Intracellular pH regulation in primary rat astrocytes and C6 glioma cells.
We used the pH-sensitive fluorescent dye BCECF to study intracellular pH (pHi) regulation in primary cultures of rat astrocytes and C6 glioma cells. Both cell types contain three pH-regulating transporters: 1) alkalinizing Na+/H+ exchange; 2) alkalinizing Na+ + HCO3-/Cl- exchange; and 3) acidifying Cl-/HCO3- exchange. Na+/H+ exchange was most evident in the absence of CO2; recovery from acidification was Na+ dependent and amiloride sensitive. Exposure to CO2 caused a cell alkalinization that was inhibited by DIDS, dependent on external Na+, and inhibited 75% in the absence of Cl- (thus mediated by Na+ + HCO3-/Cl- exchange). When pHi was increased above the normal steady-state pHi, a DIDS-inhibitable and Na(+)-independent acidifying recovery was evident, indicating the presence of Cl-/HCO3- exchange. Astrocytes, but not C6 cells, contain a fourth pH-regulating transporter, Na(+)-HCO3- cotransport; in the presence of CO2, depolarization caused an alkalinization of 0.12 +/- 0.01 (n = 8) and increased the rate of CO2-induced alkalinization from 0.23 +/- 0.02 to 0.42 +/- 0.03 pH unit/min. Since C6 cells lack the Na(+)-HCO3- cotransporter, they are an inferior model of pHi regulation in glia. Our results differ from previous observations in glia in that: 1) Na+/H+ exchange was entirely inhibited by amiloride; 2) Na+ + HCO3-/Cl- exchange was present and largely responsible for CO2-induced alkalinization; 3) Cl-/HCO3- exchange was only active at pHi values above steady state; and 4) depolarization-induced alkalinization of astrocytes was seen only in the presence of CO2.
Shrode
L D
LD
Department of Physiology and Biophysics, Wright State University School of Medicine, Dayton, Ohio 45435.
Putnam
R W
RW
D000818:Animals; D017920:Antiporters / Q000378:metabolism; D001253:Astrocytes / Q000378:metabolism* / Q000502:physiology; D002245:Carbon Dioxide / Q000494:pharmacology; D002352:Carrier Proteins / Q000378:metabolism; D027963:Chloride-Bicarbonate Antiporters; D004594:Electrophysiology; D005910:Glioma / Q000378:metabolism* / Q000473:pathology / Q000503:physiopathology; D006706:Homeostasis; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000378:metabolism*; D011188:Potassium / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism; D014407:Tumor Cells, Cultured
D017920:Antiporters; D002352:Carrier Proteins; D027963:Chloride-Bicarbonate Antiporters; D027982:Sodium-Bicarbonate Symporters; D017923:Sodium-Hydrogen Exchangers; D002245:Carbon Dioxide; D011188:Potassium