Total Adenine Nucleotide Research Articles

Recovery of adenosine triphosphate tissue levels of grafts preserved by the two-layer method after reperfusion.

Recovery of tissue adenosine triphosphate (ATP) levels after transplantation is very important for graft survival. We examined whether the pancreas grafts preserved by the two-layer method have the ability to synthesize ATP after reperfusion, and this is one of the mechanisms of action for the two-layer method in successful pancreas preservation. After preservation by the two-layer method using Euro-Collins' solution (EC) for 48 h (Group 1, n = 5) or simple cold storage in EC for 48 h (Group 2, n = 5), canine pancreas grafts were autotransplanted. In controls (Group 3, n = 5), canine pancreas grafts were autotransplanted without preservation. Graft viability was judged from graft survival after transplantation. Tissue adenine nucleotide concentrations were measured using high performance liquid chromatography after preservation, before reperfusion, and after 1 and 2 h of reperfusion. Graft survival rates were 5 of 5, 0 of 5, and 5 of 5, in Groups 1, 2, and 3, respectively. However ATP tissue levels in Group 1 were significantly higher compared with those in Group 2 after preservation and before reperfusion, respectively (10.95 +/- 1.52 vs. 2.75 +/- 0.33 and 2.90 +/- 0.51 vs. 2.03 +/- 0.68 mumol/g dry weight, p < 0.01 and p < 0.05, respectively). Total tissue adenine nucleotide levels in Group 1 before reperfusion were 7.41 +/- 1.47 mumol/g dry weight, and there was no significant difference compared with Group 2, 6.64 +/- 2.23 mumol/g dry weight. After reperfusion, there was no significant difference of ATP tissue levels between Groups 1 and 2 (4.07 +/- 1.18 vs. 4.48 +/- 1.32, not significant [NS]) after 1 h of reperfusion. However, after 2 h of reperfusion, tissue ATP levels in Group 1 (6.71 +/- 1.19 mumol/g dry weight) were significantly higher than were those in Group 2 (4.51 +/- 0.51 mumol/g dry weight, p < 0.01) and almost at the same levels as control (6.32 +/- 1.62 mumol/g dry weight). It was clear that recovery of ATP after reperfusion did not depend on the residual nucleotides pool but on the ability of the pancreas graft to synthesize ATP 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 maintaining the graft's ability to synthesize ATP promptly after reperfusion. This is one of the mechanisms of action of the two-layer method in successful pancreas preservation.

Oxygen Stress in Wetland Plants: Comparison of De-Oxygenated and Reducing Root Environments

1. Growth, photosynthesis and root adenine nucleotides were compared in two wetland plants, Phalaris arundinacea and Glyceria maxima, grown in aerated, de-oxygenated or reduced (redox potential = -250 mV) nutrient solutions, to test the hypothesis that the stress of de-oxygenated conditions is mild compared with that of naturally reducing sediments. 2. Relative growth rate (RGR) was not significantly different between plants in the aerated and de-oxygenated treatments. However, plants in the reducing treatment stopped growing and some lost mass (RGR negative). Differences in root porosity were not significant between treatments. 3. Rates of net photosynthesis in both species were stable in the de-oxygenated and aerated treatments, at 3-8 μmol CO 2 m -2 s -1 . However, net photosynthesis in the reducing treatment declined over 5 days, becoming negative in P. arundinacea and falling below 1 μmol CO 2 m -2 s -1 in G. maxima. 4. Concentrations of adenine nucleotides in the roots of both species were significantly but only slightly lower in the de-oxygenated treatment than in the aerated treatment (0.65 times as much ATP and 0.70-0.87 times as much total adenine nucleotides). However, nucleotide concentrations were much lower in the reducing treatment (0.10-0.19 times as much ATP and total adenine nucleotides as the aerated treatment). Both species tolerated the de-oxygenated treatment but were sensitive to the reducing treatment.

Why is liver preservation performed at 4°C?

Abstract To establish the most suitable temperature for liver preservation, we preserved rat livers at various temperatures (0, 5, 30, and 15 °C) in UW solution and investi gated, biochemically, the proton ATPase activity, ATP metabolites in mitochondria, and phosphatidyl-choline hydroperoxide (PC-OOH) in liver tissue. Liver specimens were taken every 6 h up to 24 h. The proton ATPase activity and the concentration of ATP, ADP, AMP, and adenosine in livers preserved at 0°C showed the best results. The total adenine nucleotide (TAN) in livers preserved for 18 and 24 h had significantly higher concentrations compared with those at other temperatures (5, 10, and 15 °C). In the livers preserved at 5 °C, TAN was degraded to hypoxanthine. On the other hand, those preserved at both 10 and 15 °C showed changes from hypoxanthine to xanthine. The concentration of xanthine in both groups preserved at 10 and 15 °C showed high values at 6 and 12 h, respectively, and similar changes in PC-OOH concentrations at both 10 and 15 °C were observed. However, the changes in PC-OOH concentration at various temperatures were not significant for any length of preservation time. In light mi croscopical examinations, there were no morphological changes in the hepatocytes. From these results, we conclude that the capability of ATP synthesis of mitochondria in livers preserved at 0 °C keep them in the best condition compared with livers preserved at 15, 10, and 5°C.

Effects of adenine nucleotide analogues on myocardial dysfunction during reperfusion after ischemia in dogs.

We examined effects of adenine nucleotide on ischemic myocardial stunning in dogs. Pentobarbitalanesthetized open-chest dogs were subjected to 20-min ligation of the left anterior descending coronary artery (LAD), followed by reperfusion for 30 min. Either saline, 5 mM 8-bromo-5'-AMP (tributyryl-AMP), or 30 mM N6, 2', 3'-tributyryl-5'-AMP (tributyryl-AMP), 5 mM 5-amino-4-imidazole carboxamide riboside (AICAr) as a positive reference, was infused at 0.1 ml/kg/min in the left femoral vein throughout the experiment. The myocardial contractile function was measured by ultrasonometry. The tissue levels of high-energy phosphates in the reperfused heart were determined. Myocardial contractile function assessed by % segment shortening (%SS) in the saline-infused group decreased during ischemia and returned toward the preischemic level during reperfusion but incompletely. A significant improvement in the %SS during reperfusion was observed in the 8-bromo-AMP- and AICAr-infused groups but not in the tributyryl-AMP-infused group. The magnitude of the protective effect of the drugs on myocardial contractility during reperfusion was 8-bromo-AMP > AICAr > tributyryl-AMP = saline. Only in the 8-bromo-AMP-infused group were the levels of ATP, ADP, and total adenine nucleotides in the reperfused heart significantly higher than those in the saline-infused group. The present result indicates that 8-bromo-AMP improves the ability of the heart to recover from ischemia and reperfusion associated with a significant restoration of ATP.

The effects of phosphate- and substrate-free incubation conditions on glycolysis in Ehrlich ascites tumour cells

The influence of phosphate-free medium buffered with synthetic organic buffers, and of a preliminary incubation of cells in medium lacking added substrate (‘pre-incubation’) was investigated with mouse-cultured Ehrlich ascites tumour cells. In comparison to phosphate-containing bicarbonate-buffered balanced-salts medium, organic-buffered medium, without a preliminary substrate-free pre-incubation, was associated with 20–30% reduction in the rate of glycolysis, the 3- to 4-fold accumulation of fructose 1,6-bisphosphate and the halving of both ATP and total adenine nucleotide levels. These perturbations were reversed by the inclusion of 5 mM sodium phosphate in the organic-buffered medium. Pre-incubation for up to 90 min, before inclusion of glucose, resulted in greater depression of the glycolytic rate and concentrations of adenine nucleotides. This occurred in both the balanced-salts medium and the organic-buffered medium. During pre-incubation cells were lysed, releasing lactate dehydrogenase, when physically agitated too vigorously. It was concluded that the use of phosphate-free medium and pre-incubation are not advisable procedures for routine metabolic investigations with this cell line.

Effects of Different Cold Preservation Solutions on Restoration of Hepatic Energy Metabolism during Cold Reperfusion

Rat livers were flushed with different preservation solutions and stored at 4°C for 24 h before being reperfused with a synthetic air-equilibrated, water-based solution. Four solutions were tested using this isolated rat liver model: Marshall's hypertonic citrate (HC); modified University of Wisconsin solution (Mod UW); a histidine-based solution (HIS); and a histidine–lactobionate–raffinose-based solution (HLR). After storage, livers were perfused at 4°C for a period of 2 h and biopsy samples taken, at different time points, to investigate energy metabolism. Livers stored in HLR and HIS had higher 24-h storage levels of ATP (0.41 and 0.24 μmol/g respectively;P< 0.05) than those stored in Mod UW and HC. On reperfusion, all groups regenerated ATP by 1–2 h. However, significantly greater levels of ATP regeneration occurred in livers stored in the HLR (1.6 ± 0.08 μmol/g) and Mod UW (1.3 ± 0.18) than HC (0.58 ± 0.19) and HIS (0.96 ± 0.12);P< 0.05. Energy charge (1) (EC) recovered in all groups but was significantly higher in HLR and Mod UW (0.79 and 0.68, respectively;P< 0.05) than HC and HIS. These represent 95% (HLR) and 80% (Mod UW) of values observed in FIL. During the reperfusion period, total adenine nucleotide levels (TA) did not vary significantly within each storage group, except in the HIS solution. However, TAs were greater with livers stored for 24 h in HLR (2.5 ± 0.25) and Mod UW (2.7 ± 0.20) than those the other two storage groups (P< 0.05 in each case). This study has demonstrated that it was possible to resuscitate liver energetics after prolonged hypothermic ischemia by a period of cold reperfusion, and the method can differentiate between preservation solutions. The livers stored in each solution showed varying degrees of success in regeneration of ATP and EC, demonstrating that oxygen was not a limiting factor when using an air-equilibrated perfusate. The solutions providing the better preservation conditions gave the greater resuscitation of liver energetics (Mod UW and HLR). Overall, livers stored in HLR had the greatest resuscitation of energy metabolism, which correlates with survival data from other studies (29–31, 33).

Hepatic resuscitation after warm anoxia: one approach for increasing the donor pool for liver transplantation

Abstract The deleterious effects of warm anoxia on the liver are seen to be irreversible if cooling and transplantation (LT) follow immediately after. The aim of our study is to demonstrate that livers subjected to anoxia may be suitable for LT if a period of resuscitation is interposed before the cooling process. Forty female Large White pigs were used. Preservation (Euro-Collins solution) and LT technique were the same in all 20 procedures. All donors underwent clamping of the porta hepatis at the end of harvesting dissection. In the so-called 19 resuscitate groups (AR and BR), the clamp was released for a period of time before the liver was cooled. Then, all livers underwent 2 h of cold ischemia followed by LT. -trastructural study showed better maintenance of mitochondria and sinusoidal cell integrity in resuscitated livers after LT. Liver synthesis of total adenine nucleotides, graft function and recipient survival were found to be better in the 19resuscitate groups. In conclusion, anoxic livers may be retrieved for LT if a resuscitation period (i. e. aerobic perfusion) is allowed prior to cold preservation. Longer periods of warm anoxia are needed to further support these preliminary results.

Metabolic compartmentalization in neonatal swine myocytes.

The transport of metabolites across the mitochondrial membrane is regulated by specific exchange and shuttle systems that are often dependent on the mitochondrial membrane potential. Thus, metabolite concentrations in the cytosol and mitochondrial compartments are largely determined by the energy state of the cardiac muscle cell. The purpose of this study was to investigate metabolic compartmentalization in ventricular myocytes isolated from newborn (< 24 h) swine hearts. Furthermore, the effect of respiratory inhibition on these distribution patterns was examined. Freshly isolated cells contained 33 nmol of ATP and 37 nmol of total adenine nucleotides (AN) per mg of myocyte protein. Rapid digitonin fractionation indicated that 95% of ATP and 86% of AN were cytosolic, whereas > 50% of the pyridine nucleotides were mitochondrial. With 11 mM added glucose, myocytes treated with the respiratory inhibitor, rotenone, maintained ATP at 88% of that of aerobic myocytes, but phosphocreatine declined by 50% over 30 min. Rotenone treatment caused the mitochondrial NAD/NADH ratio to decline from 1.2 to 0.06, whereas the cytosolic pyridine nucleotides remained > 90% oxidized. Total adenine and pyridine nucleotide content and their compartmentalization were unaffected by respiratory inhibition. Comparisons of metabolite content and respiratory activity between isolated piglet mitochondria and the mitochondrial compartment of piglet myocytes indicated that mitochondria account for approximately 30% of total myocyte protein. A similar value (29%) was obtained for the aqueous volume fraction of the in situ mitochondrial matrix using the 4000 Mr 14C-labeled polyethylene glycol-impermeable 3H2O spaces of intact and lysed myocytes. These results are comparable to literature values for myocardium from other species and age groups.

Cardioprotective effect of nicorandil in histidine-tryptophan-ketoglurate solution during the cold storage of isolated hearts.

We compared the efficacy of using histidine-tryptophan-ketoglurate (HTK) solution with that of University of Wisconsin (UW) solution for heart preservation in an isolated rat heart preparation. Nicorandil (NCR) exerts its action as an ATP-sensitive potassium channel opener at low extracellular potassium concentrations, and HTK solution has a low potassium concentration. Therefore, we also investigated the efficacy of using HTK solution with NCR following 12-hr preservation. Hearts isolated from male Wistar rats were mounted on a Langendorff apparatus to estimate baseline aortic flow (AF), coronary flow (CF), cardiac out-put (CO), heart rate (HR), systolic pressure (SP), aortic mean pressure, and the rate-pressure product (RPP). The hearts were divided into four groups: group 1, 8-hr storage in UW solution; groups 2 and 3, 8- or 12-hr storage in HTK solution, respectively; and group 4, 12-hr storage in HTK solution with NCR. They were arrested and stored at 4 degrees C in each preservation solution. Following storage, they were reperfused and postpreservative function was measured to assess cardiac functional recovery. Concentrations of creatine phosphokinase, troponin-T, and lactate in the coronary perfusate were measured. Frozen tissue samples from groups 3 and 4 were analyzed for adenylate content and cGMP. The myocardial water content was also measured. The recovery of AF, CF, CO, SP, and RPP in group 2 was significantly improved compared with that in group 1 (P<0.05). The recovery of AF, CF, CO and HR in group 4 was significantly better than that in group 3 (P<0.05). Creatine phosphokinase leakage in group 2 and troponin-T leakage in group 4 were significantly reduced (P<0.05 vs. groups 1 and 3, respectively). Total adenine nucleotides and the adenylate energy charge in group 4 were well sustained (P<0.05 vs. group 3). These results suggest that HTK solution is more effective than UW solution for cardiac preservation, and that NCR provides still better protection.

The Beneficial Effects of Heat-Shock for Prolonged Hypothermic Storage

Heat-shock has been reported to induce tolerance for subsequent ischemia. We wished to determine whether thermal stress is protective for hypothermic storage. Three groups of Sprague-Dawley rats were studied (n= 8 per group). Control animals received routine care (CONT) while anaesthetized rats were either warmed to 42°C for 20 min (HEAT) or maintained at room temperature (SHAM). Twenty-four hours later, hearts were mounted on a Langendorff apparatus with an intraventricular balloon. Hearts were then flushed and stored in UW solution for 8 hr at 0°C and reperfused for 45 min. Data are reported as a percentage of the prestorage results or as the absolute value (mean ± SD). Recovery of developed pressure was significantly greater (P< 0.05) in the heat-shocked animals (79.5 ± 10.2%) than in the SHAM (63.6 ± 17.2%) or CONT groups (59.0 ± 10.8%). Coronary flow was similarly enhanced (P< 0.05) in the HEAT group (86.8 ± 5.5%) vs the CONT (77.0 ± 12.3%) or SHAM hearts (74.5 ± 10.2%). Diastolic compliance as assessed by evaluation of the end-diastolic pressure-volume curves was reduced in all groups (P< 0.0001) but not different among groups. Cardiac creatine kinase release during reperfusion was greater in the CONT and SHAM groups (CONT: 726.9 ± 297.8 IU/g; SHAM: 548.9 ± 420.9 IU/g) than in the heat-shocked rodents (282.3 ± 175.5 IU/g,P< 0.05 HEAT vs CONT). Cardiac biopsies were performed sequentially in separate animals (n= 6 per group). Tissue levels of ATP and total adenine nucleotides were greater in the heat-shocked or SHAM hearts following reperfusion compared with controls (P< 0.05). Thermal stress provides additional protection for prolonged hypothermic storage.

Adenine nucleotide metabolism during anoxia and postanoxic recovery in insects

Severe hypoxia (anoxia), if maintained for more than a few minutes, causes irreversible damage in humans and other mammals. Why mammals are so vulnerable to anoxia is not fully understood. It is therefore of interest to study animals that are more tolerant of anoxia in order to identify physiological and metabolic properties that are correlated with a high tolerance of anoxia. Insects have high metabolic rates and their energy metabolism is dependent on aerobic ATP production. In insects, as in mammals, anoxia causes a rapid breakdown of physiological function, resulting in a state similar to rigor mortis. This is accompanied by a precipitous decrease in metabolic rate. In contrast to mammals, however, insects can survive anoxia for many hours and recover spontaneously and completely when air is again available. We have followed the metabolism of adenine nucleotides in locust tissues (mainly in the flight muscle) over 3 h of anoxia and during recovery from 1 h of anoxia. The content of ATP in the flight muscle dropped to 1% of normal during 2 h of anoxia. The main product was AMP which increased in content more than 20-fold. Some of the AMP was deaminated to IMP and this was further dephosphorylated to inosine. Altogether less than 30% of the total adenine nucleotides were degraded during 3 h of anoxia and this may contribute to the amazing ability of insects to recover from prolonged anoxia.

L-type Ca2+ channel and Na+/Ca2+ exchange inhibitors reduce Ca2+ accumulation in reperfused skeletal muscle.

It is known that extracellular Ca2+ accumulates within skeletal muscle after prolonged periods of ischemia and reperfusion. In this study, we determined whether the L-type Ca2+ channel and the Na+/Ca2+ exchanger mediated Ca2+ influx and whether Ca2+ accumulation limited the metabolic and contractile recovery of reperfused skeletal muscle. Contracting rat hindlimbs (1-Hz twitch) exposed to 40 min of no-flow ischemia were reperfused with diltiazem (500 microM) or 3,4-dichlorobenzamil (300 microM) to block the Na+/Ca2+ exchanger and/or the L-type Ca2+ channel. High inhibitor concentrations were used to counter the binding of diltiazem and 3,4-dichlorobenzamil to albumin and red blood cells. Muscle Ca2+ accumulation, contractile function, and energy metabolism were assessed by measuring intracellular Ca2+ concentration ([Ca2+]i), Ca2+ influx, twitch tension, and high-energy phosphagens [ATP, total adenine nucleotides (TAN) and phosphocreatine (PCr)]. Compared with control reperfusion, diltiazem and 3,4-dichlorobenzamil reduced Ca2+ influx and attenuated the rise in [Ca2+]i in the fast-oxidative glycolytic plantaris (Pl) and the fast-glycolytic white gastrocnemius (WG). The inhibitor-induced decrease in Ca2+ influx was 1.5- to 2-fold greater with 3,4-dichlorobenzamil than with diltiazem. Coinciding with the reduced Ca2+ accumulation, diltiazem and 3,4-dichlorobenzamil enhanced the resynthesis of ATP (Pl and WG), PCr (Pl and WG), and TAN (Pl) compared with control reperfusion. 3,4-Dichlorobenzamil also augmented twitch-tension recovery. We conclude that Ca2+ accumulation during reperfusion 1) arises from L-type Ca2+ channel and Na+/Ca2+ exchange activation; and 2) impairs the metabolic and contractile recovery of skeletal muscle.

NEUTROPENIA PRESERVES ENERGY METABOLITES DURING CEREBRAL HYPOXIA-ISCHEMIA IN THE NEONATAL RAT. † 2256

We found that neutropenia induced before cerebral hypoxia-ischemia (HI) in the 7day old rat is neuroprotective. However, neutrophil depletion achieved by 6-8hrs of recovery is not protective. Neutrophils can plug microvessels and reduce red cell velocity when flow is reduced during ischemia. The aims of this study were to determine the effect of neutropenia on cerebral energy metabolism during the HI insult and at 6hrs of recovery. We produced a HI insult to the R cerebral hemisphere (RH) of 7day old rat pups by R common carotid artery ligation and 2.25h hypoxia in 8% oxygen. This insult causes cerebral ischemia and injury to the right hemisphere. We made some rats neutropenic with antineutrophil serum before HI. Other rats received normal rabbit serum as control. Sixteen rats of each group were killed by immersion in liquid nitrogen at the end of the HI insult and 10-12 of each group at 6h of recovery from HI. From perchloric acid brain extracts we measured L and R hemisphere levels of P-Cr, ATP, ADP, AMP, and calculated Total Adenine Nucleotides (TAN). We compared the energy metabolite levels of the neutropenic and nonneutropenic rats at 0 and 6h recovery. Normal levels of ATP were 2.29±0.18mmol/kg (Mean ±SD). In the nonneutropenic rats at 0 recovery ATP levels were 0.88±.59 while in the neutropenic rats it was 1.3±0.76. Seven of 16 neutropenic rats had ATP levels at 0 recovery within 3SD of normal compared to only 2 of 14 nonneutropenic rats, p=.04 Chi Square. TAN levels were 2.7±0.22 in the normal rats, at 0h recovery they were 2.02±0.56 mmol/kg in neutropenic rats and only 1.55±0.56 in the nonneutropenic rats, p =0.02 Mann-Whitney U test. At 6 hours of recovery ATP levels had returned to within 2SD of the Mean(1.93-2.65mmol/kg) in all 12 neutropenic rats and 7/10 nonneutropenic rats. There was not a significant difference between the neutropenic and nonneutropenic rats at 6hrs of recovery for any metabolite. Average levels were normal for all metabolites at 6h recovery. In conclusion neutropenia induced before cerebral HI preserves energy metabolism during the insult. Cerebral energy metabolism recovers by 6 hrs of recovery following a hypoxic ischemic insult.

Hepatic adenine nucleotides and DNA synthesis during the regenerative and atrophic process of the liver lobes after selective portal vein ligation.

Selective portal vein occlusion prior to aggressive hepatic resection is now an alternative way to decrease postoperative morbidity and mortality rates. However, the detailed changes in the hepatic energy status and DNA synthesis rate in both portal vein ligated (PVL) and nonligated (PVNL) lobes of the liver are not clear. In rats, the portal branch that supplies 70% of the liver volume was ligated, and changes in arterial ketone body ratio (AKBR), liver weight, histology, DNA synthesis rate and adenine nucleotides of the PVL and PVNL liver lobes were determined before and 1, 2, 4 and 7 days after portal vein ligation, and compared with those in sham-operated rats. The weight of the PVL lobes decreased, while that of the PVNL lobes increased depending on time. The DNA synthesis rates of the PVNL lobes were significantly higher than those in sham-operated control liver during the first 4 days with the maximal value on the 2nd day, while those of PVL lobes were essentially similar to the control values. Energy charge (EC) in both PVL and PVNL lobes significantly decreased on day 1 and recovered gradually, but with less extent in the regenerating PVNL lobes. The concentrations of total adenine nucleotides (TAN) in both the PVL and PVNL lobes were essentially similar during the first 2 days, but became significantly lower in PVL lobes after day 4. A decrease in EC preceded an increase in DNA synthesis only in the PVNL lobes, in contrast to the PVL lobes. Mitosis of hepatocytes on day 2 and subsequently enlarged lobules with an increased number of hepatocytes were histologic features in the PVNL liver. The AKBR was not correlated with hepatic energy charge of the liver. In conclusion, PVNL liver regenerates preceded by a decrease in EC and a subsequent increase in DNA synthesis keeping TAN constant, while PVL liver becomes atrophic, with a similar change in EC of the PVNL liver but ultimately decreased TAN without any change in DNA synthesis. AKBR is not a parameter reflecting the hepatic EC after portal branch ligation.

Why is liver preservation performed at 4 degrees C?

To establish the most suitable temperature for liver preservation, we preserved rat livers at various temperatures (0, 5, 10, and 15 degrees C) in UW solution and investigated, biochemically, the proton ATPase activity, ATP metabolites in mitochondria, and phosphatidyl-choline hydroperoxide (PC-OOH) in liver tissue. Liver specimens were taken every 6 h up to 24 h. The proton ATPase activity and the concentration of ATP, ADP, AMP, and adenosine in livers preserved at 0 degree C showed the best results. The total adenine nucleotide (TAN) in livers preserved for 18 and 24 h had significantly higher concentrations compared with those at other temperatures (5, 10, and 15 degrees C). In the livers preserved at 5 degrees C, TAN was degraded to hypoxanthine. On the other hand, those preserved at both 10 and 15 degrees C showed changes from hypoxanthine to xanthine. The concentration of xanthine in both groups preserved at 10 and 15 degrees C showed high values at 6 and 12 h, respectively, and similar changes in PC-OOH concentrations at both 10 and 15 degrees C were observed. However, the changes in PC-OOH concentration at various temperatures were not significant for any length of preservation time. In light microscopical examinations, there were no morphological changes in the hepatocytes. From these results, we conclude that the capability of ATP synthesis of mitochondria in livers preserved at 0 degree C keep them in the best condition compared with livers preserved at 15, 10, and 5 degrees C.

Role of adenine nucleotides in the activation of microsomal cholesterol ester hydrolase by fructose or adenosine in rat hepatocytes

In the present study we have analysed the potential relationship between the cellular level of adenine nucleotides and the activity of microsomal cholesterol ester hydrolase by treating rat hepatocyte suspensions with fructose or adenosine. Fructose raised the microsomal hydrolysis of cholesteryl esters as a function of the dose. This ketose led to marked decreases in the cell level of ADP, ATP and total adenine nucleotide whereas that of AMP increased slightly, thus giving a rise in the cellular AMP/ATP ratio. The effects remained virtually constant over a period of 60 min. Incubation of hepatocytes in a Ca 2+-free medium with or without ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid blocked by 40% the fructose-induced activation of cholesterol esterase whereas the rise in AMP/ATP was unaffected. Adenosine caused dose-dependent activations of cholesterol ester hydrolase and raised AMP, ADP and ATP concentrations as well as the AMP/ATP ratio. 2-Chloro-adenosine and N 6-[ l-2-phenyl-isopropyl] adenosine, non-metabolizable analogues of adenosine, did not mimic the effects of the nucleoside. A positive linear correlation exists between the percentage rises in the activity of microsomal cholesterol ester hydrolase and those in the intracellular AMP/ATP ratio in fructose- or adenosine-treated cells. These results indicate that, in microsomes from intact hepatocytes, the breakdown of cholesteryl esters to yield cholesterol and fatty acids is stimulated by fructose and adenosine and this can be explained in part by the increase in the cellular AMP/ATP ratio. In the case of fructose, also a Ca 2+-dependent mechanism is involved.

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.

Purine ribonucleotide content in infected HIV-RT + and HIV-RT − lymphoblastoid cell lines

We have studied the purine nucleotide metabolism in the following cell lines: a), H9 (continuous human T-cell line) and H9/HTLV-III (H9 cell line, infected with RT+ HIV-I virus); b), A3.01 (human lymphoblastoid cell line CD4+) and 8E51 (line A3.01 permanently transfected with RT-HIV-I virus). Purine metabolism was studied by evaluating the content of the most important ribonucleotides (AMP-GMP-IMP-NAD-ADP-GDP-ATP-GTP) and their ratios. We determined several differences between the cell lines before and after viral infection. All nucleotides except triphosphates were reduced in H9/HTLV-III with respect to H9 cells; in 8E51, however, triphosphates were markedly reduced, while monophosphates increased with respect to A3.01 uninfected cells. Also the ratios exhibited different behaviors, for example the total adenine nucleotides total guanine nucleotides ratio (sigma A/sigma G) was enhanced in H9/HTLV-III cells with respect to H9 and unaltered in 8E51 with respect to A3.01 cells. We may conclude that the HIV-I virus strongly influences the purine nucleotide metabolism of the host cells and that the changes are different when induced either by RT+ or RT- virus.

Hepatic resuscitation after warm anoxia: one approach for increasing the donor pool for liver transplantation.

The deleterious effects of warm anoxia on the liver are seen to be irreversible if cooling and transplantation (LT) follow immediately after. The aim of our study is to demonstrate that livers subjected to anoxia may be suitable for LT if a period of resuscitation is interposed before the cooling process. Forty female Large White pigs were used. Preservation (Euro-Collins solution) and LT technique were the same in all 20 procedures. All donors underwent clamping of the porta hepatis at the end of harvesting dissection. In the so-called "resuscitated" groups (AR and BR), the clamp was released for a period of time before the liver was cooled. Then, all livers underwent 2 h of cold ischemia followed by LT. Ultrastructural study showed better maintenance of mitochondria and sinusoidal cell integrity in resuscitated livers after LT. Liver synthesis of total adenine nucleotides, graft function and recipient survival were found to be better in the "resuscitated" groups. In conclusion, anoxic livers may be retrieved for LT if a resuscitation period (i.e. aerobic perfusion) is allowed prior to cold preservation. Longer periods of warm anoxia are needed to further support these preliminary results.

Effect of glibenclamide on ischemic canine myocardium with glucose infusion

Glibenclamide, an ATP-sensitive K (K ATP) channel blocker, worsens the ischemia-induced metabolic derangement in the heart through inhibition of K ATP channels. We examined whether the hypoglycemic effect of glibenclamide was involved in the worsening of myocardial energy metabolism during ischemia. Pentobarbital-anesthetized dogs were subjected to 15-min ligation of the left anterior descending coronary artery. Either vehicle (dimethyl sulfoxide, DMSO) or glibenclamide (1 mg/kg) was injected i.v. 10 min before the ligation. In half of the animals given glibenclamide, glucose was continuously infused at 3 mg/kg per min immediately after glibenclamide injection. Glibenclamide increased the serum insulin level and decreased the blood glucose level. Glucose infusion completely abolished the hypoglycemia due to glibenclamide. Glibenclamide enhanced the decrease in ATP and total adenine nucleotides and increase in tissue lactate caused by ischemia. Glucose infusion did not cancel the augmentation of ischemia-induced alterations of myocardial energy metabolism caused by glibenclamide. These results suggest that K ATP channels directly play an important role in endogenous mechanisms of myocardial protection against ischemic damage.