Overstimation of verestimation of NADH-driven vascular oxidase activity due to lucigenin artifacts

Janiszewski, Mariano; Souza, Heraldo Possolo de; Li, Xiaoping; Pedro, Marcelo A.; Zweíer, Jay L.; Laurindo, Francisco Rafael Martins

doi:10.1016/s0891-5849(01)00828-0

Cited by 67 publications

(42 citation statements)

References 30 publications

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“…Furthermore, there are many enzymatic sources of ROS, in addition to NOXs, that could be concurrently altered (7,72), perhaps in directions opposite to NOXs. Moreover, detection of ROS is fraught with problems, and all methods have come under close scrutiny (15,29,68).…”

Section: Discussionmentioning

confidence: 99%

NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets

Dennis

Aschner

Milatović

et al. 2009

American Journal of Physiology-Lung Cellular and Molecular Phys

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Fike CD. NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets. Am J Physiol Lung Cell Mol Physiol 297: L596 -L607, 2009. First published July 10, 2009 doi:10.1152/ajplung.90568.2008.-Recently, we reported that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) contribute to aberrant responses in pulmonary resistance arteries (PRAs) of piglets exposed to 3 days of hypoxia (Am J Physiol Lung Cell Mol Physiol 295: L881-L888, 2008). An objective of the present study was to determine whether NOX-derived ROS also contribute to altered PRA responses at a more advanced stage of pulmonary hypertension, after 10 days of hypoxia. We further wished to advance knowledge about the specific NOX and antioxidant enzymes that are altered at early and later stages of pulmonary hypertension. Piglets were raised in room air (control) or hypoxia for 3 or 10 days. Using a cannulated artery technique, we found that treatments with agents that inhibit NOX (apocynin) or remove ROS [an SOD mimetic (M40403) ϩ polyethylene glycol-catalase] diminished responses to ACh in PRAs from piglets exposed to 10 days of hypoxia. Western blot analysis showed an increase in expression of NOX1 and the membrane fraction of p67phox. Expression of NOX4, SOD2, and catalase were unchanged, whereas expression of SOD1 was reduced, in arteries from piglets raised in hypoxia for 3 or 10 days. Markers of oxidant stress, F 2-isoprostanes, measured by gas chromatographymass spectrometry, were increased in PRAs from piglets raised in hypoxia for 3 days, but not 10 days. We conclude that ROS derived from some, but not all, NOX family members, as well as alterations in the antioxidant enzyme SOD1, contribute to aberrant PRA responses at an early and a more progressive stage of chronic hypoxiainduced pulmonary hypertension in newborn piglets. superoxide dismutase enzymes; SOD1; SOD2; NOX4; NOX1; p67phox; catalase; F 2-isoprostanes; M40403 SCIENTIFIC AND CLINICAL ADVANCES implicate disruption of enzymatic systems that produce and regulate reactive oxygen species (ROS) in the pathogenesis of a number of vascular diseases, including pulmonary hypertension (11,37,40,53,64,65). In particular, there is growing evidence that NADPH oxidase (NOX) family members are important sources for ROS generation in vascular diseases. Yet, compared with the systemic circulation, little is known about the role of the various NOX family members in the pulmonary circulation (10,22,28,39,45,62). Even less is known about the role of NOX family members in regulating vascular tone and reactivity in the neonatal compared with the adult pulmonary circulation. Fundamental differences in the regulation of pulmonary vascular tone in newborns and adults caution against extrapolation of findings regarding NOX signaling in adult lungs to the newborn (52, 74). Regulation of vascular tone is known to differ between larger (conduit-level) pulmonary arteries and smaller (resistance-level) pulmonary arteries (PRAs) (2...

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Section: Discussionmentioning

confidence: 99%

NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets

Dennis

Aschner

Milatović

et al. 2009

American Journal of Physiology-Lung Cellular and Molecular Phys

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“…Thus, our findings with APO may reflect contribution from ROS other than NADPH oxidase, such as uncoupled NOS. Likewise, findings with the lucigenin-enhanced chemiluminescence technique must be interpreted with caution (24,48). This is partly because regardless of the concentration used, lucigenin may undergo redox cycling (24).…”

Section: Discussionmentioning

confidence: 99%

Reactive oxygen species from NADPH oxidase contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension

Fike¹,

Slaughter²,

Kaplowitz³

et al. 2008

American Journal of Physiology-Lung Cellular and Molecular Phys

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Ϫ ) and hydrogen peroxide (H2O2), contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. The effect of the cell-permeable superoxide dismutase mimetic (SOD; M40403) and/or PEG-catalase (PEG-CAT) on responses to acetylcholine (ACh) was measured in endotheliumintact and denuded pulmonary resistance arteries (PRAs; 90-to-300-m diameter). To determine whether NADPH oxidase is an enzymatic source of ROS, PRA responses to ACh were measured in the presence and absence of a NADPH oxidase inhibitor, apocynin (APO). A Western blot technique was used to assess expression of the NADPH oxidase subunit, p67phox. A lucigenin-derived chemiluminescence technique was used to measure ROS production stimulated by the NADPH oxidase substrate, NADPH. ACh responses, which were dilation in intact control arteries but constriction in both intact and denuded hypoxic arteries, were diminished by M40403, PEG-CAT, the combination of M40403 plus PEG-CAT, as well as by APO. Although total amounts were not different, membrane-associated p67phox was greater in PRAs from hypoxic compared with control piglets. NADPH-stimulated lucigenin luminescence was nearly doubled in PRAs from hypoxic vs. control piglets. We conclude that ROS generated by NADPH oxidase contribute to the aberrant pulmonary arterial responses in piglets exposed to 3 days of hypoxia.superoxide; superoxide dismutase; p67phox; hydrogen peroxide; M40403; M40401 THERE IS INCREASING APPRECIATION that reactive oxygen species (ROS), such as O 2 Ϫ and H 2 O 2 , may act as essential participants in normal cell signaling and be involved in regulation of tone and reactivity in a number of vascular beds including the pulmonary circulation (8,29,33,42,49,50). NADPH oxidases are enzymes that have been shown to be prominent sources of ROS in many vascular beds (5,21,29). Moreover, evidence is accumulating that ROS, derived at least in part from NADPH oxidases, play a role in abnormal cell signaling and reactivity and thereby contribute to a number of vascular diseases, including pulmonary hypertension (5,7,21,29,37,43,47). For example, there is evidence that ROS produced by NADPH oxidase are involved in the development of chronic hypoxiainduced pulmonary hypertension in adult animals (20,31,37,38,53). However, the potential contribution of ROS derived from NADPH oxidase to the altered regulation of pulmonary vascular tone in newborns with chronic hypoxia-induced pulmonary hypertension is not yet known. Awareness of the fundamental differences in the regulation of pulmonary vascular tone in newborns and adults limits the ability to extrapolate to the newborn findings on the role of ROS in altered cell signaling in adult lungs (41,55).Regulation of pulmonary vascular tone is also known to differ between large, conduit level arteries and smaller, resistance level arteries (1, 2, 45). The vast majority of studies to date have evaluated the potential contribution from ...

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“…Because lucigeninenhanced chemiluminescence favors redox cycling of lucigenin at high concentrations (250 mol/L) and when NADH is used as a substrate, 21 in the present study, we used low lucigenin concentration (5 mol/L) and NADPH as a substrate. 22 …”

Section: Determination Of Vascular Superoxide Productionmentioning

confidence: 99%

“…21 In addition, lucigenin redox recycling is detectable even at lucigenin concentrations as low as 5 mol/L when NADH is used as a substrate, a phenomenon that is further reduced when NADPH is used as a substrate. 22 Therefore, by using low lucigenin concentrations and NADPH as a substrate in our studies, we reduce these artifacts to the minimum.…”

Section: Antoniades Et Al 5-mthf Versus Hcy and Vascular Redox In Humansmentioning

confidence: 99%

MTHFR 677 C>T Polymorphism Reveals Functional Importance for 5-Methyltetrahydrofolate, Not Homocysteine, in Regulation of Vascular Redox State and Endothelial Function in Human Atherosclerosis

et al. 2009

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Background-The role of circulating homocysteine as an atherosclerosis risk factor has recently been questioned.However, 5-methyl-tetrahydrofolate (5-MTHF), the circulating metabolite of folic acid participating in homocysteine metabolism, has direct effects on vascular function. We sought to distinguish the effects of plasma versus vascular tissue 5-MTHF and homocysteine on vascular redox and endothelial nitric oxide bioavailability in human vessels. Methods and Results-We used the methyl tetrahydrofolate reductase (MTHFR) gene polymorphism 677CϾT as a model of chronic exposure of the vascular wall to varying 5-MTHF levels in 218 patients undergoing coronary artery bypass graft surgery. Vascular superoxide, vascular 5-MTHF, and total homocysteine were determined in saphenous veins and internal mammary arteries obtained during surgery. Nitric oxide bioavailability was evaluated by organ bath studies on saphenous vein rings. MTHFR genotype was a determinant of vascular 5-MTHF (not vascular homocysteine). Both MTHFR genotype and vascular 5-MTHF were associated with vascular nitric oxide bioavailability and superoxide generated by uncoupled endothelial nitric oxide synthase. In contrast, vascular homocysteine was associated only with NADPH-stimulated superoxide. Conclusions-Genetic polymorphism 677 CϾT on MTHFR affects vascular 5-MTHF (but not homocysteine) and can be used as a model to distinguish the chronic effects of vascular 5-MTHF from homocysteine on vascular wall. Vascular 5-MTHF, rather than plasma or vascular homocysteine, is a key regulator of endothelial nitric oxide synthase coupling and nitric oxide bioavailability in human vessels, suggesting that plasma homocysteine is an indirect marker of 5-MTHF rather than a primary regulator of endothelial function. Key Words: antioxidants Ⅲ atherosclerosis Ⅲ homocysteine Ⅲ nitric oxide synthase Ⅲ nitric oxide Ⅲ superoxide P lasma homocysteine is associated with cardiovascular risk 1,2 and surrogate markers such as oxidative stress 3 and endothelial dysfunction. 4 However, the potential causative role of homocysteine in vascular disease pathogenesis remains unclear. 5,6 Although homocysteine may have direct effects on important biological processes such as thrombosis and production of reactive oxygen species, 1,2,7 interpretation of these effects is confounded by corresponding changes in folate status. Low plasma folate levels are strongly associated with elevated plasma homocysteine, 8 and intervention studies to reduce homocysteine typically use folate supplementation. 9,10 Recent evidence suggests that plasma folate may play important direct roles in the pathogenesis of atherosclerosis that are independent of homocysteine. 11 Indeed, 5-methyltetrahydrofolate (5-MTHF), the circulating form of folate, has a number of direct antiatherogenic effects by scavenging peroxynitrite radicals, 12 augmenting nitric oxide (NO) bioavailability, [13][14][15] and decreasing vascular superoxide generation in the vascular wall. 12 The potential beneficial effects of homocystein...

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Overstimation of verestimation of NADH-driven vascular oxidase activity due to lucigenin artifacts

Cited by 67 publications

References 30 publications

NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets

NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets

Reactive oxygen species from NADPH oxidase contribute to altered pulmonary vascular responses in piglets with chronic hypoxia-induced pulmonary hypertension

MTHFR 677 C>T Polymorphism Reveals Functional Importance for 5-Methyltetrahydrofolate, Not Homocysteine, in Regulation of Vascular Redox State and Endothelial Function in Human Atherosclerosis

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