EVIDENCE FOR ELECTROGENIC SODIUM-BICARBONATE COTRANSPORT IN CULTURED RAT CEREBELLAR ASTROCYTES

We have studied the regulation of intracellular pH (pH(i)), 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 pH(i) 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 pH(i) 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 pH(i), and addition of CO2/HCO3- in Na+-free saline evoked a further fall of pH(i), while the outward current was reduced or even reversed. The stilbene 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS, 0.3 mM) reduced the pH(i) recovery from the CO2/HCO3--evoked acidification ion, and blocked the prominent intracellular acidification upon removal of CO2/HCO3-. Removal of external Cl- had little effect on these pH(i) 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 cut-rent is partly due to a reversible bidirectional, electrogenic Na+-HCO3- cotransporter, which helps to regulate pH(i) in these cells. In addition, a prominent Na+/H+ exchanger contributes to extrude acid equivalents from these astrocytes to maintain the steady-state pH(i).