摘要:

Freeze‐fracture electron microscopy reveals intramembrane particle arrays in basal membranes of granular epithelial cells as well as both upper and lower plasma membranes of the underlying basal cells in the toad urinary bladder. These particle arrays are morphologically indistinguishable from the luminal membrane aggregates which are known to be associated with antidiuretic hormone (ADH)‐stimulated water transport. In both granular and basal cells particle arrays are frequently located in and/or around the openings of vesicular and/or tubular structures fused to the plasma membranes, suggesting that they may be transferred from the cytoplasm by membrane fusion. Quantification of cytoplasmic aggrephores in control granular cells shows that they can be numerous and as close to the basolateral membrane as they are with the luminal membrane, to which they are known to fuse and deliver aggregates upon ADH stimulation. Aggrephore‐like tubules were also found in the basal cells. Particle array densities were quantified for 6 pairs of control and ADH‐stimulated hemibladders. At least 1440 μm2area of plasma membrane for each membrane domain was examined. Results indicate that the presence of these particle arrays in granular and basal cell membranes is highly variable and that exposure to ADH does not cause a statistically significant increase in their f

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00818.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Microtubules and microfilaments were localized by an immunocytochemical method in the granular cells of the frog bladder after fixation and isolation.An extensive array of microtubules was observed in the granular cells with an orientation towards the luminal plasma membrane in the supranuclear zone. Actin filaments formed a continuous bundle that underlined the cellular membrane.After incubation in the presence of colchicine, nocodazole, or tubulozole, the microtubular network appeared fragmented but did not disappear completely.These observations are related to the role of the cytoskeleton in the permeability response of the frog bladder epithelium to vasopressin.

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00819.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

It is now generally accepted that the increase in water permeability induced by antidiuretic hormone (ADH) in responsive epithelia is accompanied by the insertion of specific structures in the apical membrane of epithelial cells. There are strong indications that these particles, probably proteic in nature, represent water channels.In order to evaluate the nature and role of such proteins, plasma membranes were isolated by the affinity chromatography technique. The method is based on the firm attachment of the external face of the membrane to polycations covalently bound to the surface of polyacrylamide beads, followed by shearing of the rest of the cells. Maximal binding of epithelial cells to beads was achieved in a medium of low ionic strength and pH 5.2 (i.e.sucrose‐MES buffer). By this procedure plasma membranes were obtained from both cAMP‐stimulated cells and control cells.Membranes isolated on beads were enriched in the activity of typical membrane marker enzymes (LAP; H+ATPase; Na+, K+ATPase) with respect to a whole cell homogenate, whereas contamination of plasma membrane fraction by endoplasmic reticulum, lysosomes, and mitochondria was relatively low.Analysis by SDS polyacrylamide gel electrophoresis showed an interesting difference between cAMP‐treated and control sa

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00820.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

In the amphibian urinary bladder, water permeability is correlated with the insertion of intramembrane particle aggregates (IMPAs) into the apical plasma membrane (AM) of the granular cells. These aggregates are believed to contain water channels. Characterization of the IMPAs by comparing AM fractions of antidiuretic hormone (ADH)‐treated and resting epithelia requires isolation and purification of AM‐rich material, free of other cytoplasmic aggregate‐containing organelles, in both cases. A technique derived from freeze‐fracture was chosen to isolate large sheets of apical membrane material from frog (Rana esculenta) urinary bladder epithelium. The apical side was plated on a polylysine‐coated glass slide, frozen with liquid nitrogen, and fractured. A nylon mesh was inserted between the glass slide and the bladder, in order to bring the fracture plane back to the AM periodically. Fluorescent markers were used to characterize the material having fractured with the glass slide. Samples were observed by fluorescence and phase contrast microscopy. We obtained evidence that numerous patches of fractured AM remained on the glass surface without nuclei. A phase contrast picture was obtained only at a high magnification, indicating a low thickness of the recovered material. Further characterization was made with SDS‐PAGE. Protein contents of samples were extracted under various experimental conditions and the patterns of ADH‐treated, resting AM samples, or whole epithelial cell crude homogenates, were compared. Staining of some bands increased under certain conditions, whereas many others disappeared. Both morphological and biochemical approaches demonstrate that the recovered material was api

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00821.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

We have developed a techique for recovering apical membranous sheets from aphibian urinary bladders by gelatin stripping. The tissue is mounted on a lucite support and the apical surface is first stuck onto a gelatin‐coated glass slide at 30°C. This sandwich is then chilled on ice and the bladder is pulled away from the slide. Preliminary results indicate that this simple technique could be used to remove membranous apical sheets of various sizes, almost devoid of cytoplasmic contamination and without significant damage to the underlying cell structures. The method could also be adapted to prepare perforated cells and to study the cohesive forces between the different layers of the tiss

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00822.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

We have undertaken the analytical fractionation of epithelial cells from toad urinary bladder, a tissue extensively used to study epithelial transport of ions and water. In an attempt to establish markers for the main subcellular organelles, a number of enzymes were assayed in cell homogenates. The nearly ubiquitous plasma membrane marker 5′‐nucleotidase, and the transferases that donateN‐acetylglucosaminyl, galactosyl, and sialyl residues to glycoproteins and glycolipids in the Golgi complex were not detectable. Glucose‐6‐phosphatase activity was low in relation to that of nonspecific phosphatases and, therefore, not suitable for identifying the endoplasmic reticulum. Like the cytosolic enzyme lactate dehydrogenase, catalase was essentially found in the high‐speed supernatant, with a noteworthy part of aminopeptidase (substrate, leucyl‐ß‐naphthylamide) and NAD glycohydrolase. Other enzymes, including cytochromecoxidase, acid phosphatase, acidN‐acetyl‐ß‐glucosaminidase, alkaline phosphatase, alkaline phosphodiesterase I, nucleoside diphosphatase (substrate ADP), oligomycin‐resistant Mg++‐ATPase, and mannosyltransferase (acceptor, dolichylphosphate) were fairly active and largely sedimentable. After differential centrifugation, cytochrome oxidase, acid phosphatase, and acidN‐acetyl‐ß‐glucosaminidase were typically associated with the large granule fraction, whereas the other sedimentable enzymes exhibited a broad distribution profile overlapping the nuclear, large granule, and microsome fractions. Their behavior in density equilibrium centrifugatio

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00823.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Cytoplasmic granules obtained from toad urinary bladder epithelial cells were brought to buoyancy in a linear sucrose gradient. The gradient was loaded either with untreated cytoplasmic granules, or with granules treated with Na pyrophosphate (PPi), with digitonin, or with PPi and digitonin in succession. The following enzymes were assayed in the gradient subfractions: oligomycin‐insensitive Mg++‐ATPase, alkaline phosphodiesterase I, alkaline phosphatase, acidN‐acetyl‐β‐glucosaminidase, cytochrome oxidase, nucleoside diphosphatase (substrate, ADP), aminopeptidase (substrate, leucyl‐β‐naphthylamide), and mannosyltransferase (acceptor, dolichylphosphate). Comparison of the density distributions of enzymes in untreated and treated preparations led to the characterization of 4 distinct subcellular entities. In agreement with the properties of mitochondria from other cell types, cytochrome oxidase buoys at 1.18 within a narrow density range and its behavior is not significantly altered by PPi or digitonin. Under all conditions, acidN‐acetyl‐β‐glucosaminidase is recovered over a broad density range in the lower part of the gradient and appears as a qualified lysosomal marker. Mg++‐ATPase, alkaline phosphodiesterase I, and alkaline phosphatase belong to a group with the distinguishing features of a low equilibrium density in native cytoplasmic granules and a marked shift (+0.03 density units) after digitonin treatment. Such properties are typical of the plasma membranes. Part of the aminopeptidase activity probably also belongs to plasma membrane‐derived elements. Minor differences between alkaline phosphatase and the other 2 members of that group make it possible that their distribution domains in the membrane do not overlap or coincide. Finally, mannosyltransferase is identified as an endoplasmic reticulum marker: its distribution is shifted to lower densities after PPi. This treatment releases ribosomes from rough microsomes and accordingly decreases the density of these vesicles. The subcellular localization of nucleoside diphosphatase, which displays distinct pr

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00824.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Antidiuretic hormone induces, in the apical plasma membrane of amphibian urinary bladder epithelial cells, the exocytotic insertion of intramembranous particle aggregates that probably contain water channels. Purification of the apical membrane is a way to characterize the aggregates. The isolation of such purified membranous fractions involves the use of specific exogenous or endogenous markers. One of them could be the neutral aminopeptidase (AP), whose activity was detected in urinary bladder. Enrichment in AP activity was observed in plasma membrane preparations compared to cell homogenates (×2.7). However, a large part of the enzyme activity was also recovered in the soluble fraction of the preparation, suggesting large proteolysis of the protein. The enzyme presents a low optimal pH (6.4) and a high specificity for proline‐p‐nitroanilide as compared to the AP present in kidneys and intestines. To localize the protein in the amphibian bladder epithelium, an immunological approach was necessary due to the low activity of the enzyme in this tissue. The low enzymatic activity also prevented the purification of sufficient amounts of the urinary bladder AP as antigen, and we prepared antibodies against purified AP from frog or toad kidneys where the activity is 60 tims higher than in the bladder. The serum specificity was verified by spot immunodetection, Western blot, inhibition capacity of antibodies, and immunoadsorption on a solid support with the renal enzyme. The sera were found to be able to react with native as well as denatured forms of the kidney enzyme. Antibodies cross‐reacted with several peptides of low molecular weight (40–60 kDa) from urinary bladder plasma membrane proteins (Western blot). Immunocytochemical localization of AP was observed on the apical side of toad bladder epithelial cells.In conclusion, neutral aminopeptidase is a potential marker for amphibian urinary bladder apical membranes. However, due to the considerable proteolysis during the membrane preparation, rigorous precautions, such as the use of protease inhibitor to avoid its release in a soluble fraction, should

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00825.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

Several experimental conditions such as antidiuretic hormone (ADH) challenge, apical treatment with phorbol myristate acetate (PMA), and mechanical stretching of the tissue are known to increase the insertion of intramembrane particle aggregates and/or granule exocytosis at the apical border of epithelial cells of amphibian urinary bladders. A constant release of 2 peptides of 76 and 14 kDa apparent molecular mass, respectively, was associated with these treatments. The localization of these 2 polypeptides was assessed by immunofluorescence and electron microscopy immunocytochemistry using fluorescent, peroxidase, and colloidal gold probes. The 76 kDa polypeptide appeared to be associated with the cell coat and with the granule content which is released at the apical cell surface. The 14 kDa peptide was also found in the cell coat, and postembedding immunocytochemistry indicates its presence in cytoplasmic subapical vesicles (aggrephores and/or granules).The migration of these 76 and 14 kDa polypeptides in SDS‐polyacrylamide gel electrophoresis was modified neither by a treatment at 90°C, nor by the presence or absence of calcium in the medium. Treatment with EGTA did not modify the fluorescence emission of the two peptides and, consequently, they are probably not among the major calcium binding proteins.The addition to the mucosal medium of the stretch extract or of antibodies raised against the 76 and 14 kDa peptides did not modify ADH‐induced water permeability. However, a significant decrease of the hydrosmotic response to ADH occurred in subsequent stimulation‐washout cycles when the anti‐14 kDa peptide antiserum was applied to the mucosal bath. When the bladders were incubated with a stretch extract, we observed a slight alteration of the short‐circuit current (Isc), an increase of the basal Na+transport, and a decrease of the maximal Iscin response to ADH. The 76 kDa protein, released in the apical medium, could play a protective role in the cellular plasma membrane and could participate in the formation of the thick cell coat lining the apical membrane of the granular cells. The 14 kDa protein might be one of the proteins associated with the aggregates, but further studies will be necessary to clarify its exact role in the ADH‐induced permeability modifications observed in amphibian urin

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00826.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY

摘要:

The water permeability properties of a series of epithelial barriers (the toad urinary bladder [TUB], the rat caecum [RC], the distal human colon [DHC], and the human amnion [HA]were studied in different experimental conditions. Three parameters were simultaneously determined: the water permeability coefficient in the presence of a transepithelial hydrostatic gradient (Phydr); the water permeability coefficient in the presence of an osmotic gradient (Posm); and the transepithelial potential difference (dV). All experiments were performed with the same experimental device, allowing comparison of the permeability properties of the barriers tested. The results obtained were: (1)TUB(N=8): Phydr= 0.079±0.008 cm/s; Posm= 0.0004±0.0002 cm/s; dV=31±5 mV; (2)TUB after ADH(N=8): Phydr= 0.093±0.012 cm/s; Posm= 0.0065±0.0011 cm/s; dV=52±8; (3)RC(N=10): Phydr= 0.18±0.02 cm/s; Posm= 0.0019±0.0004 cm/s; dV=3.9±0.1 mV; (4)RC adapted to a high K diet(N=10): Phydr= 0.21±0.02 cm/s; Posm= 0.0018±0.0006 cm/s; dV=4.5±0.5 mV; (5)DHC(N=6): Phydr= 0.22±0.03 cm/s; Posm= 0.002±0.05 cm/s; dV=15±3 mV; (6) HA (N=10): Phydr= 0.32±0.05 cm/s; Posm= 0.0154±0.0015; dV=0. The results show a good correlation between Phydrand dV, but not between dV and Posmor betwe

ISSN:0248-4900    

DOI:10.1111/j.1768-322X.1989.tb00827.x

出版商:Blackwell Publishing Ltd    

年代:1989

数据来源: WILEY