Intracellular pH regulation in single cultured astrocytes from rat forebrain

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 CO₂/HCO₃⁻ -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% CO₂ and 25 mM HCO₃⁻ 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 ∼ 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 ∼ 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 CO₂/HCO₃⁻-free solutions, and by ∼ 0.1 in CO₂/HCO₃⁻-containing solutions, although the initial pH_i 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 pH_i is sensitive to changes in either membrane voltage or [K⁺]₀ per se.