Acute pancreatitis is a human disease in which cytosolic Ca2+ overload and mitochondrial dysfunction have been implicated as the initial triggers. Sustained elevations in cytosolic Ca2+ result in intracellular activation of digestive enzymes, mitochondrial dysfunction and cellular necrosis. This leads to autodigestion of the pancreas and subsequent inflammation, which can prove fatal. Intracellular Ca2+ release from stores is coupled to the activation of store-operated Ca2+ entry (SOCE) channels such as the Ca2+ release activated Ca2+ (CRAC) channel or the canonical transient receptor potential (TRPC) 3 channels in the plasma membrane. These channels drive cytosolic Ca2+ overload. Targeting these channels is an appealing therapeutic strategy. One of aim of this study was to determine if there are any inhibitors, in addition to GSK-7975A, that can specifically block these channels and prevent cytosolic Ca2+ overload. Analogues of 2-APB, a well know but problematic inhibitor of SOCE channels, DPB163-AE and DPB162-AE were found to inhibit SOCE channels efficiently at a low micromolar concentration. This was similar to the effect seen with the inhibitor GSK-7975A. However, increasing concentrations of DPB163 resulted in impaired clearance of Ca2+ from the cytosol, whereas DPB162 inhibited SOCE further. RO2959, another reported CRAC channel inhibitor, was found to be less effective at inhibiting SOCE in pancreatic acinar cells than in other cell types. This phenomenon was true for the DPB compounds. RO2959 was effective at inhibiting SOCE in dose dependent manner when applied acutely or when pre-incubated with cells. In addition, there were observed effects of RO2959 on Ca2+ clearance from the cytosol. This likely contributed to the observed finding that RO2959 also increased levels of necrosis, due to impaired Ca2+ clearance. Targeting TRPC3 channels, the non-specific cation channels, has been difficult as it is lacking a specific inhibitor. Treatment with Pyr3, initially thought to be a specific TRPC3 inhibitor was particularly effective at inhibiting SOCE. However, during the time course of this work it came to light that TRPC3 at low micromolar concentration does not discriminate between TRPC3 and CRAC channels. Treatment with a reportedly specific inhibitor, Pyr10, resulted in a reduction in Ca2+ influx. However, in a similar manner to DPB163 and RO2959 there appeared to be an effect of Pyr10 on the Ca2+ clearance from the cell. A second aim of this thesis was to investigate the role of calmodulin in SOCE in pancreatic acinar cells. Calmodulin is thought to regulate CRAC channels by mediating Ca2+-dependent inactivation of the channel. Inhibitors of calmodulin: calmidazolium and W-7 were used. Both inhibitors resulted in an inhibition of SOCE. However, this inhibition was not as remarkable as CRAC channel inhibition. This could have been because the calmodulin inhibitors were not directly inhibiting the Orai1 protein of the CRAC channel rather were targeting one of the channels regulating proteins - calmodulin. A cell permeable activator of calmodulin was also utilised in this study - CALP3. CALP3 had previously been shown to protect acinar cells from cytosolic Ca2+ overload and also to block non-specific cation channels in T cells. At the concentration found to protect against Ca2+ overload, no effect on SOCE was observed. Increasing the concentration resulted in significant inhibition, but it was likely due to a non-specific effect of a supramaximal concentration. CRAC channel inhibition was the more effective as a target for reducing SOCE and subsequent Ca2+ overload than targeting calmodulin-regulation of CRAC channels or targeting TRPC3 channels. However, several of the 'specific' inhibitors available to target this channel, including DPB163 and RO2959 had a multitude of off-target effects including a significant effect on the Ca2+ extrusion mechanism of pancreatic acinar cells. GSK-7975A had the fewest undesired effects and also inhibited SOCE more than any other inhibitor. It is an ideal candidate for future drug development. When the work in this study is viewed in the context of the wider published work, the idea of focusing on CRAC channel inhibition as a potential therapeutic for acute pancreatitis is a promising future avenue.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:704938 |
| Date | January 2016 |
| Creators | Stapleton, Eloise |
| Publisher | Cardiff University |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://orca.cf.ac.uk/98542/ |
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