REGULATION OF INTRACELLULAR PH IN SINGLE-RAT CORTICAL-NEURONS IN-VITRO - A MICROSPECTROFLUOROMETRIC STUDY

Intracellular pH (pH(i)) and the mechanisms of pH(i) regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pH(i) was 7.00 +/- 0.17 (mean +/- SD) and 7.09 +/- 0.14 in nominally HCO3--free and HCO3--containing solutions, respectively, and was dependent on extracellular Na+ and Cl-. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tension, pH(i) decreased and then rapidly returned to baseline, with an average net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally HCO3--free and HCO3--containing solutions, respectively. The recovery was completely blocked by removal of extracellular Na+ and was partially inhibited by amiloride or 5-N-methyl-N-isobutylamiloride. In most cells pH(i) recovery was completely blocked in the presence of harmaline. The recovery of pH(i) was not influenced by addition of 4,4-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or removal of Cl-. The rapid regulation of pH(i) seen following a transient alkalinization was not inhibited by amiloride or by removal of extracellular Na+, but was partially inhibited by DIDS and by removal of extracellular Cl-. The results are compatible with the presence of at least two different pH(i)-regulating mechanisms: an acid-extruding Na+/H+ antiporter, possibly consisting of different subtypes, and a passive Cl-/HCO3 exchanger, mediating loss of HCO3- from the cell.