Inhibitors of ORAI1 Prevent Cytosolic Calcium-Associated Injury of Human Pancreatic Acinar Cells and Acute Pancreatitis in 3 Mouse Models.
OBJECTIVE
Sustained activation of the cytosolic calcium concentration induces injury to pancreatic acinar cells and necrosis. The calcium release-activated calcium modulator ORAI1 is the most abundant Ca(2+) entry channel in pancreatic acinar cells; it sustains calcium overload in mice exposed to toxins that induce pancreatitis. We investigated the roles of ORAI1 in pancreatic acinar cell injury and the development of acute pancreatitis in mice.


METHODS
Mouse and human acinar cells, as well as HEK 293 cells transfected to express human ORAI1 with human stromal interaction molecule 1, were hyperstimulated or incubated with human bile acid, thapsigargin, or cyclopiazonic acid to induce calcium entry. GSK-7975A or CM_128 were added to some cells, which were analyzed by confocal and video microscopy and patch clamp recordings. Acute pancreatitis was induced in C57BL/6J mice by ductal injection of taurolithocholic acid 3-sulfate or intravenous' administration of cerulein or ethanol and palmitoleic acid. Some mice then were given GSK-7975A or CM_128, which inhibit ORAI1, at different time points to assess local and systemic effects.


RESULTS
GSK-7975A and CM_128 each separately inhibited toxin-induced activation of ORAI1 and/or activation of Ca(2+) currents after Ca(2+) release, in a concentration-dependent manner, in mouse and human pancreatic acinar cells (inhibition >90% of the levels observed in control cells). The ORAI1 inhibitors also prevented activation of the necrotic cell death pathway in mouse and human pancreatic acinar cells. GSK-7975A and CM_128 each inhibited all local and systemic features of acute pancreatitis in all 3 models, in dose- and time-dependent manners. The agents were significantly more effective, in a range of parameters, when given at 1 vs 6 hours after induction of pancreatitis.


CONCLUSIONS
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.
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.
Li
L
Wen
Department of Molecular and Cellular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
Svetlana
S
Voronina
Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom; Department of Molecular and Cellular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
Muhammad A
MA
Javed
Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom.
Muhammad
M
Awais
Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom; Department of Molecular and Cellular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
Peter
P
Szatmary
Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom.
Diane
D
Latawiec
Department of Molecular and Cellular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
Michael
M
Chvanov
Department of Molecular and Cellular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
David
D
Collier
Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom.
Wei
W
Huang
Respiratory Therapy Area Unit, Medicines Research Centre, GlaxoSmithKline, Stevenage, United Kingdom.
John
J
Barrett
Respiratory Therapy Area Unit, Medicines Research Centre, GlaxoSmithKline, Stevenage, United Kingdom.
Malcolm
M
Begg
CalciMedica, La Jolla, California.
Ken
K
Stauderman
CalciMedica, La Jolla, California.
Jack
J
Roos
CalciMedica, La Jolla, California.
Sergey
S
Grigoryev
CalciMedica, La Jolla, California.
Stephanie
S
Ramos
CalciMedica, La Jolla, California.
Evan
E
Rogers
CalciMedica, La Jolla, California.
Jeff
J
Whitten
CalciMedica, La Jolla, California.
Gonul
G
Velicelebi
CalciMedica, La Jolla, California.
Michael
M
Dunn
Department of Molecular and Cellular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
Alexei V
AV
Tepikin
Department of Molecular and Cellular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, United Kingdom.
David N
DN
Criddle
Pancreas Biomedical Research Unit, National Institute for Health Research Liverpool, Royal Liverpool University Hospital, Liverpool, United Kingdom. Electronic address: r.sutton@liverpool.ac.uk.
Robert
R
Sutton
D061354:Acinar Cells / Q000166:cytology / Q000187:drug effects*; D000208:Acute Disease; D000818:Animals; D001549:Benzamides / Q000494:pharmacology*; D001647:Bile Acids and Salts / Q000633:toxicity; D002118:Calcium / Q000378:metabolism* / Q000633:toxicity; D015220:Calcium Channels / Q000187:drug effects* / Q000378:metabolism*; D002478:Cells, Cultured; D004195:Disease Models, Animal; D057809:HEK293 Cells; D006801:Humans; D007211:Indoles / Q000633:toxicity; D051379:Mice; D008810:Mice, Inbred C57BL; D000071740:ORAI1 Protein; D010195:Pancreatitis / Q000139:chemically induced / Q000188:drug therapy* / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology*; D019284:Thapsigargin / Q000633:toxicity; D013997:Time Factors; D016896:Treatment Outcome
C583716:2,6-difluoro-N-(1-(4-hydroxy-2-(trifluoromethyl)benzyl)-1H-pyrazol-3-yl)benzamide; D001549:Benzamides; D001647:Bile Acids and Salts; D015220:Calcium Channels; D007211:Indoles; D000071740:ORAI1 Protein; C510746:ORAI1 protein, human; D011720:Pyrazoles; D019284:Thapsigargin; D002118:Calcium; C000543:cyclopiazonic acid
Calcium Entry Inhibition; Drug Development; Experimental Pancreatitis; SOCE; STIM1