31P NMR studies of ATP synthesis and hydrolysis kinetics in the intact myocardium

The origin of the nuclear magnetic resonance (NMR)-measurable ATP ↔ P/sub i/ exchange and whether it can be used to determine net oxidative ATP synthesis rates in the intact myocardium were examined by detailed measurements of ATP ↔ P/sub i/ exchange rates in both directions as a function of the myocardial oxygen consumption rate (MVO2) in (1) glucose-perfused, isovolumic rat hearts with normal glycolytic activity and (2) pyruvate-perfused hearts where glycolytic activity was reduced or eliminated either by depletion of their endogenous glycogen or by use of the inhibitor iodoacetate. In glucose-perfused hearts, the P/sub i/ → ATP rate measured by the conventional two-site saturation transfer (CST) technique remained constant while MVO2 was increased approximately 2-fold. When the glycolytic activity was reduced, the P/sub i/ → ATP rate decreased significantly, demonstrating the existence of a significant glycolytic contribution. The ATP → P/sub i/ rates and rate:MVO ratios measured by the multiple-site saturation transfer method at two MVO2 levels were equal to the corresponding P/sub i/→ ATP rates and rate:MVO ratios obtained in the absence of a glycolytic contribution. The following conclusions are drawn from these studies: (1) unless the glycolytic contribution to the ATP ↔ P/sub i/ exchange is inhibited or is specifically shown not to exist, the myocardial P/sub i/ ↔ ATP exchange due to oxidative phosphorylation cannot be studied by NMR; (2) at moderate MVO2 levels, the reaction catalyzed by the two glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase is near equilibrium; (3) the ATP synthesis by the mitochondrial H+-ATPase occurs unidirectionally (i.e., the reaction is far out of equilibrium); (4) the operative P:O ratio in the intact myocardium under our conditions is significantly less than the canonically accepted value of 3