MECHANISMS OF PH(I) REGULATION STUDIED IN INDIVIDUAL NEURONS CULTUREDFROM MOUSE CEREBRAL-CORTEX

Maintenance and regulation of intracellular pH (pH(i)) was studied in single cultured mouse neocortical neurons using the fluorescent probe 2',7'-bis-(2-carboxyethyl)-5,6-carboxyfluorescein (BCECP). Reversal of the Na+ gradient by reduction of the extracellular Na+ concentration ([Na+](o)) resulted in rapid intracellular acidification, inhibited by 5'-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of Na+/H+ exch ange, In the presence of EIPA and/or 4',4'-diisothiocyano-stilbene-2', 2'-sulfonic acid (DIDS), an inhibitor of Na+-coupled anion exchangers and Na+-HCO3- cotransport, a slow decline of pH(i) was seen, Following intracellular acidification imposed by an NH4Cl prepulse, pH(i) recov ered at a rapid rate, which was reduced by reduction of [Na+](o) and w as virtually abolished by EIPA and DIDS in combination, Creating an ou tward Cl- gradient by removal of extracellular Cl- significantly incre ased the rate of pH(i) recovery, In HCO3--free media, the pH(i) recove ry rate was reduced in control cells and was abolished at zero [Na+](o ) and by EIPA, After intracellular alkalinization imposed by an acetat e prepulse, pH(i) recovery was unaffected by DIDS but was significantl y reduced in the absence of extracellular Cl-, as well as in the prese nce of Zn2+, which is a blocker of proton channels, Together, this poi nts toward a combined role of DIDS-insensitive Cl-/HCO3- and passive H + influx in the recovery of pH(i) after alkalinization. (C) 1998 Wiley -Liss, Inc.