ABSTRACT
The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca(2+) storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca(2+) that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca(2+) store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca(2+) uptake. Direct measurements with ER-targeted Ca(2+)-sensitive fluorescent "cameleon" proteins revealed that Bcl-2 decreased the free Ca(2+) concentration within the lumen of the ER, [Ca(2+)](ER). Analysis of the kinetics of Ca(2+) store depletion in response to the Ca(2+)-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca(2+) concentration within the ER lumen by increasing the Ca(2+) permeability of the ER membrane. The increased ER Ca(2+) permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.
Subject(s)
Calcium/metabolism , Endoplasmic Reticulum/metabolism , Proto-Oncogene Proteins c-bcl-2/physiology , Animals , Apoptosis/physiology , Calcium Signaling/physiology , Cell Line , Fura-2/analogs & derivatives , Fura-2/metabolism , Genes, bcl-2 , Humans , Intracellular Fluid/metabolism , Mice , Permeability , Recombinant Fusion Proteins/physiology , Transfection , Tumor Cells, CulturedABSTRACT
This study addresses the role of store-operated Ca2+ influx in the regulation of exocytosis in inflammatory cells. In HL-60 granulocytes, which do not possess voltage-operated Ca2+ channels, the chemotactic peptide fMet-Leu-Phe (fMLP) was able to stimulate store-operated Ca2+ influx and to trigger exocytosis of primary granules. An efficient triggering of exocytosis by fMLP required the presence of extracellular Ca2+ and was inhibited by blockers of store-operated Ca2+ influx. However, receptor-independent activation of store-operated Ca2+ influx through thapsigargin did not trigger exocytosis. fMLP was unable to stimulate exocytosis in the absence of cytosolic free Ca2+ concentration [Ca2+]c elevations. However, a second signal generated by fMLP synergized with store-operated Ca2+ influx to trigger exocytosis and led to a left shift of the exocytosis/[Ca2+]c relationship in ionomycin-stimulated cells. The synergistic fMLP-generated signaling cascade was long-lasting, involved a pertussis toxin-sensitive G protein and a phosphatidylinositol 3-kinase. In summary, store-operated Ca2+ influx is crucial for the efficient triggering of exocytosis in HL-60 granulocytes, but, as opposed to Ca2+ influx through voltage-operated Ca2+ channels in neurons, it is not a sufficient stimulus by itself and requires synergistic receptor-generated signals.
Subject(s)
Calcium/metabolism , Exocytosis , Granulocytes/metabolism , Androstadienes/pharmacology , Cytoplasmic Granules/drug effects , Cytoplasmic Granules/metabolism , Drug Synergism , Enzyme Activation , Enzyme Inhibitors/pharmacology , Estrenes/pharmacology , Exocytosis/drug effects , GTP-Binding Proteins/metabolism , Genistein/pharmacology , Granulocytes/cytology , Granulocytes/drug effects , HL-60 Cells , Humans , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Pertussis Toxin , Phosphatidylinositol 3-Kinases/metabolism , Phosphodiesterase Inhibitors/pharmacology , Phospholipase D/metabolism , Pyrrolidinones/pharmacology , Signal Transduction , Thapsigargin/pharmacology , Type C Phospholipases/antagonists & inhibitors , Virulence Factors, Bordetella/pharmacology , WortmanninABSTRACT
The role of the cytosolic free Ca2+ concentration ([Ca2+]c) and its relationship to other second messengers in the signalling between chemoattractant [e.g. N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)] receptors and the NADPH oxidase is still poorly understood. In this study, we have used thapsigargin, an inhibitor of the Ca2+-ATPase of intracellular stores, as a tool to selectively manipulate Ca2+ release from intracellular stores and Ca2+ influx across the plasma membrane. We thereby temporarily separated the Ca2+ signal from other signals generated by fMLP and analysed the consequences on the respiratory burst. Under all conditions investigated, the extent of fMLP-induced respiratory burst activation was critically determined by [Ca2+]c elevation. fMLP was unable to activate the respiratory burst without [Ca2+]c elevation. Thapsigargin-induced Ca2+ influx activated the respiratory burst in the absence of fMLP, but only to approx. 20% of the values observed in the presence of fMLP. The second signal generated by fMLP did not activate the respiratory burst by itself, but acted in synergy with [Ca2+]c elevation. The second signal was long lasting (>15 min) provided that there was no rise in [Ca2+]c and that the receptor was continuously occupied. The second signal was inactivated by high [Ca2+]c elevation. Our results demonstrate that [Ca2+]c elevations are an essential step in the signalling between the fMLP receptor and NADPH oxidase. They also provide novel information about the properties of the second Ca2+-independent signal that activates the respiratory burst in synergy with [Ca2+]c.
