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Abstract
Objective Caffeine reduces toxic Ca2+ signals in pancreatic acinar cells via inhibition of inositol 1,4,5-trisphosphate receptor (IP3R)-mediated signalling, but effects of other xanthines have not been evaluated, nor effects of xanthines on experimental acute pancreatitis (AP). We have determined effects of caffeine and its xanthine metabolites on pancreatic acinar IP3R-mediated Ca2+ signalling and experimental AP.
Design Isolated pancreatic acinar cells were exposed to secretagogues, uncaged IP3 or toxins that induce AP and effects of xanthines, non-xanthine phosphodiesterase (PDE) inhibitors and cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP/cGMP) determined. The intracellular cytosolic calcium concentration ([Ca2+]C), mitochondrial depolarisation and necrosis were assessed by confocal microscopy. Effects of xanthines were evaluated in caerulein-induced AP (CER-AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) or palmitoleic acid plus ethanol-induced AP (fatty acid ethyl ester AP (FAEE-AP)). Serum xanthines were measured by liquid chromatography-mass spectrometry.
Results Caffeine, dimethylxanthines and non-xanthine PDE inhibitors blocked IP3-mediated Ca2+ oscillations, while monomethylxanthines had little effect. Caffeine and dimethylxanthines inhibited uncaged IP3-induced Ca2+ rises, toxin-induced Ca2+ release, mitochondrial depolarisation and necrotic cell death pathway activation; cAMP/cGMP did not inhibit toxin-induced Ca2+ rises. Caffeine significantly ameliorated CER-AP with most effect at 25 mg/kg (seven injections hourly); paraxanthine or theophylline did not. Caffeine at 25 mg/kg significantly ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines peaked at >2 mM with 25 mg/kg caffeine but at <100 µM with 25 mg/kg paraxanthine or theophylline.
Conclusions Caffeine and its dimethylxanthine metabolites reduced pathological IP3R-mediated pancreatic acinar Ca2+ signals but only caffeine ameliorated experimental AP. Caffeine is a suitable starting point for medicinal chemistry.
- ACUTE PANCREATITIS
- CALCIUM
- DRUG DEVELOPMENT
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Contributors WH and MCC are co-first authors. WH: acquisition of data; analysis and interpretation of data; drafting of the manuscript. MCC, RM, PS, XZ, VE, YO, MC, DL and LW: acquisition of data; analysis and interpretation of data. DMB: technical support; acquisition of data; analysis and interpretation of data; critical revision of the manuscript. ACH: material support; critical revision of the manuscript. OHP and AVT: critical revision of the manuscript; obtained funding. DNC: study concept and design; acquisition of data; analysis and interpretation of data; critical revision of the manuscript; study supervision. RS: study concept and design; analysis and interpretation of data; critical revision of the manuscript; obtained funding; study supervision.
Funding This work was supported by the Medical Research Council (UK), the Biomedical Research Unit funding scheme from the National Institute for Health Research and a State Administration of Traditional Chinese Medicine Key Discipline Construction Project.
Competing interests OHP is a MRC Professor; WH was a recipient of a UK/China Postgraduate Scholarship for Excellence and State Administration of Traditional Chinese Medicine Key Discipline Construction Project, China; MCC and DB were awarded MRC scholarships; RM was supported by an Amelie Waring Clinical Research Fellowship from CORE; PS was supported by The Royal Colleague of Surgeons of England Fellowship; XZ, YO and LW were supported by the China Scholarships Council.
Ethics approval Animal experiments were performed after ethical review and appropriate approval from the UK Home Office (PPL 40/3320) in accordance with the Animals (Scientific Procedures) Act 1986.
Provenance and peer review Not commissioned; externally peer reviewed.
Data sharing statement Upon publication raw data from individual experiments will be made available by the corresponding author to interested researchers requesting data for bona fide scientific purposes.