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We used the fluorescent pH‐sensitive dye 2′,7′‐bis(carboxyethyl)‐5,6‐carboxyfluorescein (BCECF) to monitor intracellular pH (pHi) 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°C, the cells had a mean pHi of 6.89. Switching to a medium buffered to pH 7.40 with 5% CO2 and 25 mM HCO3− caused the steady‐state pHi 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 pHi to fall to a value substantially below the initial one. pHi 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 pHi to decrease rapidly by 0.5. Returning the Na+ caused pHi to increase rapidly, indicating the presence of an Na+‐dependent/HCO3−‐independent acid‐extrusion mechanism; the final pHi after returning Na+ was ∼ 0.08 higher than the initial value. This pHi 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 pHi to increase reversibly by ∼ 0.2 in nominally CO2/HCO3−‐free solutions, and by ∼ 0.1 in CO2/HCO3−‐containing solutions, although the initial pHi was ∼ 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 pHi is sensitive to changes in either membrane voltage or (K+)0 per se. © 1993 Wiley‐Liss, Inc.
Glia – Wiley
Published: Jan 1, 1993
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