Intracellular pH regulation in single cultured astrocytes from rat forebrain

Abstract

Abstract We used the fluorescent pH‐sensitive dye 2′,7′‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) to monitor intracellular pH (pH i ) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally CO 2 /HCO 3 − ‐free medium buffered to pH 7.40 with HEPES at 37 ° C, the cells had a mean pH i of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO 2 and 25 mM HCO 3 − caused the steady‐state pH i to increase by an average of 0.35, suggesting the presence of a HCO 3 − ‐dependent acid‐extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO 3 − ‐free (HEPES‐buffered) solutions, the application and withdrawal of 20 mM extracellular NH 4 + caused pH i to fall to a value substantially below the initial one. pH i spontaneously recovered from this acid load, stabilizing at a value ∼ 0.1 higher than the one prevailing before the application of NH 4 + . In other experiments conducted on cells bathed in HEPES‐buffered solutions, removing extracellular Na + caused pH i to decrease rapidly by 0.5. Returning the Na + caused pH i to increase rapidly, indicating the presence of an Na + ‐dependent/HCO 3 − ‐independent acid‐extrusion mechanism; the final pH i after returning Na + was ∼ 0.08 higher than the initial value. This pH i recovery elicited by returning Na + was not substantially affected by 50 μM ethylisopropylamiloride (EIPA), but was speeded up by 50 μM 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate (DIDS). Increasing [K + ] − from 5 to 25 mM caused pH i to increase reversibly by ∼ 0.2 in nominally CO 2 /HCO 3 − ‐free solutions, and by ∼ 0.1 in CO 2 /HCO 3 − ‐containing solutions, although the initial pH i was ∼ 0.17 higher in the presence of CO 2 /HCO 3 ‐ . These results suggest the presence of a depolarization‐induced alkalinization. Our results suggest the presence of both HCO 3 − dependent and ‐independent acid‐base transport systems in cultured mammalian astrocytes, and indicate that astrocyte pH i is sensitive to changes in either membrane voltage or [K + ] 0 per se. © 1993 Wiley‐Liss, Inc.