Intracellular alkalinization inhibits ER SERCA activity in HeLa cells.

(A) Kinetics of cytosolic Ca²⁺ increases induced by histamine (10 µM), NH₄Cl (4 mM), DIEA.HBr (4 mM), and thapsigargin (1 µM) in Fura-2 loaded HeLa cells. Data quantifications of rise ratio (340/380 per seconds) after drug treatment were expressed as mean ± S.E., n = 30–50 cells. The * symbols indicate the results of t Test analysis, p<0.05, compared with cells treated with histamine. (B) Kinetics of pHi changes induced by DIEA.HBr (4 mM) and NH₄Cl (4 mM) in HeLa cells. Data quantifications of indicated pHi changes after drug treatment were expressed as mean ± S.D., p<0.05. (C) ER Ca²⁺ concentration, indicated by the thapsigargin (10 µM)-induced Ca²⁺ increase, was inhibited by pretreatment of Fura-2 loaded HeLa cells with DIEA.HBr (4 mM) or NH₄Cl (4 mM) for 7 min or 25 min, but was not affected by ATP (100 µM) or sodium acetate (4 mM) pretreatment. Quantifications of thapsigargin-induced Ca²⁺ peaks were expressed as mean ± S.E., n = 30–50 cells, p<0.05 (*) or p<0.01 (**). (D) Alkaline pH inhibited Ca²⁺ uptake capability, whereas thapsigargin (1 µM) abolished Ca²⁺ uptake in Fluo-3 loaded permeabilized HeLa cells. Quantifications of Fluo-3 fluorescence at 20 min after drug additions and the decay rate of Fluo-3 fluorescence were expressed as mean ± S.D., p<0.05. All graphs represent data from three independent experiments.