Cytosolic NADPH may regulate differences in basal Nox oxidase-derived superoxide generation in bovine coronary and pulmonary arteries

Because systems controlled by basal NAD(P)H oxidase activity appear to contribute to differences in responses of endothelium-removed bovine coronary (BCA) and pulmonary (BPA) arteries to hypoxia, we characterized the Nox oxidases activities present in these vascular segments and how cytosolic NAD(P)H redox systems could be controlling oxidase activity. BPA generated approximately 60-80% more lucigenin (5 microM) chemiluminescence detectable superoxide than BCA. Apocynin (10 microM), a NAD(P)H oxidase inhibitor, and 6-aminonicotinamide (1 mM), a pentose phosphate inhibitor (PPP), both attenuated (approximately by 50-70%) superoxide detected in BPA and BCA. There was no significant difference in the expression of Nox2 or Nox4 mRNA or protein detected by Western blot analysis. NADPH and NADH increased superoxide in homogenates and isolated microsomal membrane fractions in a manner consistent with BPA and BCA having similar levels of oxidase activity. BPA had 4.2-fold higher levels of NADPH than BCA. The activity and protein levels of glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting PPP enzyme generating cytosolic NADPH, were 1.5-fold higher in BPA than BCA. Thus BPA differ from BCA in that they have higher levels of G6PD activity, NADPH, and superoxide. Because both arteries have similar levels of Nox expression and activity, elevated levels of cytosolic NADPH may contribute to increased superoxide in BPA.     

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.     

An increase in the redox state during reperfusion contributes to the cardioprotective effect of GIK solution

This study aimed at determining whether glucose-insulin-potassium (GIK) solutions modify the NADH/NAD(+) ratio during postischemic reperfusion and whether their cardioprotective effect can be attributed to this change in part through reduction of the mitochondrial reactive oxygen species (ROS) production. The hearts of 72 rats were perfused with a buffer containing glucose (5.5 mM) and hexanoate (0.5 mM). They were maintained in normoxia for 30 min and then subjected to low-flow ischemia (0.5% of the preischemic coronary flow for 20 min) followed by reperfusion (45 min). From the beginning of ischemia, the perfusate was subjected to various changes: enrichment with GIK solution, enrichment with lactate (2 mM), enrichment with pyruvate (2 mM), enrichment with pyruvate (2 mM) plus ethanol (2 mM), or no change for the control group. Left ventricular developed pressure, heart rate, coronary flow, and oxygen consumption were monitored throughout. The lactate/pyruvate ratio of the coronary effluent, known to reflect the cytosolic NADH/NAD(+) ratio and the fructose-6-phosphate/dihydroxyacetone-phosphate (F6P/DHAP) ratio of the reperfused myocardium, were evaluated. Mitochondrial ROS production was also estimated. The GIK solution improved the recovery of mechanical function during reperfusion. This was associated with an enhanced cytosolic NADH/NAD(+) ratio and reduced mitochondrial ROS production. The cardioprotection was also observed when the hearts were perfused with fluids known to increase the cytosolic NADH/NAD(+) ratio (lactate, pyruvate plus ethanol) compared with the other fluids (control and pyruvate groups). The hearts with a high mechanical recovery also displayed a low F6P/DHAP ratio, suggesting that an accelerated glycolysis rate may be responsible for increased cytosolic NADH production. In conclusion, the cardioprotection induced by GIK solutions could occur through an increase in the cytosolic NADH/NAD(+) ratio, leading to a decrease in mitochondrial ROS production.     

Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response

Glucose-stimulated insulin secretion is a multistep process dependent on beta-cell metabolic flux. Our previous studies on intact pancreatic islets used two-photon NAD(P)H imaging as a quantitative measure of the combined redox signal from NADH and NADPH (referred to as NAD(P)H). These studies showed that pyruvate, a non-secretagogue, enters beta-cells and causes a transient rise in NAD(P)H. To further characterize the metabolic fate of pyruvate, we have now developed one-photon flavoprotein microscopy as a simultaneous assay of lipoamide dehydrogenase (LipDH) autofluorescence. This flavoprotein is in direct equilibrium with mitochondrial NADH. Hence, a comparison of LipDH and NAD(P)H autofluorescence provides a method to distinguish the production of NADH, NADPH, or both. Using this method, the glucose dose response is consistent with an increase in both NADH and NADPH. In contrast, the transient rise in NAD(P)H observed with pyruvate stimulation is not accompanied by a significant change in LipDH, which indicates that pyruvate raises cellular NADPH without raising NADH. In comparison, methyl pyruvate stimulated a robust NADH and NADPH response. These data provide new evidence that exogenous pyruvate does not induce a significant rise in mitochondrial NADH. This inability likely results in its failure to produce the ATP necessary for stimulated secretion of insulin. Overall, these data are consistent with either a restricted pyruvate dehydrogenase-dependent metabolism or a buffering of the NADH response by other metabolic mechanisms.     

Relative importance of redox buffers GSH and NAD(P)H in age‐related neurodegeneration and Alzheimer disease‐like mouse neurons

Aging, a major risk factor in Alzheimer's disease (AD), is associated with an oxidative redox shift, decreased redox buffer protection, and increased free radical reactive oxygen species (ROS) generation, probably linked to mitochondrial dysfunction. While NADH is the ultimate electron donor for many redox reactions, including oxidative phosphorylation, glutathione (GSH) is the major ROS detoxifying redox buffer in the cell. Here, we explored the relative importance of NADH and GSH to neurodegeneration in aging and AD neurons from nontransgenic and 3xTg‐AD mice by inhibiting their synthesis to determine whether NADH can compensate for the GSH loss to maintain redox balance. Neurons stressed by either depleting NAD(P)H or GSH indicated that NADH redox control is upstream of GSH levels. Further, although depletion of NAD(P)H or GSH correlated linearly with neuron death, compared with GSH depletion, higher neurodegeneration was observed when NAD(P)H was extrapolated to zero, especially in old age, and in the 3xTg‐AD neurons. We also observed an age‐dependent loss of gene expression of key redox‐dependent biosynthetic enzymes, NAMPT (nicotinamide phosphoribosyltransferase), and NNT (nicotinamide nucleotide transhydrogenase). Moreover, age‐related correlations between brain NNT or NAMPT gene expression and NADPH levels suggest that these genes contribute to the age‐related declines in NAD(P)H. Our data indicate that in aging and more so in AD‐like neurons, NAD(P)H redox control is upstream of GSH and an oxidative redox shift that promotes neurodegeneration. Thus, NAD(P)H generation may be a more efficacious therapeutic target upstream of GSH and ROS.     

The redox switch/redox coupling hypothesis

We provide an integrative interpretation of neuroglial metabolic coupling including the presence of subcellular compartmentation of pyruvate and monocarboxylate recycling through the plasma membrane of both neurons and glial cells. The subcellular compartmentation of pyruvate allows neurons and astrocytes to select between glucose and lactate as alternative substrates, depending on their relative extracellular concentration and the operation of a redox switch. This mechanism is based on the inhibition of glycolysis at the level of glyceraldehyde 3-phosphate dehydrogenase by NAD(+) limitation, under sufficiently reduced cytosolic NAD(+)/NADH redox conditions. Lactate and pyruvate recycling through the plasma membrane allows the return to the extracellular medium of cytosolic monocarboxylates enabling their transcellular, reversible, exchange between neurons and astrocytes. Together, intracellular pyruvate compartmentation and monocarboxylate recycling result in an effective transcellular coupling between the cytosolic NAD(+)/NADH redox states of both neurons and glial cells. Following glutamatergic neurotransmission, increased glutamate uptake by the astrocytes is proposed to augment glycolysis and tricarboxylic acid cycle activity, balancing to a reduced cytosolic NAD(+)/NADH in the glia. Reducing equivalents are transferred then to the neuron resulting in a reduced neuronal NAD(+)/NADH redox state. This may eventually switch off neuronal glycolysis, favoring the oxidation of extracellular lactate in the lactate dehydrogenase (LDH) equilibrium and in the neuronal tricarboxylic acid cycles. Finally, pyruvate derived from neuronal lactate oxidation, may return to the extracellular space and to the astrocyte, restoring the basal redox state and beginning a new loop of the lactate/pyruvate transcellular coupling cycle. Transcellular redox coupling operates through the plasma membrane transporters of monocarboxylates, similarly to the intracellular redox shuttles coupling the cytosolic and mitochondrial redox states through the transporters of the inner mitochondrial membrane. Finally, transcellular redox coupling mechanisms may couple glycolytic and oxidative zones in other heterogeneous tissues including muscle and tumors.     

Preferential utilization of NADPH as the endogenous electron donor for NAD(P)H:quinone oxidoreductase 1 (NQO1) in intact pulmonary arterial endothelial cells

The goal was to determine whether endogenous cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1) preferentially uses NADPH or NADH in intact pulmonary arterial endothelial cells in culture. The approach was to manipulate the redox status of the NADH/NAD(+) and NADPH/NADP(+) redox pairs in the cytosolic compartment using treatment conditions targeting glycolysis and the pentose phosphate pathway alone or with lactate, and to evaluate the impact on the intact cell NQO1 activity. Cells were treated with 2-deoxyglucose, iodoacetate, or epiandrosterone in the absence or presence of lactate, NQO1 activity was measured in intact cells using duroquinone as the electron acceptor, and pyridine nucleotide redox status was measured in total cell KOH extracts by high-performance liquid chromatography. 2-Deoxyglucose decreased NADH/NAD(+) and NADPH/NADP(+) ratios by 59 and 50%, respectively, and intact cell NQO1 activity by 74%; lactate restored NADH/NAD(+), but not NADPH/NADP(+) or NQO1 activity. Iodoacetate decreased NADH/NAD(+) but had no detectable effect on NADPH/NADP(+) or NQO1 activity. Epiandrosterone decreased NQO1 activity by 67%, and although epiandrosterone alone did not alter the NADPH/NADP(+) or NADH/NAD(+) ratio, when the NQO1 electron acceptor duroquinone was also present, NADPH/NADP(+) decreased by 84% with no impact on NADH/NAD(+). Duroquinone alone also decreased NADPH/NADP(+) but not NADH/NAD(+). The results suggest that NQO1 activity is more tightly coupled to the redox status of the NADPH/NADP(+) than NADH/NAD(+) redox pair, and that NADPH is the endogenous NQO1 electron donor. Parallel studies of pulmonary endothelial transplasma membrane electron transport (TPMET), another redox process that draws reducing equivalents from the cytosol, confirmed previous observations of a correlation with the NADH/NAD(+) ratio.     

The energy state of tumor-bearing rats

Rats bearing the Walker-256 carcinosarcoma have a profoundly altered liver metabolite content with significant increases in the concentrations of glucose 6-phosphate, fructose 1,6-bisphosphate, citrate, lactate, and alanine, while the concentrations of glucose, pyruvate, dihydroxyacetone phosphate, and glutamine are decreased. As a result of these changes both the cytosolic NAD+/NADH ratio and the cytosolic phosphorylation potential are significantly lowered while no changes are detected in either the cytosolic NADP+/NADPH ratio or the mitochondrial NAD+/NADH ratio. These hepatic changes are accompanied by marked increases in the circulating concentrations of lactate, non-esterified fatty acids, and triacylglycerols. The activities of both liver hexokinase and phosphofructokinase are also significantly elevated in the tumor-bearing rats. The changes observed both in the redox state and phosphorylation potential are in agreement with the energy imbalance associated with tumor burden.     

Biochemical issues in estimation of cytosolic free NAD/NADH ratio

Cytosolic free NAD/NADH ratio is fundamentally important in maintaining cellular redox homeostasis but current techniques cannot distinguish between protein-bound and free NAD/NADH. Williamson et al reported a method to estimate this ratio by cytosolic lactate/pyruvate (L/P) based on the principle of chemical equilibrium. Numerous studies used L/P ratio to estimate the cytosolic free NAD/NADH ratio by assuming that the conversion in cells was at near-equilibrium but not verifying how near it was. In addition, it seems accepted that cytosolic free NAD/NADH ratio was a dependent variable responding to the change of L/P ratio. In this study, we show (1) that the change of lactate/glucose (percentage of glucose that converts to lactate by cells) and L/P ratio could measure the status of conversion between pyruvate + NADH and lactate + NAD that tends to or gets away from equilibrium; (2) that cytosolic free NAD/NADH could be accurately estimated by L/P only when the conversion is at or very close to equilibrium otherwise a calculation error by one order of magnitude could be introduced; (3) that cytosolic free NAD/NADH is stable and L/P is highly labile, that the highly labile L/P is crucial to maintain the homeostasis of NAD/NADH; (4) that cytosolic free NAD/NADH is dependent on oxygen levels. Our study resolved the key issues regarding accurate estimation of cytosolic free NAD/NADH ratio and the relationship between NAD/NADH and L/P.     

Antioxidant pyruvate inhibits cardiac formation of reactive oxygen species through changes in redox state

Myocardial ischemia-reperfusion is associated with bursts of reactive oxygen species (ROS) such as superoxide radicals (O(2)(-).). Membrane-associated NADH oxidase (NADHox) activity is a hypothetical source of O(2)(-)., implying the NADH concentration-to-NAD(+) concentration ratio ([NADH]/[NAD(+)]) as a determinant of ROS. To test this hypothesis, cardiac NADHox and ROS formation were measured as influenced by pyruvate or L-lactate. Pre- and postischemic Langendorff guinea pig hearts were perfused at different pyruvate/L-lactate concentrations to alter cytosolic [NADH]/[NAD(+)]. NADHox and ROS were measured with the use of lucigenin chemiluminescence and electron spin resonance, respectively. In myocardial homogenates, pyruvate (0.05, 0.5 mM) and the NADHox blocker hydralazine markedly inhibited NADHox (16 +/- 2%, 58 +/- 9%). In postischemic hearts, pyruvate (0.1-5.0 mM) dose dependently inhibited ROS up to 80%. However, L-lactate (1.0-15.0 mM) stimulated both basal and postischemic ROS severalfold. Furthermore, L-lactate-induced basal ROS was dose dependently inhibited by pyruvate (0.1-5.0 mM) and not the xanthine oxidase inhibitor oxypurinol. Pyruvate did not inhibit ROS from xanthine oxidase. The data suggest a substantial influence of cytosolic NADH on cardiac O(2)(-). formation that can be inhibited by submillimolar pyruvate. Thus cytotoxicities due to cardiac ischemia-reperfusion ROS may be alleviated by redox reactants such as pyruvate.     

NADH/NAD redox state of cytoplasmic glycolytic compartments in vascular smooth muscle

The cytoplasmic NADH/NAD redox potential affects energy metabolism and contractile reactivity of vascular smooth muscle. NADH/NAD redox state in the cytosol is predominately determined by glycolysis, which in smooth muscle is separated into two functionally independent cytoplasmic compartments, one of which fuels the activity of Na(+)-K(+)-ATPase. We examined the effect of varying the glycolytic compartments on cystosolic NADH/NAD redox state. Inhibition of Na(+)-K(+)-ATPase by 10 microM ouabain resulted in decreased glycolysis and lactate production. Despite this, intracellular concentrations of the glycolytic metabolite redox couples of lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate (thus NADH/NAD) and the cytoplasmic redox state were unchanged. The constant concentration of the metabolite redox couples and redox potential was attributed to 1) decreased efflux of lactate and pyruvate due to decreased activity of monocarboxylate B-H(+) transporter secondary to decreased availability of H(+) for cotransport and 2) increased uptake of lactate (and perhaps pyruvate) from the extracellular space, probably mediated by the monocarboxylate-H(+) transporter, which was specifically linked to reduced activity of Na(+)-K(+)-ATPase. We concluded that redox potentials of the two glycolytic compartments of the cytosol maintain equilibrium and that the cytoplasmic NADH/NAD redox potential remains constant in the steady state despite varying glycolytic flux in the cytosolic compartment for Na(+)-K(+)-ATPase.     

Renal metabolism during four types of lactic acidosis in the dog including anoxia

The present study was undertaken to evaluate the metabolic response of the kidney to lactic acidosis. Four types of lactic acidosis were induced in the dog: infusion of lactic acid, infusion of lactic acid with phenformin, administration of phenformin alone, and hypoxia by breathing 95% nitrogen. In all groups of animals, the same degree of acidosis was observed with plasma bicarbonate ranging from 12.8 to 14.9 mM. Plasma lactate concentration ranged from 3.0 to 8.1 mumol/mL. Renal ammoniagenesis failed to be influenced by lactic acidosis. As a matter of fact, it fell during anoxia. The extraction of glutamine by the kidney rose except during anoxia where it fell. The renal production of alanine rose during the infusion of lactic acid with and without phenformin. This coincided with the extraction of glutamine. The renal extraction of lactate rose in all forms of acidosis as well as the production of pyruvate. In the renal cortical tissue, the concentration of malate, pyruvate, and lactate rose. Alanine also rose except during anoxia. An important fall in cytosolic redox potential (NAD+/NADH lactate dehydrogenase) was observed, as well as a fall in mitochondrial redox (NAD+/NADH beta-hydroxybutyrate dehydrogenase). Lactate also accumulated in the liver and in the muscle. We propose that the kidney is unable to respond to lactic acidosis in terms of ammonia production and that this phenomenon is explained by transamination of pyruvate and glutamate into alanine and also by the observed fall in cytosolic redox potential. It is likely that renal gluconeogenesis is also inhibited and this is reflected by the rise in the concentration of malate in the kidney.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of NADH/NAD+ transport activity and glycogen store on skeletal muscle energy metabolism during exercise: in silico studies

Skeletal muscle can maintain ATP concentration constant during the transition from rest to exercise, whereas metabolic reaction rates may increase substantially. Among the key regulatory factors of skeletal muscle energy metabolism during exercise, the dynamics of cytosolic and mitochondrial NADH and NAD+ have not been characterized. To quantify these regulatory factors, we have developed a physiologically based computational model of skeletal muscle energy metabolism. This model integrates transport and reaction fluxes in distinct capillary, cytosolic, and mitochondrial domains and investigates the roles of mitochondrial NADH/NAD+ transport (shuttling) activity and muscle glycogen concentration (stores) during moderate intensity exercise (60% maximal O2 consumption). The underlying hypothesis is that the cytosolic redox state (NADH/NAD+) is much more sensitive to a metabolic disturbance in contracting skeletal muscle than the mitochondrial redox state. This hypothesis was tested by simulating the dynamic metabolic responses of skeletal muscle to exercise while altering the transport rate of reducing equivalents (NADH and NAD+) between cytosol and mitochondria and muscle glycogen stores. Simulations with optimal parameter estimates showed good agreement with the available experimental data from muscle biopsies in human subjects. Compared with these simulations, a 20% increase (or approximately 20% decrease) in mitochondrial NADH/NAD+ shuttling activity led to an approximately 70% decrease (or approximately 3-fold increase) in cytosolic redox state and an approximately 35% decrease (or approximately 25% increase) in muscle lactate level. Doubling (or halving) muscle glycogen concentration resulted in an approximately 50% increase (or approximately 35% decrease) in cytosolic redox state and an approximately 30% increase (or approximately 25% decrease) in muscle lactate concentration. In both cases, changes in mitochondrial redox state were minimal. In conclusion, the model simulations of exercise response are consistent with the hypothesis that mitochondrial NADH/NAD+ shuttling activity and muscle glycogen stores affect primarily the cytosolic redox state. Furthermore, muscle lactate production is regulated primarily by the cytosolic redox state.     

The decrease of NAD(P)H has a prominent role in dopamine toxicity

We characterized dopamine toxicity in human neuroblastoma SH-SY5Y cells as a direct effect of dopamine on cell reductive power, measured as NADH and NADPH cell content. In cell incubations with 100 or 500 microM dopamine, the accumulation of dopamine inside the cell reached a maximum after 6 h. The decrease in cell viability was 40% and 75%, respectively, after 24 h, and was not altered by MAO inhibition with tranylcypromine. Dopamine was metabolized to DOPAC by mitochondrial MAO and, at 500 microM concentration, significantly reduced mitochondrial potential and oxygen consumption. This DA concentration caused only a slight increase in cell peroxidation in the absence of Fe(III), but a dramatic decrease in NADH and NADPH cell content and a concomitant decrease in total cell NAD(P)H/NAD(P)+ and GSH/GSSG and in mitochondrial NADH/NAD+ ratios. Dopaminechrome, a product of dopamine oxidation, was found to be a MAO-A inhibitor and a strong oxidizer of NADH and NADPH in a cell-free system. We conclude that dopamine may affect NADH and NADPH oxidation directly. When the intracellular concentrations of NAD(P)H and oxidized dopamine are similar, NAD(P)H triggers a redox cycle with dopamine that leads to its own consumption. The time-course of NADH and NADPH oxidation by dopamine was assessed in cell-free assays: NAD(P)H concentration decreased at the same time as dopamine oxidation advanced. The break in cell redox equilibrium, not excluding the involvement of free oxygen radicals, could be sufficient to explain the toxicity of dopamine in dopaminergic neurons.     

Cytosolic NADH redox and thiol oxidation regulate pulmonary arterial force through ERK MAP kinase

An ERK MAP kinase-mediated contractile mechanism previously reported to be activated by peroxide and stretch in bovine coronary arteries is shown in this study to be present in endothelium-denuded bovine pulmonary arteries and subject to regulation by modulation of cytosolic NAD(H) redox through the lactate dehydrogenase reaction. Although our previous work identified an acute PO2-dependent peroxide-mediated relaxation of bovine pulmonary arteries on exposure to lactate, a 30-min treatment with 10 mM lactate enhanced ERK phosphorylation and increased force generation to 30 mM KCl. Hypoxia inhibited these responses to lactate. Increases in ERK phosphorylation and the enhancement of force generation by lactate and stretch are attenuated in the presence of inhibitors of Nox oxidase (0.1 mM apocynin) or ERK activation (10 microM PD-98059) and by 0.1 mM ebselen. Additionally, incubation of pulmonary arteries with 10 mM pyruvate lowered basal levels of ERK phosphorylation, and it inhibited both the ERK phosphorylation and the enhancement in force generation to 30 mM KCl caused by stretch. Treatment of pulmonary arteries with the thiol oxidant diamide (1 microM) elicited what appears to be a peroxide-independent increase in force and ERK phosphorylation that were both attenuated by PD-98059. Thus pulmonary arteries possess a peroxide-elicited contractile mechanism involving ERK MAP kinase, which is stimulated by stretch and regulated through the control of Nox oxidase activity by the availability of cytosolic NADH.     

Aminooxyacetic acid inhibits the malate-aspartate shuttle in isolated nerve terminals and prevents the mitochondria from utilizing glycolytic substrates

Aminooxyacetate, an inhibitor of pyridoxal-dependent enzymes, is routinely used to inhibit gamma-aminobutyrate metabolism. The bioenergetic effects of the inhibitor on guinea-pig cerebral cortical synaptosomes are investigated. It prevents the reoxidation of cytosolic NADH by the mitochondria by inhibiting the malate-aspartate shuttle, causing a 26 mV negative shift in the cytosolic NAD+/NADH redox potential, an increase in the lactate/pyruvate ratio and an inhibition of the ability of the mitochondria to utilize glycolytic pyruvate. The 3-hydroxybutyrate/acetoacetate ratio decreased significantly, indicating oxidation of the mitochondrial NAD+/NADH couple. The results are consistent with a predominant role of the malate-aspartate shuttle in the reoxidation of cytosolic NADH in isolated nerve terminals. Aminooxyacetate limits respiratory capacity and lowers mitochondrial membrane potential and synaptosomal ATP/ADP ratios to an extent similar to glucose deprivation. Thus, the inhibitor induces a functional 'hypoglycaemia' in nerve terminals and should be used with caution.     

Imaging cytosolic NADH-NAD(+) redox state with a genetically encoded fluorescent biosensor

NADH is a key metabolic cofactor whose sensitive and specific detection in the cytosol of live cells has been difficult. We constructed a fluorescent biosensor of the cytosolic NADH-NAD(+) redox state by combining a circularly permuted GFP T-Sapphire with a bacterial NADH-binding protein, Rex. Although the initial construct reported [NADH] × [H(+)] / [NAD(+)], its pH sensitivity was eliminated by mutagenesis. The engineered biosensor Peredox reports cytosolic NADH:NAD(+) ratios and can be calibrated with exogenous lactate and pyruvate. We demonstrated its utility in several cultured and primary cell types. We found that glycolysis opposed the lactate dehydrogenase equilibrium to produce a reduced cytosolic NADH-NAD(+) redox state. We also observed different redox states in primary mouse astrocytes and neurons, consistent with hypothesized metabolic differences. Furthermore, using high-content image analysis, we monitored NADH responses to PI3K pathway inhibition in hundreds of live cells. As an NADH reporter, Peredox should enable better understanding of bioenergetics.     

Studies on the formation of lactate and pyruvate from glucose in cultured skin fibroblasts: implications for detection of respiratory chain defects

We investigated the time course of the formation of lactate and pyruvate from glucose in cultured skin fibroblasts from controls, from a patient with a cytochrome c oxidase deficiency and from controls treated with inhibitors of the individual respiratory chain complexes. Fibroblasts from the patient and inhibitor treated fibroblasts produced more lactate and less pyruvate; this resulted in a significant increase in the lactate to pyruvate ratio, reflecting an increased cytosolic NADH/NAD+ redox state. We conclude that measurement of lactate and pyruvate production from glucose in cultured skin fibroblasts can be of value in the diagnosis of inherited diseases of the mitochondrial respiratory chain.     

Determinants of MTT reduction in rat hepatocytes

The determinants of reduction of the dye MTT (3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) in rat hepatocytes have been investigated. NADH, NADPH, and succinate were substrates for MTT reduction in rat liver homogenate, activity being greatest with NADH and least with succinate. Similar results were obtained with submitochondrial particles isolated from rat liver. NAD(P)Hdependent reduction of MTT was also detected in rat liver microsomes and cytosol. Rotenone, at a concentration that inhibited NAD(P)H-dependent MTT reduction in submitochondrial particles, did not inhibit MTT reduction in rat hepatocytes. Malonate, at a concentration that inhibited succinate-dependent MTT reduction in liver homogenate, did not inhibit MTT reduction in rat hepatocytes. Incubation of rat hepatocytes with ethanol or lactate (increase NADH levels), dicoumarol (inhibitor of DT-diaphorase), aminopyrine or hexobarbitone (substrates for the NADPH-requiring cytochrome P450-dependent microsomal monooxygenase) led to significant increases in the level of cellular MTT reduction. From these data, it is concluded that extramitochondrial NAD(P)H is the principal reductant for MTT reduction in rat hepatocytes, with mitochondrial dehydrogenase activity being only a minor contributor. It is also possible that cellular generation of superoxide (as might be expected on redox cycling of endogenous quinones following inhibition of DT diaphorase by dicoumarol) may be another source of MTT reduction. Caution should be exercised in ascribing an alteration in the level of cellular MTT reduction to a change in mitochondrial performance in the absence of corroborating evidence.     

Brain Levels of NADH and NAD+ Under Hypoxic and Hypoglycaemic Conditions In Vitro

The effects of hypoxia and hypoglycaemia on the redox state in vitro have been studied. NADH and NAD+ were extracted simultaneously from superfused cerebral cortex slices and assayed by bioluminescence. The results show a nonsignificant increase in NADH and the redox ratio in "mild hypoxia," whereas "severe hypoxia" produced an increase of over 200% in NADH and in the NADH/NAD+ ratio. When the glucose in the incubation medium was reduced from its control value of 10 mM to 0.5 mM, significant decreases in NADH and the redox ratio to 60% of control value were observed. Further decreasing the glucose to 0.2 mM gave lower levels of NADH and the redox ratio (40% of control). The effects on the redox state of alternative substrates to glucose were also tested. Replacement of glucose by 10 mM pyruvate decreased the NADH by 77% and the NADH/NAD+ ratio by 79%. Replacement of glucose with 10 mM lactate gave decreases of 70% and 71%, respectively, whereas in the presence of 15 mM 2-deoxyglucose and 5 mM glucose, the NADH was decreased by 56% and the ratio by 50%. The results are discussed in relation to levels of creatine phosphate and ATP, as well as evoked action potentials, observed from parallel studies.