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.

Quantitative electron microscope autoradiographs of 125I-cholecystokinin in pancreatic acini

1982 ◽  
Vol 243 (4) ◽  
pp. G291-G296 ◽  
Author(s):  
J. A. Williams ◽  
H. Sankaran ◽  
E. Roach ◽  
I. D. Goldfine
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Specific Binding ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Pancreatic Acini ◽  
Time Points ◽  
Basolateral Plasma Membrane ◽  
Specific Receptors ◽  
Silver Grains

To morphologically evaluate the interaction of cholecystokinin (CCK) with its receptors on pancreatic acinar cells, we incubated isolated mouse acini at 37 degrees C with radioiodinated CCK and then prepared quantitative electron microscope autoradiographs. Specific binding of CCK to acini was one-half maximal at 2 min of incubation and maximal after 10 min. The cell-associated radioactivity was extracted and analyzed on Sephadex G-50. After 2 min, 90% of the total cellular radioactivity remained as intact CCK; after 30 min, the intact radioactivity decreased to 65% of total. At 2 min, the fraction of bound hormone that fixed to acini was 84% of total; this amount decreased to 78% after 30 min. Thus, the majority of radioactivity in the autoradiographs at both time points was intact CCK; however, at 30 min, a small amount was also degraded hormone. After both 2 and 30 min of incubation, silver grains were highly concentrated over the basolateral plasma membrane. A significant number of grains were in the cell interior at both time points, increasing from 13% of total grains at 2 min to 42% at 30 min. At both times, the largest fraction of internalized grains was localized over the endoplasmic reticulum. At 30 min, a significant concentration of CCK grains was observed over multivesicular bodies. The present study demonstrates, therefore, that CCK binds to specific receptors on the basolateral surface of pancreatic acinar cells. After binding, the hormone is internalized, locates predominantly on the endoplasmic reticulum, and is then degraded.

Calcium oscillations in parotid acinar cells induced by microsomal Ca(2+)-ATPase inhibition

1992 ◽  
Vol 262 (3) ◽  
pp. C656-C663 ◽  
Author(s):  
J. K. Foskett ◽  
D. Wong
Keyword(s):  
Plasma Membrane ◽  
Strong Support ◽  
Cyclopiazonic Acid ◽  
Acinar Cells ◽  
Calcium Oscillations ◽  
Atpase Inhibitor ◽  
Parotid Acinar Cells ◽  
Atpase Inhibitors ◽  
Rat Parotid ◽  
Atpase Inhibition

Previous studies have demonstrated in single rat parotid acinar cells that the microsomal Ca(2+)-ATPase inhibitor thapsigargin mobilizes Ca2+ specifically from the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store, activates plasma membrane Ca2+ permeability, and induces intracellular Ca2+ concentration ([Ca2+]i) oscillations that are quite similar to those activated by carbachol. Nevertheless, the IP3-sensitive Ca2+ store remains continuously depleted during thapsigargin-induced oscillations, indicating that this pool is not involved in the oscillation mechanism. To determine the specificity of thapsigargin's effects, in the present study we have examined the effects on [Ca2+]i in single rat parotid acinar cells of two other microsomal Ca(2+)-ATPase inhibitors, cyclopiazonic acid (CPA) and 2,5-di-tert-butyl-1,4-benzohydroquinone (BHQ), and compared them with the effects of thapsigargin in the same cells. Our results demonstrate that thapsigargin, CPA, and BHQ all similarly deplete the IP3-sensitive Ca2+ store specifically, activate plasma membrane Ca2+ influx, and induce [Ca2+]i oscillations, strongly suggesting that these agents have a specific inhibitory action on microsomal Ca(2+)-ATPase activity. BHQ, in addition, inhibits plasma membrane Ca2+ influx. The data lend strong support to a model in which the state of Ca2+ filling of the IP3-sensitive store regulates plasma membrane Ca2+ influx. These results suggest either that a Ca2+ pump is involved which is insensitive to structurally dissimilar inhibitors or that a Ca2+ pump is not involved in refilling of the Ca2+ pool involved in [Ca2+]i oscillations in these cells.

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.

Ethanol sensitizes NF-κB activation in pancreatic acinar cells through effects on protein kinase C-ε

2006 ◽  
Vol 291 (3) ◽  
pp. G432-G438 ◽  
Author(s):  
Akihiko Satoh ◽  
Anna S. Gukovskaya ◽  
Joseph R. Reeve ◽  
Tooru Shimosegawa ◽  
Stephen J. Pandol
Keyword(s):  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
High Dose ◽  
Pancreatic Acini ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Ethanol Abuse ◽  
Pkc Ζ ◽  
Sensitizing Effect

Although ethanol abuse is the most common cause of pancreatitis, the mechanism of alcohol's effect on the pancreas is not well understood. Previously, we demonstrated that in vitro ethanol treatment of pancreatic acinar cells augmented the CCK-8-induced activation of NF-κB, a key signaling system involved in the inflammatory response of pancreatitis. In the present study, we determine the role for individual PKC isoforms in the sensitizing effect of ethanol on NF-κB activation. Dispersed rat pancreatic acini were treated with and without ethanol and then stimulated with CCK-8; 100 nM CCK-8 caused both NF-κB and PKC-δ, -ε, and -ζ activation, whereas 0.1 nM CCK-8 did not increase PKC-ε, PKC-ζ, or NF-κB activity. CCK-8 (0.1 nM) did activate PKC-δ. PKC-ε activator alone did not cause NF-κB activation; however, together with 0.1 nM CCK-8, it caused NF-κB activation. Ethanol activated PKC-ε without affecting other PKC isoforms or NF-κB activity. Of note, stimulation of acini with ethanol and 0.1 nM CCK-8 resulted in the activation of PKC-δ, PKC-ε, and NF-κB. The NF-κB activation to 0.1 nM CCK-8 in ethanol-pretreated acini was inhibited by both PKC-δ inhibitor and PKC-ε inhibitor. Taken together, these results demonstrate the different modes of activation of PKC isoforms and NF-κB in acini stimulated with ethanol, high-dose CCK-8, and low-dose CCK-8, and furthermore suggest that activation of both PKC-ε and -δ is required for NF-κB activation. These results suggest that ethanol enhances the CCK-8-induced NF-κB activation at least in part through its effects on PKC-ε.

Transport and interaction of nitrogen oxides and NO2 with CO2-HCO3- transporters in pancreatic acini

1994 ◽  
Vol 267 (2) ◽  
pp. C385-C393 ◽  
Author(s):  
H. Zhao ◽  
X. Xu ◽  
K. Ujiie ◽  
R. A. Star ◽  
S. Muallem
Keyword(s):  
Nitric Oxide ◽  
Nitrogen Oxides ◽  
Acinar Cells ◽  
Cellular Proteins ◽  
Pancreatic Acinar Cells ◽  
Disulfonic Acid ◽  
No Production ◽  
Pancreatic Acini ◽  
Cytosolic Acidification ◽  
Stimulation Of

Recently, we showed that NO2- increases gap junction (GJ) permeability and synchronizes intracellular Ca2+ concentration oscillations in pancreatic acini (Loessburg et al., J. Biol. Chem. 268: 19769-19775, 1993). NO2- is also an end product of nitric oxide (NO) production and metabolism. Because of the effect of NO2- on GJ permeability and the possible importance of NO2- in NO metabolism and cytotoxicity, we used pancreatic acinar cells and intracellular pH (pHi) measurements to study the interaction of nitrogen oxides and NO2- with cellular proteins. Exposing cells to NO2- resulted in a concentration-dependent cytosolic acidification. The acidification did not require the transport of NO2- and was not mediated by diffusion of HNO2. Because the acidification was prevented by CO2-HCO3- and inhibition of carbonic anhydrase, it is possible that other nitrogen oxides present in a solution containing NO2- enter the cells by diffusion and interact with OH- or H2O to stably acidify the cytosol. NO2- itself is shown to be transported by the HCO3- transporters present in the plasma membrane. Thus manipulation of the cellular Cl- gradient and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) were used to show Cl-/NO2- exchange, whereas stimulation of external Na(+)-dependent amiloride-insensitive and DIDS-sensitive pHi increase in acidified cells was used to demonstrate a Na(+)-(NO2-)n cotransport. Hence NO2- can be a convenient substitute for HCO3- when studying HCO3- transport in an open system. The studies also show that cellular levels of nitrogen oxides and NO2- can be modulated by the cellular HCO3(-)-buffering system.(ABSTRACT TRUNCATED AT 250 WORDS)

Crambene induces pancreatic acinar cell apoptosis via the activation of mitochondrial pathway

2006 ◽  
Vol 291 (1) ◽  
pp. G95-G101 ◽  
Author(s):  
Yang Cao ◽  
Sharmila Adhikari ◽  
Abel Damien Ang ◽  
Marie Véronique Clément ◽  
Matthew Wallig ◽  
...  
Keyword(s):  
Acinar Cell ◽  
Cell Apoptosis ◽  
Caspase 3 ◽  
Fas Ligand ◽  
Annexin V ◽  
Acinar Cells ◽  
Mitochondrial Pathway ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Pancreatic Acini

We investigated the apoptotic pathway activated by crambene (1-cyano-2-hydroxy-3-butene), a plant nitrile, on pancreatic acinar cells. As evidenced by annexin V-FITC staining, crambene treatment for 3 h induced the apoptosis but not necrosis of pancreatic acini. Caspase-3, -8, and -9 activities in acini treated with crambene were significantly higher than in untreated acini. Treatment with caspase-3, -8, and -9 inhibitors inhibited annexin V staining, as well as caspase-3 activity, pointing to an important role of these caspases in crambene-induced acinar cell apoptosis. The mitochondrial membrane potential was collapsed, and cytochrome c was released from the mitochondria in crambene-treated acini. Neither TNF-α nor Fas ligand levels were changed in pancreatic acinar cells after crambene treatment. These results provide evidence for the induction of pancreatic acinar cell apoptosis in vitro by crambene and suggest the involvement of mitochondrial pathway in pancreatic acinar cell apoptosis.

CRHSP-24 phosphorylation is regulated by multiple signaling pathways in pancreatic acinar cells

2003 ◽  
Vol 285 (4) ◽  
pp. G726-G734 ◽  
Author(s):  
Claus Schäfer ◽  
Hanna Steffen ◽  
Karen J. Krzykowski ◽  
Burkhard Göke ◽  
Guy E. Groblewski
Keyword(s):  
Signaling Pathways ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Dependent Manner ◽  
Calyculin A ◽  
Concentration Dependent Manner ◽  
Pancreatic Acini ◽  
Heat Stable ◽  
Heat Stable Protein ◽  
Mrna Binding

Ca2+-regulated heat-stable protein of 24 kDa (CRHSP-24) is a serine phosphoprotein originally identified as a physiological substrate for the Ca2+-calmodulin regulated protein phosphatase calcineurin (PP2B). CRHSP-24 is a paralog of the brain-specific mRNA-binding protein PIPPin and was recently shown to interact with the STYX/dead phosphatase protein in developing spermatids (Wishart MJ and Dixon JE. Proc Natl Acad Sci USA 99: 2112–2117, 2002). Investigation of the effects of phorbol ester (12- o-tetradecanoylphorbol-13-acetate; TPA) and cAMP analogs in 32P-labeled pancreatic acini revealed that these agents acutely dephosphorylated CRHSP-24 by a Ca2+-independent mechanism. Indeed, cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A, indicating that the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca2+-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B-selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration-dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca2+ dephosphorylated CRHSP-24 on at least three serine sites, whereas cAMP and TPA partially dephosphorylated the protein on at least two sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 μM), suggesting that CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells, including Ca2+, cAMP, and diacylglycerol.

scholarly journals Regulation of transforming growth factor β-induced responses by protein kinase A in pancreatic acinar cells

2008 ◽  
Vol 295 (1) ◽  
pp. G170-G178 ◽  
Author(s):  
Huibin Yang ◽  
Cheong J. Lee ◽  
Lizhi Zhang ◽  
Maria Dolors Sans ◽  
Diane M. Simeone
Keyword(s):  
Protein Kinase ◽  
Protein Kinase A ◽  
Transforming Growth Factor ◽  
Acinar Cells ◽  
Lung Epithelial Cells ◽  
Pancreatic Acinar Cells ◽  
Intracellular Camp ◽  
Physical Interaction ◽  
Pancreatic Acini ◽  
Kinase A

TGF-β is an important regulator of growth and differentiation in the pancreas and has been implicated in pancreatic tumorigenesis. We have recently demonstrated that TGF-β can activate protein kinase A (PKA) in mink lung epithelial cells (Zhang L, Duan C, Binkley C, Li G, Uhler M, Logsdon C, Simeone D. Mol Cell Biol 24: 2169–2180, 2004). In this study, we sought to determine whether TGF-β activates PKA in pancreatic acinar cells, the mechanism by which PKA is activated, and PKA's role in TGF-β-mediated growth regulatory responses. TGF-β rapidly activated PKA in pancreatic acini while having no effect on intracellular cAMP levels. Coimmunoprecipitation experiments demonstrated a physical interaction between a Smad3/Smad4 complex and the regulatory subunits of PKA. TGF-β also induced activation of the PKA-dependent transcription factor CREB. Both the specific PKA inhibitor H89 and PKI peptide significantly blocked TGF-β's ability to activate PKA and CREB. TGF-β-mediated growth inhibition and TGF-β-induced p21 and SnoN expression in pancreatic acinar cells were blocked by H89 and PKI peptide. This study demonstrates that this novel cross talk between TGF-β and PKA signaling pathways may play an important role in regulating TGF-β signaling in the pancreas.

Multiple isoforms of the ryanodine receptor are expressed in rat pancreatic acinar cells

2000 ◽  
Vol 351 (1) ◽  
pp. 265-271 ◽  
Author(s):  
Timothy J. FITZSIMMONS ◽  
Ilya GUKOVSKY ◽  
James A. McROBERTS ◽  
Edward RODRIGUEZ ◽  
F. Anthony LAI ◽  
...  
Keyword(s):  
Western Blot ◽  
Ryanodine Receptor ◽  
Acinar Cell ◽  
Acinar Cells ◽  
Protein Band ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Rt Pcr ◽  
Pancreatic Acini ◽  
Cell Functions

Regulation of cytosolic Ca2+ is important for a variety of cell functions. The ryanodine receptor (RyR) is a Ca2+ channel that conducts Ca2+ from internal pools to the cytoplasm. To demonstrate the presence of the RyR in the pancreatic acinar cell, we performed reverse transcriptase (RT)-PCR, Western blot, immunocytochemistry and microscopic Ca2+-release measurements on these cells. RT-PCR showed the presence of mRNA for RyR isoforms 1, 2 and 3 in both rat pancreas and dispersed pancreatic acini. Furthermore, mRNA expression for RyR isoforms 1 and 2 was demonstrated by RT-PCR in individual pancreatic acinar cells selected under the microscope. Western-blot analysis of acinar cell immunoprecipitates, using antibodies against RyR1 and RyR2, showed a high-molecular-mass (> 250kDa) protein band that was much less intense when immunoprecipitated in the presence of RyR peptide. Functionally, permeablized acinar cells stimulated with the RyR activator, palmitoyl-CoA, released Ca2+ from both basolateral and apical regions. These data show that pancreatic acinar cells express multiple isoforms of the RyR and that there are functional receptors throughout the cell.

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