Free Cytosolic Adenosine Research Articles

Heterogeneity of metabolic parameters in the left ventricular myocardium and its relation to local blood flow.

It is well established that myocardial blood flow is heterogeneous on the local level. During recent years comprehensive studies have been undertaken to assess the relation between myocardial metabolism and spatial blood flow heterogeneity. Based on the type of measurements two major groups of studies have been performed: enzyme activity and tissue metabolite level assessments. Enzyme activity measurements have provided only limited insight into the coupling of local metabolism and flow. This is probably due to the fact that, in addition to estimated Vmax values, local substrate affinity (Km values) and substrate concentrations affect the metabolite fluxes. However, the latter two variables remain normally unknown. In contrast, valuable insight has been obtained concerning flow-metabolism matching from tissue metabolite measurements, especially when connected with mathematical model analyses. The latter permitted the calculation of metabolic flux rates (e.g., production of oxidation water, citric acid cycle flux, glucose uptake, fatty acid uptake) or the translation of the metabolic indexes into physiologically meaningful local metabolite concentrations (e.g., free cytosolic adenosine). The bottom line of the studies reported to date is that the broad range of myocardial flows observed under resting control conditions correlates with local metabolism possibly affected by spatial differences in adrenergic stimulation. Thus, high flow samples exhibit a higher oxidative metabolism than low flow samples. As a result the flow threshold below which local myocardial ischemia ensues is higher in control high flow samples. The importance of these findings with respect to local flow-metabolism matching is underlined by the finding that the probability of developing an infarction following ischemia/reperfusion is related to the functional state of the myocardium under control conditions, i.e., the local level of flow-metabolism matching.

Nitric oxide reduces energy supply by direct action on the respiratory chain in isolated cardiomyocytes.

To investigate the effect of nitric oxide (NO) on cardiac energy metabolism, isolated cardiomyocytes of Wistar rats were incubated in an Oxystat system at a constant ambient PO2 (25 mmHg) and oxygen consumption (VO2); free intracellular Ca(2+) (fura 2), free cytosolic adenosine [S-adenosylhomocysteine (SAH) method], and mitochondrial NADH (autofluorescence) were measured after application of the NO donor morpholinosydnonimine (SIN-1). In Na(+)-free medium (contracting cardiomyocytes), VO2 increased from 7.9 +/- 1.2 to 26.4 +/- 3.1 nmol x min(-1) x mg protein(-1). SIN-1 (100 micromol/l) decreased VO2 in contracting (-21 +/- 3%) and in quiescent cells (-24 +/- 7%) by the same extent. Inhibition of VO2 was dose dependent (EC(50): 10(-7) mol/l). S-nitroso-N-acetyl-penicillamine, another NO donor, also inhibited VO2, whereas SIN-1C (100 micromol/l), the degradation product of SIN-1, displayed no inhibitory effect. Intracellular Ca(2+) remained unchanged, and inhibition of protein kinases G, A, or C did not antagonize the effect of NO. Mitochondrial NADH increased with NO, indicating a reduced flux through the respiratory chain. In quiescent but not in contracting cardiomyocytes, NO significantly increased adenosine, indicating a reduced energy status. These data suggest the following. 1) NO decreases cardiac respiration, most likely via direct inhibition of the respiratory chain. 2) Whereas in quiescent cardiomyocytes the inhibition of aerobic ATP formation by NO causes reduction in energy status, contracting cells are able to compensate for the NO-induced inhibition of oxidative phosphorylation, maintaining energy status constant.

Quantification of Extracellular and Intracellular Adenosine Production

Inhibitors of adenosine membrane transport cause vasodilation and enhance the plasma adenosine concentration. However, it is unclear why the plasma adenosine concentration rises rather than falls when membrane transport is inhibited. We tested the hypothesis that the cytosolic adenosine concentration exceeds the interstitial concentration under well-oxygenated conditions. In isolated, isovolumically working guinea pig hearts (n=50), the release rate of adenosine and accumulation of S-adenosylhomocysteine (after 20 minutes of 200 micromol/L homocysteine), a measure of the free cytosolic adenosine concentration, were determined in the absence and presence of specific and powerful blockers of adenosine membrane transport (nitrobenzylthioinosine 1 micromol/L), adenosine deaminase (erythro-9-hydroxy-nonyl-adenine 5 micromol/L), and adenosine kinase (iodotubericidine 10 micromol/L). Data analysis with a distributed multicompartment model revealed a total cardiac adenosine production rate of 2294 pmol. min-1. g-1, of which 8% was produced in the extracellular region. Because of a high rate of intracellular metabolism, however, 70.3% of extracellularly produced adenosine was taken up into cellular regions, an effect that was effectively eliminated by membrane transport block. The resulting approximately 2.8-fold increase of the interstitial adenosine concentration evoked near-maximal coronary dilation. We rejected the hypothesis that the cytosolic adenosine concentration exceeds the interstitial. Rather, there is significant extracellular production, and the parenchymal cell represents a sink, not a source, for adenosine under well-oxygenated conditions.

Spatial heterogeneity of myocardial perfusion and metabolism.

Left ventricular myocardium is characterized by a substantial spatial heterogeneity of both perfusion and metabolism. Under resting conditions, the transmural gradient of myocardial oxygen consumption (MVO2) from the subepi- to the subendocardial layer exceeds that of coronary flow, resulting in a lower subendocardial PO2, altered kinetics of oxidative phosphorylation, and enhanced free cytosolic adenosine. Within each layer, there is a major spatial variability of perfusion: Local flow rates in individual myocardial samples (200 mg) range from 20-250% of the mean myocardial blood flow. Low flow areas (< 50% of mean flow) display a rather low uptake of fatty acids and glucose; the uptake of these substrates increases in proportion to local flow. There is also a close relationship between local perfusion and the local turnover of the tricarboxylic acid cycle and, thus, MVO2 as was recently demonstrated using 13C NMR techniques. Consequently, within the well perfused left ventricular myocardium local MVO2 and, thus, energy turnover varies more than 3-fold between low and high flow areas. Low flow areas are not ischemic, since local lactate, adenosine, and ATP are comparable to mean flow areas. When coronary perfusion pressure is reduced, the transmural perfusion gradient reverses resulting in impaired energy status and enhanced adenosine predominantly in the subendocardium. This rise in local adenosine or lactate requires a decrease of the individual local flow by more than 50% of its preischemic value. It, thus, appears that not the absolute level of local flow predicts the impact of ischemia but its relative change.

Sites and mechanism of adenosine formation in the cardiovascular system

This review summarizes recent developments on the mechanisms by which adenosine is formed in the cardiovascular system. The concept is developed that AMP is both a precursor of adenosine but also a product when rephosphorylated by adenosine kinase. The functional significance of this metabolic cycle is the amplification of changes in free cytosolic adenosine, which translates a minor decrease of cardiac energetics in a substantial rise in adenosine. Furthermore, the metabolism of adenosine is highly compartmentalized, and the vascular endothelium forms an important metabolic barrier, which significantly influences the dose-response curve for adenosine when this nucleoside is applied intervascularly. Inadequate supply of oxygen (critical Po 2 : 3 mm Hg) is the most important physiologic trigger for the formation of adenosine. In the range above the critical Po 2 , cardiomyocytes have the remarkable ability to down-regulate their oxygen consumption before metabolic signs of hypoxia occur. Therefore, a reduction in oxygen supply as such is not a sufficient cause for the formation of adenosine but the imbalance between ATP formation and consumption. Future studies must resolve which factors govern the in-vivo activity of adenosine kinase and 5'-nucleotidase and how this is linked to the known physiologic effects of adenosine. Furthermore, the molecular mechanisms by which ATP can permeate through cell membranes and gives rise to the extracellular formation of adenosine need to be resolved.

Role of adenosine kinase and AMP deaminase in the regulation of cardiac purine release

To analyze the relation between cardiac energy status, adenosine formation, and purine release, reliable measurements of the cytosolic concentrations of ATP, ADP, AMP, and adenosine are required. Based on the creatine kinase and myokinase equilibrium, ADP and AMP are determined by 31 P nuclear magnetic resonance spectroscopy, whereas free cytosolic adenosine is measured by the S-adenosyl-homocysteine (SAH) technique. Combining these methods with efflux measurements, selective enzyme blockade and a comprehensive model analysis enables a description of both concentrations and flux rates in purine metabolism. In the well-oxygenated heart, adenosine is predominantly formed intracellularly from AMP, but also from S-adenosyl-homocysteine. Net adenosine formation (2.3 nmol/min per g) exceeds coronary venous release (0.07 nmol/min per g) more than 30-fold, because most of the adenosine formed is rephosphorylated by adenosine kinase. This enzyme maintains a low intracellular adenosine and limits both adenosine release and deamination to inosine. In fact, inosine is mainly formed from IMP (1.8 nmol/ min per g) the product of AMP deaminase. Inosine, hypoxanthine, xanthine, and uric acid (1.1, 0.4, 0.2, 1.4 nmol/min per g) are the main purine catabolites released. In the oxygen-limited heart, energy status is impaired and AMP increased. Under these conditions, a linear relation between AMP (200-3,000 nmol/liter), net adenosine formation, as well as net inosine formation is observed. It is, thus, the AMP substrate concentration that directly controls adenosine formation by cytosolic 5'-nucleotidase and most likely flux through AMP deaminase. Hypoxia-induced inhibition of adenosine kinase shunts adenosine from the salvage pathway to venous release and causes the amplification of small changes in AMP into a major rise in adenosine. This mechanism plays an important role in the high sensitivity of the cardiac adenosine system to impaired oxygenation.

Hypoxia-induced inhibition of adenosine kinase potentiates cardiac adenosine release.

To elucidate the physiological role of the AMP-adenosine metabolic cycle and to investigate the relation between AMP and adenosine formation, the O2 supply of isolated guinea pig hearts was varied (95% to 10% O2). The net adenosine formation rate (AMP-->adenosine) and coronary venous effluent adenosine release rate were measured; free cytosolic AMP was determined by 31P-nuclear magnetic resonance. Switching from 95% to 40% O2 increased free AMP and adenosine formation 4-fold, whereas free cytosolic adenosine and venous adenosine release rose 15- to 20-fold. In the AMP range from 200 to 3000 nmol/L, there was a linear correlation between free AMP and adenosine formation (R2 = .71); however, adenosine release increased several-fold more than formation. At 95% O2, only 6% of the adenosine formed was released; however, this fraction increased to 22% at 40% O2, demonstrating reduced adenosine salvage. Selective blockade of adenosine deaminase and adenosine kinase indicated that flux through adenosine kinase decreased from 85% to 35% of adenosine formation in hypoxia. Mathematical model analysis indicated that this apparent decrease in enzyme activity was not due to saturation but to the inhibition of adenosine kinase activity to 6% of the basal levels. The data show (1) that adenosine formation is proportional to the AMP substrate concentration and (2) that hypoxia decreases adenosine kinase activity, thereby shunting myocardial adenosine from the salvage pathway to venous release. In conclusion, because of the normal high turnover of the AMP-adenosine metabolic cycle, hypoxia-induced inhibition of adenosine kinase causes the amplification of small changes in free AMP into a major rise in adenosine. This mechanism plays an important role in the high sensitivity of the cardiac adenosine system to impaired oxygenation.

Determinants of the S-Adenosylhomocysteine (SAH) Technique for the Local Assessment of Cardiac Free Cytosolic Adenosine

The S-adenosylhomocysteine (SAH) technique allows the estimation of the free cytosolic adenosine concentration using the kinetic properties of the enzyme SAH-hydrolase (adenosine+homocysteine→SAH+H2O). Besides the cytosolic adenosine concentration, the local SAH signal may also depend on the local homocysteine availability, the continuous production of SAH from S-adenosylmethionine (SAM→SAH+CH3) and the activity of the enzyme SAH-hydrolase. These variables were studied with high spatial resolution (sample dry mass 25 mg) in left ventricular myocardium from 26 anesthetized open-chest dogs in which heart rate averaged 86±14 beats/min and mean aortic pressure 96±17 mmHg. Homocysteine infusion (48 mg/kg i.v.) increased the normal plasma homocysteine concentration from 5.0± 0.8 to 586±40μmafter 30 min when the average tissue concentration was 94% of the plasma concentration and similar in low and high flow areas (flow range 0.04 to 1.91 ml/min/g). Local SAH content was 1.18±0.48 nmol/g under control conditions and increased to 4.33±0.59 nmol/g within 60 min following competitive blockage of the SAH-hydrolase by adenosine dialdehyde (10μmol/kg i.v.). This increase of the SAH content was slightly more in high than in low-flow areas (P<0.01). Regional SAH-hydrolase activity (9.0±0.5 nmol/min/g) was comparable in high and low flow areas. All three variables exhibited an observed variability which was larger than the methodical variability suggesting significant spatial heterogeneity in the myocardium. A regrouping analysis indicated that between four and five samples taken from distant sites should be averaged to obtain a robust estimate of the above metabolic parameters. Reconciling the measurements with a mathematical model of cardiac adenosine metabolism and fitting of the measured SAH tissue levels gave an estimate of 72 pmol/min/g for the mean transmethylation rate. Estimates of the cytosolic adenosine concentration of cardiomyocytes and endothelial cells under control physiological conditions were 24 and 7μm, respectively. Thus, the present measurements provide a basis for the quantitative assessment of the local cytosolic adenosine concentration in relation to blood flow.

Spatial heterogeneity of blood flow in the dog heart. I. Glucose uptake, free adenosine and oxidative/glycolytic enzyme activity.

The spatial heterogeneity of myocardial perfusion and metabolism was studied in 11 anaesthetized dogs under resting conditions. In each heart local myocardial blood flow was assessed using the tracer microsphere technique in 256 samples (mean mass: 83.1 mg) taken from the left anterior ventricular wall. In the same samples, the following biochemical parameters were determined: accumulation of [3H]-deoxyglucose (a measure of glucose uptake), free cytosolic adenosine (S-adenosylhomocysteine accumulation technique, a measure of tissue oxygenation and a possible mediator of blood flow regulation), and the specific activities of oxidative (citrate synthase, cytochrome-c-oxidase) and glycolytic (hexokinase, phosphoglycerate kinase) enzymes. Capillary density and mitochondrial and myofibril volume densities were determined by morphometry. Myocardial perfusion in each sample (average 0.77 ml min-1 g-1) varied between 0.1 and 2.5 times the mean (coefficient of variation 0.30+/-0.02). [3H]-deoxyglucose was deposited locally in proportion to perfusion. Samples showing low flow (<0.2 ml min-1 g-1) did not exhibit increased levels of cytosolic adenosine. The specific activities of the oxidative and glycolytic enzymes, however, were uniformly distributed between low and high flow areas. Furthermore, capillary density and mitochondrial and myofibril densities were similar in high and low flow regions. The results show firstly that local glucose metabolism in the heart occurs in proportion to local blood flow, suggesting that high flow regions have a higher than average metabolic rate. Secondly, regions of low flow are not compromized by critical oxygenation and most likely have a lower than average oxygen demand and finally, the homogeneous distribution of oxidative and glycolytic enzymes, as well as the homogeneous myocardial ultrastructure, suggest that areas with high and low blood flow under resting conditions may increase their metabolic rate to similar levels when required.

S-adenosylhomocysteine hydrolase activity in human myocardium.

Measurement of S-adenosylhomocysteine (SAH) accumulation in the heart reflects the concentration of free cytosolic adenosine and is thus a sensitive indicator of regional myocardial ischaemia. To evaluate the possibility of applying this method in combination with 11C-SAH positron emission tomography (PET) to patients with ischaemic heart disease the activity of SAH hydrolase in human heart muscle and its regional distribution were studied. Myocardium from patients with dilated cardiomyopathy (n = 4), hypertrophic obstructive cardiomyopathy (HOCM, n = 6), and mitral stenosis (n = 3) was analysed. Additional studies were performed in myocardium, isolated cardiomyocytes, and endothelial cells from dog and guinea pig hearts. Enzyme activity in synthetic and hydrolytic direction including kinetic data (Vmax, KM values, pH dependency) was measured in the cytosolic fraction of myocardial tissue and cell extracts, using high performance liquid chromatography and photometric methods, respectively. Rates of SAH synthesis (Vmax) in the left ventricle in dilated cardiomyopathy, mitral stenosis, and HOCM were 0.8 (SEM 0.1), 1.0(0.2), and 1.7(0.1) nmol.min-1.mg-1 protein respectively. KM values for DL-homocysteine, adenosine, and SAH in HOCM were 187, 2.2, and 3.4 microM, respectively. Enzyme activity was homogeneously distributed among right and left atria, right and left ventricles, and septum. Additional studies in homogenated muscle and isolated cardiomyocytes of guinea pig and canine hearts showed activities similar to man. (1) SAH hydrolase activity in the human heart is quantitatively comparable to that found in other mammals but certain myocardial diseases may go along with changes in SAH hydrolase activity; (2) the kinetic properties, absolute amounts, and homogeneous distribution of the enzyme may permit the non-invasive determination of free adenosine in the human heart by PET.

Cardiac adenosine production is linked to myocardial pO 2

Experiments were performed on isolated perfused guinea-pig hearts ( n=45) to further evaluate the stimulus that triggers cardiac adenosine production. Stimulation of hearts with isoproterenol (4 n m, 20 min) enhanced left ventricular dP dt max , heart rate and myocardial oxygen consumption within 1 min to new steady state values, whereas coronary venous adenosine concentration only transiently increased reaching its maximum between 1 and 3 min of stimulation. Rate of accumulation of S-adenosylhomocysteine (SAH), a measure of the free cytosolic adenosine concentration, was steepest immediately following onset of stimulation and then progressively declined. Similar to adenosine, changes in coronary venous pO 2 were phasic and adenosine release and pO 2 closely correlated. Norepinephrine (20 n m) which increased myocardial oxygen consumption to a comparable extent as isoproterenol (4 n m) further decreased coronary venous pO 2 and increased coronary venous adenosine. When myocardial oxygen supply was systematically varied by changing coronary perfusion pressure from 60 to 90 and 35 cmH 2O, respectively, the adenosine release during isoproterenol (2n m) was markedly enhanced at 35 cmH 2O but blunted at 90 cmH 2O. Similarly SAH accumulation was greatest at 35 cmH 2O and smallest at 90 cmH 2O. It is concluded that changing myocardial oxygen consumption is not a sufficient cause to enhance adenosine formation. Myocardial oxygenation as reflected by changes in coronary venous pO 2 closely correlates with changes in free cardiac adenosine as evidenced by two independent indices: tissues SAH and coronary venous adenosine concentration. The stimulus triggering cardiac adenosine formation is most likely the imbalance of oxygen supply and oxygen demand.

Formation of S-adenosylhomocysteine in the heart. II: A sensitive index for regional myocardial underperfusion.

Rate of accumulation of myocardial S-adenosylhomocysteine (SAH) was used in an open-chest dog preparation as an index of free cytosolic adenosine levels. Following 30 minutes of coronary artery ligation and infusion of L-homocysteine thiolactone (10 mumol/kg/min i.v.) SAH levels increased from 1.3 (control) to 3.3 nmoles/g in the nonischemic and to values over 100 nmoles/g in the ischemic region. Compared with regional myocardial blood flow the enhanced rate of SAH accumulation was strictly confined to the ischemic area. As long as blood flow was 0.6-1.2 ml/min/g, SAH levels remained unchanged. However, they steeply increased when regional myocardial blood flow decreased below 60% of control. Tissue levels of adenine nucleotides, adenosine, and lactate were not significantly affected in the flow range of 0.4-0.6 ml/min/g but rate of SAH accumulation was enhanced by 400%. In the nonischemic myocardium, SAH accumulation was 60% higher in the subendocardium than in the subepicardium. Decreasing coronary perfusion pressure from 110 to 60, 45, and 35 mm Hg was associated with an exponential increase in coronary venous adenosine release only when perfusion pressure was below 60 mm Hg. Transmural mapping of SAH revealed that at 110 mm Hg SAH was homogeneously distributed, while at a perfusion pressure of 60 mm Hg SAH accumulation was enhanced only in the subendocardial layers. Decreasing perfusion pressure further to 40 and 30 mm Hg not only enhanced subendocardial SAH levels to 120 and 170 nmoles/g, respectively, but also considerably steepened the transmural gradient of SAH. SAH-hydrolase exhibited a broad pH-optimum and its activity in different parts of ventricular myocardium was identical. Our findings provide evidence that 1) measurement of SAH accumulation is a sensitive metabolic index for the assessment of regional myocardial ischemia, 2) significant formation of SAH occurs only when regional myocardial blood flow is less than 0.6 ml/min/g, and 3) transmural SAH gradient, a measure of free cytosolic adenosine, and coronary venous adenosine release significantly increase only when the autoregulatory reserve is exhausted.