ANF and exocrine pancreas: ultrastructural autoradiographic localization in acinar cells

1988 ◽  
Vol 254 (3) ◽  
pp. E301-E309 ◽  
Author(s):  
J. G. Chabot ◽  
G. Morel ◽  
M. Belles-Isles ◽  
L. Jeandel ◽  
S. Heisler
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Binding Sites ◽  
Exocrine Pancreas ◽  
Plasma Membranes ◽  
Acinar Cells ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Zymogen Granules ◽  
Sites Of Action

Atrial natriuretic factor (ANF) binding sites have been recently demonstrated to be present in exocrine pancreas by an in vitro autoradiographic approach. An autoradiographic study was carried out to identify the exocrine cells containing ANF binding sites and to monitor the fate of 125I-labeled ANF in acinar cells after removal of pancreas at specific time intervals (1-30 min) after intravenous administration. At the light microscopic level, silver grains were found over acinar and centroacinar cells. Concomitant injection of an excess of unlabeled ANF inhibited the binding of labeled peptide by approximately 60%. At the electron microscopic level, the time-course study in acinar cells has revealed that of the cell compartments examined, plasma membrane, Golgi apparatus, mitochondria, and zymogen granules, the nucleus had distinct labeling patterns. Plasma membrane was maximally labeled 1 and 2 min after injection with 125I-ANF. Golgi apparatus was significantly labeled from 2 to 30 min after injection, mitochondria from 1 to 30 min after injection, zymogen granules at 1 and 15 min, and the nucleus only at 30 min. The lysosomal compartment was not labeled during the 30-min observation period. These results suggest that after binding to the plasma membrane, ANF is rapidly internalized and distributed to the intracellular organelles as a function of time. Labeling of the zymogen granules suggests that they may bind ANF and that the atrial peptide may be secreted by acinar cells. The significance of association of radioactivity with mitochondria and nuclei remains to be elucidated but may represent intracellular sites of action of ANF complementary to those on plasma membranes.

scholarly journals The fine structure produced in cells by primary fixatives 2. Potassium dichromate

1965 ◽  
Vol s3-106 (73) ◽  
pp. 15-21
Author(s):  
JOHN R. BAKER
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Fine Structure ◽  
Golgi Apparatus ◽  
Nuclear Membrane ◽  
Osmium Tetroxide ◽  
Potassium Dichromate ◽  
Potassium Dichromate Solution ◽  
Zymogen Granules ◽  
Mouse Pancreas

The exocrine cells of the mouse pancreas were fixed in potassium dichromate solution, embedded in araldite or other suitable medium, and examined by electron microscopy. Almost every part of these cells is seriously distorted or destroyed by this fixative. The ergastoplasm is generally unrecognizable, the mitochondria and zymogen granules are seldom visible, and no sign of the plasma membrane, microvilli, or Golgi apparatus is seen. The contents of the nucleus are profoundly rearranged. It is seen to contain a large, dark, irregularly shaped, finely granular object; the evidence suggests that this consists of coagulated histone. The sole constituent of the cell that is well fixed is the inner nuclear membrane. The destructive properties of potassium dichromate are much mitigated when it is mixed in suitable proportions with osmium tetroxide or formaldehyde.

Agonist-induced internalisation of the angiotensin II-binding sites from plasma membranes of isolated rat hepatocytes

1997 ◽  
Vol 152 (3) ◽  
pp. 407-412 ◽  
Author(s):  
M Montiel ◽  
M C Caro ◽  
E Jiménez
Keyword(s):  
Plasma Membrane ◽  
Angiotensin Ii ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Binding Capacity ◽  
Rat Hepatocytes ◽  
Receptor Complex ◽  
Ang Ii ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

Angiotensin II (Ang II) provokes rapid internalisation of its receptor from plasma membranes in isolated rat hepatocytes. After 10 min stimulation with Ang II, plasma membrane lost about 60% of its 125I-Ang II-binding capacity. Internalisation was blocked by phenylarsine oxide (PhAsO), whereas okadaic acid, which markedly reduced the sustained phase of calcium mobilization, did not have a preventive effect on Ang II–receptor complex sequestration. These data suggest that Ang II receptor internalisation is probably independent of a phosphorylation/dephosphorylation cycle of critical serine/threonine residues in the receptor molecule. To establish a relationship between sequestration of the Ang II receptor and the physical properties of the Ang II-binding sites, 125I-Ang II–receptor complex profiles were analysed by isoelectric focusing. In plasma membrane preparations two predominant Ang II-binding sites, migrating to pI 6·8 and 6·5 were found. After exposure to Ang II, cells lost 125I-Ang II-binding capacity to the Ang II–receptor complex migrating at pI 6·8 which was prevented in PhAsO-treated cells. Pretreatment of hepatocytes with okadaic acid did not modify Ang II–receptor complex profiles, indicating that the binding sites corresponding to pI 6·5 and pI 6·8 do not represent a phosphorylated and/or non-phosphorylated form of the Ang II receptor. The results show that the Ang II–receptor complex isoform at pI 6·8 represents a functional form of the type-1 Ang II receptor. Further studies are necessary to identify the Ang II-related nature of the binding sites corresponding to pI 6·5. Journal of Endocrinology (1997) 152, 407–412

scholarly journals Partial purification and characterization of oestrogen receptors in subfractions of hepatocyte plasma membranes

1980 ◽  
Vol 191 (3) ◽  
pp. 743-760 ◽  
Author(s):  
Richard J. Pietras ◽  
Clara M. Szego
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Wheat Germ Agglutinin ◽  
Wheat Germ ◽  
Plasma Membranes ◽  
Specific Binding ◽  
Low Density ◽  
Marker Enzymes ◽  
Low Density Plasma ◽  
Density Plasma

To assess the subcellular distribution of oestrogen-binding components in their native state, plasma membrane and other cell fractions were prepared from hepatocytes in the absence of [3H]oestradiol-17β. Cells from livers of ovariectomized rats were disrupted, with submaximal homogenization in buffered isotonic sucrose with CaCl2 and proteinase inhibitor, and fractionated by using isotonic media. Fractions were characterized by determinations of enzyme activities, biochemical constituents and ligand binding. Specific binding of 2nm-[3H]oestradiol-17β to intact cells and their fractions was detemined after equilibration for 1.5h at 4°C. More than 92% of the radioactivity from representative preparations was verified as authentic oestradiol by thin-layer chromatography. Activities of plasma-membrane marker enzymes as well as binding sites for oestrogen and for wheat germ agglutinin were present principally in particulate fractions, rather than in 105000g-supernatant fractions. However, by using alternative homogenization procedures (i.e. hypotonic media), known to fragment and strip structural components, oestradiol-binding sites and activities of plasma-membrane marker enzymes were distributed predominantly into cytosol. By using the more conservative procedures, plasma membranes of low (ρ=1.13–1.16) and high (ρ=1.16–1.18) density were purified from crude nuclear fractions. A second low-density subfraction of plasma membrane was prepared from microsome-rich fractions. Activities of plasma-membrane marker enzymes were enriched to about 28 and four times that of the homogenate in plasma membranes of low and high density respectively. Binding sites for wheat germ agglutinin and oestradiol were concentrated in low-density plasma membranes to 46–63 times that of the homogenate. Specific binding of oestrogen in low-density plasma membranes purified from crude nuclei was saturable, with an apparent association constant of 3.5nm. At saturation, such oestradiol receptors corresponded to 526fmol/mg of membrane protein. A Hill plot showed a moderate degree of positive co-operativity in the interaction of hormone with plasma membranes. Specific binding of [3H]oestradiol-17β was reduced by a 200-fold molar excess of unlabelled oestradiol-17β, oestriol or diethylstilbestrol, but not by oestradiol-17α, cortisol, testosterone or progesterone. Binding was also blocked by prior exposure of membranes to trypsin or to 60°C, but remained essentially undiminished by extraction of membranes with either hypotonic or high-salt buffers. Extraction with 0.1% (v/v) Triton X-100 partially solubilized the oestrogen-binding component(s) of plasma membranes. Particle-free extracts were resolved on 5–20% (w/v) sucrose density gradients with either 0.01m- or 0.4m-KCl, and the fractions were analysed by adsorption to hydroxyapatite. In low-salt gradients macromolecule-bound oestrogen sedimented at predominantly 7.4S and binding was 1560 times that of the homogenate. Under high-salt conditions oestradiol-binding activity occurred at both 3.6S and 4.9S.

scholarly journals Localization of 125I-atrial natriuretic factor (ANF)-binding sites in rat renal medulla. A light and electron microscope autoradiographic study.

1987 ◽  
Vol 35 (2) ◽  
pp. 149-153 ◽  
Author(s):  
C Bianchi ◽  
J Gutkowska ◽  
R Garcia ◽  
G Thibault ◽  
J Genest ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Binding Sites ◽  
Atrial Natriuretic Factor ◽  
Renal Medulla ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Natriuretic Factor ◽  
Vasa Recta ◽  
Collecting Ducts ◽  
Atrial Natriuretic

Using light and electron microscope autoradiography in vivo, the localization of 125I-(Arg 101-Tyr 126) atrial natriuretic factor (ANF)-binding sites was studied in the renal medulla of rats. At the light microscopic level, the autoradiographic reaction was mainly distributed in patches in the outer medulla, and followed the tubular architecture in the innermost part of the inner medulla. At the electron microscopic level, binding sites were mainly found in the outer medullary descending vasa recta and inner medullary collecting ducts. These results suggest that, in rats, the renal medulla may participate in the natriuresis and diuresis produced by ANF through vascular and tubular effects; the former by changing medullary blood flow at the level of descending vasa recta and the latter by acting on electrolyte and water transport at the level of collecting ducts.

scholarly journals CHANGES IN PANCREATIC ACINAR CELLS DURING PROTEIN DEPRIVATION

1962 ◽  
Vol 12 (2) ◽  
pp. 313-327 ◽  
Author(s):  
Bernard Weisblum ◽  
Lawrence Herman ◽  
Patrick J. Fitzgerald
Keyword(s):  
Golgi Apparatus ◽  
Nuclear Matrix ◽  
Pancreatic Enzyme ◽  
Acinar Cells ◽  
Normal Structure ◽  
Pancreatic Acinar Cells ◽  
Zymogen Granules ◽  
Protein Deprivation ◽  
Rat Pancreas ◽  
Protein Free Diet

After 10 days of a protein-free diet the acinar cells of the rat pancreas showed a coarsening of nuclear matrix, depletion of zymogen granules, some loss of ribosomes, and a widening of the spaces between ergastoplasmic membranes. In addition, there could be found, but rarely, a lesion of the ergastoplasm consisting of vacuoles of agranular, disoriented membranes, which was similar to a lesion produced by ethionine. Thereafter, a return toward normal structure occurred which was characterized by beginning increase in the size of the Golgi apparatus at 12 days, appearance of zymogen granules at 18 days, and a relatively normal appearing but smaller cell at 28 days. After 10 to 12 days of protein deprivation a reversal of many of the morphologic effects of protein deprivation was accompanied by a return toward normal of some pancreatic enzyme activities. Possibly this spontaneous return toward normal levels represented a raiding of protein stores, or it may have been an adaptive phenomenon.

scholarly journals Identification and localization of cholecystokinin-binding sites on rat pancreatic plasma membranes and acinar cells: a biochemical and autoradiographic study.

1983 ◽  
Vol 96 (5) ◽  
pp. 1288-1297 ◽  
Author(s):  
S A Rosenzweig ◽  
L J Miller ◽  
J D Jamieson
Keyword(s):  
Acinar Cell ◽  
Binding Sites ◽  
Plasma Membranes ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Cross Linking ◽  
Dependent Manner ◽  
Electron Microscopic ◽  
Electron Microscopic Autoradiography ◽  
Pancreatic Acini

Using the combined approaches of affinity labeling and light and electron microscopic autoradiography, we investigated the identification and localization of cholecystokinin (CCK)-binding sites on rat pancreatic acinar cells. To define the molecular properties of the CCK-binding site, we incubated rat pancreatic plasma membranes with 125-I-CCK-33 for 15 min at 23 degrees C followed by washing and cross-linking with disuccinimidyl suberate. Specific labeling of a major Mr 85,000 component was revealed as assessed by SDS PAGE under reducing conditions and autoradiography of the dried gels. Components of Mr greater than 200,000, Mr 130,000-140,000, and, Mr 55,000 were labeled under maximal cross-linking conditions. The labeling of all components was specifically inhibited by CCK-8 in a dose-dependent manner (Kd approximately 9 nM). The Mr 85,000 component had identical electrophoretic mobilities under reducing and nonreducing conditions indicating that it likely does not contain intramolecular disulfide bonds. The larger labeled species may be cross-linked oligomers of this binding protein or complexes between it and neighboring polypeptides. For studies on the distribution of CCK-binding sites, pancreatic acini were incubated with 125I-CCK-33 (0.1 nM) in the absence or presence of CCK-8 (1 microM) for 2 or 15 min at 37 degrees C, washed, and fixed in 2% glutaraldehyde. Quantitative autoradiographic analysis indicated that approximately 60% of the total grains were located within +/- 1 HD (1 HD = 100 nm) of the lateral and basal plasmalemma with little or no labeling of the apical plasmalemma. From these data, it was estimated that each acinar cell possesses at least 5,000-10,000 CCK-binding sites on its basolateral plasmalemma. The remaining grains showed no preferential concentration over the cytoplasm or nucleus. Together, these data indicate that CCK interacts with a Mr 85,000 protein located on the basolateral plasmalemma of the pancreatic acinar cell.

Nonparallel secretion of GP-2 from exocrine pancreas implies luminal coupling between acinar and duct cells

1994 ◽  
Vol 267 (1) ◽  
pp. G40-G51 ◽  
Author(s):  
S. D. Freedman ◽  
K. Sakamoto ◽  
G. A. Scheele
Keyword(s):  
Exocrine Pancreas ◽  
Acinar Cells ◽  
Zymogen Granule ◽  
Zymogen Granules ◽  
Pancreatic Acini ◽  
Glycosyl Phosphatidylinositol ◽  
Cellular Processes ◽  
Rat Exocrine Pancreas

The in vivo and in vitro secretion of glycoprotein-2 (GP-2), a glycosyl phosphatidylinositol (GPI)-anchored protein from the rat exocrine pancreas, was characterized. GP-2 was secreted in a nonparallel manner compared with amylase, a marker of secretory enzymes. Attenuated GP-2 secretion correlated with hormones that stimulated exocytosis in acinar cells. Augmented GP-2 secretion correlated with hormones that stimulated fluid and bicarbonate secretion from ductal elements. Immunofluorescence studies identified an enriched pool of GP-2 tightly bound to the apical membranes of acinar cells in addition to zymogen granules. This non-zymogen granule pool appears to represent the source of GP-2 released from acinar cells in a nonparallel manner. With the use of dispersed pancreatic acini largely devoid of ductal elements, GP-2 release was found to be augmented by alkaline pH. Thus GP-2 secretion appears to be modulated by two discrete cellular processes: 1) delivery of prereleased GP-2 within zymogen granules to the ductal lumen by exocytic mechanisms and 2) enzymatic release of GPI-anchored GP-2 from the luminal membranes, a kinetic process that appears to be regulated by secretin- or carbachol-induced secretion of bicarbonate.

An Electron Microscopical Study of Neutral Red Granules in Mouse Exocrine Pancreas

1964 ◽  
Vol s3-105 (70) ◽  
pp. 219-226
Author(s):  
JENNIFER M. BYRNE
Keyword(s):  
Endoplasmic Reticulum ◽  
Electron Microscope ◽  
Golgi Apparatus ◽  
Normal Tissue ◽  
Exocrine Pancreas ◽  
Ultrastructural Level ◽  
Neutral Red ◽  
Microscopical Study ◽  
Zymogen Granules ◽  
Electron Microscopical

The neutral red granule cycle in the mouse exocrine pancreas was studied with the electron microscope in order to discover what changes appear at an ultrastructural level in cells treated with neutral red. There are no changes in the endoplasmic reticulum, the nucleus, the Golgi apparatus, the zymogen granules, or the mitochondria of stained cells when compared with normal tissue. Osmiophil inclusions are found which in their size and distribution correspond to the neutral red granules seen under the light microscope. Such inclusions are not seen in normal tissue. They resemble morphologically the lysosomes of various tissues.

scholarly journals Efficient Trafficking of MDR1/P-Glycoprotein to Apical Canalicular Plasma Membranes in HepG2 Cells Requires PKA-RIIα Anchoring and Glucosylceramide

2006 ◽  
Vol 17 (8) ◽  
pp. 3638-3650 ◽  
Author(s):  
Kacper A. Wojtal ◽  
Erik de Vries ◽  
Dick Hoekstra ◽  
Sven C.D. van IJzendoorn
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Hepg2 Cells ◽  
Plasma Membranes ◽  
De Novo ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Surface Transport ◽  
P Glycoprotein ◽  
Dpp Iv

In hepatocytes, cAMP/PKA activity stimulates the exocytic insertion of apical proteins and lipids and the biogenesis of bile canalicular plasma membranes. Here, we show that the displacement of PKA-RIIα from the Golgi apparatus severely delays the trafficking of the bile canalicular protein MDR1 (P-glycoprotein), but not that of MRP2 (cMOAT), DPP IV and 5′NT, to newly formed apical surfaces. In addition, the direct trafficking of de novo synthesized glycosphingolipid analogues from the Golgi apparatus to the apical surface is inhibited. Instead, newly synthesized glucosylceramide analogues are rerouted to the basolateral surface via a vesicular pathway, from where they are subsequently endocytosed and delivered to the apical surface via transcytosis. Treatment of HepG2 cells with the glucosylceramide synthase inhibitor PDMP delays the appearance of MDR1, but not MRP2, DPP IV, and 5′NT at newly formed apical surfaces, implicating glucosylceramide synthesis as an important parameter for the efficient Golgi-to-apical surface transport of MDR1. Neither PKA-RIIα displacement nor PDMP inhibited (cAMP-stimulated) apical plasma membrane biogenesis per se, suggesting that other cAMP effectors may play a role in canalicular development. Taken together, our data implicate the involvement of PKA-RIIα anchoring in the efficient direct apical targeting of distinct proteins and glycosphingolipids to newly formed apical plasma membrane domains and suggest that rerouting of Golgi-derived glycosphingolipids may underlie the delayed Golgi-to-apical surface transport of MDR1.

scholarly journals Differential filipin labeling of the luminal membranes lining the pancreatic acinus.

1983 ◽  
Vol 31 (7) ◽  
pp. 952-955 ◽  
Author(s):  
L Orci ◽  
A Perrelet ◽  
R Montesano
Keyword(s):  
Acinar Cell ◽  
Exocrine Pancreas ◽  
Plasma Membranes ◽  
Basolateral Membrane ◽  
Acinar Cells ◽  
Pancreatic Acinus ◽  
Luminal Membrane

The cytochemical labeling of cholesterol by filipin in plasma membranes of the exocrine pancreas has shown a difference in the reactivity to filipin between the acinar and centro-acinar cells. The acinar cell has a poorly labeled luminal membrane but numerous filipin-cholesterol complexes on the basolateral membrane. In contrast, the centro-acinar cell shows a comparable degree of filipin labeling on both luminal and basolateral membrane. This difference in the distribution of filipin labeling suggests underlying differences in the organization of the respective membranes, probably related to their specific functions.

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