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.

Role of PKC isozyme III (γ) in water transport in amphibian urinary bladder

1992 ◽  
Vol 50 (1) ◽  
pp. 796-797
Author(s):  
A.J Mia ◽  
L.X. Oakford ◽  
P.D. Thompson ◽  
Z.H. Ning ◽  
T. Yorio
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Phorbol Esters ◽  
Apical Membrane ◽  
Protein A ◽  
Water Channels ◽  
Renal Epithelia ◽  
Amphibian Urinary Bladder ◽  
Pkc Isozymes

Vasopressin stimulated water flow across renal epithelia is thought to occur through a V2 receptor coupled to adenylcyclase. The increase in water flow occurs as a result of a fusion of water channels with the apical membrane and is indicative of an increase in membrane capacitance following hormone addition.What controls the cycling of water channels and their insertion into the membrane is uncertain. Our laboratory has demonstrated that renal epithelia as well as amphibian urinary bladder membranes, contain a vasopressin V1 receptor which upon activation results in the breakdown of phosphoinositide and the formation of inositol triphosphate and diacylglycerol, the latter an activator of protein kinase C (PKC). The initiation of transepithelial water flow also appears to involve V1 receptors and possibly activation of PKC. To test this hypothesis, we have been using activators of PKC, such as phorbol esters and mezerein, as pharmacological tools to determine if PKC activation results in similar physiological responses as the hormone. Several PKC isozymes, upon activation, are known to be translocated to the apical membrane as visualized by FITC immunofluorescence. Previously, we reported co-localization of PKC subtypes I (γ) and II (β) in toad urinary bladders using monoclonal antibodies and protein A-gold probes. This report includes the localization of PKC subtype III (α) and its distribution pattern using immunogold labeling.

Cloning of an aquaporin homologue present in water channel containing endosomes of toad urinary bladder

1996 ◽  
Vol 270 (1) ◽  
pp. C372-C381 ◽  
Author(s):  
J. Siner ◽  
A. Paredes ◽  
C. Hosselet ◽  
T. Hammond ◽  
K. Strange ◽  
...  
Keyword(s):  
Urinary Bladder ◽  
Water Channel ◽  
Structural Features ◽  
Xenopus Laevis Oocytes ◽  
Sequence Motif ◽  
Toad Urinary Bladder ◽  
Cytoplasmic Vesicles ◽  
Terrestrial Habitats ◽  
Total Body

Regulation of total body water balance in amphibians by antidiuretic hormone (ADH) contributed to their successful colonization of terrestrial habitats approximately 200-300 million years ago. In the mammalian kidney, ADH modulates epithelial cell apical membrane water permeability (Pf) by fusion and retrieval of cytoplasmic vesicles containing water channel proteins called aquaporins (AQPs). To determine the role of AQPs in ADH-elicited Pf in amphibians, we have identified and characterized a unique AQP from Bufo marinus called AQP toad bladder (AQP-TB). AQP-TB possesses many structural features common to other AQPs, AQP-TB is expressed abundantly in ADH-responsive tissues, including toad urinary bladder and skin as well as lung, skeletal muscle, kidney, and brain. In a manner identical to that reported for the mammalian ADH-elicited water channel AQP2, AQP-TB expression is increased significantly by intervals of dehydration or chronic ADH stimulation. However, expression of AQP-TB protein in Xenopus laevis oocytes does not significantly increase oocyte Pf. The lack of expression of functional AQP-TB water channels in oocytes may result from intracellular sequestration of AQP-TB due to the presence of a YXRF sequence motif present in its carboxyterminal domain.

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.

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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