Ca2+ Signalling, Organelle Dysfunction and Pancreatitis

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences

Abstract

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Technical Summary

The acinar cells of the exocrine pancreas secrete a battery of zymogens essential for digestion, but premature intracellular digestive enzyme activation, mostly from cholelithiasis or excessive alcohol intake, leads to the common, devastating disease pancreatitis. The lack of specific treatment of pancreatitis, with over 200 almost entirely negative randomized clinical trials to date, demonstrates the need to fully understand the pathogenesis. We have discovered that local apical increases (repetitive spikes) in the cytosolic Ca2+ concentration ([Ca2+]i), elicited by acetylcholine or cholecystokinin, regulate normal acinar secretion, whereas sustained and global [Ca2+]i elevations, which can be induced by bile acids or non-oxidative ethanol metabolites, result in intracellular digestive enzyme activation, vacuole formation and cell death. Our aim is to understand how alcohol and bile damage the pancreas and how such damage and its consequent impact can be prevented or limited. We will use our combined strengths as internationally recognized investigators of Ca2+ signalling and leaders within the UK?s foremost centre for clinical pancreatology to investigate the mechanisms underlying toxic Ca2+ release from the various subcellular pools, the roles of mitochondrial inhibition, of reactive oxygen and nitrogen species (ROS and RNS), vacuole formation and the pathological digestive enzyme activation that causes the disease. We will employ patch clamp technology with confocal, two-photon and TIRF microscopy using isolated acinar cells and cell clusters including our two-photon permeabilized preparations, as well as intact pancreata, isolated or in vivo. Novel methods include those for measuring and changing intracellular ATP concentrations ([ATP]i), nucleofection and assessing vacuole formation in vivo. Various pancreatitis models will be used, including transgenics and KOs. We will examine the effects of bile and ethanol metabolites on changes in [Ca2+]i, ROS/RNS, mitochondrial membrane potential and [ATP]i, as well as the balance of oxidative and non-oxidative ethanol metabolism in the switch from apoptosis to necrosis. We will monitor Ca2+ changes simultaneously with premature intracellular enzyme activation in zymogen granules, lysosomes, endosomes and vacuoles within intact or permeabilised cells, or isolated organelles. We will determine the contribution of failure of exocytic pore expansion and of the formation, transport and rupture of endocytic vacuoles, including release of intravacuolar digestive enzymes. Key findings made in rodent cells will be explored in human cells. Specific interventions in the pathological signalling cascade will be tested, such as inhibition of Ca2+ release or Ca2+ entry, changes in FAEE metabolism and/or oxidant defence, to pave the way for eventual rational treatment.

Publications

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Gerasimenko JV (2011) Calmodulin protects against alcohol-induced pancreatic trypsinogen activation elicited via Ca2+ release through IP3 receptors. in Proceedings of the National Academy of Sciences of the United States of America

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Gerasimenko JV (2009) Pancreatic protease activation by alcohol metabolite depends on Ca2+ release via acid store IP3 receptors. in Proceedings of the National Academy of Sciences of the United States of America

 
Description MRC Programme Grant
Amount £1,425,607 (GBP)
Funding ID MR/J002771/1 
Organisation Medical Research Council (MRC) 
Sector Public
Country United Kingdom
Start 04/2012 
End 03/2017
 
Description Characterization of the actions of CRAC channel blockers on pancreatic acinar cells 
Organisation GlaxoSmithKline (GSK)
Department GlaxoSmithKline Medicines Research Centre
Country United Kingdom 
Sector Private 
PI Contribution We are characterizing the actions and investigating the mechanisms of action of GSK-identified molecules on the functional properties of pancreatic acinar cells (Calcium signalling, intracellular protease activation, cell death). We are particularly interested in potential protective effects against agents indicing pancreatitis
Collaborator Contribution GSK are supplying us with GSK-identified molecules as well as information about their chemical properties and their whole-body handling
Impact A manuscript is being prepared
Start Year 2012
 
Description Identification of IP3 receptor subtypes involved in initiation of alcohol-related pancreatitis 
Organisation RIKEN
Department RIKEN Brain Science Institute
Country Japan 
Sector Public 
PI Contribution The principal experimental work measuring changes in intracellular calcium stores
Collaborator Contribution Supply of transgenic mice with deleted specific IP3 receptor subtypes
Impact Gerasimenko et al PNAS 106, 10758-10763, 2009
Start Year 2008
 
Description Knock-out of IP3 receptors 
Organisation RIKEN
Department RIKEN Brain Science Institute
Country Japan 
Sector Public 
PI Contribution We showed the importance of functonal IP3 receptors for induction of pancreatitis by fatty acid ethyl esters or alcohol
Collaborator Contribution Provision of transgenic mice
Impact Two papers, both published in PNAS (Gerasimnko et al 2009, 2011 - listed uner publications) have so far appeared in he scienif literature
Start Year 2008
 
Description A Pancreas-protecting protein 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Type Of Presentation Paper Presentation
Geographic Reach International
Primary Audience Media (as a channel to the public)
Results and Impact Many requests for interviews with different media (incl BBC, magazines and newspapers)

Difficult to evaluate. Certainly increased awareness nationally and internationally of our work. May have contributed to increasing number of invitations to speak
Year(s) Of Engagement Activity 2011
 
Description BBC Interview 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Primary Audience Media (as a channel to the public)
Results and Impact Following Press Release by MRC and Cardiff University, interviewed live by BBC Wales

Coverage of press release and ineview in several newspapers
Year(s) Of Engagement Activity 2011
 
Description British Association Annual Meeting Liverpool 2008 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact Ole H Petersen chaired and gave introductory talk in session on Cystic Fibrosis organized by The Physiological Society. The new findings from MRC supported research on human pancreatic acinar cells published shortly before the meeting in the top-rated journal Gastroenterology (PMID: 18555802) were highlighted.

University of Liverpool issued Press release about our work, which resulted in Newspaper articles
Year(s) Of Engagement Activity 2008
 
Description Inteview published in THE 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Primary Audience Media (as a channel to the public)
Results and Impact Interview published in THE describing my research following Life Time Achievement Award fromThe European Pancreatic Club (EPC).

Invited Award Lecture at EPC Annual Meeting in Stockholm 2010
Year(s) Of Engagement Activity 2010
 
Description Invted talk at British Association Annual Meeting in Liverpool 2008 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact Ole H Petersen chaired and gave introductory talk in session on Cystic Fibrosis organized by The Physiological Society. The new findings from MRC supported research on human pancreatic acinar cells published shortly before the meeting in the top-rated journal Gastroenterology (PMID: 18555802) were highlighted.

The University of Liverpool issued a Press Release, which gave rise to reports in Newspapers.
Year(s) Of Engagement Activity 2008
 
Description Research described in MRC Annual Review 2009/2010 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact Brief description of results from our PNAS 2009 article

So far none
Year(s) Of Engagement Activity 2010