Activation of the phagocytic NADPH oxidase-2 (NOX-2) in neutrophils is a critical process in the innate immune system and is associated with elevated local concentrations of superoxide, hydrogen peroxide (H 2 O 2 ) and hypochlorous acid. Under pathological conditions, NOX-2 activity has been implicated in the development of autoimmunity, indicating a role in modulating lymphocyte effector function. Notably, T-cell clonal expansion and subsequent cytokine production requires a metabolic switch from mitochondrial respiration to aerobic glycolysis. Previous studies demonstrate that H 2 O 2 generated from activated neutrophils suppresses lymphocyte activation but the mechanism is unknown. We hypothesized that activated neutrophils would prevent the metabolic switch and suppress the effector functions of T-cells through a H 2 O 2 -dependent mechanism. To test this, we developed a model co-culture system using freshly isolated neutrophils and lymphocytes from healthy human donors. Extracellular flux analysis was used to assess mitochondrial and glycolytic activity and FACS analysis to assess immune function. The neutrophil oxidative burst significantly inhibited the induction of lymphocyte aerobic glycolysis, caused inhibition of oxidative phosphorylation and suppressed lymphocyte activation through a H 2 O 2 -dependent mechanism. Hydrogen peroxide and a redox cycling agent, DMNQ, were used to confirm the impact of H 2 O 2 on lymphocyte bioenergetics. In summary, we have shown that the lymphocyte metabolic switch from mitochondrial respiration to glycolysis is prevented by the oxidative burst of neutrophils. This direct inhibition of the metabolic switch is then a likely mechanism underlying the neutrophil-dependent suppression of T-cell effector function.

The non-steroidal anti-inflammatory drugs (NSAIDs) are a widely used group of drugs in clinical medicine. However, their propensity to cause gastrointestinal damage limits their clinical utility. The pathogenesis of this toxicity is not well established. It has been postulated that an early event in the development of damage is an effect of these drugs on mitochondrial function. The present paper sets out to evaluate the effects of indomethacin, a commonly used NSAID, on energy metabolism in vivo . Indomethacin was administered to male Sprague-Dawley rats, either intrajejunally or orally, and indices of mitochondrial function were determined. The parameters chosen for this purpose were oxygen uptake by, lactate levels in and the energy charge of jejunal tissue. Oxygen uptake by and energy charge in jejunal tissue were unaffected at 1 and 3h after dosing by gavage with indomethacin. The drug significantly affected the tissue lactate/pyruvate ratio at 3h (but not at 1h) after oral dosing. Effects of indomethacin on jejunum incubated ex vivo were found to be reversible. The data suggest that indomethacin affects mitochondrial function in vivo , but that compensatory changes in glycolytic rate maintain energy charge.

The increase in the intracellular Na + concentration ([Na + ] i ) during myocardial ischaemia is crucial for ischaemia/reperfusion cell injury, and the cardiac subtype of the Na + /H + exchanger (NHE-1) has been shown to be a major pathway for Na + loading. While the importance of glycolytically derived ATP for the optimal functioning of membrane transporters and channels has been suggested, whether NHE-1 is actually activated during myocardial ischaemia remains controversial. Here we examined whether the activity of NHE-1 is predominantly dependent on intracellular ATP generated by glycolysis, and whether the additional inhibition of glycolysis can affect the increase in [Na + ] i during the inhibition of oxidative phosphorylation in intact guinea pig ventricular myocytes. The selective inhibition of glycolysis by 2-deoxyglucose prevented the recovery of intracellular pH and the transient increase in [Na + ] i following intracellular acidosis induced by a NH 4 Cl pre-pulse. During severe metabolic inhibition (SMI; induced by amobarbital and carbonyl cyanide m-chlorophenylhydrazone in a glucose-free perfusate), most myocytes changed from rod-shaped to contracted forms by ~ 15 min. [Na + ] i increased linearly until rigor contracture occurred, but after rigor contracture the rate of increase was blunted. The increase in [Na + ] i during SMI was suppressed significantly by an inhibitor of NHE-1, hexamethylene amiloride. The increase in the intracellular Mg 2+ concentration, which can reciprocally indicate depletion of intracellular ATP, was small during the initial 10 min of SMI, but became larger from just a few minutes before rigor contracture. In the presence of 2-deoxyglucose, the time to rigor during SMI was shortened, but the increase in [Na + ] i before rigor contracture was not significant, and was much less than that in the absence of 2-deoxyglucose. It is concluded that ATP generated by glycolysis is essential to activate NHE-1, and that the dependence of NHE-1 on glycolysis might affect the increase in [Na + ] i observed during myocardial ischaemia.

1. During metabolic acidosis, significant fluxes of inorganic phosphate (P i ) may occur from cellular to extracellular fluid. In this study P i was measured in erythrocytes of uraemic patients before and after haemodialysis and was related to their plasma pH (acidosis), plasma P i (hyperphosphataemia) and cellular organic phosphate concentrations. 2. Before dialysis, the ratio of cellular to extracellular P i concentration correlated inversely with plasma pH, increasing 2.5-fold as pH fell from 7.4 to 7.2. 3. An increase in cellular P i similar to that seen in the patients was observed within 90 min of adding acid to normal erythrocytes in vitro . 4. The total P i content of the cell suspension increased 25% on decreasing plasma pH from 7.4 to 7.2, largely as a result of generation of P i from 2,3-bisphosphoglycerate in the cells. This was accompanied by net efflux of P i into plasma. 5. In addition, the increase in the steady-state cellular P i concentration on adding a constant extracellular P i load was 50% greater at pH 7.2 than at 7.4, implying that alterations in the regulation of the transmembrane P i gradient also contribute to the rise in cellular P i observed at low pH. 6. At normal plasma P i concentration (1 mM), glycolytic flux (lactate production) was inhibited by 20% when pH was lowered from 7.4 to 7.2. However, this inhibition was blocked when cellular P i was increased by adding P i to the plasma in vitro . 7. Metabolic acidosis is therefore a potent stimulus for P i generation in erythrocytes, and this P i may serve to stimulate glycolysis which is normally inhibited by low pH.

1. In sepsis various processes of carbohydrate metabolism, such as hepatic gluconeogenesis and glycolysis, are altered. Phosphofructokinase-1, a key glycolytic enzyme, is controlled in the long term via regulation of synthesis and degradation of the protein itself, while in the short term it is regulated by allosteric effectors, such as fructose 2,6-bisphosphate (the most potent). In the present study hepatic phosphofructokinase-1 activity as well as phosphofructokinase-2 activity and the concentration of fructose 2,6-bisphosphate were assayed to determine if they might contribute to the derangement of carbohydrate metabolism seen commonly in sepsis. 2. The levels of glycogen and fructose 2,6-bisphosphate and the activity of phosphofructokinase-1 and phosphofructokinase-2 were determined in hepatic biopsies obtained at laparotomy from six patients with and seven patients without abdominal septic foci. 3. A significant increase in plasma lactate concentration was observed in the septic patients, whereas no significant differences in tissue glycogen content or plasma glucose concentration were seen between the groups. 4. No significant change in plasma insulin concentration was observed. However, levels of the counter-regulatory hormones (glucagon, cortisol and adrenaline) were elevated in the septic patients. 5. A 60% decrease in hepatic phosphofructokinase-1 activity was seen in the septic patients. However, no significant changes in hepatic phosphofructokinase-2 activity and fructose 2,6-bisphosphate content were observed in the septic patients. 6. The present results demonstrate that the decrease in hepatic phosphofructokinase-1 activity occurring in sepsis does not appear to reflect alterations in the concentration of fructose 2,6-bisphosphate.

1. In order to explain the vulnerability of medullary thick ascending limb of Heme's loop (mTAL) during hypoxia, adenosine 5′-triphosphate (ATP) content was measured in isolated rat mTAL cells during control conditions and chemically induced hypoxia and compared with those in medullary collecting duct (MCD) cells. 2. Basal ATP levels in mTAL and MCD were estimated as 3.6 and 2.1 mmol/l, respectively. Antimycin A (5 μmol/l) decreased the ATP content by 41% of the control value in the mTAL cells, but failed to reduce that of the MCD cells. Administration of sodium cyanide (5 mmol/l) drastically depleted ATP in the mTAL cells within 5 min (2–3% of control). On the other hand, ATP levels in MCD cells were sustained for at least 60 min after cyanide administration (64% of control). 3. When tubules were made permeable to sodium by the addition of nystatin, the effects of chemical hypoxia on the cell ATP levels were intensified in both segments, and this was partially blocked by pretreatment with ouabain, or by lowering the sodium concentration of the medium. 4. Higher doses of nystatin in mTAL caused a reduction in ATP levels even under control conditions, but its effect was prevented in low sodium medium. 5. The present study suggests that cell ATP levels can be altered by sodium, potassium-dependent adenosine triphosphatase activity, and that due to their high sodium-transporting activity, mTAL cells are more sensitive to reductions in ATP levels during hypoxia than are MCD cells.

1. The capacity for glycolysis in muscle biopsies obtained from long-term heavy alcohol drinking patients has been compared with tissue from control subjects by assay in vitro of the total activities of glycogen phosphorylase, phosphofructokinase and fructose 1,6-bisphosphatase, key regulatory enzymes in the anaerobic glycolytic pathway. 2. Biopsies from 13 of 22 patients had type II fibre atrophy, and the activities of all three enzymes were reduced in these biopsies, when expressed in terms of DNA content, the most striking reduction being in phosphofructokinase activity. 3. The amount of glycogen in the tissue correlated closely with these enzyme activities and was slightly lower in the most atrophic tissue, when expressed in terms of DNA content. 4. The activities of acid and neutral α-glucosidases were similar in biopsies from control subjects and patients with various severities of alcohol myopathy. 5. The reduced activities are consistent with a reduced proportion of type II fibre muscle mass in these patients, and suggest that there may be a reduced capacity for glycolysis with resultant reduced lactate production. Whether the changes in enzyme activities are primary to the selective atrophy remains to be established.

1. Some effects of sodium salicylate upon anaerobic glycolysis have been studied in normal human erythrocytes incubated for up to 6 h at 37°C in autologous sera. 2. Both glucose consumption and lactate production were stimulated by concentrations of salicylate up to 60 mmol/l but at the highest concentration used (90 mmol/l) an initial stimulus was followed by inhibition of glycolysis. 3. Losses occurred of adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP) and adenosine 5′-phosphate (AMP) at higher concentrations of salicylate and there was a concomitant increase of inorganic phosphate. 4. Other phosphate esters underwent concentration changes at higher concentrations of salicylate that reflected inadequate concentrations of ATP for glycolysis. 5. The rates of sodium efflux from, and potassium influx into, erythrocytes were unaffected by the presence of salicylate at concentrations sufficient to stimulate glycolysis.