Effects of acute hypoxia on intracellular-pH regulation in astrocytes cultured from rat hippocampus

We used the pH-sensitive dye BCECF to evaluate the effect of acute (5–10 min) hypoxia (∼ 3% O2) on the regulation of intracellular pH (pHi) in astrocyte populations cultured from rat hippocampus. For cells in the nominal absence of CO2/HCO3− at an extracellular pH of 7.40 (37 °C), acute hypoxia caused a small (0.05) decrease in steady-state pHi, but increased the pHi recovery rate from an acid load during all but the late phase of the pHi recovery. During such pHi recoveries, the total acid extrusion rate (φE, the product of dpHi/dt and proton buffering power) decreased with increasing pHi. Hypoxia alkali shifted the plot of φE vs. pHi; over the upper ∼ 85% of the φE range, this shift was 0.15–0.30. Hypoxia also stimulated the pHi recovery rate from an alkali load. Under normoxic conditions, switching the extracellular buffer to 5% CO2/22 mM HCO−3 also alkali shifted the φE–pHi plot (upper ~ 85%) by 0.4–0.5. Superimposing hypoxia on CO2/HCO−3 further alkali shifted the φE–pHi plot (upper ∼ 85% of the φE range) by 0.05–0.15. The SITS-insensitive component of φE was alkali shifted by 0.20–0.30, whereas the SITS-sensitive component of φE was depressed in the low pHi range. Thus, in the nominal absence of CO2/HCO3−, acute hypoxia has little effect on steady-state pHi but stimulates acid extrusion and acid loading, whereas in the presence of CO2/HCO−3, hypoxia stimulates the SITS-insensitive but inhibits the SITS-sensitive acid extrusion.