A time course study of water permeability and morphological alterations induced by mucosal hyperosmolarity in frog urinary bladder

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23 degrees C, and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2 degrees C (group C), or with vasopressin plus sodium azide at 23 degrees C (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 +/- 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of greater than 0.1 cm/s (18 degrees C) and activation energy of 3.9 +/- 0.8 kcal/mol (16 kJ/mol). Pf was inhibited reversibly by greater than 60% by 1 mM HgCl2. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pf in the vasopressin-induced endocytic vesicles is the highest Pf reported for any biological or artificial membrane.

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Cellular pH and water permeability control in frog urinary bladder a possible action on the water pathway

Biochimica et Biophysica Acta (BBA) - Biomembranes ◽

10.1016/0005-2736(81)90043-2 ◽

1981 ◽

Vol 648 (2) ◽

pp. 267-274 ◽

Cited By ~ 14

Author(s):

Mario Parisi ◽

Ricardo Montoreano ◽

Jacques Chevalier ◽

Jacques Bourguet

Keyword(s):

Urinary Bladder ◽

Water Permeability ◽

Frog Urinary Bladder ◽

Water Pathway

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The Effect of Hypertonic Media on Water Permeability of Frog Urinary Bladder

The Journal of General Physiology ◽

10.1085/jgp.61.1.110 ◽

1973 ◽

Vol 61 (1) ◽

pp. 110-124 ◽

Cited By ~ 36

Author(s):

P. Ripoche ◽

J. Bourguet ◽

M. Parisi

Keyword(s):

Epithelial Cells ◽

Urinary Bladder ◽

Cyclic Amp ◽

Antidiuretic Hormone ◽

Water Permeability ◽

Prostaglandin E1 ◽

Physiological State ◽

Solution Temperature ◽

Bathing Solution ◽

Frog Urinary Bladder

The frog urinary bladder undergoes, in some conditions, a marked increase of its water permeability when incubated in hypertonic media. This increase was observed with various nonpermeant solutes. It seems to result from the shrinkage of an osmo-sensitive compartment of the tissue, probably the epithelial cells. Many similarities were found between this effect and the physiological increase in water permeability (hydrosmotic response) elicited by antidiuretic hormone (ADH): both were dependent on the physiological state of the animals, and although the response was slower after hyperosmolar than after hormonal challenge, the patterns of response were similar, and in both cases markedly dependent on bathing solution temperature. Norepinephrine and prostaglandin E1, which in this tissue reduce the hydrosmotic action of ADH, presumably by inhibiting the adenyl cylase also reduced the effect of hyperosmolarity. Conversely this effect was potentiated by incubation in the presence of oxytocin, exogenous cyclic AMP, and theophylline, conditions in which the intracellular concentration of cyclic AMP is increased. These data demonstrate that the response to hyperosmolarity is elicited, at least partly, by mechanisms also involved in the physiological hydrosmotic response to ADH.

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Prostaglandin E2 inhibits vasotocin-induced osmotic water permeability in the frog urinary bladder by EP1-receptor-mediated activation of NO/cGMP pathway

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00811.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. R528-R537 ◽

Cited By ~ 9

Author(s):

Vera Bachteeva ◽

Ekaterina Fock ◽

Elena Lavrova ◽

Svetlana Nikolaeva ◽

Stepan Gambaryan ◽

...

Keyword(s):

Epithelial Cells ◽

Urinary Bladder ◽

Water Permeability ◽

Inhibitory Effect ◽

No Synthase ◽

Arginine Vasotocin ◽

Osmotic Water Permeability ◽

Frog Urinary Bladder ◽

Osmotic Water ◽

Cgmp Pathway

PGE2 is a well-known inhibitor of the antidiuretic hormone-induced increase of osmotic water permeability (OWP) in different osmoregulatory epithelia; however, the mechanisms underlying this effect of PGE2 are not completely understood. Here, we report that, in the frog Rana temporaria urinary bladder, EP1-receptor-mediated inhibition of arginine-vasotocin (AVT)-induced OWP by PGE2 is attributed to increased generation of nitric oxide (NO) in epithelial cells. It was shown that the inhibitory effect of 17-phenyl-trinor-PGE2 (17-ph-PGE2), an EP1 agonist, on AVT-induced OWP was significantly reduced in the presence of 7-nitroindazole (7-NI), a neuronal NO synthase (nNOS) inhibitor. NO synthase (NOS) activity in both lysed and intact epithelial cells measured as a rate of conversion of l-[3H]arginine to l-[3H]citrulline was Ca2+ dependent and inhibited by 7-NI. PGE2 and 17-ph-PGE2, but not M&B-28767 (EP3 agonist) or butaprost (EP2 agonist), stimulated NOS activity in epithelial cells. The above effect of PGE2 was abolished in the presence of SC-19220, an EP1 antagonist. 7-NI reduced the stimulatory effect of 17-ph-PGE2 on NOS activity. 17-ph-PGE2 increased intracellular Ca2+ concentration and cGMP in epithelial cells. Western blot analysis revealed an nNOS expression in epithelial cells. These results show that the inhibitory effect of PGE2 on AVT-induced OWP in the frog urinary bladder is based at least partly on EP1-receptor-mediated activation of the NO/cGMP pathway, suggesting a novel cross talk between AVT, PGE2, and nNOS that may be important in the regulation of water transport.

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Epithelial organization and hormone sensitivity of toad urinary bladder cells in culture

AJP Renal Physiology ◽

10.1152/ajprenal.1981.241.2.f129 ◽

1981 ◽

Vol 241 (2) ◽

pp. F129-F138 ◽

Cited By ~ 1

Author(s):

J. P. Johnson ◽

R. E. Steele ◽

F. M. Perkins ◽

J. B. Wade ◽

A. S. Preston ◽

...

Keyword(s):

Urinary Bladder ◽

Cell Line ◽

Cell Lines ◽

Water Permeability ◽

Sodium Transport ◽

Time Course ◽

Cultured Cells ◽

Transepithelial Transport ◽

Seeding Density ◽

Toad Urinary Bladder

Two continuous cell lines (TB-M and TB-6c) derived from epithelial cells of the toad urinary bladder form epithelia in culture that manifest hormone-sensitive transepithelial transport. Development of transepithelial electrical resistance (R) and transport rate (ISC) are dependent on time and density of cells seeded, but steady-state ISC and R are characteristic for each cell line and independent of seeding density. Some responses of intact toad bladder are preserved in culture, whereas others are altered or absent. Neither cell line responds to vasopressin. Analogues of cAMP increase sodium transport and urea permeability in both cell lines but do not affect water permeability. The intramembrane particle aggregates associated with the vasopressin- and cAMP-induced increase in water permeability of the intact bladder could not be detected in the cell lines. Aldosterone increases sodium transport in both cell lines, and the time course and concentration dependence of the response to aldosterone are similar to those of the intact bladder. The relative effect of a series of steroids on ISC reveals corticosterone to be a more potent mineralocorticoid in cultured cells than in the intact bladder.

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ADH or theophylline-induced changes in intracellular free and membrane-bound calcium

AJP Renal Physiology ◽

10.1152/ajprenal.1984.247.6.f939 ◽

1984 ◽

Vol 247 (6) ◽

pp. F939-F945 ◽

Cited By ~ 1

Author(s):

R. M. Burch ◽

P. V. Halushka

Keyword(s):

Plasma Membrane ◽

Epithelial Cells ◽

Urinary Bladder ◽

Antidiuretic Hormone ◽

Water Permeability ◽

Time Course ◽

Toad Bladder ◽

Toad Urinary Bladder ◽

Membrane Bound ◽

Induced Changes

Ca2+ is thought to play a role in the enhancement of water permeability of toad urinary bladder epithelial cells by antidiuretic hormone (ADH) or theophylline. This study examined the effects of ADH and theophylline on intracellular free Ca2+ ([Ca2+]i) and total cellular exchangeable Ca2+ in isolated toad bladder epithelial cells. ADH or theophylline enhanced water permeability maximally by 15-25 min after a 4-min lag. 45Ca2+ efflux, a probe for total cellular exchangeable (plasma membrane plus intracellular) Ca2+, was enhanced by ADH within 2 min and returned to control by 8 min. Chlortetracycline fluorescence, a probe for intracellular Ca2+ only, was not affected, suggesting that ADH released only plasma membrane-bound Ca2+. Theophylline enhanced 45Ca2+ efflux and decreased chlortetracycline fluorescence, suggesting release of Ca2+ from intracellular sources. Both agents decreased [Ca2+]i as assessed by quin-2 fluorescence with a time course similar to the enhancement in water permeability. The results suggest that the changes in membrane-bound Ca2+ and [Ca2+]i induced by ADH and theophylline may play a role in the enhanced permeability to water in response to these agents.

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