FIGURE 5.

| Regulation of intracellular pH in isolated HAT-7 spheroids. (A–C) Recovery of pH_i from intracellular acidification evoked by a 3-min exposure to extracellular NH₄ ⁺ (20 mM) followed by substitution of extracellular Na⁺ with NMDG⁺ (0 Na ⁺): (A) in HEPES-buffered bath solution in the absence and presence of the Na⁺/H⁺ exchange inhibitor amiloride (0.3 mM); (B) in HCO₃ ^--buffered bath solution in the absence and presence of amiloride (0.3 mM); (C) in HCO₃ ^--buffered bath solution in the absence and presence of amiloride (0.3 mM) together with the Na⁺-HCO₃ ⁻ cotransport inhibitor H₂DIDS (0.5 mM). Dashed lines show the rate of recovery of pH_i when Na⁺ was restored to the perfusate. (D) Averaged data (mean ± SEM) from 4–6 experiments showing the inhibitory effects of amiloride and H₂DIDS on the rate of recovery of pH_i from acidification in the absence and presence of HCO₃ ⁻. Percentage inhibition was calculated with reference to the internal control in each experiment. ####p < 0.0001, ###p < 0.001 compared with control (one-sample t test). **p < 0.01, ***p < 0.001 (ANOVA and Tukey post-hoc test). (E) Changes in pH_i evoked by substitution of extracellular Cl⁻ with gluconate^- (0 Cl ⁻) in HCO₃ ⁻-buffered bath solution in the absence and presence of DIDS (0.1 mM). Dashed lines show the rate of recovery of pH_i when Na⁺ or Cl⁻ was restored to the perfusate. Measurements of BCECF fluorescence were pooled from 20–50 HAT-7 spheroids isolated from Matrigel matrix by treatment with 0.25% trypsin/EDTA. Traces are representative of 4–6 experiments.