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.

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.

ADH-induced recycling of fluid-phase marker from endosomes to the mucosal surface in toad bladder

1994 ◽  
Vol 267 (1) ◽  
pp. C32-C38 ◽  
Author(s):  
R. A. Coleman ◽  
J. B. Wade
Keyword(s):  
Urinary Bladder ◽  
Horseradish Peroxidase ◽  
Antidiuretic Hormone ◽  
Water Permeability ◽  
Apical Membrane ◽  
Fluid Phase ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Bathing Solution ◽  
Apical Surface

In the toad urinary bladder, the reversal of antidiuretic hormone (ADH) stimulation results in the endocytosis of apical membrane water channels, along with any fluid-phase marker present in the mucosal bathing solution. We have loaded vesicles with horseradish peroxidase (HRP), then restimulated the bladders and measured the reappearance of endocytosed HRP in the mucosal bath. HRP-loaded bladders that were restimulated showed HRP release that peaked sharply within 15 min after restimulation. Varying the interval between loading and restimulation did not vary HRP release significantly. Restimulation with forskolin gave HRP release values similar to ADH. The amount of HRP released correlated with the magnitude of water permeability induced. The demonstration that fluid-phase markers can be recycled from endosomes to the apical surface in a hormone-dependent fashion indicates that endocytosed membrane, containing water channels, is able to rapidly recycle back to the surface in response to hormone restimulation. In addition, marker release declined progressively with repeated restimulation, totaling < 30% of the retrieved amount. This result indicates that a relatively large proportion of the retrieved marker reaches a nonrecycling compartment.

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.

Effect of an osmotic gradient on antidiuretic hormone-induced endocytosis and hydroosmosis in the toad urinary bladder

1984 ◽  
Vol 247 (2) ◽  
pp. F370-F379
Author(s):  
S. K. Masur ◽  
S. Cooper ◽  
M. S. Rubin
Keyword(s):  
Antidiuretic Hormone ◽  
Apical Membrane ◽  
Surface Characteristics ◽  
Active Role ◽  
Ruthenium Red ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Apical Surface ◽  
Osmotic Permeability

The luminal (apical) border of the epithelium of the bladder in the well-hydrated toad is relatively impermeable, so the bladder usually stores hyposmotic urine. When antidiuretic hormone (ADH) increases apical membrane osmotic permeability dramatically, water is resorbed from hyposmotic mucosal solution; in the presence of hyposmotic or isosmotic mucosal solutions, ADH concomitantly induces exocytosis at the apical border of granule-rich (G) cells. Then ADH induces endocytosis at this border. We describe how an osmotic gradient affects ADH-induced endocytosis and hydroosmosis in vitro. We can assess ADH-induced endocytosis in gradient and no-gradient bladders by applying a double-marker technique that distinguishes among endocytosis, completed internalization of previously surface-attached membrane, and surface invagination by comparing the number of horseradish peroxidase (HRP) uptake bodies (endocytosis) with the number of ruthenium red (RR)-delineated bodies (surface invaginations). With this approach we find that gradient bladders have approximately six times more ADH-induced endocytosis than no-gradient bladders during 45-60 min of ADH stimulation. Furthermore, at 60 min approximately 50% of the HRP-containing structures in no-gradient bladders remain surface connected compared with approximately 1% in gradient bladders. In parallel physiological studies, no-gradient bladders reach and maintain higher induced osmotic permeabilities than gradient bladders. These findings support the hypothesis that endocytosis plays an active role in reestablishing impermeable apical surface characteristics in toad bladder.

Antidiuretic hormone-dependent membrane capacitance and water permeability in the toad urinary bladder

1983 ◽  
Vol 244 (2) ◽  
pp. F195-F204
Author(s):  
L. G. Palmer ◽  
M. Lorenzen
Keyword(s):  
Antidiuretic Hormone ◽  
Water Flow ◽  
Time Course ◽  
Apical Membrane ◽  
Constant Current ◽  
Apical Plasma Membrane ◽  
Voltage Response ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Capacitance Change

Antidiuretic hormone (ADH) increased the electrical capacitance of apical membrane of the toad bladder; this effect was modulated by the osmotic gradient across the tissue. Capacitance was measured from the transepithelial voltage response to constant-current pulses using bladders depolarized with KCl-sucrose serosal solution to reduce basolateral resistance and with Na-free mucosal solution to increase apical membrane resistance. Addition of ADH (20 mU/ml) increased capacitance by 28 +/- 9% (mean +/- SD) in the absence and by 8 +/- 3% in the presence of an osmotic gradient (200 mosM, mucosal side hypotonic). With bladders stimulated in the absence of an osmotic gradient, rapidly imposing a gradient resulted in a peak rate of water flow that declined to 40% of the peak value after 15-20 min. ADH-dependent capacitance also decreased with a similar time course. Removal of ADH reversed the capacitance change (t1/2 = 10-15 min), but the reversal was slower than the decline in water flow to basal levels (t1/2 less than 5 min). Colchicine and cytochalasin B also inhibited the ADH-induced capacitance increase. The capacitance change was also inhibited when the mucosal solution was made hypertonic with raffinose. The results are interpreted within the framework of a previously proposed model of ADH-stimulated water transport in which cytoplasmic vesicular structures fuse with the apical plasma membrane.

Dynamics of apical membrane responses to ADH in amphibian bladder

1989 ◽  
Vol 257 (5) ◽  
pp. R998-R1003 ◽  
Author(s):  
J. B. Wade
Keyword(s):  
Water Permeability ◽  
Apical Membrane ◽  
Function Analysis ◽  
Membrane Dynamics ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Apical Surface ◽  
Dynamic Feedback ◽  
Particle Aggregates ◽  
Intracellular Vesicles

The dynamic insertion and retrieval of membrane at the apical surface plays an important role in the action of antidiuretic hormone (ADH). The addition of membrane with water channels is a crucial event in initiating the water permeability response. ADH-stimulated bladders display distinctive differentiations in the apical membrane that represent sites where intracellular vesicles carrying intramembrane particle aggregates have fused with the apical surface. In the absence of an osmotic gradient these fusion sites appear to be relatively stable structures, but in the presence of an osmotic gradient there seems to be continuous addition and retrieval of membrane during sustained exposure to ADH. It is now clear that a dynamic feedback process is present, such that the water permeability of the apical membrane is adjusted by retrieval or addition of membrane depending on the magnitude of the transepithelial osmotic gradient. Removal of ADH leads to a striking retrieval of apical membrane, and intact aggregates have been demonstrated in the membrane of the vesicles that form in the apical cytoplasm after reversal of the response. Structure-function analysis has provided unique information, demonstrating that membrane dynamics is central to the mechanism whereby ADH regulates osmotic permeability in the 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.

Cytoplasmic dilution induces antidiuretic hormone water channel retrieval in toad urinary bladder

1992 ◽  
Vol 263 (1) ◽  
pp. F163-F170 ◽  
Author(s):  
H. W. Harris ◽  
B. Botelho ◽  
M. L. Zeidel ◽  
K. Strange
Keyword(s):  
Urinary Bladder ◽  
Antidiuretic Hormone ◽  
Apical Cell ◽  
Water Channel ◽  
Fluid Phase ◽  
Fluorescein Isothiocyanate ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Apical Cell Membrane

Antidiuretic hormone (ADH) increases the osmotic water permeability (Pf) of the toad urinary bladder by insertion of water channels into the apical cell membrane. Transepithelial water flow (Jv) reduces Pf by inducing endocytosis of apical water channels despite continuous ADH stimulation. This phenomenon is termed flux inhibition. We wished to determine whether cytoplasmic dilution or transcellular Jv causes flux inhibition because both have been proposed previously as a primary regulatory mechanism for this process. Apical membrane endocytosis was quantified by monitoring the uptake of the fluid phase marker fluorescein isothiocyanate dextran (FITC-dextran). FITC-dextran fluorescence was monitored in Triton X-100 extracts of epithelial cells as the ratio of total tissue fluorescence compared with background fluorescence. The background was defined as cellular autofluorescence and nonspecific tissue staining due to the presence of small amounts of free fluorescein contaminating the FITC-dextran. FITC-dextran uptake measured under symmetric isotonic (220 mosmol/kgH2O) conditions in either the absence (1.0 +/- 0.4 SD; n = 14) or presence (1.3 +/- 0.3; n = 4) of ADH was not statistically different from that of background. In contrast, flux inhibition induced by a 180 mosmol/kgH2O apical-to-basolateral osmotic gradient increased FITC-dextran uptake to 3.4 +/- 1.3 (n = 7). FITC-dextran uptake was identical in bladders exposed to symmetric hypotonic (150 mosmol/kgH2O) solutions during ADH (3.6 +/- 0.9; n = 6) or adenosine 3',5'-cyclic monophosphate (3.1 +/- 0.4 fold; n = 3) stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

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