Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.99.6 (NADPH oxidase)
10,295 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Protoporphyrin IX inhibits citrulline formation by all three nitric oxide synthase isoforms in a manner reversible by dilution. Zinc protoporphyrin IX, by contrast, produces a time- and concentration-dependent inactivation of all three nitric oxide synthase isoforms, not reversible by dilution. The inhibition of citrulline formation by protoporphyrin IX occurs with IC50 values of 0.8, 4, and 5 microM for the nNOS, iNOS, and eNOS isoforms, respectively. Inhibition by N-methyl-protoporphyrin IX occurs at IC50 values of 6, 5, and 8 microM for the nNOS, iNOS, and eNOS isoforms, respectively. Inhibition of nitric oxide synthase by protoporphyrin IX is a multisite, positively cooperative inhibition that exhibits a Hill coefficient of 2.3 for the iNOS isoform. Protoporphyrin IX reduces the maximal velocity of citrulline formation for both the iNOS and nNOS isoforms without altering the K(m) for the arginine substrate or the EC50 value for the tetrahydrobiopterin cofactor. Protoporphyrin IX inhibits the arginine-independent NADPH oxidase activity of nNOS with an IC50 value of 1 microM but has no effect on cytochrome c reductase activity at concentrations as high as 30 microM. At concentrations of 10 and 20 microM, protoporphyrin IX inhibits NO formation by cytokine-induced murine RAW 264.7 cells; however, these inhibitions are accompanied by significant cellular cytotoxicity. Coproporphyrins I and III, uroporphyrins I and III, and porphobilinogen, intermediates in the biosynthesis of heme that accumulate in hepatic porphyrias, are ineffective as inhibitors of the nitric oxide synthase isoforms. Since protoporphyrin IX is the immediate biosynthetic precursor of heme that accumulates in hepatic protoporphyria, iron deficiency anemia, and lead poisoning, protoporphyrin IX inhibition of nitric oxide synthase may contribute to the pathophysiology of these conditions.
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PMID:Inhibition of nitric oxide synthase isoforms by porphyrins. 880 50

Nitric oxide synthase (NOS) catalyzes the NADPH- and O2-dependent conversion of L-arginine to nitric oxide (NO) and citrulline; three isoforms, the neuronal (nNOS), endothelial, and inducible, have been identified. Because overproduction of NO is known to contribute to several pathophysiological conditions, NOS inhibitors are of interest as potential therapeutic agents. Inhibitors that are potent, mechanism-based, and relatively selective for the NOS isoform causing pathology are of particular interest. In the present studies we report that vinyl-L-NIO (N5-(1-imino-3-butenyl)-L-ornithine; L-VNIO) binds to and inhibits nNOS in competition with L-arginine (Ki = 100 nM); binding is accompanied by a type I optical difference spectrum consistent with binding near the heme cofactor without interaction as a sixth axial heme ligand. Such binding is fully reversible. However, in the presence of NADPH and O2, L-VNIO irreversibly inactivates nNOS (kinact = 0.078 min-1; KI = 90 nM); inactivation is Ca2+/calmodulin-dependent. The cytochrome c reduction activity of the enzyme is not affected by such treatment, but the L-arginine-independent NADPH oxidase activity of nNOS is lost in parallel with the overall activity. Spectral analyses establish that the nNOS heme cofactor is lost or modified by L-VNIO-mediated mechanism-based inactivation of the enzyme. The inducible isoform of NOS is not inactivated by L-VNIO, and the endothelial isoform requires 20-fold higher concentrations to attain approximately 75% of the rate of inactivation seen with nNOS. Among the NOS inactivating L-arginine derivatives, L-VNIO is the most potent and nNOS-selective reported to date.
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PMID:N5-(1-Imino-3-butenyl)-L-ornithine. A neuronal isoform selective mechanism-based inactivator of nitric oxide synthase. 953 69

Hereditary argininemia manifests as neurological disturbance and mental retardation, features not observed in other amino acidemias. The cytotoxic effect of a high concentration of L-arginine (L-Arg) was investigated using NB9 human neuroblastoma cells (NB9), which express neuronal nitric oxide synthase (nNOS). When the concentration of L-Arg in the medium increased from 50 microM to 2 mM after incubation for 48 hr, the intracellular concentration of L-Arg increased from 68.0 +/- 1 pmol/10(6) cells to 1310.0 +/- 5 pmol/10(6) cells and that of L-citrulline (L-Cit) from undetectable levels to 47.1 +/- 0.2 pmol/10(6) cells (mean +/- SD of three independent analyses). This increase in intracellular L-Arg levels caused a decrease in NOS activity by approximately 71%. Flow cytometric analysis showed that reactive oxygen species (ROS) are produced in NB9 exposed to 2 mM L-Arg. The production of ROS was abolished by a NOS inhibitor, NG-nitro-L arginine-methylester. Production of ROS was also observed when NB9 were treated with L-Cit for 48 hr. To investigate the effect of L-Cit on the activity of NOS, a kinetic study on nNOS was conducted using cellular extracts from NB9. The apparent Km value of nNOS for L-Arg was 8.4 microM, with a Vmax value of 8.2 pmol/min/mg protein. L-Cit competitively inhibited NOS activity, as indicated by an apparent Ki value of 65 nM. These results suggest that L-Cit formed by nNOS in L-Arg-loaded neuronal cells inhibits NOS activity and nNOS in these L-Arg-loaded cells functions as a NADPH oxidase to produce ROS, which may cause neurotoxicity in argininemia.
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PMID:High concentration of L-arginine suppresses nitric oxide synthase activity and produces reactive oxygen species in NB9 human neuroblastoma cells. 974 7

We had previously shown NADPH diaphorase activity in fixed tissue slices of the insular cortex of the Syrian golden hamster (Mesocricetus auratus). The objective of this work was to determine the chemical identity of agents responsible for the observed NADPH diaphorase activities. Three different enzymatic NADPH diaphorase activities were distinguished in the insular cortex. (a) The activity seen in endothelial cells was not characterized histochemically, but it co-localized with eNOS-like immunoreactivity. (b) The neuronal Type I activity showed little sensitivity to 10(-5) M dicoumarol, could use either alpha- or beta-NADPH with almost equal facility, and co-localized with nNOS-like immunoreactivity. This activity was primarily attributable to nNOS. (c) The neuronal Type II activity was greatly attenuated by 10(-5) M dicoumarol, had a strong preference for beta-NADPH (rather than alpha-NADPH), and did not co-localize with any NOS-like immunoreactivity. These characteristics also apply to the NADPH diaphorase activity observed in the diffuse blue band in Layers II and III of agranular and dysgranular insular cortex and in the meshwork of cortical fibers. This staining was due primarily to a dicoumarol-sensitive dehydrogenase(s), either an isozyme of DT diaphorase (EC 1.6.99.2), or NADPH dehydrogenase (quinone) (EC 1.6. 99.6), or to a novel dicoumarol-sensitive NADPH dehydrogenase.
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PMID:NOS- and non-NOS NADPH diaphorases in the insular cortex of the Syrian golden hamster. 988 55

A series of compounds (7, 8, 10-17, 23) containing new functional groups derived by the combination of the substrate, intermediate, product, and known inhibitors of nitric oxide synthase (NOS) were prepared and evaluated against NOS. While none of the compounds assayed acted as a nitric oxide-producing substrate, the sulfur-containing arginine derivatives 10-12 were competitive inhibitors of iNOS with Ki's of 202, 7, and 58 microM, respectively. Compound 11 demonstrated the greatest potency against NOS-mediated citrulline formation for each of the three isoforms with IC50's of 6. 7, 19.7, and 13 microM for nNOS, eNOS, and iNOS, respectively. Compounds 10-12 each demonstrated a slight selectivity for inhibition of the neuronal isoform compared to the endothelial and inducible isoforms. These compounds also influenced the NADPH oxidase activity and heme iron spin state in a manner similar to structurally related compounds. Compound 10, a thiocarbonyl-containing compound, decreased the NADPH oxidase activity of the enzyme (EC50 = 190 microM) and shifted the heme iron spin state toward a low-spin configuration, similar to that of L-thiocitrulline. Compounds 11 and 12, S-alkylthiocitrulline derivatives, decreased the NADPH oxidase activity of the enzyme (EC50 = 6.6 and 180 microM, respectively) and shifted the heme iron spin state toward a high-spin configuration, similar to that of L-S-methylisothiocitrulline. Carbonyl-containing amino acid (7, 8, 23) and non-amino acid (13-17) analogues did not interact well with the enzyme.
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PMID:Synthesis and evaluation of new sulfur-containing L-arginine-derived inhibitors of nitric oxide synthase. 1034 37

Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310-329) we studied the interaction of the receptor with purified eNOS and neuronal nitric-oxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC(50) values of 10.6 +/- 0.4 microM and 7.1 +/- 0.6 microM, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC(50) of 3.7 +/- 0.6 microM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC(50) >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310-329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310-329 peptide blocks flavin to heme electron transfer. Co-immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway.
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PMID:Interaction of endothelial and neuronal nitric-oxide synthases with the bradykinin B2 receptor. Binding of an inhibitory peptide to the oxygenase domain blocks uncoupled NADPH oxidation. 1068 1

In the present study, we examined the role and the mechanism of poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) activation in zinc-induced cell death in cortical culture. After brief exposure to 400 microM zinc, cortical cells exhibited DNA fragmentation, increased poly(ADP-ribosyl)ation, and decreased levels of nicotinamide adenine dinucleotide (NAD) and ATP and subsequently underwent cell death. Inhibitors of PARP/PARG attenuated both zinc-induced NAD/ATP depletion and cell death, thereby implicating the PARP/PARG cascade in these processes. The zinc-inducible enzymes NADPH oxidase and neuronal nitric oxide synthase (nNOS) contributed to PARP activation as their inhibitors attenuated zinc-induced poly(ADP-ribosyl)ation. Levels of nitric oxide and nitrites increased following zinc exposure, consistent with NOS activation. In addition, Western blots and RT-PCR analysis revealed that protein and mRNA levels of nNOS specifically increased following zinc exposure in a manner similar to that of NADPH oxidase. The present study demonstrates that induction of NADPH oxidase and nNOS actively contributes to PARP/PARG-mediated NAD/ATP depletion and cell death induced by zinc in cortical culture.
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PMID:The role of NADPH oxidase and neuronal nitric oxide synthase in zinc-induced poly(ADP-ribose) polymerase activation and cell death in cortical culture. 1242 87

We have previously shown that prolonged exposure to neurotrophins induces oxidative neuronal death. In the present study, we further examined the cascades involved in neurotrophin-4/5 (NT-4/5)-induced neuronal death. Exposure of mature cortical cultures for 48 h to NT-4/5 induced neuronal death through TrkB activation. The NT-4/5-induced neuronal death was largely attenuated by addition of MK-801, indicating a critical role for NMDA receptors. Western blots revealed the induction of NR2A by NT-4/5. In addition, levels of phospho-NR2A and 2B increased, suggesting the upregulation of the NMDA receptor function. Whereas glutamate levels in the media changed little, levels of D-serine and L-glycine, co-agonists at NMDA receptors, increased significantly following NT-4/5 treatment. Exposure to NT-4/5 resulted in the activation of Src and extracellular signal-regulated kinase-1/2 (Erk-1/2). Their inhibitors blocked NR2A induction and phosphorylation as well as neuronal death induced by NT-4/5. In addition, Egr-1 was induced in an Src- and Erk-1/2-dependent manner. Anti-sense oligodeoxynucleotides to egr-1 attenuated NR2A induction as well as neuronal death. Although induction of NADPH oxidase and neuronal nitric oxide synthase (nNOS) contributes to NT-4/5-induced neuronal death, inhibition of their activity did not reduce NR2A induction. Conversely, blockade of NMDA receptors did not attenuate induction of NADPH oxidase or nNOS. These results indicate that two events are largely independent of each other. Our results demonstrate that the signaling cascade of TrkB leads to increase in NMDA receptor activity. Whereas this cascade may play an important role in the modulation of NMDA receptors in physiologic conditions, in the context of TrkB overactivation, it may contribute to neuronal death.
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PMID:NR2A induction and NMDA receptor-dependent neuronal death by neurotrophin-4/5 in cortical cell culture. 1472 Feb 20

Accumulating evidence indicates that vascular dysfunction in atherosclerosis, hypertension, and diabetes is either caused by or accompanied by oxidative stress in the vessel wall. In particular, the role of redox processes as mediators of vascular repair and contributors to post-angioplasty restenosis is increasingly evident. Yet the pathophysiology of such complex phenomena is still unclear. After vascular injury, activation of enzymes such as NADPH oxidase leads to a marked increase in superoxide generation, proportional to the degree of injury, which rapidly subsides. Such early superoxide production is significantly greater after stent deployment, as compared to balloon injury. Recent data suggest the persistence of low levels of oxidant stress during the vascular repair reaction in neointimal and medial layers. Despite the compensatory increase in expression of iNOS and nNOS, nitric oxide bioavailability is reduced because of increased reaction rates with superoxide, yielding as by-products reactive nitrogen/oxygen species that induce protein nitration. Concurrently, the activity of vascular superoxide dismutases exhibits a sustained decrease following injury. This decreased activity appears to be a key contributor to vasoconstrictive remodeling and a major determinant of the occurrence of nitrative/oxidative stress. Replenishment of superoxide dismutase (SOD), as well as treatment with vitamins C and E or the lipid-lowering drug probucol and its analogs, led to decrease in constrictive remodeling and improved vessel caliber. Better understanding of the redox pathophysiology of vascular repair should help clarify the pathogenesis of many other vascular conditions and may provide novel therapeutic strategies to prevent vascular lumen loss.
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PMID:Redox processes underlying the vascular repair reaction. 1496 Nov 89

AT(1) double receptor (AT(1A) and AT(1B)) knockout mice have lower blood pressure, impaired growth, and develop early renal microvascular disease and tubulointerstitial injury. We hypothesized that there would be an increased expression of vasoactive, profibrotic, and inflammatory mediators expressed in the kidneys of AT(1) double-knockout mice. We examined the renal expression of various mediator systems in control (n = 6) vs. double-knockout mice (n = 6) at 3-5 mo of age by real-time PCR, immunohistochemistry, and Western blot analysis. AT(1) double-knockout mice show activation of Th1-dependent pathways (with increased expression of IFN-alpha, IL-2 mRNA) with increased expression of both monocyte (MCP-1 mRNA) and T cell (RANTES mRNA) chemokines, infiltration of CD4(+) and CD11b(+) cells, increased fibrosis-associated mediators (CTGF, TGF-beta and TNF-alpha mRNA) and extracellular matrix (collagens I and III mRNA and protein) deposition compared with controls (P < 0.05 for all markers). These changes were associated with increased mRNA expression of endothelin (ET)-1 and ET-A receptor (P < 0.05), cyclooxygenase (COX)-2/TXA2 synthase (P < 0.05), NADPH oxidase (p40-phox, p67-phox, P < 0.05) and iNOS and nNOS (P < 0.05). COX-2 and nNOS protein were also increased in the kidneys of AT(1) double-knockout mice by Western blot analysis (P < 0.05). Although renin and angiotensinogen mRNA expression were increased in the knockout mice, AT(2) receptor mRNA expression was not significantly different from wild-type mice. In conclusion, the absence of the AT(1) receptor is associated with marked renal alterations in vasoactive, profibrotic, and immune mediators with an inflammatory pattern favoring a Th1 phenotype.
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PMID:Th1 inflammatory response with altered expression of profibrotic and vasoactive mediators in AT1A and AT1B double-knockout mice. 1592 10


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