Voltage-dependent Na(+)-HCO3- cotransporter and Na+/H+ exchanger are involved in intracellular pH regulation of cultured mature rat cerebellar oligodendrocytes

Abstract

Intracellular pH (pHi) was measured at 37 degrees C in mature rat cerebellar oligodendrocytes dissociated in culture by using the pH-sensitive probe BCECF. Cells were identified by anti-galactocerebroside antibody. The mean steady-state pHi was 7.02 in the absence of CO2/bicarbonate (Hepes-buffered solution) at an external pH of 7.40 and 7.04 in 5% CO2/25 mM bicarbonate-buffered solution at the same external pH; this value was modified neither by the removal of external chloride nor by the addition of the chloride-coupled transport blocker DIDS. In both external solutions steady-state pHi values were strongly dependent on external pH. In Hepes-buffered solution pHi recovery following an acid load required external Na+ and was completely inhibited by amiloride, indicating the presence of a Na+/H+ exchanger. In CO2/bicarbonate-buffered solution amiloride partially reduced the pHi recovery rate, indicating the presence of a bicarbonate-dependent pHi regulating mechanism. Membrane depolarization induced by increasing external K+ concentration elicited an alkalinization only in the presence of external Na+ and bicarbonate. Analysis of the calculated HCO3- fluxes with respect to membrane potential indicated that these fluxes were mediated by a Na(+)-HCO3- cotransport with a stoichiometry of 1:3. These results demonstrate that a Na+/H+ exchanger and a Na(+)-HCO3- cotransporter are involved in pHi regulation of mature oligodendrocytes.