Ion selectivity of the apical membrane Na channel in the toad urinary bladder

1982 ◽  
Vol 67 (1) ◽  
pp. 91-98 ◽  
Author(s):  
Lawrence G. Palmer
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Ion Selectivity ◽  
Na Channel ◽  
Toad Urinary Bladder

scholarly journals Relationship of the aldosterone-induced protein, GP70, to the conductive Na+ channel.

1991 ◽  
Vol 2 (6) ◽  
pp. 1108-1114
Author(s):  
H Szerlip ◽  
P Palevsky ◽  
M Cox ◽  
B Blazer-Yost
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Na Channel ◽  
Toad Urinary Bladder ◽  
Apical Surface ◽  
Large Protein ◽  
Channel Modulation ◽  
Relationship Of

Although one of the primary effects of aldosterone is to increase apical membrane Na+ conductance, as yet none of the proteins induced by the hormone in renal epithelia have been shown to be related to the conductive Na+ channel. Because the toad urinary bladder aldosterone-induced glycoprotein, GP70, has recently been localized to the apical surface of this Na+ transporting epithelium, whether GP70 is associated with the Na+ channel was examined. The specificities of a monoclonal antibody used to characterize GP70 (mAb 20) and a polyclonal antibody raised against the purified bovine renal papillary Na+ channel (anti-CH) were compared: GP70 was specifically immunoprecipitated by both mAb 20 and anti-CH. Moreover, the sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of mAb 20 purified toad urinary bladder membrane preparations was similar to those reported for bovine and A6 cell Na+ channels. Under nonreducing conditions, a single, very large protein was evident; reduction yielded GP70, a 140-kd polypeptide, and a number of minor bands. Interestingly, only GP70 was induced by aldosterone. Thus, GP70 appears to be associated with the toad urinary bladder conductive Na+ channel; whether GP70 is an integral subunit of the channel or whether it functions as a regulatory moiety remains to be determined. Whatever the case, because GP70 is induced by aldosterone, it likely has a central role in Na+ channel modulation.

Functional groups of the Na+ channel: role of carboxyl and histidyl groups

1983 ◽  
Vol 245 (6) ◽  
pp. F716-F725 ◽  
Author(s):  
C. S. Park ◽  
D. D. Fanestil
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Dissociation Constants ◽  
Specific Binding ◽  
Ringer Solution ◽  
Acid Dissociation ◽  
Na Channel ◽  
Toad Urinary Bladder ◽  
Carboxyl Group ◽  
Na Transport

Two titratable groups, with effect on Na+ transport and with apparent acid dissociation constants (pKaS) of 4.2 and 6.7, were found in the apical membrane of toad urinary bladder and are tentatively identified as a carboxyl and an imidazole. N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), a reagent selective for carboxyl residues, inhibits Na+ transport in the urinary bladder of toads. The underlying chemical reaction whereby EEDQ produces inhibition through potential modification of carboxyl residues was studied. The inhibitory action of EEDQ on Na+ transport was dependent on pH of reaction media and availability of nucleophile, indicating that formation of a covalent acyl-nucleophile bond is probably involved in the irreversible inhibition of Na+ transport. The kinetics of the inhibition showed a stoichiometry of formation of one acyl-nucleophile bond per closure of one Na+ transport site, presumably the Na+ channel. The nucleophile that appears to be involved in the formation of the acyl-nucleophile bond was tentatively identified as having an apparent pKa of 6.7. Amiloride and two analogues of amiloride added to the mucosal Ringer solution (but not serosal amiloride) protected against inhibition of Na+ transport by EEDQ--a finding consistent with the hypothesis that the EEDQ-activated carboxyl group undergoes reaction with a nucleophile at or near the site of specific binding of amiloride onto the apical membrane, most likely at the Na+ channel. Our findings led us to postulate that amiloride must interact with at least two sites on the Na+ channel in order to block the channel. One of the two sites appears to be an ionic interaction between the anionic carboxyl group at the Na+ channel and the cationic guanidinium group of amiloride.

Role of carboxyl group in Na+-entry step at apical membrane of toad urinary bladder

1983 ◽  
Vol 245 (6) ◽  
pp. F707-F715 ◽  
Author(s):  
C. S. Park ◽  
J. Kipnowski ◽  
D. D. Fanestil
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Complete Recovery ◽  
Na Channel ◽  
Toad Urinary Bladder ◽  
Carboxyl Group ◽  
Na Transport ◽  
Tight Epithelia

Mucosal addition of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) and some lipid-soluble carbodiimides, agents which are selective for carboxyl groups, irreversibly inhibited Na+ transport as measured by short-circuit current (SCC) in the urinary bladder of the toad. The inhibition of Na+ transport by EEDQ had the following characteristics: 1) the inhibition was accompanied by a significant increase in the transepithelial electrical resistance; 2) the decrease in SCC was accounted for by a comparable decrease in 22Na+ influx without effect on Na+ efflux; 3) amphotericin B produced complete recovery of SCC inhibited with EEDQ but not with antimycin A or ouabain; 4) mucosal EEDQ decreased the amiloride-sensitive reversal of Na+ current that is induced by serosal nystatin in the absence of mucosal Na+; 5) vasopressin and acid mucosal pH caused an increase in SCC in proportion to the SCC remaining after EEDQ inhibition; and 6) Vmax of the SCC was decreased without alteration in the apparent Km for Na+. Based on these characteristics of EEDQ inhibition of Na+ transport, we infer that a carboxyl group of the Na+ channel is involved in the Na+-entry step across the apical membrane of “tight” epithelia. The inhibition of Na+ transport with EEDQ most likely involves closing the Na+ channel through a chemical reaction involving a carboxyl group of the channel.

Role of PKC isozymes in water transport in toad urinary bladder

1991 ◽  
Vol 49 ◽  
pp. 302-303
Author(s):  
A.J. Mia ◽  
L.X. Oakford ◽  
T. Yorio
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Membrane Surface ◽  
Protein A ◽  
Cell Types ◽  
Leukemic Cells ◽  
Toad Urinary Bladder ◽  
Pkc Isozymes ◽  
Antibody Labeling

Protein kinase C (PKC) isozymes, when activated, are translocated to particulate membrane fractions for transport to the apical membrane surface in a variety of cell types. Evidence of PKC translocation was demonstrated in human megakaryoblastic leukemic cells, and in cardiac myocytes and fibroblasts, using FTTC immunofluorescent antibody labeling techniques. Recently, we reported immunogold localizations of PKC subtypes I and II in toad urinary bladder epithelia, following 60 min stimulation with Mezerein (MZ), a PKC activator, or antidiuretic hormone (ADH). Localization of isozyme subtypes I and n was carried out in separate grids using specific monoclonal antibodies with subsequent labeling with 20nm protein A-gold probes. Each PKC subtype was found to be distributed singularly and in discrete isolated patches in the cytosol as well as in the apical membrane domains. To determine if the PKC isozymes co-localized within the cell, a double immunogold labeling technique using single grids was utilized.

Fluorescent markers to study membrane retrieval in antidiuretic hormone-treated toad urinary bladder

1986 ◽  
Vol 251 (2) ◽  
pp. C274-C284 ◽  
Author(s):  
H. W. Harris ◽  
J. B. Wade ◽  
J. S. Handler
Keyword(s):  
Electron Microscopy ◽  
Urinary Bladder ◽  
Horseradish Peroxidase ◽  
Antidiuretic Hormone ◽  
Apical Membrane ◽  
Granular Cell ◽  
Cell Uptake ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Apical Surface

Antidiuretic hormone (ADH) stimulation of toad urinary bladder causes fusion of intracellular vesicles called aggrephores with the apical plasma membrane of granular cells. Aggrephores contain intramembrane particle aggregates whose appearance in the apical membrane is believed to produce a large increase in its water permeability. ADH removal (ADH washout) is thought to cause the retrieval of aggrephores into granular cell cytoplasm. We studied granular cell uptake of dextran and horseradish peroxidase conjugated with fluorescein, rhodamine, or both during ADH washout. Granular cell uptake of fluorescent dextran was dependent on prior exposure to ADH, a linear function of dextran concentration, and increased by a transepithelial osmotic gradient. Immediately after removal of ADH, granular cell fluorescence was finely dispersed and located near the apical surface. Subsequently, it coalesced into larger bodies. This change was most apparent when a single bladder was subjected to two cycles of ADH stimulation and removal using a dextran containing a different fluorophore for each cycle. The ultrastructural correlate for these fluorescent patterns was identified using rhodamine-labeled horseradish peroxidase. Electron microscopy showed that after detachment from the apical membrane, label was initially in tubular-shaped vesicles near the apical surface. Later, these vesicles clustered near multivesicular bodies and transferred their label to these structures. These tubular vesicles closely resemble the morphology of aggrephores visualized by freeze-fracture electron microscopy. We conclude that these fluorescent compounds can be used as markers for the luminal contents of membrane retrieved during ADH washout and allow detailed study of its intracellular processing.

Acidification of vasopressin-induced endosomes in toad urinary bladder

1994 ◽  
Vol 267 (1) ◽  
pp. F106-F113
Author(s):  
F. Emma ◽  
H. W. Harris ◽  
K. Strange
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Apical Membrane ◽  
Fluid Phase ◽  
Endocytic Pathway ◽  
Toad Urinary Bladder ◽  
Acidic Ph ◽  
Terminal Event ◽  
Ultimate Fate

It is well established that water channels (WC) are removed from the apical membrane of vasopressin-sensitive epithelia by endocytosis. The processing and the ultimate fate of endocytosed WC is, however, incompletely understood. In many cells, endosome acidification plays an important role in the processing and sorting of endocytosed proteins. Endosome acidification in the toad urinary bladder was therefore examined in vivo by fluorescence ratio video microscopy after induction of endocytosis by vasopressin removal and transepithelial water flow in the presence of the pH-sensitive fluid phase marker 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-dextran. Fifteen minutes after induction of endocytosis, the majority of endosomes had a neutral or slightly acidic pH. The number of acidic endosomes increased progressively with time. Two hours after endocytosis began, 98% of the endosomes had a pH < 6.0. Bafilomycin completely blocked endosome acidification, indicating that H+ transport is mediated by a vacuolar H(+)-adenosinetriphosphatase. Bafilomycin had no effect on transepithelial water flow in bladders repetitively stimulated by vasopressin. These findings, as well as the work of other investigators, suggest that if WC recycling occurs, it is not dependent on acidification of the endosomal compartment. Acidification of vasopressin-induced endosomes most likely represents a terminal event in the endocytic pathway.

Water and nonelectrolyte permeabilities of apical membranes of toad urinary bladder granular cells

1992 ◽  
Vol 262 (5) ◽  
pp. C1109-C1118 ◽  
Author(s):  
E. B. Grossman ◽  
H. W. Harris ◽  
R. A. Star ◽  
M. L. Zeidel
Keyword(s):  
Urinary Bladder ◽  
Plasma Membranes ◽  
Magnetic Beads ◽  
Apical Membrane ◽  
Granular Cell ◽  
Structural Features ◽  
Toad Urinary Bladder ◽  
Apical Surface ◽  
General Applicability ◽  
Apical Membranes

Certain types of epithelial cells such as those lining the toad urinary bladder have been classified as "tight" because their apical membranes exhibit low permeabilities to water, ions, and small nonelectrolytes. However, the permeability properties and structural features of these specialized apical membranes remain unclear because these membranes have never been purified. To isolate toad bladder granular cell apical membranes, we derivatized the bladder apical surface with the membrane-impermeant bifunctional reagent N-hydroxysulfosuccinimydyl-S,S-biotin (NHS-SS-biotin). After cell disruption, these derivatized apical membranes were purified using streptavidin-coated magnetic beads in a magnetic field. With the use of lactoperoxidase-mediated radioiodination as a marker for apical membrane, this preparative procedure purified apical membrane 48- or 72-fold as compared with homogenate. Thin section electron microscopy revealed unilamellar vesicles with some nonvesiculated membranes, while fragments of organelles such as mitochondria were absent. Water and nonelectrolyte permeabilities of purified apical membrane vesicles were similar to those obtained in intact bladders in the absence of antidiuretic hormone stimulation. The results demonstrate that isolated apical vesicles do not contain water channels and confirm the applicability of Overton's rule to the apical membrane of the toad urinary bladder. The technique has general applicability to isolation of other plasma membranes, and the apical membranes obtained are suitable for structural analysis.

Dual action of aldosterone on toad bladder: Na+ permeability and Na+ pump modulation

1984 ◽  
Vol 246 (4) ◽  
pp. F517-F525 ◽  
Author(s):  
C. S. Park ◽  
I. S. Edelman
Keyword(s):  
Urinary Bladder ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Electrical Conductance ◽  
Na Channel ◽  
Metabolic State ◽  
Na Pump ◽  
Na Permeability ◽  
Basolateral Side ◽  
Dual Action

The effects of aldosterone on the functional characteristics of the Na+ entry step across the apical membrane and on the Na+ exit step across the basolateral membrane of the urinary bladder of toads were examined using amiloride and ouabain as probes of the respective surfaces of the cell. Aldosterone stimulated Na+ transport with a concurrent increase in the transepithelial electrical conductance as did two other agents, vasopressin (ADH) and p-chloromercuriphenylsulfonate (PCMPS), primarily active on the apical membrane. Unlike the effects of ADH and PCMPS, however, the effect of aldosterone on Na+ conductance was blocked by actinomycin D and was associated with a decreased sensitivity of the apical Na+ channel to amiloride. In addition, aldosterone increased the sensitivity of the Na+ pump on the basolateral side to ouabain, an effect that was dependent on the metabolic state of the urinary bladder. These results support the inference of coordinate effects on Na+ permeability of the apical membrane and the Na+ pump of the basolateral membrane. Both effects of aldosterone appear to be dependent on the metabolic state of the transporting epithelium.

scholarly journals Anion-sensitive sodium conductance in the apical membrane of toad urinary bladder.

1980 ◽  
Vol 76 (1) ◽  
pp. 69-81 ◽  
Author(s):  
J Narvarte ◽  
A L Finn
Keyword(s):  
Membrane Potential ◽  
Urinary Bladder ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Membrane Potentials ◽  
Toad Urinary Bladder ◽  
Bladder Epithelium ◽  
Anionic Composition ◽  
Before And After ◽  
Cl Conductance

Membrane potentials and the electrical resistance of the cell membranes and the shunt pathway of toad urinary bladder epithelium were measured using microelectrode techniques. These measurements were used to compute the equivalent electromotive forces (EMF) at both cell borders before and after reductions in mucosal Cl- concentration ([Cl]m). The effects of reduction in [Cl]m depended on the anionic substitute. Gluconate or sulfate substitutions increased transepithelial resistance, depolarized membrane potentials and EMF at both cell borders, and decreased cell conductance. Iodide substitutions had opposite effects. Gluconate or sulfate substitutions decreased apical Na conductance, where iodide replacements increased it. When gluconate or sulfate substitutions were brought about the presence of amiloride in the mucosal solution, apical membrane potential and EMF hyperpolarized with no significant changes in basolateral membrane potential or EMF. It is concluded that: (a) apical Na conductance depends, in part, on the anionic composition of the mucosal solution, (b) there is a Cl- conductance in the apical membrane, and (c) the electrical communication between apical and basolateral membranes previously described is mediated by changes in the size of the cell Na pool, most likely by a change in sodium activity.

Modulation of antidiuretic hormone-dependent capacitance and water flow in toad urinary bladder

1984 ◽  
Vol 246 (4) ◽  
pp. F501-F508
Author(s):  
L. G. Palmer ◽  
N. Speez
Keyword(s):  
Urinary Bladder ◽  
Antidiuretic Hormone ◽  
Water Flow ◽  
Water Permeability ◽  
Apical Membrane ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Apical Surface ◽  
Membrane Area ◽  
Close Relationship

To test the hypothesis that antidiuretic hormone- (ADH) dependent water permeability is associated with changes in apical membrane area, hormone-dependent water flow and capacitance changes were measured in the toad urinary bladder under a number of different conditions. Dose-response relationships for water flow (Jv) and capacitance increases (delta C) were similar from 1 to 20 mU/ml ADH. At higher concentrations, Jv reached a plateau, while delta C decreased. The decrease in delta C was prevented by elimination of the osmotic gradient across the tissue. Serosal hydrazine (10 mM) increased Jv sevenfold and delta C threefold in the presence of 1 mU/ml ADH. Mucosal NH4Cl, at constant mucosal pH, increased Jv by 50-100%, but did not significantly change delta C. In the absence of an osmotic gradient, mucosal NH+4 increased delta C by 50%. NH4Cl had no effect on hydroosmotic response to 8-bromo-adenosine 3',5'-cyclic monophosphate (cAMP). Mucosal CO2 (9%) decreased Jv by greater than 90%, and delta C by 60% with 20 mU/ml ADH. Mucosal CO2 also inhibited the hydroosmotic response to 8-bromo-cAMP. Removal of serosal Na diminished cAMP-dependent Jv and delta C. The results confirmed the close relationship between ADH-dependent water permeability and membrane capacitance. They indicate, however, that under some circumstances membrane may be retrieved from the apical surface without affecting water permeability.

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