title: Magnesium activated adenosine formation in intact perfused heart: predominance of ecto 5'-nucleotidase during hypermagnesemia.
abstract: Magnesium ion is an allosteric effector of 5'-nucleotidase and thus activates adenosine production from AMP. Two distinct 5'-nucleotidase systems, the membrane-bound ecto and the soluble cytosolic isoforms, exist in mammalian myocardium. The aim of this study was to delineate the contributions of the ecto vs. cytosolic isoforms to Mg2+-stimulated cardiac purine nucleoside formation and release. Isolated guinea pig hearts were retrogradely perfused at their physiological aortic pressure with Krebs-Henseleit bicarbonate buffer fortified with 10 mM glucose. AMP and the adenylate degradatives adenosine and inosine were measured in coronary venous effluent and in epicardial transudate, which was sampled to estimate concentrations of adenylate degradatives in the interstitium. When perfusate Mg2+ was increased from 0.6 to 6 mM, coronary vascular resistance and spontaneous heart rate fell, and steady-state coronary venous release of adenosine + inosine rose severalfold. Cytosolic free magnesium, as estimated by 31P-NMR after 15 min of perfusion with 6 mM Mg2+ or from chemically measured indicator metabolites after 30 min, rose 60 and 144% respectively (P < 0.05). Excess Mg2+ stimulated purine nucleoside release nearly threefold in coronary venous effluent and four- to sevenfold in epicardial transudate. 50 microM, alpha,beta-methylene adenosine 5'-diphosphate (AOPCP), a selective inhibitor of ecto 5'-nucleotidase, elevated interstitial AMP concentration tenfold, did not attenuate basal nucleoside release, but completely inhibited Mg2+-stimulated coronary venous purine nucleoside release and blunted Mg2+-stimulated interstitial purine nucleoside formation by 69%. During perfusion with exogenous 1 microM [8-14C]AMP, excess perfusate MgCl2 increased [14C]adenosine release by 63% in coronary effluent and 133% in epicardial transudate. AOPCP decreased baseline [14C]adenosine release in coronary effluent and epicardial transudate by 85-90%, caused equilibration of arterial and epicardial AMP, and attenuated MgCl2 activation of p[14C]adenosine formation by approx. 75%, in both the vascular and interstitial compartments. Intramyocytic concentrations of allosteric regulators of the cytosolic 5'-nucleotidases were evaluated in stop-frozen myocardium. Excess magnesium did not appreciably alter intracellular pH and ATP concentration, but lowered free cytosolic ADP and AMP concentrations by 50 and 70%, respectively. A simplified model of compartmentalized adenosine metabolism is proposed in which magnesium ion-activated cardiac purine release originates predominantly from the ecto 5'-nucleotidase; magnesium ion stimulation of metabolic flux through the cytosolic isoforms was constrained by concomitant reductions in intracellular AMP substrate and allosteric activator ADP. Magnesium ion-enhanced adenosine formation by 5'-nucleotidase could contribute to the known cardioprotective effects of this clinically used cation.
authors.affiliation: Department of Physiology, University of North Texas Health Science Center, Fort Worth 76107-2699, USA.
authors.forename: R T
authors.initials: RT
authors.lastname: Mallet
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: W L
authors.initials: WL
authors.lastname: Fan
authors.forename: Y H
authors.initials: YH
authors.lastname: Kang
authors.forename: R
authors.initials: R
authors.lastname: Bünger
mesh_terms: D015720:5'-Nucleotidase / Q000378:metabolism*; D000227:Adenine Nucleotides / Q000378:metabolism; D000241:Adenosine / Q000378:metabolism*; D000818:Animals; D003600:Cytosol / Q000378:metabolism; D004734:Energy Metabolism; D006168:Guinea Pigs; D008274:Magnesium / Q000378:metabolism* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D009206:Myocardium / Q000378:metabolism*; D010477:Perfusion
chemical_list: D000227:Adenine Nucleotides; D015720:5'-Nucleotidase; D008274:Magnesium; D000241:Adenosine