Contribution of cytosolic and membrane-bound 5'-nucleotidases to cardiac adenosine production

The purpose of this study was to evaluate the relative contributions of AMP-specific cytosolic 5'-nucleotidase and ecto-5'-nucleotidase to cardiac adenosine production and its regulation by ADP and Mg2+. 5'-Nucleotidase activity was measured spectrophotometrically in the total homogenate, the 150,000-g supernatant fraction (cytosolic 5'-nucleotidase), and the membrane pellet fraction (ecto-5'-nucleotidase) of dog left ventricles. Increasing [MgCl2] over a range from 0 to 6 mmol/l increased 5'-nucleotidase activity in both the supernatant and pellet; only cytosolic 5'-nucleotidase exhibited an absolute requirement for Mg2+. ADP (20-480 mu mol/l) activated supernatant and inhibited membrane-bound 5'-nucleotidase activity. At 80 mu mol/l ADP, 5 mmol/l MgCl2, 100 mu mol/l AMP, and pH 7.3, the average 5'-nucleotidase activities of the supernatant vs, pellet were 74% of total and 26% of total, respectively. Total adenosine production in unfractionated samples of ventricular homogenates decreased an average of 73% by specific inhibition of cytosolic 5'-nucleotidase, using antibodies against the cytosolic enzyme, and 46% by specific inhibition of ecto-5'-nucleotidase with alpha,beta-methylene adenosine 5'-diphosphate (AOPCP). These findings support the hypotheses that 1) both cytosolic and ecto-5'-nucleotidase contribute to cardiac adenosine production in dog heart homogenates; 2) AMP-specific cytosolic 5'-nucleotidase activity exceeds ecto-5'-nucleotidase activity at physiological concentrations of ADP, AMP, and Mg2+; and 3) Mg2+ is an important regulator of cardiac adenosine production via activation of both ecto- and AMP-specific cytosolic 5'-nucleotidases.