Partial inhibition of SERCA is responsible for extracellular Ca2+ dependence of AlF–4-induced [Ca2+]i oscillations in rat pancreatic

2003 ◽  
Vol 285 (5) ◽  
pp. C1142-C1149 ◽  
Author(s):  
Seon Ah Chong ◽  
Soo Young Hong ◽  
Seok Jun Moon ◽  
Jee Won Park ◽  
Jeong-Hee Hong ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
G Proteins ◽  
Acinar Cells ◽  
Cell Types ◽  
Pancreatic Acinar Cells ◽  
Protein Activation ◽  
Partial Inhibition ◽  
G Protein Activation ◽  
Inositol 1,4,5 Trisphosphate ◽  
Intracellular Ca

AlF4-is known to generate oscillations in intracellular Ca2+ concentration ([Ca2+]i) by activating G proteins in many cell types. However, in rat pancreatic acinar cells, AlF4--evoked [Ca2+]i oscillations were reported to be dependent on extracellular Ca2+, which contrasts with the [Ca2+]i oscillations induced by cholecystokinin (CCK). Therefore, we investigated the mechanisms by which AlF4- generates extracellular Ca2+-dependent [Ca2+]i oscillations in rat pancreatic acinar cells. AlF4--induced [Ca2+]i oscillations were stopped rapidly by the removal of extracellular Ca2+ and were abolished on the addition of 20 mM caffeine and 2 μM thapsigargin, indicating that Ca2+ influx plays a crucial role in maintenance of the oscillations and that an inositol 1,4,5-trisphosphate-sensitive Ca2+ store is also required. The amount of Ca2+ in the intracellular Ca2+ store was decreased as the AlF4--induced [Ca2+]i oscillations continued. Measurement of 45Ca2+ influx into isolated microsomes revealed that AlF4-directly inhibited sarco/endoplasmic reticulum Ca2+-ATPase (SERCA). The activity of plasma membrane Ca2+-ATPase during AlF4- stimulation was not significantly different from that during CCK stimulation. After partial inhibition of SERCA with 1 nM thapsigargin, 20 pM CCK-evoked [Ca2+]i oscillations were dependent on extracellular Ca2+. This study shows that AlF4- induces [Ca2+]i oscillations, probably by inositol 1,4,5-trisphosphate production via G protein activation but that these oscillations are strongly dependent on extracellular Ca2+ as a result of the partial inhibition of SERCA.

Calcium sensitization produced by G protein activation in airway smooth muscle

2001 ◽  
Vol 281 (3) ◽  
pp. L631-L638 ◽  
Author(s):  
Hayashi Yoshimura ◽  
Keith A. Jones ◽  
William J. Perkins ◽  
Tetsuya Kai ◽  
David O. Warner
Keyword(s):  
Smooth Muscle ◽  
G Proteins ◽  
Myosin Light Chain ◽  
Calcium Sensitization ◽  
Protein Activation ◽  
Maximal Force ◽  
Acute Response ◽  
Regulatory Myosin Light Chain ◽  
G Protein Activation ◽  
Intracellular Ca

We determined whether activation of G proteins can affect the force developed for a given intracellular Ca2+ concentration ([Ca2+]; i.e., the Ca2+ sensitivity) by mechanisms in addition to changes in regulatory myosin light chain (rMLC) phosphorylation. Responses in α-toxin-permeabilized canine tracheal smooth muscle were determined with Ca2+ alone or in the presence of ACh, endothelin-1 (ET-1), or aluminum fluoride (AlF[Formula: see text]; acute or 1-h exposure). Acute exposure to each compound increased Ca2+sensitivity without changing the response to high [Ca2+] (maximal force). However, chronic exposure to AlF[Formula: see text], but not to chronic ACh or ET-1, increased maximal force by increasing the force produced for a given rMLC phosphorylation. Studies employing thiophosphorylation of rMLC showed that the increase in force produced by chronic AlF[Formula: see text] exposure required Ca2+during activation to be manifest. Unlike the acute response to receptor agonists, which is mediated solely by increases in rMLC phosphorylation, chronic direct activation of G proteins further increases Ca2+ sensitivity in airways by additional mechanisms that are independent of rMLC phosphorylation.

Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles

2007 ◽  
Vol 293 (6) ◽  
pp. G1333-G1338 ◽  
Author(s):  
Svetlana G. Voronina ◽  
Mark W. Sherwood ◽  
Oleg V. Gerasimenko ◽  
Ole H. Petersen ◽  
Alexei V. Tepikin
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Acinar Cells ◽  
Cell Types ◽  
Living Cells ◽  
Pancreatic Acinar Cells ◽  
Extracellular Solution ◽  
Whole Cell Patch Clamp ◽  
Patch Pipette ◽  
Cellular Organelles

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.

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

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.

scholarly journals Direct Interactions with Gαi and Gβγ Mediate Nongenomic Signaling by Estrogen Receptor α

2007 ◽  
Vol 21 (6) ◽  
pp. 1370-1380 ◽  
Author(s):  
Premlata Kumar ◽  
Qian Wu ◽  
Ken L. Chambliss ◽  
Ivan S. Yuhanna ◽  
Susanne M. Mumby ◽  
...  
Keyword(s):  
Estrogen Receptor ◽  
G Protein ◽  
G Proteins ◽  
Cell Types ◽  
Estrogen Receptor Α ◽  
Erk Activation ◽  
Protein Activation ◽  
C Terminus ◽  
G Protein Activation ◽  
Oxide Synthase

Abstract Estrogen induces G protein-dependent nongenomic signaling in a variety of cell types via the activation of a plasma membrane-associated subpopulation of estrogen receptor α (ERα). Using pull-down experiments with purified recombinant proteins, we now demonstrate that ERα binds directly to Gαi and Gβγ. Mutagenesis and the addition of blocking peptide reveals that this occurs via amino acids 251–260 and 271–595 of ERα, respectively. Studies of ERα complexed with heterotrimeric G proteins further show that estradiol causes the release of both Gαi and Gβγ without stimulating GTP binding to Gαi. Moreover, in COS-7 cells, the disruption of ERα-Gαi interaction by deletion mutagenesis of ERα or expression of blocking peptide, as well as Gβγ sequestration with β-adrenergic receptor kinase C terminus, prevents nongenomic responses to estradiol including src and erk activation. In endothelial cells, the disruption of ERα-Gαi interaction prevents estradiol-induced nitric oxide synthase activation and the resulting attenuation of monocyte adhesion that contributes to estrogen-related cardiovascular protection. Thus, through direct interactions, ERα mediates a novel mechanism of G protein activation that provides greater diversity of function of both the steroid hormone receptor and G proteins.

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

2021 ◽  
Vol 134 (3) ◽  
pp. jcs244012 ◽  
Author(s):  
Miguel Burgos ◽  
Reginald Philippe ◽  
Fabrice Antigny ◽  
Paul Buscaglia ◽  
Emmanuelle Masson ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Chronic Pancreatitis ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Endoplasmic Reticulum Calcium ◽  
Ef Hand ◽  
Pump Activity ◽  
Intracellular Ca ◽  
Serca Pump ◽  
Main Regulator

ABSTRACTSince deregulation of intracellular Ca2+ can lead to intracellular trypsin activation, and stromal interaction molecule-1 (STIM1) 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) conformational change and enhanced SERCA pump activity leading to increased store-operated Ca2+ entry (SOCE). In 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.This article has an associated First Person interview with the first author of the paper.

Direct G protein activation reverses impaired CCK signaling in human gallbladders with cholesterol stones

1995 ◽  
Vol 269 (5) ◽  
pp. G659-G665 ◽  
Author(s):  
P. Yu ◽  
Q. Chen ◽  
K. M. Harnett ◽  
J. Amaral ◽  
P. Biancani ◽  
...  
Keyword(s):  
G Protein ◽  
G Proteins ◽  
High Performance ◽  
Second Messengers ◽  
Aluminum Fluoride ◽  
Maximal Contraction ◽  
Protein Activation ◽  
G Protein Activation ◽  
Inositol 1,4,5 Trisphosphate ◽  
Layer Chromatography

Human gallbladders were used to investigate the mechanisms of the impaired contraction induced by cholecystokinin (CCK) associated with cholesterol stones. Single muscle cells were isolated enzymatically with collagenase. Inositol 1,4,5-trisphosphate was measured by high-performance liquid chromatography. Diacylglycerol was assayed by thin-layer chromatography. CCK stimulation showed decreased muscle contraction and production of inositol 1,4,5-trisphosphate and diacylglycerol in gallbladders with cholesterol stones compared with those with pigment stones. Exogenous calmodulin induced maximal contraction of 22.4 +/- 0.5 and 21.0 +/- 0.6% in gallbladders with cholesterol and pigment stones, respectively. Similar findings were observed with a synthetic diacylglycerol analogue. Two G protein activators, aluminum fluoride and guanosine 5'-O-(3-thiotriphosphate), evoked similar responses in these two types of gallbladders, with maximal contractions of 21.3 +/- 0.4 and 23.3 +/- 0.5%, respectively, in those with cholesterol stones and 20.9 +/- 0.8 and 22.6 +/- 0.4%, respectively, in those with pigment stones. These results suggest that receptor-dependent ligands like CCK cannot fully activate the intracellular pathways, which, however, can be fully stimulated by circumventing receptors with G protein activators or second messengers. After G protein activation, the pathways appear to be functionally intact. The defect might then reside in the receptor or in the interaction between receptors and G proteins.

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