Adenine nucleotide metabolism during cardiac hypertrophy and ischemia in rats.

The effects of ischemia on the adenine nucleotides, and the production of adenosine, inosine and hypoxanthine were studied in 200 rat hearts made hypertrophic by swimming exercise, by thyroxine treatment, or by operative constriction of the abdominal dominal aorta. The results were the same irrespective of the method used for cardiac hypertrophy. In the normal heart ischemia produced a decrease in ATP and an increase in ADP, AMP, adenosine, inosine, and hypoxanthine. In the hypertrophied heart changes in ATP, AMP, adenosine, inosine and hypoxanthine were in the same direction as in the normal heart but significantly greater. ADP first increased and then fell to very low levels in the hypertrophied heart. With prolonged ischemia (5 to 20 min) adenosine, inosine, and hypoxanthine appeared in the 0.9% NaCl bathing solution of the hypertrophied hearts. When the amounts of these compounds in the medium were added to the myocardial tissue levels, the sum tended to remain constant. During the development of cardiac hypertrophy, the ATP levels, and to a lesser extent the total adenine nucleotide pool rose from the first to the third day after aortic constriction but declined from the fourth to the tenth day. These data suggest that either the “de novo” synthesis and utilization of the “salvage” pathway for the adenine nucleotides is reduced or the degradative activities dominate these synthetic pathways during cardiac hypertrophy.     

Biochemical and functional effects of nucleoside transport inhibition in the isolated cat heart

With increasing periods of normothermic ischemia, increasing amounts of mainly inosine and hypoxanthine are released in reperfusates of isolated working cat hearts. Nucleoside transport inhibition (soluflazine; 1 x 10(-7) M) markedly reduces the total release, but increases the release of adenosine. Tissue levels of adenine nucleotides are reduced by 50% after 32 min of ischemia with an almost equivalent, parallel, rise in inosine and hypoxanthine. Subsequent reperfusion leads to a complete removal of the catabolites and a restoration of the energy charge, but not to any recovery of the sum of nucleotides. Nucleoside transport inhibition has no effect on the changes in the nucleotides, but induces a marked accumulation of adenosine during ischemia and prevents the rapid escape of the nucleosides--not of hypoxanthine--upon reperfusion. Cardiac function markedly deteriorates after 20 and 32 min of ischemia. Transport inhibition completely prevents the decrease in cardiac output and pressure-rate product without any effect on normoxic performance. It is suggested that the prolonged presence of adenosine and of inosine may exert a protective effect.     

Metabolic fate of hypoxanthine and inosine in cultured cardiomyocytes.

The metabolic fate of labeled hypoxanthine and inosine, degradation products of adenine nucleotides, was studied in cultured beating cardiomyocytes, in order to assess the physiological significance of their contribution to salvage nucleotide synthesis in the heart. Inosine and hypoxanthine were found to be incorporated into nucleotides by a similar rate, but in the presence of 8-aminoguanosine, a potent inhibitor of purine nucleoside phosphorylase (EC 2.4.2.1), the rate of inosine incorporation into nucleotides was markedly reduced (by 75%), indicating that inosine incorporation to IMP (inosinic acid) occurs following its degradation to hypoxanthine. The proportion of hypoxanthine converted to IMP by hypoxanthine-guanine phosphoribosyltransferase (EC 2.4.2.8) is markedly greater than that degraded to xanthine and uric acid by xanthine oxidase (EC 1.3.2.3). However, close to 50% of the IMP formed was degraded to inosine by IMP 5'-nucleotidase (EC 3.1.3.5). The results demonstrate the activity of the following futile cycle in the cardiomyocytes: hypoxanthine----IMP----inosine----hypoxanthine. The rational for the activity of this energy consuming cycle is yet unclear.     

Purine metabolism in human lymphocytes

Summary In peripheral human blood lymphocytes the uptake and metabolism of adenine, guanine, and hypoxanthine was investigated. This was achieved by incubation of purified lymphocytes with¹⁴C-purine bases, separation of cells from the incubation medium by a rapid filtration technique, and subsequent separation of the acid soluble material by thin-layer chromatography. No preferential uptake for one of the purine bases was observed. In all cases only traces of¹⁴C-purine bases not added originally and labeled nucleosides could be demonstrated. Approximately 2/3 of adenine and 1/2 of guanine or hypoxanthine were converted to nucleotides. Separation of formed nucleotides showed that adenine and guanine were metabolized mainly to their corresponding nucleotides; hypoxanthine was converted to a considerable amount to adenine nucleotides and only to a small proportion into its own nucleotides. These results demonstrate the predominance of adenine nucleotide formation in normal human lymphocytes.The study was supported by a grant of Fonds zur Förderung der wissenschaftlichen Forschung Österreichs (project No. 3038)     

Metabolism of circulating adenosine by the porcine isolated perfused lung

Adenosine uptake was studied in the piglet isolated perfused lung by means of the single-circulation paired-tracer dilution technique. Adenosine was efficiently taken up from the pulmonary vascular bed, and the process was potently inhibited by dipyridamole. Following uptake, adenosine was incorporated into intracellular nucleotides, and at low perfusate concentrations, little or none of the incorporated radioactivity returned to the circulation. At higher concentrations, cellular uptake was saturable and products of intracellular catabolism (inosine and hypoxanthine) were returned to the circulation. Perfusion of low concentrations of adenosine after inhibition of pulmonary adenosine kinase led to a proportional decrease in the retention of nucleotides and to a release of inosine and hypoxanthine. A small proportion of adenosine was metabolised extracellularly by adenosine deaminase; this activity was not released from perfused lungs and is apparently an ecto-enzyme.     

Adenine pool catabolism in the ischemic, the calcium-depleted ischemic, and the substrate free anoxic isolated rat heart: Relationship to contracture development

Metabolic changes in the myocardial adenine and hypoxanthine pools of isolated rat hearts subjected to global ischemia, hypocalcemic global ischemia, and global substrate-free anoxia were compared. At timed intervals between 0 and 60 min separate aliquots of extracts of the ventricles were used to determine either tissue pH, or the components of the adenine pool and their catabolites by reverse phase high performance liquid chromatography (HPLC). The coronary perfusate draining from anoxically perfused hearts was collected over perchloric acid, neutralised and chromatographed by HPLC. The development of left ventricular resting tension (contracture) was recorded in the three groups of hearts. After 60 min ischemia the major catabolites, (AMP, inosine and hypoxanthine) comprised 70% of the total pool (11, 7 and 4 mumol/g dry wt, respectively). After the same period of anoxia 50% of the total pool, comprising adenosine, inosine, hypoxanthine and uric acid in approximately equal proportions, was recovered from the coronary perfusate. The major products remaining in the tissue were IMP and, to a lesser extent AMP (8 and 5 mumol/g dry wt, respectively). Left ventricular contracture developed at different rates in the three groups of hearts but always correlated closely with the maximum rate of adenine pool catabolism. The loss of components from the tissue and the divergence in pathway from adenosine to IMP production which occurs during anoxic perfusion should possibly be considered when assessing the biochemical events occurring in regionally ischemic heart muscle with significant residual flow.     

Transendothelial transport and metabolism of adenosine and inosine in the intact rat aorta

This study was aimed at defining the role of vascular endothelium in the transport and metabolism of adenosine. For this purpose, endothelium-intact and endothelium-denuded isolated rat aortas, perfused at constant flow (2 ml/min), were prelabeled with 3H-adenosine or 3H-inosine for 10 minutes at concentrations of 0.012-100 microM. Sequestration of adenosine by endothelium was determined from radioactivity recovered during selective endothelial cell removal with deoxycholic acid (0.75% for 15 seconds). In the physiological concentration range of adenosine (0.012-1 microM), fractional sequestration by endothelium was 90-92% of the total adenosine incorporation by the aorta. Endothelial sequestration of inosine at 0.1 microM was 85%. At 100 microM adenosine or inosine, fractional sequestration by aortic endothelium was 33% and 39%, respectively. Analysis of the specific radioactivity of adenine nucleotides extracted from prelabeled aortas indicated that most of the adenosine was incorporated into endothelial adenine nucleotides. Incorporation of inosine into endothelial ATP was approximately 15% that of adenosine. Inhibition of aortic adenosine deaminase with erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) did not influence sequestration of 0.1 microM adenosine, but resulted in a 49% reduction of total endothelial incorporation at 100 microM adenosine. Transfer of radioactive purines from the endothelium to underlying smooth muscle after prelabeling was equivalent to only 1%/hr of total endothelial radioactivity. Our findings suggested that 1) macrovascular endothelium of the aorta constitutes a highly effective metabolic barrier for circulating adenosine and inosine; 2) transfer of labeled adenine nucleotides from endothelium to underlying smooth muscle is rather small and most likely proceeds via dephosphorylated purine compounds; and 3) measurement of adenosine trapping in endothelial and smooth muscle compartments overestimates the transendothelial adenosine concentration gradient.     

Adenine nucleotide release from isolated perfused guinea pig hearts and extracellular formation of adenosine.

The quantification of adenine nucleotides released from the heart is hampered by their rapid dephosphorylation to adenosine in the extracellular space catalyzed by highly active ectonucleotidases. To determine the total release of adenine nucleotides from isolated Langendorff-perfused guinea pig hearts, ecto 5'-nucleotidase was effectively blocked by infusion of alpha, beta-methylene-ADP (AOPCP, 50 microM). Adenine nucleotides were measured in the coronary venous effluent by the luciferin-luciferase method after enzymatic rephosphorylation to ATP. In hearts perfused at a constant flow rate (10 ml/min) with normoxic buffer (95% O2, 5% CO2) the release +/- SEM of adenine nucleotides and adenosine was 0.06 +/- 0.01 (n = 11) and 0.04 +/- 0.01 (n = 13) nmol/min. In the presence of AOPCP, the release of adenine nucleotides increased to 0.43 +/- 0.04 nmol/min (n = 9; p less than 0.05), whereas adenosine remained unchanged. Hypoxic perfusion (10% O2, 85% N2, 5% CO2) caused a threefold increase in adenine nucleotide release but a 40-fold increase in adenosine. In contrast, global ischemia (30 seconds) caused adenine nucleotide and adenosine release to rise to similar values of 1.06 +/- 0.10 and 0.80 +/- 0.14 nmol/min (n = 9). Stimulation of hearts with isoproterenol (4 nM) likewise increased the release of adenine nucleotides (0.50 +/- 0.04 nmol/min) and adenosine (0.87 +/- 0.21 nmol/min) (n = 6). To determine the cellular source of adenine nucleotides released from the heart, the coronary endothelial adenine nucleotide pool was selectively prelabeled by [3H]adenosine. Global ischemia increased the specific radioactivity of released adenine nucleotides by 57%. The findings indicate that 1) adenine nucleotides and adenosine are released at the same order of magnitude from the well-oxygenated heart; 2) beta-adrenergic stimulation and ischemia stimulate the release of adenine nucleotides and adenosine, both purines reaching vasoactive concentrations in the effluent perfusate; 3) during hypoxic perfusion only the release of adenosine is greatly enhanced; and 4) the coronary endothelium preferentially contributes to the ischemia-induced adenine nucleotide release.     

Adenosine deaminase inhibition and myocardial purine release during normoxia and ischaemia

Quantitative determination of myocardial adenosine formation and breakdown is necessary to gain insight into the mechanism and regulation of its physiological actions. Deamination of adenosine was studied in isolated perfused rat hearts by infusion of adenosine (1 to 20 mumol X litre-1). All catabolites in the perfusates (inosine, hypoxanthine, xanthine and uric acid) were measured, as well as unchanged adenosine. Apparent uptake of adenosine was determined; it increased linearly with the concentration of adenosine infused. Adenosine was predominantly deaminated, even at low (1 mumol X litre-1) concentration. The inhibitory capacity of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was determined, while 5 mumol X litre-1 adenosine was infused. EHNA inhibited the apparent adenosine deaminase activity for 62 and 92% at 5 and 50 mumol X litre-1, respectively. When 50 mumol X litre-1 EHNA was infused into normoxic hearts, release of adenosine was significantly elevated, as was coronary flow. Induction of ischaemia increased total purine release four-to fivefold. Infusion of EHNA into ischaemic hearts did not alter total purine release, but adenosine release increased from 15 to 60% of total purines. However, when EHNA was present, a large part of total purine release still existed of inosine, hypoxanthine, xanthiner and uric acid. This was 83% during normoxia and 40% during ischaemia. These results suggest significant contribution of IMP and GMP breakdown to purine release from isolated perfused rat hearts.     

Effects of uridine on the performance and the metabolism of oxygenated and hypoxic rabbit hearts.

Since uridine is involved in the synthesis of tissue glycogen, which is the major source of energy for hypoxic myocardium, we investigated effects of this nucleoside on myocardial performance and metabolism in isolated rabbit hearts perfused under oxygenated and hypoxic conditions. Uridine (10^?5m) increased myocardial performance, glucose uptake, glycolysis, glycogen content and the breakdown of ATP in oxygenated hearts. While myocardial performance declined, glucose uptake with glycolysis, glycogenolysis and the breakdown of adenine nucleotides increased in rabbit hearts exposed to hypoxia. Uridine increased myocardial performance, glucose uptake and glycolysis, as well as diminished the disappearance of glycogen and adenine nucleotides from hypoxic hearts. Uridine also increased glucose uptake, glycolysis, contents of ATP and glycogen, as well as myocardial performance in propranolol-treated hearts. The activity of cardiac membrane Na⁺, K⁺-activated ATPase was increased by 10^?4 to 10^?6m uridine, even in the presence of 10^?5m ouabain. Increased glucose uptake and glycolysis in isolated rabbit atria due to 10^?5m ouabain were inhibited in the presence of 10^?4m uridine. The uptake of ouabain H³ (G) by non-contracting rabbit atria was unaffected by 10^?4 to 10^?5m uridine.While the mechanism of action of uridine still remains unknown, it seems to be different from the one of catecholamines or cardiac glycosides. Since greater stores of glycogen or higher supply of glucose is known to protect the heart against anoxia or ischemia, present results suggest that uridine might have similar salutary effects.     

Degradation of perfused adenine compounds up to uric acid in isolated rat heart

Cells are generally impermeable to nucleotides like ATP, ADP and AMP while nucleosides and bases readily cross the plasma membrane. A release of adenosine and of its catabolic derivatives by the myocardium of different animal species has been demonstrated in physiological and physiopathological conditions [2, 4, 13]. Furthermore there are many studies on the uptake of adenosine and inosine by the myocardial cells and their incorporation into tissue nucleotides [7–9, 15]. Taking into account the extracellular localization of adenosine receptors [5] and the role of this nucleoside as regulator of coronary blood flow and modulator of the positive inotropic effects of catecholamines [4] it was of interest to study a possible extracellular formation of adenosine from adenine nucleotides. Most mammalian tissues like muscle, liver and adipose tissue and cells like platelets, leukocytes and lymphocytes possess some enzyme activities associated with the cell surface membrane. Ecto-ATPase (EC 3.6.1.3/15) and ecto-5′-nucleotidase (EC 3.1.3.5) activities have been demonstrated in myocardial, smooth-muscle and endothelial cell membranes [3,4,8,12].In the present paper we report a comparative study on the ability of the isolated rat heart to breakdown exogenous adenine nucleotides, nucleosides and bases: we have determined in the coronary perfusate all the degradation products in basal conditions and after perfusion with ATP, ADP, AMP, adenosine, inosine and hypoxanthine. All the perfused compounds were degraded up to uric acid that amounted to about 10%. Nucleotides were catabolized at a higher rate than nucleosides and so adenosine may accumulate outside the cells.     

Adenine nucleotide degradation in the human myocardium during cardioplegia

The tissue content of adenine nucleotides and their metabolites, inosine monophosphate, adenosine, hypoxanthine, and uric acid, were determined in biopsy specimens from the left ventricle of six patients during cardioplegia for open heart surgery. Biopsy specimens were collected immediately after the induction and at the end of cardioplegia (51-82 min) and were analysed by high performance liquid chromatography. After the induction of cardioplegia (cold potassium enriched solution) the left coronary artery was continuously perfused with cold (10 degrees C), potassium enriched, diluted blood. The adenosine triphosphate concentration decreased from 13 to 8 mmol.kg-1 dry muscle (p less than 0.01) during cardioplegia. Adenosine diphosphate and adenosine monophosphate concentrations were 6 and 3 mmol.kg-1 dry muscle respectively and remained unaffected. The adenosine concentration (0.3 mmol.kg-1 dry muscle) was three times higher than that of inosine monophosphate. Inosine concentrations increased from 0.8 to 2.7 mmol.kg-1 dry muscle (p less than 0.01) in parallel with the increase in hypoxanthine from 0.1 to 0.4 mmol.kg-1 dry muscle (p less than 0.01). The total adenine nucleotide pool decreased by 5 mmol.kg-1 dry muscle (p less than 0.01), whereas the corresponding increase in nucleotides and bases only was 2 mmol.kg-1 dry muscle. In conclusion, the adenosine triphosphate content and the adenine nucleotide pool were appreciably reduced during continuous cold blood cardioplegia as used in the present study. The tissue content of adenosine and further metabolites was considerably increased.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of ischemia and infarction on regional content of adenine nucleotides and derivatives in canine left ventricle.

Samples of myocardium from four areas of ischemic and infarcted canine ventricle were examined over a 20-day period for content of the three adenine nucleotides as well as inosine, hypoxanthine, adenosine, and inosine monophosphate. The adenosine triphosphate (ATP) content of central and peripheral areas within the infarct fell to 11% and 8% of control, respectively, 1 day after coronary occlusion. The total adenine nucleotide (TAN) content in these areas fell to 17% of control and showed no significant recovery during the period of study. In the functional myocardium immediately surrounding the infarct the ATP content was depressed to 58% control after 1 day, and the TAN content was also depressed. In the healthy myocardium near the apex, the ATP content was significantly depressed only at the 3-day sample period. Adenine nucleotide derivatives were detected only at 30 min in the central ischemic area. The absence of the nucleoside and nucleobase compounds formed during adenine nucleotide degradation is attributed to their high membrane diffusibility. Loss of these compounds is considered a contributing factor in the prolonged depression of adenine nucleotide content in both ischemic and non-ischemic regions.     

Adenine nucleotide metabolites are beneficial for recovery of cardiac contractile force after hypoxia

In a previous study, we demonstrated a significant release of adenosine, inosine and hypoxanthine during hypoxia and subsequent reoxygenation. The present study was designed to determine whether or not exogenous adenosine, inosine and hypoxanthine are beneficial for the recovery of hypoxia-induced loss of cardiac contractile force. Hearts were perfused for 20 min under hypoxic conditions, followed by 45 min-perfusion under reoxygenated conditions, and changes in contractile force, resting tension and metabolic parameters of the perfused heart were examined. When either adenosine, inosine or hypoxanthine were exogenously infused during hypoxia at the rate of 3 mumol/min, remarkable recovery (61 to 68%) of cardiac contractile force was observed upon reoxygenation. The recovery was accompanied by a significant restoration of myocardial ATP (90 to 100%) and CP contents (80 to 86%), suggesting that exogenous metabolites are utilized for the restoration of myocardial ATP during reoxygenation, which may lead to a beneficial recovery of hypoxia-induced loss of cardiac contractile force upon reoxygenation. Infusion of exogenous metabolites also resulted in an almost complete inhibition of hypoxia- and reoxygenation-induced release of creatine phosphokinase from the perfused heart as well as a significant depression of hypoxia-induced calcium accumulation in the cardiac tissue. Since these phenomena are considered to represent increases in cell membrane permeability, protection of the myocardium against hypoxia- and reoxygenation-induced changes in cell membrane permeability may be an alternative mechanism for the beneficial effect of adenosine, inosine and hypoxanthine on the hypoxic myocardium.     

Perfluorochemical perfusion of the rat heart: in vivo and in vitro

Perfluorochemical (PFC) perfusates were evaluated in this study for inherent differences obtained by perfusion in vivo and of the heart in vitro. The sources of the PFC's were commercial products: Fluosol DA 20%, Fluosol DA 35%, and Fluosol-43. The first two contain both perfluorodecalin (PD) and perfluorotripropylamine (PTPA), while the third utilizes only perfluorotributylamine (PTBA). The hearts studied were excised from control animals or from animals that were first exchange perfused in vivo with Fluosol-43. For studies in vitro hearts perfused in the presence of PTBA beat at a normal rate for 10 to 12 h and then gradually slowed. By comparison, hearts perfused with Krebs-Henseleit solution (KHS) or PD-PTPA containing perfusates maintained a normal heart rate only briefly and ceased beating within 5 to 10 h. However, excised hearts from animals perfused previously in vivo with the PD-PTPA (35%) mixture showed an initial increase followed by a rapid drop in cAMP and cGMP concentrations of the left ventricle. When the other two PFC-containing perfusates were used, no significant changes were found. Only the PTBA-containing mixture maintained normal levels of the two nucleotides and Na⁺, K⁺ and Ca²⁺ levels of the left ventricle in vitro. Also, for studies in vitro when linoleic (0.156 μm) and palmitic (0.086 μm) acids were added simultaneously to all PFC preparations the PTBA-containing perfusate maintained a normal heart rate for over 10 h. Beating hearts were maintained most effectively when hydroxyethylstarch, an oncotic component, was present at 3% (w/v). These studies demonstrate that suitable PFC-containing perfusates can maintain beating rat hearts for many hours at 37°C.     

Preservation of high-energy phosphates by verapamil in reperfused myocardium

To determine whether verapamil prevents depletion of adenine nucleotides during and after severe myocardial ischemia, dogs were subjected to 15 min occlusions of the left anterior descending coronary artery followed by 240 min of reperfusion. One hour before occlusion, dogs were randomly assigned to a treatment group (n = 10) to which an infusion of intravenous verapamil was given until the onset of reperfusion or to an untreated saline group (n = 9). Verapamil reduced mean aortic pressure and heart rate. After 15 min of ischemia, endocardial adenosine triphosphate (ATP) level, determined by needle biopsy, decreased in the untreated group from 34.7 +/- 2.0 to 24.4 +/- 2.7 nmol X mg protein-1 (p less than .005 vs preocclusion) and in the verapamil group from 32.8 +/- 1.5 to 30.3 +/- 1.5 nmol X mg protein-1 (NS vs preocclusion). Dogs receiving verapamil had significantly higher ATP levels than untreated animals after 90 and 240 min of reperfusion. In untreated animals the sum of inosine and hypoxanthine levels increased during occlusion from very low levels to 4.6 +/- 1.1 nmol X mg protein-1 in the epicardium and to 6.8 +/- 1.5 nmol X mg protein-1 in the endocardium (p less than .05 compared with preocclusion values). In verapamil-treated dogs inosine and hypoxanthine levels increased to only 1.2 +/- 0.3 (epicardium) and 1.9 +/- 0.6 nmol X mg protein-1 (endocardium) (both NS compared with preocclusion values). After 90 min of reperfusion the sum of ATP, adenosine diphosphate, adenosine monophosphate, inosine, and hypoxanthine levels was decreased in the endocardium by 10.2 nmol X mg protein-1 in the untreated group, but no change was observed in verapamil-treated animals. We conclude that breakdown of ATP to inosine and hypoxanthine during severe ischemia is reduced by verapamil, resulting in higher ATP concentrations during occlusion and reperfusion and decreased washout of the diffusible purines inosine and hypoxanthine during reperfusion.     

Uridine preserves ATP during hypoxic perfusion of the rat heart

Background: The pyrimidine precursor, orotic acid, by minimising ischaemia-induced ATP loss, improves the functional performance of recently infarcted hearts that have been subjected to global ischaemia. However, we have also previously shown that orotic acid is not directly active in the heart but is preferentially taken up by the liver where it is metabolised to uridine. Aim: To investigate whether uridine itself can minimise hypoxia-induced ATP loss. Methods: Isolated Langendorff-mode perfused rat hearts were subjected to 4 protocols after 20 min normoxic stabilisation: normoxia for 30 min (n=12); hypoxia for 30 min (n=12); hypoxia in the presence of 17μM uridine for 30 min (n=12); and [U-¹⁴C]-uridine added directly to the hypoxic perfusate reservoir just prior to 30 min hypoxia (n=4). [U-¹⁴C]-uridine was used to assess the contribution of radiolabel to adenosine formation from adenine nucleotide hydrolysis. Coronary effluent was collected and hearts were freeze-clamped for metabolite assay. Results: Hypoxia reduced ATP, from 21.1±1.1 to 4.1±0.6 μmol/g dry weight (p<0.05), and reduced total adenine nucleotides (TAN) from 30±1.2 to 10.2±0.9 μmol/g dry weight (p<0.05). Uridine during hypoxia increased myocardial ATP by 94% to 8±0.9 μmol/g dry weight and TAN by 50% to 15.3±1.1 pmol/g dry weight (p<0.05). Uridine plus hypoxia increased total lactate release by 52% from 768.1± 86 μmol/g dry weight to 1148.4 ±146 μmol/g dry weight compared with hypoxia alone (p<0.05). Although the salvage of purine bases did occur, it was calculated that less than 0.01% of labelled ribose was transferred for salvage of purines. Conclusion: In the present experimental model, uridine protects the hypoxic heart by predominantly enhancing glycolytic energy production.     

Glycolytic and tricarboxylic acid cycle intermediates during cardiac arrest and recovery in eu-, hyper- and hypothyroid rats

The effects of contractile activity on intermediates of the Embden-Meyerhof and citric acid cycle pathways were studied using perfused hearts from rats in various thyroid states. Hearts were perfused with a normal and 16 mm-potassium Krebs solution containing glucose; most metabolic intermediates were measured by fluorometric, enzymatic assays. Two minutes of cardiac arrest in euthyroid hearts resulted in an increase in the intermediate levels before phosphofructokinase (PFK) step and no change in the intermediate levels below this step, indicating the PFK control of glycolytic flux. Citrate and isocitrate were increased at the same time, whereas there was no change in the adenine nucleotides. The $\text{NAD}\text{NADH}$ ratio was decreased. Upon resumption of heart beats, the raised isocitrate level and PFK control of glycolytic flux remained for at least 2 min. The level of 2-phosphoglycerate was also increased during this period. Most metabolites returned to within normal ranges after 10 min. In hyperthyroid rats, the rate of glycolytic flow during cardiac arrest was again controlled by PFK, but inhibition of this enzyme was not the result of changes in levels of adenine nucleotides or citrate. Metabolic alterations observed in hypothyroid hearts were essentially similar to those in the euthyroid animals. This study demonstrates the major role of PFK in the control of glycolytic flux in various thyroid states. However, the mechanism of inhibition of the enzyme activity in hyperthyroid rats is different from that in euthyroid animals. The turnover of adenine nucleotides and phosphocreatine responds promptly to changing cardiac activity, whereas that of tricarboxylic acid cycle intermediates and pyridine nucleotides does not.     

Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells

Summary Incorporation of adenosine and adenine into hypoxanthine nucleotides of fresh red blood cells was monitored using 8-¹⁴C-adenosine and 8-¹⁴C-adenine added to the incubation medium containing adenosine, pyruvate and inorganic phosphate (APP medium). Using 8-¹⁴C-adenosine it was shown that 21.7% of the isotope contained in the incubation medium penetrated red blood cells. Of that quantity about 50% becomes incorporated into nucleotides. Of the isotope 5.3% was found in hypoxanthine nucleotides (1.3% in ITP and 4.0% in IMP). During incubation of red blood cells in APP medium fortified with the 8-¹⁴C-adenine about 95% of isotope penetrated into cells and 60% of that quantity became incorporated into nucleotides. In hypoxanthine nucleotides only trace amounts of isotope were found (0.12% in IMP and 0.13% in ITP).     

Adenine and hypoxanthine metabolism in phythohemagglutinin-stimulated and unstimulated human lymphocytes

Summary The uptake and subsequent metabolism of adenine and hypoxanthine in phytohemagglutinin-stimulated and unstimulated peripheral human blood lymphocytes, freshly prepared or cultured, were studied. To investigate the initial step of nucleic acid metabolism the incorporation of¹⁴C-purines into the acid soluble material was examined. No preferential uptake of adenine or hypoxanthine was observed in freshly prepared and cultured lymphocytes during an incubation of 1 h. However, cultured cells utilized approximately 1/3 of the purines compared to freshly drawn cells. Within the cells 2/3 of adenine and 1/2 of hypoxanthine were metabolized to nucleotides (mainly AMP and ADP). Incubation of lymphocytes with PHA for 1 h produced in the freshly prepared cells an increase of adenine- and hypoxanthine-uptake to 191% and 153%, in 48 h stimulated cells to 158% and 132%. There was, however, no change in the relative rates of the metabolic routes though the intracellular concentrations of nucleotides formed increased with adenine as substrate to 152% and with hypoxanthine to 161% during a 1 h stimulation. In contrast no enhanced formation of acid soluble nucleotide formation could be observed with PHA stimulation during 48 h. The increased rates of purine uptake and metabolism apparent 1 h after addition of mitogen may be due to an altered transport mechanism at the beginning of the transformation as an adaptive response to the increased requirements for the synthetic processes soon to follow. Once the lymphocytes are transformed no demand of purines is necessary and the uptake and metabolism is switched off.This work has been supported by grant No. 9796 of the Fonds zur Förderung der wissenschaftlichen Forschung