INTRACELLULAR PH REGULATION IN SINGLE CULTURED ASTROCYTES FROM RAT FOREBRAIN

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 CO2/HCO3--free medium buffered to pH 7.40 with HEPES at 37-degrees-C, the cells had a mean pH(i) of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO2 and 25 mM HCO:, caused the steady-state pH(i) to increase by an average of 0.35, suggesting the presence of a HCO3--dependent acid-extrusion mechanism. The sustained alkalinization was sometimes preceded by a small transient acidification. In experiments in which astrocytes were exposed to nominally HCO3--free (HEPES-buffered) solutions, the application and withdrawal of 20 mM extracellular NH4+ 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 approximately 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(i) to decrease rapidly by 0.5. Returning the Na+ caused pH(i) to increase rapidly, indicating the presence of an Na+-dependent/HCO3--independent acid-extrusion mechanism; the final pH(i) after returning Na+ was approximately 0.08 higher than the initial value. This pH(i) recovery elicited by returning Na+ was not substantially affected by 50 muM ethylisopropylamiloride (EIPA), but was speeded up by 50 muM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS). Increasing [K+]o from 5 to 25 mM caused pH(i) to increase reversibly by approximately 0.2 in nominally CO2/HCO3--free solutions, and by approximately 0.1 in CO2/HCO3--containing solutions, although the initial pH(i) was approximately 0.17 higher in the presence of CO2/HCO3-. These results suggest the presence of a depolarization-induced alkalinization. Our results suggest the presence of both HCO3- 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+]o per se. (C) 1993 Wiley-Liss, Inc.