Permeability properties of the Bufo bufo bladder as affected by isoprenaline and vasopressin

Isoprenaline, a beta adrenergic agonist, strongly increases both transepithelial fluxes across the urinary bladder of Bufo bufo; this effect is dose dependent, 10(-6)M being necessary for the maximal action. This effect is less selective than that of vasopressin: the ratio J urea/J thiourea is 3.8 under isoprenaline and 30.4 under vasopressin treatment. Both hormones differently affect the permeability of a mainly liposoluble molecule, i.e. antipyrine: vasopressin increases antipyrine permeability, while isoprenaline decreases it. Moreover diethylpyrocarbonate treatment of the luminal membrane strongly inhibits vasopressin effect on urea permeability leaving unmodified that of isoprenaline. However, the actions of both hormones are not additive. These results allows to assume that the tissue has a feedback mechanism which inhibits other hormonal action while the bladder is stimulated by a particular hormone.     

Surface hydrophobicity and water transport of the toad urinary bladder: Effects of vasopressin

Summary The present study investigated whether the hydrophobic properties (wettability) of the luminal surface of the toad urinary bladder might play a role in modulating water transport across this epithelium. In the absence of vasopressin (ADH), water transport across the tissue was low, while luminal surface hydrophobicity (water contact angle) was relatively high. Following stimulation by ADH, water transport increased and surface hydrophobicity decreased. The addition of indomethacin to inhibit ADH-induced prostaglandin synthesis did not reduce these actions of ADH. In an attempt to alter water transport in this tissue, a liposomal suspension of surface-active phospholipids was administered to the luminal surface. This addition had no detectable influence on the low basal rates of water transport, but blocked the ADH-induced stimulation of water transport. We suggest that surface-active phospholipids on the toad bladder luminal membrane may contribute to the hydrophobic characteristics of this tissue. ADH may act to decrease surface hydrophobicity, facilitating the movement of water molecules across an otherwise impermeable epithelium. This surface alteration may be associated with the appearance of water channels in the apical membrane.     

Role of calmodulin in antidiuretic hormone mediated water transport

Several classes of tricyclic antidepressants inhibit the action of antidiuretic hormone (ADH) and cyclic adenine monophosphate (cAMP) on osmotic water flow across toad urinary bladder without any effect on sodium transport. This finding suggests that calmodulin is involved in the hydroosmotic action of ADH (and of serosal hypertonicity), possibly in inducing exocytosis at the luminal border of vesicles rich in water channels.     

Regulation of protein phosphorylation and sodium transport in toad bladder

It is well established that active sodium-ion transport and water flow across isolated toad bladder are increased by antidiuretic hormone (ADH) and by cAMP. These agents were also observed in previous studies to cause changes in the amount of radioactive phosphate in a specific protein in the toad bladder. This protein, found by SDS-polyacrylamide gel electrophoresis of toad bladder epithelial preparations, had an apparent molecular weight of 49,000 daltons. In the present study, a correlation was found between the ability of a variety of substances to affect the amount of radioactive phosphate in this 40,000-dalton protein and their ability to alter the rate of sodium transport. Thus several agents (ADH, cAMP, theophylline, adenine, prostaglandin E1, and Mn Cl-2) caused a decrease in the amount of radioactive phosphate in the 49,000-dalton protein and also stimulated active sodium transport across the bladder. Conversely, ZnCl-2 produced an increase in the amount of radioactive phosphate in this protein and an inhibition of sodium transport. With each of these agents, the time-course of change in phosphorylation of this protein was, in general, similar to that for sodium transport. A second phosphoprotein, with an apparent molecular weight of about 42,000 daltons, showed changes in parallel with, but less extensive than, those observed in the 49,000 dalton protein. There was no consistent relationship between changes in level of phosphorylation of either in the 49,000- or 42,000- dalton protein and changes in osmotic water permeability. The results are compatible with the possibility that regulation by ADH and by cAMP of sodium transport in the toad bladder epithelium may be mediated through regulation of the amount of phosphate in a specific protein.     

Regulation of ADH-stimulated water flow at a post-luminal barrier in toad bladder

Recent studies show that ADH-stimulated water flow across toad bladder may be regulated at a site other than the luminal membrane. In these studies luminal membrane particle aggregate frequency has been used as a measure of luminal membrane water permeability. In fully stretched bladders the relationship between total tissue permeability and aggregate frequency is curvilinear, rather than linear. This implies a resistance in series with the luminal membrane that can become rate-limiting for water flow during ADH stimulation. The possibility that transtissue water movement is actually regulated at such a post-luminal membrane resistance is suggested by the finding that within 30 min following exposure to hormone, water flow becomes attenuated without any change in aggregate frequency. Supporting this possibility, recent data from follow-up studies suggest that the apparent water permeability per luminal membrane aggregate is not reduced with time. Finally, for bladders in which prostaglandin synthesis is inhibited (by naproxen), increases in both base-line water flow and water flow consequent to treatment with a submaximal dose of ADH (0.125 mU/ml), are much less than expected from simultaneously observed changes in luminal membrane aggregate frequency. In parallel experiments to these, moreover, direct measurements of luminal membrane water permeability from the rate of change of cell volume consequent to a transluminal membrane osmotic challenge, confirm that luminal membrane water permeability increases to the extent expected from changes in aggregate frequency. All of the data taken together argue for a post-luminal membrane barrier in toad bladder which regulates tissue permeability during ADH stimulation.     

The nature of urea transport across the luminal membrane of Bufo bufo urinary bladder

By using the washing-out technique, counterflow acceleration for urea was demonstrated on the luminal membrane of Bufo bufo urinary bladder, in the absence of ADH. This phenomenon completely disappears in the presence of phloretin 10-4 M on the luminal side and is consistent with the presence of a mobile carrier mechanism for urea transport across the luminal membrane, in basal conditions. In the presence of ADH, counterflow acceleration is completely absent. This result is in agreement with the presence of urea selective channels, induced by ADH, as proposed by Levine & Worthington (1976).     

Evaluation by capacitance measurements of antidiuretic hormone induced membrane area changes in toad bladder

A technique for estimating effective transepithelial capacitance in vitro was used to investigate changes in epithelial cell membrane area in response to antidiuretic hormone (ADH) exposure in toad bladder. The results indicate that transepithelial capacitance increases by about 30% within 30 min after serosal ADH addition and decreases with ADH removal. This capacitance change is not blocked by amiloride and occurs whether or not there is a transepithelial osmotic gradient. It is blocked by methohexital, a drug which specifically inhibits the hydro-osmotic response of toad bladder to ADH. We conclude that the hydro-osmotic response of toad bladder to ADH is accompanied by addition of membrane to the plasmalemma of epithelial cells. This new membrane may contain channels that are permeable to water. Stimulation of Na⁺ transport by ADH is not related to membrane area changes, but appears to reflect activation of Na⁺ channels already present in the cell membrane before ADH challenge.     

The membrane action of antidiuretic hormone (ADH) on toad urinary bladder.

Radioactive tracer and electrical techniques were used to study the transport of nonelectrolytes and sodium, respectively, across toad urinary bladders in the presence and absence of ADH. The permeability of lipophilic molecules was roughly proportional to bulk phase oil/water partition coefficients both in the presence and absence of hormone; i.e., ADH elicited a general nonselective increase in the permeation of all nine solutes tested. The branched nonelectrolyte, isobutyramide, was less permeable than its straight-chain isomer, n-butyramide, in control tissues. ADH reduced the discrimination between these structural isomers. Hydrophilic solutes permeated more rapidly than expected. In the presence of hormone, there was no change in the permeation of large hydrophilic solutes considered to move via an extracellular pathway, but there was a marked increase in the permeability of water and other small hydrophilic solutes. Collectively, these results suggest that ADH acts to increase the motional freedom or fluidity of lipids in the cell membrane which is considered to be the preferred pathway for the permeation of lipophilic and small hydrophilic molecules. At concentrations of cAMP and ADH which elicit equivalent increments in the shortcircuit current, the effects of these agents on nonelectrolyte transport and membrane electrical conductance are divergent. Such observations suggest that some membrane effects of ADH may not be directly dependent upon cAMP. ADH in the mucosal solution increased the permeability of the toad bladder when the surface charge on the outer surface of the apical membrane was screened with the polyvalent cation, La-3+. These experiments emphasize that interaction of ADH with membranes of toad urinary bladder may account for at least some effects of this hormone.     

Serosal and mucosal facilitated transport of urea in urinary bladder of of Bufo bufo: evidence for an alleged water uptake

In Bufo bufo urinary bladder an urea facilitated transport has been localised on the luminal membrane. The transport fulfils the criteria for such a mechanism, i.e. is saturable and is inhibited by phloretin, a specific inhibitor for urea transport. Similarly to that of Bufo marinus and Rana esculenta the luminal membrane of Bufo bufo urinary bladder shows an ADH stimulated facilitated transport. Experiments wtih Amphotericin B, serosal phloretin (with and without ADH), have demonstrated the presence of a facilitated urea transport localised on basolateral membrane. Urea uptake on the isolated epithelial cells of Bufo bufo urinary bladder shows a characteristic feature, different from molecules passively transported such as glycerol yet inhibited by phloretin. Allegedly with urea, water flows in to the cells by a dragging or osmotic effect.     

Modulation of cytoskeletal organization and cytosolic granule distribution by verapamil in amphibian urinary epithelia

The present study examines the role of calcium in modulating epithelial cytomorphology by using verapamil, a calcium antagonist, and considering its effects on cytosolic granule distribution and exocytosis in toad urinary bladder. The effect of verapamil on the detection and distribution of microfilaments in toad urinary bladder using immunogold labeling techniques in toad urinary bladder epithelial cells was also examined. Verapamil, which inhibits antidiuretic hormone (ADH)-mediated water flow, increased the number, size and distribution of dense calcium-containing secretory granules in bladder epithelial cells. This calcium antagonist prevented granule exocytosis, such that, six-times the number of granules were present in verapamil-treated tissues. The normal cytomorphological changes that accompany the actions of ADH were attenuated by verapamil, including ADH-induction of microvilli. ADH increased the number of actin microfilaments as determined using protein A-gold by immunolabeling, whereas, verapamil treatment was unremarkable as compared to control. The results suggest that calcium may play a prominent role in mediating granule exocytosis and membrane fusion events that normally accompany hormone action.     

Localization of barriers to water flow in toad urinary bladder

Although it is well accepted that vasopressin (ADH) increases the permeability to water of the toad bladder granular cell's luminal membrane, recent studies have suggested that regulation also takes place at an additional "postluminal" site within the epithelial granular cell. These studies are based upon the observation that a number of experimental maneuvers can alter tissue permeability to water, but do not change the number of particle aggregates observed on the protoplasmic face of the granular cell's luminal membrane with freeze-fracture electron microscopy. These aggregates are believed by many investigators to mediate the transport of water across the luminal membrane. The dissociation between permeability and aggregate frequency described above has been variously interpreted as the consequence of changes in the permeability of the aggregates themselves, or of changes in the permeability of a "postluminal" barrier that is functionally in series with the luminal membrane. We attempted to distinguish between these 2 possibilities by studying paired toad bladders during 3 protocols that alter vasopressin-stimulated water flow across the intact tissue without altering aggregate frequency. Estimates of the permeability of postluminal barriers were obtained by exposing the luminal surface to amphotericin B, an antibiotic that forms water-permeant channels in the luminal membrane. Of the 3 protocols, only diminishing bladder filling volume decreased the water flow elicited by luminal amphotericin B, suggesting that only that protocol indeed decreased the permeability of some postluminal barrier. The other 2 protocols, increasing PCO2 and repeatedly stimulating the bladder with vasopressin, did not alter amphotericin B-elicited flow, suggesting that postluminal barriers were not altered by these 2 protocols.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative analysis of exocytosis and endocytosis in the hydroosmotic response of toad bladder

Summary This study concerns the timing and magnitude of exocytosis and endocytosis in the granular cells of toad bladder during the hydroosmotic response to antidiuretic hormone. Granule exocytosis at the luminal cell surface is extensive within 5 min of the administration of a physiological dose of hormone. Hydroosmosis becomes detectable during this time period. The amount of membrane added to the luminal surface by exocytosis during 60 min of exposure to hormone can be of the same order of magnitude as the extent of the luminal plasma membrane. Endocytosis, demonstrated by peroxidase uptake from the luminal surface, becomes extensive during the period 15–45 min after hormone administration. Thus, maximal endocytic activity occurs later than the period of most extensive exocytosis and seems to correlate with the onset of the decline in water movement. The amount of membrane retrieved from the luminal surface by endocytosis during 60 min of stimulation is at least three quarters of that added by exocytosis. The bulk membrane movement in ADH stimulated preparations does not require the presence of an osmotic gradient. Colchicine inhibits the hydroosmotic response, the exocytosis of granules, and endocytosis at the luminal surface. These results strengthen our view that the bulk circulation of membrane at the cell surface, via exocytosis and endocytosis, is closely related to the permeability changes occuring at the surface.     

Effect of distension on ADH-induced osmotic water flow in toad urinary bladder

Summary We recently described a method by which the resistance to water flow of the luminal membrane of ADH-stimulated toad bladder can be quantitatively distinguished from that of barriers lying in series with it. This method requires estimates of both total bladder water permeability (assessed by transbladder osmotic water flow at constant gradient) and luminal membrane water permeability (assessed by quantitation of the frequency of ADH-induced luminal membrane particle aggregates). In the present study we examined the effect of bladder distension on transepithelial osmotic water flow before and during maximal ADH stimulation. Base-line water flow was unaffected by bladder distension, but hormonally stimulated flow increased systematically as bladders became more distended. Distension had no effect on the frequency of ADH-induced intramembranous particle aggregates. By comparing the relationships between aggregate frequency and hormonally induced water permeability in distended and undistended bladders, we found that distension appeared to enhance ADH-stimulated water flow by decreasing the resistance of the series permeability barrier while the apparent water permeability associated with each single luminal membrane aggregate was unaffected. In that bladder distension causes tissue thinning, the series resistance limiting ADH-stimulated water flow appears to be accounted for by deformable barriers within the bladder tissue itself, probably unstirred layers of water.     

Antidiuretic response in the urinary bladder of Xenopus laevis: Presence of typical aggrephores and apical aggregates

The urinary bladder of the aquatic toad Xenopus laevis is known to exhibit a low permeability to water and a poor sensitivity to antidiuretic hormone. In order to precise the characteristics and the specific cellular mechanisms of this reduced hydroosmotic response we used a sensitive volumetric technique to monitor net water flow and studied the correlation between the anti-diuretic hormone (ADH)-induced net water flow and the fine ultrastructural appearence of the urinary bladder epithelium. Transmural net water flow was entirely dependent on the osmotic gradient across the preparation and not on the hydrostatic pressure difference. We observed the existence of a low but significant hydro-osmotic response to arginine vasopressin. Freeze-fracture electron microscopy demonstrated the presence of typical aggrephores in the subapical cytoplasm. The response to the hormone was accompanied by the appearance of typical intramembrane aggregates into the apical plasma membrane. Water permeability increase and apical aggregate insertion were both slowly but fully reversible. Except for the multilayered structure of the epithelium and the particularly low response to antidiuretic hormone, all the studied permeability and ultrastructural characteristics of the bladder were thus very similar to those observed in other sensitive epithelia such as the amphibian bladder and skin and the mammalian collecting duct which exhibit a high hydro-osmotic response to the hormone.     

The water permeability of toad urinary bladder. I. Permeability of barriers in series with the luminal membrane

Antidiuretic hormone (ADH) induces a large increase in the water permeability of the luminal membrane of toad urinary bladder. Measured values of the diffusional water permeability coefficient, Pd(w), are spuriously low, however, because of barriers within the tissue, in series with the luminal membrane, that impede diffusion. We have now determined the water permeability coefficient of these series barriers in fully stretched bladders and find it to be approximately 6.3 X 10(- 4) cm/s. This is equivalent to an unstirred aqueous layer of approximately 400 microns. On the other hand, the permeability coefficient of the bladder to a lipophilic molecule, hexanol, is approximately 9.0 X 10(-4) cm/s. This is equivalent to an unstirred aqueous layer of only 100 microns. The much smaller hindrance to hexanol diffusion than to water diffusion by the series barriers implies a lipophilic component to the barriers. We suggest that membrane-enclosed organelles may be so tightly packed within the cytoplasm of granular epithelial cells that they offer a substantial impediment to diffusion of water through the cell. Alternatively, the lipophilic component of the barrier could be the plasma membranes of the basal cells, which cover most of the basement membrane and thereby may restrict water transport to the narrow spaces between basal and granular cells.     

8-P-Chlorophenylthio-cyclic AMP: a potent partial simulator of antidiuretic hormone action.

In the toad urinary bladder 8-p-chlorophenylthio-cyclic AMP mimics the stimulatory effects of antidiuretic hormone on osmotic water permeability, 3H2O diffusion, and transepithelial sodium transport; but unlike the hormone does not cause an increase in urea permeability. Trheshold activation for the hydroosmotic response is observed at 1 micrometer and full activation at 100 micrometer. These results suggest that cyclic AMP may not mediate all the physiological effects of antidiuretic hormone and that this highly potent cyclic AMP analog may be useful in elucidating the precise role of cyclic AMP in other biomediate hormone action.     

Amiloride-sensitive trypsinization of apical sodium channels. Analysis of hormonal regulation of sodium transport in toad bladder

Incubation of the mucosal surface of the toad urinary bladder with trypsin (1 mg/ml) irreversibly decreased the short-circuit current to 50% of the initial value. This decrease was accompanied by a proportionate decrease in apical Na permeability, estimated from the change in amiloride-sensitive resistance in depolarized preparations. In contrast, the paracellular resistance was unaffected by trypsinization. Amiloride, a specific blocker of the apical Na channels, prevented inactivation by trypsin. Inhibition of Na transport by substitution of mucosal Na, however, had no effect on the response to trypsin. Trypsinization of the apical membrane was also used to study regulation of Na transport by anti-diuretic hormone (ADH) and aldosterone. Prior exposure of the apical surface to trypsin did not reduce the response to ADH, which indicates that the ADH-induced Na channels were inaccessible to trypsin before addition of the hormone. On the other hand, stimulation of short-circuit current by aldosterone or pyruvate (added to substrate-depleted, aldosterone-repleted bladders) was substantially reduced by prior trypsinization of the apical surface. Thus, the increase in apical Na permeability elicited by aldosterone or substrate involves activation of Na channels that are continuously present in the apical membrane in nonconductive but trypsin-sensitive forms.     

Comparative effect of metals on antidiuretic hormone induced transport in toad bladder: specificity of mercuric inhibition of water channels

We previously reported that HgCl₂ inhibits water and urea flux in tissues fixed with glutaraldehyde after antidiuretic hormone (ADH) stimulation and suggested that the ADH-induced water channel may share characteristics of the red blood cell and proximal tubule water transport pathway. To determine the specificity of mercury's action, we examined the effect of numerous other metals. In tissues fixed after ADH stimulation, water flow and urea and sucrose permeabilities are maintained from mucosal bath pH 2.5 through pH 12. Several metals including Ba, Co, Fe, Sr and Zn did not alter flux. Al, Cd, La, Li, Pb and U inhibited urea permeability but not water flow. At pH 2.8, Cu inhibited water flow by 30% and urea permeability by 50%. At pH 4.9–7.4, Cu inhibited urea permeability but not water flow. At pH 3.0, Pt inhibited flow in ADH-pretreated tissues. The inhibitory effect was not present at pH>3.0. At pH<3.0, Au inhibited flow by 90% in tissues fixed after pretreatment with ADH but increased the permeability of tissues fixed in the absence of ADH. Ag inhibited flow by 70% but also increased sucrose, urea, and basal permeabilities. This suggests that Ag and Au disrupt epithelial integrity. These results indicate that at physiologic pH, the ADH-induced water channel is specifically blocked by Hg but not by other metals. This specificity may reflect the presence of a large number of sulfhydryl groups in the water channel.     

Effect of guanosine 3′:5′-monophosphate on the hydroosmotic activity of adenosine 3′:5′-monophosphate in toad urinary bladder

Guanosine 3′:5′-monophosphate has a slight hydroosmotic effect on toad urinary bladder. Furthermore, this nucleotide strongly inhibits the responses to 3′:5′-adenosine monophosphate and oxytocin. The response to an increase in medium tonicity is not modified by the guanosine nucleotide. A role for guanosine 3′:5′-monophosphate in the regulation of water permeability in toad urinary bladder is proposed.     

Phorbol myristate acetate induces endocytosis as well as exocytosis and hydroosmosis in toad urinary bladder

The induction of the hydroosmotic response in the toad urinary bladder is considered to be associated with membrane addition mediated by exocytosis at the affected luminal membrane and reversed by endocytic retrieval at that surface. The permeability, exocytosis and endocytosis are initiated by antidiuretic hormone (ADH) receptor interaction on the basolateral membrane. In other hormone responsive systems, phorbol ester (phorbol myristate acetate, PMA), a tumor promoter, has been implicated in the regulation of various transport processes through the activation of protein kinase C and cytoskeletal protein phosphorylation. We found that addition of 10(-6) M PMA to the mucosa induces an hydroosmotic response which is gradual and which reaches a maximum within 60 min, equal to about 1/3 the maximal ADH response. Morphologically, PMA causes rapid exocytosis of the granules, endocytosis of horseradish peroxidase from the mucosal medium into tubules and multivesicular bodies and elongation of apical microvilli. Controls treated with mucosal 0.1% dimethylsulfoxide (DMSO) or an inactive PMA isomer on the mucosal surface, or PMA on the serosal surface lack the hydroosmotic, exocytic, endocytic and cytoskeletal changes. Addition of serosal ADH to PMA-treated bladders results in a precocious hydroosmotic and exocytic ADH response, but a lowering of the maximal response. Also pretreatment of bladders with PMA prevented the ADH-induced increase in transepithelial potential difference. Thus, apical events mediating the PMA hydroosmotic response are correlated with exo- and endocytosis and elongation of apical microvilli.