CONTRIBUTION OF CYTOSOLIC AND MEMBRANE-BOUND 5'-NUCLEOTIDASES TO CARDIAC ADENOSINE PRODUCTION

The purpose of this study was to evaluate the relative contributions o f AMP-specific cytosolic 5'-nucleotidase and ecto-5'-nucleotidase to c ardiac adenosine production and its regulation by ADP and Mg2+. 5'-Nuc leotidase activity was measured spectrophotometrically in the total ho mogenate, the 150,000-g supernatant fraction (cytosolic 5'-nucleotidas e), and the membrane pellet fraction (ecto-5'-nucleotidase) of dog lef t ventricles. Increasing [MgCl2] over a range from 0 to 6 mmol/l incre ased 5'-nucleotidase activity in both tile supernatant and pellet; onl y cytosolic 5'-nucleotidase exhibited an absolute requirement for Mg2. ADP (20-480 mu mol/l) activated supernatant and inhibited membrane-b ound 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 t he supernatant vs, pellet were 74% of total and 26% of total, respecti vely. Total adenosine production in unfractionated samples of ventricu lar homogenates decreased an average of 73% by specific inhibition of cytosolic 5'-nucleotidase, using antibodies against the cytosolic enzy me, and 46% by specific inhibition of ecto-5'-nucleotidase with alpha, beta-methylene adenosine 5'-diphosphate (AOPCP). These findings suppor t the hypotheses that 1) both cytosolic and ecto-5'-nucleotidase contr ibute to cardiac adenosine production in dog heart homogenates; 2) AMP -specific cytosolic 5'-nucleotidase activity exceeds ecto-5'-nucleotid ase activity at physiological concentrations of ADP, AMP, and Mg2+; an d 3) Mg2+ is an important regulator of cardiac adenosine production vi a activation of both ecto- and AMP-specific cytosolic 5'-nucleotidases .