Studies on pH Regulatory Mechanisms in Cultured Astrocytes of DBA and C57 Mice

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Abstract
Summary: 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 ∼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 pH, 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 pHi. 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 pH, or the response of pHi to changing ionic concentration in media in both DBA and C57 astrocytes.