Coronary Venous Adenosine Concentrations Research Articles

Myocardial oxygenation and adenosine release in isolated guinea pig hearts during changes in contractility

Previous work from this laboratory using near-infrared optical spectroscopy of myoglobin has shown that approximately 20% of the myocardium is hypoxic in buffer-perfused hearts that are perfused with fully oxygenated buffer at 37 degrees C. The present study was undertaken to determine cardiac myoglobin saturation in buffer-perfused hearts when cardiac contractility was increased with epinephrine and decreased during cardiac arrest with KCl. Infusion of epinephrine to achieve a doubling of contractility, as measured by left ventricular maximum pressure change over time (dP/dt), resulted in a decrease in mean myoglobin saturation from 79% at baseline to 65% and a decrease in coronary venous oxygen tension from 155 mmHg at baseline to 85 mmHg. Cardiac arrest with KCl increased mean myoglobin saturation to 100% and coronary venous oxygen tension to 390 mmHg. A previously developed computer model of oxygen transport in the myocardium was used to calculate the probability distribution of intracellular oxygen tension and the hypoxic fraction of the myocardium with an oxygen tension below 0.5 mmHg. The hypoxic fraction of the myocardium was approximately 15% at baseline, increased to approximately 30% during epinephrine infusion, and fell to approximately 0% during cardiac arrest. The coronary venous adenosine concentration changed in parallel with the hypoxic fraction of the myocardium during epinephrine and KCl. It is concluded that catecholamine stimulation of buffer-perfused hearts increases hypoxia in the myocardium and that the increase in venous adenosine concentration is a reflection of the larger hypoxic fraction of myocardium that is releasing adenosine.

Coronary microembolization does not induce acute preconditioning against infarction in pigs-the role of adenosine.

After coronary microembolization (ME) adenosine is released from ischemic areas of the microembolized myocardium. This adenosine dilates vessels in adjacent nonembolized myocardium and increases coronary blood flow. For ischemic preconditioning (IP) to protect the myocardium against infarction, an increase in the interstitial adenosine concentration (iADO) prior to the subsequent ischemia/reperfusion is necessary. We hypothesized that the adenosine release after ME is sufficient to increase iADO and protect the myocardium against infarction from subsequent ischemia/reperfusion. We have therefore compared myocardial protection by either coronary microembolization or ischemic preconditioning prior to ischemia/reperfusion. In anesthetized pigs, the left anterior descending (LAD) was cannulated and perfused from an extracorporeal circuit. In 11 pigs, sustained ischemia was induced by 85% inflow reduction for 90 min (controls). Two other groups of pigs were subjected either to IP (n = 8; 10-min ischemia/15-min reperfusion) or coronary ME (n = 9; i.c. microspheres; 42 microm Ø; 3000 x ml(-1) x min inflow) prior to sustained ischemia. Coronary venous adenosine concentration (vADO) and iADO (microdialysis) were measured. Infarct size was determined after 2-h reperfusion by triphenyl tetrazolium chloride staining. In pigs subjected to IP, infarct size was reduced to 2.6 +/- 1.1% (mean +/- S.E.M.) vs. 17.0 +/- 3.2% in controls. iADO was increased from 2.4 +/- 1.3 to 13.1 +/- 5.8 micromol x l(-1) during the reperfusion following IP. In pigs subjected to ME, at 10 min after ME, coronary blood flow (38.6 +/- 3.6 to 53.6 +/- 4.3 ml x min(-1)) and vADO (0.25 +/- 0.04 to 0.48 +/- 0.07 micromol x l(-1)) were increased. However, iADO (2.0 +/- 0.5 at baseline vs. 2.3 +/- 0.6 micromol x l(-1) at 10 min after ME) did not increase. Infarct size induced by sustained ischemia following ME (22.5 +/- 5.2%) was above that of controls for any given subendocardial blood flow. ME released adenosine into the vasculature and increased coronary blood flow. The failure of iADO to increase with ME possibly explains the lack of protection against infarction after ME.

Comparison of Interstitial Fluid and Coronary Venous Adenosine Levels inIn VivoPorcine Myocardium

There are numerous reports of interstitial fluid (ISF) and coronary venous adenosine measurements in isolated perfused hearts. This study was designed to simultaneously compare ISF and coronary venous adenosine concentrations during various interventions inin vivoporcine myocardium. In anesthetized, open-chest pigs, ISF adenosine, inosine, and hypoxanthine were sampled with cardiac microdialysis. Coronary sinus or venous purines were sampled with a metabolism-stop solution. During basal conditions, ISF adenosine was greater than coronary venous adenosine, but vascular inosine and hypoxanthine were greater than corresponding ISF levels. Dobutamine (20μg/kg/min, i.v.) and systemic hypoxia produced three- and two-fold increases in ISF adenosine, but had no significant effect on coronary sinus adenosine concentration. Hypoxia, but not dobutamine, increased coronary sinus total purines 50%. In contrast to these interventions, intracoronary adenosine infusion (0.5–50μg/kg/min) was associated with significantly greater coronary venous adenosine concentrations than ISF levels. Only during a coronary artery occlusion/reperfusion protocol were ISF and coronary venous adenosine concentrations comparable. The results of this study thus providein vivoevidence of the powerful endothelial and red blood cell metabolic barriers to both exogenous and endogenous adenosine. These results also illustrate the differences in adenosine concentrations in the ISF and vascular spaces.

Characterisation of left ventricular relaxation in the isolated guinea pig heart.

The time constant of left ventricular pressure fall, tau, has frequently been used as a measure of myocardial relaxation in the blood-perfused, ejecting heart. The aim of the present study was to characterise tau in relation to beta-adrenergic activation, coronary perfusion pressure and flow as well as cardiac oxygen supply and demand in the isolated, isovolumically beating heart. Therefore, tau was analysed from digitised left ventricular pressure data in a total of 23 guinea pig hearts perfused with saline at constant pressure (60 cmH2O). The coronary venous adenosine concentration ([ADO]) served as an index of myocardial oxygenation. Isoprenaline (0.4-3.2 nmol l-1) decreased and propranolol (3-9 mumol l-1) increased tau dose-dependently (linear regression tau vs lg([isoprenaline]), r = 0.74; tau vs. lg([propranolol]), r = 0.66, both P < 0.05). During graded reductions in cardiac oxygen supply from 96.1 +/- 12.6 (SEM) to 44.4 +/- 4.4 microliters min-1 g-1, tau was prolonged from 61.5 +/- 12.7 to 109.9 +/- 22.6 ms while left ventricular developed pressure (LVDP) decreased from 90.7 +/- 7.2 to 40.7 +/- 5.1 mmHg. In parallel, [ADO] increased from 23.7 +/- 9.1 to 58.0 +/- 19.1 pmol ml-1 (P < 0.05). Increasing oxygen supply to 165.4 +/- 32.4 microliters min-1 g-1 augmented LVDP to 102.7 +/- 7.3 mmHg but did not change tau or [ADO]. There was a dual response of tau to changes in cardiac oxygen supply or demand. As long as oxygen supply and demand matched, tau remained constant. However, when the oxygen supply was less than 100 microliters min-1 g-1, left ventricular relaxation was prolonged in parallel to the reduction in oxygen supply. In addition, a close relationship was observed between [ADO] as an indicator of myocardial oxygenation and tau (Spearman correlation, r = 0.99, P < 0.005). We conclude that the time constant of left ventricular pressure fall, tau, sensitively reflects myocardial relaxation in the isolated, isovolumically beating guinea pig heart. Moreover, in this model left ventricular relaxation is not influenced by alterations in coronary perfusion pressure or flow as long as cardiac oxygen demand is matched by an adequate supply. Rather relaxation is strictly coupled to myocardial oxygenation as reflected by coronary venous adenosine concentrations.

Adenosine kinetics in canine coronary circulation.

Adenosine kinetics in the coronary circulation were investigated in anesthetized closed-chest dogs by analysis of multiple-indicator dilution experiments. During simultaneous intracoronary bolus injections of 125I-labeled albumin, [14C]sucrose, and [3H]adenosine, dilution curves were measured by automated sampling of coronary venous blood, using high-performance liquid chromatography and isotope detection techniques. Under control conditions, only 1% of the injected [3H]adenosine was detected in coronary venous samples, compared with the reference tracer, [14C]sucrose, and the peak of the adenosine dilution curve preceded those of the reference tracers by 2-4 s. Optimized model fits to the control adenosine curves required the combination of high ratios of the capillary endothelial cell membrane permeability-surface area product to flow (5.3) and endothelial cell consumption capacity to flow (23), in addition to a broad heterogeneity of flow. Dilution curves were measured under control conditions and during increased coronary flow (nitroglycerin), inhibition of the enzyme adenosine kinase (iodotubercidin), and blockade of membrane adenosine transport (dipyridamole). Reliability of the parameter estimates was confirmed using residual analysis, sensitivity function analysis, and Monte Carlo simulation techniques. Accuracy of the model was confirmed in separate experiments in which nontracer adenosine was infused into the coronary artery and the model prediction of coronary venous adenosine concentrations was compared with measured values. Using published measurements of coronary blood flow and arterial and coronary venous plasma adenosine concentrations in open-chest dogs, the estimated in vivo interstitial adenosine concentration is 100-220 nM.

Bimakalim, an ATP-sensitive potassium channel opener, mimics the effects of ischemic preconditioning to reduce infarct size, adenosine release, and neutrophil function in dogs.

The primary goal of the present study was to determine whether the infarct size-reducing effect of preconditioning is associated with an increase in adenosine release from the ischemic myocardium during a prolonged occlusion period or the subsequent reperfusion period and by a decrease in neutrophil infiltration. A second objective was to determine whether bimakalim, a KATP channel opener, mimics the effects of ischemic preconditioning. Barbital-anesthetized open-chest dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion followed by 3 hours of reperfusion. In the preconditioning group, 5 minutes of LAD occlusion followed by 10 minutes of reperfusion was elicited before the 60-minute occlusion period. In two other groups, bimakalim 1 microgram/kg bolus followed by a 0.05 micrograms.kg-1.min-1 infusion or an equivalent volume of saline was administered intravenously 15 minutes before occlusion and continued until the time of reperfusion. In a final group, bimakalim was administered 10 minutes before reperfusion and continued until the end of the experiment. To measure the release of adenosine from the ischemic region, coronary venous blood samples were collected at various times during ischemia and after reperfusion, and the concentration of adenosine was measured. Myocardial infarct size was determined by triphenyl tetrazolium chloride; transmural myocardial blood flow, by radioactive microspheres. Transmural myeloperoxidase (MPO) activity, an index of neutrophil infiltration in the area at risk, was also measured. Preconditioning produced a marked reduction in infarct size (9.8 +/- 3.0% versus 28.6 +/- 5.2% in the control group, mean +/- SEM); adenosine release at 5, 10, 15, and 30 minutes of the 3-hour reperfusion period; and transmural MPO activity in the risk area. Similarly, pretreatment with bimakalim resulted in reductions in infarct size, adenosine release, and transmural MPO activity to an extent almost identical to that of preconditioning. When bimakalim was administered 10 minutes before reperfusion, the drug also produced a significant reduction in infarct size and transmural MPO activity; however, no significant reduction in coronary venous adenosine concentrations was observed. There were no significant differences in collateral blood flow between groups. These results indicate that myocardial preconditioning in the canine heart produced by a short period of ischemia or a KATP channel opener is not mediated by an increase in adenosine release, as measured by coronary venous adenosine concentrations, during 60 minutes of occlusion or the initial 30 minutes of reperfusion. A significant reduction in transmural MPO activity in the ischemic area also appears to result from KATP channel activation and may play a role, at least in part, in the reduction in infarct size observed, particularly when a KATP channel opener is administered just before reperfusion.

Effects of nicorandil and glyceryl trinitrate on infarct size, adenosine release, and neutrophil infiltration in the dog

The major aim of this study was to determine if nicorandil, a potassium channel opener nitrate, produces a reduction in myocardial infarct size at a non-hypotensive dose in dogs and to determine if this effect is the result of an increase in adenosine release or reduction in neutrophil infiltration into the ischaemic area. Glyceryl trinitrate was used for purposes of comparison. Barbitone anaesthetised dogs were subjected to 60 min of left anterior descending coronary artery occlusion followed by 3 h of reperfusion. Nicorandil (100 microgram.kg-1 bolus followed by a 10 microgram.kg-1.min-1 infusion; NC/pre group), glyceryl trinitrate (10 microgram.kg-1 bolus followed by a 1 microgram.kg-1.min-1 infusion; GTN/pre group), or an equivalent volume of saline (control group) were given intravenously 15 min before occlusion and continued to the time of reperfusion. In two other groups, nicorandil (NC/post group) or glyceryl trinitrate (GTN/post group) were given 10 min before reperfusion and continued until the end of the experiment. To measure the release of adenosine from the ischaemic region, coronary venous blood samples were collected before occlusion, during occlusion, and at various times following reperfusion. Myocardial infarct size was determined by triphenyltetrazolium chloride and transmural myocardial blood flow by radioactive microspheres. Transmural myeloperoxidase activity, an index of neutrophil infiltration, was measured in biopsies obtained from the area at risk. Pretreatment with nicorandil and glyceryl trinitrate caused a marked reduction in myocardial infarct size expressed as percent of the area at risk [NC/pre group, 7.8(SEM 1.6)%; GTN/pre group, 11.9(2.3)%; control group, 31.0(5.6)%]. When nicorandil and glyceryl trinitrate were given before reperfusion, both drugs still produced a significant reduction in infarct size [NC/post group, 13.8(2.0)%; GTN/post group, 18.9(4.3)%]. Coronary venous adenosine concentrations during reperfusion were significantly lower in both nicorandil and glyceryl trinitrate pretreated groups, but not in the post-treated groups. Transmural myeloperoxidase activity was significantly lower in both nicorandil treated groups. Pretreatment with a non-hypotensive dose of nicorandil or glyceryl trinitrate markedly reduces myocardial infarct size and adenosine release from the ischaemic-reperfused area. These agents were also effective, but to a lesser degree, when given just before reperfusion. The cardioprotective actions of nicorandil appear to be related not only to its potassium channel opening activity but also in part to its nitrate activity.

Determination of Plasma Adenosine by High-Performance Liquid Chromatography with Column Switching and Fluorometric Detection

A new method for the rapid determination of plasma adenosine concentrations was developed by using high-performance liquid chromatography with a column switching technique and fluorometric detection. Several "stop solutions" were used to prevent the enzymatic degradation and cellular uptake and release of adenosine in blood samples. Red blood cells and certain denatured proteins were separated by centrifugation. Subsequently, the supernatant was transferred directly into autosample vials and adenosine was reacted with chloroacetaldehyde to form a strong fluorescent, 1-N6-ethenoadenosine. The adenosine derivative was injected directly and separated on a shielded hydrophobic phase column coupled with a C18 reverse-phase column using a column switching valve. Macromolecules and other interfering substances were excluded by the shielded hydrophobic phase column and bypassed to waste. Then, the adenosine derivative and other retained compounds were switched onto the reverse-phase column for further separation and subsequently to the fluorescence detector. The system reduces the analysis time and contamination of the column and hence allows a shorter cleanup time and a longer column lifetime. Adenosine as low as 30 fmol (signal-to-noise ratio, S/N = 3) can be detected by this method. The percentage of recovery of adenosine in plasma treated with adenosine deaminase was above 90%. This method is very rapid (without tedious sample preparation) and sensitive for determining adenosine in canine blood and should prove to be useful in analyzing the effects of ischemia and reperfusion on arterial and coronary venous adenosine concentrations in blood or perfusate samples released from the ischemic or hypoxic myocardium.

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.

Alpha 2-adrenoceptor stimulation can augment coronary vasodilation maximally induced by adenosine in dogs

To determine whether alpha 2-adrenoceptor stimulation can augment adenosine-induced coronary vasodilation, 34 open-chest dogs were studied. When a small dose of clonidine (up to 0.24 micrograms.kg-1.min-1 ic) was administered under beta-adrenoceptor blockade, coronary blood flow [312 +/- 16 (SE) ml.100 g-1.min-1] maximally induced by intracoronary infusion of adenosine was further increased (P less than 0.05) by 66 +/- 16 ml.100 g-1.min-1, despite no significant changes in coronary perfusion pressure, myocardial oxygen consumption, and coronary venous adenosine concentration. However, when a larger dose of clonidine (0.36-0.60 micrograms.kg-1.min-1) was infused, adenosine-induced flow progressively decreased. This biphasic action of the alpha 2-adrenoceptor activity was also observed when the dose of norepinephrine was increased during alpha 1-adrenoceptor blockade with prazosin. Norepinephrine up to 0.24 micrograms.kg-1.min-1 (ic) further increased adenosine-induced coronary blood flow by 24 +/- 5% (P less than 0.001), whereas hyperemic flow was decreased by a larger dose of norepinephrine. In contrast to the alpha 2-adrenoceptor stimulation, the alpha 1-adrenoceptor stimulation (norepinephrine with yohimbine) progressively decreased coronary blood flow. Furthermore, with a small dose of clonidine, reactive hyperemic flow significantly increased compared with that without clonidine (303 +/- 13 vs. 355 +/- 13 ml.100 g-1.min-1, P less than 0.001), but a larger dose of clonidine adversely reduced reactive flow (254 +/- 18 ml.100 g-1.min-1, P less than 0.001). Adenosine release during reactive hyperemia with and without intracoronary infusions of clonidine were not altered significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Quantitation of coronary venous adenosine in patients: limitations evaluated by radioimmunoassay.

Experimental studies have shown that adenosine is rapidly released in response to myocardial ischaemia. To evaluate whether coronary venous adenosine release is a metabolic characteristic of myocardial ischaemia in patients, adenosine concentrations were measured by a highly sensitive and specific radioimmunoassay. In three patients with normal coronary arteries and in seven with obstructive coronary artery disease coronary venous adenosine content was measured at rest and during atrial pacing. When whole blood or plasma were extracted immediately with perchloric acid the adenosine content was found to be lower than that previously reported. Recovery studies showed that the importance of time and temperature at low adenosine concentrations had been underestimated in preceding studies. In patients with coronary artery disease coronary venous adenosine concentration increased from 106.3(48.8) nmol.litre-1 to 114.9(57.0) nmol.litre-1 (NS) during pacing and was 130.4(63.3) nmol.litre-1 (NS) 2 min after pacing. Even in the presence of lactate production enhanced adenosine release was not consistently evidenced. Furthermore, venous adenosine content did not increase in five patients undergoing coronary artery occlusion during angioplasty of the left anterior descending coronary artery. The extremely short half life of coronary venous adenosine appears to preclude its use as an index of myocardial ischaemia in patients.