Studies on pH regulatory mechanisms in cultured astrocytes of DBA and C57 mice.
pH regulatory mechanisms in primary cultures of astrocytes from the cerebral cortex of neonatal audiogenic-seizure-susceptible DBA/2J (DBA) and genetically controlled C57BL/6J (C57) mice were studied with [14C]dimethyloxazolidine-2-4-dione (DMO) and [3H]-methyl-D-glucose (MDG). Effects of changing the concentration of Na+, K+, HCO3- or Cl- in medium, and/or of different transport blockers and metabolite inhibitor on intracellular pH (pHi) of cultured astrocytes were also studied. In nominal HCO3(-)-free HEPES-buffered Hanks' balanced salt solution (HEPES HBSS), when the pH of medium (pHo) was maintained at 7.4, the steady-state pHi of cultured astrocytes from DBA mice was 6.98 +/- 0.03, and that from C57 mice was 7.01 +/- 0.03. When the cells were incubated in HBSS containing 25 mM HCO3- and equilibrated with 5% CO2 (HCO3- HBSS, pHo = 7.4), pHi of both DBA and C57 astrocytes was approximately 0.1-0.15 pH units higher than that in HEPES HBSS. Reducing the pH or the Na+ concentration in media (pHo, [Na+]o) of either HEPES HBSS or HCO3- HBSS, pHi of both DBA and C57 astrocytes decreased markedly (0.25-0.45 pH units lower than the controls). The decrease in pHi was greater in HEPES HBSS than in HCO3- HBSS. Reducing the Cl- concentration ([Cl-]o) in either HEPES or HCO3- HBSS, pHi of astrocytes increased by 0.05-0.1 pH units. Increasing the K+ concentration ([K+]o) of or adding Ba2+ to the media increased the pHi of both DBA and C57 astrocytes accordingly. SITS, an anion transport inhibitor, decreased the pHi of both DBA and C57 astrocytes in HCO3- HBSS but not in HEPES HBSS. It enhanced the response of pHi to reduction in pHo. Amiloride, a Na(+)-H+ exchange inhibitor, decreased the pHi of both DBA and C57 astrocytes more in HEPES HBSS than in HCO3- HBSS. It enhanced the response of pHi to reduction in pHo and [Na+]o. Ouabain, an Na+,K(+)-ATPase inhibitor, decreased the pHi of cultured astrocytes in HEPES HBSS, but not in HCO3- HBSS. It also enhanced the response of pHi to changing pHo and [Na+]o in HEPES HBSS. Acetazolamide, a carbonic anhydrase inhibitor, decreased the pHi of astrocytes in both HEPES and HCO3- HBSS. Both bumetanide, an Na+,K+/Cl- cotransport blocker, and KCN, a metabolic inhibitor, produced no significant effect on the steady-state pHi or the response of pHi to changing ionic concentration in media in both DBA and C57 astrocytes.
Chow
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Department of Physiology, University of Utah School of Medicine, Salt Lake City 84108.
Yen-Chow
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YC
Woodbury
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D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000086:Acetazolamide / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000378:metabolism; D002712:Chlorides / Q000378:metabolism; D003470:Culture Media; D004114:Dimethadione / Q000494:pharmacology; D004195:Disease Models, Animal; D006531:HEPES; D006863:Hydrogen-Ion Concentration; D008759:Methylglycosides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL / Q000235:genetics; D008811:Mice, Inbred DBA / Q000235:genetics; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D011190:Potassium Cyanide / Q000494:pharmacology; D012640:Seizures / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism
D001639:Bicarbonates; D002712:Chlorides; D003470:Culture Media; D008759:Methylglycosides; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D010042:Ouabain; D000584:Amiloride; D012964:Sodium; D004114:Dimethadione; D011190:Potassium Cyanide; D000086:Acetazolamide; D011188:Potassium; D006531:HEPES