Expression Of NADPH Oxidase Subunits Research Articles

Increased NADPH-oxidase activity and Nox4 expression during chronic hypertension is associated with enhanced cerebral vasodilatation to NADPH in vivo.

We examined the importance of NADPH-oxidase in reactive oxygen species production in cerebral arteries and its effect on vascular tone in vivo. Furthermore, we investigated whether chronic hypertension affects function or expression of this enzyme in cerebral vessels. Superoxide generation was detected in isolated rat basilar arteries with the use of lucigenin-enhanced chemiluminescence. mRNA expression of NADPH-oxidase subunits was assessed by real-time polymerase chain reaction. Basilar artery diameter was measured with the use of a cranial window preparation in anesthetized rats. NADPH-stimulated superoxide production was 2.3-fold higher in arteries from spontaneously hypertensive rats (SHR) versus normotensive Wistar-Kyoto rats (WKY) and could be blocked by the NADPH-oxidase inhibitor diphenyleneiodonium. Higher NADPH-oxidase activity was also reflected at the molecular level as mRNA expression of the NADPH-oxidase subunit Nox4 was 4.1-fold higher in basilar arteries from SHR versus WKY. In contrast, expression of Nox1, gp91phox, p22phox, and p47phox did not differ between strains. Application of NADPH to basilar arteries caused larger vasodilatation in SHR than WKY. Vasodilatation to NADPH could be attenuated by diphenyleneiodonium, as well as diethyldithiocarbamate (Cu(2+)/Zn(2+)-superoxide dismutase inhibitor), catalase (H(2)O(2) scavenger), or tetraethylammonium (BK(Ca) channel inhibitor). Activation of NADPH-oxidase in cerebral arteries generates superoxide, which is dismutated by Cu(2+)/Zn(2+)-superoxide dismutase to H(2)O(2). H(2)O(2) then elicits vasodilatation via activation of BK(Ca) channels. Upregulation of Nox4 during chronic hypertension is associated with elevated cerebral artery NADPH-oxidase activity.

Increased myocardial NADPH oxidase activity in human heart failure

ObjectivesThis study was designed to investigate whether nicotinamide adenine dinucleotide 3-phosphate (reduced form) (NADPH) oxidase is expressed in the human heart and whether it contributes to reactive oxygen species (ROS) production in heart failure. BackgroundA phagocyte-type NADPH oxidase complex is a major source of ROS in the vasculature and is implicated in the pathophysiology of hypertension and atherosclerosis. An increase in myocardial oxidative stress due to excessive production of ROS may be involved in the pathophysiology of congestive heart failure. Recent studies have suggested an important role for myocardial NADPH oxidase in experimental models of cardiac disease. However, it is unknown whether NADPH oxidase is expressed in the human myocardium or if it has any role in human heart failure. MethodsMyocardium of explanted nonfailing (n = 9) and end-stage failing (n = 13) hearts was studied for the expression of NADPH oxidase subunits and oxidase activity. ResultsThe NADPH oxidase subunits p22phox, gp91phox, p67phox, and p47phoxwere all expressed at messenger ribonucleic acid and protein level in cardiomyocytes of both nonfailing and failing hearts. NADPH oxidase activity was significantly increased in end-stage failing versus nonfailing myocardium (5.86 ± 0.41 vs. 3.72 ± 0.39 arbitrary units; p < 0.01). The overall level of oxidase subunit expression was unaltered in failing compared with nonfailing hearts. However, there was increased translocation of the regulatory subunit, p47phox, to myocyte membranes in failing myocardium. ConclusionsThis is the first report of the presence of NADPH oxidase in human myocardium. The increase in NADPH oxidase activity in the failing heart may be important in the pathophysiology of cardiac dysfunction by contributing to increased oxidative stress.

Platelet-derived exosomes: a new vascular redox signaling pathway

Cells release microparticles following apoptosis (apoptotic bodies) or for signaling purposes (exosomes). In contrast to the bigger apoptotic bodies (> 400 nm), exosomes (diameter ~100 nm) do not present phosphatidylserine on their surface and expose major histocompatibility complex components, CD9 and CD63. Prognosis of some thrombotic-inflammatory diseases has been related to microparticle release. Sepsis is an abnormal immuno-inflammatory response to an infection, including dysregulation of apoptotic mechanisms in vascular cells. The major redox signaling pathway in vessels involves the enzymatic complex superoxide generating NADPH oxidase. In previous work we showed that, in sepsis, there is augmented platelet release of microparticles when compared with healthy controls. Those septic microparticles also possess greater NADPH oxidase activity, which can be responsible for vascular cell apoptosis. Our objective was to better characterize those microparticles obtained from septic patients and determine possible pathways related to their release. Through sequential filtration and centrifugation we separated microparticles from septic plasma (n = 16, 24 hours of diagnosis accordingly to ACCP/SCCM 1992 criteria) or from healthy controls (n = 6). Apoptotic bodies were obtained from the medium of cultured endothelial cells exposed to ultraviolet light for 30 min. Laser light scattering revealed particles with diameter between 82 and 112 nm. In contrast to the microparticles, apoptotic bodies do not have NADPH oxidase activity as assayed by lucigenin 5 μM luminescence. Western blot analysis revealed greater NADPH oxidase subunit expression in septic particles when compared with healthy controls, and none on apoptotic bodies. Flow cytometry disclosed positive phosphatidylserine exposure on apoptotic bodies, while microparticles were positive to CD9 and CD63. Washed, fresh platelets from single donors were stimulated with thrombin (0.1 U/ml), tumor necrosis factor alpha (10 μg/ml), lipopolysaccharide (LPS) (0.1 μg/ml) and with the nitric oxide (NO) donor sodium nitroprusside (2 mM) for 30 min. Particles obtained from the tumor necrosis factor-stimulated and thrombin-stimulated platelets were similar to the apoptotic bodies, while those obtained from NO-stimulated or LPS-stimulated platelets shared characteristics with the septic microparticles, with low phosphatidylserine exposure, high CD9 and CD63 expression, and oxidase activity. In conclusion, in sepsis there is a platelet release of exosomes, which possess a vascular pro-apoptotic NADPH oxidase activity. Furthermore, we showed that LPS and NO, agents related to the pathophysiology of severe sepsis, induce similar superoxide producing exosome release from healthy platelets, suggesting the existence of a new redox signaling pathway.

Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart

<h2>Abstract</h2> J. K. Bendall, C. Heymes, T. J. F. Wright, S. Wheatcroft, D. J. Grieve, A. M. Shah and A. C. Cave. Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart. <i>Journal of Molecular and Cellular Cardiology</i> (2002)<b>34</b> , 1325–1333. Numerous studies in the literature have employed gene-modified mice to investigate vascular function. However, only very limited information exists on baseline murine vascular physiology or on potential variations between different strains. We therefore compared coronary and aortic vascular responses to endothelium-derived vasodilators and exogenous nitric oxide (NO) in three commonly used mouse strains and correlated these data with expression of eNOS, NADPH oxidase subunits, gp91^phox and p67^phox, and superoxide production. Isolated perfused hearts from MF1, 129sv and C57BL/6J mice were subjected to: (a) increasing doses of bradykinin, acetylcholine and sodium nitroprusside, and (b) bolus doses of adenosine and the NO synthase inhibitor, N^G-monomethyl-L -arginine. Vascular responses of thoracic aortic rings were assessed for comparison. Expression of eNOS and NADPH oxidase subunits was assessed by immunoblotting, and superoxide production by lucigenin-enhanced chemiluminescence. Coronary vasodilator responses to bradykinin, acetylcholine and sodium nitroprusside were significantly attenuated in MF1 compared with C57BL/6J and 129sv hearts. Similarly, aortic relaxation to acetylcholine was significantly impaired in MF1 aortic rings compared with in C57BL/6J aortae; these differences were reversed by Tiron. N^G-monomethyl-L -arginine induced significantly less vasoconstriction in MF1 and 129sv hearts compared with C57BL/6J. No differences in aortic relaxation to A23187 or sodium nitroprusside were observed. Cardiac and aortic superoxide production and cardiac expression of p67^phox and gp91^phox were significantly greater in MF1 mice compared with the other strains. There is significant strain-dependent variation in coronary and aortic vascular responsiveness in mice, which may reflect differences in the balance between NO and superoxide generation.

Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart

J. K. Bendall, C. Heymes, T. J. F. Wright, S. Wheatcroft, D. J. Grieve, A. M. Shah and A. C. Cave. Strain-Dependent Variation in Vascular Responses to Nitric Oxide in the Isolated Murine Heart. Journal of Molecular and Cellular Cardiology (2002)34 , 1325–1333. Numerous studies in the literature have employed gene-modified mice to investigate vascular function. However, only very limited information exists on baseline murine vascular physiology or on potential variations between different strains. We therefore compared coronary and aortic vascular responses to endothelium-derived vasodilators and exogenous nitric oxide (NO) in three commonly used mouse strains and correlated these data with expression of eNOS, NADPH oxidase subunits, gp91phox and p67phox, and superoxide production. Isolated perfused hearts from MF1, 129sv and C57BL/6J mice were subjected to: (a) increasing doses of bradykinin, acetylcholine and sodium nitroprusside, and (b) bolus doses of adenosine and the NO synthase inhibitor, NG-monomethyl-L -arginine. Vascular responses of thoracic aortic rings were assessed for comparison. Expression of eNOS and NADPH oxidase subunits was assessed by immunoblotting, and superoxide production by lucigenin-enhanced chemiluminescence. Coronary vasodilator responses to bradykinin, acetylcholine and sodium nitroprusside were significantly attenuated in MF1 compared with C57BL/6J and 129sv hearts. Similarly, aortic relaxation to acetylcholine was significantly impaired in MF1 aortic rings compared with in C57BL/6J aortae; these differences were reversed by Tiron. NG-monomethyl-L -arginine induced significantly less vasoconstriction in MF1 and 129sv hearts compared with C57BL/6J. No differences in aortic relaxation to A23187 or sodium nitroprusside were observed. Cardiac and aortic superoxide production and cardiac expression of p67phox and gp91phox were significantly greater in MF1 mice compared with the other strains. There is significant strain-dependent variation in coronary and aortic vascular responsiveness in mice, which may reflect differences in the balance between NO and superoxide generation.

Expression and Cellular Localization of Classic NADPH Oxidase Subunits in the Spontaneously Hypertensive Rat Kidney

Phagocytes generate superoxide anion (O(2)(-)) by a classic, 5-component NADPH oxidase. O(2)(-) contributes to hypertension in spontaneously hypertensive rats (SHR). Therefore, we tested the hypothesis that NADPH oxidase expression is enhanced in the SHR kidney. We also analyzed the localization of NADPH oxidase components in SHR kidney. Renal NADPH oxidase was quantified by reverse transcription-polymerase chain reaction and Western blotting and was localized in SHR and Wistar Kyoto rat (WKY) kidney by immunohistochemistry. The mRNA for 5 subunits of phagocyte NADPH oxidase, and also for MOX1 and RENOX (NOX4), was detected in adult rat kidney. Kidneys of adult (10 weeks old) SHR had a significantly (P<0.01) greater mRNA for p47phox (SHR 0.81 +/- 0.05 versus WKY 0.37 +/- 0.01, arbitrary unit), which was confirmed by Western blotting (SHR 0.58 +/- 0.04 versus WKY 0.42 +/- 0.04, arbitrary unit; P<0.05) and by immunohistochemistry. This higher p47phox protein expression was also detected in young prehypertensive SHR (SHR 0.61 +/- 0.05 versus WKY 0.39 +/- 0.04, arbitrary unit; P<0.01). The 10-week-old SHR contained more modest but significantly (P<0.05) greater protein for p67phox (SHR 0.54 +/- 0.02 versus WKY 0.46 +/- 0.02). Immunostaining localized p47phox, p67phox, and p22phox in vasculature, macula densa, distal convoluted tubule, cortical collecting duct, and outer and inner medullary collecting ducts. The kidney of SHR expresses genes for all the main components of phagocyte NADPH oxidase, RENOX, and MOX1. There is a prominent increase in the SHR kidney of the mRNA, and protein expression of p47phox in the vasculature, macula densa, and distal nephron, which precedes development of hypertension.

Identification of a functional leukocyte-type NADPH oxidase in human endothelial cells :a potential atherogenic source of reactive oxygen species.

Cultured human endothelial cells (EC) exposed to atherogenic low-density lipoprotein levels have increased reactive oxygen species (ROS) generation. The enzyme responsible for this ROS production elevation is unknown. We have examined for the presence of a functional leukocyte-type NADPH oxidase in EC to elucidate whether this enzyme could be the ROS source. The plasma membrane fraction of disrupted EC showed a reduced-minus-oxidized difference spectra with absorption peaks identical to those observed in the spectra of the leukocyte NADPH oxidase component, cytochrome b558. Western-blot analysis, using anti-gp91 -phox. anti -p22-phox. anti -p47-phox. and anti -p67-phox antibodies, demonstrated the protein expression of NADPH oxidase subunits in EC. Reverse transcriptase-polymerase chain reaction (RT-PCR) showed the mRNA expression of gp91-phox, p22-phox, p47-phox, and p67-phox in EC. Sonicates from unstimulated EC produced no measurable superoxide; whereas, exogenously applied arachidonic acid activated superoxide generation in a manner that was dependent upon the presence of NADPH and both membrane and cytosolic fractions combined. Apocynin, a specific leukocyte NADPH oxidase inhibitor, was shown by Western-blot analysis of membrane and cytoplasmic fractions to inhibit the translocation of p47-phox to the membrane of stimulated EC. These findings support the presence of a functionally active leukocyte-type NADPH oxidase in EC. NADPH oxidase could be the major cellular ROS source in EC perturbation, which has been hypothesized to be a major contributing factor in the pathogenesis of atherosclerosis.