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.

Contexte : Les maladies pulmonaires comme l'asthme ou la mucoviscidose représentent des problèmes majeurs de santé publique. Elles se manifestent par une inflammation chronique avec une production accrue de cytokines pro-inflammatoires et à terme une dégradation de la fonction respiratoire. Les efforts thérapeutiques tentent, d'un côté, de contrôler la réaction inflammatoire et aussi d'améliorer la biodisponibilité médicamenteuse. Objectif : Notre objectif est d'explorer l'implication des phospholipases dans l'inflammation et le rôle des transporteurs peptidiques dans le transport des antibiotiques dans la mucoviscidose. Nous avons aussi cherché à comprendre l'effet d'une supplémentation en cobalamine sur l'efficacité de la dexaméthasone dans un contexte inflammatoire. Méthodes : Des techniques immunologiques, électrophorétiques, de culture cellulaire, d'immunoprécipitation et d'expression génique sont utilisées sur des lignées bronchiques humaines normales ou mucoviscidosiques. Résultats : 1) La PC-PLC est constitutivement suractivée dans les cellules mucoviscidosiques et conduit à une surproduction d'arachidonate, à une surexpression de Cox-2, une surproduction de PGE2, une surexpression d'interleukine-8, et au défaut de régulation beta-adrénergique de la sécrétion. L'inhibition de cette enzyme par le D609 permet de corriger tous ces défauts. 2) L'activité du transporteur peptidique, impliquée dans le transport d'antibiotiques, PEPT2, a été caractérisée dans les cellules bronchiques normales (Vm = 115 pmol/106 cellules/min ; Km = 15µM). Ce transporteur n'est pas influencé par un contexte inflammatoire. Ce transporteur est inactif dans les cellules CF. 3) La cobalamine potentialise l'effet de la déxaméthasone sur la sécrétion et l'expression des cytokines pro-inflammatoires induite par le TNFa et l'histamine. Conclusions/perspectives : Cette étude devrait permettre 1) de mettre en lumière l'importance de la PC-PLC comme cible pharmacologique potentielle dans la mucoviscidose. 2) de comprendre la relative faible efficacité de l'antibiothérapie dans cette maladie et 3) de mettre en évidence une possible participation du cycle de la méthionine dans le processus inflammatoire / Background: Lung diseases such as asthma or cystic fibrosis are major public health problems. They are manifested by chronic inflammation with increased production of proinflammatory cytokines leading to respiratory failure. Current therapeutic is aimed at controling the inflammatory response and also at improving drug bioavailability. Objective: The objective is to explore the involvement of phospholipases in inflammation and the role of peptide transporters in the transport of antibiotics in cystic fibrosis. We also sought to understand the effect of cobalamin supplementation on the effectiveness of dexamethasone in an inflammatory context. Methods: immunological techniques, electrophoresis, cell culture, immunoprecipitation and gene expression are used on normal or cystic fibrosis human bronchial cell lines. Results : 1) PC-PLC is constitutively overactivated in cystic fibrosis cells and leads to overproduction of arachidonate, to overexpression of Cox-2 , an overproduction of PGE2 , an interleukin -8 overexpression , and to alteration of beta-adrenergic secretion. Inhibition of this enzyme by D609 corrects these defects. 2) The activity of the dipeptide carrier involved in the transport of antibiotics, PEPT2, was characterized in normal bronchial cells (Vm = 115 pmol/106 cells / min, Km = 15µM). This transporter is not affected by an inflammatory context. However, it was shown to be inactive in CF cells. 3) Cobalamin potentiates the effect of dexamethasone on the expression and secretion of pro-inflammatory cytokines induced by TNFa and histamine. Conclusions : This study should help 1) to highlight the importance of PC-PLC as a potential pharmacological target in cystic fibrosis. 2) to understand the relative ineffectiveness of antibiotics in this disease , and 3) to highlight a possible involvement of methionine cycle in the inflammatory process