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Title: Cellular mechanisms of L-arginine induced experimental acute pancreatitis
Author: Masood, Omar
ISNI:       0000 0004 2740 4355
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2013
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Introduction: Impairment of cytosolic calcium ([Ca2+]i) signaling and in particular calcium overload has emerged as a possible unifying mechanism for precipitating acute pancreatitis (AP.) In the L-arginine (L-arg) experimental model of AP, nitric oxide (NO) has been implicated however the disease progression is largely unaffected by nitric oxide synthase (NOS) inhibitors (8). Additionally, L-ornithine (L-orn), a NOS-independent metabolite of L-arg, has been shown to be potent at inducing AP (28). Both L-arg and L-orn activate calcium-sensing like receptors (CaSR) (31) such as the GPRC6a which may be responsible for initiating the [Ca2+]i overload. The aim of this study is to investigate the effects of L-arg and L-orn on pancreatic acinar cells that maybe linked to the pathophysiology of AP. Furthermore to provide an alternative theory to the NO mediated ones, in particular that L-arg induces toxic changes in [Ca2+]i via a GPRC6a like receptor. Methods: Whole pancreata were harvested from male Sprague Dawley rats. Pancreatic acinar cells were isolated by collagenase digestion. [Ca2+]i was measured using fura-2 imaging, and cell viability assessed using physiological CCK. Oxidative stress was measured using dichlorofluorescein (DCF) and cell death was quantified using trypan blue exclusion. Results: L-arg and L-orn (100mM) induced spike-like, reversible increases in [Ca2+]i in 46% and 74% of cells and Ca2+ overload in 11% and 26% respectively. At 500 mM both induced Ca2+ overload in all cells however this was also seen with the osmotic control, mannitol. Isosmotic L-arg and L-orn (100mM) induced only reversible increases in [Ca2+]i. Neither L-arg nor L-orn had significant effects on CCK-evoked [Ca2+]i oscillations. Both L-arg and L-orn induced significant oxidative stress responses (22% and 37% of a maximum response seen with 3mM H202, respectively). Both L-arg and L-orn caused cell death in 76% +/- 4 and 89% +/- 7 at 3 hours respectively, compared to 35% +/- 4 and 40% +/- 3 with controls (Hepes, Glycine). Conclusion: The data suggests that the L-arg and L-orn causes significant increase in oxidative stress and cell death. The data suggests that although changes in [Ca2+]i were induced by both L-arg and L-orn the large concentrations used experimentally are likely to induce significant osmotic effects.
Supervisor: Bruce, Jason; Siriwardena, Ajith Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: experimental acute pancreatitis ; L-arginine induced acute pancreatitis