To assess the concentration of free intracellular adenosine in the heart the kinetic properties of cytosolic S-adenosylhomocysteine (SAH) hydrolase were utilized at elevated levels of L-homocysteine (adenosine + L-homocysteine in equilibrium with SAH + H2O). Global hypoxia was induced in the isolated perfused guinea pig heart by graded reduction of perfusion medium PO2 in the presence of saturating concentrations of homocysteine (0.2-1.0 mM). Reduction of PO2 from 660 to 165 mm Hg increased the steady-state concentration of total tissue adenosine from 2.0 +/- 0.2 to 2.8 +/- 0.2 nmoles/g, while the rate of SAH formation increased linearly from 0.22 +/- 0.03 to 2.50 +/- 0.13 nmoles/min/g. When adenosine was exogenously applied at a concentration of 100 microM together with homocysteine (1 mM), SAH accumulation rates were much greater: 23.34 +/- 3.31 and 42.11 +/- 1.73 nmoles/min/g with normoxic (95% O2) and hypoxic (30% O2) perfusion, respectively. The apparent Km and Vmax values for SAH-hydrolase in vivo were estimated to be 20 microM and 59 nmoles/min/g wet wt, respectively. Since the relation between SAH formation and adenosine in the physiological concentration range is linear, the measured rate of SAH accumulation during normoxia and hypoxia permitted the calculation of the free intracellular adenosine level, which was 0.061 nmoles/g (0.08 microM) in the normoxic heart. With hypoxia (PO2 165 mm Hg), this value increased to 1.57 nmoles/g (2.0 microM). Free intracellular adenosine closely correlated with the hypoxia-induced changes in coronary flow. The data reveal that measurement of the rate of SAH accumulation during homocysteine infusion can be used for sensitive assessment of free intracellular adenosine levels. Assuming that the intracellular adenosine concentration equals that in the interstitial space, the results furthermore indicate that the degree of intracellular adenosine formation during hypoxic perfusion is quantitatively sufficient to account for most of the observed increases in coronary flow.
Adenosine Animals