Adenine Nucleotide Content Research Articles

Influence of ageing on functional recovery and guanine nucleotide levels of the heart following cold cardioplegic arrest.

The effect of age on metabolism and mechanical recovery of the heart after cardioplegic arrest is important, but remains a relatively unexplored subject. In this study, functional recovery and nucleotide levels were compared in the heart at different ages subjected to prolonged hypothermic cardioplegic arrest. Three different age groups of rats: 1 (A); 4 (B); and 16 months (C) were perfused in working mode and subjected to cardioplegic arrest (St. Thomas' No. 1) and ischemia for 4 h at 4 degrees C, followed by reperfusion for 35 min. Cardiac function (cardiac output and aortic pressure) was recorded before and after ischemia. Another series of hearts in all three age groups underwent 5 min of normoxic perfusion to obtain pre-ischemic baseline metabolite concentrations. Hearts were freeze-clamped at the end of each experiment and used for determination of nucleotide and creatine metabolites by HPLC. The post-ischemic recovery (% of the pre-ischemic value) of the cardiac power was 48.9 +/- 7.8% for group A, which was significantly higher than the functional recovery of group B (24.1 +/- 3.5%) or C (21.4 +/- 4.7%, P < 0.05, respectively). There was no difference in ATP or the total adenine nucleotide or creatine metabolite concentrations between the three age groups. In contrast, both GTP and the total guanine nucleotide concentration was highest in A (P < 0.05). Total guanylate pool was 1.52 +/- 0.10 1 micromol/g dry wt. in A, as compared to B (1.05 +/- 0.04) or C (1.12 +/- 0.04). NAD was significantly higher in B (4.1 +/- 0.1. P < 0.05), when compared to A (3.6 +/- 0.1) and C (3.8 +/- 0.1). Best post-ischemic functional recovery after cardioplegic arrest was observed in the 1-month-old hearts (A) and was associated with highest guanine nucleotide concentration; preservation of guanine nucleotide pool in the youngest hearts may be an important mechanism for improved cardioprotection due to the important role of GTP in signalling pathways.

Characteristics of cardiac hypertrophy in the juvenile visceral steatosis mouse with systemic carnitine deficiency.

The juvenile visceral steatosis (JVS) mouse exhibits hereditary systemic carnitine deficiency and develops cardiac hypertrophy. The aim of this study was to clarify the characteristics of cardiac hypertrophy in the JVS mouse. Total carnitine content in IVS mouse heart was about 10% of that of control mouse heart at 4 and 8 weeks of age. The heart weight/body weight ratio was bigger in JVS mice than that in control mice at 2 weeks of age, and this difference in ratio increased with age. The wall areas of both ventricles and septum in JVS mice were larger than those of the control mice at 2 and 8 weeks. The myocyte diameter in both ventricular walls and septum in JVS mice was longer than that of the control mice. On electron microscopy, the percent of mitochondria in the myocyte was 66% in JVS mice, and 37% in control mice. The percent of lipid fraction in JVS mice was six-fold higher than that in control mice. Total content of adenine nucleotides in JVS mouse heart was about 60% of that in control mouse heart. Adenylate energy charge in JVS mouse heart was 63 and 45% of that in the control mouse heart at 4 and 8 weeks, respectively. Overall, the cardiac enlargement observed in this animal model could be accounted for by a proportional increase in the myocyte diameter in the ventricles and septum, accompanied by an increase in mitochondria. Furthermore, this cellular growth is associated with decreases in the levels of ATP and ADP, and adenylate energy charge.

Prediction of the resistance to hypoxia on the basis of modifications of the adenine nucleotide system

An empirical dependence allowing one to identify rhythmic fluctuations of stable and unstable state in respect of hypoxia in the process of adaptation has been developed on the basis of long-term investigation of changes in brain content of adenine nucleotides upon “hypobaric training” of rabbits. The function approximating this dependence enables one to predict the effective number of “hypobaric training” sessions providing for the adaptive reaction.

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.

Periodate-oxidized ATP stimulates the permeability transition of rat liver mitochondria

Periodate-oxidized ADP (oADP) and periodate-oxidized ATP (oATP) stimulate the permeability transition in energized rat liver mitochondria measured as the Ca 2+-efflux induced by Ca 2+ and P i. In the presence of Mg 2+ and P i, mitochondria lose intramitochondrial adenine nucleotides at a slow rate. oATP induces a strong decrease of the matrix adenine nucleotides which is inhibited by carboxyatractyloside. Under these conditions, Mg 2+ prevents the opening of the permeability transition pore. EGTA prevents the P i-induced slow efflux of adenine nucleotides, but is without effect on the oATP-induced strong decrease of adenine nucleotides. This oATP-induced strong adenine nucleotide efflux is inhibited by ADP. oATP reduces the increase of matrix adenine nucleotides occurring when the mitochondria are incubated with Mg 2+ and ATP. This effect of oATP is also prevented by carboxyatractyloside. oATP is not taken up by the mitochondria. It is suggested that oATP induces a strong efflux of matrix adenine nucleotides by the interaction with the ADP/ATP carrier from the cytosolic side. The induction of the mitochondrial permeability transition by oADP and oATP is attributed to two mechanisms—a strong decrease in the intramitochondrial adenine nucleotide content, especially that of ADP, and a stabilization of the c-conformation of the ADP/ATP carrier.

Elevated inosine monophosphate levels in resting muscle of patients with stable chronic obstructive pulmonary disease.

In order to investigate disturbances in energy metabolism in resting muscle of patients with stable chronic obstructive pulmonary disease (COPD), concentrations of adenine nucleotides and related compounds were examined comparing 34 COPD patients with eight age-matched healthy control subjects. Biopsies were taken from the anterior tibialis muscle. Special attention was paid to the muscle content of inosine monophosphate (IMP), a deamination product of adenosine monophosphate (AMP), because IMP formation is thought to reflect an imbalance between resynthesis and utilization of adenosine triphosphate (ATP). The absolute concentrations of high-energy phosphate compounds did not differ between patients and control subjects, but the ATP/ADP and the phosphocreatine/creatine ratio were significantly lower in the patients. IMP (detection level = 0.06 mmol/kg dry weight) was detected in 25 of 34 patients versus one of eight control subjects (p = 0.001). Mean (SD) IMP level in these patients was 0.18 (0.14) versus 0.06 mmol/kg dry weight in the one control subject. Based on the presence of detectable levels of muscle IMP, the patient group was divided into two subgroups. In IMP-positive patients, ATP/ADP and phosphocreatine/creatine ratios were significantly lower than in IMP-negative patients. IMP-positive patients were furthermore characterized by a significantly lower DL(CO). The results of this study indicate an imbalance between the utilization and resynthesis of ATP in resting muscle of patients with stable COPD.

Calcium uptake by the sarcoplasmic reticulum, high energy content and histological changes in ischemic cardiomyopathy.

Sarcoplasmic reticulum (SR) Ca2+ uptake, myocardial high energy content and histology were examined in different zones of hearts from patients with ischemic cardiomyopathy. Unfractionated homogenates were prepared from left ventricular samples obtained in three zones of each heart: an infarct-remote zone, an outer peri-infarct zone, and an inner peri-infarct zone. Oxalate-supported 45Ca2+ uptake was measured at 37 degrees C using a filtration method. Maximum rate (Vmax) of uptake in absence or in presence of ryanodine was lower in inner peri-infarct (7.4 +/- 0.7 and 9.5 +/- 0.8 nmol min-1 mg-1 of protein, respectively; mean +/- SEM) and outer peri-infarct tissues (8.8 +/- 0.8 and 12.0 +/- 0.8 nmol min-1 mg-1) than in infarct-remote myocardium (12.7 +/- 2.1 and 15.8 +/- 2.2 nmol min-1 mg-1). The apparent affinity constants for Ca2+ (KCa) as well as the Hill coefficients were not different. Homogenate DNA (1.6 +/- 0.1, 1.6 +/- 0.1 and 1.7 +/- 0.1 mg/g of remote, inner peri-infarct and outer peri-infarct myocardium, respectively) and adenine nucleotides contents (ATP: 15 +/- 1.3, 14 +/- 0.8 and 15 +/- 1.0 mumol/g dry weight, respectively) were similar in all tissues. Fibrosis was increased in inner peri-infarct tissue (37 +/- 6%; vs. 13 +/- 2% and 12 +/- 2% in both remote and outer peri-infarct tissues, respectively), but the number of abnormal cells was not significantly different. The decrease of Ca2+ uptake in ischemic cardiomyopathy is not homogeneous in the ventricular wall, and reflects a decreased number/activity of SR Ca(2+)-ATPase, without altered Ca(2+)-affinity or increased Ca2+ leakage through ryanodine receptors.

Metabolic adaptation to long term changes in gravity environment

Biochemical analyses of the brain of Cichlid fish larvae, exposed during their very early development for 7 days to an increased acceleration of 3g (hyper-gravity), revealed a decrease in brain nucleoside diphosphate kinase (NDPK) as well as creatine kinase (BB-CK) activity. Using high performance liquid chromatography (HPLC) the concentrations of adenine nucleotides (AMP, ADP, ATP), phosphocreatine (CP), as well as of nicotineamide adenine dinucleotides (NAD, NADP) were analyzed in the brain of hyper-g exposed larvae vs. 1g controls. A slight reduction in the total adenine nucleotides (TAN) as well as the adenylate energy charge (AEC) was found. In parallel a significant increase in the NAD concentration and a corresponding decrease in NADP concentration occurred in larva's hyper-g brains vs. 1g controls. These results give further evidence for an influence of gravity on cellular level and furthermore contribute to a clarification of the cellular signal-response chain for gravity perception.

Mechanism of cardioprotective effect of orotic acid.

The pyrimidine base, orotic acid (OA), markedly improves the function of recently infarcted hearts subjected to global ischemia. The mechanism of cardiac action of OA is unclear, but it has been proposed that OA acts by correcting a relative deficiency of nucleotide precursors required for RNA synthesis in the stressed myocardium or by improving myocardial energy supply. The aim of this study was to investigate the mechanism of action of OA by (1) determining whether a high dose of OA can raise the concentration of pyrimidine metabolites in plasma, liver, and heart; (2) examining the effects of OA on adenine nucleotide (AN) concentrations in normal and infarcted hearts, before and after global ischemia; and (3) determining the effect of uridine, an important metabolite of OA, on myocardial energy metabolism. Three studies were performed: (1) The time course of changes in tissue and plasma concentrations of pyrimidine compounds was examined in unoperated rats after the administration of 100 mg/kg OA. (2) Rats were given OA (30 mg/kg/d) for 2 days after experimental infarction, and tissue and plasma pyrimidine concentrations were examined; the hearts were removed for perfusion in the isolated working rat heart model (37 degrees C), subjected to 30 minutes of global ischemia, and recovery of function was assessed. AN content was assessed in the noninfarcted myocardium before and after ischemia. Isolated hearts were subjected to 30 minutes of hypoxic perfusion and the effect of adding 17 microM uridine to the perfusate was examined. Study 1 showed that OA administration produced an increase in hepatic uridine and cytidine, followed by increased plasma uridine and cytidine (cytidine, +55%, P < 0.001; uridine, +124%, P = 0.011). Myocardial uracil nucleotides increased temporarily after 4 hours (+21%, P < 0.01). In infarcted hearts after 2 days of OA administration, there were no significant changes in myocardial uracil or cytosine nucleotides or total RNA. Infarction significantly reduced functional recovery after global ischemia (sham = 62%; infarct = 26% of preischemic level; P < 0.05). OA improved the recovery of preischemic function by 133% (P < 0.05) in infarcted, but not sham-operated, hearts. Preischemic ATP and total adenine nucleotides (TAN) were decreased in the surviving myocardium of infarcted hearts (ATP reduced from 21.7 +/- 0.8 to 14.7 +/- 0.7 mumol/g dry wt, P < 0.001; TAN decreased from 30.3 +/- 0.8 to 22.4 +/- 1.1 mumol/g dry wt, P < 0.001). OA treatment prevented these reductions. Study 3 showed that uridine improved myocardial ATP and TAN levels, and decreased purine loss in hypoxic hearts. The increased AN levels were accompanied by evidence of enhancement of anerobic glycolysis. We conclude: (1) That OA acts on the heart via the liver by increasing the availability of plasma uridine and cytidine. (2) Uridine is capable of increasing myocardial ATP production by stimulating anerobic glycolysis. (3) OA treatment improves tolerance to global ischemia in infarcted but not normal hearts by preventing depletion of AN in the surviving myocardium.

Nucleotide metabolism in the heart subjected to heat stress.

Heat shock increases cardiac resistance to ischemia but the mechanism of this effect is unknown.1 Whole body hyperthermia has been recognised to induce synthesis of a group of proteins, termed “heat shock proteins” (HSP). The predominant function of HSP is protection against protein denaturation and facilitation of desired protein folding at the time of ribosomal polypepide synthesis. Furthermore HSP are able to unfold the polipeptide chains, by associating with proteins being incorporated into mitochondria and placing them in a translocation-competent configuration. These roles are referred to as “chaperone” functions.2 There is strong evidence to suggest that increased expression of heat shock protein HSP70 is directly involved in the cardioprotective effect of heat stress.3,4 However it is not yet known which enzymes or structural proteins and which metabolic pathways are protected by HSP70. To investigate the possible role of alterations in nucleotide metabolism in this beneficial effect, we analysed the concentrations of adenine, guanine, pyrimidine and pyridine nucleotides and phosphocreatine concentration in the rat heart subjected to heat shock. The concentrations of nucleotide metabolites were evaluated under normoxic conditions and after 5 min of global normothermic ischemiaKeywordsHeat StressHeat Shock ProteinPyridine NucleotideNucleotide MetabolismHeat Shock Protein HSP70These 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.

Evaluation of various solutions for small bowel graft preservation.

AIM:To elucidate the effect of various solutions for small bowel graft preservation in pigs under hypothermic storage.METHODS:The swine segmental small bowel graft was autotransplanted after it was preserved with lactated Ringer's (LR), Euro-Collins (EC), hyperosmolarity citrate adenine (HC-A) and WMO-1 solutions for 10,18 and 24 hours,respectively.The recipient survival rate, morphological structure, graft mucosal energy substances and Na( +) -K(+) ATPase activity were studied,and graft absorption was estimated with D-xylose absorption test.RESULTS:The morphological study of the grafts preserved with LR or HC-A solution for 10 hours or with EC and WMO-1 solution for 18 hours was normal 6days after operation. Mucosal ATP,total adenine nucleotides (TAN) contents and Na( +) -K(+)ATPase activity of the graft preserved with EC or WMO solution were higher than that of the graft preserved with LR or HC-A solution.Serum level of D-xylose was higher in EC and WMO-1 groups than in LR and HC-A groups when the graft was preserved for 24 hours.CONCLUSION:EC and WMO-1 solutions can preserve the swine small bowel up to 18 hours, which are superior to LR and HC-A solutions.

Cardioprotection by orotic acid: metabolism and mechanism of action.

The pyrimidine base, orotic acid (OA), improves the function of recently infarcted hearts subjected to global ischemia but its mechanism of action is unclear. Our aims were to examine (i) in normal rats, the effect of OA on pyrimidine levels in plasma, liver and heart; (ii) in rats with normal or infarcted hearts, the effect of OA on adenine nucleotide metabolism and mechanical function, before and after global ischemia. To investigate the metabolism of OA, normal rats received 100 mg/kg OA, and changes in plasma and tissue concentrations of pyrimidines were examined. The effects of OA were also studied in rats receiving OA for 2 days after coronary ligation or sham operations, and plasma and tissue pyrimidine concentrations examined. Their hearts were isolated and perfused, then subjected to 30 min of global ischemia. Mechanical function and adenine nucleotide content were assessed pre- and post-ischemia. In normal, unoperated rats, administration of 100 mg/kg OA significantly increased hepatic uracil and cytosine nucleotide concentrations, then increased plasma uridine (+124%) and cytidine (+55%), and transiently increased myocardial uracil nucleotides (+21%). Infarction significantly reduced recovery of cardiac work after global ischemia (sham=62%; infarct=26%; P<0.05), and OA treatment in infarcted hearts increased post-ischemic work by 192% (P<0.05), but not in shams. Pre-ischemic ATP was reduced in the surviving myocardium of infarcted hearts from 21.7+/-0.8 to 14.7+/-0. 7 micromol/g dry weight (P<0.001) and total adenine nucleotides (TAN) from 30.3+/-0.8 to 22.4+/-1.1 micromol/g dry weight (P<0.001). OA treatment prevented these reductions in infarcted hearts (ATP 20. 7+/-0.5; TAN, 29.1+/-0.6 micromol/g dry weight). We conclude that OA protects the infarcted heart against global ischemia by enhancing hepatic release of pyrimidine nucleosides into the plasma, which then prevent depletion of adenine nucleotides in the surviving myocardium.

Modification of Sulfhydryl Groups in the [gamma]-Subunit of Chloroplast-Coupling Factor 1 Affects the Proton Slip through the ATP Synthase.

A large proton leak not coupled to ATP synthesis (slip) occurs at alkaline pH through the chloroplast ATP synthase (Y. Evron, M. Avron [1990] Biochim Biophys Acta 1019: 115-120). The involvement of the ATP synthase [gamma]-subunit in the regulation of proton conductance was analyzed by measuring the effect of thiolalkylating agents on proton slip. Alkylation by N-ethylmaleimide of [gamma]-cysteine (Cys)-89, which is exposed upon energization of thylakoids, increases the slip only at alkaline pH. The slip is partially suppressed by low concentrations of adenine nucleotides and is completely eliminated by venturicidin, a blocker of the hydrophobic polypeptide complex of the chloroplast ATP synthase (CF0). Conversely, cross-linking of [gamma]-Cys-89 with [gamma]-Cys-322 renders the ATP synthase leaky to protons and insensitive to ATP also at neutral pH. The accessibility of [gamma]-Cys-89 to alkylation by fluorescein maleimide is completely suppressed by N,N-dicyclohexylcarbodiimide and by venturicidin, which block proton conductance through CF0 and increase the pH gradient. These results suggest that the [gamma]-subunit has a dominant role in proton gating through the ATP synthase and responds to changes in pH and ligands taking place on either side of the thylakoid membrane. It is proposed that the conformational changes that induce the proton slip and the exposure of [gamma]-Cys-89 reflect the conversion of the enzyme from a catalytically latent to an active state, and depend on the deprotonation of a stromal site at alkaline pH and on protonation of an intrathylakoid inner site upon energization. Therefore, conditions that induce the conformational activation also provide the driving force for ATP synthesis.

CAMP and Ca2+Involvement in the Mitochondrial Response of Cultured Fetal Rat Hepatocytes to Adrenaline

The effect of adrenaline on the control of respiratory activity of mitochondria from fetal hepatocytes in primary culture was studied. In the absence of adrenaline, the respiratory control ratio (RCR) of mitochondria increased during the first 3 days of culture due to a decrease in the rate of state 4 respiration. The presence of adrenaline in the incubation medium further increased the mitochondrial RCR through a decrease in the rate of respiration in state 4 and to an increase in the respiration rate in state 3. The effect of adrenaline was mimicked by dibutyryl-cAMP, forskolin, and isobutyl methyl xanthine. All these compounds increased cAMP concentrations, suggesting that cAMP may be involved in the effect of adrenaline. The increase in intracellular free Ca2+concentrations caused by phenylephrine, vasopressin, or thapsigargin was also accompanied by an increase in the RCR, suggesting that both phenomena are associated. Dibutyryl-cAMP also increased free Ca2+concentrations, suggesting that the effects of cAMP may be mediated by free Ca2+concentrations. Adrenaline, dibutyryl-cAMP, phenylephrine, vasopressin, and thapsigargin promoted adenine nucleotide accumulation in mitochondria; this may be an intermediate step in the activation of mitochondrial respiratory function. These results suggest that the stimulatory effect of adrenaline on mitochondrial maturation in cultured fetal rat hepatocytes may be exerted through a mechanism in which both cAMP and Ca2+act as second messengers. It is concluded that the effect of adrenaline on mitochondrial maturation is exerted by both α- and β-adrenergic mechanisms and is mediated by the increase in adenine nucleotide contents of mitochondria.

Coincubation of rat renal proximal tubules with hepatic subcellular fractions potentiates the effects of para-aminophenol.

Treatment of rats with para-aminophenol (PAP) (300 mg/kg ip) produced decreases in renal nonprotein sulfhydryl (NPSH) content, oxygen consumption, and adenine nucleotide concentrations 2-4 hr following administration. In contrast, incubation of rat renal tubules with up to 1 mm PAP for 4 hr produced inconsistent changes in renal tubules. This discrepancy suggested that extrarenal metabolism of PAP may be involved in PAP bioactivation and nephrotoxicity. We designed the present studies to test the hypothesis that hepatic metabolism of PAP potentiates the effects of PAP on renal tubules. Incubation of renal tubules with 0.5 mm PAP and 10 mg protein from hepatic postmitochondrial supernatant (S9 fraction) in the absence of glutathione (GSH) for 4 hr did not alter renal oxygen consumption or adenine nucleotide metabolite concentrations. We observed no changes when we incubated tubules with 0.5 mm PAP and 1 mm GSH in the absence of hepatic S9 fraction. However, incubation of renal tubules with 0.5 mm PAP, 1 mm GSH, and 10 mg hepatic S9 protein for 4 hr significantly decreased renal oxygen consumption and adenosine triphosphate and total nucleotide concentrations. These data suggest that the effects of PAP in renal tubules may be potentiated by enzymatic metabolism of PAP, possibly involving oxidation and GSH conjugation. From experiments using hepatic microsomes or cytosol instead of S9 fraction, we found that changes were produced when we incubated tubules with PAP in the presence of hepatic microsomes, but not cytosol. These data suggest that hepatic microsomal metabolism of PAP may contribute to the production of changes in renal tubules in vitro. PAP-induced changes in renal proximal tubules were prevented when we included a beta-nicotinamide adenine dinucleotide phosphate (NADPH) generating system in the incubation medium. The protective effect of NADPH persisted when microsomes were inactivated by incubation with 1-aminobenzotriazole, a cytochrome P450 inhibitor. These data suggest that cytochrome P450-dependent oxidation is not involved in the production or prevention of PAP-induced changes in renal tubules. The enzyme(s) responsible for PAP bioactivation and the mechanism(s) by which NADPH protects renal tubules from PAP-induced decrements in oxygen consumption and adenine nucleotide concentrations are currently unclear.

Energy metabolism disturbances induced by multiple ischemic preconditioning worsen the recovery of cardiac function in reperfusion

Single ischemic preconditioning improves functional recovery of isolated rat heart subjected to global ischemia and reperfusion. Two preconditioning cycles abolish this protective effect, while 4 cycles impair functional recovery of the heart. These phenomena are due to the fact that repetitive ischemic preconditioning reduces the content of phosphocreatine and adenine nucleotides in the heart before long-term ischemia, which prevents the recovery of energy metabolism and induces additional damage to cardiomyocyte membranes during reperfusion.

Characterization of Type 3 Ryanodine Receptor (RyR3) of Sarcoplasmic Reticulum from Rabbit Skeletal Muscles

We investigated type 3 isoform (RyR3) of ryanodine receptor in rabbit skeletal muscles using an antibody specific for RyR3. By Western blot analysis and by immunoprecipitation, a single polypeptide for RyR3 was detected in sarcoplasmic reticulum vesicles from rabbit diaphragm but not in those from back muscle. The molecular mass was slightly smaller than that of RyR1, the major isoform in skeletal muscles. Each of RyR1 and RyR3 formed a homotetramer in rabbit diaphragm. RyR3 had a single class of [3H]ryanodine binding sites of high affinity (KD = 1.6 nM). From the Bmax of the binding, the content of RyR3 was estimated to be only 0.6% of RyR1 in rabbit diaphragm. -3H-Ryanodine binding to RyR3 was biphasically dependent on Ca2+, as is true of RyR1, and was stimulated further by adenine nucleotide, caffeine, or high salt concentration. Procaine and ruthenium red inhibited the binding. RyR3 was more resistant to Mg2+ inhibition than RyR1. Interestingly, RyR3 showed about a 7-fold lower Ca2+ sensitivity for activation than RyR1. Comparison with the counterparts in bullfrog skeletal muscles indicates that the Ca2+ sensitivities of RyR3 homologs are similar to each other, whereas those of RyR1 homologs are species-specific.

In vivo analysis of metabolic dynamics inSaccharomyces cerevisiae : I. Experimental observations

The goal of this work was to obtain rapid sampling technique to measure transient metabolites in vivo. First, a pulse of glucose was added to a culture of the yeast Saccharomyces cerevisiae growing aerobically under glucose limitation. Next, samples were removed at 2 to 5 s intervals and quenched using methods that depend on the metabolite measured. Extracellular glucose, excreted products, as well as glycolytic intermediates (G6P, F6P, FBP, GAP, 3-PG, PEP, Pyr) and cometabolites (ATP, ADP, AMP, NAD(+), NADH) were measured using enzymatic or HPLC methods. Significant differences between the adenine nucleotide concentrations in the cytoplasm and mitochondria indicated the importance of compartmentation for the regulation of the glycolysis. Changes in the intra- and extracellular levels of metabolites confirmed that glycolysis is regulated on a time scale of seconds. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 305-316, 1997.

Response of myocardial cellular energy metabolism to variation of buffer composition during open-chest experimental cardiopulmonary resuscitation in the pig.

The aim of the present study was to investigate possible relationships in piglets between myocardial energy-related metabolites and intracellular electrolytes during open-chest cardiopulmonary resuscitation (OCCPR) supplemented by the administration of alkaline buffers with varying sodium content. Our hypothesis was that an increasing myocardial intracellular sodium content would decrease the intracellular energy stores. In addition to haemodynamics, acid-base and blood gas variables were analysed, and myocardial biopsies were collected before and during OCCPR as well as after the return of spontaneous circulation. After a period of 4 min of untreated ventricular fibrillation (VF). 25 piglets were randomly allocated to one of four groups: OCCPR with normal saline (n = 5); OCCPR with sodium bicarbonate (SB) (n = 7); OCCPR with Tris buffer mixture (TBM) (n = 7); and a totally untreated control group (n = 6). The results showed that 4 min of untreated VF almost eradicated creatine phosphate (CrP) and that the ATP/ADP ratio decreased to 1.5-2.0. During OCCPR with normal saline, the myocardial content of CrP increased, whereas lactate, ATP and ADP levelled off and AMP decreased, causing an increased ATP/ADP ratio. The adenosine and inosine contents increased, whereas inosine monophosphate was unchanged at a low level, the adenosine and inosine contents being inversely correlated with the total content of adenine nucleotides. In both buffered groups, the increase in most energy-related metabolites (CrP, ATP, ADP, AMP and the ATP/ADP quotient) was less and in lactate more pronounced than in the group not being buffered, with no difference between the groups receiving SB or TBM. Although the intracellular potassium content was unaltered, the sodium, chloride and calcium concentration increased, more so in the group receiving SB. The intracellular content of sodium was correlated with that of calcium. Thus, buffering increased the myocardial AMP degradation during OCCPR by increasing the flux via the 5'-nucleotidase reaction, and SB increased the intracellular contents of sodium and calcium to a greater extent than did TBM.

Protective effects of succinic aciddimethyl ester infusion in experimental endotoxemia

In rats injected with bacterial lipopolysaccharide (LPS; 5 γmg/g body weight [BWT]), the toxin provokes death within 24 h in 23% of the animals and, in surviving rats, causes a decrease in BWT, hyperlactacidemia, hyperlipacidemia, and hyperketonernia, as well as depletion of both liver and muscle glycogen content. In the liver, LPS severely lowers the ATP and total adenine nucleotide content, ATP/ADP ratio, and adenylate charge. In hepatocytes from LPS-injected rats, the oxidation of d-glucose is first increased 2 h after administration of the toxin, despite close-to-normal phosphorylation of the hexose. In hepatocytes prepared from rats killed 24 h after injection of LPS, the phosphorylation of d-glucose, its incorporation into glycogen, and its oxidation are all severely impaired. This sequence of changes, which coincides with a decreased ratio between pyruvate and lactate production from exogenous d-glucose, is comparable to that found with agents that uncouple oxidative phosphorylation. The injection of LPS also alters the metabolic response of hepatocytes to the dimethyl ester of succinic acid (SAD), in terms, for instance, of the sparing action of the ester upon both the production of 14CO 2 by hepatocytes prelabeled with l-[U- 14C]glutamine and the output of NH 4 +, and its inhibitory action on glycogenolysis and futile cycling in the reactions catalyzed by glucokinase and glucose-6-phosphatase. Nevertheless, the infusion of SAD protects the rats against the deleterious effect of LPS upon such variables as the plasma concentration of free fatty acids and β-hydroxybutyrate, the liver ATP content, and the oxidation of D-glucose, as well as the pyruvate/lactate ratio, in hepatocytes prepared from the LPS-injected rats. The infusion of SAD also virtually suppresses lethality in the LPS-injected animals. It is proposed, therefore, that the infusion of succinic acid esters may represent a novel therapeutic approach in endotoxemia and multiple-organ failure.