Critical role for NHE1 in intracellular pH regulation in pancreatic acinar cells

2003 ◽  
Vol 285 (5) ◽  
pp. G804-G812 ◽  
Author(s):  
David A. Brown ◽  
James E. Melvin ◽  
David I. Yule
Keyword(s):  
Intracellular Ph ◽  
Ph Regulation ◽  
Critical Role ◽  
Acinar Cells ◽  
Pancreatic Tissue ◽  
Pancreatic Acinar Cells ◽  
Muscarinic Agonist ◽  
Targeted Disruption ◽  
General Role ◽  
Pancreatic Acini

The primary function of pancreatic acinar cells is to secrete digestive enzymes together with a NaCl-rich primary fluid which is later greatly supplemented and modified by the pancreatic duct. A Na+/H+ exchanger(s) [NHE(s)] is proposed to be integral in the process of fluid secretion both in terms of the transcellular flux of Na+ and intracellular pH (pHi) regulation. Multiple NHE isoforms have been identified in pancreatic tissue, but little is known about their individual functions in acinar cells. The Na+/H+ exchange inhibitor 5-( N-ethyl- N-isopropyl) amiloride completely blocked pHi recovery after an NH4Cl-induced acid challenge, confirming a general role for NHE in pHi regulation. The targeted disruption of the Nhe1 gene also completely abolished pHi recovery from an acid load in pancreatic acini in both [Formula: see text]-containing and [Formula: see text]-free solutions. In contrast, the disruption of either Nhe2 or Nhe3 had no effect on pHi recovery. In addition, NHE1 activity was upregulated in response to muscarinic stimulation in wild-type mice but not in NHE1-deficient mice. Fluctuations in pHi could potentially have major effects on Ca2+ signaling following secretagogue stimulation; however, the targeted disruption of Nhe1 was found to have no significant effect on intracellular Ca2+ homeostasis. These data demonstrate that NHE1 is the major regulator of pHi in both resting and muscarinic agonist-stimulated pancreatic acinar cells.

scholarly journals The ryanodine receptor is expressed in human pancreatic acinar cells and contributes to acinar cell injury

2014 ◽  
Vol 307 (5) ◽  
pp. G574-G581 ◽  
Author(s):  
Christopher M. Lewarchik ◽  
Abrahim I. Orabi ◽  
Shunqian Jin ◽  
Dong Wang ◽  
Kamaldeen A. Muili ◽  
...  
Keyword(s):  
Ryanodine Receptor ◽  
Acinar Cell ◽  
Cell Injury ◽  
Acinar Cells ◽  
Pancreatic Tissue ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Muscarinic Agonist ◽  
Basal Region ◽  
Release Channel

Physiological calcium (Ca2+) signals within the pancreatic acinar cell regulate enzyme secretion, whereas aberrant Ca2+ signals are associated with acinar cell injury. We have previously identified the ryanodine receptor (RyR), a Ca2+ release channel on the endoplasmic reticulum, as a modulator of these pathological signals. In the present study, we establish that the RyR is expressed in human acinar cells and mediates acinar cell injury. We obtained pancreatic tissue from cadaveric donors and identified isoforms of RyR1 and RyR2 by qPCR. Immunofluorescence staining of the pancreas showed that the RyR is localized to the basal region of the acinar cell. Furthermore, the presence of RyR was confirmed from isolated human acinar cells by tritiated ryanodine binding. To determine whether the RyR is functionally active, mouse or human acinar cells were loaded with the high-affinity Ca2+ dye (Fluo-4 AM) and stimulated with taurolithocholic acid 3-sulfate (TLCS) (500 μM) or carbachol (1 mM). Ryanodine (100 μM) pretreatment reduced the magnitude of the Ca2+ signal and the area under the curve. To determine the effect of RyR blockade on injury, human acinar cells were stimulated with pathological stimuli, the bile acid TLCS (500 μM) or the muscarinic agonist carbachol (1 mM) in the presence or absence of the RyR inhibitor ryanodine. Ryanodine (100 μM) caused an 81% and 47% reduction in acinar cell injury, respectively, as measured by lactate dehydrogenase leakage ( P < 0.05). Taken together, these data establish that the RyR is expressed in human acinar cells and that it modulates acinar Ca2+ signals and cell injury.

Pilocarpine and carbachol exhibit markedly different patterns of Ca2+ signaling in rat pancreatic acinar cells

1993 ◽  
Vol 264 (4) ◽  
pp. G786-G791 ◽  
Author(s):  
D. I. Yule ◽  
T. E. Essington ◽  
J. A. Williams
Keyword(s):  
Partial Agonist ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Muscarinic Agonist ◽  
Dependent Manner ◽  
Maximal Concentration ◽  
Concentration Dependent Manner ◽  
Amylase Release ◽  
Pancreatic Acini ◽  
Pi Hydrolysis

The effects of the partial muscarinic agonist pilocarpine on physiological responses were investigated in rat pancreatic acinar cells and compared with carbachol, a full muscarinic agonist, together with previous results using JMV-180, a partial agonist of CCK-A receptors. Pilocarpine was found to stimulate amylase release from isolated pancreatic acini in a concentration-dependent manner. At a maximal concentration (10 microM), pilocarpine was only capable of stimulating 63% of the secretion stimulated by a maximal concentration of carbachol. Moreover pilocarpine did not induce a decrease in secretion at supramaximal concentrations as does carbachol. In acini loaded with fura-2, superfusion of pilocarpine resulted exclusively in generation of intracellular Ca2+ concentration ([Ca2+]i) oscillations at all concentrations tested (0.3 microM-1 mM), in marked contrast to high concentrations of full agonists, which result in a biphasic sustained increase in [Ca2+]i. In common with low concentrations of other secretagogues that stimulate [Ca2+]i oscillations, pilocarpine at all concentrations was only able to stimulate a very small increase in phosphoinositide (PI) hydrolysis. In acini previously incubated with [3H]inositol, pilocarpine was shown to stimulate PI hydrolysis 27% above basal, compared with 872% for carbachol. To ascertain if this small degree of PI hydrolysis seen with pilocarpine is responsible for the generation of [Ca2+]i oscillations, an inhibitor of phospholipase C-linked processes, U-73122, which has been shown to inhibit Ca2+ oscillations induced by carbachol and CCK but not JMV-180 was tested. This agent rapidly inhibited pilocarpine-stimulated oscillations, indicating that in contrast to JMV-180, oscillations induced by pilocarpine are the result of PI hydrolysis.

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)

Effect of intracellular pH on acetylcholine-induced Ca2+ waves in mouse pancreatic acinar cells

1998 ◽  
Vol 275 (3) ◽  
pp. C810-C817 ◽  
Author(s):  
Antonio González ◽  
Fatima Pfeiffer ◽  
Andreas Schmid ◽  
Irene Schulz
Keyword(s):  
Intracellular Ph ◽  
Acinar Cells ◽  
Propagation Rate ◽  
Spreading Speed ◽  
Pancreatic Acinar Cells ◽  
Acidic Ph ◽  
Basolateral Side ◽  
Inositol 1,4,5 Trisphosphate ◽  
The Relationship ◽  
Cytosolic Acidification

We have used fluo 3-loaded mouse pancreatic acinar cells to investigate the relationship between Ca2+ mobilization and intracellular pH (pHi). The Ca2+-mobilizing agonist ACh (500 nM) induced a Ca2+ release in the luminal cell pole followed by spreading of the Ca2+ signal toward the basolateral side with a mean speed of 16.1 ± 0.3 μm/s. In the presence of an acidic pHi, achieved by blockade of the Na+/H+exchanger or by incubation of the cells in a Na+-free buffer, a slower spreading of ACh-evoked Ca2+ waves was observed (7.2 ± 0.6 μm/s and 7.5 ± 0.3 μm/s, respectively). The effects of cytosolic acidification on the propagation rate of ACh-evoked Ca2+ waves were largely reversible and were not dependent on the presence of extracellular Ca2+. A reduction in the spreading speed of Ca2+ waves could also be observed by inhibition of the vacuolar H+-ATPase with bafilomycin A1 (11.1 ± 0.6 μm/s), which did not lead to cytosolic acidification. In contrast, inhibition of the endoplasmic reticulum Ca2+-ATPase by 2,5-di- tert-butylhydroquinone led to faster spreading of the ACh-evoked Ca2+ signals (25.6 ± 1.8 μm/s), which was also reduced by cytosolic acidification or treatment of the cells with bafilomycin A1. Cytosolic alkalinization had no effect on the spreading speed of the Ca2+ signals. The data suggest that the propagation rate of ACh-induced Ca2+ waves is decreased by inhibition of Ca2+ release from intracellular stores due to cytosolic acidification or to Ca2+ pool alkalinization and/or to a decrease in the proton gradient directed from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool to the cytosol.

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.

scholarly journals Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. II. Subcellular localization of the fluorescent probe chlorotetracycline.

1978 ◽  
Vol 76 (2) ◽  
pp. 386-399 ◽  
Author(s):  
D E Chandler ◽  
J A Williams
Keyword(s):  
Divalent Cation ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Ionophore A23187 ◽  
Pancreatic Acini ◽  
Fluorescent Cell ◽  
Mitochondrial Inhibitors ◽  
Cell Compartments ◽  
Large Numbers ◽  
Stimulus Secretion Coupling

Subcellular distribution of the divalent cation-sensitive probe chlorotetracycline (CTC) was observed by fluorescence microscopy in isolated pancreatic acinar cells, dissociated hepatocytes, rod photoreceptors, and erythrocytes. In each cell type, areas containing membranes fluoresced intensely while areas containing no membranes (nuclei and zymogen granules) were not fluorescent. Cell compartments packed with rough endoplasmic reticulum or Golgi vesicles (acinar cells) or plasma membrane-derived membranes (rod outer segments) exhibited a uniform fluorescence. In contrast, cell compartments having large numbers of mitochondria (hepatocytes and the rod inner segment) exhibited a punctate fluorescence. Punctate fluorescence was prominent in the perinuclear and peri-granular areas of isolated acinar cells during CTC efflux, suggesting that under these conditions mitochondrial fluorescence may account for a large portion of acinar cell fluorescence. Fluorometry of dissociated pancreatic acini, preloaded with CTC, showed that application of the mitochondrial inhibitors antimycin A, NaCN, rotenone, or C1CCP, or of the divalent cation ionophore A23187 (all agents known to release mitochondrial calcium) rapidly decreased the fluorescence of acini. In the case of mitochondrial inhibitors, this response could be elicited before but not following the loss of CTC fluorescence induced by bethanechol stimulation. Removal of extracellular Ca2+ and Mg2+ or addition of EDTA also decreased fluorescence but did not prevent secretagogues or mitochondrial inhibitors from eliciting a further response. These data suggest that bethanechol acts to decrease CTC fluorescence at the same intracellular site as do mitochondrial inhibitors. This could be due to release of calcium from either mitochondria or another organelle that requires ATP to sequester calcium.

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