Relationships between cytosolic [ATP], [ATP]/[ADP] and ionic fluxes in the perfused rat heart: A 31P, 23Na and 87Rb NMR study.
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 hearts were treated with 2 mM DG in the presence of 10IU/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 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 8.9 kJ/mol] and an increase in [ADP] (five-fold) and free [Mg2+] (two-fold). Subsequent perfusion with DG--insulin resulted in slow recovery of PCr, [ATP]/[ADP] and A(ATP) 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+] = 3.76 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]/[ADP]) 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].
Stewart
L C
LC
Institute for Biodiagnostics, National Research Council, Winnipeg, MB, Canada.
Deslauriers
R
R
Kupriyanov
V V
VV
D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D002451:Cell Compartmentation; D003600:Cytosol / Q000378:metabolism; D003847:Deoxyglucose / Q000378:metabolism / Q000494:pharmacology; D004734:Energy Metabolism / Q000187:drug effects; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D010477:Perfusion; D010759:Phosphorus Isotopes; D051381:Rats; D017207:Rats, Sprague-Dawley; D012414:Rubidium Radioisotopes; D012964:Sodium / Q000378:metabolism*; D012975:Sodium Isotopes
D010759:Phosphorus Isotopes; D012414:Rubidium Radioisotopes; D012975:Sodium Isotopes; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003847:Deoxyglucose; D012964:Sodium; D008274:Magnesium