Energy Charge Potential Research Articles

Excessive liver oxidant stress causes mortality in response to burn injury combined with endotoxin and is prevented with antioxidants.

We studied the effect of the oral administration of a water-soluble antioxidant solution containing ascorbic acid, glutathione, and a precursor for glutathione synthesis, N-Acetyl-L-cysteine, on liver antioxidant activity, liver cell energetics, and mortality in rats in response to a 20% third-degree burn injury challenged 5 days later with an intraperitoneal injection of 30 mg/kg endotoxin. Rats with burns were fluid-resuscitated with subcutaneous Ringer's lactate solution according to the Parkland formula (4 cc/kg/%burn). Rats challenged with endotoxin 5 days after burn were given an additional 100 ml/kg of subcutaneous Ringer's lactate solution immediately after the injection of endotoxin. A group of rats with burns challenged with endotoxin 5 days after burn were given an oral antioxidant solution beginning after burn injury. Liver cell energetics were measured as tissue energy charge potential (ECP), adenosine triphosphate (ATP) content, and total adenine nucleotides. The levels of endogenous liver glutathione, catalase, vitamin C, and vitamin E were measured to monitor antioxidant status. We found that burn injury alone did not produce any mortality over the 6-day period despite a 35% decrease in liver energy charge potential resulting from a decrease in ATP, a 34% decrease in liver catalase activity, and a 20% decrease in liver vitamin C. It was interesting that glutathione increased and vitamin E remained unchanged. We found that endotoxin injury combined with burn injury produced a 61% mortality rate with a 63% decrease in liver energy charge potential, again resulting from a decrease in ATP, a 74% decrease in liver catalase activity, a 16% decrease in vitamin C, and a 29% decrease in vitamin E. Glutathione was significantly decreased compared with burn alone. We compared the liver antioxidant status of survivors with that of nonsurvivors who were killed when appearing moribund and found that glutathione was decreased by 51% and vitamin C by 73% in nonsurvivors over that in survivors, whereas catalase and vitamin E levels were comparable between the two groups. The oral administration of the antioxidants prevented mortality and the decrease in antioxidant activity and attenuated the decrease in energy charge potential. We conclude that a 20% burn produces a modest decrease in liver energy charge potential and antioxidant defenses without producing mortality. The addition of endotoxin further decreases liver antioxidant defenses, liver energy charge potential, and markedly increases mortality. Antioxidants, given post-burn, restored antioxidant defenses, attenuated the altered cell energetics, and prevented mortality, indicating oxidants to be the cause of mortality. This data also suggests that a critical value of decreases in antioxidant defenses and ATP exists, resulting in mortality.

Effects of thoracic epidural anesthesia on changes in ischemic myocardial metabolism induced by intracoronary injection of endothelin in dogs

Thoracic epidural anesthesia (TEA) has been reported to alleviate ischemic damage to the myocardium. Endothelin, an endothelium-derived peptide and a potent coronary vasoconstrictor, may contribute to poor cardiac perfusion and ischemia. The objective was to examine regional myocardial metabolism during ischemia caused by intracoronary injection of endothelin with and without TEA. The three experimental groups and three treatments were randomized. All studies were conducted in a university research laboratory. Thirty anesthetized dogs comprised the study groups. Study animals were divided into three groups of 10 animals each identified as normal saline (NS); TEA; and TEA + blood pressure controlled (TEA + BPC). The NS group had 0.5 mL/kg of normal saline injected into the T4-5 epidural space. The TEA group had 0.5 mL/kg of saline containing 1% lidocaine injected into the T4-5 space. The TEA + BPC group had blood pressure and heart rate maintained at pre-epidural injection values by partially occluding the descending aorta and by atrial pacing. Endothelin (15 pmol/kg) was bolus injected into the left anterior descending (LAD) artery of each heart. Systolic and diastolic blood pressure, heart rate, and LAD coronary blood flow (CBF) were monitored. Three minutes after injection of endothelin, myocardial tissue was sampled from the distribution of the LAD artery and from the control, left circumflex (LCx) artery. ATP, ADP, AMP, lactate, and pyruvate were measured by enzymatic methods. It was found that in each group endothelin consistently decreased LAD CBF, but the decrease was less in the TEA + BPC group. In the tissue distribution of the LAD, the levels of ATP and energy charge potential were lower, and the level of lactate was higher in the NS group than in the TEA or the TEA + BPC groups (p < 0.01). These results confirm that (1) endothelin injected into the LAD artery decreases CBF and causes selective myocardial ischemia in a fashion similar to intravascular stenosis of the LAD rather than to mechanical occlusion and (2) TEA, with or without pressure support, lessens the degree of regional ischemia induced by injection of endothelin in the LAD.

Adenine nucleotide metabolism of canine pancreas graft preserved by the two-layer (Euro-Collins' solution/perfluorochemical) method after reperfusion

We have demonstrated that oxygenation of a pancreas during preservation by the two-layer method leads continued ATP production to maintain cellular integrity and produces an extended period of preserved pancreatic viability. In this study, we examined whether the pancreas grafts preserved by the two-layer method maintained adenine nucleotides pool as substrates for ATP synthesis before reperfusion and had the ability to synthesize ATP promptly and to recover energy charge potential (ECP) of the pancreas graft on reperfusion. After preservation by the two-layer cold storage method using EC for 48 hrs (group 1, n = 5), or simple cold storage in EC for 48 hrs (group 2, n = 5), canine pancreas grafts were autotransplanted. In control (group 3, n = 5), canine pancreas grafts were autotransplanted without preservation. Tissue adenine nucleotides concentrations were measured using high performance liquid chromatography (HPLC) before and after preservation, before reperfusion and after 1 hr- and 2 hr-reperfusion. Graft survival rates were 5/5, 0/5, and 5/5, in groups 1, 2 and 3, respectively. Total adenine nucleotides before reperfusion in group 1 was almost same levels as group 2 (7.41 +/- 1.47 vs 6.64 +/- 2.23 mumol/g dry weight, N.S.). Furthermore, ATP tissue levels in group 1 before reperfusion was 2.90 +/- 0.51 mumol/g dry weight and almost same levels as group 2 (2.03 +/- 0.68 mumol/g dry weight, N.S.). However, tissue ATP levels in group 1 after 2 hrs of reperfusion (6.71 +/- 1.19 mumol/g dry weight) were significantly higher than group 2 (4.51 +/- 0.51 mumol/g dry weight, P < 0.05), and almost same levels as control (group 3) (6.32 +/- 1.62 mumol/g dry weight, N.S.). In addition, ECP in group 1 and 3 (0.90 +/- 0.03 and 0.83 +/- 0.09, respectively) after 2 hr-reperfusion, were significantly higher than the value in group 2 (0.73 +/- 0.09, P < 0.01 and P < 0.05, respectively). It was clear that recovery of ATP on reperfusion was not depend on the residual nucleotides pool before reperfusion but the ability of the pancreas graft to recover tissue ATP levels and energy charge potential after reperfusion. We conclude that oxygenation of the pancreas graft during preservation by the two-layer method allows for ATP synthesis which is essential in maintaining cellular integrity and leads to maintain the graft's ability to recover ATP levels and energy charge potential promptly after reperfusion. Consequently the pancreas graft survives.

Acute ischaemic preconditioning protects against skeletal muscle infarction in the pig

The aims were to investigate the efficacy of acute ischaemic preconditioning for protection of skeletal muscles against infarction and its effect on muscle blood flow and ischaemic muscle metabolism. The efficacy of preconditioning was tested by subjecting pig latissimus dorsi and gracilis muscles to different numbers and durations of ischaemia/reperfusion cycles before 4 h of global ischaemia. Infarction was assessed at 48 h of reperfusion, using nitroblue tetrazolium dye. Blood flow in the latissimus dorsi was measured at the end of preconditioning and 1.5 and 3.0 h of reperfusion, using the radioactive microsphere (15 microns) technique. Muscle biopsies were taken from the latissimus dorsi before ischaemia, at the end of 2 and 4 h of ischaemia, and 1.5 h of reperfusion. At least three cycles of 10 min ischaemia and 10 min reperfusion were required for preconditioning of latissimus dorsi and gracilis muscles for protection against infarction. Preconditioning reduced the total infarct size by 44% and 62% in latissimus dorsi and gracilis muscles, respectively. Preconditioning did not affect preischaemia muscle blood flow but it reduced the muscle content (preischaemia reserve) of phosphocreatine and ATP and the muscle energy charge potential (ECP) by 13.5%*, 27.5%*, and 8%* (*P < 0.05), respectively. In spite of a lower preischaemia reserve of phosphocreatine and ATP, the muscle contents of phosphocreatine and ATP and muscle ECP were maintained higher and the lactate lower (*P < or = 0.05) in the preconditioned than in the non-preconditioned (control) muscles at the end of 4 h of ischaemia [phosphocreatine 8.0(SEM 0.4) v 3.2(0.3)*; ATP 9.8(0.7) v 7.8(0.3); ECP 0.72(0.02) v 0.66(0.01)*; lactate 115.4(8.6) v 160.5(11.8)* mumol.g-1 dry muscle]. The level of ATP and ECP also remained significantly higher and the level of lactate significantly lower in the preconditioned than in the non-preconditioned latissimus dorsi muscles at 1.5 h of reperfusion. Hyperaemia was seen in the preconditioned latissimus dorsi muscles at 1.5 h of reperfusion and it subsided by the end of 3h of reperfusion. The protective effect of preconditioning can be induced in pig skeletal muscle but at a higher threshold than reported previously in pig cardiac muscle (one cycle). Preconditioning of pig skeletal muscle is associated with a lower energy metabolism during sustained ischaemia. At the present time, it is not known if this energy sparing effect is a major mechanism of ischaemic preconditioning against infarction in skeletal muscles.

The ratio of reduced glutathione/oxidized glutathione is maintained in the liver during short-term hepatic hypoxia.

Controversy persists as to whether reperfusion-induced injuries actually occur in the hepatocyte. The liver is the major source of glutathione, a scavenger of hydrogen peroxide. The aim of this study was to evaluate the sensitivity of the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) [GSH:GSSG] as an index of hepatic metabolic stress. A total of 121 rats were studied. The superior mesenteric vein (SMV) was occluded for 30 min, and this was followed by 0, 10, or 120 min of reperfusion. Total glutathione and GSSG levels in the liver, bile, and plasma were quantified, using glutathione reductase-coupled enzymatic assays. Results indicated that the hepatic GSH/GSSG ratio was maintained after an occlusion of the SMV, despite a decrease in adenosine triphosphate (ATP) level and energy charge potential. However, plasma levels of total glutathione and GSSG in the inferior vena cava increased after SMV occlusion and continued to increase after reperfusion. Biliary GSSG efflux decreased during 30-min occlusion of the SMV, and remained low even after reperfusion. The liver maintains homeostasis despite a decrease in biliary GSSG efflux, probably by secreting excess GSSG into the hepatic vein when the SMV is occluded. We conclude that the total amount of glutathione and GSSG in the plasma is directly correlated with oxidative stress in the liver.

Surgical Approach for Maintaining Nonischemic Conditions of the Liver in Acute Hepatic Vein Obstruction

To establish a possible surgical approach for preventing warm ischemic injury to the liver followed by hepatic vein occlusion (HVO), the hepatic hemodynamics and energy metabolism were investigated in an acute canine HVO model with and without hepatic arterial blood flow. Arterial blood ketone body ratio (AKBR; acetoacetate/3-hydroxybutyrate) and adenylate energy charge potential (ECP = [ATP + 1/2 ADP]/[ATP + ADP + AMP]) of the liver tissue were measured during and after 60 min of HVO. In the group with hepatic arterial blood flow, in which arterial blood was drained by hepatofugal portal flow via the venovenous bypass, total hepatic blood flow, portal vein pressure, ECP, and AKBR were maintained at almost normal level after the termination of HVO, resulting in the survival of all animals for 3 days or longer. By contrast, in the group without hepatic arterial blood flow (warm ischemic group), total hepatic blood flow was maintained at less than 60% of preischemic value, and portal vein pressure gradually increased up to twice the preischemic value. ECP decreased from 0.81 ± 0.06 to 0.71 ± 0.07 along with increasing portal venous pressure, and AKBR also decreased from 1.23 ± 0.12 to 0.63 ± 0.23, resulting in no survival longer 6 hr. It was concluded that hepatic arterial blood flow during HVO, if drained as hepatofugal portal flow, could maintain nonischemic conditions in the liver, despite vena cava obstruction, by providing an alternate outflow via reversed flow in the portal vein.

Superoxide dismutase and leupeptin prevent delayed reperfusion injury in the rat small intestine during burn shock.

Delayed fluid resuscitation during burn shock is thought to compromise the integrity of gut mucosa and allow enteric bacteria to cross the luminal wall and infect other sterile organ systems. Superoxide dismutase, a free-oxygen radical scavenger; leupeptin, a protease inhibitor; and verapamil, a calcium channel blocker, were studied to evaluate their efficacy in maintaining cellular integrity in the gut of thermally burned rats whose fluid resuscitation had been delayed. Fifty male rats weighting 280 to 320 gm were given a full-thickness scald burn covering 50% total body surface area. Ten received early fluid resuscitation beginning half an hour after burn, and 40 received fluid resuscitation delayed by 6 hours. Those receiving delayed resuscitation were given superoxide dismutase (n = 10), leupeptin (n = 10), verapamil (n = 10), or a placebo of normal saline solution (n = 10) at the time of fluid resuscitation. Ileal mucosa samples were harvested, and adenosine triphosphate, diphosphate, and monophosphate were measured. Adenosine triphosphate, total nucleotides, and energy charge potential were significantly lower in the placebo group without therapy compared with those of the early resuscitation group. Superoxide dismutase and leupeptin therapy prevented this drop in cellular energy. Total water content was significantly increased in the placebo group compared with that of the early resuscitation group; superoxide dismutase was able to prevent this increase. Data indicate that intestinal reperfusion injury in burned rats can be effectively modulated with superoxide dismutase or leupeptin therapy.

Inhibition of glyceraldehyde 3-phosphate dehydrogenase in boar spermatozoa by bromohydroxypropanone.

In the presence of 3-bromo-1-hydroxypropanone (BOP), cauda epididymal sperm obtained from mature boars produced a carbonyl compound which is assumed to be (S)-3-bromolactaldehyde. Glyceraldehyde 3-phosphate dehydrogenase was rapidly inhibited which resulted in the accumulation of dihydroxyacetone phosphate and fructose-1,6-bisphosphate, and no accumulation of lactate when fructose was the substrate. The energy charge potential of the cells declined in the presence of BOP when either fructose or glycerol were substrates. It is suggested that BOP is transformed into (S)-3-bromolactaldehyde, which is the actual inhibitor of glyceraldehyde 3-phosphate dehydrogenase, thus demonstrating BOP to be the first brominated chemical to have an anti-glycolytic action on mature sperm in vitro.

Glutamate efflux via the reversal of the sodium-dependent glutamate transporter caused by glycolytic inhibition in rat cultured astrocytes

[ 3H] l-Glutamate uptake in cultured rat astrocytes was completely reduced by 30 min preincubation with 1 mM of iodoacetic acid, a glycolytic inhibitor. This treatment significantly reduced the energy charge potential, but did not cause membrane destruction in the cultured astrocytes. To examine the effect of iodoacetic acid on the glutamate release, [ 3H] l-glutamate was preloaded into astrocytes in the presence of methionine sulfoximine, a glutamine synthetase inhibitor, and the total intracellular radioactivity was measured after 30-min treatment with 1 mM iodoacetic acid for comparison with non-treated astrocytes. During the treatment, about 40% of the total intracellular glutamate content was effluxed. This efflux could be decreased by reducing the extracellular potassium ion concentration. The intracellular sodium concentration, measured with a sodium ion-sensitive fluorescent probe (sodium-binding benzofuran isophtalate), gradually increased to 30 mM on addition of 1 mM iodoacetic acid. These results indicate that the glutamate efflux via reversal of the Na +-dependent transporter occurred during glycolytic inhibition, and which may be caused by intracellular Na + overload. Such an iodoacetic acid-induced Na + overload could be completely diminished by pretreatment with 1 μ M 5-[ N-ethyl- N-isopropyl]amiloride, a selective Na +-H + antiporter inhibitor, but even this did not stop the iodoacetic acid-induced glutamate efflux. The intracellular pH, measured by a pH-sensitive fluorescent probe [2′,7′-bis(carboxyethyl)-5,6-car-boxy-fluorescein], was gradually decreased to 7.1 by the iodoacetic acid treatment. On the other hand, iodoacetic acid-induced intracellular acidosis was more rapid and severe in the presence of5-[ N-ethyl- N-isopropyl]amiloride. These results suggest that the reversal of the Na +-dependent glutamate transporter may be caused by not only intracellular Na + overload but also intracellular acidosis.

The Effects of Isoflurane on Regional Myocardial Contractility and Metabolism in Stunned Myocardium in Acutely Instrumented Dogs

The sustained dysfunction of myocardial contractility after short periods of coronary artery occlusion and reperfusion is termed "stunned myocardium." Isoflurane may improve the recovery of regional myocardial contractility in stunned myocardium. The purpose of the present study was to determine the effects of isoflurane on 1) the restoration of regional myocardial contractility and 2) the high-energy phosphate metabolism in an acute canine model. To evaluate the effects of isoflurane on the recovery of regional myocardial contractility in stunned myocardium, the left anterior descending coronary artery (LAD) was occluded for 15 min and then reperfused for 60 min during isoflurane (1.5% end-tidal concentration) anesthesia. Regional myocardial contraction was evaluated by ultrasonic segment shortening (SegS), systolic shortening (SysS), and postsystolic shortening (PSS) measurements. High-energy phosphate metabolites were measured at 60 min after reperfusion in another group using full-thickness samples of myocardium obtained from the reperfused area (supplied by the LAD), and the nonischemic area (supplied by the left circumflex coronary artery). The concentrations of adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), creatine phosphate (CP), and lactate in the endocardial portion of the myocardium were measured and the energy charge potential (ECP) was calculated. After 60 min of reperfusion percent SegS was 67.7% +/- 6.6% vs 23.9% +/- 9.4%, isoflurane and control, respectively; percent SysS was 67.4% +/- 6.3% isoflurane vs 29.3% +/- 7.4% control; and percent PSS was 10.5% +/- 4.6% isoflurane vs 40.5% +/- 8.8% control. ATP in the reperfused area decreased in both anesthetic groups but was higher in the isoflurane (88.2%) compared to the control group (77.4%).(ABSTRACT TRUNCATED AT 250 WORDS)

Age-related alterations of skeletal muscle metabolism by intermittent hypoxia and trhanalogue treatment

The characteristics of the energy metabolism were evaluated in the gastrocnemius muscle from 3- and 24-month-old rats in normoxia or subjected to either mild or severe chronic (4 weeks) intermittent normobaric hypoxia. Furthermore, 4-week treatment with saline or the TRH-analogue posatireline was performed. The muscular concentration of the following metabolites related to the energy metabolism was evaluated: glycogen, glucose, glucose 6-phosphate, pyruvate, lactate, lactate-to-pyruvate ratio; citrate, a-ketoglutarate, succinate, malate; aspartate, glutamate, alanine; ammonia; ATP, ADP, AMP, creative phosphate; energy charge potential. Furthermore the maximum rate of the following muscular enzymes was evaluated: hexokinase, phosphofructokinase, pyruvate kinase, lactate dehydrogenase; citrate synthase, malate dehydrogenase; total NADH cytochrome c reductase; cytochrome oxidase. The age-related decrease in muscular glucose 6-phosphate, pyruvate and alanine concentrations and increase in citrate concentration were consistent with the age-related decreased hexokinase and increased citrate synthase activities. Ageing was characterized by a decrease in muscular creative phosphate concentration, while the energy mediators and the energy charge potential were unchanged. The chronic (4 weeks) intermittent normobaric mild and severe hypoxia-induced alterations of the components in the anaerobic glycolytic pathway, tricarboxylic acid cycle and energy storage, that were magnified in the skeletal muscle from the oldest animals. The effect of the chronic treatment with the TRH-analogue posatireline suggests that the action of central nervous system-acting drugs could also be related to their direct influence on the muscular biochemical mechanisms related to the energy transduction.

Short-term or long-term intracerebroventricular (i.c.v.) infusion of insulin exhibits a discrete anabolic effect on cerebral energy metabolism in the rat

Evidence for an involvement of insulin in the regulation of cerebral glycolytic glucose catabolism is becoming more and more convincing. To investigate whether short-term or long-term administration of insulin to the brain influences brain energy metabolism, we determined tissue concentrations of energy-rich phosphates in cerebral parietotemporal cortex and in hippocampus of 1-(adult) and 2-year-old (aged) male Wistar rats treated with intracerebroventricular (i.c.v.) infusion of the hormone for 1, 7, and 21 days. In cerebral parietotemporal cortex, tissue concentrations of ATP, PCr, ADP, ATP/ADP ratio and energy charge potential were all unaltered. In hippocampus, however, the concentration of PCr was significantly increased. Thus, short-term and long-term i.c.v. administration of insulin are assumed to have a discrete anabolic effect on the cerebral energy pool.

Hemodynamics and Hepatic Energy Metabolism in Canine Model of Acute Hepatic Venous Occlusion with Mesocaval Shunt

The relationship between portal hemodynamics and the energy metabolism of the liver with acute hepatic venous occlusion (HVO) was investigated by assessing the changes in the hepatic blood flow, arterial blood ketone body ratio (AKBR) and adenylate energy charge potential (ECP) of the liver tissue in canine model. Acute HVO was induced by the ligation of both the supra- and infrahepatic inferior vena cava (IVC) over the protruding ends of a heparin-coated polyethylene cannula inserted into the IVC. All dogs with only HVO (n = 5) died within 30 min. HVO dogs with additional mesocaval (MC) shunt (n = 10) survived longer than 7 days, during which time their AKBR were maintained in the normal range (over 1.0). ECP was also maintained above the normal level (over 0.850) during the 28-day period. Along with increasing portal pressure caused by the narrowing of the shunt anastomosis, the hepatic blood flow decrease gradually, resulting in a sudden decrease in AKBR and ECP when the portal pressure increased over 11 mm Hg. It is suggested that the normalization of portal pressure is one of the most important factors for maintaining the hepatic energy metabolism and that MC shunt is an effective therapy for maintaining the function of the liver with HVO, as long as portal pressure can be kept within normal range.

Effects of K + channel openers on ischemic dysfunction and metabolic disturbance in isolated perfused rat heart

The effects of two structurally different K + channel openers, KRN2391 and cromakalim, on cardiac mechanisms during ischemia and reperfusion were studied in isolated perfused rat hearts. Isolated buffer-perfused rat hearts pretreated with KRN2391, cromakalim and vehicle were subjected to 25 min of ischemia followed by 30 min of reperfusion. Before ischemia, KRN2391 (1–10 ωM) and cromakalim (1–10 ωM) increased coronary flow, but did not modify cardiac function or biochemical parameters (adenine nucleotides, energy charge potential: ECP, lactate). During ischemia, KRN2391 (3, 10 ωM) and cromakalim (10 ωM) significantly accelerated the reduction in cardiac function and attenuated the decreased levels of ATP and ECP, but did not change the lactate content. After 30 min of reperfusion, pretreatment with KRN2391 and cromakalim resulted in a significant improvement in cardiac function, ischemic contracture and biochemical parameters. Thus, both KRN2391 and cromakalim have beneficial effects on biochemical parameters during ischemia and reperfusion, effects which may be related to cardiodepression during ischemia.

Effects of iloprost, a PGI2 derivative, on ischemic myocardial energy and carbohydrate metabolism in dogs.

Effects of iloprost, which is a stable prostacyclin analogue, on the ischemic myocardium were examined in the open-chest dog heart in terms of biochemical parameters. Ischemia was initiated by ligating the left anterior descending coronary artery. When the coronary artery was ligated for 3 min, the levels or glycogen, fructose-1,6-diphosphate (FDP), adenosine triphosphate and creatine phosphate decreased, and the levels of glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), lactate, adenosine diphosphate and adenosine monophosphate increased. During ischemia, therefore, energy charge potential was significantly decreased from 0.89 +/- 0.01 to 0.82 +/- 0.01, and ([G6P] + [F6P])/[FDP] and [lactate]/[pyruvate] ratios were significantly increased from 1.75 +/- 0.30 to 29.05 +/- 5.70 and 13 +/- 3 to 393 +/- 112, respectively. Iloprost (0.1, 0.3, or 1 microgram.kg-1) was injected intravenously 5 min before the onset of ischemia. Iloprost (0.1, 0.3, and 1 micrograms.kg-1) reduced the ischemia-induced decrease in energy charge potential to 94, 74, and 86%, respectively, the increase in ([G6P] + [F6P])/[FDP] to 38, 29, 32%, respectively, and the increase in [lactate]/[pyruvate] to 67, 45, 65%, respectively. These results suggest that iloprost lessens the myocardial metabolic derangements produced by ischemia, and the most potent effect was obtained at the dose of 0.3 microgram.kg-1.

Metabolic and functional effects of the nucleoside transport inhibitor R75231 in the ischaemic and blood reperfused rabbit heart

The ability of R75231, a nucleoside transport inhibitor, to influence adenine nucleotide metabolism and enhance postischaemic functional recovery was assessed in the blood perfused rabbit heart. Hearts (n = 8 per group) from donor animals were excised and perfused with blood at 37 degrees C from a support rabbit. After 20 min of aerobic perfusion hearts were arrested with St Thomas' Hospital cardioplegic solution (2 min at 37 degrees C) and rendered globally ischaemic for 60 min. This was followed by 60 min of reperfusion. R75231 (0.1 mg.kg-1, intravenously) was given to donor and support rabbits 1 h before the experiment, control rabbits receiving the same volume of vehicle. Treatment with R75231 resulted in a 45% reduction in coronary vascular resistance in aerobically perfused control hearts, an effect that was absent during postischaemic reperfusion. Thus, before ischaemia, coronary flow was greater in R75231 treated hearts [6.6(SEM 0.8) ml.min-1] than in controls [4.3(0.6) ml.min-1; p < 0.05] but during reperfusion no significant difference was observed [4.0(0.6) v 3.6(0.3) ml.min-1]. The mean time to onset and extent of contracture during ischaemia was similar in R75231 treated and control groups, at 42(4) v 41(4) min and 27(3) v 26(6) mm Hg, respectively. Left ventricular developed pressure recovered to approximately 50% of its preischaemic value during the first 40 min of reperfusion in both groups; however, after longer durations of reperfusion, it tended to deteriorate in the R75231 treated group whereas it was maintained at a constant level in the controls [37(10) v 53(6) mm Hg, respectively; NS]. At the end of reperfusion, tissue adenosine content was 13-fold greater in the R75231 treated group, at 0.40(0.09) v 0.03(0.01) mumol.g-1 dry wt in controls; p < 0.05; the nucleotide pool, nicotinamide adenine dinucleotide phosphate content, and the energy charge potential were similar in groups. R75231 decreased coronary vascular resistance and increased coronary flow during aerobic perfusion in control hearts, an effect that was lost after ischaemia and reperfusion. R75231 also increased greatly the tissue content of adenosine but, despite this, failed to improve either the recovery of cardiac contractile function or the replenishment of the adenine nucleotide pool.

The relation of proton motive force, adenylate energy charge and phosphorylation potential to the specific growth rate and efficiency of energy transduction in Bacillus licheniformis under aerobic growth conditions.

The magnitude of the proton motive force (delta p) and its constituents, the electrical (delta psi) and chemical potential (-Z delta pH), were established for chemostat cultures of a protease-producing, relaxed (rel-) variant and a not protease-producing, stringent (rel+) variant of an industrial strain of Bacillus licheniformis (respectively referred to as the A- and the B-type). For both types, an inverse relation of delta p with the specific growth rate mu was found. The calculated intracellular pH (pHin) was not constant but inversely related to mu. This change in pHin might be related to regulatory functions of metabolism but a regulatory role for pHin itself could not be envisaged. Measurement of the adenylate energy charge (EC) showed a direct relation with mu for glucose-limited chemostat cultures; in nitrogen-limited chemostat cultures, the EC showed an approximately constant value at low mu and an increased value at higher mu. For both limitations, the ATP/ADP ratio was directly related to mu. The phosphorylation potential (delta G'p) was invariant with mu. From the values for delta G'p and delta p, a variable -->H+/ATP-stoichiometry was inferred: -->H+/ATP = 1.83 +/- 0.52 mu, so that at a given -->H+/O-ratio of four (4), the apparent P/O-ratio (inferred from regression analysis) showed a decline of 2.16 to 1.87 for mu = 0 to mu max (we discuss how more than half of this decline will be independent of any change in internal cell-volume). We propose that the constancy of delta G'p and the decrease in the efficiency of energy-conservation (P/O-value) with increasing mu are a way in which the cells try to cope with an apparent less than perfect coordination between anabolism and catabolism to keep up the highest possible mu with a minimum loss of growth-efficiency. Protease production in nitrogen-limited cultures as compared to glucose-limited cultures, and the difference between the A- and B-type, could not be explained by a different energy-status of the cells.

Absence of mitochondrial enhancement in the remnant liver after partial hepatectomy in cirrhotic rats

Changes in energy metabolism after 70% partial hepatectomy were investigated in normal and carbon-tetrachloride-(CCl4)-induced-cirrhotic rats by evaluating hepatic mitochondrial ATP synthesizing activity as well as liver tissue levels of adenine nucleotides and lipid peroxide. Preoperative concentrations of ATP and total adenine nucleotides (TAN: ATP + ADP + AMP) in mumol/g dry weight (dw) and the energy charge potential (ECP) in the cirrhotic livers were 8.53 SEM 0.25, 14.73 SEM 0.54, and 0.74 SEM 0.01, respectively, and were significantly lower than those of normal livers (12.04 SEM 0.34, 15.75 SEM 0.12, and 0.86 SEM 0.01, P less than 0.01 in TAN). There was no difference in the preoperative mitochondrial phosphorylation rate (PR = x 10(-10) mol ATP/sec per mg mitochondrial protein) between normal and cirrhotic livers (21.01 SEM 0.95 and 21.55 SEM 1.03, respectively). After hepatectomy, in the normal livers these values decreased slightly after 12 h, remained low until 48 h, and returned to the preoperative value at 72 h. PR was remarkably enhanced and reached the maximum level of 32.54 SEM 2.07 at 24 h (P less than 0.001, compared to the sham-operated rats) and gradually returned to the preoperative value at 72 h. In the cirrhotic livers, ATP and ECP levels were drastically decreased at 12 h and recovered to the preoperative levels within 24 h, while TAN level remained unchanged. Enhancement of PR was not observed in any of the cirrhotic livers during the experiment. Lipid peroxidation was transiently increased postoperatively with no difference between normal and cirrhotic livers both in the sham-operated and the hepatectomized rats. These findings suggest that the energy status was more depressed in the cirrhotic livers than in normal livers both before and after hepatectomy. This depressed energy status might be attributed to the low preoperative tissue levels of adenine nucleotides and ECP level in the cirrhotic livers as well as to the absence of the enhancement of PR in the remnant livers.

Efficacy of Leupeptin, Superoxide Dismutase, and Verapamil in Modulating Delayed Reperfusion Damage After Burn Injury

Reperfusion damage has been identified as an important factor in multiorgan failure after severe burn injury. We wondered if leupeptin, a protease inhibitor, superoxide dismutase (SOD), a scavenger of free oxygen radicals, or verapamil, a calcium antagonist, would protect the cellular energy metabolism when they were given with fluid resuscitation that was delayed 6 hours after a severe burn injury. Fifty male rats weighing 280 to 300 gm received a 50% third-degree scald burn. Ten of these received fluid resuscitation at 30 minutes and 1 1/2 hours after injury, and 40 received delayed fluid resuscitation at 6 and 7 hours after injury. Thirty of these 40 rats were given leupeptin (n = 10), SOD (n = 10), or verapamil (n = 10). Heart, liver, and kidney tissue samples were obtained 8 hours after injury; adenosine triphosphate, adenosine diphosphate, and adenosine monophosphate were measured; and the energy charge potential was calculated. Tissue water content (TWC) in lung and skeletal muscle was also determined. The adenine nucleotide pool and the energy charge potential in heart, liver, and kidney tissue were all significantly decreased (p < 0.01) in rats receiving delayed fluid resuscitation compared with those receiving early resuscitation. Leupeptin was effective in protecting the heart against reperfusion damage, and verapamil and leupeptin showed some efficacy in protecting kidney tissue. Liver tissue, however, showed no protective response with therapy. TWC was significantly decreased (p < 0.01) in skeletal muscle with SOD treatment, and though all treatments appeared to keep lung water content reduced, none was significant at p < 0.01. We thus conclude that both the decreases in heart and kidney adenine nucleotides and the increase in TWC that are caused by delayed fluid resuscitation can be attenuated with appropriate pharmacologic agents.

Adenosine formation and myocardial energy status during graded hypoxia.

Previous studies using hypoperfusion and 2-deoxyglucose infusion have revealed a biphasic relationship between myocardial energy status and adenosine release (RADO). As energy charge ([ATP] + 1/2[ADP])/([ATP] + [ADP] + [AMP]) or phosphorylation potential ([ATP]/[ADP][Pi]) is lowered there is an initial increase in RADO, but RADO declines from peak levels during severe energy depletion. This study examined the hypothesis that the same pattern of RADO exists during graded hypoxia. Isolated guinea-pig hearts were perfused at constant flow and exposed to mild (30% O2) and severe (0% O2) hypoxia in the presence of norepinephrine (NE, 6 x 10(-8) M). Phosphorylation potential and energy charge were determined using 31P-NMR spectroscopy and adenosine release into coronary venous effluent was measured. Graded hypoxia lowered energy charge and phosphorylation potential, and raised RADO. Although severe hypoxia plus NE lowered energy charge and phosphorylation potential to levels equivalent to those associated with decreased RADO during hypoperfusion or 2-deoxyglucose treatment, RADO during severe hypoxia was greater than during mild hypoxia. HCl was infused during severe hypoxia in order to reproduce the low intracellular pH seen during hypoperfusion, but HCl increased RADO rather than decreasing it. We conclude that during hypoxia, RADO does not have a biphasic relationship to phosphorylation potential or energy charge, suggesting that the regulation of adenosine formation cannot be explained solely in terms of these variables. Furthermore, intracellular acidosis is not responsible for inhibiting RADO at low phosphorylation potential and energy charge during hypoperfusion because it has no effect on RADO during severe hypoxia.