Glycolysis in the rat kidney shortly after ischemia and administration of calmodulin inhibitors, AMP and NAD

It is shown in experiments on rats that the early postischemic period after 1- and 1.5-hour ischemia of kidneys is characterized by a decrease in the damage of the glycolytic system site which induces glucose-6-phosphate transformation into lactate and by an increase in the inhibition intensity of the initial hexokinase reaction of glycolysis. In the postischemic period after more prolonged (2-, 3-hour) ischemia the damage of the glycolytic system develops also at the site of glucose-6-phosphate transformation into lactate. Administration either of the nucleotide complex (NAD and AMP) or calmodulin inhibitors (aminazine and zinc sulphate) to rats prior to two-hour occlusion of kidneys vessels promotes a decrease in the inhibition of the glycolytic system activity in the postischemic period. At the same time the separate and combined application of zinc sulphate and triftazin (the most intensive calmodulin inhibitor) is not efficient. The positive effect of NAD, AMP and aminazine on the state of the glycolytic kidney system in the postischemic period correlates with the improvement of the blood microcirculation processes in them.     

Effect of mitochondria and microsomal fractions on glycolytic activity of rat brain and liver tissues under hypoxia

It is found that acute hypoxia inhibits the glycolytic activity of postmitochondrial fraction in the liver, activates in the brain, but has no effect on glycolysis under conditions of a preliminary administration of diethylaminoethylamide of parachlorophenoxyacetic acid--antihypoxic preparation. In the processes of two- and four-week interrupted training of adaptation to hypoxia the activity of the liver glycolytic system rises. Suspensions of the mitochondric and microsomal fraction added in definite ratios to the postmitochondrial fraction of the brain and liver intensify its glycolytic activity both in control and hypoxic animals. The activating effect of mitochondria is higher as compared with the control when glycolysis is decreased; when glycolysis is increased the phenomenon is not observed. A mechanism of the found changes in glycolysis and the validity of the tissue glycolysis estimation from the activity of the postmitochondrial fraction are discussed.     

Effect of glycolysis inhibition on mitochondrial function in rat brain

Inhibition of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase enhances the neural vulnerability to excitotoxicity both in vivo and in vitro through an unknown mechanism possibly related to mitochondrial failure. However, as the effect of glycolysis inhibition on mitochondrial function in brain has not been studied, the aim of the present work was to evaluate the effect of glycolysis inhibition induced by iodoacetate on mitochondrial function and oxidative stress in brain. Mitochondria were isolated from brain cortex, striatum and cerebellum of rats treated systemically with iodoacetate (25 mg/kg/day for 3 days). Oxygen consumption, ATP synthesis, transmembrane potential, reactive oxygen species production, lipoperoxidation, glutathione levels, and aconitase activity were assessed. Oxygen consumption and aconitase activity decreased in the brain cortex and striatum, showing that glycolysis inhibition did not trigger severe mitochondrial impairment, but a slight mitochondrial malfunction and oxidative stress were present.     

Inhibiting 3-phosphoglycerate kinase by EDTA stimulates the development of the cleavage stage mouse embryo

Addition of EDTA to the medium significantly enhances mouse embryo development in culture. Embryos cultured in the absence of EDTA exhibit abnormal increases in glycolytic activity that result in reduced development. Culture with EDTA was able to prevent this increase in glycolysis and, therefore, maintain developmental competence. EDTA was shown to inhibit the activity of the glycolytic enzyme, 3-phosphoglycerate kinase. Additionally, the effect of EDTA on maintaining high rates of embryo development in culture could be mimicked by the addition of Cibacron blue, an inhibitor of 3-phosphoglycerate kinase. The inhibition of 3-phosphoglycerate kinase by EDTA could be overcome by the addition of exogenous magnesium, indicating that the effect of EDTA was to reduce the availability of this co-factor to the glycolytic kinases. Embryos cultured with EDTA had significantly lower levels of intracellular magnesium compared to embryos cultured without EDTA. Therefore, the effect of EDTA appears to be as a chelator of divalent cations such as magnesium, that are required for normal activity of kinases such as 3-phosphoglycerate kinase.     

Cardiac ATP-sensitive K+ channels. Evidence for preferential regulation by glycolysis

The ability of glycolysis, oxidative phosphorylation, the creatine kinase system, and exogenous ATP to suppress ATP-sensitive K+ channels and prevent cell shortening were compared in patch-clamped single guinea pig ventricular myocytes. In cell-attached patches on myocytes permeabilized at one end with saponin, ATP-sensitive K+ channels were activated by removing ATP from the bath, and could be closed equally well by exogenous ATP or substrates for endogenous ATP production by glycolysis (with the mitochondrial inhibitor FCCP present), mitochondrial oxidative phosphorylation, or the creatine kinase system. In the presence of an exogenous ATP-consuming system, however, glycolytic substrates (with FCCP present) were superior to substrates for either oxidative phosphorylation or the creatine kinase system at suppressing ATP-sensitive K+ channels. All three groups of substrates were equally effective at preventing cell shortening. In 6 of 38 excised inside-out membrane patches, ATP-sensitive K+ channels activated by removing ATP from the bath were suppressed by a complete set of substrates for the ATP-producing steps of glycolysis but not by individual glycolytic substrates, which is consistent with the presence of key glycolytic enzymes located near the channels in these patches. Under whole-cell voltage-clamp conditions, inclusion of 15 mM ATP in the patch electrode solution dialyzing the interior of the cell did not prevent activation of the ATP-sensitive K+ current under control conditions or during exposure to complete metabolic inhibition. In isolated arterially perfused rabbit interventricular septa, selective inhibition of glycolysis caused an immediate increase in 42K+ efflux rate, which was prevented by 100 microM glyburide, a known blocker of ATP-sensitive K+ channels. These observations suggest that key glycolytic enzymes are associated with cardiac. ATP-sensitive K+ channels and under conditions in which intracellular competition for ATP is high (e.g., in beating heart) that act as a preferential source of ATP for these channels.     

Metabolic Plasticity in Resting and Thrombin Activated Platelets

Platelet thrombus formation includes several integrated processes involving aggregation, secretion of granules, release of arachidonic acid and clot retraction, but it is not clear which metabolic fuels are required to support these events. We hypothesized that there is flexibility in the fuels that can be utilized to serve the energetic and metabolic needs for resting and thrombin-dependent platelet aggregation. Using platelets from healthy human donors, we found that there was a rapid thrombin-dependent increase in oxidative phosphorylation which required both glutamine and fatty acids but not glucose. Inhibition of fatty acid oxidation or glutamine utilization could be compensated for by increased glycolytic flux. No evidence for significant mitochondrial dysfunction was found, and ATP/ADP ratios were maintained following the addition of thrombin, indicating the presence of functional and active mitochondrial oxidative phosphorylation during the early stages of aggregation. Interestingly, inhibition of fatty acid oxidation and glutaminolysis alone or in combination is not sufficient to prevent platelet aggregation, due to compensation from glycolysis, whereas inhibitors of glycolysis inhibited aggregation approximately 50%. The combined effects of inhibitors of glycolysis and oxidative phosphorylation were synergistic in the inhibition of platelet aggregation. In summary, both glycolysis and oxidative phosphorylation contribute to platelet metabolism in the resting and activated state, with fatty acid oxidation and to a smaller extent glutaminolysis contributing to the increased energy demand.     

Glycolysis in human erythrocytes containing elevated concentrations of 2, 3-P2-glycerate.

In studies on the mechanism of the inhibitory effect of 2, 3-diphosphoglycerate on glycolysis in human erythrocytes, the following results were obtained: 1) Glucose consumption and lactate production are reduced by 70 and 40 per cent relative to normal erythrocytes in red blood cells containing five times the normal amount of 2, 3, -P2-glycerate ("high-diphosphoglycerate" cells) at an extracellular pH of 7.4. The marked dependency of glycolysis on the extracellular pH observed in normal erythrocytes is almost completely lost in the "high-diphosphoglycerate" cells. 2) About 50 per cent of the inhibition of glycolysis in "high-diphosphoglycerate" cells can be accounted for by the 2, 3-P2-glycerate-induced decrease of the red-cell pH. This fall of the red-cell pH which occurs as a conswquence of the Donnan effect of the non-pentrating 2, 3-P2-glycerate anion leads to a reduction of the glycolytic rate due to the properties of the enzyme phosphofructokinase. 3) The remaining part of the inhibitory effect must be attributed to an inhibition by 2, 3-P2-glycerate of glycolytic enzymes. From measurements of glycolytic rates and of the concentrations of glycolytic intermediates in the absence and presence of methylene blue it is concluded that the hexokinase reaction is inhibited by an elevation of 2, 3-P2-glycerate concentration in "high-diphosphoglycerate" cells suggests that also the enzyme pyruvate kinase is inhibited by 2, 3-P2-glycerate. 4) The dependencies of net-change of 2, 3-P2-glycerate concentration on the red-cell pH are identical in normal and "high-diphosphoglycerate" cells indicating that the balance between formation and decomposition of 2, 3-P2-glycerate is the same in erythrocytes with normal and very high compositions of 2, 3-P2-glycerate.     

A glycolytic marker test for individualizing the selection of chemical compounds for antitumor activity]

Based on literature data on effects of various preparations on the glycolysis in tumor and normal cells, a glycolytic molecular biochemical marker is proposed to screen chemical substances as potential antitumor drugs. A glycolytic specificity was noted in tumor cells which was regarded as a criterion for distinction of tumor cells from normal ones and among various histotypes of tumor cells as well as for the selective sensitivity of tumor cells to a substance. 17 of 38 substances tested were observed to inhibit glycolysis in tumor cells. The testing chemical substances for an antitumor activity with application of the glycolytic marker is recommended. A possibility is discussed of applying the marker for testing potential antitumor drugs, their individualization, and genetic typing.     

Effect of calcium on brain metabolism in vitro

In attempts to distinguish between direct and indirect effects of Ca on brain cell metabolism, respiration, glycolysis, ATP, phosphocreatine, incorporation of [¹⁴C] leucine into protein, and accumulation of⁴⁵Ca was determined in brain slices. Incubation was carried out in normal salt-balanced medium, in high-potassiumor ouabain-containing medium under aerobic and anaerobic conditions. Calcium ions inhibited slightly glycolysis and respiration in normal medium and activated amino acid incorporation into proteins. Levels of ATP and phosphocreatine remained normal. These effects were interpreted as due to a stabilization of plasma membranes by Ca ions to prevent their spontaneous depolarization. Incubation of slices in high-potassium and ouabain media in aerobic conditions in the presence of Ca resulted in activation of respiration and glycolysis, decrease of ATP and phosphocreatine levels, and inhibition of amino acid incorporation into proteins. The disturbances in energy metabolism, caused by the respiration-linked Ca uptake in brain mitochondria and concomitant inhibition of oxidative phosphorylation, may lead to the inhibition of amino acid incorporation into proteins. An increase in Ca levels in the cytoplasm may only be expected in anaerobic conditions during the incubation in high-potassium and ouabain media. This is manifested by a direct inhibition of glycolysis by Ca ions and a drastic decrease of ATP and phosphocreatine in slices. The results suggest that stimulation of aerobic glycolysis and inhibition of anaerobic glycolysis by Ca may explain the unknown mechanism of the so-called reversed Pasteur effect of brain slices incubated in high-potassium media.     

Characteristics of rabbit muscle adenylate kinase inhibition by ascorbate

Earlier studies [1-3] showed that of the glycolytic enzymes, the muscle isozymes PFK-1, LDH, and AK were inhibited by ascorbic acid. These studies on the characteristics of the inhibition of RMAK by ascorbate are part of a hypothesis [3] that ascorbate facilitates the storage of skeletal muscle glycogen by inhibiting glycolysis when the muscle is at rest. These studies examine conditions for RMAK inhibition, prevention of inhibition, and reversal of ascorbate inhibition. We found that the concentration of RMAK was an important condition for inhibition. Above 200 nM RMAK, inhibition by ascorbate could not be demonstrated and below that concentration RMAK became increasingly sensitive to ascorbate inhibition. Associated with increased sensitivity to inhibition by ascorbate is a deviation from a linear to a concave relationship between low RMAK concentrations and enzyme activity. At low RMAK concentrations, the concave relationship becomes convex in the presence of muscle aldolase. In addition, aldolase reverses inhibitions by ascorbate. A comparison of inhibition of RMAK byascorbate and inhibition of LDH-m4 [3] is discussed. Other proteins prevent RMAK inhibition but do not reverse inhibition by ascorbate. The role of RMAK as a factor in the control of the rate of glycolysis is presented as is the role of compartmentalization with respect to the proposed role for ascorbate inhibition.     

The relation between inhibition of glycolysis and stimulation of oxygen uptake due to glucagon in livers from rats in different metabolic conditions

The relation between the effects of glucagon on oxygen consumption and glycolysis in livers from rats under different metabolic conditions was examined. Respiration of substrate-free perfused livers with different glycolytic fluxes, induced by changes in the pattern of food intake, responds differently to the infusion of 1 nM glucagon. The increases in oxygen uptake caused by 1 nM glucagon correlate reasonably well with the absolute decreases in glycolysis. The degree of inhibition of glycolysis is approximately constant (58 per cent) for all metabolic conditions. When no recovery of glycolysis occurs upon cessation of glucagon infusion, the same happens with oxygen consumption, which remains stimulated. It is concluded that in livers with no appreciable biosynthetic activities, the action of glucagon on respiration and glycolysis may be interpreted in terms of an interaction of interpreted in terms of an interaction of cytosolic and mitochondrial ATP generating processes.     

Oocyte control of metabolic cooperativity between oocytes and companion granulosa cells: energy metabolism

Intercellular communication between oocytes and granulosa cells is essential for normal follicular differentiation and oocyte development. Subtraction hybridization was used to identify genes more highly expressed in cumulus cells than in mural granulosa cells of mouse antral follicles. This screen identified six genes involved in glycolysis: Eno1, Pkm2, Tpi, Aldoa, Ldh1, and Pfkp. When oocytes were microsurgically removed from cumulus cell-oocyte complexes, the isolated cumulus cells exhibited decreased expression levels of genes encoding glycolytic enzymes, glycolysis and activity of the tricarboxylic acid (TCA) cycle. These decreases were prevented by culturing the cumulus cells with paracrine factors secreted by fully grown oocytes. Paracrine factors from fully grown oocytes exhibited greater ability than those from growing oocytes to promote expression of genes encoding glycolytic enzymes and glycolysis in the granulosa cells of preantral follicles. However, neither fully grown nor growing oocytes secreted paracrine factors affecting activity of the TCA cycle. These results indicate that oocytes regulate glycolysis and the TCA cycle in granulosa cells in a manner specific to the population of granulosa cells and to the stage of growth and development of the oocyte. Oocytes control glycolysis in granulosa cells by regulating expression levels of genes encoding glycolytic enzymes. Therefore, mouse oocytes control the intercellular metabolic cooperativity between cumulus cells and oocytes needed for energy production by granulosa cells and required for oocyte and follicular development.     

Metabolic activity of the Sticker's lymphosarcoma

Some parameters (glycolysis, respiration, levels of glycolytic enzymes) of the lymphoid cells from the Sticker's lymphosarcoma were established in order to better define the biochemical behavior of the venereal tumor of the dog. For comparative purposes lymphocytes from peripheral blood of normal and tumor-bearing dogs were also studied. Lactic acid produced by the tumor cells during aerobic glycolysis is liberated in the reaction medium. Oxygen uptake is enhanced in the presence of succinate, but not with pyruvate, alpha-ketoglutarate, or malate as substrates. Higher levels of some of the enzymes from the glycolytic pathways as well as differences on the physicochemical and kinetic properties of the glycolytic regulatory enzymes are found in Sticker's tumor cells, when compared with the lymphocytes from peripheral blood of normal and tumor-bearing dogs. A fructose-bisphosphate positively modulated pyruvatekinase is found in the tumor cells.     

Reactive changes in the capillaries of the mammillary bodies of the brains of aging animals

As electron microscopic investigation of the capillary wall in the mammillary bodies has demonstrated, in old animals the number of organells in the endothelial cells decreases; discomplexion and reduction of mitochondrial crists occur, their matrix becomes cleared; myelin-like structures are formed. In pericytes pigment inclusions are accumulating. Noncellular component of the basal layer loses its regular structure and foci of hydratation appear in it. When pharmacological loadings (adrenaline, aminazine) are applied to old animals, distrophic processes in the vascular wall increase; that results in disturbance of permeability, sharp hydratation of the basal layer, edema of the tissue elements around the brain and, hence, in a more prolonged restorative period.     

Mechanism of inhibition of the glycolytic activity of the myocardium during the early postnatal period]

Reduction of the activity of glycolysis and glycogenolysis regularly observed in the developing cardiac muscle was not associated with reduction of the glycolytic system, but was due to the regulatory inhibition of the phosphofructokinase link in the glycolytic link. This was indicated by a 4.5-fold increase in the rat heart of the ratio of the active masses of the phosphofructokinase reaction in the course of 2 weeks of the postnatal development.     

Balancing of mitochondrial and glycolytic ATP production and of the ATP-consuming processes of Ehrlich mouse ascites tumour cells in a high phosphate medium

A balance of energy budgeting of Ehrlich mouse ascites tumour cells including mitochondrial and glycolytic ATP production and about 80% of ATP consumption in a high phosphate medium is presented. In the share of glycolysis was about one-third of the total ATP production, more than twice that found in a low phosphate medium. The extent of a single energy reaction was assessed from the decrease of coupled oxygen consumption and lactate formation following the specific inhibition of this process. The inhibitory effects on coupled respiration and glycolysis were identical for the energy consuming processes measured: protein turnover, Na+/K(+)-ATPase, Ca2(+)-transport and RNA synthesis.     

Rat brain Mg2+-ATPase activity and Ca2+ and Mg2+ concentration in the presence of amizil and arecoline]

The effect of benactyzine (the central cholinolytic) in a dose of 40 mg/kg and arecoline (cholinomimetic) in a dose of 2.5 mg/kg on the activity of Mg2+-dependent ATP-ase and the content of Ca2+ and Mg2+ ions in the brain was studied in rats. It was shown that benactyzine and arecoline evoked a biphasic change in the activity of the enzyme and the electrolyte content. A conclusion was drawn that the enzyme inhibition was connected with the accumulation of Ca2+ ions in the brain tissue, whereas its inhibition--with the Mg2+ ion accumulation. It is supposed that throught these effects benactyzine and arecoline influenced the release and retention of the neuromediators in the tissue depot.     

Modeling cancer glycolysis

Most cancer cells exhibit an accelerated glycolysis rate compared to normal cells. This metabolic change is associated with the over-expression of all the pathway enzymes and transporters (as induced by HIF-1α and other oncogenes), and with the expression of hexokinase (HK) and phosphofructokinase type 1 (PFK-1) isoenzymes with different regulatory properties. Hence, a control distribution of tumor glycolysis, modified from that observed in normal cells, can be expected. To define the control distribution and to understand the underlying control mechanisms, kinetic models of glycolysis of rodent AS-30D hepatoma and human cervix HeLa cells were constructed with experimental data obtained here for each pathway step (enzyme kinetics; steady-state pathway metabolite concentrations and fluxes). The models predicted with high accuracy the fluxes and metabolite concentrations found in living cancer cells under physiological O(2) and glucose concentrations as well as under hypoxic and hypoglycemic conditions prevailing during tumor progression. The results indicated that HK≥HPI>GLUT in AS-30D whereas glycogen degradation≥GLUT>HK in HeLa were the main flux- and ATP concentration-control steps. Modeling also revealed that, in order to diminish the glycolytic flux or the ATP concentration by 50%, it was required to decrease GLUT or HK or HPI by 76% (AS-30D), and GLUT or glycogen degradation by 87-99% (HeLa), or decreasing simultaneously the mentioned steps by 47%. Thus, these proteins are proposed to be the foremost therapeutic targets because their simultaneous inhibition will have greater antagonistic effects on tumor energy metabolism than inhibition of all other glycolytic, non-controlling, enzymes.     

Combined enhancement of microtubule assembly and glucose metabolism in neuronal systems in vitro: decreased sensitivity to copper toxicity.

Brain cell-free extract greatly stimulates the polymerization rate of purified tubulin with a reduction of the nucleation period and without a significant alteration of the final assembly state. This effect is mimicked by neuroblastoma extract at 10-fold lower extract concentration, but not by excess muscle extract. Copper inhibits microtubule assembly in vitro but in the presence of brain extract the copper effect is suspended. Electron microscopic images showed that intact microtubules are formed and decorated by cytosolic proteins in the absence and presence of copper, while the copper alone induces the formation of S-shaped sheets and oligomeric threads. The flux of triosephosphate formation from glucose is enhanced by microtubules in brain extract, but not in muscle extract. Copper inhibits the glycolytic flux; however, the presence of microtubules not only suspends the inhibition by copper but the activation of glycolysis by microtubules is also preserved. We conclude that the organization of neuronal proteins modifies both the rates of microtubule assembly and glycolysis, and reduces their sensitivities against the inhibition caused by copper.