Evidence for electrogenic sodium-bicarbonate cotransport in cultured rat cerebellar astrocytes

We have studied the regulation of intracellular pH (pHi), and HCO3(-)-dependent membrane currents in cultured astrocytes from neonatal rat cerebellum, using the fluorescent pH-sensitive dye 2,7'-bis(carboxyethyl)-5,6-carboxyfluorescein (BCECF) and the whole-cell patch-clamp technique. The steady-state pHi was 6.96 in both nominally CO2/HCO3(-)-free, HEPES-buffered saline (6.96 +/- 0.14; n = 48) and in a saline containing 5% CO2/24 mM HCO3- (6.96 +/- 0.18; n = 48) (at pH 7.4). Inhibition of the Na+/H+ exchange by amiloride (2 mM) caused a significant decrease of pHi in nominally CO2/HCO3(-)-free saline. Addition of CO2/HCO3- in the continuous presence of amiloride induced a large and fast intracellular alkalinization. Removal of external Na+ also caused a fall of pHi, and addition of CO2/HCO3- in Na(+)-free saline evoked a further fall of pHi, while the outward current was reduced or even reversed. The stilbene 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3 mM) reduced the pHi recovery from the CO2/HCO3(-)-evoked acidification, and blocked the prominent intracellular acidification upon removal of CO2/HCO3-. Removal of external Cl- had little effect on these pHi changes. Lowering the external pH from 7.4 to 6.6 in CO2/HCO3(-)-containing saline produced a large and rapid intracellular acidification and inward current, which were both greatly reduced by DIDS and in the absence of CO2/HCO3-. The results suggest that the CO2/HCO3(-)-dependent current is partly due to a reversible bidirectional, electrogenic Na(+)-HCO3- cotransporter, which helps to regulate pHi in these cells. In addition, a prominent Na+/H+ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steady-state pHi.