Intracellular pH regulation in single cultured astrocytes from rat forebrain

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 (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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glia Wiley

Intracellular pH regulation in single cultured astrocytes from rat forebrain

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Publisher
Wiley
Copyright
Copyright © 1993 Wiley‐Liss, Inc.
ISSN
0894-1491
eISSN
1098-1136
DOI
10.1002/glia.440080404
pmid
8406681
Publisher site
See Article on Publisher Site

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. © 1993 Wiley‐Liss, Inc.

Journal

GliaWiley

Published: Jan 1, 1993

References

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