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 cul tured rat cortical neurons were studied with microspectrofluorometry a nd the pH-sensitive fluorophore 2',7'-bis(carboxyethyl)-5,6-carboxyflu orescein. Steady-state pH(i) was 7.00 +/- 0.17 (mean +/- SD) and 7.09 +/- 0.14 in nominally HCO3--free and HCO3--containing solutions, respe ctively, and was dependent on extracellular Na+ and Cl-. Following an acid transient, induced by an NH1 prepulse or an increase in CO2 tensi on, pH(i) decreased and then rapidly returned to baseline, with an ave rage net acid extrusion rate of 2.6 and 2.8 mmol/L/min, in nominally H CO3--free and HCO3--containing solutions, respectively. The recovery w as completely blocked by removal of extracellular Na+ and was partiall y inhibited by amiloride or 5-N-methyl-N-isobutylamiloride. In most ce lls pH(i) recovery was completely blocked in the presence of harmaline . The recovery of pH(i) was not influenced by addition of 4,4-diisothi ocyanatostilbene-2,2'-disulfonic acid (DIDS) or removal of Cl-. The ra pid 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, possibl y consisting of different subtypes, and a passive Cl-/HCO3 exchanger, mediating loss of HCO3- from the cell.