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:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We tested the hypothesis that angiotensin II-induced hypertension is associated with an increase in vascular .O2- production, and characterized the oxidase involved in this process. Infusion of angiotensin II (0.7 mg/kg per d) increased systolic blood pressure and doubled vascular .O2- production (assessed by lucigenin chemiluminescence), predominantly from the vascular media. NE infusion (2.75 mg/kg per d) produced a similar degree of hypertension, but did not increase vascular .O2- production. Studies using various enzyme inhibitors and vascular homogenates suggested that the predominant source of .O2- activated by angiotensin II infusion is an NADH/NADPH-dependent, membrane-bound oxidase. Angiotensin II-, but not NE-, induced hypertension was associated with impaired relaxations to acetylcholine, the calcium ionophore A23187, and nitroglycerin. These relaxations were variably corrected by treatment of vessels with liposome-encapsulated superoxide dismutase. When Losartan was administered concomitantly with angiotensin II, vascular .O2- production and relaxations were normalized, demonstrating a role for the angiotensin type-1 receptor in these processes. We conclude that forms of hypertension associated with elevated circulating levels of angiotensin II may have unique vascular effects not shared by other forms of hypertension because they increase vascular smooth muscle .O2- production via NADH/NADPH oxidase activation.
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PMID:Angiotensin II-mediated hypertension in the rat increases vascular superoxide production via membrane NADH/NADPH oxidase activation. Contribution to alterations of vasomotor tone. 862 76

The human neutrophil NADPH oxidase is a multi-component complex composed of membrane-bound and cytosolic proteins. During activation, cytosolic proteins p47(phox), p67(phox), Rac2, and possibly p40(phox) translocate to the plasma membrane and associate with flavocytochrome b to form the active superoxide-generating system. To further investigate the role of p67(phox) in this complex assembly process, experiments were performed to identify possible regions of interaction between p67(phox) and other NADPH oxidase proteins. Using random sequence peptide phage-display library analysis of p67(phox), we identified a novel region in p47(phox) encompassing residues 323-332 and a previously identified SH3 binding domain encompassing p47(phox) residues 361-370 as p67(phox) binding sites. Synthetic peptides mimicking p47(phox) residues 323-332 inhibited the p47(phox)-p67(phox) binding interaction in an affinity binding assay; however, peptides mimicking flanking regions were inactive. Surprisingly, this same region of p47(phox) was found previously to represent a site of binding interaction for flavocytochrome b (DeLeo, F. R., Nauseef, W. M., Jesaitis, A. J., Burritt, J. B., Clark, R. A., and Quinn, M. T.(1995) J. Biol. Chem. 270, 26246-26251), and this observation was confirmed in the present report using two different in vitro assays that were not evaluated previously. Using affinity binding assays, we also found that p67(phox) and flavocytochrome b competed for binding to p47(phox)after activation, suggesting that prior to full NADPH oxidase assembly the 323-332 region of p47(phox) is associated with p67(phox) and at some point in the activation process is transferred to flavocytochrome b. Thus, taken together our data demonstrate that both p67(phox) and flavocytochrome b utilize a common binding site in p47(phox), presumably at distinct stages during the activation process, and this p47(phox) region plays a key role in regulating NADPH oxidase assembly.
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PMID:Assembly of the human neutrophil NADPH oxidase involves binding of p67phox and flavocytochrome b to a common functional domain in p47phox. 866 33

The possible mechanism of activation of the NADPH oxidase by fluoride was investigated in the cell-free system. It is shown that the stimulatory effect of fluoride is inhibited by guanosine 5'-O-(2-thiodiphosphate) (GDP[S]) and potentiated by GTP. The effect of fluoride is not additive with GTP[S]. Fluoride activation requires the presence of Mg2+ in millimolar concentration but is independent of Al3+. The activating effect of fluoride is preserved in solubilized membrane extract after removal of the majority of heterotrimeric GTP-binding proteins by immunoadsorption. Fluoride has no direct action either on the nucleotide exchange of GTP hydrolysis of the isolated Rac protein. In contrast, fluoride effectively inhibits Rac-GTPase activity enhanced by a membrane component. In this way, fluoride could prolong the prevalence of Rac in the GTP-bound state and, as a consequence, activate NADPH oxidase. The possibility of the involvement of a membrane-bound Rac GTPase-activating protein activity in the physiological regulation of the enzyme is raised.
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PMID:In vitro activation of the NADPH oxidase by fluoride. Possible involvement of a factor activating GTP hydrolysis on Rac (Rac-GAP). 870 42

Superoxide anion formation is vital to the microbicidal activity of phagocytes. Recently, however, there is accumulating evidence that it is also involved in cell growth in vascular smooth muscle cells (VSMCs). We have shown that the hypertrophic agent angiotensin II stimulates superoxide production by activating the membrane-bound NADH/NADPH oxidase and that inhibition of this oxidase attenuates vascular hypertrophy. However, the molecular identity of this oxidase in VSMCs is unknown. We have recently cloned the cytochrome b558 alpha-subunit, p22(phox) (one of the key electron transfer elements of the NADPH oxidase in phagocytes), from a rat VSMC cDNA library, but its role in VSMC oxidase activity remains unclarified. Here we report that the complete inhibition of p22(phox) mRNA expression by stable transfection of antisense p22(phox) cDNA into VSMCs results in a decrease in cytochrome b content, which is accompanied by a significant inhibition of angiotensin II-stimulated NADH/NADPH-dependent superoxide production, subsequent hydrogen peroxide production, and [3H]leucine incorporation. We provide the first evidence that p22(phox) is a critical component of superoxide-generating vascular NADH/NADPH oxidase and suggest a central role for this oxidase system in vascular hypertrophy.
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PMID:p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. 879 32

The effect of high temperatures (39, 41, and 43 degrees C) on acetaminophen (AM-) induced inhibition of the oxidative respiratory burst of polymorphonuclear leukocytes (PMNs) in vitro has been examined. Whole blood or isolated human PMNs were exposed to various temperatures in vitro in the presence or absence of AM for 0-90 min. Phagocyte membrane-bound NADPH oxidase was studied using the luminol chemiluminescence (CL) response and the superoxide dismutase inhibitable reduction of ferricytochrome C. The NADPH oxidase was stimulated by phorbol myristate acetate (PMA). The results showed that high temperatures (39-43 degrees C) potentiate the AM inhibitory effect on CL peak response of phagocytes in a temperature-dependent manner. Furthermore, the inhibition of superoxide (O2-) production induced by AM was potentiated by incubating the cells at 39 or 43 degrees C at different time intervals. These studies suggest that high temperatures significantly potentiate the AM inhibitory effect on oxidative metabolism of PMNs in vitro. These actions of AM may influence the outcome in patients with infectious febrile conditions.
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PMID:The thermal potentiation of acetaminophen-inhibited PMN oxidative metabolism in vitro. 886 41

The NADPH oxidase complex of activated neutrophils consists of a membrane-bound flavocytochrome b and cytosolic activation factors. Despite its ability to react with O2, the heme b component of the flavocytochrome is insensitive to cyanide and CO2, and slowly reactive to butyl isocyanide. We report here that arachidonic acid, an anionic amphophil which elicits oxidase activation in a cell-free system induces a transition of the heme iron of the neutrophil flavocytochrome b from a low-spin hexacoordinated state to a high-spin pentacoordinated state and promotes the binding of butyl isocyanide to the heme b. Low-temperature EPR spectra of air-oxidized flavocytochrome b either purified or in its membrane-bound form showed a low-spin signal at g = 3.26 and a high-spin signal at g = 6.0. Upon addition of arachidonic acid, the g = 3.26 signal vanished; a low-spin signal at g = 2.23 appeared, and the signal at g = 6.0 progressively increased. The subsequent addition of butyl isocyanide resulted in the decrease of the g = 6.0 and g = 2.23 signals and in the appearance of a new low-spin signal at g = 2.33. Consistent with the EPR results, upon addition of arachidonic acid to oxidized flavocytochrome b, a 2.5 nm blue shift of the Soret peak was detected in low-temperature optical spectra. The subsequent addition of butyl isocyanide resulted in the emergence of a peak at 432 nm reflecting the formation of a butyl isocyanide-oxidized heme b complex. In the case of sodium dithionite-reduced flavocytochrome b, arachidonic acid promoted the binding of butyl isocyanide to the reduced heme b, as shown by the emergence of a peak at 434 nm and the decrease of the alpha band at 558 nm. The same promoting effect was encountered with sodium dodecyl sulfate, an anionic amphophil capable of eliciting oxidase activation like arachidonic acid. In contrast to arachidonic acid, arachidonic acid methyl ester was ineffective and counteracted the effect of arachidonic acid. Butyl isocyanide added to intact neutrophils was found to bind to heme b, only after the cells have been activated. These data demonstrate the transient accumulation of a pentacoordinated form of the heme iron of flavocytochrome b under in vitro and in vivo conditions; the pentacoordinated form of the reduced heme b is postulated to react with O2 to generate the superoxide anion.
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PMID:Electron transfer across the O2- generating flavocytochrome b of neutrophils. Evidence for a transition from a low-spin state to a high-spin state of the heme iron component. 887 8

TH1-type proinflammatory cytokines induce the expression of phagocytic nitric oxide synthase (NOS) and prime the membrane-bound NADPH oxidase of neutrophils and monocytes of mice so as to attain an activated state, which upon a second stimulus releases up to 6-fold increased levels of reactive oxygen species (ROS) than do unprimed phagocytes. Enhanced levels of ROS and NO deregulate inflammatory signal transduction pathways, which play a crucial role in the pathogenesis of arthritis. The antiarthritic reactivity of diphenylene iodoniumchloride (DPI), an irreversible inhibitor of NADPH oxidase and NOS, was tested in male DBA/1xB10A(4R) hybrid mice suffering from potassium peroxochromate-induced arthritis. Daily doses of 2.8 mu mol/kg of DPI sufficed to inhibit the arthritis by 50%. A complete inhibition was obtained with 10 mu mol/kg of DPI. The reduction of overt arthritic symptoms correlated well with both the reduced levels of ROS and NO in plasma of DPI-treated mice. Our data support the hypothesis that oxidative stress and nitric oxides play a pivotal role in the pathology of arthritis, which can be therapeutically targetted by NADPH oxidase- and NO synthase-inhibitors.
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PMID:Suppression of inflammatory arthritis by simultaneous inhibition of nitric oxide synthase and NADPH oxidase. 890 81

Thymocyte apoptosis is one of the best characterized experimental models of apoptosis that can be induced by a variety of stimuli such as glucocorticoids, ionizing radiation, antibodies, and toxins. Recently, it has been suggested that oxidative stress is a common mediator of apoptosis. However, little is known about the production and possible function of reactive oxygen intermediates (ROI) in thymocytes. We used a highly sensitive flow cytometric assay with the hydrogen peroxide-sensitive dye, 2',7'-dichlorofluorescin diacetate (DCFH-DA), to measure intracellular ROI production in rat thymocytes, to study its primary sources, and to compare ROI levels in normal and apoptotic thymocytes. Apoptosis was induced by incubating the cells in the presence or absence of dexamethasone (Dex) at 37 degrees C in vitro. Normal thymocytes spontaneously produced significant amounts of ROI. Catalase or superoxide dismutase did not affect this intracellular fluorescence, presumably due to their failure to penetrate into the cells. However, N-acetyl-L-cysteine significantly attenuated the fluorescence in a dose-dependent manner. Significant inhibition of the intracellular fluorescence was also observed by addition of N-nitro-L-arginine methyl ester (L-NAME), that could not be reversed by L-arginine. The addition of N-nitro-D-arginine methyl ester (D-NAME) also caused considerable inhibition. This indicates that the inhibition by L-NAME or D-NAME is due to a direct scavenging effect, and nitric oxide production is not likely to be involved. In contrast to neutrophils and macrophages whose superoxide anions are released from membrane-bound NADPH oxidase, the production of ROI in thymocytes is likely to originate mainly from mitochondria, as indicated by the inhibitory effect of the addition of rotenone or antimycin A. The addition of lymphocyte simulators phytohemagglutinin (PHA), concanavalin A (Con A), or phorbol 12-myristate 13-acetate (PMA) enhanced intracellular fluorescence of thymocytes. This increase was abrogated by addition of rotenone or antimycin A. The ROI production was decreased with time after incubation of the thymocytes for 1, 3, and 6 h in vitro. The appearance of apoptosis of thymocytes in vitro, as indicated by DNA content of cells by flow cytometry and DNA ladder formation in agarose gel electrophoresis, was delayed, as compared to the time course of the decreased ROI production. The addition of Dex to the culture medium accelerated both of these processes. The results suggest that a decreased spontaneous production of ROI in thymocytes precedes the spontaneous in vitro apoptosis and Dex exaggerates these changes.
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PMID:Decreased production of reactive oxygen intermediates is an early event during in vitro apoptosis of rat thymocytes. 890 94

The superoxide (O2-)-generating NADPH oxidase of human neutrophils consists of membrane-bound and cytosolic proteins that assemble in the plasma membrane of activated cells. To date, most of our understanding of the assembly of the NADPH oxidase has been obtained through the use of a cell-free assay, and a number of peptides that mimic regions of NADPH oxidase proteins have been shown to block oxidase assembly using this assay. However, the cell-free assay provides an incomplete representation of the assembly and regulation of the NADPH oxidase in vivo, and it has become necessary to develop methods for introducing biomolecules, such as peptides, into intact neutrophils where their effects can be investigated. One such method is electropermeabilization. Although this method has been used previously with human neutrophils, it has not been well characterized. We report here a detailed characterization of the electropermeabilized neutrophil assay system, including optimal conditions for membrane electropermeabilization with maximal retention of functional capacity, optimal conditions for analyzing the effects of experimental peptides, quantification of internalized peptide concentration, and molecular size limits for diffusion of molecules into these cells. Our results demonstrate that optimal neutrophil permeabilization (98-100%) can be achieved using significantly lower electrical fields than previously reported, resulting in the retention of higher levels of O2(-)-generating activity. We also found that biomolecules as large as 2.3 kDa readily diffuse into permeabilized cells. Analysis of flavocytochrome b peptides that were shown previously to inhibit NADPH oxidase activity in a cell-free assay demonstrated that these peptides also blocked O2- production in electropermeabilized human neutrophils; although at higher effective concentrations than in the cell-free system. Thus, electropermeabilized neutrophils provide a model system for evaluating the effects of peptides and other pharmacological agents in intact cells which closely mimic neutrophils in vivo.
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PMID:Characterization of peptide diffusion into electropermeabilized neutrophils. 891 95

Phagocytes such as neutrophils play a key role in the body's innate immune response to infection. These cells travel throughout the body in search of pathogens and are rapidly mobilized to sites of inflammation where they phagocytose these pathogens and subsequently release a variety of toxic oxygen radical species and proteolytic enzymes to directly destroy the engulfed particle. The generation of microbicidal oxidants by neutrophils results from the action of a multi-protein enzymatic complex known as the NADPH oxidase. Altogether, there are currently seven proteins reported to be associated with the NADPH oxidase assembly. In resting neutrophils, these NADPH oxidase protein components are segregated into cytoplasmic and plasma membrane compartments. However, during assembly and activation of the NADPH oxidase, the cytosolic protein components translocate to the plasma membrane or phagosomal membrane where they assemble around a central membrane-bound protein known as flavocytochrome b. This assembly process is highly regulated and involves multiple binding interactions between the individual NADPH oxidase proteins, resulting in an active oxidase complex. Over the past few years, a number of these sites of binding interaction between the oxidase proteins have been identified, leading to a clearer understanding of the intermolecular interactions occurring among protein components during the assembly process. In addition, this information has contributed to our understanding of the roles played by each protein during the activation and assembly process. In this review, we describe the key features of each NADPH oxidase protein and then summarize our current understanding of the specific molecular interactions occurring between these proteins, focusing on the role these protein:protein binding interactions play in the NADPH oxidase assembly process.
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PMID:Assembly of the phagocyte NADPH oxidase: molecular interaction of oxidase proteins. 897 69


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