Figure 4.

Effect of cytosolic pH changes on pH_ER and on Golgi pH. (A) Cytosolic pH (pH_C), Golgi pH (pH_G) and pH_ER were measured in separate, parallel experiments by using BCECF, FITC-labeled VT1B and DTAF-conjugated B-Glyc-KDEL, respectively. Cells were Na⁺-loaded by preincubation in medium with 1 mM ouabain for 45 min. Recording was initiated upon transfer of the cells to a Na⁺-free medium, pH 5.6, intended to induce cytosolic acidification. (B) HeLa cells labeled with DTAF-conjugated B-Glyc-KDEL were permeabilized with streptolysin O and perfused sequentially with media of pH 6.0 or 7.0, as indicated, while recording pH_ER. Where noted, 10 μM heparin was added to the solutions. (Inset) effect of NH₄⁺ (40 mM) on pH_ER in permeabilized cells. (C) Intact cells labeled with DTAF-conjugated B-Glyc-KDEL were bathed in either Na⁺-rich or Na⁺-free, isoosmotic N-methyl-d-glucammonium (NMG⁺) solution, as indicated. Where specified, 40 mM NH₄Cl was added to the medium. Representative of six experiments.