Keith Kroll

Rapid turnover of the AMP-adenosine metabolic cycle in the guinea pig heart

The intracellular flux rate through adenosine kinase (adenosine-->AMP) in the well-oxygenated heart was investigated, and the relation of the AMP-adenosine metabolic cycle (AMP<-->adenosine) to transmethylation (S-adenosylhomocysteine [SAH]-->adenosine) and coronary flow was determined. Adenosine kinase was blocked in isolated guinea pig hearts by infusion of iodotubercidin in the presence of the adenosine deaminase blocker erythro-9-(2-hydroxy-3-nonyl)adenine (5 mumol/L). Iodotubercidin (1 nmol/L to 4 mumol/L) caused graded increases in venous effluent concentrations of adenosine, from 8 +/- 3 to 145 +/- 32 nmol/L (mean +/- SEM, n = 3), and in coronary flow, which increased to maximal levels. Flow increases were completely abolished by adenosine deaminase (5 to 10 U/mL). Interstitial adenosine concentrations, estimated using a mathematical model, increased from 22 nmol/L during control conditions to 420 nmol/L during maximal vasodilation. The possibility that iodotubercidin caused increased venous adenosine by interfering with myocardial energy metabolism was ruled out in separate 31P nuclear magnetic resonance experiments. To estimate total normoxic myocardial production of adenosine (AMP-->adenosine<--SAH), the time course of coronary venous adenosine release was measured during maximal inhibition of adenosine kinase with 30 mumol/L iodotubercidin. Adenosine release increased more than 15-fold over baseline, reaching a new steady-state value of 3.4 +/- 0.3 nmol.min-1 x g-1 (n = 5) after 4 minutes. In parallel experiments, the relative roles of AMP hydrolysis and transmethylation (SAH hydrolysis) were determined, using adenosine dialdehyde (10 mumol/L) to block SAH hydrolase. In these experiments, adenosine release increased to similar levels of 3.4 +/- 0.5 nmol.min-1 x g-1 (n = 6) during inhibition of adenosine deaminase and adenosine kinase. It is concluded that (1) maximal increases in coronary flow are elicited by increases in interstitial adenosine concentration to approximately 400 nmol/L, (2) more than 90% of the adenosine produced in the heart is normally rephosphorylated to AMP without escaping into the venous effluent, (3) AMP hydrolysis is the dominant pathway for cardiac adenosine production under normoxic conditions, and (4) the high rate of adenosine salvage is due to rapid turnover of a metabolic cycle between AMP and adenosine. Rapid cycling may serve to amplify the relative importance of AMP hydrolysis over transmethylation in controlling cytosolic adenosine concentrations.

Quantitative Relation Between Interstitial Adenosine Concentration and Coronary Blood Flow

The effect of exogenous and endogenous adenosine in controlling coronary flow was determined using an axially distributed mathematical model of the myocardium to estimate interstitial adenosine concentration from coronary arterial and venous adenosine values. The left main coronary artery was perfused at constant pressure in closed-chest, anesthetized dogs, and exogenous adenosine was infused intracoronary to increase coronary flow. Basal interstitial adenosine was 92 nmol/L, just at the threshold for increasing coronary flow. An increase in interstitial adenosine concentration of only 62% was sufficient to increase coronary flow from 5% to 50% of maximal flow. The possible contribution of an endothelial dilator secondary to activation of adenosine receptors on endothelial cells was tested by comparing the response to exogenous intracoronary adenosine infusion with increases in endogenous adenosine produced by inhibition of adenosine kinase and adenosine deaminase. If adenosine increases coronary flow by an endothelial mechanism, then the interstitial ED50 of exogenous adenosine would be lower than that for endogenous adenosine due to the postulated additional endothelial dilator. The interstitial ED50 for exogenous adenosine was 156 nmol/L, not different from the endogenous ED50 of 150 nmol/L. In conclusion, basal interstitial adenosine concentration is at the threshold of a remarkably steep dose-response curve for increasing coronary blood flow. No evidence was found for an endothelium-mediated vasodilator mechanism secondary to adenosine receptor activation of endothelial cells in vivo. The steep adenosine dose-response curve indicates that measurements of adenosine concentration should be interpreted with caution, because small changes in adenosine concentration cause large changes in coronary flow.

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.

Role of adenosine in local metabolic coronary vasodilation.

Adenosine has been postulated to mediate the increase in coronary blood flow when myocardial oxygen consumption is increased. The aim of this study was to evaluate the role of adenosine when myocardial oxygen consumption was augmented by cardiac paired-pulse stimulation without the use of catecholamines. In 10 anesthetized closed-chest dogs, coronary blood flow was measured in the left circumflex coronary artery, and myocardial oxygen consumption was calculated using the arteriovenous oxygen difference. Cardiac interstitial adenosine concentration was estimated from coronary venous and arterial plasma adenosine measurements using a previously described multicompartmental, axially distributed mathematical model. Paired stimulation increased heart rate from 55 to 120 beats/min, increased myocardial oxygen consumption 104%, and increased coronary blood flow 92%, but the estimated interstitial adenosine concentration remained below the threshold for coronary vasodilation. After adenosine-receptor blockade with 8-phenyltheophylline (8-PT), coronary blood flow and myocardial oxygen consumption were not significantly different from control values. Paired-pulse pacing during adenosine-receptor blockade resulted in increases in myocardial oxygen consumption and coronary blood flow similar to the response before 8-PT. Coronary venous and estimated interstitial adenosine concentration did not increase to overcome the adenosine blockade by 8-PT. These results demonstrate that adenosine is not required for the local metabolic control of coronary blood flow during pacing-induced increases in myocardial oxygen consumption.Adenosine has been postulated to mediate the increase in coronary blood flow when myocardial oxygen consumption is increased. The aim of this study was to evaluate the role of adenosine when myocardial oxygen consumption was augmented by cardiac paired-pulse stimulation without the use of catecholamines. In 10 anesthetized closed-chest dogs, coronary blood flow was measured in the left circumflex coronary artery, and myocardial oxygen consumption was calculated using the arteriovenous oxygen difference. Cardiac interstitial adenosine concentration was estimated from coronary venous and arterial plasma adenosine measurements using a previously described multicompartmental, axially distributed mathematical model. Paired stimulation increased heart rate from 55 to 120 beats/min, increased myocardial oxygen consumption 104%, and increased coronary blood flow 92%, but the estimated interstitial adenosine concentration remained below the threshold for coronary vasodilation. After adenosine-receptor blockade with 8-phenyltheophylline (8-PT), coronary blood flow and myocardial oxygen consumption were not significantly different from control values. Paired-pulse pacing during adenosine-receptor blockade resulted in increases in myocardial oxygen consumption and coronary blood flow similar to the response before 8-PT. Coronary venous and estimated interstitial adenosine concentration did not increase to overcome the adenosine blockade by 8-PT. These results demonstrate that adenosine is not required for the local metabolic control of coronary blood flow during pacing-induced increases in myocardial oxygen consumption.

Role of Capillary Endothelial Cells in Transport and Metabolism of Adenosine in the Heart: An Example of the Impact of Endothelial Cells on Measures of Metabolism

Endothelial cells, lying between the blood stream and the parenchymal cells of an organ, are a part of the set of signaling paths for the organ. Sensing blood solute concentrations or sensing intravascular shear can lead to the endothelial production of substances sensed or taken up by other cells. The interactions between endothelium and smooth muscle fall into a special class relating to the regulation of vasomotion. A component of the vasoregulatory system concerns the regulation of interstitial adenosine; understanding of adenosine in endothelial cells and myocytes has come slowly from early beginnings (Berne et al., 1983) and from studies of transport and exchange (Bassingthwaighte et al., 1985a,b; Gorman et al., 1986). In this chapter we provide a further set of ideas on relationships between endothelial cells and cardiac myocytes in vivo, using adenosine as the substrate of interest. These ideas hold for a variety of solutes, substrates, agonists, and pharmacologic agents, which one can choose to contemplate while reading about this local adenosine story.

Myocardial adenosine stimulates release of cyclic adenosine monophosphate from capillary endothelial cells in guinea pig heart

Activation of coronary endothelial cell adenylate cyclase was studied in the isolated guinea pig heart by prelabelling endothelial adenine nucleotides using intracoronary infusion of [3H]-adenosine, and measuring the coronary efflux of [3H]-cyclic adenosine monophosphate (cAMP). Hypoxia (30 % O2) caused a 4-fold increase in coronary release of [3H]-cAMP, which was decreased by 63 % by infusion of the adenosine receptor antagonist, theophylline (50 μM). During normoxic control conditions, degrading adenosine to non-vasoactive inosine by intracoronary infusion of adenosine deaminase (1.7 U/ml) caused a 20 % decrease in the release of [3H]-cAMP. The effect of adenosine deaminase was reversed by a specific enzyme inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride. Coronary efflux of [3H]-cAMP during intracoronary infusion of 1 μM adenosine triphosphate (ATP), adenosine diphosphate or adenosine monophosphate (AMP) (plus adenosine deaminase 8 U/ml) was only 13 % of that due to 1 μM adenosine. Adenosine receptor blockers theophylline and CGS 15943A caused equivalent inhibition of the coronary vasodilator actions of adenosine and ATP. Intracoronary infusion of prostaglandin E1 and the β2-adrenergic agonist procaterol caused parallel, dose-dependent increases in coronary conductance and the venous release of [3H] cAMP. It is concluded that (1) under both normoxic and hypoxic conditions, adenosine formed by the heart may activate endothelial cell adenylate cyclase via membrane adenosine receptors, (2) coronary receptors for adenosine and ATP share common ligand affinities but ATP receptors are not coupled to adenylate cyclase, and (3) other vasodilators known to activate endothelial adenylate cyclase in vitro cause parallel increases in coronary conductance and adenylate cyclase activity in the beating heart.

Strategies for Uncovering the Kinetics of Nucleoside Transport and Metabolism in Capillary Endothelial Cells

For the analysis of signals obtained by external detection techniques such as positron tomographic imaging (PET), magnetic resonance imaging (MRI), and X-ray computed tomography (X-ray CT), investigators and diagnosticians usually obtain a sequence of images. For physiological interpretation in terms of the underlying physical and chemical events, it is essential to use models when one wants to learn more than the simplest measures. Among the simplest measures one can often include volume and flow estimates, but not always, for it commonly occurs that the distinctive estimation of these two parameters simultaneously requires using knowledge of the anatomy or of other properties of the tissue. The two most accessible measures of indicator transport are the areas under dilution curves and their mean transit times following a pulse injection. Mass conservation for substances that are not destroyed, such as radioactive tracers, relies on the general expression:

A Multisubstrate Blood-Tissue Exchange Model for the Analysis of Indicator Dilution Curves from Whole Organs

q(t){\text{ = }}F\int_0^t {{C_{in}}} \;dt{\text{ - }}F\int_0^t {{C_{out}}} \;dt,