scholarly journals The effects of ammonia and glutamine on mitochondrial respiration of rat pancreatic acinar cells

2021 ◽  
pp. 105-112
Author(s):  
A. Zub ◽  
◽  
O.V. Manko ◽  
B.O. Manko ◽  
◽  
...  
Keyword(s):  
Respiratory Rate ◽  
Respiration Rate ◽  
Mitochondrial Respiration ◽  
Repeated Measures ◽  
Mitochondrial Permeability Transition ◽  
Statistical Significance ◽  
Acinar Cells ◽  
Permeability Transition ◽  
Pancreatic Acinar Cells ◽  
Pancreatic Acini

During glutamine catabolism is produced ammonia, which can be toxic to cells. In hepatic encephalopathy neuron mitochondria ammonia causes the formation of free radicals, the opening of the mitochondrial permeability transition pore, oxidative phosphorylation disruption and swelling. It is still unknown whether the utilization of glutamine in the mitochondria of acinar cells of the pancreas produces toxic concentrations of ammonia. The experiments were performed on male Wistar rats weighing 250–300 g. Pancreatic acini were isolated using collagenase. Cells were incubated for 30 min with glucose (10 mM) in the control and additionally NH4Cl (5 mM) or glutamine (2 mM) in the experiment. Acetylcholine (10 μM) or cholecystokinin (0.1 nM) was used to stimulate secretion. Respiration rate of isolated rat pancreatic acini was measured using a Clark electrode. Maximum respiration rate was stimulated by addition to the FCCP. Statistical significance (P) of difference between the groups was determined with two-way repeated-measures ANOVA followed by a Holm-Bonferroni corrected post-hoc t tests. The secretagogues acetylcholine and cholecystokinin did not affect basal and FCCP-stimulated respiratory rate. The basal respiratory rate of pancreatic acinar cells decreased with NH4Cl compared to the basal respiratory rate with glucose oxidation, and this decrease was observed both at normal condition and under the action of secretagogues. Glutamine did not affect basal respiratory rate. During glutamine oxidation, the maximum respiratory rate increased compared to the control, regardless of the effect of acetylcholine or cholecystokinin. NH4Cl reduced the maximum rate of FCCP-stimulated respiration in rest or upon stimulation with secretagogues compared to glucose control. Therefore, NH4Cl causes a negative effect mitochondrial respiration regardless of secretory stimulation with acetylcholine or cholecystokinin. The toxic amount of ammonia required for inhibition of mitochondrial respiration is apparently not formed due to glutamine oxidation even when stimulated by acinar cells by secretagogues.

scholarly journals Effects of insulin on adaptive capacity of rat pancreatic acinar cells mitochondria

2020 ◽  
pp. 24-30
Author(s):  
O Bilonoha ◽  
◽  
B.O. Manko ◽  
V Manko ◽  
◽  
...  
Keyword(s):  
Adaptive Capacity ◽  
Repeated Measures ◽  
Energy Demand ◽  
Statistical Significance ◽  
Acinar Cells ◽  
Basal Respiration ◽  
Pancreatic Acinar Cells ◽  
Standard Diet ◽  
Pancreatic Acini ◽  
Pyruvate Oxidation

Insulin increases the basal and agonist-stimulated secretion of pancreatic acinar cells, which leads to increase of energy demand and requires sufficient oxidative substrates supply. Cholecystokinin substantially increases the respiration rate of pancreatic acinar cells upon pyruvate oxidation. However, it is not clear how insulin affects mitochondrial oxidative processes at rest and upon secretory stimulation. Experiments were carried out on male Wistar rats (250–300 g) kept on standard diet. Animals were fasted 12 h before the experiment. Pancreatic acini were isolated with collagenase. Basal and FCCP-stimulated respiration of rat pancreatic acini was measured with Clark electrode. Adaptive capacity of mitochondria was assessed by the maximal rate of uncoupled respiration. Statistical significance (P) of differenced between the means was assessed either with a paired t-test or with repeated measures two-way ANOVA and post-hoc Turkey test. Adaptive capacity of pan­creatic acinar mitochondria was significantly higher when pyruvate (2 mM) was used as oxidative substrate comparing with glucose (10 mM). Incubation with insulin (100 nM) for 20 minutes elevated the basal respiration and adaptive capacity of pancreatic acinar mitochondria upon glucose, but not pyruvate, oxidation. Cholecystokinin (0.1 nM, 30 min) stimulated the rate of basal and maximal uncoupled respiration of acinar cells upon pyruvate oxidation, but insulin completely negated this increase of mitochondrial adaptive capacity. Thus, insulin increases the glucose oxidation in pancreatic acinar cells at resting state, but suppresses pyruvate oxidation upon secretory stimulation with cholecystokinin. The mechanisms of insulin action of pyruvate metabolism in pancreatic acinar cells require further elucidation.

Caspase-8-mediated apoptosis induced by oxidative stress is independent of the intrinsic pathway and dependent on cathepsins

2007 ◽  
Vol 293 (1) ◽  
pp. G296-G307 ◽  
Author(s):  
Heidi K. Baumgartner ◽  
Julia V. Gerasimenko ◽  
Christopher Thorne ◽  
Louise H. Ashurst ◽  
Stephanie L. Barrow ◽  
...  
Keyword(s):  
Mitochondrial Permeability Transition ◽  
Cathepsin L ◽  
Permeability Transition Pore ◽  
Acinar Cells ◽  
Permeability Transition ◽  
Pancreatic Acinar Cells ◽  
Caspase 8 ◽  
Caspase 9 ◽  
Calcium Dependent ◽  
Apoptotic Pathways

Cell-death programs executed in the pancreas under pathological conditions remain largely undetermined, although the severity of experimental pancreatitis has been found to depend on the ratio of apoptosis to necrosis. We have defined mechanisms by which apoptosis is induced in pancreatic acinar cells by the oxidant stressor menadione. Real-time monitoring of initiator caspase activity showed that caspase-9 (66% of cells) and caspase-8 (15% of cells) were activated within 30 min of menadione administration, but no activation of caspase-2, -10, or -12 was detected. Interestingly, when caspase-9 activation was inhibited, activation of caspase-8 was increased. Half-maximum activation ( t0.5) of caspase-9 occurred within ∼2 min and was identified at or in close proximity to mitochondria, whereas t0.5 for caspase-8 occurred within ∼26 min of menadione application and was distributed homogeneously throughout cells. Caspase-9 but not caspase-8 activation was blocked completely by the calcium chelator BAPTA or bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore. In contrast, caspase-8 but not caspase-9 activation was blocked by the destruction of lysosomes (preincubation with Gly-Phe β-naphthylamide, a cathepsin C substrate), loss of lysosomal acidity (bafilomycin A1), or inhibition of cathepsin L or D. Using pepstatin A-BODIPY FL conjugate, we confirmed translocation of cathepsin D out of lysosomes in response to menadione. We conclude that the oxidative stressor menadione induces two independent apoptotic pathways within pancreatic acinar cells: the classical mitochondrial calcium-dependent pathway that is initiated rapidly in the majority of cells, and a slower, caspase-8-mediated pathway that depends on the lysosomal activities of cathepsins and is used when the caspase-9 pathway is disabled.

Activated Pancreatic Stellate Cells Promote Acinar Duct Metaplasia by Disrupting Mitochondrial Respiration and Releasing Reactive Oxygen Species

2021 ◽  
Vol 01 ◽  
Author(s):  
Hong Xiang ◽  
Fangyue Guo ◽  
Qi Zhou ◽  
Xufeng Tao ◽  
Deshi Dong
Keyword(s):  
Reactive Oxygen Species ◽  
Mitochondrial Respiration ◽  
Reactive Oxygen ◽  
Acinar Cells ◽  
Stellate Cells ◽  
Pancreatic Fibrosis ◽  
Pancreatic Stellate Cells ◽  
Pancreatic Acinar Cells ◽  
Oxygen Species

Background: Chronic pancreatitis (CP) is a long-term risk factor for pancreatic ductal adenocarcinoma (PDAC), and both diseases share a common etiology. The activation of Pancreatic stellate cells (PaSCs) caused by inflammation of the chronic pancreas plays a pivotal role in the pathology of pancreatic fibrosis and the malignant phenotype of PDAC. However, the central role of activated PaSCs in acinar-to-ductal metaplasia (ADM) remains unknown. Objective: In the present study, we investigated the link between pancreatic fibrosis and ADM and the possible underlying mechanism. Methods: A caerulein-treated mouse CP model was established, and Masson trichrome histochemical stain and transmission electron microscope (TEM) were used to observe stromal fibrosis and cell ultrastructure, respectively. The expression of amylase and cytokeratin 19 (CK19), mitochondria respiration, and reactive oxygen species (ROS) were detected in vitro in the co-culture model of primary pancreatic acinar cells and PaSCs. Results: The activation of PaSCs and pancreatic fibrosis were accompanied by ADM in pancreatic parenchyma in caerulein-treated mice, which was verified by the co-cultivation experiment in vitro. Furthermore, we showed that activated PaSCs promote ADM by disrupting mitochondrial respiration and releasing ROS. The expression of inflammation-and ADM-related genes, including S100A8, S100A9, and CK19, was observed to be up-regulated in pancreatic acinar cells in the presence of activated PaSCs. The expression of S100A9 and CK19 proteins was also up-regulated in acinar cells co-cultured with activated PaSCs. Conclusion: The manipulation of mitochondrial respiration and ROS release is a promising preventive and/or therapeutic strategy for PDAC, and S100A9 is expected to be a therapeutic target to block the ADM process induced by the activation of PaSCs.

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.

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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