REGULATION OF INTRACELLULAR PH IN GUINEA-PIG CEREBRAL-CORTEX EX-VIVO STUDIED BY P-31 AND H-1 NUCLEAR-MAGNETIC-RESONANCE SPECTROSCOPY - ROLE OF EXTRACELLULAR BICARBONATE AND CHLORIDE

The role of transmembrane processes that are dependent on external anions in the regulation of cerebral intracellular pH (pH(i)), high-energy metabolites, and lactate was investigated using P-31 and H-1 NMR spectroscopy in an ex vivo brain slice preparation. During oxygenated superfusion, removal of external HCO3-/CO2 in the presence of Na+ led to a sustained split of the inorganic phosphate (P-i) peak so that the pH(l) indicated by one part of the peak was 0.38 pH units more alkaline and by the other part 0.10 pH units more acidic at 5 min than in the presence of HCO3-. The pH in the compartment with a higher pH(l) value returned to 7.29 +/- 0.04 by 10.5 min of superfusion in a HCO3--free medium, whereas the pH(l) in an acidic compartment was reduced to 7.02. In the presence of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid or the absence of external Cl-, removal of HCO3- caused alkalinization without split of the P-l peak. Both treatments reduced the rate of pH(i) normalization following alkalinization. Simultaneous omission of external HCO3- and Na+ did not inhibit alkalinization of the pH(i) following CO2 exit. All these data show that the acid loading mechanism at neutral pH(l) is mediated by an Na+-independent anion transport. During severe hypoxia, pH(l) dropped from 7.29 +/- 0.05 to 6.13 +/- 0.16 and from 7.33 +/- 0.03 to 6.67 +/- 0.05 in the absence and presence of HCO3-, respectively, in Na+-containing medium. Lactate accumulated to 18.7 +/- 2.8 and 19.6 +/- 1.5 mmol/kg under the respective conditions. In the HCO3--free medium supplemented with 1 mM amiloride, the pH(l) fell only to 6.94 +/- 0.08 despite the lactate concentration of 18.9 +/- 2.4 mmol/kg. Acidification caused by hypoxia was also small in the slice preparations superfused in the absence of both HCO3- and Cl-, as the pH(i) was 7.01 +/- 0.12 at a lactate concentration of 24.5 +/- 2.4 mmol/kg. These data indicate that apart from anaerobic glucose metabolism, separate acidifying mechanisms are functioning during hypoxia under these conditions. Recovery of phosphocreatine levels following reoxygenation was >75% relative to the prehypoxic level in the slice preparations superfused in the absence of HCO3- but <47% in those preparations superfused without HCO3- and Cl-. This indicates that either neutral pH(i) or absence of Cl- during hypoxia was deleterious to the energy metabolism. The present data indicate that Cl-/HCO3-( )exchange mechanisms have distinct roles in cerebral H+ homeostasis depending on the level of pH(i) and energy state.