76 and 14 kDa polypeptides, two major components released from amphibian urinary bladder epithelium. Purification and characterization

Exocytotic processes play a major role in the hormonal control of water permeability in the amphibian urinary bladder. Different treatments such as antidiuretic hormone (ADH) stimulation, incubation with phorbol ester or mild detergent and mechanical stretch of the bladder, consistently induce a liberation of two major polypeptides of 76 and 14 kDa molecular mass into the luminal medium. Each of these polypeptides represents 3 to 5% of the total protein of epithelial cell homogenates and 20 to 50% of the released material. Proportions of released 76 kDa polypeptide in urinary bladders of toads (Bufo marinus) and frogs (Rana esculenta) were similar but, in the frog extracts, two bands ("doublet") were resolved at the level of 76 kDa. In high performance liquid chromatography (HPLC), using gel filtration and ion exchange chromatography, the frog 76 kDa protein was resolved into two polypeptides of 80,000 to 100,000 and 60,000 to 80,000 daltons while the 14 kDa protein included two polypeptides, each with a molecular mass of approximately 14,000 daltons. Isoelectric focusing of the material released during a mechanical stretch of the tissue ("stretch extract") or of isolated purified proteins from the frog urinary bladder showed that the 14 kDa polypeptides were resolved in two major groups of polypeptides, one in the range of pH 7.4 to 7.8, the other at pH 5.6. The lower band of the 76 kDa doublet also comprised some diffuse bands (5.0 less than pI less than 5.2) while the other polypeptide of the doublet presented a sharp band at pH 6.2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maximal flux responses after multiple challenges with vasopressin

Antidiuretic hormone (ADH) increases transepithelial flux of water and particular solutes across the amphibian urinary bladder and mammalian collecting duct by increasing the permeability of the apical surface. We find that if each challenge with ADH is ended by replacing the medium bathing both the mucosal and serosal surfaces of the toad bladder, then rechallenge with the same supramaximal dose of ADH 36-100 min later produces flux equivalent to or greater than the original response, but rechallenge after 15 min produces only 68% of the original response. If the medium bathing the mucosal surface is neither replaced nor returned to its original volume, complete recovery of the osmotic flux response to ADH does not occur. Maximal restimulation by ADH occurs with transepithelial osmotic gradients between 119 and 180 mosmol/kg during both challenges (the serosal bath is always isotonic amphibian Ringers). In addition, ADH-containing serosal baths that have maximally activated transport across bladders for 30-60 min can be reused and again produce maximal activation of ADH responses in fresh bladders or in the original bladders after washing. These results are in contradistinction to reports of desensitization of transepithelial flux upon rechallenge with ADH after an initial stimulation under many conditions. Our findings suggest that desensitization in vitro may result from experimental design rather than intrinsic biological characteristics of the system.     

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.     

Selective fixation with glutaraldehyde of ADH-induced urea permeability sites in toad bladder

Toad bladders exposed to vasopressin (ADH) and then fixed on the mucosal surface with 1% glutaraldehyde were highly permeable to water and to urea compared to control bladders fixed in the absence of hormone. When identical conditions of fixation were were used, but the concentration of glutaraldehyde was decreased to 0.25%, the ADH-induced increase in membrane permeability to urea was preserved whereas water permeability was not. About 74% of the hormone-induced urea permeability sites were preserved by glutaraldehyde and were stable to changes in temperature as suggested by a constant value for the activation energy of urea movement of 5.4 kcal/mole (4-33 degrees C). In other studies bladders were exposed at low temperatures to 0.17% glutaraldehyde applied either to the serosal or the mucosal surface. The ADH-induced increase in membrane permeability to urea, bulk water, and tritiated water was well preserved with serosal fixation, but not with mucosal fixation. The observation that the urea pathway can be selectively preserved with 0.25% glutaraldehyde applied to the mucosa indicates that this structure is more accessible and (or) more sensitive to low-dose glutaraldehyde than is the ADH-induced water pathway. The observation that glutaraldehyde is more effective in stabilizing the ADH-induced urea channels from the serosal than from the mucosal surface indicates that these channels are not fixed at the extracellular surface of the apical plasma membrane. It appears, rather, that glutaraldehyde exerts its effects from an intracellular position, where it cross-links components of the urea channels at the cytoplasmic surface of the apical membrane and (or) inactivates the intracellular machinery responsible for the removal or dispersal of the ADH-induced urea permeability sites.     

Wheat germ agglutinin (WGA) reduces ADH-induced water flow and induces cell surface changes in epithelial cells of frog urinary bladder

The functional and structural changes induced by apical wheat germ agglutinin (WGA) 100 micrograms/ml exposure on frog urinary bladder have been investigated and the possible correlations between these effects discussed. Bladders, apically exposed to WGA for 30 min to 3 hr exhibit a marked reduction of their response to antidiuretic hormone (ADH) challenge and of their hydrosmotic reactivity. Structural changes triggered by WGA treatment are: 1. apical invaginations of the plasma membrane, interpreted as endocytotic in nature, taking into account the results of carbohydrate cytochemical detection and horseradish peroxidase (HRP) exposure: 2. cytoskeleton disorganization and microvilli collapse. These phenomena do not interfere with cortical granule traffic and are independent of ADH challenge: they occur in ADH-stimulated bladders as well as in bladders at rest. These findings could be interpreted as follows: binding of the divalent lectin WGA to its coat specific receptors would induce changes in the apical membrane structure which in turn could provoke disorganization and disruption of apical cytoskeletal elements associated with plasma membrane. Reduction of bladder response to ADH challenge could result from a reduced recycling of aggrephores, as they are associated with cytoskeletal elements in the subapical cytoplasm. Collapse of microvilli and endocytotic events also could result from apical cytoskeleton disruption, as microvilli are sustained by bundles of actin filaments interconnected with apical cytoskeletal filaments and as plasma membrane is associated with apical cytoskeleton. However, these two last events evidently occur in ADH-challenged or non-challenged bladders.     

The electron microscopic analysis of the mechanism of the insertion of high water permeability domains into the apical membrane of epithelial cells

Fusion processes of cell organelles with granular cell apical membranes under ADH-induced water permeability has been analysed. The data obtained suggest that specific granule membranes inserted in the apical membrane may change its water permeability. This occurs due to the fusion of the granule membrane with the apical membrane without exocytosis, the granule contents remaining in the cytoplasm.     

Possible role of Ca2+-binding sites in the regulation of Na+ transport in toad urinary bladder

La3+ was used to assess the role of membrane-bound Ca2+ in the regulation of basal and antidiuretic hormone (ADH)-induced Na+ transport by the isolated toad urinary bladder. Na+ transport was monitored by means of a short-circuit current (Isc) device. Mucosal La3+ (0.5-5 mM) increased Isc, while serosal La3+ (5 mM) produced a biphasic response (stimulation followed by inhibition). The stimulatory effects of La3+ were additive when present on both sides and were suppressed by mucosal amiloride or serosal ouabain. The action of mucosal La+ was reversible but the inhibition produced by serosal La3+ was not. In the presence of serosal La3+ the natriferic effect of ADH was abolished, but Theophylline, dibutyryl-cAMP, Amphotericin B, mucosal La3+, mucosal low pH, and phospho(enol) pyruvate, were able to increase Isc. These results suggest that Ca2+ binding sites in apical and basolateral membranes may play a key role in the modulation of both basal and ADH-induced Na+ transport. Serosal La3+ apparently inactivates the hormone-receptor interaction and/or the link between the ADH-receptor complex and the activation of adenylate cyclase, but does not interfere with the operation of the Na+ "pump", the basal activity of adenylate cyclase or any of the intracellular events that mediate the effect of ADH on Na+ transport.     

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.     

Fluorescence labeling of proteins related to ADH-induced change in frog bladder luminal membrane

Sulfhydryl (SH) reactive reagents, such as eosin derivatives, have been found to be useful in labeling water pathways in red cells. In the present study we used an impermeable SH-reagent, a fluorescent maleimide analogue EMA (eosin-5'-maleimide), in order to identify proteins involved in water permeability response to antidiuretic hormone (ADH). We observed that: 1) EMA (1 mM) mucosal pretreatment did not modify either the basal water flux or the subsequent ADH-induced hydrosmotic response; 2) EMA added to the mucosal bath at the maximum response to ADH, significantly decreased net water flux by about 40%; similar results were obtained when 10(-5) M forskolin was used as a hydrosmotic agent. These results suggest that the inhibitory effect of EMA occurs at a post cAMP step, possibly at the level of the sulfhydryl groups of the water channels themselves. Fluorescence distribution in SDS-PAGE of Triton X-100 extracted proteins from bladder labeled with EMA in both control conditions and under ADH stimulation allowed us to identify apical membrane proteins, labeled during ADH stimulation and not labeled in water impermeable controls. Of particular importance are four proteins of 52, 32-35, 26, 17, kDa. These polypeptides are probably involved in ADH-stimulated water transport and may be components of the water channels.     

Glutaraldehyde fixation preserves the permeability properties of the ADH-induced water channels

Summary Unidirectional and net water movements were determined, in frog urinary bladders, before and after glutraldehyde fixation. Experiments were performed in three experimental conditions: 1) in nonstimulated preparations, 2) after the action of antidiuretic hormone (ADH) and 3) in nonstimulated preparations to which amphotericin B was incorporated from the luminal bath. As previously observed for net water fluxes, the increase in the unidirectional water movement induced by ADH was well preserved by glutaraldehyde fixation. After correction for the effects of unstirred layers and nonosmotic pathways, the observed correlation between the ADH-induced increases in the osmotic (Pf) and diffusional (Pd) permeability coefficients was not modified by the fixative action (before glutaraldehyde: slope 11.19,r:0.87±0.07;n=12; after glutaraldehyde: slope 10.67,r:0.86±0.04,n=39). In the case of amphotericin B, Pf/Pd=3.08 (r: 0.83±0.08), a value similar to that observed in lipid bilayers or in nonfixed toad urinary bladders. It is concluded that 1) The experimental approach previously employed to study water channels in artificial lipid membranes and in amphibian urinary bladders, can be applied to the glutaraldehyde-fixed frog urinary bladder. 2) Glutaraldehyde fixation does not modify the permeability properties of the ADH-induced water channels. 3) Any contribution of exo-endocytic processes or cell regulatory mechanisms to the observed permeability parameters can probably be excluded. 4) Glutaraldehyde-fixed preparations are a good model to characterize these water pathways.     

Antidiuretic response: what markers for water channel components?

Antidiuretic hormone increases the water permeability of its target epithelial tissues by triggering the insertion into the apical cell membrane of aggregated intramembrane particles that contain channels specific for water. Little is known about the chemical composition of these membrane particles and of the water channel components. Present work describes a procedure for obtaining selected antibodies that specifically recognize ADH-induced components of the apical membrane in the amphibian urinary bladder epithelial cells.     

ADH-induced water permeability: what role for the microtubular network?

1. ADH-induced intramembrane particle aggregates in the apical membrane of the epithelial cells are specifically related to water permeability in the epithelium. 2. Colchicine and nocadozole (both of which bind to tubulin) inhibit ADH-induced osmotic water flow in the amphibian bladder. 3. Microtubules may be involved in the translocation of the aggrephores prior to their insertion into the plasma membrane.     

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.     

The single file hypothesis and the water channels induced by antidiuretic hormone

Unidirectional and net water movements were determined at minute intervals in frog urinary bladders. The changes in both parameters were followed, during the action of antidiuretic hormone (ADH), at different temperatures and stirring conditions. After correction for external unstirred layer effects, the ratio of the osmotic (Pf) and diffusional (Pd) permeability coefficients was remarkably constant, at different times and in different experimental conditions. In the presence of ADH the delta Pf/delta Pd ratio in the mucosal border was probably greater than 9. On the other hand, in nonstimulated preparations the ratio was smaller, and probably not different from 1. These results, together with previous observations indicating that other small molecules (like urea) are excluded from the ADH-induced channel, might indicate that single-file water movement can occur through this structure. Alternatively, the delta Pf/delta Pd ratio could result from a complex geometric arrangement in series with the aqueous pore.     

Requirement of potassium for the action of anti-diuretic hormone (ADH) on frog skin

The aim of the present study is to study whether the presence of K(+) in bathing media is required for the action of ADH to the ionic transport across the skin in the frog species Rana hexadactyla. lonic transport was measured as transepithelial potential difference (TEPD) and short circuit current (SCC) by using an indigenously developed computer based voltage-clamp technique. Addition of ADH (40 nM) on the serosal side significantly increased the TEPD and SCC with Normal Ringer (NR) on both sides. ADH had no effect subsequent to amiloride (100 μM) pre-treatment, which confirmed the ADH-induced Na(+) transport. Chloride also has a significant role in the development of TEPD. To determine the role of K(+), Potassium-free Ringer (KFR) was placed on both sides; addition of ADH had no effect consequently. Further experiments were carried out to find out which side of K(+) was required for the action of ADH. There was a lack of ADH effect with apical NR and serosal KFR, demonstrating that serosal K(+) is essential to activate Na(+), K(+)- ATPase. Similarly, the ADH effect was lacking with apical KFR and serosal NR that was the novel finding of this study. Due to the concentration gradient, the K(+) was secreted from serosal side to apical side through barium (1 mM) blockable K(+) channel. This study provides evidence that serosal as well as apical K(+) are necessary for the action of ADH.     

Detergent extraction of membrane proteins related to the action of antidiuretic hormone

Antidiuretic hormone (ADH) induces, in the apical plasma membrane of target cells, the insertion of intramembranous particle aggregates that probably contain water channels. A mild attack of this membrane by a polyoxyethylene nonylphenyl detergent, which reversibly depressed ADH-induced water permeability, has been found to modify aggregate structure while extracting additional proteins. This simple procedure could be a valuable approach to the problem of aggregate isolation and characterization.     

Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone

Summary Antidiuretic hormone (ADH) increases the apical (external facing) membrane water permeability of granular cells that line the toad urinary bladder. In response to ADH, cytoplasmic vesicles called aggrephores fuse with the apical plasma membrane and insert particle aggregates which are visualized by freeze-fracture electron microscopy. Aggrephores contain particle aggregates within their limiting membranes. It is generally accepted that particle aggregates are or are related to water channels. High rates of transepithelial water flow during ADH stimulation and subsequent hormone removal decrease water permeability and cause the endocytosis of apical membrane and aggrephores which retrieve particle aggregates. We loaded the particle aggregate-rich endocytic vesicles with horseradish peroxidase (HRP) during ADH stimulation and removal. Epithelial cells were isolated and homogenized, and a subcellular fraction was enriched for sequestered HRP obtained. The HRP-enriched membrane fraction was subjected to a density shifting maneuver (Courtoy et al.,J. Cell Biol. 98:870, 1984), which yielded a purified membrane fraction containing vesicles with entrapped HRP. The density shifted vesicles were composed of approximately 20 proteins including prominent species of 55, 17 and 7 kD. Proteins of these molecular weights appear on the apical surface of ADH-stimulated bladders, but not the apical surface of control bladders. Therefore, we believe these density shifted vesicles contain proteins involved in the ADH-stimulated water permeability response, possibly components of particle aggregates and/or water channels.     

Cardiac overexpression of catalase antagonizes ADH-associated contractile depression and stress signaling after acute ethanol exposure in murine myocytes.

Alcohol dehydrogenase (ADH), which oxidizes ethanol into acetaldehyde, exacerbates ethanol-induced cardiac depression, although the mechanism of action remains unclear. This study was designed to examine the impact of antioxidant catalase (CAT) on cardiac contractile response to ethanol and activation of stress signaling. ADH-CAT double transgenic mice were generated by crossing CAT and ADH lines. Mechanical, intracellular Ca(2+) properties and reactive oxygen species generation were measured in ventricular myocytes. ADH-CAT, ADH, CAT and wild-type FVB myocytes exhibited similar mechanical and intracellular Ca(2+) properties. ADH or ADH-CAT myocytes had higher acetaldehyde-producing ability. Ethanol (80-640 mg/dl) suppressed FVB cell shortening and intracellular Ca(2+) transients with maximal inhibitions of 43.5 and 45.2%, respectively. Ethanol-induced depression on cell shortening and intracellular Ca(2+) was augmented in ADH group with maximal inhibitions of 66.8 and 69.6%, respectively. Interestingly, myocytes from CAT-ADH mice displayed normal ethanol response with maximal inhibitions of 46.0 and 47.2% for cell shortening and intracellular Ca(2+), respectively. CAT transgene lessened ethanol-induced inhibition on cell shortening (maximal inhibition of 30.3%) but not intracellular Ca(2+). ADH amplified ethanol-induced reactive oxygen species generation, which was nullified by the CAT transgene. Western blot analysis showed that ethanol reduced ERK phosphorylation and enhanced JNK phosphorylation without affecting p38 phosphorylation. The ethanol-induced changes in phosphorylation of ERK and JNK were amplified by ADH. CAT transgene itself did not affect ethanol-induced response in ERK and JNK phosphorylation, but it cancelled ADH-induced effects. These data suggest that antioxidant CAT may effectively antagonize ADH-induced enhanced cardiac depression in response to ethanol.