Contribution of cytosolic and membrane bound, 5_-nucleotidases to
cardiac adenosine production.
Darvish, Ahmad, Richard W. Pomerantz, Peter G. Zografides, and
Patricia J. Metting.
Laboratory of Cardiovascular Research, Department of Physiology and
Molecular Medicine, and Division of Cardiology, Department of
Medicine, Medical College of Ohio, Toledo, Ohio 43699
APStracts 3:0205H, 1996.
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 150,000xg 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 mmoles/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]moles/L) activated
supernatant and inhibited membrane-bound 5_-nucleotidase activity. At
80 [mu]moles/L ADP, 5 mmoles/L MgCl2, 100 [mu]moles/L AMP, and pH
7.3, the average 5_-nucleotidase activities of the supernatant vs.
pellet were 147 (74% of total) and 51 (26% of total) nmoles/min/gm
tissue, respectively. Total adenosine production in unfractionated
samples of ventricular homogenates was 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.
Received 21 October 1994; accepted in final form 15 April 1996.
APS Manuscript Number H942-4.
Article publication pending Am. J. Physiol. (Heart Circ. Physiology).
ISSN 1080-4757 Copyright 1996 The American Physiological Society.
Published in APStracts on 19 May 96