Intracellular alkalinization induces cytosolic Ca2+ increases by inhibiting sarco/endoplasmic reticulum Ca2+-ATPase (SERCA)

Abstract

Intracellular pH (pHi) and Ca(2+) regulate essentially all aspects of cellular activities. Their inter-relationship has not been mechanistically explored. In this study, we used bases and acetic acid to manipulate the pHi. We found that transient pHi rise induced by both organic and inorganic bases, but not acidification induced by acid, produced elevation of cytosolic Ca(2+). The sources of the Ca(2+) increase are from the endoplasmic reticulum (ER) Ca(2+) pools as well as from Ca(2+) influx. The store-mobilization component of the Ca(2+) increase induced by the pHi rise was not sensitive to antagonists for either IP(3)-receptors or ryanodine receptors, but was due to inhibition of the sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA), leading to depletion of the ER Ca(2+) store. We further showed that the physiological consequence of depletion of the ER Ca(2+) store by pHi rise is the activation of store-operated channels (SOCs) of Orai1 and Stim1, leading to increased Ca(2+) influx. Taken together, our results indicate that intracellular alkalinization inhibits SERCA activity, similar to thapsigargin, thereby resulting in Ca(2+) leak from ER pools followed by Ca(2+) influx via SOCs.

Figure 1. Intracellular alkalinization induces cytosolic Ca²⁺ increases in HeLa cells.

(A) DIEA.HBr, similar to NH₄Cl, induced cytosolic Ca²⁺ increases in a dose-dependent manner in HeLa cells as measured by the Ca²⁺-indicator, Fura-2 AM. (B) Intracellular alkalinization induced by DIEA.HBr (10 mM) and NH₄Cl (10 mM) were inhibited by sodium acetate (40 mM) as measured by the pH-indicator, BCECF AM. (C) Cytosolic Ca²⁺ increases induced by DIEA.HBr (10 mM) and NH₄Cl (10 mM) were markedly inhibited by sodium acetate (40 mM). The graphs represent data from three independent experiments. Data quantifications of the time to reach pH_i peak (B) or [Ca²⁺]_i peak (C) after drug treatment were expressed as mean ± S.E., n = 30–50 cells.

Figure 2. Intracellular alkalinization releases Ca²⁺ from ER pools in HeLa cells and PC12 cells.

(A) DIEA.HBr (4 mM)-induced Ca²⁺ increase in Fura-2 loaded HeLa cells was abolished by thapsigargin (1 µM) pretreatment. This Ca²⁺ increase was inhibited by removal of external Ca²⁺ (Ca²⁺-free HBSS with 4 mM EGTA). (B) Pretreatment of Fura-2 loaded HeLa cells with either Xestospongin C (XeC) (10 µM) or U73122 (10 µM) did not inhibit the DIEA.HBr-induced Ca²⁺ increase compared with untreated cells. The graphs represent data from three independent experiments. (C) Pretreatment of Fura-2 loaded PC12 cells with ryanodine (20 µM) or 8-Br-cADPR (100 µM) did not inhibit the DIEA.HBr-induced Ca²⁺ increase compared with untreated cells. The graphs represent data from three independent experiments. (D) Pretreatment of Fura-2 loaded HeLa cells with glycyl-l-phenylalanine 2-naphthylamide (GPN) (50 µM) did not inhibit the DIEA.HBr-induced Ca²⁺ increase compared with untreated cells while completely blocked GPN or bafilomycin A1 (0.5 µM)-induced Ca²⁺ increase. The graphs represent data from three independent experiments.

Figure 3. 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.

Figure 4. Intracellular alkalinization inhibits ER Ca²⁺ store filling after histamine and ATP treatment in HeLa cells.

(A) Thapsigargin (10 µM) or DIEA.HBr (4 mM) inhibited ER Ca²⁺ refilling after histamine (10 µM) treatment in Fura-2 loaded HeLa cells. (B) Thapsigargin (10 µM) or DIEA.HBr (4 mM) diminished ATP (100 µM)-induced Ca²⁺ oscillations in Fura-2 loaded HeLa cells. The graphs represent data from three independent experiments, and data quantification are presented as mean ± S.E., n = 9–36 cells. The * symbols indicate the results of t Test analysis, p<0.05.

Figure 5. Intracellular alkalinization induces extracellular Ca²⁺ influx through SOCs in NIH 3T3 cells.

(A) DIEA.HBr (4 mM) induced-Ca²⁺ influx was inhibited by La³⁺ (100 µM), a SOC blocker, treatment in Fura-2 loaded NIH3T3 cells incubated in regular HBSS. (B) Immunoblot analysis of Stim1-knockdown in NIH3T3 cells. MEK1 immunoblot was used as the internal control. (C) Quantitative real-time RT-PCR analysis of Orai1-knockdown in NIH3T3 cells. GAPDH was used as the internal control. Data are expressed as means ± S.D., n = 3. (D) and (E) Stim1 or Orai1 knockdown abolished the sustained Ca²⁺ influx induced by thapsigargin (10 µM) (D) and by DIEA.HBr (4 mM) (E) in Fura-2 loaded NIH3T3 cells incubated in regular HBSS. (F) Stim1 and Orai1 knockdown diminished the Ca²⁺ influx induced by DIEA.HBr (4 mM) in Fura-2 loaded NIH3T3 cells. Cells were initially treated with thapsigargin (1 µM) in Ca²⁺-free HBSS to deplete ER Ca²⁺ pool, followed by 2 mM Ca²⁺ addition. All graphs represent data from three independent experiments. Data quantification in (A), (D), (E), and (F) are presented as mean ± S.E., n = 30–50 cells. The * symbols indicate the results of t Test analysis, p<0.05.

Figure 6. Intracellular alkalinization induces Stim1 and Orai1 colocalization in HeLa cells.

HeLa cells, co-transfected with plasmids, Stim1-mCherry and Orai1-EGFP, were incubated in Ca²⁺ free HBSS for 15 min as control or treated with thapsigargin (10 M) or DIEA.HBr (4 mM) for 15 min in Ca²⁺ free HBSS. Confocal imaging of both mCherry and EGFP were taken. The graphs represent data from three independent experiments. Scale bar: 5 µm.

Figure 7. Extracellular alkalinization induces cytosolic Ca²⁺ increases in μHeLa cells and NIH3T3 cells.

(A) Extracellular alkaline buffers induced pHi rise in HeLa cells as measured by the pH-indicator, BCECF AM. Data are expressed as means ± S.D., n = 3. (B) Extracellular alkaline buffers induced cytosolic Ca²⁺ rises in Fura-2 loaded HeLa cells. (C) Extracellular alkaline buffer-induced Ca²⁺ increase in Fura-2 loaded HeLa cells was abolished by thapsigargin (1 µM) pretreatment or was inhibited by removal of external Ca²⁺ (Ca²⁺-free HBSS with 4 mM EGTA). (D) ER Ca²⁺ concentration, indicated by the thapsigargin (10 µM)-induced Ca²⁺ increases, was reduced by extracellular alkaline buffers in HeLa cells. (E) Stim1 or Orai1 knockdown inhibited external Ca²⁺ influx induced by extracellular alkaline buffer in NIH3T3 cells. Cells were initially incubated in Ca²⁺-free HBSS with pH adjusted as indicated, followed by 2 mM Ca²⁺ addition. All graphs represent data from three independent experiments. Data quantification in (B), (C), (D), and (E) are presented as mean ± S.E., n = 30–50 cells. The * symbols indicate the results of t Test analysis, p<0.05.