MECHANISMS OF PH(I) RECOVERY FROM NH4CL-INDUCED ACIDOSIS IN ANOXIC ISOLATED TURTLE HEART - A P-31-NMR STUDY

Mechanisms of intracellular pH (pH(i)) recovery from NH4Cl-induced aci dosis were investigated onisolated perfused hearts of the turtle, Chry semys picta bellii, using P-31 nuclear magnetic resonance (NMR) spectr oscopy at 20 degrees C. A major goal was to assess the activity of the se mechanisms under anoxic conditions. Based on calculated buffer capa city and a pH(i) recovery range at 20 degrees C of 6.75-6.95 (normal p H(i) 7.2-7.4), mean H+ efflux rate during perfusion with CO2-free N-tr is(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES)-buffered Ring er was only 15% (normoxia) and 25% (anoxia) of that with HCO3--buffere d Ringer. With HCO3- solution, anoxic H+ efflux rate was similar to 50 % of normoxia (0.333 vs 0.645 mmol . l(-1). min(-1)), but in TES solut ion, H+ efflux rare 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 substit ution, and temperature change. EIPA (10 mu M) inhibited H+ efflux rate by 40% in anoxic TES solution; DIDS (0.5 mM) blocked H+ efflux rate b y 85% in anoxic HCO3- solution. No pH(i) recovery was observed in eith er normoxic or anoxic Na+-free solutions, but normal recovery was obse rved in the absence of extracellular Cl-. Recovery of pH(i) occurred 2 -3 times faster at 30 degrees C than at 20 degrees C. ATP was unaffect ed by any manipulation in this study, whereas creatine phosphate (CP) fell during anoxia, and both CP and mechanical performance changed in parallel to pH(i). We conclude that pH(i) 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+ a nd HCO3--dependent mechanism, whereas EIPA-sensitive Na+-H+ antiport p lays a less important role.