Regulation of intracellular pH in single rat cortical neurons in vitro: a microspectrofluorometric study

Intracellular pH (pHi) and the mechanisms of pHi regulation in cultured rat cortical neurons were studied with microspectrofluorometry and the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5,6-carboxyfluorescein. Steady-state pHi 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, pHi 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 pHi recovery was completely blocked in the presence of harmaline. The recovery of pHi was not influenced by addition of 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) or removal of Cl-. The rapid regulation of pHi 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 pHi-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.