Vasopressin decreases immunogold labeling of apical actin in the toad bladder granular cell

1992 ◽  
Vol 263 (4) ◽  
pp. C908-C912 ◽  
Author(s):  
Y. Gao ◽  
N. Franki ◽  
F. Macaluso ◽  
R. M. Hays
Keyword(s):  
Actin Cytoskeleton ◽  
Arginine Vasopressin ◽  
Apical Membrane ◽  
Water Channel ◽  
Granular Cell ◽  
Immunogold Labeling ◽  
Toad Bladder ◽  
Apical Region ◽  
Electron Microscopic ◽  
Relative Extent

Studies with the confocal microscope have shown that arginine vasopressin (AVP) depolymerizes F-actin in the apical region of the toad bladder granular cell. However, the resolution of the fluorescence microscope is not great enough to reveal the exact pattern of depolymerization or the relative extent to which microvillar and subapical membrane actin pools contribute to overall depolymerization. We have developed an electron microscopic immunogold method that shows a significant decrease in immunogold labeling of actin in the region just below the apical membrane, with the decrease most pronounced in regions adjacent to the microvilli. There was no significant change of immunogold labeling within the microvilli themselves. Our studies show a reorganization of the actin cytoskeleton in the region of the granular cell, where water channel-carrying vesicles are positioned and fuse in response to AVP.

Vasopressin depolymerizes apical F-actin in rat inner medullary collecting duct

1993 ◽  
Vol 265 (3) ◽  
pp. C757-C762 ◽  
Author(s):  
H. Simon ◽  
Y. Gao ◽  
N. Franki ◽  
R. M. Hays
Keyword(s):  
Collecting Duct ◽  
Water Channel ◽  
Granular Cell ◽  
Immunogold Labeling ◽  
Target Cells ◽  
Apical Region ◽  
Rhodamine Phalloidin ◽  
Consistent Finding ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

In amphibian bladder, arginine vasopressin (AVP) depolymerizes F-actin in the apical region of the granular cell, promoting fusion of water channel-carrying vesicles with the apical membrane. We now report the effect of AVP on F-actin in the mid- and terminal segments of rat inner medullary collecting duct (IMCD2 and IMCD3). In IMCD3, 5 min of stimulation by 2.5-250 nM AVP significantly depolymerized F-actin by 13-24% in whole cell assays employing the rhodamine-phalloidin binding technique. The IMCD2 was more sensitive, responding to subnanomolar (0.25 nM) AVP with 6 +/- 2% depolymerization. Depolymerization occurred as early as 2 min after 2.5 and 25 nM but not 250 nM AVP. 8-Bromoadenosine 3',5'-cyclic monophosphate depolymerized F-actin in IMCD3 at both 2 and 5 min. Immunogold labeling of the apical actin pool in IMCD3 principal cells was reduced by 26 +/- 5% (P < 0.05) by 2.5 nM AVP; the lateral and basal pools showed no significant changes. Capillary endothelial, thin limb of Henle, and intercalated cells showed no changes in immunogold labeling after AVP. Thus reorganization of the apical actin network by AVP is a consistent finding in both mammalian and amphibian target cells.

Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder

1991 ◽  
Vol 261 (1) ◽  
pp. C143-C153 ◽  
Author(s):  
H. W. Harris ◽  
M. L. Zeidel ◽  
C. Hosselet
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Apical Membrane ◽  
Water Channel ◽  
Protein S ◽  
Water Channels ◽  
Toad Bladder ◽  
Western Blots ◽  
Vesicle Protein

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

Water channel-carrying vesicles in the rat IMCD contain cellubrevin

1995 ◽  
Vol 269 (3) ◽  
pp. C797-C801 ◽  
Author(s):  
N. Franki ◽  
F. Macaluso ◽  
W. Schubert ◽  
L. Gunther ◽  
R. M. Hays
Keyword(s):  
Electron Microscopy ◽  
Membrane Protein ◽  
Arginine Vasopressin ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Immunogold Electron Microscopy ◽  
Cyclic Process ◽  
Docking Proteins ◽  
Medullary Collecting Duct

Antidiuretic hormone (arginine vasopressin) induces a cyclic process of docking, fusion, and endocytosis of water channel-containing vesicles in the collecting duct. There is now evidence that docking and endocytosis are mediated by an array of proteins associated with vesicles and target membranes. In recent studies, we have shown that cellubrevin, a member of the vesicle-associated membrane protein family, as well as other docking proteins, are expressed in the rat inner medullary collecting duct. We now show by immunogold electron microscopy that cellubrevin is present on vesicles containing water channels, that it is associated with both coated and uncoated vesicles, and that it is present on the apical membrane. Cellubrevin, therefore, is in a position to mediate one or more steps in arginine vasopressin-induced water channel cycling.

Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells

2001 ◽  
Vol 281 (6) ◽  
pp. F1092-F1101 ◽  
Author(s):  
Grazia Tamma ◽  
Enno Klussmann ◽  
Kenan Maric ◽  
Klaus Aktories ◽  
Maria Svelto ◽  
...  
Keyword(s):  
Actin Cytoskeleton ◽  
Kinase Inhibitor ◽  
Apical Membrane ◽  
Water Channel ◽  
Small Gtpases ◽  
Fusion Toxin ◽  
Cd8 Cells ◽  
Actin Depolymerization ◽  
Aquaporin 2 ◽  
Water Permeability Coefficient

First published August 8, 2001; 10.1152/ajprenal.00091.2001.—We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29). The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton. In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C. limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin. Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient. Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin. Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation. Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton.

Electron-microscopic study of the apical region of the toad bladder epithelial cell

1984 ◽  
Vol 247 (3) ◽  
pp. C268-C281 ◽  
Author(s):  
J. Sasaki ◽  
S. Tilles ◽  
J. Condeelis ◽  
J. Carboni ◽  
L. Meiteles ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Tannic Acid ◽  
Toad Bladder ◽  
Apical Region ◽  
Electron Microscopic ◽  
Bladder Epithelial Cell ◽  
Luminal Membrane ◽  
Microfilament Bundles ◽  
Major Mode ◽  
The Matrix

Antidiuretic hormone (ADH) promotes fusion of cytoplasmic tubules with the luminal membrane and delivery of particles from the tubules to the membrane. The particles are believed to be the water-conducting elements in the membrane. We have employed several scanning (SEM) and transmission electron-microscopic (TEM) techniques to study the relationship of the cytoplasmic tubules to the luminal membrane and to the apical cytoskeleton of the toad bladder epithelial cell. This paper reports the results of freeze-crack SEM and tannic acid-fixed TEM studies, as well as studies with a resinless method of embedding. Freeze-cracked epithelial cells reveal that the tubules are anchored in a matrix of cytoskeleton and granules just below the luminal membrane, and many, if not all, retain their anchorage to the matrix after ADH-induced fusion. Tannic acid-fixed specimens show that the tubules in unstimulated cells lie horizontally. Fusion appears to involve an angulation of the tubules, and this may be the major mode of ADH-induced tubule movement. There are suggestions in the tannic acid sections of filamentous attachments of tubules to the surrounding cytoskeleton. In addition there are prominent microfilament bundles running down the microvilli and a dense concentration of filaments just below the luminal membrane. The presence of these filaments is confirmed in the resinless sections, and their possible role in ADH action is discussed.

ADH-induced depolymerization of F-actin in the toad bladder granular cell: a confocal microscope study

1992 ◽  
Vol 262 (3) ◽  
pp. C672-C677 ◽  
Author(s):  
K. Holmgren ◽  
K. E. Magnusson ◽  
N. Franki ◽  
R. M. Hays
Keyword(s):  
Confocal Microscopy ◽  
Apical Membrane ◽  
Granular Cell ◽  
Toad Bladder ◽  
Rhodamine Phalloidin ◽  
Actin Depolymerization ◽  
Cytoplasmic Vesicles ◽  
Hormone Sensitive ◽  
And Control ◽  
Good Agreement

Antidiuretic hormone (ADH) induces the fusion of cytoplasmic vesicles containing water channels with the apical membrane of the toad bladder granular cell. Fusion is accompanied by a 30% depolymerization of F-actin. We have used confocal microscopy to determine the region in the cell that undergoes depolymerization. Bladders were mounted in a split chamber, and control halves and halves stimulated by ADH for 15 min were fixed and then stained with rhodamine phalloidin. Vertical sections through the cells were obtained by confocal microscopy, and the fluorescence intensity of the apical and side regions of the cells was determined. To normalize the data, the apex-side intensity was determined for each cell, and these ratios measured for control and ADH-treated halves. In six paired experiments, the ratio for control halves was 3.69 +/- 0.50 and for ADH-treated halves was 2.61 +/- 0.33; the decrease was significant and in good agreement with earlier studies. Thus actin depolymerization takes place in a hormone-sensitive apical pool where vesicle fusion occurs and supports the view that actin depolymerization may be required for fusion.

Fate of antidiuretic hormone water channel proteins after retrieval from apical membrane

1993 ◽  
Vol 265 (3) ◽  
pp. C822-C833 ◽  
Author(s):  
M. L. Zeidel ◽  
T. G. Hammond ◽  
J. B. Wade ◽  
J. Tucker ◽  
H. W. Harris
Keyword(s):  
Membrane Proteins ◽  
Antidiuretic Hormone ◽  
Apical Membrane ◽  
Water Channel ◽  
Fluid Phase ◽  
Water Channels ◽  
Endocytic Pathway ◽  
Electron Microscopic ◽  
Channel Proteins ◽  
Fluorescence Measurements

In toad bladder granular cells, antidiuretic hormone (ADH) stimulates insertion of vesicles containing water channels (WCV), markedly increasing apical membrane osmotic water permeability (Pf). After withdrawal of ADH stimulation, WCV are removed from the apical membrane and fluid-phase markers endocytosed from the apical solution appear predominantly in endosomes at 10-15 min and multivesicular bodies at 30-60 min. Although the luminal contents of this endocytic pathway have been well characterized, the fate of membrane proteins, including functional ADH water channels in these vesicles remains unclear. Using electron microscopic, flow cytometric, and stopped-flow fluorescence measurements and characterization of labeled vesicle proteins, we examined the fate of membrane proteins contained within WCV. The protein complements of endosomes harvested after 10, 30, and 60 min of ADH withdrawal were similar. Selective covalent labeling of apical proteins during ADH stimulation followed by ADH reversal for 30 or 60 min showed that apical proteins colocalize with fluid-phase marker-labeled endosomes at all times, and most apically labeled protein bands present in the 10-min fraction were also present in the 30- and 60-min endosome fractions. Endosomes at 10 and 30 min but not at 60 min contained functional water channels revealed by high Pf and proton permeability, low activation energy of Pf, and sensitivity of Pf to mercurial reagents. We conclude that a portion of apically exposed membrane proteins, including candidate water channel proteins, travel together with fluid-phase markers from 10-min endosomes into later endosomal compartments. Functional water channels may be inactivated or some essential protein component selectively sorted away between 30 and 60 min after ADH withdrawal.

Silver Staining of Pancreatic Islets as Revealed by Electron Microscopy

1967 ◽  
Vol 25 ◽  
pp. 44-45
Author(s):  
Kazuaki Misugi ◽  
Nobuko Misugi ◽  
Hiroshi Yamada
Keyword(s):  
Pancreatic Islet ◽  
Silver Staining ◽  
Islet Cells ◽  
Severe Hypoglycemia ◽  
Granular Cell ◽  
Electron Microscopic Level ◽  
Staining Reaction ◽  
Electron Microscopic ◽  
Pancreatic Islet Cell ◽  
D Cell

The authors had described the fine structure of a type of pancreatic islet cell, which appeared different from typical alpha and beta cells, and tentatively considered that this third type of granular cell probably represents the D cell (Figure 1).Since silver staining has been widely used to differentiate different types of pancreatic islet cells by light microscopy, an attempt to examine this staining reaction at the electron microscopic level was made.Material and Method: Surgically removed specimens from three infants who suffered from severe hypoglycemia were used. The specimens were fixed and preserved in 20% neutral formalin. Frozen sections, 30 to 40 micron thick, were prepared and they were stained by Bielschowsky's method as modified by Suzuki (2). The stained sections were examined under a microscope and islet tissues were isolated. They were fixed in 1% osmium tetroxide in phosphate buffer for one hour and embedded in Epon 812 following dehydration through a series of alcohols and propylene oxide.

scholarly journals An artificial test substrate for evaluating electron microscopic immunocytochemical labeling reactions.

1987 ◽  
Vol 35 (8) ◽  
pp. 909-916 ◽  
Author(s):  
G D Gagne ◽  
M F Miller
Keyword(s):  
Electron Microscope ◽  
Hepatitis B ◽  
Horseradish Peroxidase ◽  
Polyclonal Antibodies ◽  
Hepatitis B Surface Antigen ◽  
Immunogold Labeling ◽  
Artificial Substrate ◽  
Chemical Fixation ◽  
Electron Microscopic ◽  
Immunocytochemical Studies

We describe an artificial substrate system for optimization of labeling parameters in electron microscope immunocytochemical studies. The system involves use of blocks of glutaraldehyde-polymerized BSA into which a desired antigen is incorporated by a simple soaking procedure. The resulting antigen-impregnated artificial substrate can then be fixed and embedded identically to a piece of tissue. The BSA substrate can also be dried and then sectioned for immunolabeling with or without chemical fixation and without exposing the antigen to dehydrating agents and embedding resins. The effects of various fixation and embedding procedures can thus be evaluated separately. Other parameters affecting immunocytochemical labeling, such as antibody and conjugate concentration, can also be evaluated. We used this system, along with immunogold labeling, to determine quantitatively the optimal fixation and embedding conditions for labeling of hepatitis B surface antigen (HbsAg), human IgG, and horseradish peroxidase. Using unfixed and unembedded HBsAg, we were able to detect antigen concentrations below 20 micrograms/ml. We have shown that it is not possible to label HBsAg within resin-embedded cells using conventional aldehyde fixation protocols and polyclonal antibodies.

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