Cytosolic NADPH Oxidation Research Articles

Dehydroepiandrosterone promotes pulmonary artery relaxation by NADPH oxidation‐elicited subunit dimerization of protein kinase G1α (1175.9)

The activity of glucose‐6‐phosphate dehydrogenase (G6PD) controls a vascular smooth muscle relaxing mechanism promoted by the oxidation of cytosolic NADPH, which has been associated with activation of the 1α form of protein kinase G (PKG1α) by a thiol oxidation‐elicited subunit dimerization. This PKG1α‐activation mechanism appears to contribute to responses of isolated endothelium‐removed bovine pulmonary arteries (BPA) elicited by peroxide, cytosolic NADPH oxidation resulting from G6PD inhibition and hypoxia. Dehydroepiandrosterone (DHEA) is a steroid hormone with pulmonary vasodilator activity, which has beneficial effects in treating pulmonary hypertension. Since one of the known actions of DHEA is inhibiting G6PD, we investigated if it promoted relaxation associated with NADPH oxidation, PKG1α dimerization and PKG activation detected by increased vasodilator‐stimulated phosphoprotein (VASP) phosphorylation. Relaxation of BPA to DHEA under aerobic or hypoxic conditions was associated with NADPH oxidation, PKG1α dimerization and increased VASP phosphorylation. The vasodilator activity of DHEA was markedly attenuated in pulmonary arteries and aorta from a PKG knockin mouse containing a serine in place of a cysteine involved in PKG dimerization. DHEA promoted increased PKG dimerization in lungs from wild type mice, which was not detected in the PKG knockin mouse model. Thus, PKG1α dimerization is a major contributing factor to the vasodilator actions of DHEA, and perhaps its beneficial effects in treating pulmonary hypertension.Grant Funding Source: Supported by NIH Grant R01HL115124

Suppression of the External Mitochondrial NADPH Dehydrogenase, NDB1, in Arabidopsis thaliana Affects Central Metabolism and Vegetative Growth

Ca(2+)-dependent oxidation of cytosolic NADPH is mediated by NDB1, which is an external type II NADPH dehydrogenase in the plant mitochondrial electron transport chain. Using RNA interference, the NDB1 transcript was suppressed by 80% in Arabidopsis thaliana plants, and external Ca(2+)-dependent NADPH dehydrogenase activity became undetectable in isolated mitochondria. This was linked to a decreased level of NADP(+) in rosettes of the transgenic lines. Sterile-grown transgenic seedlings displayed decreased growth specifically on glucose, and respiratory metabolism of (14)C-glucose was increased. On soil, NDB1-suppressing plants had a decreased vegetative biomass, but leaf maximum quantum efficiency of photosystem II and CO2 assimilation rates, as well as total respiration, were similar to the wild-type. The in vivo alternative oxidase activity and capacity were also similar in all genotypes. Metabolic profiling revealed decreased levels of sugars, citric acid cycle intermediates, and amino acids in the transgenic lines. The NDB1-suppression induced transcriptomic changes associated with protein synthesis and glucosinolate and jasmonate metabolism. The transcriptomic changes also overlapped with changes observed in a mutant lacking ABAINSENSITIVE4 and in A. thaliana overexpressing stress tolerance genes from rice. The results thus indicate that A. thaliana NDB1 modulates NADP(H) reduction levels, which in turn affect central metabolism and growth, and interact with defense signaling.

Dehydroepiandrosterone promotes pulmonary artery relaxation by NADPH oxidation-elicited subunit dimerization of protein kinase G 1α

The activity of glucose-6-phosphate dehydrogenase (G6PD) controls a vascular smooth muscle relaxing mechanism promoted by the oxidation of cytosolic NADPH, which has been associated with activation of the 1α form of protein kinase G (PKG-1α) by a thiol oxidation-elicited subunit dimerization. This PKG-1α-activation mechanism appears to contribute to responses of isolated endothelium-removed bovine pulmonary arteries (BPA) elicited by peroxide, cytosolic NADPH oxidation resulting from G6PD inhibition, and hypoxia. Dehydroepiandrosterone (DHEA) is a steroid hormone with pulmonary vasodilator activity, which has beneficial effects in treating pulmonary hypertension. Because multiple mechanisms have been suggested for the vascular effects of DHEA and one of the known actions of DHEA is inhibiting G6PD, we investigated whether it promoted relaxation associated with NADPH oxidation, PKG-1α dimerization, and PKG activation detected by increased vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Relaxation of BPA to DHEA under aerobic or hypoxic conditions was associated with NADPH oxidation, PKG-1α dimerization, and increased VASP phosphorylation. The vasodilator activity of DHEA was markedly attenuated in pulmonary arteries and aorta from a PKG knockin mouse containing a serine in place of a cysteine involved in PKG dimerization. DHEA promoted increased PKG dimerization in lungs from wild-type mice, which was not detected in the PKG knockin mouse model. Thus PKG-1α dimerization is a major contributing factor to the vasodilator actions of DHEA and perhaps its beneficial effects in treating pulmonary hypertension.

Roles for cytosolic NADPH redox in regulating pulmonary artery relaxation by thiol oxidation-elicited subunit dimerization of protein kinase G1α

The activity of glucose-6-phosphate dehydrogenase (G6PD) appears to control a vascular smooth muscle relaxing mechanism regulated through cytosolic NADPH oxidation. Since our recent studies suggest that thiol oxidation-elicited dimerization of the 1α form of protein kinase G (PKG1α) contributes to the relaxation of isolated endothelium-removed bovine pulmonary arteries (BPA) to peroxide and responses to hypoxia, we investigated whether cytosolic NADPH oxidation promoted relaxation by PKG1α dimerization. Relaxation of BPA to G6PD inhibitors 6-aminonicotinamide (6-AN) and epiandrosterone (studied under hypoxia to minimize basal levels of NADPH oxidation and PKG1α dimerization) was associated with increased PKG1α dimerization and PKG-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Depletion of PKG1α by small inhibitory RNA (siRNA) inhibited relaxation of BPA to 6-AN and attenuated the increase in VASP phosphorylation. Relaxation to 6-AN did not appear to be altered by depletion of soluble guanylate cyclase (sGC). Depletion of G6PD, thioredoxin-1 (Trx-1), and Trx reductase-1 (TrxR-1) in BPA with siRNA increased PKG1α dimerization and VASP phosphorylation and inhibited force generation under aerobic and hypoxic conditions. Depletion of TrxR-1 with siRNA inhibited the effects of 6-AN and enhanced similar responses to peroxide. Peroxiredoxin-1 depletion by siRNA inhibited PKG dimerization to peroxide, but it did not alter PKG dimerization under hypoxia or the stimulation of dimerization by 6-AN. Thus regulation of cytosolic NADPH redox by G6PD appears to control PKG1α dimerization in BPA through its influence on Trx-1 redox regulation by the NADPH dependence of TrxR-1. NADPH regulation of PKG dimerization may contribute to vascular responses to hypoxia that are associated with changes in NADPH redox.

Roles for Cytosolic NADPH Redox in Regulating Pulmonary Artery Relaxation by Thiol Oxidation‐Elicited Subunit Dimerization of Protein Kinase G 1α

The activity of glucose‐6‐phosphate dehydrogenase (G6PD) appears to control a vascular smooth muscle relaxation regulated via cytosolic NADPH oxidation. Since our studies suggest thiol oxidation‐elicited dimerization of protein kinase G 1α (PKG1α) contributes to the relaxation of endothelium‐removed bovine pulmonary arteries (BPA) to H2O2 and responses to hypoxia, we investigated if cytosolic NADPH oxidation promoted relaxation by PKG1α dimerization. Depletion of PKG1α by siRNA inhibited relaxation of BPA to 6‐aminonicotinamide (6‐AN), and attenuated the increase in vasodilator‐stimulated phosphoprotein (VASP) phosphorylation. Relaxation to 6‐AN was not altered by depletion of soluble guanylate cyclase. Depletion of G6PD in BPA with siRNA increased PKG1α dimerization and VASP phosphorylation, and inhibited force generation under hypoxia. Depletion of thioredoxin‐1 and thioredoxin reductase‐1 with siRNA elicited responses similar to G6PD depletion. Thus, cytosolic NADPH redox appears to control relaxation of BPA by PKG1α dimerization through its influence on thioredoxin‐1 redox, and these processes may contribute to vascular responses to hypoxia that are associated with changes in NADPH redox.Supported by NIH grants HL031069, HL043023 and HL066331

A constraint-based model analysis of the metabolic consequences of increased NADPH oxidation in Saccharomyces cerevisiae

Controlling the amounts of redox cofactors to manipulate metabolic fluxes is emerging as a useful approach to optimizing byproduct yields in yeast biotechnological processes. Redox cofactors are extensively interconnected metabolites, so predicting metabolite patterns is challenging and requires in-depth knowledge of how the metabolic network responds to a redox perturbation. Our aim was to analyze comprehensively the metabolic consequences of increased cytosolic NADPH oxidation during yeast fermentation. Using a genetic device based on the overexpression of a modified 2,3-butanediol dehydrogenase catalyzing the NADPH-dependent reduction of acetoin into 2,3-butanediol, we increased the NADPH demand to between 8 and 40-fold the anabolic demand. We developed (i) a dedicated constraint-based model of yeast fermentation and (ii) a constraint-based modeling method based on the dynamical analysis of mass distribution to quantify the in vivo contribution of pathways producing NADPH to the maintenance of redox homeostasis. We report that yeast responds to NADPH oxidation through a gradual increase in the flux through the PP and acetate pathways, providing 80% and 20% of the NADPH demand, respectively. However, for the highest NADPH demand, the model reveals a saturation of the PP pathway and predicts an exchange between NADH and NADPH in the cytosol that may be mediated by the glycerol–DHA futile cycle. We also reveal the contribution of mitochondrial shuttles, resulting in a net production of NADH in the cytosol, to fine-tune the NADH/NAD+ balance. This systems level study helps elucidate the physiological adaptation of yeast to NADPH perturbation. Our findings emphasize the robustness of yeast to alterations in NADPH metabolism and highlight the role of the glycerol–DHA cycle as a redox valve, providing additional NADPH from NADH under conditions of very high demand.

Roles for redox mechanisms controlling protein kinase G in pulmonary and coronary artery responses to hypoxia

We previously reported that isolated endothelium-removed bovine pulmonary arteries (BPAs) contract to hypoxia associated with removal of peroxide- and cGMP-mediated relaxation. In contrast, bovine coronary arteries (BCAs) relax to hypoxia associated with cytosolic NADPH oxidation coordinating multiple relaxing mechanisms. Since we recently found that H(2)O(2) relaxes BPAs through PKG activation by both soluble guanylate cyclase (sGC)/cGMP-dependent and cGMP-independent thiol oxidation/subunit dimerization mechanisms, we investigated if these mechanisms participate in BPA contraction and BCA relaxation to hypoxia. The contraction of BPA (precontracted with 20 mM KCl) to hypoxia was associated with decreased PKG dimerization and PKG-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. In contrast, exposure of 20 mM KCl-precontracted endothelium-removed BCAs to hypoxia caused relaxation and increased dimerization and VASP phosphorylation. Depletion of sGC by organoid culture of BPAs with an oxidant of the sGC heme (10 μM 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one) increased aerobic force generation, decreased VASP phosphorylation, and inhibited further contraction to hypoxia and changes in VASP phosphorylation. Thiol reduction with dithiothreitol increased aerobic force in BPAs and decreased PKG dimerization, VASP phosphorylation, and the contraction to hypoxia. Furthermore, PKG-1α and sGC β(1)-subunit small interfering RNA-transfected BPAs demonstrated increased aerobic K(+) force and inhibition of further contraction to hypoxia, associated with an attenuation of H(2)O(2)-elicited relaxation and VASP phosphorylation. Thus, decreases in both a sGC/cGMP-dependent and a dimerization-dependent activation of PKG by H(2)O(2) appear to contribute to the contraction of BPAs elicited by hypoxia. In addition, stimulation of PKG activation by dimerization may be important in the relaxation of coronary arteries to hypoxia.

Redox regulation of responses to hypoxia and NO-cGMP signaling in pulmonary vascular pathophysiology

There is much controversy in mechanisms controlling the pulmonary arterial response to acute and chronic hypoxia. Our previous work suggests bovine pulmonary arteries (BPA) contract to hypoxia via removal of a relaxation mediated by hydrogen peroxide derived from superoxide generated by Nox4 [1]. In contrast, bovine coronary arteries (BCA) relax to hypoxia via cytosolic NADPH oxidation coordinating multiple processes that lower intracellular calcium [2]. Peroxide appears to relax BPA by both stimulation of soluble guanylate cyclase (sGC) and by a cGMP-independent activation of protein kinase G (PKG) resulting from thiol oxidation-mediated subunit dimerization [3]. A removal of both of these mechanisms appears to contribute to the hypoxic pulmonary vasoconstriction (HPV) response seen in BPA. In addition, PKG dimerization appears to participate in the relaxation of coronary arteries to hypoxia. BPA secrete superoxide derived from Nox2 into the extracellular environment, and increased extracellular SOD (ecSOD) activity appears to attenuate the HPV response by increasing extracellular peroxide levels from this extracellular source of superoxide. Increased peroxide generated by Nox2 or mitochondria also appears to attenuate the relaxation of BCA to hypoxia by stimulating ERK MAP kinase [4]. Exposure of mice to 21 days of hypoxia (10% O2) promotes pulmonary hypertension associated with decreased pulmonary artery and aortic contraction to phenylephrine, NO-mediated relaxation to acetylcholine (ACh) and responses to hypoxia. While increased pulmonary arterial expression of sGC has been reported in this mouse model of pulmonary hypertension, chronic hypoxia had minimal effects on relaxation to an NO donor. Catalase markedly restored contraction to phenylephrine and the aortic relaxation to hypoxia. Chronic treatment of mice with cobalt protoporphyrin IX, which induces heme oxygenase and ecSOD, attenuated the development of pulmonary hypertension without restoring relaxation to ACh. In addition, chronic treatment of mice with delta-aminolevulinic acid, a precursor to the sGC activator protoporphyrin IX and heme needed for sGC activation and heme oxygenase activity, also attenuated pulmonary hypertension development without restoring responses to ACh. Thus, redox mechanisms regulating PKG seem to be important contributors to vascular oxygen sensing mechanisms. In addition, increased ecSOD expression and PKG-mediated vasodilation to endogenously generated peroxide and other sGC activators may function as a protective mechanism against the development of hypoxia-induced pulmonary hypertension.

Redox regulation of guanylate cyclase and protein kinase G in vascular responses to hypoxia.

The production of cGMP by the soluble form of guanylate cyclase (sGC) in bovine pulmonary arteries (BPA) is controlled by cytosolic NADPH maintaining reduced thiol and heme sites on sGC needed for activation by NO, and the levels of Nox oxidase-derived superoxide and peroxide that influence pathways regulating sGC activity. Our recent studies in BPA suggest that the activities of peroxide metabolizing pathways in vascular smooth muscle potentially determine the balance between sGC stimulation by peroxide and a cGMP-independent activation of cGMP-dependent protein kinase (PKG) by a disulfide-mediated subunit dimerization. Cytosolic NADPH oxidation also appears to function in BPA through its influence on protein thiol redox control as an additional mechanism promoting vascular relaxation through PKG activation. These processes regulating PKG may participate in decreases in peroxide and increases in NADPH associated with contraction of BPA to hypoxia and in cytosolic NADPH oxidation potentially mediating bovine coronary artery relaxation to hypoxia.

Potential Role of NADPH Redox in Regulating Thiol Oxidation‐Elicited Subunit Dimerization Activation of Protein Kinase G in the Relaxation of Bovine Pulmonary Arteries to Pentose Phosphate Pathway Inhibitors

We have previously provided evidence that inhibition of glucose‐6‐phosphate dehydrogenase (G6PD) in the pentose phosphate pathway promotes oxidation of NADPH, and elicits vascular relaxation through coordinating multiple processes that lower intracellular calcium. Since it was reported that H2O2 induces coronary relaxation associated with a NO/cGMP‐independent thiol oxidation/subunit dimerization activation of protein kinase G (PKG), we investigated if inhibition of G6PD activates PKG through disulfide‐mediated subunit dimerization under conditions where relaxation of isolated endothelium‐removed bovine pulmonary arteries (BPA) is observed. BPA precontracted with 20mM KCl under hypoxic conditions were exposed to either 1mM 6‐aminonicotinamide (6‐AN) or 0.5mM epiandrosterone (Epi) to inhibit G6PD. It was observed that both 6‐AN and Epi increased PKG dimerization under conditions where relaxation was observed. These responses were associated with increased phosphorylation of vasodilator‐stimulated phosphoprotein (VASP) at the serine‐239 site known to be mediated by PKG. Thus, inhibition of G6PD promotes thiol oxidation‐elicited dimerization activation of PKG, suggesting that PKG activation is likely to be a participant in mechanisms through which cytosolic NADPH oxidation promotes vascular relaxation.Supported by NIH grants HL31069, HL43023 and HL66331

Effects of hypoxia on relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in coronary arterial smooth muscle

Since controversy exists on how hypoxia influences vascular reactive oxygen species (ROS) generation, and our previous work provided evidence that it relaxes endothelium-denuded bovine coronary arteries (BCA) in a ROS-independent manner by promoting cytosolic NADPH oxidation, we examined how hypoxia alters relationships between cytosolic and mitochondrial NAD(P)H redox and superoxide generation in BCA. Methods were developed to image and interpret the effects of hypoxia on NAD(P)H redox based on its autofluorescence in the cytosolic, mitochondrial, and nuclear regions of smooth muscle cells isolated from BCA. Aspects of anaerobic glycolysis and cytosolic NADH redox in BCA were assessed from measurements of lactate and pyruvate. Imaging changes in mitosox and dehydroethidium fluorescence were used to detect changes in mitochondrial and cytosolic-nuclear superoxide, respectively. Hypoxia appeared to increase mitochondrial and decrease cytosolic-nuclear superoxide under conditions associated with increased cytosolic NADH (lactate/pyruvate), mitochondrial NAD(P)H, and hyperpolarization of mitochondria detected by tetramethylrhodamine methyl-ester perchlorate fluorescence. Rotenone appeared to increase mitochondrial NAD(P)H and superoxide, suggesting hypoxia could increase superoxide generation by complex I. However, hypoxia decreased mitochondrial superoxide in the presence of contraction to 30 mM KCl, associated with decreased mitochondrial NAD(P)H. Thus, while hypoxia augments NAD(P)H redox associated with increased mitochondrial superoxide, contraction with KCl reverses these effects of hypoxia on mitochondrial superoxide, suggesting mitochondrial ROS increases do not mediate hypoxic relaxation in BCA. Since hypoxia lowers pyruvate, and pyruvate inhibits hypoxia-elicited relaxation and NADPH oxidation in BCA, mitochondrial control of pyruvate metabolism associated with cytosolic NADPH redox regulation could contribute to sensing hypoxia.

Phosphatidylinositol 3-Kinase γ Signaling through Protein Kinase Cζ Induces NADPH Oxidase-mediated Oxidant Generation and NF-κB Activation in Endothelial Cells

We addressed the role of class 1B phosphatidylinositol 3-kinase (PI3K) isoform PI3Kgamma in mediating NADPH oxidase activation and reactive oxidant species (ROS) generation in endothelial cells (ECs) and of PI3Kgamma-mediated oxidant signaling in the mechanism of NF-kappaB activation and intercellular adhesion molecule (ICAM)-1 expression. We used lung microvascular ECs isolated from mice with targeted deletion of the p110gamma catalytic subunit of PI3Kgamma. Tumor necrosis factor (TNF) alpha challenge of wild type ECs caused p110gamma translocation to the plasma membrane and phosphatidylinositol 1,4,5-trisphosphate production coupled to ROS production; however, this response was blocked in p110gamma-/- ECs. ROS production was the result of TNFalpha activation of Ser phosphorylation of NADPH oxidase subunit p47(phox) and its translocation to EC membranes. NADPH oxidase activation failed to occur in p110gamma-/- ECs. Additionally, the TNFalpha-activated NF-kappaB binding to the ICAM-1 promoter, ICAM-1 protein expression, and PMN adhesion to ECs required functional PI3Kgamma. TNFalpha challenge of p110gamma-/- ECs failed to induce phosphorylation of PDK1 and activation of the atypical PKC isoform, PKCzeta. Thus, PI3Kgamma lies upstream of PKCzeta in the endothelium, and its activation is crucial in signaling NADPH oxidase-dependent oxidant production and subsequent NF-kappaB activation and ICAM-1 expression.

Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH

Vascular smooth muscle (VSM) derived from pulmonary arteries generally contract to hypoxia, whereas VSM from systemic arteries usually relax, indicating the presence of basic oxygen-sensing mechanisms in VSM that are adapted to the environment from which they are derived. This review considers how fundamental processes associated with the generation of reactive oxygen species (ROS) by oxidase enzymes, the metabolic control of cytosolic NADH, NADPH and glutathione redox systems, and mitochondrial function interact with signaling systems regulating vascular force in a manner that is potentially adapted to be involved in Po2 sensing. Evidence for opposing hypotheses of hypoxia, either decreasing or increasing mitochondrial ROS, is considered together with the Po2 dependence of ROS production by Nox oxidases as sensors potentially contributing to hypoxic pulmonary vasoconstriction. Processes through which ROS and NAD(P)H redox changes potentially control interactive signaling systems, including soluble guanylate cyclase, potassium channels, and intracellular calcium are discussed together with the data supporting their regulation by redox in responses to hypoxia. Evidence for hypothesized potential differences between systemic and pulmonary arteries originating from properties of mitochondrial ROS generation and the redox sensitivity of potassium channels is compared with a new hypothesis in which differences in the control of cytosolic NADPH redox by the pentose phosphate pathway results in increased NADPH and Nox oxidase-derived ROS in pulmonary arteries, whereas lower levels of glucose-6-phosphate dehydrogenase in coronary arteries may permit hypoxia to activate a vasodilator mechanism controlled by oxidation of cytosolic NADPH.

Two mechanisms for oxidation of cytosolic NADPH by Kluyveromyces lactis mitochondria.

Null mutations in the structural gene encoding phosphoglucose isomerase completely abolish activity of this glycolytic enzyme in Kluyveromyces lactis and Saccharomyces cerevisiae. In S. cerevisiae, the pgi1 null mutation abolishes growth on glucose, whereas K.lactis rag2 null mutants still grow on glucose. It has been proposed that, in the latter case, growth on glucose is made possible by an ability of K. lactis mitochondria to oxidize cytosolic NADPH. This would allow for a re-routing of glucose dissimilation via the pentose-phosphate pathway. Consistent with this hypothesis, mitochondria of S. cerevisiae cannot oxidize NADPH. In the present study, the ability of K. lactis mitochondria to oxidize cytosolic NADPH was experimentally investigated. Respiration-competent mitochondria were isolated from aerobic, glucose-limited chemostat cultures of the wild-type K. lactis strain CBS 2359 and from an isogenic rag2Delta strain. Oxygen-uptake experiments confirmed the presence of a mitochondrial NADPH dehydrogenase in K.lactis. This activity was ca. 2.5-fold higher in the rag2Delta mutant than in the wild-type strain. In contrast to mitochondria from wild-type K. lactis, mitochondria from the rag2Delta mutant exhibited high rates of ethanol-dependent oxygen uptake. Subcellular fractionation studies demonstrated that, in the rag2Delta mutant, a mitochondrial alcohol dehydrogenase was present and that activity of a cytosolic NADPH-dependent 'acetaldehyde reductase' was also increased. These observations indicate that two mechanisms may participate in mitochondrial oxidation of cytosolic NADPH by K. lactis mitochondria: (a) direct oxidation of cytosolic NADPH by a mitochondrial NADPH dehydrogenase; and (b) a two-compartment transhydrogenase cycle involving NADP(+)- and NAD(+)-dependent alcohol dehydrogenases.