Nucleotide Resynthesis Research Articles

Xanthine oxidase inhibitor ameliorates postischemic renal injury in mice by promoting resynthesis of adenine nucleotides.

Although oxidative stress plays central roles in postischemic renal injury, region-specific alterations in energy and redox metabolism caused by short-duration ischemia remain unknown. Imaging mass spectrometry enabled us to reveal spatial heterogeneity of energy and redox metabolites in the postischemic murine kidney. After 10-minute ischemia and 24-hour reperfusion (10mIR), in the cortex and outer stripes of the outer medulla, ATP substantially decreased, but not in the inner stripes of the outer medulla and inner medulla. 10mIR caused renal injury with elevation of fractional excretion of sodium, although histological damage by oxidative stress was limited. Ischemia-induced NADH elevation in the cortex indicated prolonged production of reactive oxygen species by xanthine oxidase (XOD). However, consumption of reduced glutathione after reperfusion suggested the amelioration of oxidative stress. An XOD inhibitor, febuxostat, which blocks the degradation pathway of adenine nucleotides, promoted ATP recovery and exerted renoprotective effects in the postischemic kidney. Because effects of febuxostat were canceled by silencing of the hypoxanthine phosphoribosyl transferase 1 gene in cultured tubular cells, mechanisms for the renoprotective effects appear to involve the purine salvage pathway, which uses hypoxanthine to resynthesize adenine nucleotides, including ATP. These findings suggest a novel therapeutic approach for acute ischemia/reperfusion renal injury with febuxostat through salvaging high-energy adenine nucleotides.

Ribose Supplementation Alone or with Elevated Creatine Does Not Preserve High Energy Nucleotides or Cardiac Function in the Failing Mouse Heart

BackgroundReduced levels of creatine and total adenine nucleotides (sum of ATP, ADP and AMP) are hallmarks of chronic heart failure and restoring these pools is predicted to be beneficial by maintaining the diseased heart in a more favourable energy state. Ribose supplementation is thought to support both salvage and re-synthesis of adenine nucleotides by bypassing the rate-limiting step. We therefore tested whether ribose would be beneficial in chronic heart failure in control mice and in mice with elevated myocardial creatine due to overexpression of the creatine transporter (CrT-OE).Methods and ResultsFour groups were studied: sham; myocardial infarction (MI); MI+ribose; MI+CrT-OE+ribose. In a pilot study, ribose given in drinking water was bioavailable, resulting in a two-fold increase in myocardial ribose-5-phosphate levels. However, 8 weeks post-surgery, total adenine nucleotide (TAN) pool was decreased to a similar amount (8–14%) in all infarcted groups irrespective of the treatment received. All infarcted groups also presented with a similar and substantial degree of left ventricular (LV) dysfunction (3-fold reduction in ejection fraction) and LV hypertrophy (32–47% increased mass). Ejection fraction closely correlated with infarct size independently of treatment (r2 = 0.63, p<0.0001), but did not correlate with myocardial creatine or TAN levels.ConclusionElevating myocardial ribose and creatine levels failed to maintain TAN pool or improve post-infarction LV remodeling and function. This suggests that ribose is not rate-limiting for purine nucleotide biosynthesis in the chronically failing mouse heart and that alternative strategies to preserve TAN pool should be investigated.

The NAD metabolome — a key determinant of cancer cell biology

NAD is a vital molecule in all organisms. It is a key component of both energy and signal transduction--processes that undergo crucial changes in cancer cells. NAD(+)-dependent signalling pathways are many and varied, and they regulate fundamental events such as transcription, DNA repair, cell cycle progression, apoptosis and metabolism. Many of these processes have been linked to cancer development. Given that NAD(+)-dependent signalling reactions involve the degradation of the molecule, permanent nucleotide resynthesis through different biosynthetic pathways is crucial for incessant cancer cell proliferation. This necessity supports the targeting of NAD metabolism as a new therapeutic concept for cancer treatment.

Lidoflazine Combined with Nucleotide Precursors Increases ATP Content and Adenosine Production in Cardiomyocytes

We have previously identified that the nucleoside transport blocker dipyridamole increases adenosine production but may cause depletion of the nucleotide pool in cardiomyocytes during extended exposure and that this effect was abolished by co-administration of adenine and ribose. The present study aimed to establish whether lidoflazine, a newer generation of nucleoside transport inhibitor with calcium antagonist properties, would cause a similar effect. We conclude that lidoflazine did not affect the nucleotide pool while the combined application of lidoflazine with precursors of nucleotide resynthesis increased ATP concentration and further enhanced adenosine production.

Effect of ribose supplementation on resynthesis of adenine nucleotides after intense intermittent training in humans

The effect of oral ribose supplementation on the resynthesis of adenine nucleotides and performance after 1 wk of intense intermittent exercise was examined. Eight subjects performed a random double-blind crossover design. The subjects performed cycle training consisting of 15 x 10 s of all-out sprinting twice per day for 7 days. After training the subjects received either ribose (200 mg/kg body wt; Rib) or placebo (Pla) three times per day for 3 days. An exercise test was performed at 72 h after the last training session. Immediately after the last training session, muscle ATP was lowered (P < 0.05) by 25 +/- 2 and 22 +/- 3% in Pla and Rib, respectively. In both Pla and Rib, muscle ATP levels at 5 and 24 h after the exercise were still lower (P < 0.05) than pretraining. After 72 h, muscle ATP was similar (P > 0.05) to pretraining in Rib (24.6 +/- 0.6 vs. 26.2 +/- 0.2 mmol/kg dry wt) but still lower (P < 0.05) in Pla (21.1 +/- 0.5 vs. 26.0 +/- 0.2 mmol/kg dry wt) and higher (P < 0.05) in Rib than in Pla. Plasma hypoxanthine levels after the test performed at 72 h were higher (P < 0.05) in Rib compared with Pla. Mean and peak power outputs during the test performed at 72 h were similar (P > 0.05) in Pla and Rib. The results support the hypothesis that the availability of ribose in the muscle is a limiting factor for the rate of resynthesis of ATP. Furthermore, the reduction in muscle ATP observed after intense training does not appear to be limiting for high-intensity exercise performance.

Purine and Pyrimidine Salvage in Whole Rat Brain: UTILIZATION OF ATP-DERIVED RIBOSE-1-PHOSPHATE AND 5-PHOSPHORIBOSYL-1-PYROPHOSPHATE GENERATED IN EXPERIMENTS WITH DIALYZED CELL-FREE EXTRACTS

The object of this work stems from our previous studies on the mechanisms responsible of ribose-1-phosphate- and 5-phosphoribosyl-1-pyrophosphate-mediated nucleobase salvage and 5-fluorouracil activation in rat brain (Mascia, L., Cappiello M., Cherri, S., and Ipata, P. L. (2000) Biochim. Biophys. Acta 1474, 70-74; Mascia, L., Cotrufo, T., Cappiello, M., and Ipata, P. L. (1999) Biochim. Biophys. Acta 1472, 93-98). Here we show that when ATP at "physiological concentration" is added to dialyzed extracts of rat brain in the presence of natural nucleobases or 5-fluorouracil, adenine-, hypoxanthine-, guanine-, uracil-, and 5-fluorouracil-ribonucleotides are synthesized. The molecular mechanism of this peculiar nucleotide synthesis relies on the capacity of rat brain to salvage purine and pyrimidine bases by deriving ribose-1-phosphate and 5-phosphoribosyl-1-pyrophosphate from ATP even in the absence of added pentose or pentose phosphates. The levels of the two sugar phosphates formed are compatible with those of synthesized nucleotides. We propose that the ATP-mediated 5-phosphoribosyl-1-pyrophosphate synthesis occurs through the action of purine nucleoside phosphorylase, phosphopentomutase, and 5-phosphoribosyl-1-pyrophosphate synthetase. Furthering our previous observations on the effect of ATP in the 5-phosphoribosyl-1-pyrophosphate-mediated 5-fluorouracil activation in rat liver (Mascia, L., and Ipata, P. L. (2001) Biochem. Pharmacol. 62, 213-218), we now show that the ratio [5-phosphoribosyl-1-pyrophosphate]/[ATP] plays a major role in modulating adenine salvage in rat brain. On the basis of our in vitro results, we suggest that massive ATP degradation, as it occurs in brain during ischemia, might lead to an increase of the intracellular 5-phosphoribosyl-1-pyrophosphate and ribose-1-phosphate pools, to be utilized for nucleotide resynthesis during reperfusion.

Base excision repair is impaired in mammalian cells lacking Poly(ADP-ribose) polymerase-1.

In mammalian cells, damaged bases in DNA are corrected by the base excision repair pathway which is divided into two distinct pathways depending on the length of the resynthesized patch, replacement of one nucleotide for short-patch repair, and resynthesis of several nucleotides for long-patch repair. The involvement of poly(ADP-ribose) polymerase-1 (PARP-1) in both pathways has been investigated by using PARP-1-deficient cell extracts to repair single abasic sites derived from uracil or 8-oxoguanine located in a double-stranded circular plasmid. For both lesions, PARP-1-deficient cell extracts were about half as efficient as wild-type cells at the polymerization step of the short-patch repair synthesis, but were highly inefficient at the long-patch repair. We provided evidence that PARP-1 constitutively interacts with DNA polymerase beta. Using cell-free extracts from mouse embryonic cells deficient in DNA polymerase beta, we demonstrated that DNA polymerase beta is involved in the repair of uracil-derived AP sites via both the short and the long-patch repair pathways. When both PARP-1 and DNA polymerase beta were absent, the two repair pathways were dramatically affected, indicating that base excision repair was highly inefficient. These results show that PARP-1 is an active player in DNA base excision repair.

Energy Metabolism Following Prolonged Hepatic Cold Preservation: Benefits of Interrupted Hypoxia on the Adenine Nucleotide Pool in Rat Liver

The ability of brief hypothermic reperfusion (HtR) to restore hepatic energy metabolism following periods of cold hypoxic preservation was studied in isolated rat livers after storage times of 5, 10, and 24 h. In addition, investigations were performed on the effects of HtR used to restore liver oxidative metabolism in the middle of a prolonged (24 h) hypoxic preservation period. A histidine–lactobionate–raffinose solution was used for the initial cold portal flush in all groups. Results showed that cold hypoxia for either 5 or 10 h yielded livers capable of similar recoveries of ATP, energy charge, and total adenine nucleotides, but that HtR after 24 h cold preservation resulted in reduced regeneration of ATP, a lower energy charge, and a fall in tissue adenine nucleotides. When livers were stored for 24 h but subjected to brief HtR after either 5 or 10 h before return to hypoxic storage, improved recoveries of the energy metabolites were seen over those recorded after 24 h hypoxia alone. The fact that these improvements were not due to an improved supply of adenine nucleotide precursors was demonstrated by studying groups which were given HtR with perfusate containing precursors of adenine nucleotides (adenosine, adenine, and inosine) after 24 h cold hypoxia. These data are consistent with the hypothesis that poor metabolic recovery after long-term hepatic cold preservation results more from decreased mitochondrial oxidative phosphorylation than from a lack of precursors for adenine nucleotide resynthesis. In addition, restoring oxidative metabolism at hypothermia for brief periods can to some extent protect final metabolic status after prolonged storage.

Drug-Induced Rhabdomyolysis

The syndrome of rhabdomyolysis is the result of skeletalmuscle injury that alters the integrity of the sarcolemmaand leads to the eventual release of intracellular contentsinto the plasma. The causes are diverse, and includemuscle trauma (for instance, from vigorous exercise, crushinjuries, battering, or seizures), inadequate bloodperfusion, heat stroke, electrolyte imbalance, hereditaryenzyme deficiencies, infections and ingestion of drugs andtoxins. Various serious medical disorders, particularlythose resulting in states of disordered energy production,are also associated with rhabdomyolysis.In general, drug toxicity involves organs, such as thekidney, liver, gastrointestinal tract and the central nervoussystem, with skeletal muscle being usually less readilyaffected. Although drug-induced rhabdomyolysis wasuncommon in the past, it is no longer rare, due to theintroduction of more and increasingly potent drugs intoclinical practice.The incidence of drug-induced rhabdomyolysis isuncertain, largely because most of it is unreported.Similarly, the mortality rates are unknown. However, ithas been suggested that rhabdomyolysis from all causesleads to 5%-25% of cases of acute renal failure.

The Pathophysiology of the Acute Compartment Syndrome

The acute compartment syndrome is a condition in which increased pressure within a limited space compromises the circulation and function of the tissues therein, resulting in tissue ischaemia, necrosis and nerve damage. This rise in tissue pressure originates in a decrease of the compartment size or increase of the intra-compartmental volume by oedema and/or haemorrhage. Following the arterio-venous gradient theory, capillary blood flow may be impaired through increased venous pressure, decreased arterial pressure and increased peripheral vascular resistance. Often, compartment syndromes develop during reperfusion following a period of ischaemia. During ischaemia, there is a gradual depletion of intracellular stores of high energy phosphate bonds and glycogen stores. There is a buildup of products of glycolysis, particularly lactic acid, with accompanying hydrogen ion accumulation as well as an increase in intracellular reducing agents. Reperfusion may, instead of restoring normal muscle metabolic activity, cause harmful effects by washing out necessary precursors for adenine nucleotide resynthesis. Production of oxygen free radicals occurs with ensuing lipid peroxidation, and calcium influx occurs upon reoxygenation with resultant disruption of oxidative rephosphorylation in the mitochondria. Furthermore, several lines of evidence suggest that white blood cells are important in the pathogenesis of reperfusion injury. Upregulation of both neutrophil receptors and endothelial leucocyte adhesion molecules leads to the sequestration of white blood cells in the muscle with prolongation of the reperfusion injury. This subsequently results in damage to remote organs such as lungs, liver, heart and kidneys.

Effect of the extramitochondrial adenine nucleotide pool size on oxidative phosphorylation in isolated rat liver mitochondria.

The effect of the concentration of extramitochondrial adenine nucleotides on oxidative phosphorylation was studied in isolated rat liver mitochondria. Mitochondria were incubated with succinate and hexokinase or creatine kinase at constant or varying extramitochondrial adenine nucleotide concentrations ranging over 0.3-5 mM. As parameters of oxidative phosphorylation, rate of respiration, membrane potential as well as intra- and extra-mitochondrial adenine nucleotide concentrations were determined. Below a threshold concentration of extramitochondrial adenine nucleotides of 2 mM, the free Gibb's energy for the adenine nucleotide transport increased but the extramitochondrial ATP/ADP ratio decreased at intermediate rates of respiration with decreasing extramitochondrial adenine nucleotide concentrations. In this range the rate of respiration was dependent on the extramitochondrial ADP concentration. No effect of the extramitochondrial adenine nucleotide concentration on the relationships between the rate of respiration and the membrane potential, the intramitochondrial adenine nucleotide pool and the intramitochondrial ATP/ADP ratio was found. This suggests that the decline of extramitochondrial ATP due to adenine nucleotide degradation and the limitation of adenine nucleotide transport may be of importance in the postischemic phase as nucleotide resynthesis and reorganization of physiological ion distribution are ATP consuming processes.

Oxidative phosphorylation and the realkalinization of intracellular pH during recovery from anoxia in Artemia franciscana embryos

The contribution of mitochondrial oxidative phosphorylation to the realkalinization of intracellular pH (pH i) and resynthesis of purine nucleotides during recovery from anoxia was investigated in embryos of Artemia franciscana by assessing the sensitivity of mitochondrial respiration to pH, calculating proton consumption by oxidative phosphorylation, and measuring changes in pH i using 31P nuclear magnetic resonance. Following short-term anoxia, pH i increased from 6.7 to 7.7 during 20 min of aerobic recovery and was temporally correlated with a large increase in ATP. State 3 respiration rates of isolated mitochondria were not substantially compromised at the acidic pH corresponding to the pH i during anoxia (pH 6.3–6.8) compared to values obtained at pH 7.7. Both state 3 respiration rates and respiratory control ratios exhibited broad, substrate-specific pH optima, whereas state 4 respiration rates increased gradually with increasing pH. P:O flux ratios were near their mechanistic limits and did not vary appreciably with pH below 7.5. Estimates of intracellular buffering capacity indicate that between 18 and 37 mmol H + (1 cytosol) −1 must be consumed to elevate pH i from 6.7 to 7.7. Phosphorylation of mono- and diphosphate purine-nucleotides during the first 20 min of recovery may account for the consumption of up to 4.79 mmol H + (1 cytosol) −1. An additional 4.77 to 8.18 mmol H + (1 cytosol) −1 may be consumed through the oxidation of mono- or dicarboxylic acids, respectively, in the Krebs cycle. Taken together, these data are consistent with a role for oxidative phosphorylation in the realkalinization of pH i and resynthesis of purine nucleotides in A. franciscana embryos during recovery from anoxia.

Purine and pyrimidine catabolite production in the postischemic rat heart--effect of adenosine supply during reperfusion.

Marked reduction in purine catabolite production is invariably observed in the course of repeated ischemic events1–3. Both beneficial and deleterious effects of this phenomenon should be taken into consideration. Inhibition of nucleotide breakdown protects the nucleotide pool particularly ATP in the heart but at the expense of the reduction of the release of endogenous protective metabolite — adenosine (ADO). Exogenous ADO exerts a significant reviving effect on the heart when supplied during reperfusion4–6 which may be partially mediated by substitution for impaired release of endogenous ADO. Exogenous ADO is also the most efficiently used substrate for resynthesis of adenine nucleotides. To obtain further information concerning the mechanism producing beneficial effect of ADO supplied during reperfusion we investigated the effects of three concentrations of ADO (optimal for vasoactive, metabolic and cardioplegic effect) on mechanical recovery, myocardial nucleotides and purine and pyrimidine catabolite production in Langendorff perfused rat heart.KeywordsMyocardial StunningNucleotide PoolDevelop TensionMyocardial Reperfusion InjuryImpaired ReleaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Adenosine's Effect on Myocardial Functional Recovery: Substrate or Signal?

During induced ischemia for cardiac surgery, nucleotides are degraded while being used to maintain myocyte integrity. The resulting nucleosides washout upon reperfusion, limiting nucleotide resynthesis resulting in poor postischemic cardiac function. We studied if the mechanism of the beneficial effect of adenosine, a nucleotide precursor, which is known to improve postischemic functional recovery is as a substrate for nucleotide resynthesis or by stimulation of adenosine A1 or A2 receptors. Isolated, retrograde-perfused rabbit hearts received cardioplegia as controls or cardioplegia containing 80 μM [R]-N6-[1-methyl-2-phenylethyl]-adenosine, an A1 receptor agonist, or 200 μM 5′-(N-ethylcarboxamido)adenosine, or 200 μM adenosine alone. To assess functional recovery developed pressure, max dP/dt, pressure-rate product, coronary flow, and myocardial oxygen consumption were compared after 120 min of 34°C global cardioplegic ischemia. Following ischemia and reperfusion, adenosine alone had better developed pressure, dP/dt , and pressure-rate product, while heart rates, wet weights, %H2O, end-diastolic volumes/pressures, and oxygen extraction were not significantly different between groups. While adenosine receptor stimulation may play a role, in this model the beneficial effect of adenosine on functional recovery appears to be mediated more by adenosine's role as a substrate for nucleotide resynthesis.

Precursor Trapping: A "Neonatal" Mechanism of Myocardial Protection

During induced ischemia for cardiac surgery, 5′-nucleotidase (5NT) catalyzes nucleotide breakdown by dephosphorylating AMP and IMP to diffusible precursors—adenosine and inosine. These precursors become unavailable upon reperfusion washout limiting nucleotide resynthesis, resulting in poor postischemic function. Neonatal hearts, which are more resistant to ischemia than adults, have low 5NT activity, trapping available precursors. Adult rabbit hearts given cardioplegia with a 5NT inhibitor, pentoxifylline, demonstrated improved postischemic contractility, compliance, and myocardial oxygen consumption after 120 min of 34°C ischemia. To determine if this improved function was a result of enhanced nucleotide precursor availability during or following ischemia, total nondiffusible nucleotides, ATP, ADP, AMP, and IMP, and total diffusible nucleotides, adenosine, inosine, hypoxanthine, and xanthine, were measured by HPLC at end ischemia, 1 and 15 min after reperfusion. While all preischemic values were equivalent, pentoxifylline-treated hearts had significantly greater total nondiffusible nucleotides at end ischemia, 1 and 15 min after reperfusion. Additionally, pentoxifylline-treated hearts had significantly greater total diffusible nucleosides at end ischemia and 1 min after reperfusion, but were equal to control at 15 min after reperfusion. Furthermore, coronary sinus effluent had a significantly higher release of total diffusible nucleosides in control vs pentoxifylline-treated hearts. The data indicate that precursor trapping with pentoxifylline prevented nucleotide catabolism to diffusible precursors and enhanced postischemic nucleotide availability. We postulate the increased precursor availability augmented myocardial nucleotide resynthesis and correlated with the improved functional recovery noted. This strategy may have application in adult cardiac surgery.

Acadesine and myocardial protection. Studies of time of administration and dose-response relations in the rat.

Although there are many factors that might contribute to tissue injury during ischemia and reperfusion, the loss of adenine nucleotides has long been considered to be of importance. This has led to the study of interventions designed to limit the loss of nucleotides or to enhance the rate of nucleotide resynthesis during reperfusion. Alternatively, the breakdown of adenosine triphosphate to adenosine might represent a protective response of the ischemic heart because adenosine is considered an anti-injury autocoid. Augmentation of endogenous adenosine levels might be beneficial. For these reasons, the protective properties of acadesine (AICAr: 5-amino-4-imidazole carboxamide riboside) were assessed in a rat model of myocardial ischemia and reperfusion. The protective properties of acadesine were studied in the isolated, perfused rat heart subjected to global hypothermic (20 degrees C) ischemia and reperfusion. When acadesine was given as an in vivo pretreatment (100 mg/kg i.v. 15 minutes before study) followed by being administered as an additive (20 mumol/l) to the St. Thomas' Hospital cardioplegic solution (single dose) and then as an additive (20 mumol/l) to the initial reperfusion (15 minutes) solution, the recovery of aortic flow after 2.5 hours of ischemia was improved from its control value of 16.5 +/- 3.9 ml/min to 28.9 +/- 4.1 ml/min (n = 8 per group; p less than 0.05). Similar protection was seen with other indexes of cardiac function. Analysis of hearts obtained at the end of 2.5 hours of ischemia and 35 minutes of reperfusion revealed no significant differences in metabolite content between control and drug-treated hearts with the exception of inosine monophosphate, which was increased from its drug-free control value of 0.10 +/- 0.01 mumol/g dry wt to 0.86 +/- 0.06 mumol/g dry wt (p less than 0.05). In further studies (n = 8 per group), with multidose (every 30 minutes) cardioplegia and extended periods (6 hours) of hypothermic ischemia, acadesine consistently led to higher mean recoveries of function and lower levels of creatine kinase leakage. Again, the only significant metabolic effect was an increase in tissue inosine monophosphate content. In studies (n = 12 per group) to determine whether acadesine was acting before, during, or after ischemia, the drug was given 1) only as pretreatment (100 mg/kg i.v.), 2) only during single-dose cardioplegia (20 mumol/l), or 3) only during reperfusion (20 mumol/l). Significant protection was observed in the first two groups (recovery of aortic flow increased from 10.6 +/- 2.6 ml/min in the acadesine-free control to 22.6 +/- 2.8 and 23.6 +/- 3.1 ml/min, respectively; p less than 0.05). No significant protection was observed when acadesine was given only during reperfusion. In dose-response studies, acadesine (0, 5, 20, 50, 200, and 1,000 mumol/l; n = 12 per group) was given only as a cardioplegic additive; the postischemic recoveries of aortic flow were 15.4 +/- 2.8, 16.9 +/- 3.6, 29.5 +/- 3.8, 27.4 +/- 3.8, 26.7 +/- 4.2, and 27.1 +/- 2.7 ml/min, respectively. Acadesine improves the ability of the heart to recover from ischemia and reperfusion when administered before ischemia or with cardioplegia. The mechanism underlying the protection remains to be resolved.

Prolonged adenine nucleotide resynthesis and reperfusion injury in postischemic skeletal muscle

Skeletal muscle ischemia results in energy depletion and intracellular acidosis. Reperfusion is associated with impaired adenine nucleotide resynthesis, edema formation, and myocyte necrosis. The purpose of these studies was to define the time course of cellular injury and adenine nucleotide depletion and resynthesis in postischemic skeletal muscle during prolonged reperfusion in vivo. The isolated canine gracilis muscle model was used. After 5 h of ischemia, muscles were reperfused for either 1 or 48 h. Lactate and creatine phosphokinase (CPK) release during reperfusion was calculated from arteriovenous differences and blood flow. Adenine nucleotides, nucleosides, bases, and creatine phosphate were quantified by high-performance liquid chromatography, and muscle necrosis was assessed by nitroblue tetrazolium staining. Reperfusion resulted in a rapid release of lactate, which paralleled the increase in blood flow, and a delayed but prolonged release of CPK. Edema formation and muscle necrosis increased between 1 and 48 h of reperfusion (P less than 0.05). Recovery of energy stores during reperfusion was related to the extent of postischemic necrosis, which correlated with the extent of nucleotide dephosphorylation during ischemia (r = 0.88, P less than 0.001). These results suggest that both adenine nucleotide resynthesis and myocyte necrosis, which are protracted processes in reperfusing skeletal muscle, are related to the extent of nucleotide dephosphorylation during ischemia.

Ischemia/Reperfusion Injury in Skeletal Muscle

Ischemia/reperfusion injury to skeletal muscle may be explained by a cascade of cellular and systemic events initiated by an ischemic period followed by reperfusion. During the period of ischemia there is a gradual reduction of intracellular energy stores. Adenosine triphosphate is gradually depleted despite the buffering effect of creatine phosphate which is present in large stores in muscles. As well, glycogen stores are depleted with resultant production of small amounts of energy and large accumulations of lactate. Upon reperfusion there is a reactive hyperemia, resulting in an overall increase in muscle blood flow, despite the fact that areas may continue to be underperfused. Results of this blood flow are mixed with the beneficial effects of removing metabolic by-products and supplying exogenous substrates and oxygen. However, this blood flow also causes harmful effects by washing out necessary precursors for adenine nucleotide resynthesis. Production of oxygen free radicals occurs with resultant membrane lipid peroxidation, and calcium influx occurs upon reoxygenation with resultant disruption of oxidative rephosphorylation in the mitochondria. The sequestration of white blood cells in the muscle due to up regulation of both neutrophil receptors and endothelial leukocyte adhesion molecules results in a prolongation of the reperfusion injury. This subsequently results in damage to remote organs, including lung, heart, and kidneys. The future for therapeutic interventions aimed at reducing this injury lie mostly in the ability to modulate the reperfusion effect.

Adenine, guanine, and inosine nucleotides of chick growth cartilage: relationship between energy status and the mineralization process.

The major aim of this investigation was to measure the nucleotide content of the developing chick epiphysis and to relate changes in nucleotide levels to chondrocyte maturation and the development of mineralization. Using a cryostat, sections of cartilage were isolated from the proximal head of the tibial growth cartilage, care being taken to preserve the metabolic integrity of the tissue. Sections were identified microscopically, pooled, and the nucleotide and nucleoside content of each sample determined by HPLC. Procedures used for the study were shown to minimize degradation of nucleotides. Their effectiveness was assessed through an evaluation of the rapid freezing technique and by examination of the effects of apatite on the recovery of endogenous and added nucleotides. Analysis of nucleotide levels in the growth cartilage indicated that chondrocytes undergo a profound change in energy metabolism during development and maturation. Thus, in the premineralized resting and proliferative zones, ATP and, to a lesser extent, GTP values were high, suggesting that the chondrocytes obtained metabolic energy through both glycolytic and mitochondrial oxidative processes. In the hypertrophic zone and in calcified cartilage, there was a profound decrease in the ATP concentration and a corresponding fall in the energy charge and the ATP/ADP ratios. The nucleotide levels in this zone indicated that there was increased reliance on nonoxidative metabolism. Measurement of nucleoside levels in premineralized cartilage suggested that there was little resynthesis of nucleotides through the salvage pathway. These observed changes in nucleotide values are consistent with earlier observations concerning chondrocyte redox and the low pO2 tension of the hypertrophic zone.2+off

Degradation and resynthesis of adenine nucleotides in adult rat heart myocytes.

The degradation and short-term resynthesis of adenine nucleotides have been examined in a preparation of isolated rat heart myocytes. These myocyte preparations are essentially free of vascular and endothelial cells, contain levels of adenine nucleotides quite comparable to those of intact heart tissue, and retain these components remarkably well for up to 2 h of aerobic incubation in the presence of 1 mM Ca2+. When the cells are rapidly and synchronously de-energized by addition of uncoupler, an inhibitor of respiration and iodoacetate, cellular ATP is degraded almost quantitatively to AMP. The AMP is then converted to either intracellular adenosine, which accumulates to high concentrations before release to the cell exterior, or to IMP. The relative contribution of these two pathways depends on the metabolic state of the cells just prior to de-energization, with IMP production favored when respiring cells are de-energized and adenosine formation predominant when glycolyzing myocytes are subjected to this treatment. Cells de-energized by anaerobiosis in the absence of glucose lose ATP and adenine nucleotides with the production of IMP and adenosine. Upon reoxygenation, these cells restore a high adenylate energy charge and about 60% of control levels of GTP. There is a net resynthesis of 5-7 nmol of adenine nucleotides.mg-1 protein with a corresponding decline in IMP. Added [14C]adenosine labels the adenine nucleotide pool, but little net resynthesis of adenine nucleotides via adenosine kinase can be detected. It therefore appears that a rapid regeneration of adenine nucleotides can occur via the enzymes of the purine nucleotide cycle in heart myocytes and is limited by the size of the IMP pool retained.