scholarly journals Histochemical methods for characterizing secretory and cell surface sialoglycoconjugates.

1985 ◽  
Vol 33 (5) ◽  
pp. 427-438 ◽  
Author(s):  
B A Schulte ◽  
S S Spicer
Keyword(s):  
Sialic Acid ◽  
Periodate Oxidation ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Surface Epithelium ◽  
Secretory Cells ◽  
Sublingual Gland ◽  
Mucous Cells ◽  
Acetylneuraminic Acid ◽  
Hamster Trachea

Paraffin sections of trachea, sublingual gland, and pancreas from rats, mice, and hamsters were stained with peanut agglutinin (PNA) or Dolichos biflorus agglutinin (DBA) conjugated to horseradish peroxidase before or after enzymatic removal of sialic acid. Adjacent sections were oxidized with periodate prior to incubation with sialidase and staining with PNA and DBA. PNA binding demonstrated terminal beta-galactose in secretions, at the basolateral plasmalemma of mouse tracheal serous cells, in or at the surface of zymogen granules, and at the apical and basolateral surface of mouse and hamster pancreatic acinar cells. Sialidase digestion revealed PNA binding, demonstrative of penultimate beta-galactose, in secretions of mucous cells in tracheal and sublingual glands and at the apical glycocalyx of ciliated and secretory cells in the tracheal surface epithelium of all the rodents studied. Sialidase also imparted PNA affinity to endothelium in all three species and to secretions and the basolateral plasmalemma of tracheal serous cells and pancreatic acinar cells in the rat. Periodate oxidation blocked the enzymatic removal of N-acetylneuraminic acid as judged by prevention of staining with the sialidase-PNA procedure. Sites in which periodate prevented sialidase-PNA staining included pancreatic islet cells and at the luminal glycocalyx of ciliated and secretory cells in tracheal surface epithelium in all three rodents, most sublingual mucous cells in the hamster, pancreatic acinar cells in the rat, and endothelium, except that of the rat. Glycoconjugate in other sites remained positive with the periodate-sialidase-PNA sequence. Resistance to periodate was interpreted as evidence for the presence of terminal sialic acid with an O-acetylated polyhydroxyl side chain. DBA binding demonstrated terminal alpha-N-acetylgalactosamine in the secretion of all mucous cells in the hamster trachea and 50-90% of those in the rat, secretion and the basolateral plasmalemma of all glandular serous cells in the mouse trachea, at the apical surface of most secretory cells lining the lumen of the rat and hamster trachea, and cilia of 5-10% of ciliated cells in the rat trachea. Periodate oxidation and sialidase digestion demonstrated N-acetylneuraminic acid and penultimate alpha-N-acetylgalactosamine in cilia in the mouse trachea and sialic acid containing O-acetylated polyhydroxyl side chains subtended by N-acetylgalactosamine in the secretion of all mucous cells in the rat and hamster trachea and of 80-90% of mucous cells in the hamster sublingual gland.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

scholarly journals Spatial aspects of Ca2+ signalling in pancreatic acinar cells

1993 ◽  
Vol 184 (1) ◽  
pp. 129-144
Author(s):  
P. Thorn
Keyword(s):  
Digestive Enzymes ◽  
Acinar Cells ◽  
Fluid Secretion ◽  
Cell Types ◽  
Cellular Mechanism ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
High Affinity ◽  
High Concentrations ◽  
Cell Changes

Secretory cells do not only respond to an agonist with a simple rise in [Ca2+]i. It is now clear that complex patterns of [Ca2+]i elevation in terms of space and time are observed in many cell types and that these patterns may be a cellular mechanism for the regulation of different responses. Ca2+ signalling in exocrine cells of the pancreas promotes the secretion of digestive enzymes and fluid. It has been shown that at high concentrations of agonist (acetylcholine or cholecystokinin) the [Ca2+]i response is initiated in the secretory pole of the cell before spreading across the whole cell. This site of initiation of the [Ca2+]i elevation is in the region where exocytotic release of enzymes occurs and is also the site of a Ca(2+)-dependent chloride channel thought to be crucially important for fluid secretion. Lower concentrations of agonist elicit [Ca2+]i oscillations with complex repetitive patterns characteristic of each agonist. At physiological agonist concentrations, we have recently described repetitive short-lasting Ca2+ spikes that are spatially restricted to the secretory pole of the cell. In addition to these spikes, cholecystokinin also promotes slow transient Ca2+ rises that result in a global rise in Ca2+. The inositol trisphosphate (InsP3) receptor plays a crucial role in all of these various agonist responses, most of which can be reproduced by the infusion of InsP3 into the cell. The high InsP3-sensitivity of the secretory pole is postulated to be due to a localization of high-affinity InsP3 receptors. We speculate that in response to cholecystokinin the short-lasting spikes elicit exocytosis from a small ‘available pool’ of vesicles and that the broader oscillations induce both exocytosis and cell changes that involve movement of vesicles into this ‘available pool’.

scholarly journals Monoclonal antibodies to the Golgi apparatus of serous exocrine cells.

1993 ◽  
Vol 41 (11) ◽  
pp. 1623-1633 ◽  
Author(s):  
S Yamashita ◽  
H Uchida ◽  
M Shiozawa ◽  
S Aiso ◽  
K Yasuda
Keyword(s):  
Monoclonal Antibodies ◽  
Golgi Apparatus ◽  
Parotid Gland ◽  
Submandibular Gland ◽  
Apical Cell ◽  
Acinar Cells ◽  
Immunohistochemical Method ◽  
Pancreatic Acinar Cells ◽  
Sublingual Gland ◽  
Exocrine Cells

We demonstrated that a common antigen (Golgi-associated antigen, GAA 108) is present in the Golgi apparatus of serous exocrine cells, using an immunohistochemical method with monoclonal antibodies (MAb) 108 (IgG1) and 18 (IgM), raised to the microsomal fractions of rat parotid gland. The MAb reacted with polypeptides of molecular weights in the 58-170 KD range in parotid gland on Western blot analysis. The Golgi apparatus of the following cells was immunostained with these MAb: acinar cells of parotid gland, pancreas, and exorbital lacrimal gland, serous cells of sublingual gland, chief cells of stomach, and epithelial cells of rat prostate. However, positive reaction occurred throughout the entire cytoplasm of submandibular gland acinar cells. Immunoelectron microscopy (IM) revealed antigen (GAA 108) localization in the medial and trans-Golgi cisternae and trans-Golgi network (TGN), including condensing vacuoles, in parotid, exorbital lacrimal, and pancreatic acinar cells, and serous acinar cells of sublingual gland. Lysosomes and apical cell membranes also stained positively in some cells. In the submandibular gland reactions were observed in the medial and trans-Golgi cisternae, condensing vacuoles, secretory granule contents, cell membrane, and in some duct lumens. These results suggest that although GAA 108 is found in the Golgi apparatus of most serous exocrine cells, it is secreted by a regulated pathway in the acinar cells of submandibular gland.

scholarly journals Dynamic Regulation of the Large Exocytotic Fusion Pore in Pancreatic Acinar Cells

2007 ◽  
Vol 18 (9) ◽  
pp. 3502-3511 ◽  
Author(s):  
Olga Larina ◽  
Purnima Bhat ◽  
James A. Pickett ◽  
Bradley S. Launikonis ◽  
Amit Shah ◽  
...  
Keyword(s):  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Fusion Pore ◽  
Secretory Cells ◽  
Zymogen Granule ◽  
Latrunculin B ◽  
Extracellular Solution ◽  
Dynamic Regulation ◽  
Pore Closure ◽  
Dependent Mechanism

Loss of granule content during exocytosis requires the opening of a fusion pore between the secretory granule and plasma membrane. In a variety of secretory cells, this fusion pore has now been shown to subsequently close. However, it is still unclear how pore closure is physiologically regulated and contentious as to how closure relates to granule content loss. Here, we examine the behavior of the fusion pore during zymogen granule exocytosis in pancreatic acinar cells. By using entry of high-molecular-weight dyes from the extracellular solution into the granule lumen, we show that the fusion pore has a diameter of 29–55 nm. We further show that by 5 min after granule fusion, many granules have a closed fusion pore with evidence indicating that pore closure is a prelude to endocytosis and that in granules with a closed fusion pore the chymotrypsinogen content is low. Finally, we show that latrunculin B treatment promotes pore closure, suggesting F-actin affects pore dynamics. Together, our data do not support the classical view in acinar cells that exocytosis ends with granule collapse. Instead, for many granules the fusion pore closes, probably as a transition to endocytosis, and likely involving an F-actin–dependent mechanism.

scholarly journals Identification of N-acetyl-4–O-acetylneuraminyl-lactose in echidna milk

1974 ◽  
Vol 139 (2) ◽  
pp. 415-420 ◽  
Author(s):  
Michael Messer
Keyword(s):  
Sialic Acid ◽  
Acid Hydrolysis ◽  
Acid Treatment ◽  
Periodate Oxidation ◽  
Thiobarbituric Acid ◽  
Chromatographic Mobility ◽  
Mild Acid Hydrolysis ◽  
Acetylneuraminic Acid ◽  
Acid Reagent ◽  
Mild Acid

The identity of a novel form of sialyl-lactose found in milk of the echidna (Tachyglossus aculeatus) was investigated. The sialyl-lactose yielded equimolar amounts of N-acetylneuraminic acid and lactose during mild acid hydrolysis but was resistant to the action of a bacterial neuraminidase. A viral neuraminidase hydrolysed it to lactose plus a form of sialic acid that reacted positively with thiobarbituric acid reagent but whose chromatographic mobility was greater than that of N-acetylneuraminic acid. Treatment with alkali converted the sialyl-lactose into a substance with the same chromatographic mobility as N-acetylneuraminyl-(2→3)-lactose and made it susceptible to the action of bacterial neuraminidase. The sialyl-lactose contained one mol of ester (identified as acetyl), and released one mol of formaldehyde during periodate oxidation, per mol of sialic acid. It did not contain N-glycollylneuraminic acid. These results indicate that the sialyl-lactose is N-acetyl-4-O-acetylneuraminyl-(2→3)-lactose. Echidna milk contained, in addition, a small amount of N-acetylneuraminyl-(2→3)-lactose.

scholarly journals The site of incorporation of sialic acid residues into glycoproteins and the subsequent fates of these molecules in various rat and mouse cell types as shown by radioautography after injection of [3H]N-acetylmannosamine. II. Observations in tissues other than liver.

1981 ◽  
Vol 88 (1) ◽  
pp. 16-28 ◽  
Author(s):  
G Bennett ◽  
F W Kan ◽  
D O'Shaughnessy
Keyword(s):  
Plasma Membrane ◽  
Sialic Acid ◽  
Golgi Apparatus ◽  
Preceding Paper ◽  
Distal Tubule ◽  
Cell Types ◽  
Pancreatic Acinar Cells ◽  
Kidney Cortex ◽  
Secretory Cells ◽  
Silver Grains

Biochemical evidence from the preceding paper indicated that [3H]N-acetylmannosamine may be used as a fairly specific precursor for the sialic acid residues of glycoproteins (and perhaps glycolipids) in radioautographs of rat liver and duodenum. In order to study the site of incorporation of this label in cell types of various tissues, we gave 40-g rats and 15-g Swiss albino mice a single intravenous injection of 8 mCi of [3H]N-acetylmannosamine and sacrificed them after 2 and 10 min. To trace the subsequent migration of the labeled glycoproteins, we injected 40-g rats with 4 mCi of [3H]N-acetylmannosamine and sacrificed them after 20 and 30 min, 1, 4, and 24 h, and 3 and 9 d. Light microscope radioautographic analysis revealed that in a great variety of cell types the label was initially localized to the Golgi region. Electron microscope radioautographic analysis of duodenal villous columnar and goblet cells, pancreatic acinar cells and Paneth cells, from rats and mice sacrificed 10 min after injection, showed that the silver grains were localized over Golgi saccules (and adjacent secretion granules). In kidney proximal and distal tubule cells reaction was initially localized to the Golgi apparatus in some areas of the kidney cortex whereas in other areas it was more diffuse. In all cells, the proportion of silver grains over the Golgi apparatus decreased with time after injection while an increasing number of grains appeared over secretion products in secretory cells or over the plasma membrane in other cell types. Lysosomes also became increasingly labeled at later time intervals. The above results suggest that in most cell types sialic acid residues are incorporated into glycoproteins (and perhaps glycolipids), primarily in the Golgi apparatus. With time, these newly synthesized molecules migrate to secretion products, to the plasma membrane, or to the lysosomes.

scholarly journals Targeting of the zymogen-granule protein syncollin in AR42J and AtT-20 cells

2000 ◽  
Vol 350 (3) ◽  
pp. 637-643 ◽  
Author(s):  
Alois HODEL ◽  
J. Michael EDWARDSON
Keyword(s):  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Secretory Granules ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Apical Region ◽  
Zymogen Granule ◽  
Ar42j Cells ◽  
Green Fluorescent

Syncollin is a 13-kDa protein associated with the membranes of pancreatic zymogen granules. Here we determine the in situ localization of syncollin in pancreatic acinar cells from adult and neonatal rats, and study the targeting of green fluorescent protein-(GFP-) and His6-tagged syncollin chimaeras in model exocrine and endocrine secretory cells. Immunocytochemical analysis of the distribution of syncollin in fully differentiated and neonatal acinar cells revealed a granular pattern that corresponded with that of the zymogen-granule markers synaptobrevin 2 and amylase. In fully differentiated acinar cells syncollin-positive vesicles were detected in the apical region of the cells, whereas in neonatal acinar cells they were found clustered near the cell nucleus. Both GFP- and His6-tagged syncollin entered the secretory pathway when transiently expressed in AR42J or AtT-20 cells. Syncollin-GFP was found predominantly in amylase-positive granules in AR42J cells and in adrenocorticotrophic hormone- (ACTH-) positive granules in AtT-20 cells. Syncollin-GFP was also present in the Golgi complex in AR42J cells. Syncollin-His6 became localized in ACTH-containing granules in the neuritic processes of AtT-20 cells. In AR42J cells syncollin-His6 did not co-localize with amylase, but was detected in acidic vesicles. These results show that the exocrine protein syncollin contains intrinsic cell-type-independent targeting information that is retained in both exocrine and endocrine cells after fusion to the GFP tag. In contrast, His6-tagged syncollin is efficiently targeted to secretory granules only in AtT-20 cells and not in AR42J cells.

scholarly journals The periodate oxidation of bovine bone sialoprotein, and some observations on its structure

1969 ◽  
Vol 111 (5) ◽  
pp. 621-627 ◽  
Author(s):  
A. T. De B. Andrews ◽  
G. M. Herring ◽  
P. W. Kent
Keyword(s):  
Sialic Acid ◽  
Periodate Oxidation ◽  
Peptide Bond ◽  
Bone Sialoprotein ◽  
Bovine Bone ◽  
Acetylneuraminic Acid ◽  
Terminal Sialic Acid ◽  
Smith Degradation ◽  
Fucose Residue ◽  
Oligosaccharide Structures

1. Bovine bone sialoprotein (mol.wt. 23000) contains N-acetylneuraminic acid and N-glycollylneuraminic acid, fucose, galactose, mannose, N-acetylgalactosamine and N-acetylglucosamine residues in the form of a very small number, perhaps one, of highly branched oligosaccharide structures linked covalently to peptide. 2. Periodate oxidation of the sialoprotein results in quantitative destruction only of the sialic acid and fucose residue consistent with the earlier findings of their positions as terminal groups. 3. Terminal sialic acid residues are attached to galactopyranose residues by 2,3-linkages, and to some N-acetylgalactosamine residues (at C-6). 4. Sequential Smith degradation indicates that N-acetylgalactosamine residues may be present as points of branching (linked in C-1, C-3 and C-6) and N-acetylglucosamine residues are located in the inner part of the structure, adjacent to the carbohydrate–peptide bond(s). 5. Mannose residues appear to be linked in the 1,3-positions.

scholarly journals InsP3 receptors and Orai channels in pancreatic acinar cells: co-localization and its consequences

2011 ◽  
Vol 436 (2) ◽  
pp. 231-239 ◽  
Author(s):  
Gyorgy Lur ◽  
Mark W. Sherwood ◽  
Etsuko Ebisui ◽  
Lee Haynes ◽  
Stefan Feske ◽  
...  
Keyword(s):  
Acinar Cells ◽  
Apical Part ◽  
Inhibitory Effect ◽  
Pancreatic Acinar Cells ◽  
Secretory Cells ◽  
Stromal Interaction Molecule 1 ◽  
Store Depletion ◽  
Insp3 Receptors ◽  
Knockout Animals ◽  
Entry Mechanism

Orai1 proteins have been recently identified as subunits of SOCE (store-operated Ca2+ entry) channels. In primary isolated PACs (pancreatic acinar cells), Orai1 showed remarkable co-localization and co-immunoprecipitation with all three subtypes of IP3Rs (InsP3 receptors). The co-localization between Orai1 and IP3Rs was restricted to the apical part of PACs. Neither co-localization nor co-immunoprecipitation was affected by Ca2+ store depletion. Importantly we also characterized Orai1 in basal and lateral membranes of PACs. The basal and lateral membranes of PACs have been shown previously to accumulate STIM1 (stromal interaction molecule 1) puncta as a result of Ca2+ store depletion. We therefore conclude that these polarized secretory cells contain two pools of Orai1: an apical pool that interacts with IP3Rs and a basolateral pool that interacts with STIM1 following the Ca2+ store depletion. Experiments on IP3R knockout animals demonstrated that the apical Orai1 localization does not require IP3Rs and that IP3Rs are not necessary for the activation of SOCE. However, the InsP3-releasing secretagogue ACh (acetylcholine) produced a negative modulatory effect on SOCE, suggesting that activated IP3Rs could have an inhibitory effect on this Ca2+ entry mechanism.

The ultrastructure of acinar cells in the external orbital gland of young and old male rats

1978 ◽  
Vol 36 (2) ◽  
pp. 276-277
Author(s):  
R. Carriere
Keyword(s):  
Young Adulthood ◽  
Acinar Cells ◽  
Cell Number ◽  
Male Rats ◽  
Polyploid Cell ◽  
Secretory Cells ◽  
Age Changes ◽  
Albino Rat ◽  
Golgi Membranes ◽  
Diploid Cells

The external orbital gland of the albino rat exhibits both sexual dimorphism and histological age changes. In males, many cells attain a remarkable degree of polyploidy and an increase of polyploid cell number constitutes the major age change until young adulthood. The acini of young adults have a small lumen and are composed of tall serous cells. Subsequently, many acini acquire a larger lumen with an irregular outline while numerous vacuoles accumulate throughout the secretory cells. At the same time, vesicular acini with a large lumen surrounded by pale-staining low cuboidal diploid cells begin to appear and their number increases throughout old age. The fine structure of external orbital glands from both sexes has been explored and in considering acinar cells from males, emphasis was given to the form of the Golgi membranes and to nuclear infoldings of cytoplasmic constituents.

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