scholarly journals Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel.

1990 ◽  
Vol 95 (5) ◽  
pp. 941-960 ◽  
Author(s):  
L B Shi ◽  
D Brown ◽  
A S Verkman
Keyword(s):  
Urinary Bladder ◽  
Permeability Coefficient ◽  
Water Channel ◽  
Water Channels ◽  
Water Proton ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Urea Transport ◽  
Proton Permeability ◽  
Quenching Assay

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.

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)

Nature of ADH-Induced Endosomes In Toad Urinary Bladder Granular Epithelial Cells

1998 ◽  
Vol 4 (S2) ◽  
pp. 1034-1035
Author(s):  
A.J. Mia ◽  
L.X. Oakford ◽  
A. Dibas ◽  
T. Yorio
Keyword(s):  
Urinary Bladder ◽  
Water Channel ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Multivesicular Bodies ◽  
Osmotic Gradient ◽  
True Nature ◽  
Golgi Bodies ◽  
Glass Tubes

Serosal ADH stimulation enhances water flow under an imposed osmotic gradient through insertion of water channels (aggrephores) into the mucosal plasma membrane of toad urinary bladder sacs. Following cessation of ADH actions, water channels are retrieved as endosomes that can be visualized by mucosal inclusion of horseradish peroxidase (HRP) into round vesicles, long tubules and multivesicular bodies within the cytosol (1,2,3). Endosomes also occur adjacent to golgi bodies or lysosomes (1,2,3). However, true nature of endosomes including their formation at the mucosal surface and their shuttling in granular cells is still unclear (4,5). Current studies were undertaken to understand the role of endosomes in water channel cycling in this renal membrane model.Urinary bladder sacs removed surgically from doubly-pithed toads, were suspended at ends of glass tubes. Control (no hormone) and experimental bladder sacs were exposed to ADH for 10 min in the absence of osmotic gradient.

Drugs activating G proteins disturb cycling of ADH-dependent water channels in toad urinary bladder

1995 ◽  
Vol 269 (2) ◽  
pp. C424-C434 ◽  
Author(s):  
A. Boom ◽  
B. Flamion ◽  
M. Abramow ◽  
R. Beauwens
Keyword(s):  
Urinary Bladder ◽  
G Proteins ◽  
Apical Membrane ◽  
Water Channel ◽  
Fluid Phase ◽  
Fluorescein Isothiocyanate ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Fluid Phase Endocytosis

In the toad urinary bladder, antidiuretic hormone (ADH)-mediated changes in water permeability depend on exocytic insertion and endocytic retrieval of water channels into and from the apical membrane, respectively. Because GTP-binding proteins (G proteins) are well-recognized regulators of vesicular trafficking throughout the cell, we tested the hypothesis that drugs interfering with G protein would modify the hydrosmotic response to ADH and the ADH-regulated formation of endosomes, as assessed by luminal incorporation of a fluid-phase marker [fluorescein isothiocyanate (FITC)-dextran, 70 kDa]. Mastoparan (4 microM) and compound 48/80 (poly-p-methoxyphenylethylmethylamine; 50 micrograms/ml), added to the luminal side of the toad urinary bladder, as well as AlF3 added to the serosal side (400 microM), inhibited ADH- and 8-bromoadenosine 3',5'-cyclic monophosphate-induced transepithelial water flow by > 50% and simultaneously enhanced cellular incorporation of FITC-dextran by > 200%. The pattern of FITC-dextran uptake observed using fluorescence microscopy both in scraped cells and in the intact bladder was granular, suggesting fluid-phase endocytosis. Mastoparan and AlF3, which are both probes of G proteins, increased FITC-dextran uptake only in the presence of ADH and a transepithelial osmotic gradient, i.e., under conditions where water channel-carrying endosomes presumably cycle. Therefore, we suggest that the ADH-dependent cycling of water channels could be controlled by one or more G proteins associated with the apical membrane and/or the water channel-carrying vesicles.

Progesterone inhibition of water permeability in Bufo arenarum oocytes and urinary bladder

1996 ◽  
Vol 270 (5) ◽  
pp. F880-F885 ◽  
Author(s):  
P. Ford ◽  
G. Amodeo ◽  
C. Capurro ◽  
C. Ibarra ◽  
R. Dorr ◽  
...  
Keyword(s):  
Urinary Bladder ◽  
Xenopus Laevis ◽  
Water Permeability ◽  
Water Channel ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Osmotic Permeability ◽  
Bufo Arenarum ◽  
Mrna Extraction

The ovarian oocytes from Bufo arenarum (BAO) but not those from Xenopus laevis (XLO) would have water channels (WC). We now report that the injection of the mRNA from BAO into the oocytes from XLO increased their water osmotic permeability (Pi) (reduced by 0.3 mM HgCl2 and reversed by 5 mM beta-mercaptoethanol). A 30-min challenge with progesterone induced, 18 h later, a reduction of the mercury-sensitive fraction of Pf in the BAO (but not in XLO). The mRNA from BAO pretreated with progesterone lost its capacity to induce WC in the XLO, but the hormone did not affect the expression of the WC in XLO previously injected with the mRNA from BAO. Pf was also measured in urinary bladders of BAO. Eighteen hours after a challenge with progesterone, a reduction in the hydrosmotic response to oxytocin was observed. Finally, the mRNA from the urinary bladder of BAO was injected into XLO. An increase in Pf was observed. This was not the case if, before the mRNA extraction, the bladders were treated with progesterone. We conclude that the BAO WC share progesterone sensitivity with the oxytocin-regulated water channel present in the toad urinary bladder.

Mechanisms and regulation of water permeability in renal epithelia

1989 ◽  
Vol 257 (5) ◽  
pp. C837-C850 ◽  
Author(s):  
A. S. Verkman
Keyword(s):  
Urinary Bladder ◽  
Proximal Tubule ◽  
Water Permeability ◽  
Permeability Coefficient ◽  
Biological Membranes ◽  
Water Channels ◽  
Proton Pumps ◽  
Toad Urinary Bladder ◽  
Water Permeability Coefficient ◽  
Collecting Tubule

Water transport occurs in all biological membranes. A few selected membranes in the kidney, amphibian urinary bladder, and erythrocyte have very high water permeability and are thought to contain specialized water transporting units termed "water channels." The known biophysical properties of membranes containing water channels are a high osmotic water permeability coefficient (Pf), an osmotic-to-diffusional water permeability coefficient ratio (Pf/Pd) greater than unity, a low activation energy (Ea), and inhibition by mercurial compounds. The biochemical and molecular characteristics of water channel pathways are not known at present. Established and new methods to measure Pf and Pd in kidney tubules and in isolated membrane vesicles from kidney cells are reviewed and evaluated. In the mammalian proximal tubule, a high Pf results from transcellular movement of water across highly permeable apical and basolateral membranes containing water channels. It has been assumed that proximal tubule Pf is unregulated; however, recent results indicate that apical water channels are retrieved by endocytosis and that Pf is decreased fivefold with increasing transepithelial osmotic gradients. In the thin and thick ascending limbs, Pf is nearly the lowest of all biological membranes and is not subject to regulation. In contrast, collecting tubule Pf is subject to hormonal regulation by vasopressin. Vasopressin binding to receptors located at the basal membrane of principal cells initiates adenosine 3',5'-cyclic monophosphate production, which is thought ultimately to activate the exocytic insertion of intracellular vesicles containing water channels into the cell apical membrane. Vasopressin-induced endosomes from kidney collecting tubule and toad urinary bladder contain functional water channels but no proton pumps or urea transporters, supporting a membrane shuttle hypothesis that is selective for water channels. Future directions for the isolation and molecular cloning of kidney water channels are evaluated.

scholarly journals Antidiuretic hormone modulates membrane phosphoproteins in toad urinary bladder and retrieved water channel containing apical membrane vesicles.

1991 ◽  
Vol 1 (9) ◽  
pp. 1114-1122
Author(s):  
H W Harris
Keyword(s):  
Membrane Proteins ◽  
Urinary Bladder ◽  
Membrane Protein ◽  
Antidiuretic Hormone ◽  
Water Permeability ◽  
Apical Membrane ◽  
Water Channel ◽  
Water Channels ◽  
Toad Urinary Bladder ◽  
Granular Cells

Antidiuretic hormone (ADH) dramatically increases the water permeability of toad urinary bladder by insertion of unique highly selective water channels into the apical membranes of granular cells. Before ADH stimulation, water channels are stored in high concentrations in the limiting membranes of large cytoplasmic vesicles called aggrephores. ADH stimulation causes aggrephore fusion with the granular cell apical membrane and increases water permeability. Transepithelial osmotic water flow causes a rapid attenuation of the ADH-elicited increase in water permeability through a process called flux inhibition. Flux inhibition is due to retrieval of ADH water channels by apical membrane endocytosis. When phosphoproteins of intact bladders are labeled with (32P)orthophosphate, the 32P content of 34-, 28-, and 17-kDa proteins is increased by ADH stimulation. When flux inhibition occurs, the 32P-labelling of a 15.5-kDa protein is reduced to approximately one half its original value (Konieczkowski M, Rudolph SA, J Pharmacol Exp Ther 1985;234:515). These observations have been confirmed, and these studies have been extended, by using a combination of subcellular fractionation and membrane protein chemistry techniques. All four of these phosphoproteins are present in membrane fractions of granular cells. Analysis of membrane proteins by a combination of Triton X-114 partitioning and an alkaline stripping technique reveals that the 28- and 17-kDa species are integral membrane proteins of unknown function. In contrast, the 32P-labeled 15.5-kDa protein is a peripheral membrane protein. It is attached to the cytoplasmic (outer) surface of highly water-permeable vesicles retrieved during flux inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)

OSMOTIC INHIBITION OF THE NATRIFERIC RESPONSE OF THE TOAD URINARY BLADDER TO VASOPRESSIN

1975 ◽  
Vol 67 (1) ◽  
pp. 119-125
Author(s):  
P. J. BENTLEY
Keyword(s):  
Urinary Bladder ◽  
Water Movement ◽  
Short Circuit ◽  
Electrical Potential ◽  
Short Circuit Current ◽  
Toad Bladder ◽  
Toad Urinary Bladder ◽  
Electrical Potential Difference ◽  
Osmotic Gradient

SUMMARY The electrical potential difference and short-circuit current (scc, reflecting active transmural sodium transport) across the toad urinary bladder in vitro was unaffected by the presence of hypo-osmotic solutions bathing the mucosal (urinary) surface, providing that the transmural flow of water was small. Vasopressin increased the scc across the toad bladder (the natriferic response), but this stimulation was considerably reduced in the presence of a hypo-osmotic solution on the mucosal side, conditions under which water transfer across the membrane was also increased. This inhibition of the natriferic response did not depend on the direction of the water movement, for if the osmotic gradient was the opposite way to that which normally occurs, the response to vasopressin was still reduced. The natriferic response to cyclic AMP was also inhibited in the presence of an osmotic gradient. Aldosterone increased the scc and Na+ transport across the toad bladder but this response was not changed when an osmotic gradient was present. The physiological implications of these observations and the possible mechanisms involved are discussed.

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.

The vasopressin-regulated urea transporter in renal inner medullary collecting duct

1990 ◽  
Vol 259 (3) ◽  
pp. F393-F401 ◽  
Author(s):  
M. A. Knepper ◽  
R. A. Star
Keyword(s):  
Apical Membrane ◽  
Collecting Duct ◽  
Water Channel ◽  
Toad Urinary Bladder ◽  
Urea Transport ◽  
Urea Transporter ◽  
Transport Pathway ◽  
Inner Medullary Collecting Duct ◽  
Urea Permeability ◽  
Medullary Collecting Duct

The terminal part of the inner medullary collecting duct (terminal IMCD) is unique among collecting duct segments in part because its permeability to urea is regulated by vasopressin. The urea permeability can rise to extremely high levels (greater than 100 x 10(-5) cm/s) in response to vasopressin. Recent studies in isolated perfused IMCD segments have established that the rapid movement of urea across the tubule epithelium occurs via a specialized urea transporter, presumably an intrinsic membrane protein, present in both the apical and basolateral membranes. This urea transporter has properties similar to those of the urea transporters in mammalian erythrocytes and in toad urinary bladder, namely, inhibition by phloretin, inhibition by urea analogues, saturation kinetics in equilibrium-exchange experiments, and regulation by vasopressin. The urea transport pathway is distinct from and independent of the vasopressin-regulated water channel. The increase in transepithelial urea transport in response to vasopressin is mediated by adenosine 3',5'-cyclic monophosphate and is associated with an increase in the urea permeability of the apical membrane. However, little is known about the physical events associated with the activation or insertion of urea transporters in the apical membrane. Because of the importance of this transporter to the urinary concentrating mechanism, efforts toward understanding its molecular structure and the molecular basis of its regulation appear to be justified.

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