Emodin attenuates calcium overload and endoplasmic reticulum stress in AR42J rat pancreatic acinar cells

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.

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Cleavage of SNAP-25 and VAMP-2 impairs store-operated Ca2+entry in mouse pancreatic acinar cells

AJP Cell Physiology ◽

10.1152/ajpcell.00241.2004 ◽

2005 ◽

Vol 288 (1) ◽

pp. C214-C221 ◽

Cited By ~ 19

Author(s):

Juan A. Rosado ◽

Pedro C. Redondo ◽

Ginés M. Salido ◽

Stewart O. Sage ◽

Jose A. Pariente

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Secretory Cell ◽

Botulinum Toxin A ◽

Acinar Cells ◽

Pancreatic Acinar Cells ◽

Secretory Proteins ◽

Amylase Secretion ◽

Cell Type ◽

Store Depletion

We recently reported that store-operated Ca2+entry (SOCE) in nonexcitable cells is likely to be mediated by a reversible interaction between Ca2+channels in the plasma membrane and the endoplasmic reticulum, a mechanism known as “secretion-like coupling.” As for secretion, in this model the actin cytoskeleton plays a key regulatory role. In the present study we have explored the involvement of the secretory proteins synaptosome-associated protein (SNAP-25) and vesicle-associated membrane protein (VAMP) in SOCE in pancreatic acinar cells. Cleavage of SNAP-25 and VAMPs by treatment with botulinum toxin A (BoNT A) and tetanus toxin (TeTx), respectively, effectively inhibited amylase secretion stimulated by the physiological agonist CCK-8. BoNT A significantly reduced Ca2+entry induced by store depletion using thapsigargin or CCK-8. In addition, treatment with BoNT A once SOCE had been activated reduced Ca2+influx, indicating that SNAP-25 is needed for both the activation and maintenance of SOCE in pancreatic acinar cells. VAMP-2 and VAMP-3 are expressed in mouse pancreatic acinar cells. Both proteins associate with the cytoskeleton upon Ca2+store depletion, although only VAMP-2 seems to be sensitive to TeTx. Treatment of pancreatic acinar cells with TeTx reduced the activation of SOCE without affecting its maintenance. These findings support a role for SNAP-25 and VAMP-2 in the activation of SOCE in pancreatic acinar cells and show parallels between this process and secretion in a specialized secretory cell type.

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Cytisine induces endoplasmic reticulum stress caused by calcium overload in HepG2 cells

Oncology Reports ◽

10.3892/or.2018.6200 ◽

2018 ◽

Cited By ~ 2

Author(s):

Lei Yu ◽

Bo Jiang ◽

Zhifeng Chen ◽

Xin Wang ◽

Dongxue Shang ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Endoplasmic Reticulum Stress ◽

Hepg2 Cells ◽

Calcium Overload

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p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis

10.1101/2020.01.22.916254 ◽

2020 ◽

Author(s):

Miguel Burgos ◽

Reginald Philippe ◽

Fabrice Antigny ◽

Paul Buscaglia ◽

Emmanuelle Masson ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Chronic Pancreatitis ◽

Acinar Cells ◽

Pancreatic Acinar Cell ◽

Pancreatic Acinar Cells ◽

Endoplasmic Reticulum Calcium ◽

Store Operated Calcium Entry ◽

Intracellular Ca ◽

Intracellular Changes ◽

Main Regulator

ABSTRACTSince deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and STIM1 (stromal interaction molecule-1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile α-motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) interactions and enhanced SERCA pump activity leading to increased Store Operated Calcium Entry (SOCE). In the pancreatic AR42J cells expressing the p.E152K variant, Ca2+-signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation.Summary statementp.E152K-STIM1 variant found in pancreatitis patients leads to intracellular changes in calcium homeostasis through SERCA interaction, enabling intracellular trypsin activation and pancreatic acinar cell death.

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