Adenine Nucleotide Content Research Articles

Heat-shock preconditioning reduces oxidative protein denaturation and ameliorates liver injury by carbon tetrachloride in rats

Membrane lipids and cytosolic proteins are major targets of oxidative injury. This study examined the effect of heat-shock preconditioning associated with the induction of heat-shock protein 72 on liver injury, from the aspect of lipid peroxidation and protein denaturation after carbon tetrachloride (CCl4) administration in rats--one of the representative oxidative injuries. Male Wistar rats were divided into two groups, group HS (preconditioned by heat exposure) and group C (not preconditioned). Expression of HSP72 in the liver tissue was confirmed by Western blot analysis. After a 48-h recovery period, all rats were given CCl4 intragastrically. Liver damage was assessed by measuring serum liver-related enzyme levels and adenine nucleotide concentration in the liver tissue. Lipid peroxidation and protein denaturation were evaluated by measuring tiobarbituric acid reactive substances (TBARS) and by immunohistochemical staining of 4-hydroxy-2-nonenal(HNE)-modified proteins in the liver. Survival rates of the rats after CCl4 administration were also compared. Expression of HSP72 was clearly detected in group HS, but not in group C. Heat-shock preconditioning significantly improved the survival rate, suppressed the increase in liver-related enzyme levels and maintained adenosine triphosphate levels (P<0.01 each). HNE-modified proteins--denatured proteins by free radical attack--were significantly less stained in group HS than in group C (P<0.05). However, TBARS levels did not differ between groups. Because heat-shock preconditioning did not alter TBARS levels but reduced HNE-modified proteins in association with the expression of HSP72, it is suggested that HSP72 did not prevent lipid peroxidation but decreased the lipid peroxidation-induced denaturation of proteins. This seemed to be a mechanism of heat-shock preconditioning to ameliorate oxidative liver injury.

Redox state and energy metabolism during liver regeneration: alterations produced by acute ethanol administration.

Ethanol metabolism can induce modifications in liver metabolic pathways that are tightly regulated through the availability of cellular energy and through the redox state. Since partial hepatectomy (PH)-induced liver proliferation requires an oversupply of energy for enhanced syntheses of DNA and proteins, the present study was aimed at evaluating the effect of acute ethanol administration on the PH-induced changes in cellular redox and energy potentials. Ethanol (5 g/kg body weight) was administered to control rats and to two-thirds hepatectomized rats. Quantitation of the liver content of lactate, pyruvate, beta-hydroxybutyrate, acetoacetate, and adenine nucleotides led us to estimate the cytosolic and mitochondrial redox potentials and energy parameters. Specific activities in the liver of alcohol-metabolizing enzymes also were measured in these animals. Liver regeneration had no effect on cellular energy availability, but induced a more reduced cytosolic redox state accompanied by an oxidized mitochondrial redox state during the first 48 hr of treatment; the redox state normalized thereafter. Administration of ethanol did not modify energy parameters in PH rats, but this hepatotoxin readily blocked the PH-induced changes in the cellular redox state. In addition, proliferating liver promoted decreases in the activity of alcohol dehydrogenase (ADH) and of cytochrome P4502E1 (CYP2E1); ethanol treatment prevented the PH-induced diminution of ADH activity. In summary, our data suggest that ethanol could minimize the PH-promoted metabolic adjustments mediated by redox reactions, probably leading to an ineffective preparatory event that culminates in compensatory liver growth after PH in the rat.

HERBICIDE-INDUCED MITOCHONDRIAL AND CELLULAR LIVER TOXICITY: A REVIEW OF PARAQUAT, DINOSEB, AND 2, 4-D EFFECTS

This review articleis a synthesis of the toxic effects induced by dinoseb, 2, 4-D, and paraquat on liver mitochondrial bioenergetics and hepatic cells. Liver mitochondria and hepatocytes were used as models for toxicological studies, owing to the similarities of the organization and function among mammalian species and the particular role of the liver in cell detoxification mechanisms. The interactions of these herbicides, all of which belong to different chemical families, on mitochondrial respiration, membrane potential, redox complex activities, ATPase, and ATP synthase activities are reviewed and analyzed in detail. Additionally, the cytotoxic effects on isolated hepatocytes regarding cell viability, glutathione status, adenine and pyridine nucleotide contents, lipid peroxidation, protein thiols, and intracellular calcium are discussed. This review article calls attention to the role of mitochondria in the development of cellular toxicity by these herbicides.

Plasma hypoxanthine and ammonia in humans during prolonged exercise.

In this study we examined the time course of changes in the plasma concentration of oxypurines [hypoxanthine (Hx), xanthine and urate] during prolonged cycling to fatigue. Ten subjects with an estimated maximum oxygen uptake (VO2(max)) of 54 (range 47-67) ml x kg(-1) x min(-1) cycled at [mean (SEM)] 74 (2)% of VO2(max) until fatigue [79 (8) min]. Plasma levels of oxypurines increased during exercise, but the magnitude and the time course varied considerably between subjects. The plasma concentration of Hx ([Hx]) was 1.3 (0.3) micromol/l at rest and increased eight fold at fatigue. After 60 min of exercise plasma [Hx] was >10 micromol/l in four subjects, whereas in the remaining five subjects it was <5 micromol/l. The muscle contents of total adenine nucleotides (TAN = ATP+ADP+AMP) and inosine monophosphate (IMP) were measured before and after exercise in five subjects. Subjects with a high plasma [Hx] at fatigue also demonstrated a pronounced decrease in muscle TAN and increase in IMP. Plasma [Hx] after 60 min of exercise correlated significantly with plasma concentration of ammonia ([NH(3)], r = 0.90) and blood lactate (r = 0.66). Endurance, measured as time to fatigue, was inversely correlated to plasma [Hx] at 60 min (r = -0.68, P < 0.05) but not to either plasma [NH(3)] or blood lactate. It is concluded that during moderate-intensity exercise, plasma [Hx] increases, but to a variable extent between subjects. The present data suggest that plasma [Hx] is a marker of adenine nucleotide degradation and energetic stress during exercise. The potential use of plasma [Hx] to assess training status and to identify overtraining deserves further attention.

Effect of adenine nucleotide pool size in mitochondria on intramitochondrial ATP levels

Net adenine nucleotide transport into and out of the mitochondrial matrix via the ATP-Mg/P i carrier is activated by micromolar calcium concentrations in rat liver mitochondria. The purpose of this study was to induce net adenine nucleotide transport by varying the substrate supply and/or extramitochondrial ATP consumption in order to evaluate the effect of the mitochondrial adenine nucleotide pool size on intramitochondrial adenine nucleotide patterns under phosphorylating conditions. Above 12 nmol/mg protein, intramitochondrial ATP/ADP increased with an increase in the mitochondrial adenine nucleotide pool. The relationship between the rate of respiration and the mitochondrial ADP concentration did not depend on the mitochondrial adenine nucleotide pool size up to 9 nmol ADP/mg mitochondrial protein. The results are compatible with the notion that net uptake of adenine nucleotides at low energy states supports intramitochondrial ATP consuming processes and energized mitochondria may lose adenine nucleotides. The decrease of the mitochondrial adenine nucleotide content below 9 nmol/mg protein inhibits oxidative phosphorylation. In particular, this could be the case within the postischemic phase which is characterized by low cytosolic adenine nucleotide concentrations and energized mitochondria.

Selective detection of adenosine A 1 receptor-dependent G-protein activity in basal and stimulated conditions of rat brain [ 35S]guanosine 5′-(γ-thio)triphosphate autoradiography

[ 35S]Guanosine 5′-(γ-thio)triphosphate autoradiography is a novel technique to detect receptor-dependent activation of G-proteins in brain tissue sections. While an increasing number of reports using this approach are beginning to appear, little effort has been directed to the identification of factors responsible for the heterogeneously distributed [ 35S]guanosine 5′-(γ-thio)triphosphate signal in basal conditions. The present study demonstrates that endogenously formed adenosine generates a widespread and prominent adenosine A 1 receptor-dependent signal in basal conditions using this technique. Treatment of rat brain tissue sections with the A 1-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine dose-dependently ( ec 50<10 nM) suppressed basal [ 35S]guanosine 5′-(γ-thio)triphosphate binding in a region-specific manner, an effect fully mimicked by the adenosine-depleting enzyme adenosine deaminase, and less so by the A 1 antagonist cirsimarin and by caffeine. That adenosine was continuously formed during the incubation is supported by the constant requirements of adenosine deaminase in order to suppress basal radioligand binding and further by the fact that low micromolar concentrations of adenine nucleotides evoked only adenosine-mimicking and fully 8-cyclopentyl-1,3-dipropylxanthine-sensitive binding responses. In the presence of adenosine deaminase, all responses to adenine nucleotides were abolished, indicating that prior conversion to adenosine was required. Upon stimulation, this technique selectively detected A 1 receptor-activated G-proteins, as the non-selective agonists adenosine and 2-chloroadenosine and the A 1-selective agonist N 6- p-sulfophenyladenosine all evoked only 8-cyclopentyl-1,3-dipropylxanthine-sensitive responses in identical gray matter areas, and also in several white matter areas such as the corpus callosum, anterior commissure, optic tract and cerebellar white matter. Dose–response studies revealed region-specific differences in the magnitude of A 1 receptor-stimulated G-protein activation, with the highest response (nine-fold over basal) detectable in the hippocampus. No response to the A 2A-selective agonist 2-[(2-aminoethylamino)carbonylethylphenylethylamino]-5′- N-ethylcarboxamidoadenosine or the A 3-selective agonist 2-chloro- N 6-(3-iodobenzyl)-adenosine-5′- N-methyluronamide was detected in any region. These data reveal that a significant amount of noise inherent to [ 35S]guanosine 5′-(γ-thio)triphosphate autoradiography can be eliminated by removal of the adenosine signal, a step likely facilitating detection of responses to other receptors. Furthermore, the data establish [ 35S]guanosine 5′-(γ-thio)triphosphate autoradiography as a novel and selective approach to directly assess A 1 receptor–G-protein coupling in anatomically defined regions of the central nervous system.

Ectonucleotidases of Avian Gizzard Smooth Muscle and Liver Plasma Membranes: A Comparative Study

Extracellular nucleotides, e.g., ATP, ADP, and UTP, are important signaling molecules which elicit various physiological responses in different tissues. Their degradation is catalyzed by ectonucleotidases which are located on cell surfaces. Most tissues have a mixed population of ectonucleotidases. In this report, the ATP and ADP hydrolyzing ectonucleotidases of chicken gizzard smooth muscle and liver plasma membranes were studied. The two membranes exhibited marked differences in the ratio of ATPase/ADPase activities, activation by divalent cations, thermal stability, responses to detergents and cross-linking agents, and sensitivity to several enzyme inhibitors. The ATPase activity of chicken gizzard membranes is (i) labile to heat and detergents; (ii) activated by concanavalin A and disuccinimidyl suberate, both cross-linking agents; (iii) inhibited by mercurials; and (iv) insensitive to high concentrations of azide, a known inhibitor of ecto-ATP diphosphohydrolases (ecto-ATP/Dase). In contrast, the liver membrane ATPase and ADPase activities are more stable to treatment by heat and detergents and insensitive to cross-linking agents and mercurials, but are inhibited by azide. A low ADP hydrolase activity in the gizzard membranes could be distinguished from both the gizzard ATPase and the liver ATPase/ADPase. This ADP hydrolase, which is markedly stimulated by NBD-Cl, accounts for most of the ADP hydrolysis activity in gizzard membranes. It is concluded that the major ectonucleotidase in the gizzard membranes is an ecto-ATPase whereas that in the liver membranes is an ecto-ATP/Dase. That both membranes contain a mixed population of the ecto-ATPase and ecto-ATP/Dase, but in different proportions, is further demonstrated by immunochemical characterization. The different composition of ectonucleotidases in the two membranes is expected to have an important effect on the regulation of hydrolysis of extracellular ATP as well as the concentration of extracellular adenine nucleotides in the gizzard and liver tissues.

Rat cultured astrocytes release guanine-based purines in basal conditions and after hypoxia/hypoglycemia

Brain ischemia stimulates release from astrocytes of adenine-based purines, particularly adenosine, which is neuroprotective. Guanosine, which has trophic properties that may aid recovery following neurological damage, is present in high local concentrations for several days after focal cerebral ischemia. We investigated whether guanine-based purines, like their adenine-based counterparts, were released from astrocytes and whether their release increased following hypoxia/hypoglycemia. HPLC analysis of culture medium of rat astrocytes showed spontaneous release of endogenous guanine-based purines at a higher rate than their adenine-based counterparts. The concentration of guanosine (approximately 120 nM) and adenosine (approximately 43 nM) in the culture medium remained constant, whereas concentrations of adenine and guanine nucleotides, particularly GMP, and their metabolites increased with time. Exposure of the cultures to hypoxia/hypoglycemia for 30 min increased the extracellular concentration of adenine-based purines by 2.5-fold and of guanine-based purines by 3.5-fold. Following hypoxia/hypoglycemia extracellular adenine nucleotide levels increased further. Adenosine concentration increased, but not proportionally to nucleotide levels. Accumulation of adenosine metabolites indicated it was rapidly metabolized. Conversely, the concentrations of extracellular guanine-based nucleotides remained elevated and the concentration of guanosine continued to increase. These data indicate that astrocytes are a major source of guanine-based purines, the release of which is markedly increased following hypoxia/hypoglycemia, permitting them to exert neurotrophic effects.

Cytoprotective actions of estrogens against tert-butyl hydroperoxide-induced toxicity in hepatocytes

Estrogens are effective antioxidants in diverse biological systems. Despite their antioxidant activities, it is not known yet whether estrogens prevent or alleviate liver toxicity induced by oxidative stress. In the present work, we studied this possibility by examining in vitro the protective potential of different estrogen compounds (17β-estradiol, 2-hydroxyestradiol, and diethylstilbestrol) against tert-butyl hydroperoxide-induced hepatocyte damage. Various parameters such as cell viability, lipid peroxidation, adenine nucleotide content, and thiol status were measured as an index of cytotoxicity. The protective effects of estrogens were compared to those of the iron chelator deferoxamine. The molecules tested prevented oxidant-induced cell death differently, showing variable degrees of protection. Deferoxamine was the most potent agent, followed by diethylstilbestrol and 2-hydroxyestradiol, 17β-estradiol being the least efficient. The inhibitory effects on lipid and thiol oxidations paralleled the effects on cell viability. The molecules also reduced the oxidant-induced ATP depletion, except for 17β-estradiol which had no effect on the decreased ATP levels. Our results suggest that the mechanisms of the preventive actions of estrogens may be related not only to their antioxidant activity against free radicals, but also and to a lesser extent to the maintenance of the normal redox status of the cell, which partially recovers the intracellular GSH levels.

Diabetes-induced changes in lens antioxidant status, glucose utilization and energy metabolism: effect of DL-alpha-lipoic acid.

The study was aimed at evaluating changes in lens antioxidant status, glucose utilization, redox state of free cytosolic NAD(P)-couples and adenine nucleotides in rats with 6-week streptozotocin-induced diabetes, and to assess a possibility of preventing them by DL-alpha-lipoic acid. Rats were divided into control and diabetic groups treated with and without DL-alpha-lipoic acid (100 mg x kg body weight(-1) x day(-1), i.p.). The concentrations of glucose, sorbitol, fructose, myo-inositol, oxidized glutathione, glycolytic intermediates, malate, alpha-glycerophosphate, and adenine nucleotides were assayed in individual lenses spectrofluorometrically by enzymatic methods, reduced glutathione and ascorbate--colorimetrically, and taurine by HPLC. Free cytosolic NAD+:NADH and NADP+:NADPH ratios were calculated from the lactate dehydrogenase and malic enzyme systems. Sorbitol pathway metabolites were found to increase, and antioxidant concentrations were reduced in diabetic rats compared with controls. The profile of glycolytic intermediates (increase in glucose 6-phosphate and fructose 6-phosphate, decrease in fructosel,6-diphosphate, increase in dihydroxyacetone phosphate, 3-phosphoglycerate, phosphoenolpyruvate, pyruvate, and no change in lactate), and 5.9-fold increase in alpha-glycerophosphate suggest diabetes-induced inhibition of glycolysis. Free cytosolic NAD+:NADH ratios, ATP levels, ATP/ADP x inorganic phosphate (Pi), and adenylate charge were reduced in diabetic rats while free cytosolic NADP+:NADPH ratios were elevated. Diabetes-induced changes in the concentrations of antioxidants, key glycolytic intermediates, free cytosolic NAD+:NADH ratios, and energy status were partially prevented by DL-alpha-lipoic acid, while sorbitol pathway metabolites and free cytosolic NADP+:NADPH ratios remained unaffected. In conclusion, diabetes-induced impairment of lens antioxidative defense, glucose intermediary metabolism via glycolysis, energy status and redox changes are partially prevented by DL-alpha-lipoic acid. The findings support the important role of oxidative stress in lens metabolic imbalances in diabetes.

Recovery of Microvascular Blood Perfusion and Energy Metabolism of the Obstructed Rabbit Urinary Bladder after Relieving Outlet Obstruction

We investigated the changes in urinary bladder microvascular blood perfusion and energy metabolism following outlet obstruction and after relieving the obstruction. We induced mild bladder outlet obstruction in male New Zealand White rabbits. Following 2 weeks of obstruction, one group of animals (n = 6) was sacrificed, while outlet obstruction was relieved in three additional groups, which were sacrificed 1 (n = 5), 2 (n = 5) and 4 (n = 5) weeks after relieving the obstruction. Seven sham-operated rabbits served as controls. Before obstruction, immediately before relieving the obstruction and preceding the sacrifice, the microvascular blood perfusion of the urinary bladder was measured using a laser Doppler blood flowmeter. The detrusor content of phosphocreatine and adenine nucleotides was determined by high-performance liquid chromatography. The results showed: (1) 2 weeks of outlet obstruction significantly decreased the bladder microvascular blood perfusion, which recovered gradually after relief of the obstruction and returned to the control level by 4 weeks of obstruction reversal; (2) outlet obstruction reduced detrusor energy charge and phosphocreatine content, which were restored in parallel after relieving the obstruction; by 4 weeks the bladder had regained their normal energy producing capability; (3) bladder microvascular perfusion has a very close correlation with detrusor energy charge (r = 0.791, p < 0.001). In conclusion, our findings of the close correlation between microvascular perfusion and energy production in bladder outlet obstruction suggest an important role for the decreased microvascular blood perfusion in reducing bladder energy production.

The permeability transition pore induced under anaerobic conditions in mitochondria energized with ATP

The role of oxygen in the induction of mitochondrial permeability transitions was studied. Oxygen consumption, swelling, membrane potential and calcium transport were recorded simultaneously in isolated rat liver mitochondria. Oxygen depletion was accomplished by saturating the medium with N 2 and allowing either mitochondrial respiration or glucose/glucose oxidase to consume the residual oxygen. Upon anaerobiosis, mitochondria were supplemented with 500 μM ATP to support succinate-driven membrane potential. Under these conditions, 100 μM Ca 2+ induced cyclosporin A-sensitive permeability transitions. To eliminate the possible inhibition of permeability transition by high concentrations of adenine nucleotides, anaerobic mitochondria were also energized by the combination of 20 μM ADP and phosphoenolpyruvate/pyruvate kinase. These mitochondria also underwent Ca 2+-induced permeability transition. Under both of these conditions, namely the addition of ATP as a single or through actions of pyruvate kinase, the respiratory components were totally reduced. Thus, oxygen is not a necessary factor for mitochondria to undergo permeability transitions.

Hepatic preconditioning in rats is defined by a balance of adenosine and xanthine.

The present work investigates the relationship between adenosine, nitric oxide (NO), and free radicals during ischemic preconditioning in rat liver. For this purpose, we evaluated: 1) the efficacy of different periods of preconditioning; 2) the changes in the concentration of adenine nucleotides during preconditioning; 3) the importance of adenosine and xanthine concentrations in the induction of preconditioning; and 4) the possible effect of xanthine oxidase-derived superoxide anion on NO during preconditioning. Results show that just a 10- to 15-minute period of ischemia followed by 10-minute reperfusion prevents the ischemic damage that would be induced by a subsequent 90 minutes of ischemia followed by 90 minutes of reperfusion. Administration of xanthine or metabolization of endogenous adenosine abolishes the protective effect of preconditioning. When rats have been subjected to a period of preconditioning not within the effective time window (10-15 minutes), and thus offering no protection, the administration of a NO donor was found to restore the protection. The dose needed to restore protection appears to be proportional to the endogenous xanthine concentration. In addition, when xanthine oxidase was inhibited, preconditioning effectively offered protection in front of ischemia and reperfusion, independently of the xanthine concentration. Altogether, this indicates that the time window of ischemia capable to induce preconditioning in liver is defined by the relative tissue concentrations of adenosine and xanthine. The lower limit of this window (10 minutes) is defined by the amount of adenosine able to induce NO generation. Its upper limit (15 minutes) is defined by the concentration of xanthine able to remove the generated NO.

Rapid and highly automated determination of adenine and pyridine nucleotides in extracts of Saccharomyces cerevisiae using a micro robotic sample preparation-HPLC system

An ion-pair reversed-phase chromatography method was adapted for the simultaneous separation and quantification of adenine and pyridine nucleotide concentrations in cell extracts of Saccharomyces cerevisiae. Microbial extracts including metabolites, macromolecular constituents and inorganic compounds were loaded onto a ODS pre-column in the presence of triethylamine phosphate (TEA-P i ) resulting in a selective binding of the nucleotides and removing of interfering compounds. After washing the enrichment cartridge with 30 mM TEA-P i buffer, adenine and pyridine nucleotides were eluted with a gradient of Mg(II), the competing hetaeron. This combined cleaning and concentration step leads to remarkable improvement of the detection limit for all nucleotides of interest and column lifetimes. The clean up and separation procedures were performed automatically with a micro robotic-system and a control software package written in PASCAL. The paper reports about the application of the proposed method to separation of adenine and pyridine nucleotides in cells extracts of S. cerevisiae grown anaerobically in a continuous culture ( D=0.1 h −1). Rapidity of analysis, high sensitivity as well as reproducibility of the system and the accurate evaluation of the adenine and pyridine nucleotide concentrations make this method particularly useful for routine analysis.

Preconditioning with cromakalim improves long-term myocardial preservation for heart transplantation

Background. Myocardial preservation for heart transplantation relies on hyperkalemic cardiac arrest and hypothermic storage. Our study investigated whether pretreatment with a potassium-channel opener (cromakalim) before prolonged storage in an extracellular fluid improves left ventricular recovery. Methods. Rabbit hearts were submitted to 6-hours’ cold storage and assessed on a blood-perfused isolated heart preparation. Hemodynamic recovery, enzyme release (creatine kinase and lactate dehydrogenase), and adenine nucleotide content were determined. Five groups were tested: control (n = 6), no ischemia; UW group (n = 7), hearts arrested with and stored in University of Wisconsin solution; STH group (n = 5), hearts arrested with and stored in St. Thomas’ Hospital solution; cromakalim group (n = 6), hearts pretreated with cromakalim (30 μg/kg) before arrest with and storage in St. Thomas’ Hospital solution; and glibenclamide group (n = 5), hearts pretreated with cromakalim followed by glibenclamide (a potassium-channel blocker) before arrest with and storage in St. Thomas’ Hospital solution. Results. Hemodynamic recovery was improved and enzyme release was lower in the UW group than in the STH group. Compared with the STH group, the group pretreated with cromakalim had significantly decreased left ventricular end-diastolic pressures, increased left ventricular developed pressures, increased maximal values of positive and negative rates of rise of left ventricular pressure, and increased time constant of isovolumetric relaxation. Hemodynamic recovery was similar in the UW group and cromakalim groups. Glibenclamide did not abolish the effects of cromakalim. None of the protocols affected myocardial energy stores. Conclusions. Pretreatment with cromakalim affords additional protection to that provided by cardioplegic arrest and prolonged cold storage using an extracellular solution. The intracellular mechanisms involved remain to be determined.

Respiration and adenine nucleotides of Baltic lamprey ( Lampetra fluviatilis l.) hepatocytes during spawning migration

Seasonal variations of lamprey hepatocyte endogenous respiration during spawning migration (November–May), when the animals are starving, were determined. In winter, the respiration is decreased 2.5–3 times as compared with the respiration of early migrants in Autumn and markedly (about twice) increased again in Spring (April). The reasons for such regulation are discussed. The respiration responsible for ATP synthesis and proton leak and non-mitochondrial respiration in hepatocytes were determined in every season. The content of adenine nucleotides in hepatocytes of the autumn migrants was close to that of mammals. However, ATP concentration in Winter decreased markedly (ten times in individual animals). Isolation of viable cells at this period is possible only in the medium with pH 6.5. In Spring, the level of adenine nucleotides again reached the Autumn value. Weight characteristics and protein content of lamprey hepatocytes are about ten times lower than those of the mammalian ones. The mass of lamprey hepatocytes decreased by 30–40% at the period from early Spring to the spawn in May, most likely this decrease being due to the lipid expenditure.

The effects of thyroid hormone modulation on rat liver injury associated with ischemia-reperfusion and cold storage.

We investigated the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion (I-R) and cold storage in rats. First, euthyroid and thyroxine (T4)-pretreated rats were exposed in vivo to 20-min global liver ischemia, then 30-min reperfusion. Liver injury was assessed by measuring serum alanine aminotransferase (ALT) levels. Liver concentrations of adenine nucleotides, reduced glutathione (GSH), and oxidized glutathione were evaluated. Second, rats were given the antithyroid drug propylthiouracil (PTU). Livers stored at 0-1 degrees C in Euro-Collins' solution for 20 h were reperfused at 37 degrees C for 15 min. Lactate dehydrogenase (LDH) in the effluent perfusate and bile flow were evaluated during reperfusion. Serum ALT levels increased after ischemia and I-R. ALT increased significantly more in T4-pretreated than in euthyroid rats after ischemia and I-R. Preischemic levels of adenosine triphosphate (ATP) were significantly lower in livers from T4-pretreated than in euthyroid rats (6.22 +/- 0.7 and 11 +/- 0.9 nmol/mg protein, respectively; P < 0.05). After ischemia, liver ATP was similarly reduced in T4-pretreated and euthyroid rats. After reperfusion, ATP partially recovered in euthyroid rats but remained low in T4-pretreated rats (6.7 +/- 1.0 and 1.91 +/- 0.7 nmol/mg protein, respectively; P < 0.05). Preischemic levels of liver GSH decreased to 44% in T4-pretreated rats. After ischemia, GSH decreased similarly in euthyroid and T4-pretreated rats. GSH recovered promptly after reperfusion in euthyroid rats but remained low in T4-pretreated rats (13.9 +/- 3.3 and 3.9 +/- 0.9 nmol/mg protein, respectively; P < 0.02). During reperfusion after cold storage, LDH in effluent perfusate was significantly lower and bile flow higher in livers from PTU-pretreated rats than from euthyroid rats. The histopathological changes observed after I-R and cold storage confirmed the biochemical findings. Our results suggest that T4 administration exacerbates pretransplant liver damage by increasing liver susceptibility to I-R, whereas PTU administration reduces the liver injury associated with cold storage. We studied the effects of thyroid hormone modulation on liver injury associated with ischemia-reperfusion and cold storage in rats. Thyroxine administration increased susceptibility to ischemia-reperfusion injury, whereas the antithyroid agent propylthiouracil reduced the deleterious effects associated with cold storage.

MUSCLE IMP ACCUMULATION IN TRAINED AND UNTRAINED MEN DURING SUBMAXIMAL CYCLING TO FATIGUE

719 This study was undertaken to compare the accumulation of IMP within the contracting muscle of endurance trained and untrained men following prolonged submaximal exercise to fatigue. Seven endurance trained (28.3 ± 1.7 yrs; 67.7 ± 2.8 kg; VO2max = 65.8 ± 2.4 ml/kg/min, mean± SEM) and six untrained (21.8 ± 1.4 yrs; 79.7 ± 4.4 kg; VO2max = 46.2 ± 1.9 ml/kg/min) men cycled to fatigue at a work rate calculated to require 70% peak oxygen uptake. Time to exhaustion was 36% longer (P < 0.01) in trained compared with untrained men (148 ± 11 and 95 ± 8 min, respectively). Glycogen content was extremely low in both TR and UT (112 ± 38 and 76 ± 35 mmol/kg dw, respectively) at fatigue and no differences for this metabolite were observed between the groups at this point. Despite the low muscle glycogen content at fatigue in TR, inosine 5'-monophosphate (IMP), a marker of metabolic stress, was not different compared with rest. In contrast, significant (P < 0.01) accumulation of IMP was observed at fatigue in UT. Additionally, the exercise-induced change in total adenine nucleotide (TAN = ATP + ADP + AMP) content tended (P = 0.06) to be greater in UT compared with TR. These data demonstrate that fatigue was associated with a similar low level of intramuscular glycogen in both trained and untrained men. Furthermore, fatigue from prolonged exercise in untrained men resulted in a compromised ADP rephosphorylation rate, which may be related to fatigue; whereas in endurance trained men, fatigue is probably related to factors other than impaired ATP resynthesis.

Advantage of preoperative portal vein occlusion for hepatectomy that exceeds portal vein occluded lobes

Background: The purpose of our study was to examine the effect of preoperative portal vein (PV) occlusion on hepatic reserve function after extended hepatectomy that is an excision of areas beyond the PV occluded lobes. Methods: Male Wistar rats were divided into three groups and underwent a two-stage operation: a PVL-hepatectomy group (ligation of the PV [PVL] of the left and median lobes followed by hepatectomy of the right lobes together with the PV occluded lobes), a sham-88% hepatectomy group (sham operation without PVL followed by hepatectomy corresponding to the lobes excised in the PVL-hepatectomy group), and a sham-67% hepatectomy group (sham operation followed by hepatectomy of the left and median lobes to approximate the volume excised in the PVL-hepatectomy group). In all subjects, hepatectomy was carried out 7 days after the PVL or sham operation. On days 0, 1, 2, and 3 after hepatectomy, liver weight, histologic elements, DNA synthesis rates, energy charge, adenine nucleotides, and lipoperoxide levels of the remaining liver were determined. Results: In the sham-88% hepatectomy group, the volume of resected liver was 88.2% ± 0.5%. In the PVL-hepatectomy group it was 69.1% ± 0.8%, although anatomically identical lobes were excised. At the time of hepatectomy, DNA synthesis, hepatic concentrations of adenine nucleotides and lipoperoxide, and serum liver function tests showed similar results in all three groups. The survival rate 3 days after hepatectomy was significantly low (53%) in the sham-88% hepatectomy group, whereas it was 100% in the PVL-hepatectomy and sham-67% hepatectomy groups. The gain in liver weight per day was significantly lower in the sham-88% hepatectomy group than in the other two groups. The decline in hepatic energy charge after hepatectomy was less, with less activated DNA synthesis, in the PVL-hepatectomy group compared with the sham-88% hepatectomy and sham-67% hepatectomy groups. Lipoperoxide concentration in the PVL-hepatectomy group was significantly lower than that in the sham-88% hepatectomy and sham-67% hepatectomy groups. Conclusions: Preoperative PV occlusion not only increases the remaining liver volume but also is advantageous to hepatic reserve after hepatectomy that exceeds PV occluded lobes. (Surgery 1998;123:545-53.)

Influence of heat stress on myocardial metabolism and functional recovery after cardioplegic arrest: a 31P N.M.R study.

Heat stress and induction of heat shock proteins confer protection against myocardial ischemia-reperfusion injury; however the precise mechanisms of this effect remain unknown. We investigated the influence of heat stress on metabolic and functional recovery after cardioplegic arrest, in a protocol mimicking clinical donor heart preservation. Langendorff perfused rat hearts in control group (C, n = 6) and heat stressed (24 h prior to experiment) group (HS, n = 6) were subjected to 4 h of ischemia at 4 degrees C following cardioplegic arrest (St. Thomas' No. 1). 31P nuclear magnetic resonance spectroscopy was used to follow changes in ATP, phosphocreatine and inorganic phosphate concentrations during the pre-ischemic, ischemic and reperfusion periods. Myocardial adenine nucleotide levels in hearts at the end of experiments and purine catabolite release in coronary effluent during reperfusion, were evaluated using high performance liquid chromatography. Mechanical function in the pre-ischemic and reperfusion periods was evaluated using an intraventricular balloon. Western immunoblotting was used to quantitate HSP70 expression. Although baseline concentrations of ATP and phosphocreatine were similar in C and HS groups, the rate of high-energy phosphate depletion was attenuated during the early phase of ischemia in HS groups. On reperfusion, recovery of ATP was 10-20% greater in HS versus C groups; phosphocreatine levels also recovered better in the HS group, transiently reaching levels 40% higher in HS versus C groups. The concentrations of adenine nucleotides in hearts were significantly higher in the HS versus C groups. These changes were associated with an attenuation of total purine catabolite release in the coronary effluent in HS versus C groups. A significant improvement in relative recovery of developed pressure was shown in HS versus C groups in the post-ischemic periods. Heat stress causes beneficial changes in high-energy phosphate metabolism in the rat heart subjected to cardioplegic arrest and ischemia. Improved mechanical recovery in HS versus C groups was associated with a decreased rate of high-energy phosphate depletion and increased recovery of ATP and phosphocreatine levels during reperfusion. Changes in energy metabolism may play a role in the mechanism of cardioprotection by heat stress during prolonged hypothermic cardiac arrest. rights reserved.