A. Tepikin

Correlation of NADH and Ca2+ signals in mouse pancreatic acinar cells

Relationships between calcium signals and NADH responses were investigated in pancreatic acinar cells stimulated with calcium‐releasing secretagogues. Cytosolic calcium signals were studied using Fura Red or calcium‐sensitive Cl− current. Mitochondrial calcium was measured using Rhod‐2. The highest levels of NADH autofluorescence were found around the secretory granule region. Stimulation of cells with physiological doses of cholecystokinin (CCK) triggered slow oscillations of NADH autofluorescence. NADH oscillations were clearly resolved in the mitochondrial clusters around secretory granules. Very fast apical calcium signals induced by acetylcholine (ACh) produced no detectable changes in NADH; slightly more extended apical (or preferentially apical) calcium transients triggered clear NADH responses. Triple combined recordings of cytosolic calcium, mitochondrial calcium and NADH revealed the sequence of development of individual signals: an increase in cytosolic calcium was accompanied by a slower mitochondrial calcium response followed by a delayed increase in NADH fluorescence. Recovery of cytosolic calcium was faster than recovery of mitochondrial calcium. NADH recovery occurred at elevated mitochondrial calcium levels. During the transient cytosolic calcium oscillations induced by intermediate doses of ACh, there was an initial increase in NADH fluorescence following the first calcium transient; each of the subsequent calcium responses produced biphasic NADH changes comprising an initial small decline followed by restoration to an elevated calcium level. During the higher‐frequency sinusoidal calcium oscillations induced by higher doses of ACh, NADH responses fused into a smooth rise followed by a slow decline. Supramaximal doses of ACh and CCK produced single large NADH transients.

PWE-209 Orai inhibition prevents cytosolic ca2+ overload and acute pancreatitis

Introduction Cytosolic calcium overload triggers pancreatic acinar injury induced by pancreatitis toxins. Sustained Ca2+elevation depends on Ca2+entry through store-operated Ca2+entry (SOCE) channel Orai1, but the role of which in experimental acute pancreatitis (EAP) and human pancreatic acinar cell injury has not been determined. Method Confocal and patch clamp technology were used to examine the effects of GSK-7975A and CM_128, inhibitors of SOCE channel Orai1 on bile acid-, hystimulation-, thapsigargin-, or cyclopiazonic acid-induced calcium entry into murine and human pancreatic acinar as well as human Orai1/STIM1-transfected HEK 293 cells. The effects of GSK-7975A and CM_128 on human necrotic pancreatic acinar cell death pathway activation induced by bile acid were monitored. EAP was induced by seven hourly intraperitoneal cerulein injections (50 mg/kg), retrograde pancreatic ductal TLCS infusion (50 mL 3 mM) or two hourly intraperitoneal injections of 150 mg/kg palmitoleic acid and 1.35g/kg ethanol. Different doses of either compound were tested in three diverse clinical representative models of EAP, begun at different time point after disease induction. EAP severity was assessed by standard biochemical parameters and blinded histopathology. Results GSK-7975A and CM_128 inhibited toxin-induced SOCE and/or Ca2+release-activated Ca2+currents in a concentration-dependent manner up to >90% of control in all cells tested and significantly inhibited murine and human necrotic pancreatic acinar cell death pathway activation (p < 0.05). Administration of GSK-7975A or CM_128 after induction of EAP had pronounced inhibitory effects on all local and systemic disease parameters in all three models (all p < 0.05), demonstrating both dose- and time-dependency, with significantly greater effectiveness in a range of parameters when given one hour rather than six hours after disease induction. Conclusion This study confirms the pivotal role of cytosolic calcium overload in the pathogenesis of acute pancreatitis and provides robust preclinical validation for Orai inhibition as a treatment for acute pancreatitis. Disclosure of interest L. Wen: None Declared, S. Voronina: None Declared, M. Javed: None Declared, M. Awais: None Declared, P. Szatmary: None Declared, D. Latawiec: None Declared, M. Chvanov: None Declared, D. Collier: None Declared, J. Barrett Employee of: GlaxoSmithKline, M. Begg Employee of: GlaxoSmithKline, K. Stauderman Employee of: CalciMedica, M. Dunn Employee of: CalciMedica, A. Tepikin: None Declared, D. Criddle: None Declared, R. Sutton: None Declared.