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

Abstract

We used the pH-sensitive dye BCECF to evaluate the effect of acute (5–10 min) hypoxia (∼ 3% O 2) on the regulation of intracellular pH (pH i) in astrocyte populations cultured from rat hippocampus. For cells in the nominal absence of CO 2/HCO 3 − at an extracellular pH of 7.40 (37 °C), acute hypoxia caused a small (0.05) decrease in steady-state pH i, but increased the pH i recovery rate from an acid load during all but the late phase of the pH i recovery. During such pH i recoveries, the total acid extrusion rate ( φ E, the product of dpH i/d t and proton buffering power) decreased with increasing pH i. Hypoxia alkali shifted the plot of φ E vs. pH i; over the upper ∼ 85% of the φ E range, this shift was 0.15–0.30. Hypoxia also stimulated the pH i recovery rate from an alkali load. Under normoxic conditions, switching the extracellular buffer to 5% CO 2/22 mM HCO − 3 also alkali shifted the φ E–pH i plot (upper ~ 85%) by 0.4–0.5. Superimposing hypoxia on CO 2/HCO − 3 further alkali shifted the φ E–pH i plot (upper ∼ 85% of the φ E range) by 0.05–0.15. The SITS-insensitive component of φ E was alkali shifted by 0.20–0.30, whereas the SITS-sensitive component of φ E was depressed in the low pH i range. Thus, in the nominal absence of CO 2/HCO 3 −, acute hypoxia has little effect on steady-state pH i but stimulates acid extrusion and acid loading, whereas in the presence of CO 2/HCO − 3, hypoxia stimulates the SITS-insensitive but inhibits the SITS-sensitive acid extrusion.