Supramaximal caerulein stimulation and ultrastructure of rat pancreatic acinar cell: early morphological changes during development of experimental pancreatitis

1984 ◽  
Vol 246 (4) ◽  
pp. G457-G467 ◽  
Author(s):  
O. Watanabe ◽  
F. M. Baccino ◽  
M. L. Steer ◽  
J. Meldolesi
Keyword(s):  
Morphological Changes ◽  
Freeze Fracture ◽  
Acinar Cells ◽  
Pancreatic Acinar Cell ◽  
Experimental Pancreatitis ◽  
Pancreatic Acinar Cells ◽  
Morphological Evidence ◽  
Lysosomal Hydrolases ◽  
Thin Section Electron Microscopy ◽  
Section Electron

Rats infused with a supramaximally stimulating dose of the cholecystokinin-pancreozymin analogue caerulein develop acute interstitial pancreatitis (M. Lampel and H.F. Kern. Virchows Arch. A 373: 97-117, 1977). We have studied the early (30-180 min) morphological changes in pancreatic acinar cells induced by infusing caerulein (2.5 micrograms X kg-1 X h-1). The techniques of thin-section electron microscopy, freeze fracture, and enzyme and immunocytochemistry were employed. Shortly (30 min) after the onset of caerulein infusion, large vacuoles appeared in the Golgi area. After longer periods of infusion, these vacuoles further enlarged (probably by fusion with other such vacuoles as well as autophagic vacuoles) and became more widely distributed in the cytoplasm. These large vacuoles were found to be acid phosphatase positive and to be labeled by antibodies directed against digestive zymogens as well as the lysosomal enzyme cathepsin D. These observations indicate that the large vacuoles contain both digestive zymogens and lysosomal hydrolases. During caerulein infusion, morphological evidence of exocytosis at the luminal plasmalemma was reduced or absent, and evidence of basolateral exocytosis was not noted. These studies suggest that secretagogue hyperstimulation with caerulein interferes with the processes involved in condensing vacuole maturation, which normally lead to the separation of digestive zymogens and lysosomal hydrolases. As a result, both types of enzymes remain within the same compartment. This may lead to the intracellular activation of digestive enzymes by lysosomal hydrolases and be an important step in the development of acute pancreatitis.

Deformation of Yeast Plasma Membrane by Ethidium Bromide

1988 ◽  
Vol 46 ◽  
pp. 254-255
Author(s):  
E. Keyhani
Keyword(s):  
Plasma Membrane ◽  
Thin Section ◽  
Ethidium Bromide ◽  
Freeze Fracture ◽  
Mutagenic Effect ◽  
Yeast Cells ◽  
Hydrophobic Region ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Deep Pits

The mutagenic effect of ethidium bromide on the mitochondrial DNA is well established. Using thin section electron microscopy, it was shown that when yeast cells were grown in the presence of ethidium bromide, besides alterations in the mitochondria, the plasma membrane also showed alterations consisting of 75 to 110 nm-deep pits. Furthermore, ethidium bromide induced an increase in the length and number of endoplasmic reticulum and in the number of intracytoplasmic vesicles.Freeze-fracture, by splitting the hydrophobic region of the membrane, allows the visualization of the surface view of the membrane, and consequently, any alteration induced by ethidium bromide on the membrane can be better examined by this method than by the thin section method.Yeast cells, Candida utilis. were grown in the presence of 35 μM ethidium bromide. Cells were harvested and freeze-fractured according to the procedure previously described.

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.

Acetylcholine Receptors of Cultured Muscle Cells Demonstrated with Ferritin-α-Bungarotoxin Conjugates

1974 ◽  
Vol 16 (2) ◽  
pp. 473-479
Author(s):  
B. T. HOURANI ◽  
B. F. TORAIN ◽  
M. P. HENKART ◽  
R. L. CARTER ◽  
V. T. MARCHESI ◽  
...  
Keyword(s):  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Surface Membrane ◽  
Freeze Fracture ◽  
Muscle Cells ◽  
Muscle Fibres ◽  
Toxin Binding ◽  
Thin Section Electron Microscopy ◽  
Section Electron ◽  
Small Clusters

α-Bungarotoxin-ferritin conjugates were used to visualize by freeze-fracture and thin-section electron microscopy toxin-binding sites, presumably acetylcholine (ACh) receptors, in membranes of muscle cells grown in tissue culture. Toxin conjugated to ferritin by a glutaraldehyde reaction and purified by column chromatography in a buffer of high ionic strength remains active in blocking the effect of iontophoretically applied ACh. The potency of the conjugates was decreased 5-10 times compared to native α-bungarotoxin. Toxin-ferritin conjugates were identified in small clusters which were not uniformly distributed over the surface membrane. Binding was inhibited by preincubation in D-tubocurare or unconjugated toxin. The relation of the clusters to the non-uniform distribution of ACh sensitivity and α-bungarotoxin binding on cultured muscle fibres is discussed.

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.

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.

scholarly journals Membrane interactions between secretion granules and plasmalemma in three exocrine glands

1980 ◽  
Vol 84 (2) ◽  
pp. 438-453 ◽  
Author(s):  
Y Tanaka ◽  
P De Camilli ◽  
J Meldolesi
Keyword(s):  
Plasma Membranes ◽  
Intact Cell ◽  
Freeze Fracture ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Membrane Interactions ◽  
Thin Sections ◽  
Secretion Granules

Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.

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.

Sign in / Sign up

Export Citation Format

Share Document