We used the fluorescent pH-sensitive dye 2',7 '-bis(carboxyethyl)-5,6carboxyfluorescein (BCECF) to monitor intracellular pH (pH,) in single astrocytes cultured from the forebrain of neonatal rats. When exposed to a nominally COz/HC03--free medium buffered to pH 7.40 with HEPES at 37"C, the cells had a mean pH, of 6.89.
Switching to a medium buffered to pH 7.40 with 5% COz and 25 mM HC03-caused the steady-state pH, to increase by an average of 0.35, suggesting the presence of a HC0,-dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HC0,--free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH4+ caused pH, to fall to a value substantially below the initial one. pH, spontaneously recovered from this acid load, stabilizing at a value -0.1 higher than the one prevailing before the application of NH4+. In other experiments conducted on cells bathed in HEPES-buffered solutions, removing extracellular Na+ caused pH, to decrease rapidly by 0.5. Returning the Na+ caused pH, to increase rapidly, indicating the presence of an Na+-dependent/HCO,--independent acid-extrusion mechanism; the final pH, after returning Na+ was -0.08 higher than the initial value. This pH, recovery elicited by returning Na+ was not substantially affected by 50 pM ethylisopropylamiloride (EIPA), but was speeded up by 50 pM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). Increasing [K+l, from 5 to 25 mM caused pH, to increase reversibly by -0.2 in nominally C02/HC03--free solutions, and by -0.1 in CO,/HCO,--containing solutions, although the initial pH, was -0.17 higher in the presence of CO,/HCO,-. These results suggest the presence of a depolarization-induced alkalinization. Our results suggest the presence of both HC03dependent and -independent acid-base transport systems in cultured mammalian astrocytes, and indicate that astrocyte pH, is sensitive to changes in either membrane voltage or [K' ], per se. Q 1993 Wiley-Liss, Inc.