Iodination (125I) of the apical plasma membrane of toad bladder epithelium: Electron-microscopic autoradiography and physiological effects

Strum, Judy M.; Edelman, Isidore S.

doi:10.1007/bf01868066

Cited by 15 publications

(11 citation statements)

References 17 publications

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“…Previous studies (29,32) (29,33), and the iodinations were performed at 2TC instead of 23TC. We measured the transepithelial potential difference and ADHinduced water flow in paired hemibladders and observed no difference between untreated hemibladders and those subjected to either of our iodination protocols (data not shown).…”

Section: Lactoperoxidase/glucose Oxidase Iodination Of the Apicalmentioning

confidence: 99%

Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.

Harris¹,

Wade²,

Handler³

1988

Proc. Natl. Acad. Sci. U.S.A.

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Antidiuretic hormone (ADH) increases the water permeability of the toad urinary bladder. The increase occurs in the apical plasma membrane of granular cells that line the urinary surface of the bladder and is produced by the insertion of water permeability units that have been identified by freeze-fracture electron microscopy as intramembrane particle aggregates. Under water-impermeable conditions, particle aggregates reside in intracellular vesicles called "aggrephores." In response to ADH, the aggrephores fuse with the apical plasma membrane and render it water permeable. When ADH is removed, intramembrane particle aggregates and aggrephores are retrieved from the apical membrane, and it returns to a water-impermeable state. To identify proteins involved in the water permeability response, we used lactoperoxidase/glucose oxidase to "MI-label external apical membrane proteins to compare control and ADH-treated bladders. Several polypeptides were consistently labeled in ADH-treated bladders and not in paired controls. After demonstrating that lactoperoxidase behaves as a fluid-phase marker and is sequestered in aggrephore-like vesicles when ADH is withdrawn, we used the technique of Mellman et al. BMl. 86, 712-7221 to label proteins endocytosed when water permeability declines after ADH is withdrawn to test whether the membrane proteins labeled in ADH-treated bladders behaved like particle aggregates. The internalized membranes contained polypeptides of the same molecular weights (55,000, 17,000-14,000, and 7,000) as those labeled on the apical surface of ADH-treated but not control bladders. These polypeptides are evidently involved in the ADH-stimulated water permeability response and may be components of particle aggregates.Antidiuretic hormone (ADH) plays a major role in the control of water balance of many vertebrates. It stimulates the conversion of specific water-impermeable epithelia to a state of high water permeability. For example, the water permeability of the urinary bladder of the toad Bufo marinus increases by >50-fold, allowing water to flow from dilute urine to blood (1). The purpose of this study was to identify membrane proteins involved in the water permeability change that occurs in the toad bladder in response to ADH.The apical plasma membranes of granular cells form >90o of the bladder surface area in contact with urine, and it is these apical membranes that undergo the remarkable change in water permeability (2). Under basal conditions, the apical cytoplasm of granular cells contains tubularshaped vesicles that have been named "aggrephores" because on freeze-fracture electron microscopy they contain distinctive particle aggregates within their limiting membranes (3-5). Aggrephores store and shuttle particle aggregates into and out of the apical plasma membrane in response to changes in the concentration of ADH (6). In response to ADH, aggrephores fuse with the apical plasma membrane and the plasma membrane becomes enriched in particle aggregates (4-8). When ADH is withdrawn, parti...

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Section: Lactoperoxidase/glucose Oxidase Iodination Of the Apicalmentioning

confidence: 99%

Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.

Harris¹,

Wade²,

Handler³

1988

Proc. Natl. Acad. Sci. U.S.A.

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“…The fixed particulates were collected by centrifugation at 100,000 • g for 1 hr. The pellets were washed three times with 0.4 ml, 200 mM sodium cacodylate, pH 7.5, post-fixed in 2% osmium tetroxide in 100 mM sodium cacodylate, pH 7.5 an d processed for electron microscopy as previously described [31].…”

Section: Morphologymentioning

confidence: 99%

“…The validity of enzymatic markers relies on the results of histochemical analysis, which assigns a given enzymatic activity to a particular organelle or recognizable membrane element [6,7]. The limitations inherent in histochemical analysis prompted attempts to develop alternative approaches, including the use of surface antigens, membrane receptors (lectins, hormones, cholera toxin), or covalently attached radioactive labels [3,5,24,31]. We elected to explore the utility of covalent, radioactive membrane markers in analysis of the biochemical properties of epithelial plasma membranes of the urinary bladder of the toad [26].…”

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confidence: 99%

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium

Rodriguez

Edelman

1979

J. Membrain Biol.

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The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Normarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with 125I. The basal-lateral components yielded a hetero-disperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochrome c reductase activities, were separated from the radio-iodine labeled by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 x g x 1 hr after removal of the mitochrondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.

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“…After dehydration in a graded series of aqueous ethanol solutions, the tissues were embedded in Epon 812 [19]. Light and electron-microscopic autoradiographs were obtained as described previously [26].…”

Section: Light and Electron-microscopic Autoradiographymentioning

confidence: 99%

“…In an earlier study, Strum and Edelman [26] reported covalent labeling of the apical plasma membrane of toad bladder with 12sI activated by the glucose oxidase-lactoperoxidase system. The availability of multiple iodine isotopes and the prospect of group specific labeling in the same membrane (i.e., the apical leaflet), with two different reagents prompted us to explore the potential utility of 125I-DDISA.…”

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confidence: 98%

Differential covalent labeling of apical and basal-lateral membranes of the epithelium of the toad bladder

1976

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The apical (luminal) plasma membrane of toad bladder epithelial cells has been labeled with (125I) diazo-diiodo sulfanilic acid (125I-DDISA) as demonstrated by electron-microscopic autoradiography. The silver grains (125I) were localized exclusively to the apical surface. At concentrations of DDISA of 10(-3) M or less, binding to the apical membrane had no significant effect on the fine structure of the epithelium. At concentrations of DDISA of 10(-6) M or less, the baseline short-circuit current (SCC), and the response to cyclic 3',5'-adenosine monophosphate (cAMP) plus theophylline were unimpaired. At 10(-5) M, baseline SCC was unchanged and the response to cyclic AMP plus theophylline was enhanced. At concentrations of 10(-4) M and greater baseline SCC was depressed and the response to the nucleotide inhibited. The basal-lateral epithelial plasma membranes were labeled by exposing the serosal side to pyridoxal phosphate and reducing the resultant Schiff base with sodium borotritide (3H-NaBH1). In electron-microscopic autoradiographs, the silver grains (3H) were found over the basal and lateral surfaces of the epithelium. At concentrations of pyridoxal phosphate of 10(-4) M and 3H-NaBH1 of 10(-3) M, there were no significant changes in the fine structure of the epithelium. Addition of pyridoxal phosphate (10(-4) M) and NaBH4 (10(-3) M) to the serosal side decreased the baseline SCC significantly but not the response to vasopressin. Covalent attachment of the 125I and the 3H was indicated by resistance to elution in the preparation of the sections for electron-microscopy and the reagent requirements for binding.

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Iodination (125I) of the apical plasma membrane of toad bladder epithelium: Electron-microscopic autoradiography and physiological effects

Cited by 15 publications

References 17 publications

Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.

Identification of specific apical membrane polypeptides associated with the antidiuretic hormone-elicited water permeability increase in the toad urinary bladder.

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium

Differential covalent labeling of apical and basal-lateral membranes of the epithelium of the toad bladder

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