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.