The role of Bcl-2 family proteins and calmodulin in calcium signalling in pancreatic acinar cells

Bcl-2 proteins are very well known regulators of the programmed cell death. \nAccumulating evidence suggests that they are also involved in regulation of calcium \nsignalling events. Bcl-2 has been reported to affect calcium release from the \nintracellular calcium stores through regulation of inositol trisphosphate receptors and \nendoplasmic reticulum calcium pumps. Physiological and pathological processes in \npancreatic acinar cells are controlled by calcium. Intracellular Ca2+ signals regulate \nnot only gene expression and trigger enzyme secretion but also might contribute to \npremature trypsinogen activation and development of pancreatitis, which is \ncharacterised by extensive necrosis of the pancreatic tissue. Detailed investigation of \nBcl-2 family-dependent mechanisms of intracellular Ca2+ regulation and its \nassociation with cell death induction is required for understanding of the basic \nphysiological signalling pathways as well as pathophysiological processes leading to \ndevelopment of severe diseases of pancreas. This study investigates the effects of \nBcl-2 family proteins on intracellular calcium homeostasis, with particular focus on \ntheir involvement in Ca2+ fluxes and CICR phenomenon. Also, the Ca2+-related \nactions of different doses of ethanol in pancreatic acinar cells and their contribution \nto pancreatitis are presented and assessed. \nThe results indicate that pharmacological inhibition of anti-apoptotic Bcl-2 and \nBcl-xL proteins with BH3 mimetics BH3I’-2' or HA14-1 sensitizes pancreatic acinar \ncells to CICR; and overexpression of Bcl-2 has the opposite effect significantly \ndecreasing rising phase of CICR-types of responses in pancreatic cell line AR42J. \nResponses to BH3 mimetics are at least partially dependent on both IP3Rs and RyRs, \n5 \nsince inhibition of either of them results in a substantial decrease in Ca2+ release \nfrom the intracellular stores. However, simultaneous blockade of IP3Rs and RyRs did \nnot completely abolish BH3 mimetic-elicited Ca2+ release, which indicates \nengagement of other factors in the development of the response. Importantly, the \neffects of BH3 mimetics on intracellular Ca2+ were effectively inhibited by loss of \nBax protein, suggesting Bax involvement in the regulation of Ca2+ release from \nthe ER. \nFurther, the results presented here demonstrate that moderate concentrations of \nethanol (10 - 100 mM), although having only a minor effect on intact cells, induce \nsubstantial Ca2+ release from both the ER and the acidic store in permeabilized cells, \nand trigger intracellular trypsinogen activation - the hallmark of acute pancreatitis. \nThe data suggest that calmodulin, which is present in intact cells but is lost in \npermeabilized cells, constitutes a part of natural defence mechanism responsible for \nthe differences in the severity of the responses to ethanol. What is more, the evidence \nindicates that specific pre-activation of calmodulin by Ca2+-like peptides boosts this \ndefence and reduces the pathological calcium responses to ethanol as well as to BH3 \nmimetics in pancreatic acinar cells decreasing necrosis. Finally, the effects of Bcl-2 \nprotein on calcium fluxes in pancreatic acinar cells were investigated. Cells lacking \nfunctional Bcl-2 showed substantially more rapid clearance of thapsigargin / \nhigh Ca2+-induced cytosolic calcium plateau as compared to wild type cells. This \neffect has been explained by increased activity of the PMCA that results in a marked \nincrease of apoptosis / necrosis ratio in oxidative stress induced cell death. \nOverexpression of Bcl-2 in AR42J cells reduces ER calcium content, while silencing \nof Bcl-2 expression by siRNA results in substantially increased releasable calcium \n6 \npool in the ER. The data indicate that at least a fraction of Bcl-2 in both AR42J cells \nand pancreatic acinar cells locates in the ER and is present in close proximity to the \nplasma membrane, making possible the direct regulation of the PMCA. \nIn conclusion, this study provides new insights into roles of Bcl-2 family proteins \nand calmodulin in intracellular calcium homeostasis. This thesis presents evidence \nfor involvement of anti-apoptotic Bcl-2 members in Ca2+-induced Ca2+ release; \ndemonstrates previously unknown regulation of the PMCA by Bcl-2; and suggests \ninvolvement of Bax protein in regulation of Ca2+ release from the intracellular stores. \nThe study also proposes that Ca2+-like peptides can boost natural protective \nmechanisms and suggests their potential applications as therapeutic agents.