scholarly journals Membrane-cytoskeleton dynamics in rat parietal cells: mobilization of actin and spectrin upon stimulation of gastric acid secretion.

1989 ◽  
Vol 108 (2) ◽  
pp. 441-453 ◽  
Author(s):  
F Mercier ◽  
H Reggio ◽  
G Devilliers ◽  
D Bataille ◽  
P Mangeat
Keyword(s):  
Gastric Acid ◽  
Dynamic Equilibrium ◽  
Gastric Gland ◽  
Cytoskeletal Proteins ◽  
Parietal Cells ◽  
Electron Microscopic Level ◽  
Apical Surface ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Acid Secreting

The gastric parietal (oxyntic) cell is presented as a model for studying the dynamic assembly of the skeletal infrastructure of cell membranes. A monoclonal antibody directed to a 95-kD antigen of acid-secreting membranes of rat parietal cells was characterized as a tracer of the membrane movement occurring under physiological stimuli. The membrane rearrangement was followed by immunocytochemistry both at the light and electron microscopic level on semithin and thin frozen sections from resting and stimulated rat gastric mucosa. Double labeling experiments demonstrated that a specific and massive mobilization of actin, and to a lesser extent of spectrin (fodrin), was involved in this process. In the resting state, actin and spectrin were mostly localized beneath the membranes of all cells of the gastric gland, whereas the bulk of acid-secreting membranes appeared diffusely distributed in the cytoplasmic space of parietal cells without any apparent connection with cytoskeletal proteins. In stimulated cells, both acid-secreting material and actin (or spectrin) extensively colocalized at the secretory apical surface of parietal cells, reflecting that acid-secreting membranes were now exposed at the lumen of the secretory canaliculus and that this insertion was stabilized by cortical proteins. The data are compatible with a model depicting the membrane movement occurring in parietal cells as an apically oriented insertion of activated secretory membranes from an intracellular storage pool. The observed redistribution of actin and spectrin argues for a direct control by gastric acid secretagogues of the dynamic equilibrium existing between nonassembled (or preassembled) and assembled forms of cytoskeletal proteins.

scholarly journals Murine endodermal cytokeratins Endo A and Endo B are localized in the same intermediate filament.

1986 ◽  
Vol 34 (6) ◽  
pp. 785-793 ◽  
Author(s):  
W E Howe ◽  
F G Klier ◽  
R G Oshima
Keyword(s):  
Immunoelectron Microscopy ◽  
Intermediate Filament ◽  
Microscopic Level ◽  
Cytoskeletal Proteins ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Label ◽  
Secondary Antibodies ◽  
Endodermal Cells ◽  
20 Nm

The intracellular distribution of extra-embryonic endodermal, cytoskeletal proteins A (Endo A) and B (Endo B) was investigated by double-label immunofluorescent microscopy and double-label immunoelectron microscopy. In parietal endodermal cells, the immunofluorescent distribution of Endo B was always coincident with that of Endo A and could be distinguished from vimentin, particularly at the periphery of the cell. At the electron microscopic level, antibodies against both Endo A and Endo B recognized both bundles and individual intermediate filaments. Double-label immunoelectron microscopy was achieved by use of two sizes of colloidal gold particles (5 nm and 20 nm) that were stabilized with secondary antibodies. These results show that Endo A and B are found in the same intermediate filament and probably co-polymerize to form such structures.

scholarly journals Metabolism and gastric acid secretion. Substrate-dependency of aminopyrine accumulation in isolated rat parietal cells

1985 ◽  
Vol 227 (1) ◽  
pp. 223-229 ◽  
Author(s):  
G P Shaw ◽  
N G Anderson ◽  
P J Hanson
Keyword(s):  
Acid Secretion ◽  
Gastric Acid Secretion ◽  
Gastric Acid ◽  
Parietal Cell ◽  
Parietal Cells ◽  
Intestinal Epithelial ◽  
Acid Secreting ◽  
High Concentrations ◽  
Isolated Rat

The substrate-dependency of gastric acid secretion was investigated in isolated rat parietal cells by using the accumulation of the weak base aminopyrine as an index of acid secretion. Exogenous substrates enhanced accumulation of aminopyrine in rat parietal cells stimulated by secretagogues, and this effect was probably directly related to the provision of energy for acid secretion. At physiological concentrations, certain of the substrates (glucose, oleate, lactate, D-3-hydroxybutyrate, L-isoleucine, L-valine and acetoacetate) could support acid secretion, with glucose being the most effective. L-Leucine and acetate were only effective stimulators of parietal-cell aminopyrine accumulation at high concentrations (5mM). L-Glutamine was unable to stimulate aminopyrine accumulation even at high concentrations, and glutaminase activity in parietal cells was estimated to be low by comparison with small-intestinal epithelial cells. Variation in the concentrations of D-3-hydroxybutyrate and L-isoleucine, but not of glucose, within the physiological range affected their ability to support aminopyrine accumulation. The presence of 5 mM-L-isoleucine, 5 mM-lactate and combinations of certain substrates at physiological concentrations produced aminopyrine accumulation in stimulated parietal cells that was greater than that obtained in cells incubated with 5 mM-glucose alone. In conclusion, fulfillment of the metabolic requirements of the acid-secreting parietal cell under physiological circumstances requires a combination of substrates, and integration of the results with previous data [Anderson & Hanson (1983) Biochem. J. 210, 451-455; 212, 875-879] suggests that after overnight starvation in vivo metabolism of glucose, D-3-hydroxybutyrate and L-isoleucine may be of particular importance.

scholarly journals Inter- and intracellular relationship of substance P-containing neurons with serotonin and GABA in the dorsal raphe nucleus: combination of autoradiographic and immunocytochemical techniques.

1986 ◽  
Vol 34 (6) ◽  
pp. 735-742 ◽  
Author(s):  
R Mâgoul ◽  
B Onteniente ◽  
A Oblin ◽  
A Calas
Keyword(s):  
Substance P ◽  
Dorsal Raphe Nucleus ◽  
Dorsal Raphe ◽  
Raphe Nucleus ◽  
Electron Microscopic Level ◽  
Gamma Aminobutyric Acid ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Relationship Of ◽  
Dorsal Raphé

Double-labeling experiments were performed at the electron microscopic level in the dorsal raphe nucleus of rat, in order to study the inter- and intracellular relationship of substance P with gamma-aminobutyric acid (GABA) and serotonin. Autoradiography for either [3H]serotonin or [3H]GABA was coupled, on the same tissue section, with peroxidase-antiperoxidase immunocytochemistry for substance P in colchicine-treated animals. Intercellular relationships were represented by synaptic contacts made by [3H]serotonin-labeled terminals on substance P-containing somata and dendrites, and by substance P-containing terminals on [3H]GABA-labeled cells. Intracellular relationships were suggested by the occurrence of the peptide within [3H]serotonin-containing and [3H]GABA-containing cell bodies and fibers. Doubly labeled varicosities of the two kinds were also observed in the supraependymal plexus adjacent to the dorsal raphe nucleus. The results demonstrated that, in addition to reciprocal synaptic interactions made by substance P with serotonin and GABA, the dorsal raphe nucleus is the site of intracellular relationships between the peptide and either the amine or the amino acid.

A combined pre- and post-embedding double labeling in the central nervous system with good tissue preservation

1991 ◽  
Vol 49 ◽  
pp. 276-277
Author(s):  
A.M. Milroy ◽  
D.D. Ralston
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Room Temperature ◽  
Osmium Tetroxide ◽  
Microscopic Level ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Highly Sensitive ◽  
The Central Nervous System

Multiple labeling at the electron microscopic level is routinely done in various parts of the central nervous system. We demonstrate that the pre-embedding tetramethylbenzidine (TMB) reaction for visualizing horseradish peroxidase (HRP) of Olucha and the slow osmication of Henry combined with a post-embedding nonetching immunogold method will also preserve good ultrastructure. Furthermore, the post-embedding immunocytochemistry of some neurotransmitters, i.e. gammaaminobutyric acid (GABA), can be done months after the tissue has been reacted for HRP and embedded in regular epon.Pre-embedding histochemistry:The use of TMB as a chromagen for the demonstration of neuronally transported HRP has both the advantage of being highly sensitive and of producing very specific needle-like crystals. Olucha et al demonstrated that one could further stabilize this reaction product with amonium heptamolybdate. Unfortunately the next step, fixation with regular osmium tetroxide, often resulted in the loss of the reaction product. However, the slow osmication with a lower pH (5.5) in the phosphate buffer at room temperature as recommended by Henry et al prevented this loss, and at the same time resulted in well preserved ultrastructure.

scholarly journals Achlorhydria by ezrin knockdown

2005 ◽  
Vol 169 (1) ◽  
pp. 21-28 ◽  
Author(s):  
Atsushi Tamura ◽  
Shojiro Kikuchi ◽  
Masaki Hata ◽  
Tatsuya Katsuno ◽  
Takeshi Matsui ◽  
...  
Keyword(s):  
Acid Secretion ◽  
Gastric Acid Secretion ◽  
Gastric Acid ◽  
Parietal Cells ◽  
Protein Levels ◽  
Acid Secreting ◽  
Apical Membranes ◽  
Gastric Parietal Cells ◽  
Neomycin Resistance

Loss of gastric acid secretion is pathologically known as achlorhydria. Acid-secreting parietal cells are characterized by abundant expression of ezrin (Vil2), one of ezrin/radixin/moesin proteins, which generally cross-link actin filaments with plasma membrane proteins. Here, we show the direct in vivo involvement of ezrin in gastric acid secretion. Ezrin knockout (Vil2−/−) mice did not survive >1.5 wk after birth, making difficult to examine gastric acid secretion. We then generated ezrin knockdown (Vil2kd/kd) mice by introducing a neomycin resistance cassette between exons 2 and 3. Vil2kd/kd mice born at the expected Mendelian ratio exhibited growth retardation and a high mortality. Approximately 7% of Vil2kd/kd mice survived to adulthood. Ezrin protein levels in Vil2kd/kd stomachs decreased to <5% of the wild-type levels without compensatory up-regulation of radixin or moesin. Adult Vil2kd/kd mice suffered from severe achlorhydria. Immunofluorescence and electron microscopy revealed that this achlorhydria was caused by defects in the formation/expansion of canalicular apical membranes in gastric parietal cells.

Gastric parietal cell H(+)-K(+)-ATPase microsomes are associated with isoforms of ankyrin and spectrin

1993 ◽  
Vol 264 (1) ◽  
pp. C63-C70 ◽  
Author(s):  
P. R. Smith ◽  
A. L. Bradford ◽  
E. H. Joe ◽  
K. J. Angelides ◽  
D. J. Benos ◽  
...  
Keyword(s):  
Apical Membrane ◽  
Immunoblot Analysis ◽  
Cytoskeletal Proteins ◽  
Parietal Cells ◽  
Apical Surface ◽  
Gastric Parietal Cell ◽  
Apical Domain ◽  
Hcl Secretion ◽  
Gastric Parietal Cells ◽  
Stimulation Of

Stimulation of HCl secretion by gastric parietal cells requires the fusion of cytoplasmic H(+)-K(+)-ATPase-bearing tubulovesicles with the apical membrane. This insertion of membrane results in a dramatic increase in apical surface area through the formation of microvilli. To elucidate the elements that may stabilize the newly inserted H(+)-K(+)-ATPase within the apical membrane, we searched for specific cytoskeletal proteins associating with the gastric enzyme. We document by immunoblot analysis that ankyrin, spectrin, and actin copurify with H(+)-K(+)-ATPase microsomes prepared from gastric parietal cells. Coprecipitation of 125I-labeled native erythrocyte ankyrin with the H(+)-K(+)-ATPase from gastric microsomes using anti-H(+)-K(+)-ATPase antibodies suggests that ankyrin associates with the H(+)-K(+)-ATPase. Indirect immunofluorescence and confocal microscopy show that ankyrin and H(+)-K(+)-ATPase cosegregate within resting and secreting parietal cells. Taken together, these data suggest that the association of the gastric H(+)-K(+)-ATPase with spectrin and actin is mediated by ankyrin and that this interaction contributes to the maintenance of the polarized distribution of the enzyme to the apical domain of gastric parietal cells during acid secretion.

scholarly journals Combination of horseradish peroxidase and lucifer yellow staining for selective labeling of neurons at the electron microscopic level.

1991 ◽  
Vol 39 (11) ◽  
pp. 1579-1583 ◽  
Author(s):  
L Nonnotte ◽  
A Buisson ◽  
F Nagy ◽  
M Moulins
Keyword(s):  
Horseradish Peroxidase ◽  
Lucifer Yellow ◽  
Electron Microscopic Level ◽  
Reaction Products ◽  
Stomatogastric Nervous System ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Selective Labeling ◽  
Experimental Neuroanatomy ◽  
Labeling Method

We have developed a new double labeling method for electron microscopy to characterize selectively two physiologically identified neurons on the same preparation. The stomatogastric nervous system of crustaceans was used to test the distinguishing staining characteristics of the two labels. Neurons were labeled on one side with horseradish peroxidase (HRP) and on the other side with Lucifer yellow (LY). After blue light irradiation of the tissue in the presence of diaminobendizine, the two labeled neurons could be easily observed and discriminated on the same section by the two different reaction products. This simple technique of double labeling is useful in experimental neuroanatomy for the detailed study of synaptic relationships.

scholarly journals Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase.

1994 ◽  
Vol 127 (3) ◽  
pp. 641-652 ◽  
Author(s):  
C N Connolly ◽  
C E Futter ◽  
A Gibson ◽  
C R Hopkins ◽  
D F Cutler
Keyword(s):  
Golgi Complex ◽  
Secretory Pathway ◽  
High Sensitivity ◽  
Electron Microscopic Level ◽  
Electron Microscopic ◽  
Double Labeling ◽  
Golgi Stack ◽  
Er Retention ◽  
Peroxidase Cytochemistry ◽  
Golgi Network

We have developed a novel technique with which to investigate the morphological basis of exocytotic traffic. We have used expression of HRP from cDNA in a variety of cells in combination with peroxidase cytochemistry to outline traffic into and out of the Golgi apparatus at the electron microscopic level with very high sensitivity. A secretory form of the peroxidase (ssHRP) is active from the beginning of the secretory pathway and the activity is efficiently cleared from cells. Investigation of the morphological elements involved in the itinerary of soluble ER proteins using ssHRP tagged with the ER retention motif (ssHRPKDEL) shows that it progresses through the Golgi stack no further than the cis-most element. Traffic between the RER and the Golgi stack as outlined by ssHRPKDEL occurs via vesicular carriers as well as by tubular elements. ssHRP has also been used to investigate the trans side of the Golgi complex, where incubation at reduced temperatures outlines the trans-Golgi network with HRP reaction product. Tracing the endosomal compartment with transferrin receptor in double-labeling experiments with ssHRP fails to show any overlap between these two compartments.

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