Mechanisms of pHi recovery from NH4Cl-induced acidosis in anoxic isolated turtle heart: a 31P-NMR study

H. Shi, P. H. Hamm, R. S. Meyers, R. G. Lawler and D. C. Jackson Department of Physiology, Brown University, Providence, Rhode Island 02912, USA. Mechanisms of intracellular pH (pHi) recovery from NH4Cl-induced acidosis were investigated on isolated perfused hearts of the turtle, Chrysemys picta bellii, using 31P nuclear magnetic resonance (NMR) spectroscopy at 20 degrees C. A major goal was to assess the activity of these mechanisms under anoxic conditions. Based on calculated buffer capacity and a pHi recovery range at 20 degrees C of 6.75-6.95 (normal pHi 7.2-7.4), mean H' efflux rate during perfusion with CO2-free N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-buffered Ringer was only 15% (normoxia) and 25% (anoxia) of that with HCO3-buffered Ringer. With HCO3 solution, anoxic H1 efflux rate was approximately 50% of normoxia (0.333 vs. 0.645 mmol.l-1.min-1), but in TES solution, H1 efflux rate was unaffected by anoxia. To further characterize the transporters, we used blockers [the Na(+)-H+ antiport inhibitor 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and the anion exchanger inhibitor 4,4'diisothiocyanostilbene-2, 2'-disulfonic acid (DIDS)], ion substitution, and temperature change. EIPA (10 microM) inhibited H+ efflux rate by 40% in anoxic TES solution; DIDS (0.5 mM) blocked H+ efflux rate by 85% in anoxic HCO3 solution. No pHi recovery was observed in either normoxic or anoxic Na(+)-free solutions, but normal recovery was observed in the absence of extracellular Cl-. Recovery of pHi occurred 2-3 times faster at 30 degrees C than at 20 degrees C. ATP was unaffected by any manipulation in this study, whereas creatine phosphate (CP) fell during anoxia, and both CP and mechanical performance changed in parallel to pHi. We conclude that pHi regulation functions during anoxia, although at a reduced rate, and that recovery from acidosis is dominated, during both normoxia and anoxia, by a DIDS-sensitive Na+ and HCO3(-)-dependent mechanism, whereas EIPA-sensitive Na(+)-H+ antiport plays a less important role.