Phosphorus-31 NMR saturation transfer measurements of phosphorus exchange reactions in rat heart and kidney in situ

Abstract

31P NMR spectra of rat kidney and heart, in situ, were obtained at 97.2 MHz by using chronically implanted radio-frequency coils. Previous investigators have used magnetization transfer techniques to study phosphorus exchange in perfused kidney and heart. In the current experiments, saturation transfer techniques were used to measure the steady-state rate of exchange between inorganic phosphate (Pi) and the .gamma.-phosphate of ATP (.gamma.ATP) in kidney, and between phosphocreatine (PCr) and .gamma.ATP, catalyzed by creatine kinase, in heart. The rate constant for the exchange detected between Pi and .gamma.ATP in kidney, presumably catalyzed by oxidative phosphorylation, was 0.12 .+-. 0.03 s-1. This corresponds to an ATP synthesis rate of 12 .mu.mol min-1 (g wet weight)-1. Comparison of previously published O2 consumption and Na+ reabsorption rates for the intact kidney with the NMR-derived rate for ATP synthesis gave flux ratios of JATP/JO2 = 1.6-3.3 and JNa+/JATP = 4-10. The rate constants for the creatine kinase reaction, assuming a simple two-site exchange, were found to be 0.57 .+-. 0.12 s-1 for the forward direction (PCr .fwdarw. ATP) and 0.50 .+-. 0.16 s-1 for the reverse direction (ATP .fwdarw. PCr). The forward rate (0.78 .+-. 0.18 intensity unit/s) was significantly larger (p < 0.05) than the reverse rate (0.50 .+-. 0.16 intensity unit/s). This difference between the forward and reverse rates of creatine kinase has been previously noted in the perfused heart. The difference was been attributed to participation of ATP in other reactions. To test this possibility, the reverse rate was measured by saturating both PCr and the .beta.-phosphate of ATP. The rate obtained in this manner was not significantly different from that derived by assuming at two-site exchange. These results suggest the possibility that compartmentation of ATP might be responsible for the difference in rates.