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Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models.

Wen L, Voronina S, Javed MA, Awais M, Szatmary P, Latawiec D, Chvanov M, Collier D, Huang W, Barrett J, Begg M, Stauderman K, Roos J, Grigoryev S, Ramos S, Rogers E, Whitten J, Velicelebi G, Dunn M, Tepikin AV, Criddle DN, Sutton R - Gastroenterology (2015)

Bottom Line: The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis.ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

View Article: PubMed Central - PubMed

Affiliation: Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom; Department of Integrated Traditional and Western Medicine, Sichuan Provincial Pancreatitis Centre, West China Hospital, Sichuan University, Chengdu, People's Republic of China.

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CM_128 concentration-dependently inhibits CRAC entry and necrosis (PI uptake). (A) Changes in mouse pancreatic acinar [Ca2+]C induced by thapsigargin (Fura-2 340:380 normalized at 2000 s), showing effect of 1 μmol/L CM_128. (B) Mean (±SEM) [Ca2+]C at 2000 and 3000 s from panel A, showing a marked reduction with 1 μmol/L CM_128 (≥62 cells/group; *P < .001, thapsigargin vs thapsigargin plus CM_128 at 3000 s). (C) CM_128 protected isolated murine pancreatic acinar cells from necrotic cell death pathway activation induced by TLCS (500 μmol/L) (mean ± SEM; ≥3 experiments/group; *P < .001, TLCS vs control; †P < .05, TLCS vs TLCS plus CM_128). (D) Concentration-dependent inhibitory effects of CM_128 on cyclopiazonic acid–induced Ca2+ influx, showing a progressive reduction of the initial rate of Ca2+ entry and plateau with increasing CM_128, with complete inhibition of Ca2+ entry at 10 μmol/L (≥17 cells/group). (E) Concentration-dependent inhibitory effects of CM_128 on the initial rate of Ca2+ entry.
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fig3: CM_128 concentration-dependently inhibits CRAC entry and necrosis (PI uptake). (A) Changes in mouse pancreatic acinar [Ca2+]C induced by thapsigargin (Fura-2 340:380 normalized at 2000 s), showing effect of 1 μmol/L CM_128. (B) Mean (±SEM) [Ca2+]C at 2000 and 3000 s from panel A, showing a marked reduction with 1 μmol/L CM_128 (≥62 cells/group; *P < .001, thapsigargin vs thapsigargin plus CM_128 at 3000 s). (C) CM_128 protected isolated murine pancreatic acinar cells from necrotic cell death pathway activation induced by TLCS (500 μmol/L) (mean ± SEM; ≥3 experiments/group; *P < .001, TLCS vs control; †P < .05, TLCS vs TLCS plus CM_128). (D) Concentration-dependent inhibitory effects of CM_128 on cyclopiazonic acid–induced Ca2+ influx, showing a progressive reduction of the initial rate of Ca2+ entry and plateau with increasing CM_128, with complete inhibition of Ca2+ entry at 10 μmol/L (≥17 cells/group). (E) Concentration-dependent inhibitory effects of CM_128 on the initial rate of Ca2+ entry.

Mentions: To determine the effect of CM_128 on SOCE into isolated murine pancreatic acinar cells, thapsigargin was used to empty Ca2+ stores and initiate STIM-mediated ORAI pore formation, while maintaining cells in zero external Ca2+ until Ca2+ was re-introduced to enable SOCE.1,3 Application of this protocol showed that CM_128 reduced SOCE markedly, at a lower dose than that of GSK-7975A (1 μmol/L) (Figure 3A and B); this same dose also was effective in significantly reducing necrotic cell death pathway activation by TLCS in these cells (Figure 3C). To confirm the effect of CM_128 on SOCE and to determine dose-dependency, cyclopiazonic acid was used to empty Ca2+ stores within murine pancreatic acinar cells10 (maintained in zero external Ca2+) to stimulate STIM-mediated ORAI opening. Upon reintroduction of external Ca2+ (1.8 mmol/L), the rate of Ca2+ entry showed concentration-dependent log proportionality, with the IC50 at approximately 0.7 μmol/L and no loss of effect at high concentrations (10 μmol/L) (Figure 3D and E).


Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models.

Wen L, Voronina S, Javed MA, Awais M, Szatmary P, Latawiec D, Chvanov M, Collier D, Huang W, Barrett J, Begg M, Stauderman K, Roos J, Grigoryev S, Ramos S, Rogers E, Whitten J, Velicelebi G, Dunn M, Tepikin AV, Criddle DN, Sutton R - Gastroenterology (2015)

CM_128 concentration-dependently inhibits CRAC entry and necrosis (PI uptake). (A) Changes in mouse pancreatic acinar [Ca2+]C induced by thapsigargin (Fura-2 340:380 normalized at 2000 s), showing effect of 1 μmol/L CM_128. (B) Mean (±SEM) [Ca2+]C at 2000 and 3000 s from panel A, showing a marked reduction with 1 μmol/L CM_128 (≥62 cells/group; *P < .001, thapsigargin vs thapsigargin plus CM_128 at 3000 s). (C) CM_128 protected isolated murine pancreatic acinar cells from necrotic cell death pathway activation induced by TLCS (500 μmol/L) (mean ± SEM; ≥3 experiments/group; *P < .001, TLCS vs control; †P < .05, TLCS vs TLCS plus CM_128). (D) Concentration-dependent inhibitory effects of CM_128 on cyclopiazonic acid–induced Ca2+ influx, showing a progressive reduction of the initial rate of Ca2+ entry and plateau with increasing CM_128, with complete inhibition of Ca2+ entry at 10 μmol/L (≥17 cells/group). (E) Concentration-dependent inhibitory effects of CM_128 on the initial rate of Ca2+ entry.
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Related In: Results  -  Collection

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fig3: CM_128 concentration-dependently inhibits CRAC entry and necrosis (PI uptake). (A) Changes in mouse pancreatic acinar [Ca2+]C induced by thapsigargin (Fura-2 340:380 normalized at 2000 s), showing effect of 1 μmol/L CM_128. (B) Mean (±SEM) [Ca2+]C at 2000 and 3000 s from panel A, showing a marked reduction with 1 μmol/L CM_128 (≥62 cells/group; *P < .001, thapsigargin vs thapsigargin plus CM_128 at 3000 s). (C) CM_128 protected isolated murine pancreatic acinar cells from necrotic cell death pathway activation induced by TLCS (500 μmol/L) (mean ± SEM; ≥3 experiments/group; *P < .001, TLCS vs control; †P < .05, TLCS vs TLCS plus CM_128). (D) Concentration-dependent inhibitory effects of CM_128 on cyclopiazonic acid–induced Ca2+ influx, showing a progressive reduction of the initial rate of Ca2+ entry and plateau with increasing CM_128, with complete inhibition of Ca2+ entry at 10 μmol/L (≥17 cells/group). (E) Concentration-dependent inhibitory effects of CM_128 on the initial rate of Ca2+ entry.
Mentions: To determine the effect of CM_128 on SOCE into isolated murine pancreatic acinar cells, thapsigargin was used to empty Ca2+ stores and initiate STIM-mediated ORAI pore formation, while maintaining cells in zero external Ca2+ until Ca2+ was re-introduced to enable SOCE.1,3 Application of this protocol showed that CM_128 reduced SOCE markedly, at a lower dose than that of GSK-7975A (1 μmol/L) (Figure 3A and B); this same dose also was effective in significantly reducing necrotic cell death pathway activation by TLCS in these cells (Figure 3C). To confirm the effect of CM_128 on SOCE and to determine dose-dependency, cyclopiazonic acid was used to empty Ca2+ stores within murine pancreatic acinar cells10 (maintained in zero external Ca2+) to stimulate STIM-mediated ORAI opening. Upon reintroduction of external Ca2+ (1.8 mmol/L), the rate of Ca2+ entry showed concentration-dependent log proportionality, with the IC50 at approximately 0.7 μmol/L and no loss of effect at high concentrations (10 μmol/L) (Figure 3D and E).

Bottom Line: The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.Cytosolic calcium overload, mediated via ORAI1, contributes to the pathogenesis of acute pancreatitis.ORAI1 inhibitors might be developed for the treatment of patients with pancreatitis.

View Article: PubMed Central - PubMed

Affiliation: Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom; Department of Integrated Traditional and Western Medicine, Sichuan Provincial Pancreatitis Centre, West China Hospital, Sichuan University, Chengdu, People's Republic of China.

Show MeSH
Related in: MedlinePlus