Cytosolic Phosphate Potential Research Articles

Energetics of isolated hepatocyte swelling induced by sodium co-transported amino acids.

This study was designed to investigate the energetics of isolated rat hepatocyte swelling due to sodium-cotransported amino acid accumulation in a medium containing either glucose or octanoate as basal substrate. We show that the size of the increase in cytosolic volume is directly correlated with the total amino acid accumulation, which depends on the difference of electrical potential across the plasma membrane. Such a change in cell volume, with either glucose or octanoate, does not modify the mitochondrial volume. Addition of sodium-cotransported amino acids for which the metabolism was avoided showed that the rise in cell volume, per se, did not change the respiratory rate, deltap, or phosphate potential in either mitochondrial or cytosolic compartments. Conversely, the large increase in oxidative phosphorylation flux was due to the metabolism of amino acids as a consequence of a rise in electron supply for the respiratory chain rather than an increase in cellular ATP demand, as indicated by the increase in cytosolic phosphate potential. Moreover, although we confirm that octanoate addition largely increases the respiratory rate by a process different from uncoupling, we observed that the same overall thermodynamic driving force through the respiratory chain and the same mitochondrial or cytosolic phosphate potential were maintained for much higher oxygen consumption when octanoate was present. We propose that these octanoate effects are due to a decrease in the actual protons/2 electrons stoichiometry as a consequence of a shift in electron supply toward a two-coupling site instead of a three-coupling site. The change in the FADH2/NADH formation flux ratio in either fatty acid or carbohydrate oxidation explains such results.

Glycolytic pathway, redox state of NAD(P)-couples and energy metabolism in lens in galactose-fed rats: effect of an aldose reductase inhibitor

PURPOSE. The present study was aimed at evaluating early changes in glycolysis, the redox state of free cytosolic NAD(P)-couples, and the adenine nucleotide system in lens in both control and 50% galactose-fed rats, with the possibility of preventing these with an aldose reductase inhibitor (ARI). METHODS. Experiments were performed on male Sprague-Dawley rats fed the galactose diet for 2–14 days. The levels of glucose, galactose, glycolytic intermediates, a-glycerophosphate, malate, NAD, ATP, ADP, AMP were assayed spectrofluorometrically in individual lenses by enzymatic procedures, while galactitol and myo -inositol were quantified by GC-MS. Free cytosolic NAD + /NADH, NADP + /NADPH, and ATP/ADP × P i (phosphate potential) were estimated from lactate dehydrogenase, malic enzyme, and triose phosphate isomerase-glyceraldehyde 3-phosphate dehydrogenase-3-phosphoglycerate kinase systems. Lactate and pyruvate production by lenses of both control and galactose-fed rats was measured in a set of in vitro incubation studies (2 hr, 37°C, Krebs bicarbonate-Hepes buffer, pH 7.45, with 5mM glucose or 5mM glucose + 30 mM galactose, respectively). RESULTS. Lens galactitol levels in 2, 4, 6, 8, 10, and 14-day galactose-fed rats were 48 ± 8, 58 ± 9, 68 ± 8, 73 ± 5, 81 ± 20, and 75 ± 11 mmol/g wet weight (mean ± SD), respectively. NAD + /NADH ratios were indistinguishable from controls after 2–6 days on the galactose diet, but fell dramatically between 8 and 10 days, and did not correlate with polyol accumulation per se. The pattern of glycolytic intermediates (no change in G6P, F6P, and 3-PG, increase in GA3P, decrease in FDP, PEP, pyruvate, and lactate), as well as reduced in vitro lactate and pyruvate production, suggest inhibition of glycolysis at the sites of phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, enolase, and pyruvate kinase. ATP levels as well as total ATP/ADP, ATP/ADP 3 P i, adenylate charge, and cytosolic phosphate potential were decreased in galactose-fed rats, while galactose 1-phosphate and a-glycerophosphate levels as well as NADP + /NADPH ratio were increased. Lens galactitol levels were reduced ~57% in 10-day galactose-fed rats treated with the ARI (tolrestat, 100 mg/kg bwt/day, 6-day pretreatment); the changes in the lower segment of glycolysis, a-glycerophosphate levels, redox state of NAD-couples, and energy metabolism were partially prevented while NADP + /NADPH ratios were unchanged and galactose 1-phosphate levels were further increased. CONCLUSIONS. Depressed glycolysis in lens in galactose-fed rats is consistent with decreased NAD + /NADH and adenine nucleotide phosphorylation. Early changes in lens glucose utilization, redox state of NAD-couples, and energy metabolism in this model of galactosemia are similar to those in diabetes, are at least in part mediated by aldose reductase involved mechanisms, and can be partially prevented by an aldose reductase inhibitor.

Control of oxidative metabolism in volume-overloaded rat hearts: effects of different lipid substrates

The relationship between intracellular energy parameters and myocardial O2 consumption (VO2) was studied in control and volume-overloaded hearts perfused with different lipid substrates and over a range of left ventricular work loads. In control hearts, a unique linear relationship between log of cytosolic [ATP]/[ADPf].[Pi] (where [ADPf] is concentration of free ADP) and myocardial VO2 was observed between low and high work loads for both fatty acids studied. In volume-overloaded hearts perfused in the presence of exogenous palmitate, the slope of the relationship between log [ATP]/[ADPf].[Pi] and myocardial VO2 was considerably depressed. It would seem that, under these conditions, much of the thermodynamic control of respiratory chain function has been lost. When myocardial VO2 was expressed as a function of cytosolic ADPf, the cytosolic ADPf was not regulatory. This may be related to a substrate limitation of the respiratory chain, as suggested by an excessive oxidation of pyridine nucleotides. When octanoate, instead of palmitate, was used, most of the above limitation of the respiration disappeared. With this substrate, the reduction of mitochondrial pyridine nucleotides in volume-overloaded hearts was similar to that in controls, and the linear relationship between log [ATP]/[ADPf].[Pi] and myocardial VO2 reappeared over the range of work loads studied. The above failure of cytosolic phosphate potential and ADPf to drive respiration when mitochondrial NADH is low fits well with the integrated model of kinetic regulation, as proposed by recent nuclear magnetic resonance studies. our results also indicate that, even at high respiratory rates, free-energy change of ATP synthesis of volume-overloaded hearts can be protected by use of an appropriate substrate. This, in turn, prevents contractile failure.

Tissue selective modulation of redox and phosphate potentials by beta,beta'-methyl-substituted hexadecanedioic acid

beta,beta'-Methyl-substituted hexadecanedioic acid (MEDICA 16) shares some of the calorigenic-hypolipidemic characteristics of thyroid hormones. In light of this similarity, MEDICA 16 was further evaluated here as a modulator of rat liver redox and phosphate potentials as well as an effector of cardiac high energy intermediates level in comparison to thyroid hormone. 1) Similarly to thyroid hormone, MEDICA 16 treatment resulted in 3.5-fold decrease in cytosolic redox potential. With both treatment modes, the induced decrease in cytosolic redox potential resulted in a concomitant increase in the liver capacity of handling an ethanol or xylitol load. 2) The apparent liver cytosolic phosphate potential calculated from the glycerol-3-phosphate 3-phosphoglyceric acid ratio evaluated under conditions of rapid equilibrium within the glycerol-3 phosphate/3-phosphoglyceric acid metabolic section was found to remain unaffected by either MEDICA 16 or T3 treatment. However, the apparent cellular phosphate potential was substantially decreased by both treatment modes, thus reflecting a decrease in the apparent liver mitochondrial phosphate potential. 3) Similarly to thyroid hormone, the effect of MEDICA 16 on liver redox and phosphate potentials could be accounted for by induction of mitochondrial glycerol-3-phosphate dehydrogenase. 4) In contrast to liver, heart high energy intermediates were affected by thyroid hormone but not by MEDICA 16 treatment. 5) The effect of T3 and MEDICA 16 with respect to liver redox and phosphate potentials may partially account for the calorigenic-hypolipidemic effect of both. MEDICA 16 may however serve as a selective liver thyromimetic agent lacking the cardiac affect induced by thyroid hormone.

Tissue selective modulation of redox and phosphate potentials by beta,beta'-methyl-substituted hexadecanedioic acid.

beta,beta'-Methyl-substituted hexadecanedioic acid (MEDICA 16) shares some of the calorigenic-hypolipidemic characteristics of thyroid hormones. In light of this similarity, MEDICA 16 was further evaluated here as a modulator of rat liver redox and phosphate potentials as well as an effector of cardiac high energy intermediates level in comparison to thyroid hormone. 1) Similarly to thyroid hormone, MEDICA 16 treatment resulted in 3.5-fold decrease in cytosolic redox potential. With both treatment modes, the induced decrease in cytosolic redox potential resulted in a concomitant increase in the liver capacity of handling an ethanol or xylitol load. 2) The apparent liver cytosolic phosphate potential calculated from the glycerol-3-phosphate 3-phosphoglyceric acid ratio evaluated under conditions of rapid equilibrium within the glycerol-3 phosphate/3-phosphoglyceric acid metabolic section was found to remain unaffected by either MEDICA 16 or T3 treatment. However, the apparent cellular phosphate potential was substantially decreased by both treatment modes, thus reflecting a decrease in the apparent liver mitochondrial phosphate potential. 3) Similarly to thyroid hormone, the effect of MEDICA 16 on liver redox and phosphate potentials could be accounted for by induction of mitochondrial glycerol-3-phosphate dehydrogenase. 4) In contrast to liver, heart high energy intermediates were affected by thyroid hormone but not by MEDICA 16 treatment. 5) The effect of T3 and MEDICA 16 with respect to liver redox and phosphate potentials may partially account for the calorigenic-hypolipidemic effect of both. MEDICA 16 may however serve as a selective liver thyromimetic agent lacking the cardiac affect induced by thyroid hormone.

Effect of thyroid hormone treatment on redox and phosphate potentials in rat liver.

In the present study rat liver cytosolic/mitochondrial redox and phosphate potentials were evaluated as a function of thyroid hormone status. T3 treatment resulted in a dose-dependent 2-fold decrease in the cytosolic redox potential, as reflected by the liver lactate/pyruvate ratio, with a concomitant increase in the liver capacity of handling an ethanol load. The effect of T3 on liver cytosolic redox potential was correlated with a T3-induced increase in mitochondrial glycerol-3-phosphate dehydrogenase activity. The apparent liver cytosolic phosphate potential was calculated from the glycerol-3-phosphate/3-phosphoglyceric acid ratio, using 31P-nmr under conditions of rapid equilibrium within the glycerol-3-phosphate/3- phosphoglyceric acid metabolic section and was found to be essentially unaffected by T3 treatment. The apparent total cellular phosphate potential measured by 31P-nmr, however, was decreased in T3-treated animals, reflecting a decrease in the apparent liver mitochondrial phosphate potential induced by T3 treatment. Also, while the apparent cellular phosphate potential of euthyroid rats was independent of ethanol administration, the reduced cellular phosphate potential of T3-treated rats was normalized by ethanol treatment. In conclusion, thyroid hormone treatment induces in vivo a dramatic decrease in the liver cytosolic redox potential, with a concomitant increase in the liver oxidizing capacity. The decrease in cytosolic redox potential induced by thyroid hormone treatment is accompanied by a decrease in mitochondrial phosphate potential. Liver mitochondrial ATP production in thyroid hormone-treated animals appears to be rate limited by the availability of cytosolic reducing equivalents.

Energy depletion and autophagy. Cytochemical and biochemical studies in isolated rat hepatocytes

In this paper, data dealing with the sensitivity of autophagy towards partial ATP depletion in isolated rat hepatocytes are reviewed. Partial reduction of intracellular ATP causes: (1) a decrease of proteolytic flux; (2) a decrease in uptake of cytosolic components into the autophagic-lysosomal compartment; (3) either a decrease or no change in the ratio between volume densities of autophagosomes and lysosomes, depending on whether or not the cytosolic phosphate potential is affected; and (4) impairment of the lysosomal proton pump. It is concluded that the consecutive steps of autophagy all respond to relatively small changes of intracellular ATP concentration.

Cytochemical and morphometric analysis of autophagy in energy depleted rat hepatocytes

The energy dependence of lysosomal enzyme acquisition by autophagosomes was studied in isolated rat hepatocytes by ultrastructural analysis for acid phosphatase activity. Reduction of the intracellular ATP content by addition of actractyloside or fructose decreased the flux through the autophagic proteolytic pathway to a similar extent (40–50%). Unexpectedly, in the presence of atractyloside the volume density of autophagosomes was reduced by 65%, whereas in the presence of fructose this reduction was only 20%. The volume density of lysosomes was not significantly affected by either of the two compounds. It is concluded that partial ATP depletion by fructose not only inhibits sequestration of cytoplasmic material in autophagosomes, but also affects the fusion between autophagosomes and lysosomes. Since fructose, in contrast to atractyloside, does not affect the cytosolic phosphate potential, it is proposed that autophagic sequestration is more sensitive to changes in the cytosolic phosphate potential whereas the fusion between autophagosomes and lysosomes is more responsive to changes in the ATP concentration.

An evaluation of the metabolite indicator method for determining the cytosolic phosphate potential in rat liver cells

The cytosolic phosphate potential was estimated in isolated rat liver parenchymal cells incubated with various gluconeogenic substrates. The value of the cytosolic [ ATP] [ ADP][P i ] ratio was either estimated directly from measurements of ATP, ADP and P i after digitonin fractionation of the cells, or calculated by the metabolite indicator method. When cells were incubated with lactate, pyruvate or alanine so that net flux through the indicator enzymes was in the gluconeogenic direction, there was excellent agreement between the values obtained by the two methods over a wide range of fluxes. However, when the cells were incubated with substrates that could be converted both to glucose and to lactate so that net flux through the indicator enzymes was in the glycolytic direction, a large difference in the values of the cytosolic [ ATP] ([ ADP][P i ] ) ratio as derived by the two methods was observed. It is concluded that the reaction catalysed by glyceraldehyde-3-phosphate dehydrogenase plus 3-phosphoglycerate kinase is out of equilibrium when flux through the reaction is in the glycolytic direction, and that use of the metabolite indicator method for the calculation of the cytosolic phosphate potential under these conditions leads to erroneous values.

A 31P-NMR study of some metabolic and functional effects of the inotropic agents epinephrine and ouabain, and the ionophore R02-2985 (X537A) in the isolated, perfused rat heart

1. Some metabolic effects of increased mechanical activity by the Langendorff-perfused rat heart have been characterized using 31P-NMR. Mechanical activity was increased by infusion of ouabain (0.9−7.0·10 −5 M), the ionophore R02-2985 (1·10 −5 M) or epinephrine (5·10 −8 M). 2. Similar metabolic changes accompanied infusion of each of the positive inotropic agents into hearts perfused with buffer containing 11 mM glucose as the substrate. In each case phosphocreatine concentrations decreased. During the period of epinephrine infusion the phosphocreatine began to recover its original concentration, although there were no significant changes in mechanical activity. 3. Comparisons of the metabolic changes accompanying the positive inotropic and chronotropic effects of epinephrine were made between hearts perfused with either glucose (11 mM), acetate (5 mM) or lactate (5 mM). A time-dependent decrease in phosphocreatine concentrations also accompanied infusion of epinephrine into hearts perfused with lactate as the sole exogenous substrate, but no statistically significant metabolite changes were observed after identical epinephrine infusions with acetate as the substrate. 4. Calculation of the concentration of free ADP assuming equilibrium in the creatine phosphokinase reaction allows estimation of the cytosolic phosphate potential ( [ ATP] [ADP][P i ] ), which appears to be dependent on a number of factors, including the nature of the exogenous substrate and the level of mechanical activity. 5. Thus, we conclude that there is no general correlation between the phosphate potential and the mitochondrial respiratory rate in the perfused rat heart.

Determination of the free-energy difference of the adenine nucleotide translocator reaction in rat-liver mitochondria using intra- and extramitochondrial ATP-utilizing reactions

During mitochondrial oxidative phosphorylation oxidative free energy is transduced for the synthesis of ATP from ADP and phosphate. The rate of oxidative phosphorylation and consequently of oxygen consumption must be geared to the ATP requirements of the cell. Extensive studies have been carried out on the control of respiration in isolated mitochondria. Chance and Williams [l] proposed that the only factor controlling the rate of: respiration is the availability of ADP, i.e., that kinetic regulation occurs. Klingenberg [2,3], however, proposed that the rate of respiration is thermodynamically controlled by the extramitochondrial phosphate potential. Slater et al. [4],, Davis and coworkers [5-71 and Kunz and coworkers [8,9] concluded that the rate of mitochondrial respiration is controlled by the extramitochondrial ATP/ADP ratio, the adenine nucleotide translocator being the rate-limiting step. Wilson and coworkers (review [lo]) concluded that the first two sites of the respiratory chain are in near-equilibrium with the cytosolic phosphate potential and that regulation of respiration occurs at the cytochrome oxidase step. The adenine nucleotide translocator,as an integral part of the nearequilibrium system, would therefore also have to operate near to thermodynamic equilibrium. operating. The results obtained have enabled us to measure the free energy difference of the adenine nucleotide translocator reaction under flux conditions.

Analysis of control of glycolysis in ischemic hearts having heterogeneous zones of anoxia

The effects of hypercapnia have been studied in rat hearts perfused with glucose and insulin. At pH 6.7 to 6.8, two different responses were observed. One was characterized by a rapid decrease in pressure development followed by a gradual recovery of contractile function. The tissue was uniformly aerobic, ATP levels were well maintained, the cytosolic and mitochondrial pyridine nucleotides were significantly more oxidized than during control conditions, and glycolytic flux was decreased due to inhibition of phosphofructokinase. In the second type of response, there was an uneven distribution of coronary perfusion with anoxic areas appearing in regions where flow was most restricted, and myocardial function remained severely impaired. Unperfused vessels and anoxic tissue were shown to exist throughout the myocardium under these conditions. Whole-tissue ATP levels were significantly decreased and NADH levels increased, reflecting impaired electron transport and oxidative phosphorylation. The problem of interpretation of total tissue metabolite contents in heterogeneously oxygenated tissue was resolved by calculation of the proportion of aerobic to anaerobic tissue in these hearts. In the anaerobic portions of ischemic hearts, glycolysis was inhibited at the level of glyceraldehyde 3-P dehydrogenase due to increased cytosolic NADH and decreased intracellular pH. Calculations of cytosolic and mitochondrial ATP/ADP ratios and phosphate potentials suggest (a) that the cytosolic free Mg2+ concentration was 0.6 to 0.7 m M in aerobic hearts, but was much lowerin anaerobic hearts, (b) that the rate of respiration did not depend solely on the cytosolic phosphate potential but was also influenced by the mitochondrial NAD redox state, and (c) that the estimated phosphorylation potential of aerobic hearts was 3.5 kcal/mol higher in the cytosol than in the mitochondria.

Regulation of Ca2+ release from mitochondria by the oxidation-reduction state of pyridine nucleotides.

Mitochondria from normal rat liver and heart, and also Ehrlich tumor cells, respiring on succinate as energy source in the presence of rotenone (to prevent net electron flow to oxygen from the endogenous pyridine nucleotides), rapidly take up Ca(2+) and retain it so long as the pyridine nucleotides are kept in the reduced state. When acetoacetate is added to bring the pyridine nucleotides into a more oxidized state, Ca(2+) is released to the medium. A subsequent addition of a reductant of the pyridine nucleotides such as beta-hydroxybutyrate, glutamate, or isocitrate causes reuptake of the released Ca(2+). Successive cycles of Ca(2+) release and uptake can be induced by shifting the redox state of the pyridine nucleotides to more oxidized and more reduced states, respectively. Similar observations were made when succinate oxidation was replaced as energy source by ascorbate oxidation or by the hydrolysis of ATP. These and other observations form the basis of a hypothesis for feedback regulation of Ca(2+)-dependent substrate- or energy-mobilizing enzymatic reactions by the uptake or release of mitochondrial Ca(2+), mediated by the cytosolic phosphate potential and the ATP-dependent reduction of mitochondrial pyridine nucleotides by reversal of electron transport.