Caffeine Protects Against Experimental Acute Pancreatitis by Inhibition Of
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Gut Online First, published on December 7, 2015 as 10.1136/gutjnl-2015-309363 Pancreas ORIGINAL ARTICLE Gut: first published as 10.1136/gutjnl-2015-309363 on 7 December 2015. Downloaded from Caffeine protects against experimental acute pancreatitis by inhibition of inositol 1,4,5-trisphosphate receptor-mediated Ca2+ release Wei Huang,1,2,3 Matthew C Cane,2 Rajarshi Mukherjee,1,2 Peter Szatmary,1,2 Xiaoying Zhang,1,3 Victoria Elliott,1 Yulin Ouyang,1,2 Michael Chvanov,1,2 Diane Latawiec,1 Li Wen,1,3 David M Booth,2 Andrea C Haynes,4 Ole H Petersen,5 Alexei V Tepikin,2 David N Criddle,1,2 Robert Sutton1 ▸ Additional material is ABSTRACT published online only. To view Objective Caffeine reduces toxic Ca2+ signals in Significance of this study please visit the journal online (http://dx.doi.org/10.1136/ pancreatic acinar cells via inhibition of inositol 1,4,5- gutjnl-2015-309363). trisphosphate receptor (IP3R)-mediated signalling, but effects of other xanthines have not been evaluated, nor 1NIHR Liverpool Pancreas What is already known on this subject? Biomedical Research Unit, effects of xanthines on experimental acute pancreatitis ▸ Acute pancreatitis is a major health problem Royal Liverpool University (AP). We have determined effects of caffeine and its without specific drug therapy. Hospital, University of xanthine metabolites on pancreatic acinar IP3R-mediated ▸ Coffee consumption reduces the incidence of Liverpool, Liverpool, UK 2+ 2 Ca signalling and experimental AP. acute alcoholic pancreatitis. Department of Cellular and ▸ 2+ Molecular Physiology, Institute Design Isolated pancreatic acinar cells were exposed to Caffeine blocks physiological intracellular Ca of Translational Medicine, secretagogues, uncaged IP3 or toxins that induce AP and oscillations by inhibition of inositol University of Liverpool, effects of xanthines, non-xanthine phosphodiesterase 1,4,5-trisphosphate receptor-(IP3R)-mediated Liverpool, UK (PDE) inhibitors and cyclic adenosine monophosphate signalling. 3Department of Integrated and cyclic guanosine monophosphate (cAMP/cGMP) ▸ Sustained cytosolic Ca2+ overload from Traditional Chinese and 2+ Western Medicine, Sichuan determined. The intracellular cytosolic calcium abnormal Ca signalling is implicated as a 2+ Provincial Pancreatitis Centre, concentration ([Ca ]C), mitochondrial depolarisation and critical trigger in the pathogenesis of acute West China Hospital, Sichuan necrosis were assessed by confocal microscopy. Effects of pancreatitis. University, Chengdu, China xanthines were evaluated in caerulein-induced AP (CER- 4 fl Immuno-In ammation AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) What are the new findings? Therapeutic Area Unit, ▸ Caffeine and its dimethylxanthine http://gut.bmj.com/ GlaxoSmithKline, Stevenage, or palmitoleic acid plus ethanol-induced AP (fatty acid UK ethyl ester AP (FAEE-AP)). Serum xanthines were metabolites inhibit IP3R-mediated, sustained 2+ 5Cardiff School of Biosciences, measured by liquid chromatography-mass spectrometry. cytosolic Ca elevations, loss of Cardiff University, Cardiff, UK Results Caffeine, dimethylxanthines and non-xanthine mitochondrial membrane potential and 2+ necrotic cell death pathway activation in Correspondence to PDE inhibitors blocked IP3-mediated Ca oscillations, Professor Robert Sutton, NIHR while monomethylxanthines had little effect. Caffeine and pancreatic acinar cells. Liverpool Pancreas Biomedical 2+ ▸ Neither specific phosphodiesterase inhibitors dimethylxanthines inhibited uncaged IP3-induced Ca on September 27, 2021 by guest. Protected copyright. Research Unit, 5th Floor UCD, rises, toxin-induced Ca2+ release, mitochondrial nor cyclic adenosine monophosphate and cyclic Royal Liverpool University depolarisation and necrotic cell death pathway activation; guanosine monophosphate inhibit sustained Hospital, Daulby Street, 2+ cAMP/cGMP did not inhibit toxin-induced Ca2+ rises. Ca elevations in pancreatic acinar cells. Liverpool L69 3GA, UK; ▸ [email protected] Caffeine significantly ameliorated CER-AP with most Serum levels of xanthines after 25 mg/kg effect at 25 mg/kg (seven injections hourly); paraxanthine caffeine administration are sufficient to inhibit Received 10 February 2015 2+ or theophylline did not. Caffeine at 25 mg/kg significantly IP3R-mediated Ca overload in experimental Revised 31 August 2015 acute pancreatitis. Accepted 22 September 2015 ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines ▸ Caffeine but not theophylline or paraxanthine peaked at >2 mM with 25 mg/kg caffeine but at administered at 25 mg/kg significantly <100 mM with 25 mg/kg paraxanthine or theophylline. ameliorated pancreatic injury in experimental Conclusions Caffeine and its dimethylxanthine acute pancreatitis through IP3R-mediated signalling inhibition. metabolites reduced pathological IP3R-mediated 2+ pancreatic acinar Ca signals but only caffeine How might it impact on clinical practice in ameliorated experimental AP. Caffeine is a suitable the foreseeable future? starting point for medicinal chemistry. ▸ These findings support an approach of inhibition of Ca2+ overload and of its consequences as novel potential therapy for To cite: Huang W, acute pancreatitis. Cane MC, Mukherjee R, ▸ et al. Gut Published Online INTRODUCTION Methylxanthine-based structures are suitable First: [please include Day Acute pancreatitis (AP) has an incidence of 30 per starting points for drug discovery and Month Year] doi:10.1136/ 100 000 per annum in the UK, commonly caused development to treat acute pancreatitis. gutjnl-2015-309363 by gallstones or alcohol excess.1 Most cases are Huang W, et al. Gut 2015;0:1–13. doi:10.1136/gutjnl-2015-309363 1 Copyright Article author (or their employer) 2015. Produced by BMJ Publishing Group Ltd (& BSG) under licence. Pancreas mild, whereas a complicated clinical course occurs in one out of inhibiting innate immunity.35 Caffeine is a non-selective inhibitor every five patients, resulting in significant morbidity, mortality of adenosine receptors, removing an endogenous brake on neural Gut: first published as 10.1136/gutjnl-2015-309363 on 7 December 2015. Downloaded from and financial burden.2 Over the last two decades, our under- activity.26 This stimulant effect of caffeine is the most familiar, standing of pathogenesis has advanced, but there is still no spe- but taken to excess may result in caffeine intoxication with major cific therapy despite many randomised trials.2 The development central nervous system hyperstimulation.26 Degradation of caf- of treatments for AP is, therefore, a priority, one strategy for feine in the liver forms the dimethylxanthines theophylline which is to follow leads from complementary laboratory and (1,3-dimethylxanthine), paraxanthine (1,7-dimethylxanthine) clinical studies, as here. and theobromine (3,7-dimethylxanthine), used variously as drugs Intracellular Ca2+ signals control normal secretion from pan- with similar actions to those of caffeine, although their actions creatic acinar cells but can become a critical trigger in pathogen- on IP3R-mediated signalling have not been clarified. As data esis. Physiological concentrations of acetylcholine (ACh) and suggest caffeine and/or related methylxanthines may be protect- cholecystokinin (CCK) generate repetitive elevations in the cyto- ive in AP,we sought to determine their actions on toxin-induced, 2+ 2+ 2+ solic Ca concentration ([Ca ]C) within the cellular apical IP3R-mediated [Ca ]C changes and cell death in vitro, and in pole that elicit stimulus metabolism coupling to generate ATP three models of AP in vivo. from mitochondria and stimulus-secretion coupling to initiate exocytosis.3 Intermittently, global extension of short-lived MATERIALS AND METHODS signals throughout the cell is necessary for nuclear signalling Animals contributing to transcription and translation.3 In contrast, toxins Adult male CD1 mice (8–12 weeks old) were housed at 23±2°C such as bile acids,4 oxidative5 and non-oxidative metabolites67 under a 12 h light/dark cycle with ad libitum access to standard of ethanol and CCK hyperstimulation89each elicit abnormal laboratory chow and water. For in vivo experiments, animals 2+ elevations of [Ca ]C that are global and sustained. These were deprived of food but were allowed access to water from abnormal elevations induce premature activation of intracellular 12 h before the start of the experiments. enzymes, mitochondrial dysfunction, impaired autophagy, vacuolisation and necrosis, all of which contribute to the patho- Measurements of Ca2+ responses, mitochondrial membrane 10 2+ genesis of AP. Ca chelation prevents zymogen activation and potential (ΔΨM) and IP3 uncaging vacuolisation through attenuation of Ca2+ overload in acinar Fresh pancreatic acinar cells were isolated as described.7 Fluo 11 12 13 cells in vitro and ameliorates the severity of AP in vivo. 4-AM (3 μM), ci-IP3/PM (2 mM) and/or tetramethyl rhodamine Blockage of the Ca2+ release-activated Ca2+ channel, also methyl ester (TMRM, 37.5 nM) were loaded for 30 min at known as the store-operated Ca2+ entry (SOCE) channel, by room temperature. Confocal images were acquired on a Zeiss Orai1 inhibitor GSK-7975A, reduces Ca2+ overload and necro- LSM510 system (Carl Zeiss Jena GmbH, Germany) with a 63× 14 15 15 sis in both mouse and human pancreatic acinar cells and C-Apochromat water immersion objective (NA 1.2). ΔΨM was prevents AP in three different mouse models. Genetic deletion recorded in the perigranular mitochondrial