NADH Redox Potential Research Articles

Mitochondrial NADH redox potential impacts the reactive oxygen species production of reverse Electron transfer through complex I.

There is substantial evidence that Reactive Oxygen Species (ROS) play a major part in cell functioning. Although their harmfulness through oxidative stress is well documented, their role in signaling and sensing as an oxidative signal still needs to be investigated. In most cells, the mitochondrial Electron Transport Chain (ETC) is the primary source of ROS. The production of ROS by reverse electron transfer through complex I has been demonstrated both in an experimental context but also in many pathophysiological situations. Therefore, understanding the mechanisms that regulate this ROS production is of great interest to control its harmful effects. We used nigericin, Pi and valinomycin as tools to modulate the pH gradient (∆pH) and the membrane potential (∆Ψ) of the protonmotive force (∆p) in liver and muscle mitochondria to accurately determine how these parameters control the ROS production. We show that a high ∆Ψ is the "sine qua none" condition for ROS production from the reverse electron transfer (RET) through the complex I. However, a high ∆Ψ is not the only condition governing ROS production. Indeed, using tools that modulate the mitochondrial NADH level, we also demonstrate that ROS production is directly related to the mitochondrial redox potential when the membrane potential is almost stable.

Imaging Redox State in Mouse Muscles of Different Ages.

Aging is the greatest risk factor for many diseases. Intracellular concentrations of nicotinamide adenine dinucleotide (NAD+) and the NAD+-coupled redox state have been proposed to moderate many aging-related processes, yet the specific mechanisms remain unclear. The concentration of NAD+ falls with age in skeletal muscle, yet there is no consensus on whether aging will increase or decrease the redox potentialof NAD+/NADH. Oxidized flavin groups (Fp) (e.g. FAD, i.e., flavin adenine dinucleotide, contained in flavoproteins) and NADH are intrinsic fluorescent indicators of oxidation and reduction status of tissue, respectively. The redox ratio, i.e., the ratio of Fp to NADH, may be a surrogate indicator of the NAD+/NADH redox potential. In this study we used the Chance redox scanner (NADH/Fp fluorescence imaging at low temperature) to investigate the effect of aging on the redox state of mitochondria in skeletal muscles. The results showed that there are borderline significant differences in nominal concentrations of Fp and NADH, but not in the redox ratio s when comparing 3.5-month and 13-month old muscles of mice (n = 6). It may be necessary to increase the number of muscle samples and study mice of more advanced age.

In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences

NAD is an essential metabolite that exists in NAD(+) or NADH form in all living cells. Despite its critical roles in regulating mitochondrial energy production through the NAD(+)/NADH redox state and modulating cellular signaling processes through the activity of the NAD(+)-dependent enzymes, the method for quantifying intracellular NAD contents and redox state is limited to a few in vitro or ex vivo assays, which are not suitable for studying a living brain or organ. Here, we present a magnetic resonance (MR) -based in vivo NAD assay that uses the high-field MR scanner and is capable of noninvasively assessing NAD(+) and NADH contents and the NAD(+)/NADH redox state in intact human brain. The results of this study provide the first insight, to our knowledge, into the cellular NAD concentrations and redox state in the brains of healthy volunteers. Furthermore, an age-dependent increase of intracellular NADH and age-dependent reductions in NAD(+), total NAD contents, and NAD(+)/NADH redox potential of the healthy human brain were revealed in this study. The overall findings not only provide direct evidence of declined mitochondrial functions and altered NAD homeostasis that accompany the normal aging process but also, elucidate the merits and potentials of this new NAD assay for noninvasively studying the intracellular NAD metabolism and redox state in normal and diseased human brain or other organs in situ.

β-Lapachone alleviates alcoholic fatty liver disease in rats

Alcohol-induced liver injury is the most common liver disease in which fatty acid metabolism is altered. It is thought that altered NAD+/NADH redox potential by alcohol in the liver causes fatty liver by inhibiting fatty acid oxidation and the activity of tricarboxylic acid cycle reactions. β-Lapachone (βL), a naturally occurring quinone, has been shown to stimulate fatty acid oxidation in an obese mouse model by activating adenosine monophosphate-activated protein kinase (AMPK). In this report, we clearly show that βL reduced alcohol-induced hepatic steatosis and induced fatty acid oxidizing capacity in ethanol-fed rats. βL treatment markedly decreased hepatic lipids while serum levels of lipids and lipoproteins were increased in rats fed ethanol-containing liquid diets with βL administration. Furthermore, inhibition of lipolysis, enhancement of lipid mobilization to mitochondria and upregulation of mitochondrial β-oxidation activity in the soleus muscle were observed in ethanol/βL-treated animals compared to the ethanol-fed rats. In addition, the activity of alcohol dehydrogenase, but not aldehyde dehydrogenase, was significantly increased in rats fed βL diets. βL-mediated modulation of NAD+/NADH ratio led to the activation of AMPK signaling in these animals. Conclusion: Our results suggest that improvement of fatty liver by βL administration is mediated by the upregulation of apoB100 synthesis and lipid mobilization from the liver as well as the direct involvement of βL on NAD+/NADH ratio changes, resulting in the activation of AMPK signaling and PPARα-mediated β-oxidation. Therefore, βL-mediated alteration of NAD+/NADH redox potential may be of potential therapeutic benefit in the clinical setting.

Malate-Aspartate Shuttle, Cytoplasmic NADH Redox Potential, and Energetics in Vascular Smooth Muscle

The effect of inhibition of the malate-aspartate shuttle on the cytoplasmic NADH/NAD ratio and NADH redox state and its corresponding effects on mitochondrial energetics in vascular smooth muscle were examined. Incubation of porcine carotid arteries with 0.4 mmol amino-oxyacetic acid an inhibitor of glutamate-oxaloacetate transaminase and, hence the malate-aspartate shuttle, inhibited O2consumption by 21%, decreased the content of phosphocreatine and inhibited activity of the tricarboxylic acid cycle. The rate of glycolysis and lactate production was increased but glucose oxidation was inhibited. These effects of amino-oxyacetic acid were accompanied by evidence of inhibition of the malate-aspartate shuttle and elevation in the cytoplasmic redox potential and NADH/NAD ratio as indicated by elevation of the concentration ratios of the lactate/pyruvate and glycerol-3-phosphate/dihydroxyacetone phosphate metabolite redox couples. Addition of the fatty acid octanoate normalized the adverse energetic effects of malate-aspartate shuttle inhibition. It is concluded that the malate-aspartate shuttle is a primary mode of clearance of NADH reducing equivalents from the cytoplasm in vascular smooth muscle. Glucose oxidation and lactate production are influenced by the activity of the shuttle. The results support the hypothesis that an increased cytoplasmic NADH redox potential impairs mitochondrial energy metabolism.

Ca2+ dependence of gluconeogenesis stimulation by glucagon at different cytosolic NAD(+)-NADH redox potentials.

The influence of Ca2+ on hepatic gluconeogenesis was measured in the isolated perfused rat liver at different cytosolic NAD(+)-NADH potentials. Lactate and pyruvate were the gluconeogenic substrates and the cytosolic NAD(+)-NADH potentials were changed by varying the lactate to pyruvate ratios from 0.01 to 100. The following results were obtained: a) gluconeogenesis from lactate plus pyruvate was not affected by Ca(2+)-free perfusion (no Ca2+ in the perfusion fluid combined with previous depletion of the intracellular pools); gluconeogenesis was also poorly dependent on the lactate to pyruvate ratios in the range of 0.1 to 100; only for a ratio equal to 0.01 was a significantly smaller gluconeogenic activity observed in comparison to the other ratios. b) In the presence of Ca2+, the increase in oxygen uptake caused by the infusion of lactate plus pyruvate at a ratio equal to 10 was the most pronounced one; in Ca(2+)-free perfusion the increase in oxygen uptake caused by lactate plus pyruvate infusion tended to be higher for all lactate to pyruvate ratios; the most pronounced difference was observed for lactate/pyruvate ratio equal to 1. c) In the presence of Ca2+ the effects of glucagon on gluconeogenesis showed a positive correlation with the lactate to pyruvate ratios; for a ratio equal to 0.01 no stimulation occurred, but in the 0.1 to 100 range stimulation increased progressively, producing a clear parabolic dependence between the effects of glucagon and the lactate to pyruvate ratio. d) In the absence of Ca2+ the relationship between the changes caused by glucagon in gluconeogenesis and the lactate to pyruvate ratio was substantially changed; the dependence curve was no longer parabolic but sigmoidal in shape with a plateau beginning at a lactate/pyruvate ratio equal to 1; there was inhibition at the lactate to pyruvate ratios of 0.01 and 0.1 and a constant stimulation starting with a ratio equal to 1; for the lactate to pyruvate ratios of 10 and 100, stimulation caused by glucagon was much smaller than that found when Ca2+ was present. e) The effects of glucagon on oxygen uptake in the presence of Ca2+ showed a parabolic relationship with the lactate to pyruvate ratios which was closely similar to that found in the case of gluconeogenesis; the only difference was that inhibition rather than stimulation of oxygen uptake was observed for a lactate to pyruvate ratio equal to 0.01; progressive stimulation was observed in the 0.1 to 100 range. f) In the absence of Ca2+ the effects of glucagon on oxygen uptake were different; the dependence curve was sigmoidal at the onset, with a well-defined maximum at a lactate to pyruvate ratio equal to 1; this maximum was followed by a steady decline at higher ratios; at the ratios of 0.01 and 0.1 inhibition took place; oxygen uptake stimulation caused by glucagon was generally lower in the absence of Ca2+ except when the lactate to pyruvate ratio was equal to 1. The results of the present study demonstrate that stimulation of gluconeogenesis by glucagon depends on Ca2+. However, Ca2+ is only effective in helping gluconeogenesis stimulation by glucagon at highly negative redox potentials of the cytosolic NAD(+)-NADH system. The triple interdependence of glucagon-Ca(2+)-NAD(+)-NADH redox potential reveals highly complex interrelations that can only be partially understood at the present stage of knowledge.

The Role of Mitochondria in Ischemic Heart Disease

Summary Mitochondria in the heart play two roles essential for cell survival: ATP synthesis and maintenance of Ca2+ homeostasis. These two processes are driven by the same energy source, the H+ electrochemical gradient (ΔμH). Under aerobic physiologic conditions, mitochondria do not contribute to the beat-to-beat regulation of cytosolic Ca2+, although a Ca2+ transient in mitochondrial matrix has been described. Micromolar increases in mitochondrial Ca2+ concentration stimulate the Krebs cycle and the NADH redox potential and, therefore, ATP synthesis. Trimetazidine has been shown to improve the calcium transient and, in so doing, the overall myocardial energy production. Under pathologic conditions, mitochondrial Ca2+ overload causes a series of vicious cycles that lead to irreversible cell damage. During ischemia, an alteration in intracellular Ca2+ homeostasis occurs and mitochondria are able to buffer cytosolic Ca2+, suggesting that they retain the Ca2+-transporting capacity. Accordingly, once isolated, even after prolonged ischemia the majority of the mitochondria are able to use oxygen for ATP phosphorylation. When isolated after reperfusion, mitochondria are structurally altered, contain large quantities of Ca2+, and produce an excess of oxygen free radicals. Their membrane pores are stimulated and the capacity for oxidative phosphorylation is irreversibly disrupted. The role of mitochondrial DNA damage in progressive human diseases such as coronary atherosclerosis is receiving growing interest. The sequence of ischemia and reperfusion, through increased production of oxygen free radicals, causes mitochondrial deletions in several areas of the mitochondrial genome. This cumulative mitochondrial DNA damage is associated with induction of nuclear oxidative phosphorylation gene mRNA. These observations support the hypothesis that mitochondria and mitochondrial DNA damage play important roles in ischemic heart disease.

The role of mitochondria in ischemic heart disease.

Mitochondria in the heart play two roles essential for cell survival: ATP synthesis and maintenance of Ca2+ homeostasis. These two processes are driven by the same energy source, the H+ electrochemical gradient (delta microH). Under aerobic physiologic conditions, mitochondria do not contribute to the beat-to-beat regulation of cytosolic Ca2+, although a Ca2+ transient in mitochondrial matrix has been described. Micromolar increases in mitochondrial Ca2+ concentration stimulate the Krebs cycle and the NADH redox potential and, therefore, ATP synthesis. Trimetazidine has been shown to improve the calcium transient and, in so doing, the overall myocardial energy production. Under pathologic conditions, mitochondrial Ca2+ overload causes a series of vicious cycles that lead to irreversible cell damage. During ischemia, an alteration in intracellular Ca2+ homeostasis occurs and mitochondria are able to buffer cytosolic Ca2+, suggesting that they retain the Ca(2+)-transporting capacity. Accordingly, once isolated, even after prolonged ischemia the majority of the mitochondria are able to use oxygen for ATP phosphorylation. When isolated after reperfusion, mitochondria are structurally altered, contain large quantities of Ca2+, and produce an excess of oxygen free radicals. Their membrane pores are stimulated and the capacity for oxidative phosphorylation is irreversibly disrupted. The role of mitochondrial DNA damage in progressive human diseases such as coronary atherosclerosis is receiving growing interest. The sequence of ischemia and reperfusion, through increased production of oxygen free radicals, causes mitochondrial deletions in several areas of the mitochondrial genome. This cumulative mitochondrial DNA damage is associated with induction of nuclear oxidative phosphorylation gene mRNA. These observations support the hypothesis that mitochondria and mitochondrial DNA damage play important roles in ischemic heart disease.

Mitochondrial energy production and cation control in myocardial ischaemia and reperfusion.

In the heart mitochondria exert two roles essential for cell survival: ATP synthesis and maintainance of Ca2+ homeostasis. These two processes are driven by the same energy source: the H+ electrochemical gradient (delta microH) which is generated by electron transport along the inner mitochondrial membrane. Under aerobic physiological condition mitochondria do not contribute to the beat to beat regulation of cytosolic Ca2+, although Ca2+ transient in mitochondrial matrix has been described. Increases in mitochondrial Ca2+ of mumolars concentration stimulate the Krebs cycle and NADH redox potential and, therefore, ATP synthesis. Under pathological conditions, however, mitochondrial Ca2+ transport and overload might cause a series of vicious cycles leading to irreversible cell damage. Mitochondrial Ca2+ accumulation causes profound alterations in permeability of the inner membrane to solutes, leading to severe mitochondrial swelling. In addition Ca2+ transport takes precedence over ATP synthesis and inhibits utilization of delta microH for energy production. These processes are important to understand the sequence of the molecular events occurring during myocardial reperfusion after prolonged ischaemia which lead to irreversible cell damage. During ischaemia an alteration of intracellular Ca2+ homeostasis occurs and mitochondria are able to buffer cytosolic Ca2+, suggesting that they retain the Ca2+ transporting capacity. Accordingly, once isolated, even after prolonged ischaemia, the majority of the mitochondria is able to use oxygen for ATP phosphorylation. When isolated after reperfusion, mitochondria are structurally altered, contain large quantities of Ca2+, produce excess of oxygen free radicals, their membrane pores are stimulated and the oxidative phosphorylation capacity is irreversibly disrupted. Most likely, reperfusion provides oxygen to reactivate mitochondrial respiration but also causes large influx of Ca2+ in the cytosol as result of sarcolemmal damage. Mitochondrial Ca2+ transport is therefore stimulated at maximal rates and, as consequence, the equilibrium between ATP synthesis and Ca2+ influx is shifted towards Ca2+ influx with loss of the ability of ATP synthesis.

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 γ-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 malateaspartate 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.

EFFECT OF INOSINE ON ADRENALINE AND VASOPRESSIN INDUCED MYOCARDIAL HYPOXIA

The authors have shown that inosine is the main component responsible for the antagonizing effect of the whale heart extract on vasopressin-induced ST depression in the anesthetized rats (1). Huckabee introduced the concept of “excess lactate” in order to distinguish between pyruvate induced changes in lactate and those related solely to a shift in NAD: NADH redox potential (2, 3). So that, the increase of “excess lactate” in heart, with the measurement of lactate and pyruvate concentrations in the coronary vein and artery, has been considered to represent anaerobic metabolism due to cellular hypoxia.The present paper reports the effect of inosine on the ischemic changes in myocardial lactate and pyruvate metabolism (increases in “excess lactate”) induced with vasopressin and adrenaline, and the effect of inosine on the ischemic electrocardiographic change induced with adrenaline in rats.

Effect of inosine on adrenaline and vasopressin induced myocardial hypoxia.

Abstract The authors have shown that inosine is the main component responsible for the antagonizing effect of the whale heart extract on vasopressin-induced ST depression in the anesthetized rats (1). Huckabee introduced the concept of “excess lactate” in order to distinguish between pyruvate induced changes in lactate and those related solely to a shift in NAD: NADH redox potential (2, 3). So that, the increase of “excess lactate” in heart, with the measurement of lactate and pyruvate concentrations in the coronary vein and artery, has been considered to represent anaerobic metabolism due to cellular hypoxia. The present paper reports the effect of inosine on the ischemic changes in myocardial lactate and pyruvate metabolism (increases in “excess lactate”) induced with vasopressin and adrenaline, and the effect of inosine on the ischemic electrocardiographic change induced with adrenaline in rats.

Beta adrenergic blockade: Hemodynamics and myocardial energy metabolism in patients with ischemic heart disease

Systemic and coronary hemodynamics and myocardial intermediary metabolism were studied before and after beta adrenergic blockade with propranolol in 9 patients with ischemic heart disease. A significant reduction was produced in heart rate, left ventricular isometric tension development (dp/dt) and pressure-time per minute, whereas left ventricular end-diastolic pressure rose. Cardiac output and left ventricular mean systolic pressure and mechanical work did not change consistently. Myocardial oxygen consumption was influenced by left ventricular external work and pressure-time per min., the latter having the predominant effect. Myocardial oxygen consumption was not closely related to the consistent reduction of dp/dt, presumably because of wide variations in stroke volume. Coronary blood flow was reduced in 5 of 8 patients, changes in coronary diastolic vascular resistance in most cases favoring the preservation of an autoregulatory mechanism. A constant or reduced coronary arteriovenous oxygen extraction in 6 of 9 patients suggested that changes in perfusion rather than in extraction accommodated the altered myocardial oxygen needs. Propranolol appeared to have no direct action on vasomotor tone but probably reduced extravascular compression resistance. Myocardial efficiency did not change consistently. External efficiency was generally reduced, but internal efficiency rose in 3 patients. When a reduction in efficiency indexes resulted from decreased cardiac work but increased coronary flow and myocardial oxygen consumption, the overstressed myocardium may have benefited. Total myocardial substrate oxygen extraction ratios exceeded 100 per cent under control conditions and after beta blockade; therefore the respective energy contributions of carbohydrates and fatty acids were obscure, and conversion and storage seemed likely. The NAD +/NADH redox potential gradient was consistently changed to a more positive value. Arterial levels of inorganic phosphate and creatine phosphokinase activity rose, and the myocardial balance of creatine phosphokinase became positive after propranolol. The antianginal action of propranolol may be related to the following mechanisms: (1) myocardial work is reduced, thus lessening the disparity between oxygen demand and supply; (2) a relative or absolute improvement in coronary perfusion and myocardial oxygen delivery may result, subject to arteriolosclerotic limitations; (3) changes in myocardial intermediary metabolism and enzyme systems favor lessened cellular hypoxia and enhanced aerobiosis.