TY - JOUR
T1 - Magnesium activated adenosine formation in intact perfused heart
T2 - Predominance of ecto 5′-nucleotidase during hypermagnesemia
AU - Mallet, Robert T.
AU - Jie, Sun
AU - Fan, Wen Lin
AU - Kang, Young Hee
AU - Bünger, Rolf
N1 - Funding Information:
This study was supported by grants (to RTM) from the National Heart, Lung and Blood Institute (R29 HL 50441) and (to RB) from the National Heart, Lung and Blood Institute (RO1 HL 29060 and RO1 HL 37067), the Veterans Administration (G276CD) and Uniformed Services University for the Health Sciences (R07638). The expert advice of Dr. Miao-Xiang He regarding 3~p-NMR spectroscopy is gratefully acknowledged.
PY - 1996/6/4
Y1 - 1996/6/4
N2 - 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 μM α, β-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 μM [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 [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.
AB - 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 μM α, β-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 μM [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 [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.
KW - (Guinea pig)
KW - (Heart)
KW - 5′-nucleotidase
KW - AMP
KW - ATP phosphorylation potential
KW - Adenosine
KW - Magnesium, free
KW - Myocardium
UR - http://www.scopus.com/inward/record.url?scp=0029974727&partnerID=8YFLogxK
U2 - 10.1016/0304-4165(96)00016-5
DO - 10.1016/0304-4165(96)00016-5
M3 - Article
C2 - 8645720
AN - SCOPUS:0029974727
VL - 1290
SP - 165
EP - 176
JO - Biochimica et Biophysica Acta - General Subjects
JF - Biochimica et Biophysica Acta - General Subjects
SN - 0304-4165
IS - 2
ER -