This thesis presents an investigation into the role of the recently identified two pore channels (TPCs) in β-cell stimulus-secretion coupling. TPCs are the receptors for calcium mobilising messenger nicotinic acid adenine dinucleotide phosphate (NAADP) located in the membrane of acidic intracellular calcium stores. It is proposed that they are responsible for the ATP-sensitive potassium channel (Katp channel) independent pathway of stimulus-secretion coupling; and that this pathway is not subordinate to the KAT? channel dependent pathway; but an alternative explanation of stimulus-secretion coupling in its own right. The first section of this thesis presents a characterisation of sub-membrane cal- cium signals observed in primary mouse β-cells in response to glucose and the membrane-permeable acetoxymethyl ester form of NAADP (NAADP-AM) using the non-ratiometric fluorescent calcium indicator fluo-4 and total internal reflection (TIRF) microscopy. These are compared to global cytosolic calcium changes observed with epifluorescence microscopy. Factors affecting the shape and time course of re- sponses are investigated, and pharmacological tools used to provide evidence for the role of intracellular calcium release from acidic stores mediated by NAADP. Having characterised the calcium responses of β-cells using TIRF; the second part of the thesis examines the effects of knocking out TPC2 (single KO), or both TPC1 and TPC2 (DKO) on these responses; after an initial assessment of pancreatic islet and β-cell morphology using electron microscopy. Gender differences in β-cell responses to glucose and NAADP are assessed in both wild type and knockout animals. Finally, the third section presents the discovery of elementary calcium release events in pancreatic β-cells. The current project visualises what are likely the triggering events for the global calcium signals examined in sections one and two. They take the form of localised calcium release in response to NAADP-AM and glucose; akin to sparks and puffs observed by stimulation with cADPR and IP3. Optical quantal analysis demonstrates the quantal nature of the events and estimates the size of the unitary calcium release unit (CRU) for NAADP. .
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:581190 |
| Date | January 2012 |
| Creators | Heister, Paula Maria |
| Contributors | Galione, Antony |
| Publisher | University of Oxford |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://ora.ox.ac.uk/objects/uuid:4bed27d2-e7e4-49ff-8168-aa02b6f9b613 |
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