Relationships Between Cytosolic [ATP], [ATP]/[ADP] and Ionic Fluxes in the Perfused Rat Heart: A 31P, 23Na and 87Rb NMR Study

Abstract

In order to assess the relationship between cytosolic [ATP] or [ATP]/[ADP] and the intracellular Na + concentration ([Na +] i), we have used the phosphate trap 2-deoxy-D-glucose (DG) to alter the high energy phosphate levels in rat cardiomyocytes. Pyruvate-perfused rat heart were treated with 2 mM DG in the presence of 10 IU/l of insulin for 28 min, followed by perfusion with DG without insulin for 60 min. The DG+insulin treatment resulted in dramatic changes in the 31P NMR spectra: phosphocreatine (PCr) and total ATP decreased (to 15 and 35%, respectively) and deoxyglucose-6-phosphate accumulated, with the little change in either inorganic phosphate or intracellular pH. These changes corresponded to a decrease in cytoplasmic [ATP] (from 7.6 to 1.8 mM). [ATP]/[ADP] (from 494 to 24) and ATP affinity [A(ATP), by 8.9kJ/mol] and an increase in [ADP] (five-fold) and free [Mg 2+] (two-fold). Subsequent perfusion with DG-insulin resulted in slow recovery of PCr. [ATP],[ATP] and A(ADP) such that the "low energy" state lasted an additional 16 min during which ATP remained low and constant. There were no detectable changes in the intracellular Na + content as assessed by shift reagent-aided 23Na NMR at the end of DG+insulin treatment (98 ± 18%, 28-36 min of the protocol). In addition, there was no change in the Rb + influx rate as measured by 87Rb NMR at the beginning of insulin washout which was achieved by replacing 20% of the KCl with RbCl ([K +] = 3.7 mM,[Rb +] = 0.94 mM). During DG+insulin treatment the pressure-rate product (PRP) decreased by half and was restored upon insulin washout to 80% of its initial value both in the presence and in the absence of the shift reagent [5 mM Dy (triethylenetetraminehexaacetate) 3-]. These data imply that unfavorable thermodynamic [low A(ATP)] and kinetic (low [ATP] and [ATP]/[ATP]) conditions induced by DG treatment do not inhibit Na +, K + -ATPase activity. We speculate that during anoxia when changes in [ATP]/[ADP] are comparable to those induced by DG treatment, the observed increase in [Na +] i is not due to inhibition of the Na + pump by reduced [ATP] or [ATP]/[ADP].