Control of aerobic glycolysis in the brain in vitro

Protoveratrine-(5 M) stimulated aerobic glycolysis of incubated rat brain cortex slices that accompanies the enhanced neuronal influx of Na⁺ is blocked by tetrodotoxin (3 M) and the local anesthetics, cocaine (0.1 mM) and lidocaine (0.5 mM). On the other hand, high [K⁺]-stimulated aerobic glycolysis that accompanies the acetylcholine-sensitive enhanced glial uptakes of Na⁺ and water is unaffected by acetylcholine (2 mM). Experiments done under a variety of metabolic conditions show that there exists a better correlation between diminished ATP content of the tissue and enhanced aerobic glycolysis than between tissue ATP and the ATP-dependent synthesis of glutamine. Whereas malonate (2 mM) and amino oxyacetate (5 mM) suppress ATP content and O₂ uptake, stimulate lactate formation, but have little effect on glutamine levels, fluoroacetate (3 mM) suppresses glutamine synthesis in glia, presumably by suppressing the operation of the citric acid cycle, with little effect on ATP content, O₂ uptake, and lactate formation. Exogenous citrate (5 mM), which may be transported and metabolized in glia but not in neurons, inhibits lactate formation by cell free acetone-dried powder extracts of brain cortex but not by brain cortex slices. These results suggest that the neuron is the major site of stimulated aerobic glycolysis in the brain, and that under our experimental conditions glycolysis in glia is under lesser stringent metabolic control than that in the neuron. Stimulation of aerobic glycolysis by protoveratrine occurs due to diminution of the energy charge of the neuron as a result of stimulation of the sodium pump following tetrodotoxin-sensitive influx of Na⁺; stimulation by high [K⁺, NH₄ ⁺, or Ca²⁺ deprivation occurs partly by direct stimulation of key enzymes of glycolysis and partly by a fall in the tissue ATP concentration.     

Acidosis,glycolysis and energy state in anaerobic heart tissue from rainbow trout

With focus on metabolism not depending on contractility in myocardial tissue from rainbow trout, Oncorhynchus mykiss, the effects of high CO₂ on lactate production, phosphocreatine, creatine, ATP, ADP, AMP and intracellular pH were examined under a blockage of cell respiration either alone or in combination with a glycolytic inhibition. Irrespective of metabolic interventions, a change in CO₂ from 1 to either 11 or 5% of the gas mixture perfusing the muscle bath with 15 mmol·l^-1 HCO _- ³ caused a drop of intracellular pH from 7.4 to either 6.5 or 7.0, respectively. An elevation of CO₂ to 11% diminished the rate of anaerobic lactate formation and slightly lowered anaerobic energy degradation. The further addition of 1 mmol·l^-1 iodoacetate to inhibit glycolysis strongly enhanced the tendency of acidosis to lower energy degradation. Moreover, iodoacetate induced a parallel decrease in ATP and total concentration of phosphorylated adenylates and an increase in resting tension. These effects were all substantially dampened by acidosis and could not immediately be related to tissue content of energy-rich phosphates. Tentatively, the depression of resting tension was the prime effect and a cause of the other effects acidosis. However, these were not affected by an inhibition of resting tension development with 2,3-butadione monoxime. The results suggest that glycolysis protects the anaerobic myocardium also by means not immediately related to tissue energy state. Acidosis exerts a similar protection, which is marginal as long as glycolysis is fully active, but substantial with an inhibited glycolysis.Abbreviations Cr _t total tissue concentration of creatine - G _PCr energy liberated per mol PCr hydrolyzed - IAA iodoacetate - PCr phosphocreatine - PE total tissue concentration of energy-rich phosphate bonds - pH _i intracellular pH - P _i inorganic phosphate - TAN total tissue concentration of phosphorylated adenylates - 2,3-BDM 2,3-butadione monoxime - SE standard error of the mean     

ATP,creatine phosphate,and mechanical activity in rainbow trout myocardium under inhibition of glycolysis and cell respiration

Summary Twitch force and resting tension of electrically stimulated ventricular strips of rainbow trout were compared with tissue contents of phosphocreatine, creatine, and ATP. The phosphocreatine/total creatine ratio, which was used to assess the cytoplasmic phosphorylation potential, fell with the fraction of cell respiration that was inhibited by sodium cyanide and N₂. Concomitantly, twitch force decreased while resting tension tended to increase. This relation between phosphocreatine/total creatine and mechanical parameters became more prominent as glycolysis was increasingly inhibited by sodium iodoacetate. Furthermore, glycolytic inhibition was followed by a decrease in the ATP/phosphocreatine ratio. The latter effect was the same in 1% and 6% CO₂. Thus, it cannot be ascribed to an action of intracellular pH on the creatine kinase catalyzed reaction. Notably, resting tension as well as twitch force relative to ATP was augmented by glycolytic inhibition. The main conclusions are that in the presence of a decreased mitochondrial activity, glycolysis protects contractility not only by counteracting a lowering in high energy phosphates but also by supporting the ATP/phosphocreatine ratio. Apparently, the creatine kinase activity is insufficient to maintain ATP in equilibrium with phosphocreatine. In addition, glycolysis seems to elevate the level of free phosphate relative to ATP, so that twitch force development as well as rigor complex formation is counteracted.     

EFFECTS OF OUABAIN ON ACID-SOLUBLE PHOSPHATES AND ELECTROLYTES OF ISOLATED CEREBRAL TISSUES IN PRESENCE OR ABSENCE OF CALCIUM

—(1) Cerebral slices were incubated in Ca²⁺-free media or in media which contained 2.8 mm -Ca²⁺. Omission of Ca²⁺ brought about a drop in creatine phosphate content of 28 per cent, as well as a drop of 3–10 per cent in non-inulin K⁺ content. There was little change in content of 10-min phosphate or of non-inulin Na⁺. (2) Ouabain in concentrations of to M increased the loss of K⁺ from the slice and caused a rise in Na⁺ content. The changes were most marked in Ca²⁺-free media. Creatine phosphate levels were depressed by ouabain both in the presence or absence of Ca²⁺. In the absence of Ca²⁺, the lowering of phosphocreatine did not occur until significant shifts in K⁺ had taken place. In contrast, slices incubated in Ca²⁺-containing media lost creatine phosphate and K⁺ at about the same rate. (3) When ouabain and labelled phosphate were added simultaneously, there was little difference in the rate of incorporation of label into creatine phosphate in media which differed in Ca²⁺ concentration. However, incorporation of ^azP-labelled phosphate into creatine phosphate was decreased by 30–40 per cent in media which lacked Ca²⁺ when ouabain was added 15 min prior to the labelled phosphate. This change was not observed when the media contained Ca²⁺. (4) Ouabain did not affect oxidative phosphorylation or respiratory control when added directly to bovine brain mitochondrial preparations. (5) The results suggest that the previously observed depression of respiration brought about by ouabain in Ca²⁺-deficient media is not a good indicator of the proportion of the cell's metabolism used for active cation transport. Under these conditions, the inhibition of cation transport is accompanied by a drop in slice content of high-energy phosphate which may represent a secondary effect of ouabain, or of cytoplasmic alterations brought about by ouabain, on energy-producing processes.     

Metabolic disturbances of synaptosomes isolated from ischemic gerbil brain

The effects of cerebral ischemia, induced for 10 min by bilateral common carotid ligation in the Mongolian gerbil, on the brain and synaptosomal content of phospholipids and free fatty acids were measured. Moreover, the incorporation of arachidonic acid and oleoyl-CoA into phospholipids, as well as the respiration and the accumulation of⁴⁵Ca, norepinephrine, dopamine, choline, glutamate, and -aminobutyrate in the ischemic brain synaptosomal fraction were studied. Analyses of lipids showed a drop in phospholipids content with concomitant increase of lysocompounds and free fatty acids in ischemic cerebral cortex. Disturbances in lipid metabolism including rapid phospholipids hydrolysis and changes in the incorporation of arachidonic acid into inositol and choline phosphoglycerides were also shown in the synaptosomal fraction of ischemic brain. The uptake of neurotransmitter substances, expressed as a percent of control value, was reduced 21% for norepinephrine, 40% for dopamine, 20% for choline, 24% for glutamate and 13% for -aminobutyrate in ischemic synaptosomes. There was no significant effect of ischemia on synaptosomal respiration and⁴⁵Ca uptake in both control and high potassium media. the inhibition of neurotransmitter uptake in ischemic brain synaptosomes may be caused by the disturbance of fatty acid metabolism.     

Effects of ethacrynic acid on ion transport and energy metabolism in slices of avian salt gland and of mammalian liver and kidney cortex

Summary Ethacrynic acid greatly inhibited net transport of ions and aerobic, energyconserving metabolism in slices of avian salt gland, rat liver, and rat and guinea-pig kidney cortex. The effects of increasing concentrations of ethacrynic acid on the transport of Na⁺, K⁺ and Cl^– ran closely parallel to its effects on tissue ATP levels and respiration. The concentration needed for maximal inhibition of transport reduced ATP levels by 80–90%. Respiration was reduced by 80–90% in salt gland and kidney cortex, and by a maximum of 30% in liver slices. The effects of low concentrations of ethacrynic acid required time to become fully manifest in some tissues, and the development of transport inhibition followed a similar course to decline of respiration and ATP levels. Ca²⁺ extrusion by liver cells was inhibited by ethacrynic acid. The concentration dependence of the inhibition was similar to that shown by the other transport systems inhibited. There was no distinction evident between the sensitivity of Na⁺ extrusion and of K⁺ accumulation to the diuretic. Lactate production increased as respiration decreased in the presence of increasing concentrations of ethacrynic acid. We conclude that ethacrynic acid acted primarily as an inhibitor of mitochondrial respiration and ATP synthesis in the tissue slices, and that inhibition of ion transport was a nonspecific consequence of the failure of the energy supply.     

CONTROL OF AEROBIC GLYCOLYSIS IN GUINEA-PIG CEREBRAL CORTEX SLICES

—The effect of glutamate on aerobic glycolysis in guinea-pig cerebral cortex slices was analysed in comparison with that of high-potassium. In contrast to the increased glycolysis in 50 mm -potassium medium which was accompanied by increases of fructose diphosphate and triose phosphates in the slices, the addition of 5 mm -d -glutamate to the medium increased the rate of glycolysis without increasing these intermediates. When increasing the concentration of potassium in the medium up to 20 mm , the rate of aerobic glycolysis was not increased although fructose diphosphate and triose phosphates in the slices were increased. At this potassium concentration in the medium ATP in the slices was highest. At 30 mm -potassium the rate of glycolysis was increased significantly, but fructose diphosphate and triose phosphates were decreased. ATP was lower at 30 mm - than at 20 mm -potassium. By increasing potassium to 40 mm and above, the rate of glycolysis was further increased, and fructose diphosphate and triose phosphates were again increased. Between 5 and 20 mm -potassium in the medium the increasing effect of glutamate on glycolysis was very pronounced. d -Glutamate decreased the amounts of ATP, fructose diphosphate and triose phosphates at any concentration of potassium in the medium. When adding cyclic AMP and 5′AMP to the slices, fructose diphosphate and triose phosphates were increased, but the rate of glycolysis was not increased. On the basis of these observations mechanisms of the control over glycolysis in guinea-pig cerebral cortex slices are discussed. It is suggested that the glycolysis is controlled by the changes in ATP concentration through their action on the glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase system. The changed patterns of the glycolytic intermediate profile in the slices when adding ATP to the medium are consistent with this suggestion. The addition of l -phenylalanine to guinea-pig cerebral cortex slices did not inhibit the rate of glycolysis, although it inhibited the activity of pyruvate kinase.     

Energy transduction in intact synaptosomes. Influence of plasma-membrane depolarization on the respiration and membrane potential of internal mitochondria determined in situ.

A method is described, based on the differential accumulation of Rb+ and methyltriphenylphosphonium, for the simultaneous estimation of the membrane potentials across the plasma membrane of isolated nerve endings (synaptosomes), and across the inner membrane of mitochondria within the synaptosomal cytoplasm. These determinations, together with measurements of respiratory rates, and ATP and phosphocreatine concentrations, are used to define the bioenergetic behaviour of isolated synaptosomes under a variety of conditions. Under control conditions, in the presence of glucose, the plasma and mitochondrial membrane potentials are respectively 45 and 148mV. Addition of a proton translocator induces a 5-fold increase in respiration, and abolishes the mitochondrial membrane potential. The addition of rotenone to inhibit respiration does not affect the plasma membrane potential, and only lowers the mitochondrial membrane potential to 128mV. Evidence is presented that ATP synthesis by anaerobic glycolysis is sufficient under these conditions to maintain ATP-dependent processes, including the reversal of the mitochondrial ATP synthetase. Addition of oligomycin under non-respiring conditions leads to a complete collapse of the mitochondrial potential. Even under control conditions the plasma membrane (Na+ + K+)-dependent ATPase is responsible for a significant proportion of the synaptosomal ATP turnover. Veratridine greatly increases respiration, and depolarizes the plasma membrane, but only slightly lowers the mitochondrial membrane potential. High K+ and ouabain also lower the plasma membrane potential without decreasing the mitochondrial membrane potential. In non-respiring synaptosomes, anaerobic glycolysis is incapable of maintaining cytosolic ATP during the increased turnover induced by veratridine, and the mitochondrial membrane potential collapses. It is concluded that the internal mitochondria must be considered in any study of synaptosomal transport.     

Control of brain slice respiration by (Na+ + K+)-activated adenosine triphosphate and the effects of enzyme inhibitors.

The involvement of membrane (Na+ + K+)-ATPase (Mg2+-dependent, (Na+ + K+)-activated ATP phosphohydrolase, E.C. 3.6.1.3) in the oxygen consumption of rat brain cortical slices was studied in order to determine whether (Na+ + K+)-ATPase activity in intact cells can be estimated from oxygen consumption. The stimulation of brain slice respiration with K+ required the simultaneous presence of Na+. Ouabain, a specific inhibitor of (Na+ + K+)-ATPase, significantly inhibited the (Na+ + K+)-stimulation of respiration. These observations suggest that the (Na+ + K+)-stimulation of brain slice respiration is related to ADP production as a result of (Na+ + K+)-ATPase activity. However, ouabain also inhibited non-K+ -stimulated respiration. Additionally, ouabain markedly reduced the stimulation of respiration by 2,4-dinitrophenol in a high (Na+ + K+)-medium. Thus, ouabain depresses brain slice respiration by reducing the availability of ADP through (Na+ + K+)-ATPase inhibition and acts additionally by increasing the intracellular Na+ concentration. These studies indicate that the use of ouabain results in an over-estimation of the respiration related to (Na+ + K+)-ATPase activity. This fraction of the respiration can be estimated more precisely from the difference between slice respiration in high Na+ and K+ media and that in choline, K+ media. Studies were performed with two (Na+ + K+)-ATPase inhibitors to determine whether administration of these agents to intact rats would produce changes in brain respiration and (Na+ + K+)-ATPase activity. The intraperitoneal injection of digitoxin in rats caused an inhibition of brain (Na+ + K+)-ATPase and related respiration, but chlorpromazine failed to alter either (Na+ + K+)-ATPase activity or related respiration.     

The effects of oligomycin on energy metabolism and cation transport in slices of rat liver Inhibition of oxidative phosphorylation as the primary action

1. In slices of rat liver, oligomycin inhibited the net transport of Na⁺ and K⁺ by a maximum of 30% and endogenous respiration by 25%. These effects were not increased by a number of modifications in the incubation conditions.2. Mitochondria isolated from the slices after incubation showed respiratory control ratios that were somewhat less than in mitochondria from fresh liver, but state 3 respiration retained normal sensitivity to oligomycin.3. Low concentrations of oligomycin or cyanide reduced respiration and ATP levels of the slices but did not affect ion transport unless these levels fell below a definite critical value. In contrast, ouabain and atractyloside each caused substantial degrees of transport inhibition at ATP levels which were in excess of the critical value.4. High concentrations of cyanide and oligomycin reduced ATP contents maximally by 90% and 65%, respectively. Studies of lactate production, and of the effects of arsenite on respiration and ATP levels, suggested that substrate-level phosphorylation in the citric-acid cycle was the major source of the oligomycin-resistant ATP synthesis.5. The results suggest that oligomycin acts in the liver slices primarily as an inhibitor of oxidative phosphorylation, and that this is the cause of the partial inhibition of ion transport. The oligomycin-resistant ion-transporting activity is consistent with the persisting level of ATP synthesis.     

Net movements of monovalent and bivalent cations, and their relation to energy metabolism, in slices of hepatoma 3924A and of a mammary tumour

1. During incubation at 1° in saline medium buffered either with phosphate or bicarbonate, slices of Morris hepatoma 3924A, and of a chemically induced tumour of rat mammary gland, lost K⁺ and gained Na⁺, Ca²⁺ and water.

2. Upon subsequent incubation at 38° in oxygenated medium, these changes were partially reversed. In the hepatoma, the reaccumulation of K⁺ was equally efficient in phosphate or bicarbonate medium, and in the presence and absence of glucose. Ca²⁺ was extruded in bicarbonate, but not in phosphate medium, and its extrusion was reduced in the presence of glucose.

3. When respiration was inhibited in the presence of glucose, K⁺ transport by the hepatoma continued to an extent which varied with the glycolytic activity of the slices, suggesting that the rate of ATP synthesis was a limiting factor under these conditions.

4. In the absence of glucose, the transport of Na⁺ and K⁺ was completely stopped by respiratory inhibition. However, more than 50% of the O₂ uptake had to be inhibited before any effect on transport was observed, suggesting that the rate of synthesis of ATP from endogenous respiration is in excess of that required to maintain transport.

5. Inhibition of transport by ouabain was accompanied by a 30% fall in the rate of endogenous respiration, and by a fall of 33% in the rate of glycolysis in the presence of cyanide plus glucose.

6. Comparison of the minimum rates of respiration and of glycolysis (in the presence of glucose plus cyanide) required to maintain the maximal extent of K⁺ transport in the hepatoma slices, suggests that ATP derived from oxidative phosphorylation or from anaerobic glycolysis is equally efficient as a source of energy for ion transport.     

Protective metabolic mechanisms during liver ischemia: Transferable lessons from long-diving animals

During periods of O₂ lack in liver of seals, mitochondrial respiration and adenosine triphosphate (ATP) synthesis are necessarily arrested. During such electron transfer system (ETS) arrest, the mitochondria are suspended in functionally protected states; upon resupplying O₂ and adenosine diphosphate (ADP), coupled respiration and ATP synthesis can resume immediately, implying that mitochondrial electrochemical potentials required for ATP synthesis are preserved during ischemia. A similar situation occurs in the rest of the cell since ion gradients also seem to be maintained across the plasma membrane; with ion-specific channels seemingly relatively inactive, ion fluxes (e.g., K⁺ efflux and Ca⁺⁺ influx) can be reduced, consequently reducing ATP expenditure for ion pumping. The need for making up energy shortfalls caused by ETS arrest is thus minimized, which is why anaerobic glycolysis can be held in low activity states (anaerobic ATP turnover rates being reduced in ischemia to less than 1/100 of typical normoxic rates in mammalian liver and to about 1/10 the rates expected during liver hypoperfusion in prolonged diving). As in many ectotherms, an interesting parallelism (channel arrest coupled with a proportionate metabolic arrest at the level of both glycolysis and the ETS) appears as the dominant hypoxia defense strategy in a hypoxia-tolerant mammalian organ.Abbreviations ADP Adenosine Diphosphate - ATP Adenosine Triphosphate - BSA Bovine Serum Albumin - ETS Electron Transfer System - RCR Respiratory Control Ratio - EGTA Ethyleneglycol-Bis-(-aminoethyl ether)N,N,N,N-Tetraacetate     

Effects of monensin on ATP levels and cell functions in rat liver and lung in vitro

Summary Effects of the proton-alkali cation-exchanging ionophore, monensin, on aspects of cellular metabolism and ionic exchanges have been studied in rat tissues in vitro. Incubation of liver slices at 38°C with 0.1 m monensin induced timedependent vesiculation, initially in the Golgi region, reduction of ATP content and of protein synthesis. At 1 m, monensin also reduced net, active movements of K⁺, Na⁺, Cl^– and water in liver slices and inhibited state 3 respiration in isolated mitochondria. The respiratory inhibitor, amytal, similarly reduced ATP content and protein synthesis at concentrations lower than those inhibiting ion transport in slices. Low concentrations of monensin (0.1–1.0 m) had similar effects on ATP and ion transport in slices of adult lung. By contrast, late-fetal liver and lung were much less sensitive to monensin; in these tissues, glycolysis sustained substantial levels of ATP. Monensin also induced vesiculation of the Golgi apparatus in fetal lung cells. It is concluded that by lowering ATP levels, monensin can markedly alter various metabolic activities in those cells which depend primarily on oxidative phosphorylation for their metabolic energy.     

Antibody-dependent cell-mediated cytotoxicity in humans. II. Energy requirement.

Antibody-dependent cytotoxicity mediated by human peripheral lymphocytes is an active energy-requiring phenomenon. In this study the relative importance of glycolysis and respiration was analyzed, by measuring the effect of various metabolic inhibitors (sodium azide, antimycin A, 2-deoxy-D-glucose, iodoacetate), alone or in combination, on effector cell efficiency and intracellular ATP level. The inhibition of both aerobic and anaerobic energy production in the effector cells completely abolished cytotoxicity. An inhibition of 50% was observed when the intracellular ATP concentration was decreased to levels corresponding to 30 to 50% of those in the untreated controls.     

BIOCHEMICAL CHANGES IN THE BRAIN IN RATS POISONED WITH AN ALKYLMERCURY COMPOUND, WITH SPECIAL REFERENCE TO THE INHIBITION OF PROTEIN SYNTHESIS IN BRAIN CORTEX SLICES

The incorporation of [U-¹⁴C]leucine into the protein of brain cortex slices from rats poisoned with methylmercury thioacetamide was markedly inhibited before the development of neurological symptoms and when the oxygen consumption, aerobic and anaerobic glycolysis and sulphydryl enzyme activities were unchanged. After the appearance of neurological symptoms, the oxygen consumption decreased significantly, while lactic acid formation did not change under anaerobic conditions, but slightly decreased under aerobic conditions. The activities of the three sulphydryl enzymes (Mg-activated ATPase, fructose- diphosphate aldolase and succinate dehydrogenase) were almost the same in visual cortex, motor cortex, cerebellum and caudate nucleus, while the activities of Mg-activated ATPase and succinate dehydrogenase in the white matter were lower than that in the grey matter. There was no difference in the activity of fructosediphosphate aldolase in grey and white matter. The activities of all three enzymes did not show any change in the earlier stage of poisoning when the animal remained free from neurological symptoms. At the more advanced stage, when neurological symptoms were present, only the activity of the succinate dehydrogenase decreased significantly, while the activities of the other two enzymes remained unchanged. The selective inhibition of protein synthesis may have a direct bearing on the poisoning by the alkylmercury compound.     

Enhancement of Anaerobic Respiration in Root Tips of Zea mays following Low-Oxygen (Hypoxic) Acclimation

Root tips (10-millimeter length) were excised from hypoxically pretreated (HPT, 4% [v/v] oxygen at 25°C for 16 hours) or nonhypoxically pretreated (NHPT, 40% [v/v] oxygen) maize (Zea mays) plants, and their rates of respiration were compared by respirometry under aerobic and anaerobic conditions with exogenous glucose. The respiratory quotient under aerobic conditions with 50 millimolar glucose was approximately 1.0, which is consistent with glucose or other hexose sugars being utilized as the predominant carbon source in glycolysis. Under strictly anaerobic conditions (anoxia), glycolysis was accelerated appreciably in both HPT and NHPT root tips, but the rate of anaerobic respiration quickly declined in NHPT roots. [U-¹⁴C]Glucose supplied under anaerobic conditions was taken up and respired by HPT root tips up to five times more rapidly than by NHPT roots. When anaerobic ethanol production was measured with excised root tips in 50 millimolar glucose, HPT tissues consistently produced ethanol more rapidly than NHPT tissues. These data suggest that a period of low oxygen partial pressure is necessary to permit adequate acclimation of the root tip of maize to subsequent anoxia, resulting in more rapid rates of fermentation and generation of ATP.     

The part of Na+K+-ATPase in total energy consumption in slices of brain and kidney cortex as well as in reticulocytes: a damaging effect of ouabain on brain cells

A progressive inhibition of the respiration of brain cortex slices by ouabain (0.2 mM) is not only caused by a specific inhibition of the Na+K+-ATPase, but is also due to damage of the cell membrane. It involves efflux and degradation of NADH or NAD. In contrast, the respiration of kidney cortex slices and reticulocytes exhibits only a rapid and constant inhibition by ouabain in concentrations of 0.2, 0.5 and 1.0 mM, respectively. Ouabain causes a decrease in O2-consumption of 12%, 45% and 23% in reticulocytes, slices of kidney cortex and brain cortex, respectively.     

The Relationship between Cell Membrane Potassium Ion Transport and Glycolysis : The effect of ethacrynic acid

Cell membrane transport of K⁺ stimulates the rate of glycolysis in Ehrlich ascites tumor cells. A study of the characteristics of this relationship indicates that the stimulation occurs under anaerobic as well as under aerobic conditions. The data suggest that glycolysis is stimulated by a K⁺ transport mechanism that is coupled to Na⁺ transport because the effect is blunted or abolished when the principal intracellular ion is lithium or choline. This stimulus to glycolysis is blocked by ouabain and ethacrynic acid, agents that have been shown to inhibit monovalent cation transport in erythrocytes. In contrast to the action of ouabain, glycolysis is inhibited by ethacrynic acid in Ehrlich ascites tumor cells in the absence of cell membrane K⁺ transport. In studies with ghost-free hemolysates of human erythrocytes and with cytosol prepared from Ehrlich ascites tumor cells, ethacrynic acid significantly blocks lactate formation from fructose diphosphate demonstrating the direct inhibitory effect of this agent on one or more enzymes of the Embden-Meyerhof pathway. Since ethacrynic acid has no influence on lactate formation in intact erythrocytes utilizing an endogenous substrate, the presumptive site of inhibition is proximal to the 3-phosphoglycerate level.     

Effects of dinitrophenol and oligomycin on the coupling between anaerobic metabolism and anaerobic sodium transport by the isolated turtle bladder

Dinitrophenol (1 x 10^-5 M) has been found to inhibit anaerobic sodium transport by the isolated urinary bladder of the fresh water turtle. Concurrently, anaerobic glycolysis was stimulated markedly. However, tissue ATP levels diminished only modestly, remaining at approximately 75% of values observed under anaerobic conditions without DNP. The utilization of glucose (from endogenous glycogen) corresponded closely to that predicted from the molar quantities of lactate formed. Thus the glycolytic pathway was completed in the presence of DNP and if ATP were synthesized normally during glycolysis, synthesis should have been increased. On the other hand, the decrease in Na transport should have decreased ATP utilization. Oligomycin did not block sodium transport either aerobically or anaerobically, but ATP concentrations did decrease. When anaerobic glycolysis was blocked by iodoacetate, pyruvate did not sustain sodium transport thus suggesting that no electron acceptors were available in the system. Two explanations are entertained for the anaerobic effect of DNP: (a) Stimulation by DNP of plasma membrane as well as mitochondrial ATPase activity; (b) inhibition of a high energy intermediate derived from glycolytic ATP or from glycolysis per se. The arguments relevant to each possibility are presented in the text. Although definitive resolution is not possible, we believe that the data favor the hypothesis that there was a high energy intermediate in the anaerobic system and that this intermediate, rather than ATP, served as the immediate source of energy for the sodium pump.