Lactate formation by rat small intestine in vitro.

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na+ in the incubating medium were replaced by K+, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Lactate formation by rat small intestine in vitro

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na⁺ in the incubating medium were replaced by K⁺, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Lactate production in the perfused rat liver

1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat.     

No effect of short-term arginine supplementation on nitric oxide production, metabolism and performance in intermittent exercise in athletes

Arginine supplementation has been shown to alleviate endothelial dysfunction and improve exercise performance through increasing nitric oxide production in patients with cardiopulmonary diseases. In addition, arginine supplementation could decrease accumulations of lactate and ammonia, metabolites involved in development of muscular fatigue. The aim of this study was to investigate the effect of short-term arginine supplementation on performance in intermittent anaerobic exercise and the underlying mechanism in well-trained male athletes. Ten elite male college judo athletes participated with a randomized crossover, placebo-controlled design. The subjects consumed 6 g/day arginine (ARG trial) or placebo (CON trial) for 3 days then performed an intermittent anaerobic exercise test on a cycle ergometer. Blood samples were collected before supplementation, before and during exercise and 0, 3, 6, 10, 30 and 60 min after exercise. ARG trial had significantly higher arginine concentrations than CON trial at the same time point before, during and after exercise. In both trials, nitrate and nitrite concentration was significantly higher during and 6 min after exercise comparing to the basal concentration. The increase in nitrate and nitrite concentration during exercise in both trials was parallel to the increase in plasma citrulline concentrations. There was no significant difference between the 2 trials in plasma nitrate and nitrite, lactate and ammonia concentrations and peak and average power in the exercise. The results of this study suggested that short-term arginine supplementation had no effect on nitric oxide production, lactate and ammonia metabolism and performance in intermittent anaerobic exercise in well-trained male athletes.     

Formation of products of anaerobic metabolism in the ischemic myocardium

The effect of ischemia on the formation of products of anaerobic metabolism and their release into the cardiac effluent in isolated perfused guinea pig hearts was studied. During 30 min normothermal ischemia, the myocardial ATP and phosphocreatine levels decreased to 34% and 15% of the initial values, respectively. The net alanine formation in ischemia was approximately a stoichiometric glutamate decrease; the increase in the tissue malate content corresponded to the aspartate----oxaloacetate----malate anaplerotic flux, the succinate production being commensurable to alpha-ketoglutaric acid formation in the alanine aminotransferase reaction. Using 1H-NMR, it was shown that the release of trace amounts of lactate, alanine, succinate, creatine and pyruvate into cardiac effluents occurred during the first 5 minutes of reperfusion. The rate of metabolite release decreased in the following order: lactate much greater than alanine greater than succinate greater than creatine. By the 30th minute of reperfusion, the decrease in the tissue levels of these metabolites to preischemic values was accompanied by the recovery of ATP and phosphocreatine to 65% and 90% of the initial levels, respectively. The data obtained suggest that the formation and release of alanine, creatine or succinate as well as lactate from ischemic myocardium may testify to significant disturbances in energy metabolism of the myocardium.     

Anaerobic metabolism during activity in lizards

Summary A new technique developed for the determination of total lactate production in small animals was used to evaluate the role of anaerobiosis during activity at different temperatures in lizards. Measurements on six species of small lizards indicate little interspecific variation or thermal effect in resting lactate levels (0.35 mg lactate/g body weight) or maximal lactate levels achieved at exhaustion (1.4 mg lactate/g). Normally activeAnolis in captivity had a lactate content of 0.5 mg lactate/g. Rates of lactate formation were most rapid during the first 30 sec of activity and had a low thermal dependence (Q₁₀=1.1–1.3 above 20 °C). The lactate formed during activity persists for long periods; e.g., for 30 to 60 min between 20 and 37 °C inAnolis carolinensis (Fig. 1). Recovery rate generally increases with temperature. Muscle lactate concentrations peak at the end of activity, but liver and blood lactate are not maximal until 10 and 30 min, respectively, after activity (Fig. 2). The decrease in the blood lactate is shown to be a poor estimator of total recovery. An estimated 80–90% of the total energy utilized during initial vigorous activity comes from anaerobic sources. Because of its low thermal dependence, anaerobiosis permits high levels of activity in lizards at all body temperatures without requiring high levels of aerobic resting metabolism.Support for this research was provided by a Miller Post-doctoral Research Felowship to AFB and NSF Grant GB 22642 to PL.     

Effect of dichloroacetate on mechanical performance and metabolism of compromised diaphragm muscle.

We investigated the effect of dichloroacetate (DCA) on tension generation and carbohydrate metabolism of the rat diaphragm in vitro. Isolated diaphragms were placed in individual organ chambers and were hooked to force-displacement transducers. Net lactate production and glucose and lactate oxidation were measured in vitro. Diaphragmatic fatigue was precipitated by in vivo endotoxemic shock, by in vitro hypoxia, or by in vitro repetitive tetanic stimulation. In diaphragms isolated from endotoxemic rats, DCA increased tension generation by 30 and 20% at stimulation frequencies of 20 and 100 Hz, respectively. Associated with changes in mechanical performance, DCA reduced net lactate production by 53% after 60 min of incubation and increased glucose oxidation 54% but had no effect on lactate oxidation. During in vitro hypoxia, DCA reduced net diaphragmatic lactate production by 30% and increased glucose oxidation by 45% but did not attenuate hypoxic fatigue. DCA had no effect on tension generation during repetitive tetanic stimulation. We conclude that DCA improves in vitro diaphragmatic fatigue due to endotoxicosis but not due to hypoxia or repetitive stimulation.     

Fasciola hepatica: Carbohydrate metabolism of the adult

Glycogen (or exogenous glucose) was the only energy source utilized by adult Fasciola hepatica under a number of different incubation conditions. When exogenous glucose was present in the incubation medium, significant amounts of lactate were excreted. Anaerobically, in the presence of glucose, lactate accounted for 20% of the total end products measured. In the absence of glucose, organic acid production accounted for approximately 60% of glycogen carbon utilized; this value was reduced to 40% in the presence of exogenous glucose. There was no appreciable Pasteur effect.     

Epinephrine infusion enhances muscle glycogenolysis during prolonged electrical stimulation

To determine the effects of epinephrine (EPI) infusion on muscle glycogenolysis and force production, the quadriceps muscles of both legs in six subjects were intermittently stimulated for 30 min. Contractions lasted 1.6 s (20 Hz) and were separated by 1.6 s of rest. EPI was infused (approximately 0.14 micrograms.kg body wt-1.min-1) in one leg during the last 15 min and the vastus lateralis was biopsied at rest (control leg only) and after 15, 18 (EPI leg only), and 30 min of stimulation. EPI infusion doubled the mole fraction of phosphorylase a (22.5 +/- 4.1 to 44.8 +/- 9.0%) and glycogenolysis (2.16 +/- 0.72 to 5.45 +/- 0.81 mmol glucosyl U.kg dry muscle wt-1.min-1) during stimulation. Muscle glucose 6-phosphate increased from 3.04 +/- 0.17 to 6.43 +/- 0.20 mmol/kg dry muscle wt, and lactate increased from 25.8 +/- 4.4 to 34.3 +/- 4.6 mmol/kg after 3 min of EPI infusion. Isometric force production was unaltered by EPI infusion. These results demonstrate a strong glycogenolytic effect of EPI infusion during prolonged electrical stimulation and suggest that the extra pyruvate formed was converted mainly to lactate. Exclusive anaerobic metabolism of the extra substrate would provide only a 10% increase in total ATP production, possibly accounting for the lack of improvement in force production. We suggest that the decrease in force production during prolonged electrical stimulation is related to decreased excitation of the contractile mechanism rather than inhibition of cross-bridge turnover caused by a shortage of energy or accumulation of hyproducts.     

The effects of pH and temperature on the kinetic properties of skeletal muscle lactate dehydrogenase from anuran amphibians

Summary Muscle LDH activities were measured in two anuran amphibians with different behaviour and ecology, Rana perezi and Bufo calamita. Both pyruvate reduction and lactate oxidation were measured at temperatures of 15, 20 and 30°C, and at pH 7.0, 7.4, and 8.0. Pyruvate and lactate muscle concentrations were determined in individuals at rest and after exercise. R. perezi muscle used anaerobic glycolysis during 3 min of exhaustive exercise, with rising pyruvate and lactate concentrations. Enforced walking for 30 min caused high variability in lactate concentration in B. calamita muscle. Temperature and pH changes affected apparent Km values for pyruvate. When these factors varied simultaneously, enzyme affinity tended not to change. Thus, the thermodynamic effect on pyruvate reduction activity is high, especially at physiological substrate concentrations. In contrast, lactate oxidation activity tended to stabilize when temperature and pH varied jointly. Inhibition by substrate, pyruvate or lactate, seemed to have no importance in vivo.During exercise there was a rise in pyruvate concentration, and a probable decrease in pH, which increased pyruvate reduction reaction and decreased lactate oxidation, contributing to lactate accumulation in Rana perezi muscle. B. calamita muscle did not show pyruvate increase after exercise and its LDH was less dependent on pH at physiological concentrations. Pyruvate reduction rate did not therefore increase. R. perezi muscle enzyme had features of anaerobic LDH while B. calamita LDH muscle was more similar to mammalian heart enzyme, with differences in accordance with the different behaviour of these anurans.Abbreviations LDH lactate dehydrogenase     

Glucose regulation of guinea-pig sperm motility.

Washed guinea-pig spermatozoa from the vas deferens re-acquired progressive motility within 1-2 min of incubation in minimal culture medium containing pyruvate and lactate. When glucose was added, either at the beginning or after 15 min of incubation, the cells showed stimulated motility (increased straight-line velocity, linearity and beat-cross frequency, P less than 0.01). Re-acquisition of progressive motility was preceded by a significant (P less than 0.005) transient increase in sperm concentration of cyclic adenosine 5'-phosphate (cAMP) with or without glucose in the medium. Papaverine caused another large significant (P less than 0.001) increase in cAMP concentration; and 5.56mM glucose with papaverine caused a further stimulation in cAMP beyond that with papaverine alone (P less than 0.005). Although 0.05 or 5.56mM glucose plus alpha-chlorohydrin stimulated sperm motility, they did not further stimulate the increase in cAMP after 30 s of incubation. Thus, there was no apparent correlation between the glucose-stimulating effect on sperm motility and the enhancement of cAMP at 30 s. However, there was a close correlation between glucose-stimulated motility and enhancement of ATP (P less than 0.05) by glucose even under incubation conditions in which glucose caused the Crabtree effect (decrease in respiration rate).     

Effect of glucose on ATP dephosphorylation in rat spermatids

Round spermatids were isolated from rat testes and the effects of different energy-yielding substrates on the cellular ATP content were estimated. The ATP content was constant and high (6-8 nmol/10(6) cells) during metabolism of exogenous lactate. During incubation for 30 min in the absence of exogenous lactate, there was a remarkably slow decline of the ATP content, indicating ATP production from other substrates. It was shown that this could reflect beta-oxidation of fatty acids, but not the mobilization of an endogenous pool of acetylcarnitine. Glucose metabolism in the absence of exogenous lactate resulted in a rapid decline of the ATP content. This effect of glucose was correlated with a high fructose 1,6-biphosphate content (6-7 nmol/10(6) cells) and could be prevented by the addition of lactate. It is suggested that metabolism of glucose (and also mannose and fructose, but not galactose) in the absence of exogenous lactate can result in ATP dephosphorylation.     

Metabolism of round spermatids: gossypol induces uncoupling of respiratory chain and oxidative phosphorylation

The effect of gossypol on energy metabolism of round spermatids of rats was examined. When spermatids were treated with various concentrations of gossypol for 30 min at 32 degrees C, a biphasic response (stimulation at low concentrations and inhibition at high concentrations) was seen in pyruvate and CO2 production from lactate. At the early period of incubation, gossypol at even high concentrations stimulated CO2 production to an extent similar to that stimulated by 2,4-dinitrophenol (DNP). At longer periods of incubation, however, the rates of CO2 production from lactate dropped to those seen in the rotenone-treated cells. The rates of oxygen consumption were not increased further by DNP when cells were pretreated with gossypol. The adenosine triphosphate (ATP) content in spermatids was reduced markedly, although lactate oxidation to CO2 and mitochondrial respiration were stimulated by gossypol. These results suggest that gossypol probably exerts its effect on spermatids by uncoupling respiratory chain and oxidative phosphorylation.     

The nucleotide metabolism in lactate perfused hearts under ischaemic and reperfused conditions

It was examined whether lactate influences postischaemic hemodynamic recovery as a function of the duration of ischaemia and whether changes in high-energy phosphate metabolism under ischaemic and reperfused conditions could be held responsible for impairment of cardiac function. To this end, isolated working rat hearts were perfused with either glucose (11 mM), glucose (11 mM) plus lactate (5 mM) or glucose (11 mM) plus pyruvate (5 mM). The extent of ischaemic injury was varied by changing the intervals of ischaemia, i.e. 15, 30 and 45 min. Perfusion by lactate evoked marked depression of functional recovery after 30 min of ischaemia. Perfusion by pyruvate resulted in marked decline of cardiac function after 45 min of ischaemia, while in glucose perfused hearts hemodynamic performance was still recovered to some extent after 45 min of ischaemia. Hence, lactate accelerates postischaemic hemodynamic impairment compared to glucose and pyruvate. The marked decline in functional recovery of the lactate perfused hearts cannot be ascribed to the extent of degradation of high-energy phosphates during ischaemia as compared to glucose and pyruvate perfused hearts. Glycolytic ATP formation (evaluated by the rate of lactate production) can neither be responsible for loss of cardiac function in the lactate perfused hearts. Moreover, failure of reenergization during reperfusion, the amount of nucleosides and oxypurines lost or the level of high-energy phosphates at the end of reperfusion cannot explain lactate-induced impairment. Alternatively, the accumulation of endogenous lactate may have contributed to ischaemic damage in the lactate perfused hearts after 30 min of ischaemia as it was higher in the lactate than in the glucose or pyruvate perfused hearts. It cannot be excluded that possible beneficial effects of the elevated glycolytic ATP formation during 15 to 30 min of ischaemia in the lactate perfused hearts are counterbalanced by the detrimental effects of lactate accumulation.     

Evaluation of cellular energetics by the Pasteur effect in intact cardiomyoblasts and isolated perfused hearts

This work aims at exploring changes in cellular energetics by exploiting the Pasteur effect. We assumed that lactate overproduction arising from antimycin A-induced inhibition of mitochondrial respiration (delta-lactate = stimulated [lactate] -basal [lactate]) is indicative of the energy provided aerobically by the cell. Rat embryonal cardiomyocytes (H9c2), incubated with 2 micromol/L antimycin A, increased about 6 fold their lactate production in a manner linear with time and cell number. Antimycin A was also delivered to Langendorff-perfused rat hearts under control aerobic conditions or after 20 min-ischemia and 30 min-reperfusion. The test started at the end of each perfusion and lactate was measured into perfusate collected for further 25 min. A cardioplegic solution was also delivered during the test to exclude that lactate production was influenced by cardiac contraction. Control delta-lactate was 20.9 +/- 2.31 (S.E.M.) microg/mL and markedly decreased after reperfusion (7.66 +/- 0.51, p < 0.001), showing that energy production was impaired of about 70%. The determination of oxygen consumption by mitochondria isolated from reperfused hearts also suggested that the damage to the respiratory chain was similar to that evaluated by lactate overproduction (Respiratory Control Index: 75% lower than control, p < 0.001). Moreover, when delta-lactate was referred to the estimated cells which remained viable at the end of reperfusion (49.9%), it was 25% lower than control (p < 0.05). Therefore, we proposed this test as a tool for quantifying both physiological and pathological energetic modifications in living intact cardiomyocytes and in isolated and perfused hearts.     

Lactic Acid efflux as a mechanism of hypoxic acclimation of maize root tips to anoxia

Hypoxic pretreatment (3 kPa oxygen) of maize (Zea mays L.) root tips improved their survival time in a subsequent anoxic incubation from 10 h to more than 3 d, provided that glucose was added to the medium to sustain metabolism. The glycolytic flux (lactate + ethanol) was the same in both pretreated and untreated root tips during the 1st h after transfer to anoxia. It was only after 2 h that it declined sharply in untreated tips, but was sustained in pretreated ones. Right after the transition from normoxia to anoxia of untreated root tips, the only fermentative product detected was lactic acid, which accumulated in a 7:1 proportion after 30 min in tissue and medium, respectively. It took 10 min before ethanol could be detected and 20 min for it to be produced at its maximum rate at the expense of lactate production, which slowed down. In contrast, in hypoxically pretreated root tips, ethanol was produced at a maximum rate right after the transfer to anoxia. Concurrently, low amounts of lactic acid were produced that accumulated in a 1:1 proportion after 30 min in tissue and medium, respectively. This large efflux of lactic acid could account for the higher cytoplasmic pH values always found in pretreated tissues. The presence of cycloheximide during pretreatment abolished this difference, suggesting that the greater efficiency of lactate efflux was linked to protein synthesis. The role of lactate in cytosolic pH regulation and in sensitivity to anoxia is discussed.     

Impact of leachate recirculation and recirculation volume on stabilization of municipal solid wastes in simulated anaerobic bioreactors

The effects of leachate recirculation and the recirculation rate on the anaerobic treatment of domestic solid waste was investigated in three simulated landfill anaerobic bioreactors. A single pass reactor was operated without leachate recirculation while the other two reactors were operated with leachate recirculation. The leachate recirculation rate was 9 l/day (13% of the reactor volume) in Reactor₉, while the recirculation rate was 21 l/day (30% of the reactor volume), in Reactor₂₁. pH, chemical oxygen demand (COD), volatile fatty acids (VFA), ammonium–nitrogen (NH₄–N) total and methane gas measurements in leachate samples were regularly monitored. After 220 days of anaerobic incubation, it was observed that the pH, COD, VFA concentrations, methane gas productions and methane percentages in Reactor₉ were better than the single pass reactor and Reactor₂₁. When the leachate recirculation rate was increased to three times a decrease in pH, and an increase in VFA and COD concentrations were observed in Reactor₂₁. The COD values were measured as 47 000, 39 000 and 52 000 mg/l while the VFA concentrations were 15 000, 13 000 and 21 000 mg/l, respectively, in single pass, Reactor₉ and Reactor₂₁ after 220 days of anaerobic incubation. The values of pH were 5.89, 6.44 and 6.16, respectively, after anaerobic incubation. The mean methane percentages of single pass reactor, Reactor₉ and Reactor₂₁ were 30, 50 and 40%, respectively, after 50 days of incubation. Leachate recirculation reduced the waste stabilization time and was effective in enhancing methane gas production and improving leachate. However, leachate recirculation was not effective in removing ammonia from the leachate. The amounts of COD recovered by methane were 62.9, 162.3 and 94.6 g for single pass, Reactor₉ and Reactor₂₁, respectively, at the end of 220 days of anaerobic incubation.     

Brain Lactate Is an Obligatory Aerobic Energy Substrate for Functional Recovery After Hypoxia: Further In Vitro Validation

Abstract: This study used the rat hippocampal slice preparation and the monocarboxylate transporter inhibitor, α-cyano-4-hydroxycinnamate (4-CIN), to assess the obligatory role that lactate plays in fueling the recovery of synaptic function after hypoxia upon reoxygenation. At a concentration of 500 µ M , 4-CIN blocked lactate-supported synaptic function in hippocampal slices under normoxic conditions in 15 min. The inhibitor had no effect on glucose-supported synaptic function. Of control hippocampal slices exposed to 10-min hypoxia, 77.8 ± 6.8% recovered synaptic function after 30-min reoxygenation. Of slices supplemented with 500 µ M 4-CIN, only 15 ± 10.9% recovered synaptic function despite the large amount of lactate formed during the hypoxic period and the abundance of glucose present before, during, and after hypoxia. These results indicate that 4-CIN, when present during hypoxia and reoxygenation, blocks lactate transport from astrocytes, where the bulk of anaerobic lactate is formed, to neurons, where lactate is being utilized aerobically to support recovery of function after hypoxia. These results unequivocally validate that brain lactate is an obligatory aerobic energy substrate for posthypoxia recovery of function.     

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.     

Stimulation of protein synthesis in cultured heart muscle cells by glucose.

Glucose stimulated the rate of incorporation of [3H]leucine into HCLO4-insoluble fraction of cultured rat heart muscle cells under both aerobic and anaerobic conditions. In the aerobic system the incorporation proceeded at a constant rate during 3h of incubation with and without glucose whereas in the anaeorbic system the incorporation ceased after approx. 60 min and could be renewed only by the addition of glucose. No correlation was found to exist between the above effect of glucose on protein synthesis and glucose-dependent changes in the intracellular ATP concentration. The extent of the stimulation of protein synthesis was related to the concentration of glucose. The effect of glucose was suppressed by cycloheximide but was not affected by actinomycin D. Glucose had no effect on the rate of transport of alpha-aminoisobutyric acid. Mannose also stimulated [3H]leucine incorporation. Substances that did not produce lactate were ineffective. Iodoacetate inhibited the stimulatory effect of glucose, but pyruvate, which by itself had no apprecialbe stimulatory action, relieved the inhibition induced by iodoacetate. There was no concomitant change in the concentration of ATP when iodoacetate inhibition was reversed by pyruvate. L-Lactate or other intermediates of energy metabolism could not relieve the inhibitory effect of iodoacetate.