Proceedings of The Physiological Society
University of Manchester (2010) Proc Physiol Soc 19, C132
Insulin protects against oxidant-induced impairment of Ca2+ homeostasis and plasma membrane Ca2+-ATPase (PMCA) inhibition in pancreatic acinar cells
P. Mankad1, A. James1, T. Leggett1, J. Bruce1
1. University of Manchester, Manchester, United Kingdom.
Pancreatitis is an inflammatory disease of the exocrine pancreas, characterised by auto-digestion of the pancreas and necrotic cell death. Oxidative stress and impairment of intracellular calcium ([Ca2+]i) homeostasis have been implicated in this disease. Previous studies have shown that oxidative stress impairs hormone-evoked Ca2+ signalling and induces an irreversible increase in [Ca2+]i (Ca2+ overload) (Bruce et al, 2007). This oxidant-induced Ca2+ overload response coincided with inhibition of the PMCA and mitochondrial depolarisation. (Baggaley et al, 2008). Several studies have demonstrated that insulin can protect from pancreatic cell injury (Hegyi et al, 2004). Therefore the aim of the current study was to test the effects of insulin on oxidant-mediated impairment of Ca2+ signalling and inhibition of the PMCA. Pancreatic acinar cells were isolated from male Sprague-Dawley rats by collagenase digestion. The effect of oxidative stress on [Ca2+]i was tested on fura-2 loaded cells treated with or without insulin (1-100nM). In addition, an in situ [Ca2+]i clearance assay was utilised in which the PMCA was pharmacologically isolated. A paired experimental design was used to directly compare clearance phases in which oxidative stress, using hydrogen peroxide (H2O2), was applied to the second clearance phase and the rate normalised to the first clearance phase. All results are expressed with standard error values. H2O2 (50-500µM) caused a concentration dependent increase in [Ca2+]i overload response, measured in three ways - maximum increase in [Ca2+]i, recovery of [Ca2+]i to baseline and area under the curve. Pre-treatment with 100nM insulin reduced both the H2O2-induced increase in [Ca2+]i (at 100µM H2O2: 437 ± 200nM, n = 7 control; 45 ± 6nM, n = 5 insulin treated) and area under the curve (at 100µM H2O2: 2491 ± 565nM.s, n = 7 control; 571 ± 202nM.s, n = 5 insulin treated). During [Ca2+]i clearance assays, H2O2 inhibited the PMCA in a concentration dependent manner (rate = 92 ± 13%, n = 8 control; rate = 18 ± 5%, n = 6 500µM H2O2). This inhibition was attenuated by pre-treatment with insulin (1-100nM) (rate = 32 ± 5%, n = 6 100µM H2O2; rate = 57 ± 3%, n = 6 100nM insulin + 100µM H2O2). Furthermore, preliminary data has found that treatment with 100nM insulin increased phosphorylation of Akt, shown by western blotting with pAkt S473 antibody. In summary, these data suggest that insulin could protect against oxidant-induced [Ca2+]i overload and PMCA inhibition; possibly via Akt mediated cell survival pathways. This may have important implications for the prevention of necrotic cell death associated with pancreatitis. Future work will involve further elucidating the molecular mechanisms of this insulin-mediated protection of oxidant-induced impairment of [Ca2+]i signalling.
Where applicable, experiments conform with Society ethical requirements