Intracellular pH regulation in single cultured astrocytes from rat forebrain

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Abstract
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.