Different sites of adenosine formation in the heart

In an attempt to further define the site of myocardial adenosine formation, isolated guinea pig hearts were perfused with potent inhibitors of 5'-nucleotidase [alpha, beta-methylene adenosine 5'-diphosphate (AOPCP)] and of nucleoside transport [4-nitrobenzyl thioinosine (NBMPR)]. AOPCP (50 microM) inhibited the activity of cardiac ecto-5'-nucleotidase by 85% but did not influence the release of adenosine, inosine, and hypoxanthine formed at an accelerated rate by the heart during hypoxic perfusion (30% O2). In contrast, NBMPR (5 microM) diminished the hypoxia-induced release of adenosine and its degradatives and greatly potentiated the increase of myocardial tissue levels of respective purine compounds. Studies carried out with 5'-deoxyadenosine, an adenosine derivative that is not metabolized, indicate NBMPR to inhibit both uptake and release of adenosine in the isolated heart and in human erythrocytes. Cell fractionation studies on guinea pig ventricular muscle revealed that 5'-nucleotidase, though mainly associated with the membrane fraction, is also found in the cardiac cytosol (200,000-g supernatant), exhibiting a different substrate specificity. Furthermore, S-adenosylhomocysteine hydrolase as well as adenosine kinase and adenosine deaminase proved to be exclusively present in the cytosolic fraction. Our findings suggest that in the hypoxic heart a) ecto-5'-nucleotidase most likely is not involved in the formation of adenosine, b) release of adenosine from the heart requires adenosine to be transported across the sarcolemma membrane, and c) adenosine is predominantly formed intracellularly, a process involving cytosolic 5'-nucleotidase and/or S-adenosylhomocysteine hydrolase.