Blockade Of Glycolysis Research Articles

In vivo glycolytic equilibria in dog gracilis muscle.

While the equilibrium assumption and the validity of using total measured concentrations for near equilibrium indicator reactions have been widely tested in liver, these have not been systematically evaluated in skeletal muscle. Vascularly isolated dog gracilis muscles were stimulated via the nerve at 4 Hz, and tissue was sampled by quick freezing at rest and after 10, 15, 30, 60, and 180 s of stimulation or after stimulation in the presence of glycolytic blockade by iodoacetate. Phosphocreatine, creatine, and several glycolytic intermediates were measured in tissue extracts. The in vivo mass action ratios for triosephosphate isomerase and aldolase were evaluated relative to substrate concentrations and compared with equilibrium constants determined in vitro. Although there was evidence of substrate binding at low substrate levels for the triosephosphate isomerase reaction, the in vivo mass action ratios for both reactions stabilized at a constant value at moderate substrate levels and in glycolytically blocked muscles. It was concluded that both enzymes are in apparent equilibrium in vivo, but the equilibrium constants are lower than those determined in vitro. The mass action ratios of the combined creatine kinase, lactate dehydrogenase, glyceraldehyde-phosphate dehydrogenase and phosphoglycerate kinase reactions were determined for resting muscles. These reactions are also at equilibrium and the equilibrium constants are consistent with in vitro values.

Outward current and repolarization in hypoxic rat myocardium.

We studied the effects of brief periods (20-30 min) of hypoxia in the presence of 5 and 50 mM glucose and of glycolytic blockade (10(-4) M iodoacetic acid, IAA) on action potentials, membrane currents, and mechanical activity in rat ventricular papillary muscles using a single sucrose gap voltage-clamp technique. Steady-state outward current (iss) was determined at the end of a 500-ms clamp to the test potential following a 600-ms clamp to a holding potential of -50 mV. In the presence of 5 mM glucose, hypoxia resulted in a decrease in action potential duration (APD) and an increase in iss (on the order of 60% at 0 mV) over the potential range studied. The increase in iss did not appear to be due to an increase in leakage current or to a change in the cable properties of the preparation. Addition of 50 mM glucose prevented the change in both APD and iss with hypoxia. In addition, glycolytic blockade with IAA did not alter iss in the presence of oxygen. We conclude that an increase in iss appears to be a major factor in the abbreviation of rat ventricular action potential seen with hypoxia. Glycolysis appears to be a sufficient (with 50 mM glucose) but not necessary source of energy for the maintenance of normal iss.

Hypoxia, calcium fluxes, and inotropic state: Studies in cultured heart cells

Methods have been developed for the study of cellular mechanisms underlying a variety of physiologic and pathophysiologic phenomena using cultured chick embryo heart cells. These spontaneously beating monolayer preparations lack appreciable diffusion barriers, and interstitial space components can be defined and controlled conveniently. These properties facilitate the measurement of transmembrane fluxes and cellular contents of sodium, potassium, and calcium. Concomitantly, methods have been developed to measure the contractile state as reflected in the amplitude and velocity of movement of individual myocytes in the culture. Experiments are described in which the contractile responses of cultured heart cells to graded hypoxia demonstrated a critical P O 2 level around 12 mm Hg, below which progressive impairment of contractile function occurred. In preparations with iodoacetate-induced glycolytic blockade, enhanced sensitivity to hypoxia occurred. Additional experiments are described, demonstrating the applicability of this model system to the study of problems including the mechanism of cardiac glycoside-induced increases in inotropic state and the definition of calcium flux pathways important in excitation-contraction coupling.

Effect of acute tobacco smoke inhalation on the mechanical responses of isolated rat cardiac muscle

To evaluate the effects of acute inhalation of tobacco smoke upon the intrinsic properties of cardiac muscle, rats were exposed to cigarette smoke from 2R1 Kentucky reference cigarettes in special smoking chambers for periods of 10Min/hr for 5 hr. Animals were sacrificed, and the mechanical properties of isolated left ventricular papillary and columnar carneae preparations were studied. Mean COHb concentration of animals exposed to tobacco smoke was 13.2 ± 0.9 vol%. No significant difference was found in rats exposed to tobacco smoke when compared to shams in (1) cardiac chamber weight or chamber wet-to-dry weight ratios, (2) cardiac muscle performance in oxygenated Krebs-Henseleit solution (95% O 2, 5% CO 2), (3) muscle performance during 60 min of hypoxia (95% N 2, 5% CO 2) followed by 30 min of reoxygenation, (4) muscle performance during graded hypoxia with and without glycolytic blockade (iodoacetic acid 10 −4 m), or (5) sensitivity of myocardial performance to isoproterenol. Thus, after acute exposure to tobacco smoke, no effects on the intrinsic mechanical properties of rat cardiac muscle were demonstrable.

Glycolytic and oxidative metabolism in relation to retinal function.

Measurements of lactate production and ATP concentration in superfused rat retinas were compared with extracellular photoreceptor potentials (Fast PIII). The effect of glucose concentration, oxygen tension, metabolic inhibition, and light were studied. Optimal conditions were achieved with 5-20 mM glucose and oxygen. The isolated retina had a high rate of lactate production and maintained the ATP content of a freshly excised retina, and Fast PIII potentials were similar to in vivo recordings. Small (less than 10%) decreases in aerobic and anaerobic lactate production were observed after illumination of dark-adapted retinas. There were no significant differences in ATP content in dark- and light-adapted retinas. In glucose-free medium, lactate production ceased, and the amplitude of Fast PIII and the level of ATP declined, but the rates of decline were slower in oxygen than in nitrogen. ATP levels were reduced and the amplitude of Fast PIII decreased when respiration was inhibited, and these changes were dependent on glucose concentration. Neither glycolysis alone nor Krebs cycle activity alone maintained the superfused rat retina at an optimal level. Retinal lactate production and utilization of ATP were inhibited by ouabain. Mannose but not galactose or fructose produced lactate and maintained ATP content and Fast PIII. Iodoacetate blocked lactate production and Fast PIII and depleted the retina of ATP. Pyruvate, lactate, and glutamine maintained ATP content and Fast PIII reasonably well (greater than 50%) in the absence of glucose, even in the presence of iodoacetate. addition of glucose, mannose, or 2-deoxyglucose to medium containing pyruvate and iodoacetate abolished Fast PIII and depleted the retina of its ATP. It is suggested that the deleterious effects of these three sugars depend upon their cellular uptake and phosphorylation during the blockade of glycolysis by iodoacetate.

On the mechanism of the glucose-induced ATP catabolism in ascites tumour cells and its reversal by pyruvate.

Addition of glucose to Ehrlich-Landschütz ascites tumour cells preincubated for 30-60 min in phosphate-buffered Krebs-Ringer salt solution ("starved cells") resulted within 1-2 min in an approx. 90% decline of their ATP content and a massive accumulation of fructose 1,6-bisphosphate. These alterations, which took place under both aerobic and anaerobic conditions, were followed by a gradual spontaneous recovery with restoration of normal ATP and fructose 1,6-bisphosphate values. The transient derangement of the energy metabolism after glucose addition to starved ascites tumour cells by preventable by simultaneous addition of pyruvate or 2-oxobutyrate, or by preincubating the cells in the presence of glucose. The protective effect of pyruvate was duplicated by addition of phenazine methosulphate or NAD+ to the incubation medium. The data seem to warrant the conclusion that the glucose-induced ATP depletion is determined by a blockade of glycolysis at the stage of glyceraldehyde phosphate dehydrogenase caused by the failure of the cells to oxidize the NADH produced in the same reaction. The continued unrestrained action of 6-phosphofructokinase results in accumulation of fructose 1,6-bisphosphate, which constitutes a trap for the high-energy phosphate bonds of ATP. The primary metabolic disturbance appears to consist of a transient inhibition of pyruvate kinase with the resultant inability of the cells to maintain an unimpaired supply of pyruvate, as required for the lactate dehydrogenase-mediated oxidation of NADH. The regulatory mechanism underlying this phenomenon is discussed.

Anion exchange and volume regulation during metabolic blockade of renal cortical slices.

1. The development of swelling of rat and guinea-pig renal cortical slices was studied after metabolic blockade (hypoxia plus glycolytic blockade with iodo-acetic acid) and/or exposure to 'isotonic' high potassium, no sodium solution. 2. Swelling was greater after exposure to oxygenated high potassium solution than after metabolic blockade in physiologic Krebs-Henseleit solution. Swelling was reduced after metabolic blockade in high potassium solution compared to incubation in oxygenated high potassium solution. Increasing periods of transient metabolic blockade in Krebs-Henseleit solution progressively blunted swelling when slices were subsequently incubated in oxygenated high potassium solution. 3. Metabolic blockade in Krebs-Henseleit solution resulted in marked reductions in potassium and increases in sodium. Incubation in high potassium solution resulted in marked increases in potassium and similar low levels of sodium regardless of associated interventions. Metabolic blockade in both media resulted in significantly greater increases in renal cortical chloride than in monovalent cations (potassium plus sodium). Incubation in oxygenated high potassium solution was associated with similar increases in renal cortical chloride and total monovalent cations. 4. Renal cortical losses of solids and protein and increases in renal cortical inulin space were greater after metabolic blockade than after incubation under oxygenated conditions regardless of the incubation media. 5. These data support the conclusion that during metabolic blockade there is a significant replacement of larger intracellular anions by extracellular chloride. The loss of osmotically active intracellular anions limits the increase in renal cortical volume during metabolic inhibition and exposure to high potassium solution.

Mechanical and structural correlates of contracture induced by metabolic blockade in cardiac muscle from the rat.

We performed morphological studies of myocardial contracture to define its nature and relationship to mechanical changes occurring during metabolic blockade. Isolated rat papillary and trabecular muscles were stretched to the apices of their length-tension curves and stimulated to contract isometrically 12 times a minute at a temperature of 28 degrees C. Incomplete and total metabolic blockade were induced by 1 hour of hypoxia (95% N2, 5% CO2) or by hypoxia plus glycolytic blockade with iodoacetic acid, 10-4M, respectively. In oxygenated control preparations, mechanical performance was stable for the 60-minute experimental period. In preparations exposed to hypoxia, developed tension fell to 7 +/- 2% of prehypoxia values at 60 minutes. Contracture tension increased progressively to 2.5 +/- 0.4 g/mm2. With total metabolic blockade, developed tension declined to zero by 10 minutes, contracture tension rose to an average peak value of 5.3 +/- 0.4 g/mm2 by 15 minutes, and subsequently slowly declined. All preparations were fixed at Lmax in the muscle bath. Light and electron microscopic studies revealed focal irregularities of A, I, and Z bands with sarcomere malalignment, hypercontraction, and fiber disruption, which increased in severity with increasing metabolic blockade. Linear densities appeared in mitochondria following total metabolic blockade, but mitochondria appeared normal otherwise. Thus, myocardial contracture after metabolic blockade is a focal process beginning within the sarcomere; morphological alterations in the contractile apparatus correlate with mechanical changes and are more severe than those in the mitochondria.

Resistance of contracting myocardium to swelling with hypoxia and glycolytic blockade.

The interrelationship of myocardial metabolism, performance and tissue hydration was examined in isolated contracting rat, guinea pig and dog myocardium. Myocardial metabolism was altered by blocking aerobic, and both aerobic and anaerobic metabolism. Myocardial water content and distribution were measured in rat myocardium using 3H-inulin and 51Cr-EDTA as extracellular markers. Myocardial hydration was also evaluated by light and electron microscopy. The relative susceptibility of non-contracting slices of rat and guinea pig myocardium and kidney to swelling secondary to these interventions was also explored. Hypoxia resulted in a partially reversible reduction in mechanical function; hypoxia plus glycolytic blockade led to irreversible severe contracture and total loss of tension development. Neither hypoxia nor hypoxia plus glycolytic blockade resulted in increased total tissue or extracellular water in previously contracting preparations or in non-contracting slices of myocardium. On the other hand, there were significant increases in cellular water in similarly treated kidney slices after each intervention. Thus, despite severe, irreversible derangements of mechanical function, myocardium did not swell under conditions which produced swelling in renal cortex.

Acute cardiac ischemia and reperfusion: contractility, relaxation, and glycolysis.

The mechanical and metabolic effects of 3 min of complete global ischemia and 25 min of reperfusion were studied in the isolated rat heart. The decrease in contracile function was biphasic; a rapid 50% decline occurred in the first 10 s of ischemia, after which contractile function transiently stabilized and then fell at a slower rate. During reperfusion, recovery of relaxation was impaired relative to recovery of contractile function. A second period of ischemia and reflow produced changes in contractility, relaxation, and lactate production virtually identical to the initial one. In the absence of glycolytic blockade, tissue lactate accumulation developed, no contracture occurred, the pacing threshold did not increase, and reperfusion after 3 min of ischemia resulted in complete recovery of contractile function. Glycolytic blockade with 0.1 mM iodoacetate (IAA) prevented ischemic lactate production, accelerated the fall in contractility, caused irreversible contracture after 30 s of ischemia, an irreversible increase in pacing threshold within 3 min of ischemia, and poor recovery of contractile function with reperfusion. Thus during the first 3 min of severe ischemia, glycolysis exerted a net beneficial effect on myocardial function despite significant tissue lactate accumulation.

The role of malonyl-coa in the coordination of fatty acid synthesis and oxidation in isolated rat hepatocytes.

Fatty acid synthesis and fatty acid oxidation were examined in rat hepatocytes under a variety of experimental conditions. In cells from fed animals, glucagon acutely switched the direction of fatty acid metabolism from synthesis to oxidation. Addition of lactate plus pyruvate had the opposite effect. The inhibitory action of glucagon on fatty acid synthesis and its stimulatory effect on fatty acid oxidation were largely, but not completely, offset by the simultaneous addition of lactate plus pyruvate. Changes in cellular citrate and malonyl-CoA levels indicated that glucagon exerted its inhibitory effect on fatty acid synthesis at two levels: (i) blockade of glycolysis; and (ii) partial inhibition of a more distal step, probably acetyl-CoA carboxylase. Under all conditions, fatty acid oxidation was related in a linear and reciprocal fashion to the rate of fatty acid synthesis and the tissue malonyl-CoA content. The latter fluctuated through a range of 1 to 6 nmol per g wet weight of cells. Since malonyl-CoA inhibits carnitine acyltransferase I of liver mitochondria with a Ki in the region of 1 to 2 micron, the present studies support the concept that this compound plays a pivotal role in the coordination of hepatic fatty acid synthesis and oxidation. The ketogenic effect of glucagon on liver appears to be manifested in large part through the ability of the hormone to reduce the tissue malonyl-CoA concentration.

Changes in in vitro myocardial hydration and performance in response to transient metabolic blockade in hypertonic, isotonic, and hypotonic media.

The effect of changes in tonicity of the bathing media on in vitro myocardial water content and mechanical performance during hypoxia and total metabolic blockade (consisting of hypoxia plus glycolytic blockade) was studied in 175 isometrically contracting rat papillary muscles. Recovery of mechanical performance after metabolic blockade in hypertonic (440 mOsm), isotonic (315 mOsm), and hypotonic (190 mOsm) media was also evaluated. Myocardial water contents were 3.2 ± 0.1 cm3·g−1 dry weight in hypertonic, 3.9 ± 0.1 cm3·g−1 in isotonic and 4.5 ± 0.1 cm3·g−1 in hypotonic media. The addition of metabolic blockade did not alter myocardial water content. Return to isotonic solution resulted in restoration of ‘normal’ myocardial water content both with and without continued metabolic blockade. Developed tension fell during hypoxia in all three media, and recovered partially with reoxygenation and return to isotonic solution. Recovery was best after hypoxia in an isotonic medium and poorest after hypoxia in a hypertonic medium. Tension development was completely lost after less than 15 min of total metabolic blockade and remained absent after removal of glycolytic blockade and reoxygenation regardless of the tonicity of the bathing solution. Thus, appropriate fluid shifts between myocardium and bathing solution in response to alterations in the tonicity of the bath were observed despite total irreversible loss of tension development. In addition, the data do not support the concept that hypertonic solutions protect the myocardium during metabolic blockade.

Isolated cardiac muscle performance during fluorocarbon immersion and effects of metabolic blockade.

SummaryThe mechanical performance of isolated trabecular muscle preparations from the left ventricle of rats was evaluated while immersed in Krebs-Henseleit and fluorocar-bon solution under oxygenated conditions and during hypoxia and total metabolic blockade with combined hypoxia and iodoa-cetic acid 10-4M. Under oxygenated conditions, mechanical performance was relatively stable in both Krebs-Henseleit and fluorocar-bon solutions for 60 minutes. With glycolytic blockade, performance in both groups was relatively stable, although it was significantly increased in fluorocarbon solution in comparison to Krebs-Henseleit solution at 60 min (P < 0.01). With total metabolic blockade, mechanical performance fell to zero within 10 min in both Krebs-Henseleit and fluorocarbon solution. With hypoxia alone, mechanical performance declined more promptly and remained at lower levels in fluorocarbon solution than in Krebs-Henseleit solution (P < 0.001).

Glycogenolysis and control of anaphylactic histamine release by cyclic adenosine monophosphate-related agents: II. Modification of histamine release by glycogenolytic metabolites

d-glucose-6-phosphate (⩽ 5 × 10 −3 M), pyruvate, lactate (⩽ 1 × 10 −2 M), and dibutyryl cyclic AMP (⩽ 5 × 10 −3 M) were capable of inhibiting anaphylactic histamine release in vitro from chopped guinea pig lung. In lower concentrations, pyruvic acid and lactate, as well as dibutyryl cyclic AMP, enhanced the release. Significant synergism was observed between pyruvate (5 × 10 −3 M) and isoproterenol (1 × 10 −8 M) in the inhibition of histamine release. The inhibitory actions of isoproterenol, glucose-6-phosphate, and pyruvate were influenced by calcium ion concentration. However, beta blockade, which diminished the isoproterenol effect, was without effect on pyruvate (1 × 10 −2 M) or glucose-6-phosphate (5 × 10 −3 M) action. Reduction of histamine release as the result of blockade of glycolysis by 2-deoxy- d-glucose was slightly but reproducibly reversed by the addition of pyruvate (1 × 10 −2 M) to the release system. Glucose-6-phosphate and isoproterenol did not have this effect. The results, together with a previous study, suggest that glycogenolysis may possess a role in the anaphylactic histamine release in vitro from sensitized lung fragments.

The explanation for the blockade of glycolysis in early mouse embryos.

The reason for the failure of early-stage mouse embryos to grow on glucose alone was investigated by measurement of glucose-6-phosphate, fructose-1,6-bisphosphate plus triose phosphates, citrate, and malate in individual embryos during starvation and refeeding with glucose or glucose plus pyruvate. The results indicate a block at the 6-phosphofructokinase (EC 2.7.1.11) step at early stages, which is later removed. Although there seems to be no early difficulty in phosphorylation of glucose, maximum glucose-6-phosphate levels (and probably fructose-6-phosphate levels) are much lower at early stages than at later stages. The increase in fructose-6-phosphate with age may be the major cause of the increase in 6-phosphofructokinase activity. Unusually high citrate levels at all ages may help to keep this enzyme strongly inhibited until the increase in fructose-6-phosphate occurs. The changes in metabolite levels also indicate an early defect in mobilization of glycogen and a probably less important defect in the citrate cycle.

Phagocytosis by human blood leucocytes during suppression of glycolysis

Phagocytosis of staphylococci by human blood leucocytes was measured during suppression of glycolysis with iodoacetate, 2 × 10 −4 M, and fluoride, 2 × 10 −2 M. Both inhibitors prevented the increased respiration usually associated with phagocytosis. When disappearance of supernatant bacteria was measured, leucocytes pretreated with iodoacetate removed as many bacteria as did normal leucocytes, indicating no alteration of phagocytic function with this inhibitor. In contrast, leucocytes pretreated with sodium fluoride showed marked inhibition of phagocytosis. Cells pretreated with iodoacetate were observed to have less granule lysis after phagocytosis than normal leucocytes. Measurements of granule-associated acid phosphatase activity confirmed this observation. Cells treated with sodium fluoride became swollen, vacuolated, permeable to eosin dye, and were rarely phagocytic. In contrast, such changes were not observed with iodoacetate in concentrations up to 5 × 10 −4 M. Thus fluoride appears to have a greater effect on cell function than can be attributed to glycolytic blockade alone. Rapid loss of acid-soluble ATP from blood leucocytes occurred after phagocytosis, and was accentuated in the presence of iodoacetate. These results indicate that active glycolysis is not essential for phagocytosis by human blood leucocytes. They suggest that some pre-formed energy source may be available to these cells for this function.