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WIREs Membr Transp Signal

Monitoring of intra‐ ER free Ca 2+

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The importance of calcium signaling in cell health and disease is the major driving force in current research of intracellular calcium homeostasis. Ca2+ release from the endoplasmic reticulum (ER) and other calcium stores seems to be the crucial factor in the activation of many cellular functions. Significant changes in ER Ca2+ content and dynamics have been implicated in the activation of the ER stress response, abnormal autophagy, and cell death which leads to a variety of pathological conditions. For example, in acute pancreatitis, an inflammatory disease of the exocrine pancreas caused primarily by bile stones or alcohol, excessive intracellular calcium overload due to Ca2+ release from internal stores followed by store operated Ca2+ entry (SOCE) leads to the premature activation of digestive proenzymes within pancreatic acinar cells. Recent data show that SOCE channel blockers are capable of substantially reducing the intracellular Ca2+ overload and subsequent cell necrosis without major alteration of ER Ca2+ content. We also demonstrate here that indirect ER measurements can be misleading and only direct intra‐ER Ca2+ monitoring offers reliable conclusions. In this respect, it is essential to summarize the methods available and provide examples of direct measurements of free Ca2+ concentration [Ca2+] in the ER lumen in pancreatic acinar cells. This article is aimed at highlighting the major techniques for monitoring ER Ca2+ with reference to their advantages, limitations, and views for future improvements. WIREs Membr Transp Signal 2014,3:63–71. doi: 10.1002/wmts.106

Ethanol elicits Ca2+ release from internal stores in permeabilized cells. (Reprinted with permission from Ref . Copyright 2014 National Academy of Sciences). (a) Application of 10 mM ethanol induces a reduction in [Ca2+]store. (b) Addition of CaM (2.5 µM) protects against intracellular Ca2+ release. (c) Comparison of Ca2+ response amplitudes induced by 10 mM ethanol or 100 mM ethanol in the presence or absence of CaM (2.5 µM) or mixture of CaM (2.5 µM) and CaM inhibitory peptide (CaMi) (20 µM). Ca2+ release induced by Ins(1,4,5)P3 (10 µM) was also inhibited by CaM and restored by mixture of CaM (2.5 µM) and CaMi (20 µM). Error bars indicate SEM.
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Two‐photon permeabilization of pancreatic acinar cells and the effects of Ca2+‐releasing messengers (Reprinted with permission from Ref . Copyright 2014 The Company of Biologists Ltd). (a) Pancreatic acinar doublet preloaded with Fluo‐5 N AM. The permeabilization point with infra red light (IR) (735 nm) is shown by the blue dot. (b) Permeabilized cell (the lower cell of the doublet) is stained with Texas Red dextran (excitation 543 nm, emission >600). (c) Perfusion of permeabilized cell with internal K+‐ rich buffer solution leads to reduction of the cytosolic component of Fluo‐5 N and Texas Red dextran fluorescence. Two different ROIs indicate nonpermeabilized (yellow circle) and permeabilized cell (green circle). (d) Transmitted light image of the same cell doublet after permeabilization. E. Ins(1,4,5)P3 (10 µM) elicited reduction in [Ca2+]stores in permeabilized cell (green trace) but not in the other cell.
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Measurements of ER Ca2+ changes using ER targeted fluorescent cameleon D1ER. (Reprinted with permission from Ref . Copyright 2014 Elsevier Inc.). (a) Cyan and yellow fluorescence of AR42J cells transfected with D1ER cameleon. (b) Comparison of relative changes of emission ratio YFP/CFP before and after treatment with 20 µM CPA, normalized to controls. (c) Average traces (including mean values and standard errors) of cytosolic calcium responses to application of 10 µM thapsigargin in Fura‐2‐loaded pancreatic acinar cells isolated from WT (blue trace) and Bcl‐2 KO (red trace) mice represented as ratio changes. The initial increase in cytosolic [Ca2+] induced by thapsigargin is followed by Ca2+ extrusion across the plasma membrane. The change in amplitude is not significant; however, comparison of the areas under the curve does show different values.
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