scholarly journals Mechanism and regulation of folate uptake by pancreatic acinar cells: effect of chronic alcohol consumption

2010 ◽  
Vol 298 (6) ◽  
pp. G985-G993 ◽  
Author(s):  
Hamid M. Said ◽  
Lisa Mee ◽  
V. Thillai Sekar ◽  
Balasubramaniem Ashokkumar ◽  
Stephen J. Pandol
Keyword(s):  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Exocrine Function ◽  
Acid Uptake ◽  
Human Pancreas ◽  
Chronic Alcohol ◽  
Transport Inhibitors ◽  
Pancreatic Exocrine ◽  
One Carbon Metabolism

Folate plays an essential role in one-carbon metabolism, and a relationship exists between methyl group metabolism and pancreatic exocrine function. Little, however, is known about the mechanism(s) and regulation of folate uptake by pancreatic acinar cells and the effect of chronic alcohol use on the process. We addressed these issues using the rat-derived pancreatic acinar cell line AR42J and freshly isolated primary rat pancreatic acinar cells as models. We found [3H]folic acid uptake to be 1) temperature and pH dependent with a higher uptake at acidic than at neutral/alkaline pH; 2) saturable as a function of substrate concentration at both buffer pH 7.4 and 6.0; 3) inhibited by folate structural analogs and by anion transport inhibitors at both buffer pH 7.4 and 6.0; 4) trans-stimulated by unlabeled folate; 5) adaptively regulated by the prevailing extracellular folate level, and 6) inhibited by modulators of the cAMP/PKA-mediated pathway. Both the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) were found to be expressed in AR42J and in primary pancreatic acinar cells, as well as in native human pancreas with expression of RFC being higher than PCFT. Chronic alcohol feeding of rats (4 wk; 36% of calories from ethanol) led to a significant decrease in folate uptake by freshly isolated primary pancreatic acinar cells compared with cells from pair-fed controls; this effect was associated with a parallel decrease in the level of expression of RFC and PCFT. These studies reveal that folate uptake by pancreatic acinar cells is via a regulated carrier-mediated process which may involve RFC and PCFT. In addition, chronic alcohol feeding leads to a marked inhibition in folate uptake by pancreatic acinar cells, an effect that is associated with reduction in level of expression of RFC and PCFT.

scholarly journals Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms

2014 ◽  
Vol 307 (9) ◽  
pp. G941-G949 ◽  
Author(s):  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
Arundhati Biswas ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Chronic Exposure ◽  
Methylation Status ◽  
Acinar Cells ◽  
Alcohol Exposure ◽  
Significant Inhibition ◽  
Pancreatic Acinar Cells ◽  
Chronic Alcohol ◽  
Wild Type ◽  
Chronic Alcohol Exposure

Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5′-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na+ dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms.

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.

MiR-155 aggravates impaired autophagy of pancreatic acinar cells through targeting Rictor

2020 ◽  
Vol 52 (2) ◽  
pp. 192-199 ◽  
Author(s):  
Xueming Zhang ◽  
Jiangtao Chu ◽  
Haijun Sun ◽  
Dali Zhao ◽  
Biao Ma ◽  
...  
Keyword(s):  
Acinar Cells ◽  
Immunofluorescence Assay ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Luciferase Reporter ◽  
Cellular Model ◽  
Protein Levels ◽  
Ar42j Cells ◽  
Direct Target ◽  
Dual Luciferase Reporter Assay

Abstract The aim of this study was to investigate the role and mechanism of miR-155 in regulating autophagy in a caerulein-induced acute pancreatitis (AP) cellular model. GFP-LC3 immunofluorescence assay was performed to detect autophagy vesicle formation in pancreatic acinar cell line AR42J. AR42J cells were transfected with miR-155 mimic, inhibitor, and corresponding controls to explore the effect of miR-155 on autophagy. The protein levels of LC3-I, LC3-II, Beclin-1, and p62 were analyzed by western blot analysis. Dual-luciferase reporter assay was performed to verify the interaction between miR-155 and Rictor (RPTOR independent companion of MTOR complex 2). The results showed that caerulein treatment induced impaired autophagy as evidenced by an increase in the accumulation of p62 together with LC3-II in AR42J cells, accompanied by miR-155 upregulation. Furthermore, miR-155 overexpression aggravated, whereas miR-155 silencing reduced the caerulein-induced impairment of autophagy. Mechanistically, Rictor was confirmed to be a direct target of miR-155, which could rescue the miR-155 overexpression-mediated aggravation of impaired autophagy. Collectively, these findings indicate that miR-155 aggravates impaired autophagy in caerulein-treated pancreatic acinar cells by targeting Rictor.

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.

Calcium signaling in rat pancreatic acinar cells: a role for Gαq, Gα11, and Gα14

1999 ◽  
Vol 276 (1) ◽  
pp. G271-G279 ◽  
Author(s):  
David I. Yule ◽  
Christopher W. Baker ◽  
John A. Williams
Keyword(s):  
Cell Line ◽  
G Protein ◽  
Cho Cells ◽  
Acinar Cells ◽  
Cell Types ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Pcr Products ◽  
Α Subunits ◽  
G Protein Α Subunits

Stimulus-secretion coupling in the pancreatic acinar cell is initiated by the secretagogues CCK and ACh and results in the secretion by exocytosis of the contents of zymogen granules. A key event in this pathway is the G protein-activated production of second messengers and the subsequent elevation of cytosolic-free Ca2+. The aim of this study was therefore to define the heterotrimeric G protein α-subunits present and participating in this pathway in rat pancreatic acinar cells. RT-PCR products were amplified from pancreatic acinar cell mRNA with primers specific for Gαq, Gα11, and Gα14 but were not amplified with primers specific for Gα15. The sequences of these PCR products confirmed them to be portions of the rat homologues of Gαq, Gα11, and Gα14. The pancreatic-derived cell line AR42J similarly expressed Gαq, Gα11, and Gα14; however, the Chinese hamster ovary (CHO) cell line only expressed Gα11 and Gαq. These data indicate that caution should be exercised when comparing signal transduction pathways between different cell types. The expression of these proteins in acinar cells was confirmed by immunoblotting samples of acinar membrane protein using specific antisera to the individual G protein α-subunits. The role of these proteins in Ca2+ signaling events was investigated by microinjecting a neutralizing antibody directed against a homologous sequence in Gαq, Gα11, and Gα14 into acinar cells and CHO cells. Ca2+ signaling was inhibited in acinar cells and receptor-bearing CHO cells in response to both physiological and supermaximal concentrations of agonists. The inhibition was >75% in both cell types. These data indicate a role for Gαq and/or Gα11 in intracellular Ca2+ concentration signaling in CHO cells, and in addition to Gαq and Gα11, Gα14 may also fulfill this role in rat pancreatic acinar cells.

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 SEC23B is required for pancreatic acinar cell function in adult mice

2017 ◽  
Vol 28 (15) ◽  
pp. 2146-2154 ◽  
Author(s):  
Rami Khoriaty ◽  
Nancy Vogel ◽  
Mark J. Hoenerhoff ◽  
M. Dolors Sans ◽  
Guojing Zhu ◽  
...  
Keyword(s):  
Acinar Cell ◽  
Cell Function ◽  
Acinar Cells ◽  
Cell Loss ◽  
Normal Function ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Total Protein Content ◽  
Pancreatic Cell ◽  
Adult Mice

Mice with germline absence of SEC23B die perinatally, exhibiting massive pancreatic degeneration. We generated mice with tamoxifen-inducible, pancreatic acinar cell–specific Sec23b deletion. Inactivation of Sec23b exclusively in the pancreatic acinar cells of adult mice results in decreased overall pancreatic weights from pancreatic cell loss (decreased pancreatic DNA, RNA, and total protein content), as well as degeneration of exocrine cells, decreased zymogen granules, and alterations in the endoplasmic reticulum (ER), ranging from vesicular ER to markedly expanded cisternae with accumulation of moderate-density content or intracisternal granules. Acinar Sec23b deletion results in induction of ER stress and increased apoptosis in the pancreas, potentially explaining the loss of pancreatic cells and decreased pancreatic weight. These findings demonstrate that SEC23B is required for normal function of pancreatic acinar cells in adult mice.

scholarly journals Intracellular divalent cation release in pancreatic acinar cells during stimulus-secretion coupling. I. Use of chlorotetracycline as fluorescent probe.

1978 ◽  
Vol 76 (2) ◽  
pp. 371-385 ◽  
Author(s):  
D E Chandler ◽  
J A Williams
Keyword(s):  
Fluorescent Probe ◽  
Spectral Properties ◽  
Divalent Cations ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Rapid Loss ◽  
Amylase Release ◽  
Membrane Bound ◽  
Pancreatic Exocrine ◽  
Stimulus Secretion Coupling

Stimulus-secretion coupling in pancreatic exocrine cells was studied using dissociated acini, prepared from mouse pancreas, and chlorotetracycline (CTC), a fluorescent probe which forms highly fluorescent complexes with Ca2+ and Mg2+ ions bound to membranes. Acini, preloaded by incubation with CTC (100 microM), displayed a fluorescence having spectral properties like that of CTC complexed to calcium (excitation and emission maxima at 398 and 527 nm, respectively). Stimulation with either bethanechol or caerulein resulted in a rapid loss of fluorescence intensity and an increase in outflux of CTC from the acini. After 5 min of stimulation, acini fluorescence had been reduced by 40% and appeared to be that of CTC complexed to Mg2+ (excitation and emission maxima at 393 and 521 nm, respectively). The fluorescence loss induced by bethanechol was blocked by atropine and was seen at all agonist concentrations that elicited amylase release. Maximal fluorescence loss, however, required a bethanechol concentration three times greater than that needed for maximal amylase release. In contrast, acini preloaded with ANS or oxytetracycline, probes that are relatively insensitive to membrane-bound divalent cations, displayed no secretagogue-induced fluorescence changes. These results are consistent with the hypothesis that CTC is able to probe some set of intracellular membranes which release calcium during secretory stimulation and that this release results in dissociation of Ca(2+)-complexed CTC.

scholarly journals Monoclonal antibodies as probes for plasma membrane domains in the exocrine pancreas.

1988 ◽  
Vol 36 (8) ◽  
pp. 1043-1051 ◽  
Author(s):  
R C De Lisle ◽  
C D Logsdon ◽  
S R Hootman ◽  
J A Williams
Keyword(s):  
Monoclonal Antibodies ◽  
Acinar Cell ◽  
Apical Membrane ◽  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Apical Surface ◽  
Membrane Domains ◽  
Monolayer Cultures ◽  
Apical Membranes

Monoclonal antibodies (mAb) were generated as probes for the plasma membrane domains of pancreatic acinar cells. Primary monolayer cultures of mouse pancreatic acinar cells, which have an expanded apical surface relative to normal pancreas, were used to immunize rats. With conventional immunization and fusion protocols, 3% of the hybridomas were positive against the acinar lumen by indirect immunofluorescence of mouse pancreas cryosections. Culturing of spleen cells from an immunized rat on the apical surface of acinar cell monolayer cultures before fusion with the myeloma (an in vitro boost) doubled the percentage of hybridomas producing apical membrane-specific mAb. Monoclonal antibodies were characterized by immunofluorescence, ultrastructural immunoperoxidase cytochemistry, immunoprecipitation, and immunoblotting. One antibody, acinar-1 (IgG2a), labeled the apical membranes of pancreatic acinar cells, hepatocytes, salivary and lacrimal gland acinar cells, and the brush border of small intestine enterocytes. This mAb precipitated and blotted a protein of 94 KD. Acinar-2 (IgM) also labeled pancreatic acinar cell apical membranes but did not label other tissues and did not precipitate or blot. Acinar-3 labeled pancreatic acinar cell lateral membranes. Duct-1 (IgM) labeled pancreatic duct apical membrane and ducts in liver and salivary glands but did not precipitate or blot. These domain-specific mAb demonstrate that common antigenic determinants occur in the apical surfaces of several exocrine epithelia and may be important in secretion.

scholarly journals Upregulation of dynamin II expression during the acquisition of a mature pancreatic acinar cell phenotype.

1996 ◽  
Vol 44 (12) ◽  
pp. 1373-1378 ◽  
Author(s):  
T A Cook ◽  
K J Mesa ◽  
B A Gebelein ◽  
R A Urrutia
Keyword(s):  
Acinar Cell ◽  
Growth Factor Receptor ◽  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Pancreatic Acinar Cells ◽  
Cell Phenotype ◽  
Receptor Mediated Endocytosis ◽  
Ar42j Cells

Members of the dynamin superfamily are GTPases which have been shown to support receptor-mediated endocytosis in vivo and bind to growth factor receptor-associated proteins in vitro. In acinar cells of the pancreas, receptor-mediated endocytosis is very important for the recycling of membranes after secretory granule release. Therefore, characterization of the molecular machinery responsible for this process is critical for a better understanding of this phenomenon. In this study we sought to determine the expression pattern of the endocytic GTPase dynamin II during pancreatic acinar cell differentiation in developing rat embryos and in dexamethasone-treated AR42J cells using Western blot, Northern blot, and immunocytochemical analyses. During pancreatic development, dynamin immunoreactivity is almost undetectable until day E17 but undergoes significant upregulation in acinar cells starting at E18. In addition, the levels of dynamin mRNA and protein in AR42J cells increase approximately threefold during dexamethasone-induced acinar differentiation. The increase in dynamin levels that occurs in both embryonic pancreatic cells and dexamethasone-treated AR42J cells correlates with the establishment of a more differentiated acinar phenotype. Therefore, these results suggest a potential role for dynamin in supporting receptor-mediated endocytosis in mature pancreatic acinar cells.

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