Polyclonal antibodies in study of ADH-induced water channels in frog urinary bladder

1991 ◽  
Vol 261 (3) ◽  
pp. F437-F442
Author(s):  
G. Valenti ◽  
G. Calamita ◽  
M. Svelto
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Apical Membrane ◽  
Polyclonal Antibodies ◽  
Immune Serum ◽  
Frog Urinary Bladder ◽  
Hormonal Stimulation ◽  
Osmotic Water ◽  
Triton X ◽  
Osmotic Water Flow

It is now generally accepted that changes in water permeability in anti-diuretic hormone (ADH)-responsive target epithelial cells result from the insertion in the plasma apical membrane of new components that contain channels for water. The specificity of these channels suggests that they are formed by intrinsic proteins having access to both facies and spanning the whole membrane. We have previously shown that Triton X-100 apical extracts from ADH-stimulated frog urinary bladder contain some proteins inserted under hormonal stimulation. In the present study we have developed polyclonal antibodies using Triton X-100 extract as an immunogen. After considering the inhibitory effect exerted by the whole immune serum on the osmotic water flow, we used different adsorption steps to select, from the immune serum, antibodies to apical membrane proteins inserted in response to the hormone. Immunoblot analysis of these selected antibodies shows that they recognize seven to eight proteins, of which 55-, 35-, 26-, and 17-kDa proteins are always present. Antibodies to these four proteins, affinity purified on nitrocellulose sheets, inhibited ADH-induced osmotic water flow. Altogether these results strongly suggest that proteins of 55, 35, 26, and 17 kDa (or at least one of them) are likely to be involved in the mechanism of water transport.

THE SWELLING OF FROG BLADDER CELLS PRODUCED BY OXYTOCIN

1965 ◽  
Vol 33 (2) ◽  
pp. 171-177 ◽  
Author(s):  
J. V. NATOCHIN ◽  
K. JANÁČEK ◽  
R. RYBOVÁ
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Osmotic Gradient ◽  
Frog Urinary Bladder ◽  
Intracellular Space ◽  
Osmotic Water ◽  
Inulin Space ◽  
Bladder Cells ◽  
Synthetic Oxytocin ◽  
Osmotic Water Flow

SUMMARY (1) Synthetic oxytocin (100 m-u./ml.) produces swelling of the isolated frog urinary bladder even in the absence of an osmotic gradient across the bladder. (2) Calculations show that the change in intracellular space does not necessarily differ significantly from that in the presence of an osmotic gradient since the inulin space is markedly affected by the osmotic water flow. (3) Part of the cellular potassium is exchanged for sodium during the swelling produced by oxytocin. (4) A possible mechanism and the significance of the swelling is discussed.

Selected polyclonal antibodies and ADH challenge in frog urinary bladder: a label-fracture study

1992 ◽  
Vol 262 (2) ◽  
pp. F267-F274
Author(s):  
G. Calamita ◽  
G. Valenti ◽  
M. Svelto ◽  
J. Bourguet
Keyword(s):  
Urinary Bladder ◽  
Binding Sites ◽  
Apical Membrane ◽  
Polyclonal Antibodies ◽  
Initial Study ◽  
Frog Urinary Bladder ◽  
Fusion Images ◽  
Fracture Study ◽  
Induced Changes ◽  
Triton X

It is clearly established that the changes induced by antidiuretic hormone (ADH) in its target epithelial cells result from the insertion in the apical membrane of new components that contain channels for water. We have already undertaken an initial study of these channels by raising polyclonal antibodies against Triton X-100 apical extracts from ADH-treated bladders and approached their purification by different adsorption steps. In the present study, we used the label-fracture technique to investigate the localization of the binding sites of the obtained polyclonal antibodies on the apical membrane of ADH-stimulated frog urinary bladder. The results obtained clearly demonstrated a preferential labeling by the selected antibodies of morphological structures such as the groove arrays and the fusion images that are generally accepted as being involved in ADH-induced changes in water permeability of the apical membrane.

scholarly journals THE EFFECT OF SMOOTH MUSCLE ON THE INTERCELLULAR SPACES IN TOAD URINARY BLADDER

1970 ◽  
Vol 46 (2) ◽  
pp. 235-244 ◽  
Author(s):  
Donald R. DiBona ◽  
Mortimer M. Civan
Keyword(s):  
Smooth Muscle ◽  
Urinary Bladder ◽  
Water Flow ◽  
Toad Urinary Bladder ◽  
Reliable Index ◽  
Intercellular Spaces ◽  
Phase Microscopy ◽  
Osmotic Water ◽  
Smooth Muscle Relaxant ◽  
Osmotic Water Flow

Phase microscopy of toad urinary bladder has demonstrated that vasopressin can cause an enlargement of the epithelial intercellular spaces under conditions of no net transfer of water or sodium. The suggestion that this phenomenon is linked to the hormone's action as a smooth muscle relaxant has been tested and verified with the use of other agents effecting smooth muscle: atropine and adenine compounds (relaxants), K+ and acetylcholine (contractants). Furthermore, it was possible to reduce the size and number of intercellular spaces, relative to a control, while increasing the rate of osmotic water flow. A method for quantifying these results has been developed and shows that they are, indeed, significant. It is concluded, therefore, that the configuration of intercellular spaces is not a reliable index of water flow across this epithelium and that such a morphologic-physiologic relationship is tenuous in any epithelium supported by a submucosa rich in smooth muscle.

Blockade of colchicine-induced inhibition of vasopressin-stimulated osmotic water flow: failure to influence microtubule formation

1985 ◽  
Vol 249 (4) ◽  
pp. F464-F469
Author(s):  
D. A. Baron ◽  
R. M. Burch ◽  
P. V. Halushka ◽  
S. S. Spicer
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Volume Fraction ◽  
Cyclooxygenase Inhibitors ◽  
Maximal Response ◽  
Prostaglandin E ◽  
Toad Urinary Bladder ◽  
Meclofenamic Acid ◽  
Osmotic Water ◽  
Osmotic Water Flow

Colchicine inhibits vasopressin-induced osmotic water flow across isolated toad urinary bladder. Concomitantly, colchicine has been shown to reduce the relative cytoplasmic volume fraction of microtubules in the apical granular cells of this epithelium that have been shown previously to mediate the hydroosmotic effect of vasopressin. Therefore, an intact cytoskeleton has been postulated to be a requirement for a full response to vasopressin. Since it has been demonstrated recently that cyclooxygenase inhibitors (meclofenamic acid) abrogate the inhibition by colchicine of vasopressin-stimulated water flow, we tested by stereological criteria the hypothesis that colchicine in the presence of meclofenamic acid does not prevent the polymerization of tubulin. Our results show that the relative cytoplasmic volume fraction of microtubules was reduced 75% by colchicine in the presence or absence of meclofenamic acid. An alternative explanation of the inhibitory action of colchicine is its ability in the toad urinary bladder to enhance the endogenous synthesis of and sensitivity to prostaglandin E, a potent negative modulator of vasopressin-stimulated water flow. An intact microtubular component of the cytoskeleton does not appear to be required for a maximal response to a physiological dose of vasopressin.

Effect of colchicine on the osmotic water flow across the toad urinary bladder

1975 ◽  
Vol 413 (2) ◽  
pp. 277-282 ◽  
Author(s):  
Shigekazu Yuasa ◽  
Shigeharu Urakabe ◽  
Genjiro Kimura ◽  
Dairoku Shirai ◽  
Yoshihiro Takamitsu ◽  
...  
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Toad Urinary Bladder ◽  
Osmotic Water ◽  
Osmotic Water Flow

Effects of prostacyclin on short-circuit current and water flow in the toad urinary bladder

1983 ◽  
Vol 244 (3) ◽  
pp. F270-F277
Author(s):  
T. Pohlman ◽  
J. Yates ◽  
P. Needleman ◽  
S. Klahr
Keyword(s):  
Urinary Bladder ◽  
Water Transport ◽  
Water Flow ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Toad Bladder ◽  
Camp Production ◽  
Acid Product ◽  
Osmotic Water ◽  
Osmotic Water Flow

The effects of prostaglandins of the E series on sodium and water transport have been studied extensively. PGE2 has been shown to inhibit the increase in osmotic water flow produced by vasopressin and to stimulate short-circuit current (SCC) in the toad bladder. On the other hand, the effects of prostacyclin (PGI2), an arachidonic acid product, on sodium and water transport have not been extensively evaluated. The present studies describe the effects of PGI2 on basal and vasopressin-stimulated osmotic water flow and on SCC in the urinary bladder of the toad. Studies were performed in the absence or presence of indomethacin. PGI2 in the absence of indomethacin had no effect on basal or vasopressin-stimulated osmotic water flow. When indomethacin was present, thereby eliminating intrinsic prostaglandin biosynthesis, PGI2 inhibited basal but not vasopressin-stimulated osmotic water flow. PGI2 increased SCC in the presence or absence of indomethacin. 6-keto PGF1 alpha, the stable metabolite of PGI2, had no effect on SCC. PGI2 stimulated cAMP production in isolated toad bladder epithelial cells. 2',5'-Dideoxyadenosine, an inhibitor of cAMP production, blocked the increase in SCC produced by PGI2, suggesting that the effects of this compound on SCC are mediated via cAMP.

ADH-induced water permeability and particle aggregates: alteration by a synthetic estrogen

1991 ◽  
Vol 261 (1) ◽  
pp. F144-F152 ◽  
Author(s):  
G. Calamita ◽  
Y. Le Guevel ◽  
J. Bourguet
Keyword(s):  
Urinary Bladder ◽  
Water Flow ◽  
Water Permeability ◽  
Glucose Transporter ◽  
Apical Membrane ◽  
Selective Inhibition ◽  
Synthetic Estrogen ◽  
Permeability Changes ◽  
Particle Aggregates ◽  
Amphibian Urinary Bladder

In the amphibian urinary bladder, the increase in water permeability induced by antidiuretic hormone (ADH) is accompanied by the appearance of apical intramembrane particle (IMP) aggregates that are believed to contain specific channels for water. In a previous work, we have shown that 3,3'-diallyldiethylstilbestrol (DADES), a synthetic estrogen which is a blocker of the glucose transporter, also inhibits the hydrosmotic response to ADH in the bladder. Our aim in the present study was to analyze the alterations of the membrane fine structure further and to correlate them with the water permeability changes. The results point to a selective inhibition of the ADH-induced net water flow, probably due to an interference with one of the last steps of the response to the hormone. This inhibition is associated with an increase in the density of the apical IMP aggregates, which are thus probably not operational. The resting net water flow is not inhibited and, surprisingly, typical IMP aggregates are frequently observed in the apical membrane after DADES treatment. The compound also induces the appearance of unusual loose IMP clusters that can only be seen on the apical membrane of the granular cells and that share several ultrastructural similarities with the ADH-induced aggregates. These results suggest that 1) apical DADES treatment stimulates the insertion of IMP aggregates in the apical membrane of the urinary bladder and 2) DADES inhibits the ADH-induced water flow by interfering with the aggregates and thus probably by blocking the specific water channels.

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.

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