uptake was accompanied by a decrease in the steady-state reduction level of coenzyme Q, and by a small decrease in the steady-state reduction level of cytochrome c. Modular kinetic analysis around coenzyme Q revealed that stimulation of the rate was due to stimulation of quinol oxidation via the cytochrome pathway (cytochrome bc1, cytochrome c and cytochrome c oxidase). It was not a consequence of any effect on quinone reduction (by external NADH or NADPH dehydrogenase). This explains the salt-induced decrease in the steady-state reduction level of coenzyme Q. Analysis around cytochrome c revealed that stimulation by salts was due to a dual effect on the respiratory chain. The kinetic curves for the oxidation and reduction pathways of cytochrome c revealed that they were both activated by salt, the simultaneity explaining the small variation observed in the steady-state reduction level of cytochrome c. A simple kinetic core model is used to show that changes in the rate of dissociation of cytochrome c from the membrane can explain the observed kinetic changes in both cytochrome c reduction and cytochrome c oxidation. The stimulation is proposed to be the result of an increase in the rate constant of cytochrome c dissociation from the membrane induced by cation screening. We conclude that this type of modular kinetic analysis is a powerful tool to identify and quantitatively characterize multiple-site effects on the mitochondrial respiratory chain.
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PMID:Identification of the site where the electron transfer chain of plant mitochondria is stimulated by electrostatic charge screening. 1065 25


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