Permeability to water in a tight epithelium: possible modulating action of gap junctions

2004 ◽  
Vol 82 (6) ◽  
pp. 417-421 ◽  
Author(s):  
Gabriel Orce ◽  
Graciela Castillo ◽  
Yolanda Chanampa ◽  
Alejandra Bellomio
Keyword(s):  
Cyclic Amp ◽  
Gap Junctions ◽  
Gap Junction ◽  
Water Flow ◽  
Apical Membrane ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Distal Nephron ◽  
Osmotic Water ◽  
Epithelial Membranes

Osmotic water flow (Jw) across tight distal nephron epithelial membranes increases upon exposure to vasopressin: following binding of the hormone to its receptors, intracellular cyclic AMP concentration increases, leading to insertion of aquaporins in the apical membrane. The involvement of intercellular communication in the process, however, has not been adequately explored. Octanol, 1.2 × 10–3 M, a gap junction inhibitor, significantly reduced Jw (expressed as mg·20 min–1) in isolated toad urinary bladders (a model of the distal nephron) subjected to a transepithelial osmotic gradient and exposed to agents mimicking the vasopressin-triggered mechanism: oxytocin, 50 mIU·mL–1 (from 185.3 ± 28.0, P < 0.001, to 69.0 ± 23.6, P < 0.05; Pdiff < 0.01, n = 6), and cyclic AMP, 2.5 × 10–3 M (from 98.0 ± 32.6, P < 0.02, to 31.0 ± 13.9, NS; Pdiff < 0.05, n = 12), without altering the effect of nystatin, 450 U·mL–1, which increases Jw via a mechanism unrelated to apical aquaporin insertion (163.2 ± 16.3, P < 0.001, in controls vs. 150.3 ± 10.4, P < 0.001, in octanol-treated bladders; Pdiff: NS, n = 6). Another gap junction blocker, carbenoxolone, 2.0 × 10–4 M (CBX), exerted similar effects on the responses to oxytocin, 100 mIU·mL–1, reducing the response from 256.7 ± 33.6, P < 0.001, to 102.7 ± 10.4, P < 0.001; Pdiff < 0.01, n = 6) and nystatin, which was unaffected (95.0 ± 20.9, P < 0.01, vs. 132.0 ± 27.0, P < 0.01; Pdiff: NS, n = 6). Our results suggest that either gap junctions or, alternatively, unapposed gap junction hemichannels, may be important in the regulation of Jw in the isolated toad bladder, by modulating a step in the physiological process leading to increased apical membrane permeability. Key words: Bufo arenarum, toad urinary bladder, water flow, epithelial permeability, n-octanol, carbenoxolone.

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.

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.

Cytochalasin B inhibition of toad bladder apical membrane responses to ADH

1988 ◽  
Vol 255 (4) ◽  
pp. C526-C530 ◽  
Author(s):  
J. B. Wade ◽  
W. A. Kachadorian
Keyword(s):  
Water Permeability ◽  
Cytochalasin B ◽  
Apical Membrane ◽  
Freeze Fracture ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Osmotic Water ◽  
Structural Responses ◽  
Freeze Fracture Electron Microscopy

The possible role of actin microfilaments in antidiuretic hormone (ADH)-induced increases in apical membrane water permeability was investigated in studies that evaluate inhibition by cytochalasin B of both permeability and membrane structural responses in the toad urinary bladder. Experiments were carried out in the absence of a transepithelial osmotic gradient to eliminate possible flow-induced distortions of the response. Measurements of osmotic water permeability after a brief tissue fixation with glutaraldehyde show that cytochalasin B reduces the permeability response to ADH by approximately one-third. Freeze-fracture electron microscopy indicates that the intramembrane particle aggregates, previously found to correlate closely with ADH-induced permeability, are reduced by about the same extent (28%) under these conditions. However, the frequency of apical membrane fusion events was not affected by cytochalasin B treatment. These results suggest that cytochalasin B treatment in the absence of an osmotic gradient alters the ADH-induced permeability through an effect on apical membrane aggregate frequency.

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.

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.

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.

Protein synthesis inhibitors attenuate water flow in vasopressin-stimulated toad urinary bladder

1988 ◽  
Vol 254 (1) ◽  
pp. F139-F144
Author(s):  
B. S. Hoch ◽  
M. B. Ast ◽  
M. J. Fusco ◽  
M. Jacoby ◽  
S. D. Levine
Keyword(s):  
Protein Synthesis ◽  
Protein Kinase ◽  
Urinary Bladder ◽  
Water Flow ◽  
Toad Urinary Bladder ◽  
Osmotic Gradient ◽  
Protein Synthesis Inhibitors ◽  
Dependent Protein Kinase ◽  
Camp Dependent Protein Kinase ◽  
Dependent Protein

Vasopressin stimulates the introduction of aggregated particles, which may represent pathways for water flow, into the luminal membrane of toad urinary bladder. It is not known whether water transport pathways are degraded on removal from membrane or whether they are recycled. We examined the effect of the protein synthesis inhibitors cycloheximide and puromycin using repeated 30-min cycles of vasopressin followed by washout of vasopressin, all in the presence of an osmotic gradient, a protocol that maximizes aggregate turnover. “High dose” cycloheximide (200 micrograms/ml) inhibited flow immediately. “Low dose” cycloheximide (1 microgram/ml) did not affect initial flow; however, flow was inhibited by the fourth restimulation. On further rechallenge, inhibition persisted but did not increase. In the absence of vasopressin, inhibition did not develop. Despite the inhibition of flow in vasopressin-treated tissues, the cAMP-dependent protein kinase ratio (-cAMP/+cAMP), an index of in vivo cAMP effect, was elevated in cycloheximide-treated tissues, suggesting modulation at a distal site in the stimulatory cascade. Cycloheximide inhibited flow when 10 microM forskolin or 0.2 mM 8-BrcAMP was substituted for vasopressin in the fourth period; however, MIX (4 mM)-stimulated flow was enhanced by 1 microgram/ml cycloheximide but inhibited by 200 micrograms/ml cycloheximide. [14C]urea permeability was not inhibited by cycloheximide. Puromycin (0.5 mM) also inhibited water flow by the fourth challenge with vasopressin. The data suggest that protein synthesis inhibitors attenuate flow at a site that is distal to cAMP-dependent protein kinase.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for transcellular osmotic water flow in rat proximal tubules

1985 ◽  
Vol 249 (1) ◽  
pp. F124-F131 ◽  
Author(s):  
P. A. Preisig ◽  
C. A. Berry
Keyword(s):  
Surface Area ◽  
Water Flow ◽  
Water Permeability ◽  
Channel Length ◽  
Osmotic Gradient ◽  
Osmotic Water Permeability ◽  
Osmotic Water ◽  
Major Route ◽  
Osmotic Water Flow

To determine the predominant pathway for transepithelial osmotic water flow, the transepithelial osmotic water permeability [Pf(TE)] and the apparent dimensions of paracellular pores and slits were determined in rat proximal convoluted tubules microperfused in vivo. To measure Pf(TE), tubules were perfused with a hyposmotic, cyanide-containing solution. Pf(TE), calculated from the observed volume flux in response to the measured log mean osmotic gradient, was 0.12-0.15 cm/s, assuming sigmaNaCl equal to 1.0-0.7, respectively. The dimensions of the paracellular pathways were determined using measured sucrose and mannitol permeabilities (nonelectrolytes confined to the extracellular space). These were 0.43 and 0.87 X 10(-5) cm/s, respectively. By using the ratio of these permeabilities, their respective free solution diffusion coefficients and molecular radii, and the Renkin equation, the radius of the nonelectrolyte-permeable pores and the total pore area/cm2 surface area/channel length were calculated to be 1.4 nm and 3.56 cm-1, respectively. Similar calculations for slits yielded a slit half-width of 0.8 nm and a total slit area/cm2 surface area/channel length of 3.16 cm-1. The osmotic water permeability of these nonelectrolyte-permeable pathways was calculated by Poiseuille's law to be 0.0018 cm/s (pores) or 0.0014 cm/s (slits), at most 2% of Pf(TE). We conclude that the nonelectrolyte-permeable pathway in the tight junctions is not the major route of transepithelial osmotic water flow in the rat proximal tubule.

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.

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