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 degrees 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 approximately 0.1 higher than the one prevailing before the application of NH4+. In other experiments conducted on cell 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(-)-dependent acid-extrusion mechanism; the final pHi after returning Na+ was approximately 0.08 higher than the initial value. This pHi recovery elicited by returning Na+ was not substantially affected by 50 microM ethylisopropylamiloride (EIPA), but was speeded up by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS).(ABSTRACT TRUNCATED AT 250 WORDS)
Boyarsky
G
G
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
Ransom
B
B
Schlue
W R
WR
Davis
M B
MB
Boron
W F
WF
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000641:Ammonia / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn / Q000502:physiology; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D001253:Astrocytes / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D002478:Cells, Cultured; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D011188:Potassium / Q000378:metabolism; D016548:Prosencephalon / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D012964:Sodium / Q000378:metabolism; D017923:Sodium-Hydrogen Exchangers; D012996:Solutions
D000889:Anti-Arrhythmia Agents; D001639:Bicarbonates; D005456:Fluorescent Dyes; D017923:Sodium-Hydrogen Exchangers; D012996:Solutions; D000641:Ammonia; D000584:Amiloride; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D011188:Potassium; C039614:ethylisopropylamiloride