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

Abstract

We have studied the regulation of intracellular pH (pH_i), and HCO ⁻₃ -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/HCO ⁻₃ -free, HEPES-buffered saline (6.96 ±0.14;n=48) and in a saline containing 5% CO₂/24 mM HCO ⁻₃ (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/HCO ⁻₃ -free saline. Addition of CO₂/HCO ⁻₃ 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/HCO ⁻₃ 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/HCO ⁻₃ -evoked acidification, and blocked the prominent intracellular acidification upon removal of CO₂/HCO ⁻₃ . Removal of external Cl⁻ had little effect on these pH_i changes. Lowering the external pH from 7.4 to 6.6 in CO₂/HCO ⁻₃ -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/HCO ⁻₃ . The results suggest that the CO₂/HCO ⁻₃ -dependent current is partly due to a reversible bidirectional, electrogenic Na⁺-HCO ⁻₃ 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 steadystate pH_i.