Recovery of intracellular pH in cortical brain slices following anoxia studied by nuclear magnetic resonance spectroscopy: role of lactate removal, extracellular sodium and sodium/hydrogen exchange.
[31P]- and [1H]nuclear magnetic resonances recorded in an interleaved fashion were used in order to quantify high-energy phosphates, intracellular pH and lactate in cortical brain slices of the guinea-pig superfused in a CO2/HCO3(-)-buffered medium during and after anoxic insults. The volume-averaged intracellular pH and energy status of the preparation following anoxia were determined. In the presence of external Na+, intracellular pH normalized in 3 min and was significantly more alkaline from 10 to 12 min of recovery, but lactate remained elevated for 12 min of reoxygenation following anoxia. The amount of lactate removed was only 40% of the quantity of acid extruded showing operation of H+ neutralizing transmembrane mechanisms other than transport of lactic acid. Amiloride (1 or 2 mM) did not prevent the recovery of intracellular pH, but it blocked the "overshoot" of the alkalinization at 10-12 min of recovery. In a medium containing 70 mM K+, 60 mM Na+ and 0.1 mM Ca2+, the recovery of pH, but not lactate washout, was significantly delayed. Removal of external Na+ caused severe energetic failure, decreases both in oxygen uptake and in N-acetyl aspartate concentration, indicating loss of viable tissue. In Na(+)-free superfusion, lactic acidosis caused a more severe drop in intracellular pH than in the presence of Na+. Complexing of extracellular Ca2+ in the Na(+)-free medium inhibited the acidification by 0.38 pH units during anoxia which is as much as the acidification caused by lactate accumulation in the absence of Na+. In Na(+)-free medium intracellular pH recovered, however, from an anoxic level to a normoxic value in 6 min. Metabolic damage of the slice preparation induced by anoxia in the absence of Na+ was as profound in the presence as in the absence of Ca2+ showing that accumulation of Ca2+ is not the only reason for the damage. It is concluded that recovery of intracellular pH from lactic-acidosis can occur independently of energetic recovery and involves acid extrusion mechanism(s) that is(are) dependent on external Na+ and sensitive to high K+.
Pirttilä
T R
TR
Department of Biochemistry and Biotechnology, University of Kuopio, Finland.
Kauppinen
R A
RA
D000140:Acidosis, Lactic / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002540:Cerebral Cortex / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D000860:Hypoxia / Q000378:metabolism*; D066298:In Vitro Techniques; D007773:Lactates / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D010101:Oxygen Consumption / Q000502:physiology; D010725:Phosphocreatine / Q000378:metabolism; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D007773:Lactates; D017923:Sodium-Hydrogen Exchangers; D010725:Phosphocreatine; D012964:Sodium; D011188:Potassium