scholarly journals Oxidant-impaired intracellular Ca2+ signaling in pancreatic acinar cells: role of the plasma membrane Ca2+-ATPase

2007 ◽  
Vol 293 (3) ◽  
pp. C938-C950 ◽  
Author(s):  
Jason I. E. Bruce ◽  
Austin C. Elliott
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Cell Injury ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Dependent Manner ◽  
Atpase Inhibitors

Pancreatitis is an inflammatory disease of pancreatic acinar cells whereby intracellular calcium concentration ([Ca2+]i) signaling and enzyme secretion are impaired. Increased oxidative stress has been suggested to mediate the associated cell injury. The present study tested the effects of the oxidant, hydrogen peroxide, on [Ca2+]i signaling in rat pancreatic acinar cells by simultaneously imaging fura-2, to measure [Ca2+]i, and dichlorofluorescein, to measure oxidative stress. Millimolar concentrations of hydrogen peroxide increased cellular oxidative stress and irreversibly increased [Ca2+]i, which was sensitive to antioxidants and removal of external Ca2+, and ultimately led to cell lysis. Responses were also abolished by pretreatment with (sarco)endoplasmic reticulum Ca2+-ATPase inhibitors, unless cells were prestimulated with cholecystokinin to promote mitochondrial Ca2+ uptake. This suggests that hydrogen peroxide promotes Ca2+ release from the endoplasmic reticulum and the mitochondria and that it promotes Ca2+ influx. Lower concentrations of hydrogen peroxide (10–100 μM) increased [Ca2+]i and altered cholecystokinin-evoked [Ca2+]i oscillations with marked heterogeneity, the severity of which was directly related to oxidative stress, suggesting differences in cellular antioxidant capacity. These changes in [Ca2+]i also upregulated the activity of the plasma membrane Ca2+-ATPase in a Ca2+-dependent manner, whereas higher concentrations (0.1–1 mM) inactivated the plasma membrane Ca2+-ATPase. This may be important in facilitating “Ca2+ overload,” resulting in cell injury associated with pancreatitis.

scholarly journals Oxidant-induced inhibition of the plasma membrane Ca2+-ATPase in pancreatic acinar cells: role of the mitochondria

2008 ◽  
Vol 295 (5) ◽  
pp. C1247-C1260 ◽  
Author(s):  
Erin M. Baggaley ◽  
Austin C. Elliott ◽  
Jason I. E. Bruce
Keyword(s):  
Oxidative Stress ◽  
Plasma Membrane ◽  
Cell Injury ◽  
Mitochondrial Permeability Transition ◽  
Acinar Cells ◽  
Atp Depletion ◽  
Pancreatic Acinar Cells ◽  
Spatiotemporal Pattern ◽  
Mitochondrial Depolarization

Impairment of the normal spatiotemporal pattern of intracellular Ca2+ ([Ca2+]i) signaling, and in particular, the transition to an irreversible “Ca2+ overload” response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca2+ overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H2O2) evokes a Ca2+ overload response and inhibition of plasma membrane Ca2+-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938–C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca2+]i clearance assay in which mitochondrial Ca2+ uptake was blocked with Ru-360, H2O2 (50 μM–1 mM) markedly inhibited the PMCA activity. This H2O2-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H2O2-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca2+ handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca2+ overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.

scholarly journals S-Allyl Cysteine Alleviates Hydrogen Peroxide Induced Oxidative Injury and Apoptosis through Upregulation of Akt/Nrf-2/HO-1 Signaling Pathway in HepG2 Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-14 ◽  
Author(s):  
Chitra Basu ◽  
Runa Sur
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Oxidative Damage ◽  
Hepg2 Cells ◽  
Liver Diseases ◽  
Heme Oxygenase 1 ◽  
Related Factor ◽  
Dependent Manner ◽  
Cell Viability Assay

Hydrogen peroxide (H2O2) mediated oxidative stress leading to hepatocyte apoptosis plays a pivotal role in the pathophysiology of several chronic liver diseases. This study demonstrates that S-allyl cysteine (SAC) renders cytoprotective effects on H2O2 induced oxidative damage and apoptosis in HepG2 cells. Cell viability assay showed that SAC protected HepG2 cells from H2O2 induced cytotoxicity. Further, SAC treatment dose dependently inhibited H2O2 induced apoptosis via decreasing the Bax/Bcl-2 ratio, restoring mitochondrial membrane potential (∆Ψm), inhibiting mitochondrial cytochrome c release, and inhibiting proteolytic cleavage of caspase-3. SAC protected cells from H2O2 induced oxidative damage by inhibiting reactive oxygen species accumulation and lipid peroxidation. The mechanism underlying the antiapoptotic and antioxidative role of SAC is the induction of the heme oxygenase-1 (HO-1) gene in an NF-E2-related factor-2 (Nrf-2) and Akt dependent manner. Specifically SAC was found to induce the phosphorylation of Akt and enhance the nuclear localization of Nrf-2 in cells. Our results were further confirmed by specific HO-1 gene knockdown studies which clearly demonstrated that HO-1 induction indeed played a key role in SAC mediated inhibition of apoptosis and ROS production in HepG2 cells, thus suggesting a hepatoprotective role of SAC in combating oxidative stress mediated liver diseases.

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

2005 ◽  
Vol 288 (1) ◽  
pp. C214-C221 ◽  
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.

Role of Pancreatitis-Associated Protein 1 on Oxidative Stress-Induced Cell Death of Pancreatic Acinar Cells

2009 ◽  
Vol 1171 (1) ◽  
pp. 545-548 ◽  
Author(s):  
Joo Weon Lim ◽  
Ji Yeon Song ◽  
Jeong Yeon Seo ◽  
Hyeyoung Kim ◽  
Kyung Hwan Kim
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Pancreatitis Associated Protein

Endoplasmic reticulum Ca2+-ATPase inhibitors stimulate membrane guanylate cyclase in pancreatic acinar cells

2000 ◽  
Vol 278 (2) ◽  
pp. C363-C371 ◽  
Author(s):  
Anna S. Gukovskaya ◽  
Sofiya Gukovsky ◽  
Stephen J. Pandol
Keyword(s):  
Endoplasmic Reticulum ◽  
Guanylate Cyclase ◽  
Cyclopiazonic Acid ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Membrane Guanylate Cyclase ◽  
Intact Cells ◽  
Pancreatic Acini ◽  
Atpase Inhibitors ◽  
Atpase Inhibition

In this study, we show that particulate guanylate cyclase (GC) is present in rat pancreatic acinar cells and is located both on plasma membrane and membranes of endoplasmic reticulum (ER). Western blot analysis indicates that the enzyme isoform GC-A is present in the acinar cell membranes. The specific inhibitors of ER Ca2+-ATPase thapsigargin, 2,5-di-( t-butyl)-1,4-hydroquinone (BHQ), and cyclopiazonic acid all activated particulate GC in pancreatic acini, both in membrane fractions and intact cells. These inhibitors also induced dephosphorylation of GC. Dose dependencies of Ca2+-ATPase inhibition and GC activation by BHQ are very similar, and those for thapsigargin partially overlap. ER Ca2+-ATPase and GC are coimmunoprecipitated both by antisera against membrane GC and by antisera against ER Ca2+-ATPase, suggesting a physical association between the two enzymes. The results suggest that thapsigargin and the other inhibitors act through ER Ca2+-ATPase to activate membrane GC in pancreatic acinar cells, although their direct effect on GC cannot be excluded.

scholarly journals Intracellular Sources of ROS/H2O2 in Health and Neurodegeneration: Spotlight on Endoplasmic Reticulum

Cells ◽  
2021 ◽  
Vol 10 (2) ◽  
pp. 233
Author(s):  
Tasuku Konno ◽  
Eduardo Pinho Melo ◽  
Joseph E. Chambers ◽  
Edward Avezov
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Endoplasmic Reticulum ◽  
Neurodegenerative Diseases ◽  
Antioxidative Enzymes ◽  
Redox Reactions ◽  
Ros Production ◽  
Oxygen Species ◽  
Specific Target

Reactive oxygen species (ROS) are produced continuously throughout the cell as products of various redox reactions. Yet these products function as important signal messengers, acting through oxidation of specific target factors. Whilst excess ROS production has the potential to induce oxidative stress, physiological roles of ROS are supported by a spatiotemporal equilibrium between ROS producers and scavengers such as antioxidative enzymes. In the endoplasmic reticulum (ER), hydrogen peroxide (H2O2), a non-radical ROS, is produced through the process of oxidative folding. Utilisation and dysregulation of H2O2, in particular that generated in the ER, affects not only cellular homeostasis but also the longevity of organisms. ROS dysregulation has been implicated in various pathologies including dementia and other neurodegenerative diseases, sanctioning a field of research that strives to better understand cell-intrinsic ROS production. Here we review the organelle-specific ROS-generating and consuming pathways, providing evidence that the ER is a major contributing source of potentially pathologic ROS.

Effect of oxidative stress on cellular functions and cytosolic free calcium of rat pancreatic acinar cells

1997 ◽  
Vol 272 (6) ◽  
pp. G1489-G1498 ◽  
Author(s):  
H. Klonowski-Stumpe ◽  
R. Schreiber ◽  
M. Grolik ◽  
H. U. Schulz ◽  
D. Haussinger ◽  
...  
Keyword(s):  
Cell Injury ◽  
Cell Damage ◽  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Free Calcium ◽  
Cellular Functions ◽  
Cytosolic Free Calcium ◽  
Catalyzed Oxidation ◽  
Sustained Increase

The present study evaluates the effect of free radicals generated by xanthine oxidase-catalyzed oxidation of hypoxanthine on cellular function of isolated rat pancreatic acinar cells. The results show that a rapid and sustained increase in intracellular Ca2+ concentration ([Ca2+]i) preceded all other morphological and functional alterations investigated. Radical-induced [Ca2+]i increase was largely inhibited by 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester, which prevents Ca2+ release from intracellular stores, but not by Ca2(+)-depleted medium. Radicals released Ca2+ from thapsigargin-insensitive, ryanodine-sensitive intracellular stores, whereas the secretagogue caerulein at physiological concentrations mainly released Ca2+ from thapsigargin-sensitive stores. In contrast to effects of the secretagogue, radical-induced Ca2+ changes did not cause luminal protein secretion but cell death. In single-cell measurements, both secretagogue and radicals induced oscillations of [Ca2+]i. Radical-induced oscillations had a lower frequency but similar amplitude when compared with caerulein-induced oscillations. 1,2-Bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid and ryanodine, which prevented the radical-induced Ca2+ increase without altering the generation of radicals, markedly reduced the radical-induced cell damage. These results suggest that the Ca2+ increase mediates the radical-induced cell injury. The studies also indicate that not only the extent and duration but also the origin of [Ca2+]i release as well as the frequency of Ca2+ oscillations may determine whether a pancreatic acinar cell will secrete or die.

scholarly journals Redistribution of a rab3-like GTP-binding protein from secretory granules to the Golgi complex in pancreatic acinar cells during regulated exocytosis

1994 ◽  
Vol 124 (1) ◽  
pp. 43-53 ◽  
Author(s):  
BP Jena ◽  
FD Gumkowski ◽  
EM Konieczko ◽  
GF von Mollard ◽  
R Jahn ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Binding Protein ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Apical Plasma Membrane ◽  
Gtp Binding Protein ◽  
Regulated Exocytosis ◽  
Gtp Binding

Regulated secretion from pancreatic acinar cells occurs by exocytosis of zymogen granules (ZG) at the apical plasmalemma. ZGs originate from the TGN and undergo prolonged maturation and condensation. After exocytosis, the zymogen granule membrane (ZGM) is retrieved from the plasma membrane and ultimately reaches the TGN. In this study, we analyzed the fate of a low M(r) GTP-binding protein during induced exocytosis and membrane retrieval using immunoblots as well as light and electron microscopic immunocytochemistry. This 27-kD protein, identified by a monoclonal antibody that recognizes rab3A and B, may be a novel rab3 isoform. In resting acinar cells, the rab3-like protein was detected primarily on the cytoplasmic face of ZGs, with little labeling of the Golgi complex and no significant labeling of the apical plasmalemma or any other intracellular membranes. Stimulation of pancreatic lobules in vitro by carbamylcholine for 15 min, resulted in massive exocytosis that led to a near doubling of the area of the apical plasma membrane. However, no relocation of the rab3-like protein to the apical plasmalemma was seen. After 3 h of induced exocytosis, during which time approximately 90% of the ZGs is released, the rab3-like protein appeared to translocate to small vesicles and newly forming secretory granules in the TGN. No significant increase of the rab3-like protein was found in the cytosolic fraction at any time during stimulation. Since the protein is not detected on the apical plasmalemma after stimulation, we conclude that recycling may involve a membrane dissociation-association cycle that accompanies regulated exocytosis.

scholarly journals Septin-dependent compartmentalization of the endoplasmic reticulum during yeast polarized growth

2005 ◽  
Vol 169 (6) ◽  
pp. 897-908 ◽  
Author(s):  
Cosima Luedeke ◽  
Stéphanie Buvelot Frei ◽  
Ivo Sbalzarini ◽  
Heinz Schwarz ◽  
Anne Spang ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Diffusion Barriers ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Protein Diffusion ◽  
Ultrastructural Studies ◽  
Bud Neck ◽  
Er Membrane

Polarized cells frequently use diffusion barriers to separate plasma membrane domains. It is unknown whether diffusion barriers also compartmentalize intracellular organelles. We used photobleaching techniques to characterize protein diffusion in the yeast endoplasmic reticulum (ER). Although a soluble protein diffused rapidly throughout the ER lumen, diffusion of ER membrane proteins was restricted at the bud neck. Ultrastructural studies and fluorescence microscopy revealed the presence of a ring of smooth ER at the bud neck. This ER domain and the restriction of diffusion for ER membrane proteins through the bud neck depended on septin function. The membrane-associated protein Bud6 localized to the bud neck in a septin-dependent manner and was required to restrict the diffusion of ER membrane proteins. Our results indicate that Bud6 acts downstream of septins to assemble a fence in the ER membrane at the bud neck. Thus, in polarized yeast cells, diffusion barriers compartmentalize the ER and the plasma membrane along parallel lines.

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