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.\ud Accumulating evidence suggests that they are also involved in regulation of calcium\ud signalling events. Bcl-2 has been reported to affect calcium release from the\ud intracellular calcium stores through regulation of inositol trisphosphate receptors and\ud endoplasmic reticulum calcium pumps. Physiological and pathological processes in\ud pancreatic acinar cells are controlled by calcium. Intracellular Ca2+ signals regulate\ud not only gene expression and trigger enzyme secretion but also might contribute to\ud premature trypsinogen activation and development of pancreatitis, which is\ud characterised by extensive necrosis of the pancreatic tissue. Detailed investigation of\ud Bcl-2 family-dependent mechanisms of intracellular Ca2+ regulation and its\ud association with cell death induction is required for understanding of the basic\ud physiological signalling pathways as well as pathophysiological processes leading to\ud development of severe diseases of pancreas. This study investigates the effects of\ud Bcl-2 family proteins on intracellular calcium homeostasis, with particular focus on\ud their involvement in Ca2+ fluxes and CICR phenomenon. Also, the Ca2+-related\ud actions of different doses of ethanol in pancreatic acinar cells and their contribution\ud to pancreatitis are presented and assessed.\ud The results indicate that pharmacological inhibition of anti-apoptotic Bcl-2 and\ud Bcl-xL proteins with BH3 mimetics BH3I’-2' or HA14-1 sensitizes pancreatic acinar\ud cells to CICR; and overexpression of Bcl-2 has the opposite effect significantly\ud decreasing rising phase of CICR-types of responses in pancreatic cell line AR42J.\ud Responses to BH3 mimetics are at least partially dependent on both IP3Rs and RyRs,\ud 5\ud since inhibition of either of them results in a substantial decrease in Ca2+ release\ud from the intracellular stores. However, simultaneous blockade of IP3Rs and RyRs did\ud not completely abolish BH3 mimetic-elicited Ca2+ release, which indicates\ud engagement of other factors in the development of the response. Importantly, the\ud effects of BH3 mimetics on intracellular Ca2+ were effectively inhibited by loss of\ud Bax protein, suggesting Bax involvement in the regulation of Ca2+ release from\ud the ER.\ud Further, the results presented here demonstrate that moderate concentrations of\ud ethanol (10 - 100 mM), although having only a minor effect on intact cells, induce\ud substantial Ca2+ release from both the ER and the acidic store in permeabilized cells,\ud and trigger intracellular trypsinogen activation - the hallmark of acute pancreatitis.\ud The data suggest that calmodulin, which is present in intact cells but is lost in\ud permeabilized cells, constitutes a part of natural defence mechanism responsible for\ud the differences in the severity of the responses to ethanol. What is more, the evidence\ud indicates that specific pre-activation of calmodulin by Ca2+-like peptides boosts this\ud defence and reduces the pathological calcium responses to ethanol as well as to BH3\ud mimetics in pancreatic acinar cells decreasing necrosis. Finally, the effects of Bcl-2\ud protein on calcium fluxes in pancreatic acinar cells were investigated. Cells lacking\ud functional Bcl-2 showed substantially more rapid clearance of thapsigargin /\ud high Ca2+-induced cytosolic calcium plateau as compared to wild type cells. This\ud effect has been explained by increased activity of the PMCA that results in a marked\ud increase of apoptosis / necrosis ratio in oxidative stress induced cell death.\ud Overexpression of Bcl-2 in AR42J cells reduces ER calcium content, while silencing\ud of Bcl-2 expression by siRNA results in substantially increased releasable calcium\ud 6\ud pool in the ER. The data indicate that at least a fraction of Bcl-2 in both AR42J cells\ud and pancreatic acinar cells locates in the ER and is present in close proximity to the\ud plasma membrane, making possible the direct regulation of the PMCA.\ud In conclusion, this study provides new insights into roles of Bcl-2 family proteins\ud and calmodulin in intracellular calcium homeostasis. This thesis presents evidence\ud for involvement of anti-apoptotic Bcl-2 members in Ca2+-induced Ca2+ release;\ud demonstrates previously unknown regulation of the PMCA by Bcl-2; and suggests\ud involvement of Bax protein in regulation of Ca2+ release from the intracellular stores.\ud The study also proposes that Ca2+-like peptides can boost natural protective\ud mechanisms and suggests their potential applications as therapeutic agents.
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