Ca2+ signalling and mitochondrial dynamics in the exocrine pancreas: physiology and pathophysiology

Cane, Matthew
Ca2+ signalling and mitochondrial dynamics in the exocrine pancreas: physiology and pathophysiology. Doctor of Philosophy thesis, University of Liverpool.

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Pancreatic acinar cells release hydrolytic enzymes into the gut where they are responsible for digestion. When this process goes awry, these enzymes can be activated within the pancreas itself, characteristic of a severe and systemic inflammatory disease: acute pancreatitis. A large body of data has highlighted that the IP3R Ca2+-release channel is a central mediator of the toxic Ca2+ signals associated with the onset of early enzyme activation and cellular necrosis. Not only is the pancreatic acinar cell a focus of investigation into the onset of this acute disease, it is also investigated as a paradigm cell type in the fields of Ca2+ signalling; stimulus secretion coupling; and cellular polarity. The mitochondria, which are the ATP-producing powerhouses and Ca2+ signal modulators of the cell, have a well documented polarised distribution in pancreatic acinar cells. This is a thesis of three main parts. Initial experiments aimed to determine the mechanisms of inhibition of IP3-mediated Ca2+ signals by xanthines. Xanthines were shown to inhibit the IP3R and did not inhibit Ca2+ influx directly. A range of methylxanthines were shown to have varied effects on Ca2+ oscillations, however another role of methylxanthines, PDE inhibition, is likely to be involved in this. No significant differences were seen between xanthines when inhibiting IP3-mediated Ca2+ signals or necrosis, but inhibition of the Ca2+ signal does protect the cell against mitochondrial dysfunction. Whilst no xanthines tested offered any superior protection against pathological Ca2+ signals, this only goes to highlight the need for better pharmacological modulators of IP3-mediated Ca2+ signals. The development of multiplex assays to record pathological criteria such as Ca2+ signals and necrosis in a rapid and more user-friendly way was also undertaken. Assays for determining ∆ΨM and necrosis were successfully developed but throughput was limited by the primary cell isolation procedure. Finally, the most fascinating part of this research involves the mitochondrial network in pancreatic tissue. We investigate the mechanisms and signals involved in how the mitochondrial distribution comes into being and what effect pathological signals have on this distribution. This mitochondrial belt is formed in a Ca2+-dependent manner upon stimulation. Redistribution is tubulin-mediated and the maintenance of subplasmalemmal mitochondria is dependent upon Ca2+-influx, highlighting a potential role for Ca2+ microdomains in inhibiting tubulin-mediated motility. Hyperstimulation induced actin-mediated redistribution of cellular contents, including mitochondria into the apical pole. Finally we determined the functional importance of mitochondrial redistribution away from the plasma membrane by determining the effects of redistribution on store-operated Ca2+ entry. This investigation strongly advocates the importance of intact tissue to bolster molecular and cellular tools in the study of organ physiology.

Item Type: Thesis (Doctor of Philosophy)
Additional Information: Date: 2013-09 (completed)
Subjects: ?? QP ??
Depositing User: Symplectic Admin
Date Deposited: 13 Feb 2014 10:43
Last Modified: 16 Dec 2022 04:39
DOI: 10.17638/00012813