Effects of colchicine and cytochalasin B on hypertonicity-induced changes in toad urinary bladder

Summary Coincident with an increase in the water permeability of toad urinary bladder induced by serosal hypertonicity, a transformation of the ridge-like surface structures of the granular cells into individual microvillous structures occurs. This study was initiated to establish whether the transformation is mediated by the cytoskeletal network and, thus, can be prevented by disruption of microtubulemicrofilament function with colchicine or cytochalasin B (CB). Scanning electron microscopy revealed the characteristic branching ridges on granular cells of control bladder incubated with colchicine or CB. In contrast, transformation of ridges to discrete microvilli was observed in experimental bladders exposed to serosal hypertonicity alone or in combination with either colchicine or CB. These results suggest that the mechanism underlying hypertonicity-induced surface changes which are associated with increased water permeability does not involve either microtubules or microfilaments.     

Antidiuretic hormone response in the amphibian urinary bladder: time course of cytochalasin-induced vacuole formation, an ultrastructural study employing ruthenium red

Cytochalasin is known to inhibit the antidiuretic hormone-induced hydro-osmotic response (bulk water flow) in the amphibian urinary bladder without altering hormone-stimulated diffusional water permeability or short-circuit current. In addition, histological studies have shown that the mold metabolite induces the formation of large intracellular vacuoles or lakes in the epithelial cells. We report here a transmission electron microscopic time-course study which indicates that during the early phases of the ADH response cytochalasin causes the formation of numerous multivesicular bodies or aggregates derived from individual basolateral pinocytotic vesicles. Because of their apparent hypertonic nature, the vesicles, as well as the vesicular aggregates, accumulate water during hormone-stimulated hydro-osmotic flow. As a result, the multivesicular bodies dilate and fuse to form the large intracellular lakes characteristic of cytochalasin treatment in the presence of both an applied osmotic gradient and vasopressin. In the presence of mucosal ruthenium red, the luminal glycocalyx was heavily stained with this tracer. At no time, however, even in the presence of hormone, was there any evidence for the uptake of this dye at the apical epithelial border. In the presence of serosal ruthenium red, the lateral intercellular spaces, basolateral pinocytotic vesicles, basal lamina, and collagen, as well as other subepithelial structures, were ruthenium positive. With cytochalasin D, vasopressin, and serosal ruthenium red, both the pinocytotic vesicles and the multivesicular bodies demonstrated an apparent membrane associated ruthenium positive coat. The tracer data indicates that the basolateral pinocytotic vesicles, increased by the presence of hormone, are indeed endocytotic in nature. The mucopolysaccharide coat associated with these structures may be involved in ionic and/or fluid transport.     

EFFECTS OF CYTOCHALASIN B ON THE RESPONSE OF TOAD URINARY BLADDER TO VASOPRESSIN

A combined physiological and morphological study of the effects of cytochalasin B (CB) on the toad urinary bladder has been carried out. CB inhibits the hydro-osmotic response to vasopressin without altering basal water permeability or diffusion, or the increase in ³H₂O diffusion observed after hormone addition. Although CB increases [²²Na]-, [³⁶Cl]-, and [¹⁴C]urea fluxes, and decreases transepithelial potential, no alteration in basal short-circuit current, the vasopressin-induced increase in this parameter, or [¹⁴C]inulin permeability occurs. In the absence of hormone, CB does not markedly alter the structure of the toad bladder. However, in the presence of vasopressin, CB induces the formation of large intracellular vacuoles. These results suggest a possible coupling of solute and water movement across the tissue.     

Mucosal surface morphology of the toad urinary bladder. Scanning electron microscope study of the natriferic and hydro-osmotic response to vasopressin

The mucosal cell surface of the toad urinary bladder was examined by scanning electron microscopy, and changes in the structure of the surface of the granular cell were correlated with specific physiological responses to vasopressin. Survey views of the mucosal surface demonstrated that there was no consistent repeating anatomical relationship between the granular cell and the mitochondria-rich cell that would support the concept of cooperativeness in the response to vasopressin. During base-line states of Na+-transport and water flux, the microvilli on the mucosal surface of the granular cell are arranged in a ridge-like network with occasional individual projections. When water flux is increased by exposing the tissue to vasopressin, in the presence of an osmotic gradient across the tissue the microvilli on the granular cell lose the ridge structure and appear, predominantly, as individual projection. Variability-of this appearance points out the necessity of examining large areas and many samples before the significance of any morphological change can be assessed. Blocking the simultaneously occurring natriferic response of the toad urinary bladder with 10(-2)M ouabain does not prevent these changes in the microvilli. When the hydro-osmotic response is blocked by eliminating the osmotic gradient, the granular cell shows no consistent change in mucosal surface morphology even when fixed at the height of the natriferic response. The mitochondria-rich and mucous cells did not show any change in morphology throughout these studies. We conclude that the changes in the mucosal surface morphology of the toad bladder seen after exposure to vasopressin are a result of the increased water flux that occurs when an osmotic gradient exists across the tissue, and are not related to the natriferic response or any specific alteration in the membrane properties.     

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)     

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.     

Electron microscopic cytochemical localization of Ca-ATPase in toad urinary bladder

The calcium-regulating enzyme calcium adenosine triphosphatase (Ca-ATPase) was localized in the epithelium of amphibian urinary bladder by the one-step electron microscopic cytochemical procedure. The enzyme was identified along the basolateral border of the epithelial cells that comprise the bladder mucosa. The electron-dense precipitate indicating Ca-ATPase activity was seen in association with the outer leaflet of the basolateral plasmalemmae. Intracellularly, Ca-ATPase activity was seen in association with the mitochondrial matrix of the mitochondria-rich cells. Ca-ATPase was not seen along the apical microvillated border. Enzyme activity was also not seen after incubation in substrate-free media, calcium-free media, or incubation in the presence of vanadate. However, Ca-ATPase activity was evident when the calcium in the standard reaction medium was deleted in favor of magnesium. Addition of antidiuretic hormone (ADH; vasopressin) increased both the basolateral Ca-ATPase reaction and the mitochondrial reaction. Such data appear to indicate further that changes in cytosolic calcium ion concentration take place during the response of amphibian urinary bladder to the polypeptide hormone vasopressin.     

Electron microscopic cytochemical localization of adenylate cyclase in amphibian urinary bladder epithelium: effects of antidiuretic hormone

A cytochemical technique for electron microscopic localization of adenylate cyclase was used to identify this enzyme in quiescent and hormone-stimulated toad urinary bladder epithelium. In the absence of vasopressin (antidiuretic hormone), adenylate cyclase was detected along the outer surface of the basolateral plasma membranes of granular cells, mitochondria-rich cells, and basal cells, the major cell types comprising the hormone-sensitive urinary epithelium. In the presence of antidiuretic hormone, the basolateral precipitates were markedly increased. The latter was true for both tissues incubated in the presence of an osmotic gradient and those stimulated in the absence of such a gradient. A significant mucosal reaction was never seen. Such data indicate that the hormone receptors for vasopressin are located along the basolateral membranes of all epithelial cells comprising the mucosal hormone-sensitive epithelium. All cells of the epithelium also demonstrate a vasopressin-sensitive adenylate cyclase. We discuss possible mechanisms that attempt to integrate the cytochemical data into an overall scheme for the physiological action of this hormone on amphibian urinary bladder.     

Studies on the Movement of Water through the Isolated Toad Bladder and Its Modification by Vasopressin

Measurements of diffusion permeability and of net transfer of water have been made across the isolated urinary bladder of the toad, Bufo marinus, and the effects thereon of mammalian neurohypophyseal hormone have been examined. In the absence of a transmembrane osmotic gradient, vasopressin increases the unidirectional flux of water from a mean of 340 to a mean of 570 µl per cm² per hour but the net water movement remains essentially zero. In the presence of an osmotic gradient but without hormone net transfer of water remains very small. On addition of hormone large net fluxes of water occur; the magnitude of which is linearly proportional to the osmotic gradient. The action of the hormone on movement of water is not dependent on the presence of sodium or on active transport of sodium. Comparison of the net transport of water and of unidirectional diffusion permeability of the membrane to water indicates that non-diffusional transport must predominate as the means by which net movement occurs in the presence of an osmotic gradient. An action of the hormone on the mucosal surface of the bladder wall is demonstrated. The effects of the hormone on water movement are most simply explained as an action to increase the permeability and porosity of the mucosal surface of the membrane.     

Membrane structural studies of the action of vasopressin

Freeze-fracture electron microscopy of the toad urinary bladder indicates that distinctive intramembrane particle aggregates are responsible for the increase in apical membrane water permeability that occurs with vasopressin (VP) stimulation. In unstimulated bladders the aggregates occur in the cytoplasm of the cells in tubular membrane structures now called aggrephores. After stimulation by VP, aggrephores are shuttled to the surface and fuse with the apical membrane. It is suggested by structural observations and by measurements of membrane capacitance that the area of aggregates inserted into the apical membrane is much greater than previously suspected because many aggregates remain in the wall of the fused aggrephores. The area of the aggregates in a stimulated bladder is sufficiently large for these structures to represent an organized array of water channels that mediates the change in apical membrane permeability. Work with antibodies supports the concept that these channels are not always resident in the apical membrane but become inserted only after stimulation by the hormone VP.     

Cytochalasin B and water transport

Summary A morpho-functional study of the effects of cytochalasin B (CB) on Na and water transport was made in amphibian epithelia. The functional studies confirmed the dissociation of the natriferic and hydrosmotic effects of vasopressin in toad urinary bladders exposed to CB and showed in addition that the block of the hydrosmotic effect was reversible and could still be induced in epithelia maximally stimulated with the hormone. Scanning electron microscopy revealed that CB, per se, did not alter the apical surface of the bladders. An almost total loss of microvilli of granular cells was seen, however, if CB was associated with vasopressin and an osmotic gradient. The results suggest two points: a) the block of the hydrosmotic flow induced by CB is due to factors beyond the apical membrane; b) microfilaments may be important mechanochemical transducers in the chain of events leading to the hydrosmotic effect of vasopressin.Supported by the grants Nos 3.1300.73 and 3.043-0.76 of the Swiss National Science FoundationThe authors are grateful to Miss C. Brücher, SEM operator of the Department of Physics, Ciba-Geigy, for skillful collaboration, to Mr. R. Mira for the illustrations and to Mrs. A. Cergneux for secretarial assistance     

Permeability of the Isolated Toad Bladder to Solutes and Its Modification by Vasopressin

Measurements have been made of the permeability of the isolated urinary bladder of the toad to a number of small solute molecules, in the presence and absence of vasopressin. Vasopressin has a strikingly specific effect on increasing permeability of the bladder to a group of small, uncharged amides and alcohols while penetration by other small molecules and ions is unaffected. The movement of urea is passive, as indicated by equal flux rates in the two directions. The reflection coefficients for chloride and thiourea indicate a high degree of impermeability of the bladder to these solutes even in the presence of large net movements of water. The low concentration of thiourea in the tissue water when this compound is added to the mucosal bathing medium indicates that the major permeability barrier to thiourea is at the mucosal surface of the bladder. The findings can be accounted for by a double permeability barrier consisting of a fine selective diffusion barrier and a porous barrier in series. The former would constitute the permeability barrier to most small solutes while the latter would be the rate-limiting barrier for water and the amides. It would be the porous barrier which is affected by vasopressin. Reasons are presented which require both barriers to be contained in or near the plasma membrane at the mucosal surface of the bladder.     

Changes in the topography of the sea urchin egg after fertilization

Changes in the topography of the sea urchin egg after fertilization were studied by scanning and transmission electron microscopy. Strongylocentrotus purpuratus eggs were treated with dithiothreitol to modify the vitelline layer and to prevent formation of a fertilization membrane. Dithiothreitol treatment caused the microvilli to become more irregular in shape, length, and diameter than those of untreated eggs. The microvilli were similarly modified by trypsin treatment. This effect did not appear to be due to disruption of cytoskeletal elements beneath the plasma membrane, for neither colchicine nor cytochalasin B altered microvillar morphology. Thus, it appears that the vitelline layer may act in the maintenance of surface form of unfertilized eggs. Since dithiothreitol-treated eggs did not elevate a fertilization membrane, scanning electron microscopy could be used to directly observe modifications in the egg plasma membrane after fertilization. The wave of cortical granule exocytosis initiated at the point of attachment of the fertilizing sperm was characterized by the appearance of pits that subsequently opened, releasing the cortical granule contents and leaving depressions upon the egg surface. The perigranular membranes inserted during exocytosis were seen as smooth patches between the microvillous patches remaining from the original egg surface. This produced a mosaic surface with more than double the amount of membrane of unfertilized eggs. The mosaic surface subsequently reorganized to accommodate the inserted membrane material by elongation of microvilli. Blebs and membranous whorls present before reorganization suggested the existence of an unstable intermediate state of plasma membrane reorganization. Exocytosis and mosaic membrane formation were not blocked by colchicine or cytochalasin B, but microvillar elongation was blocked by cytochalasin B treatment.     

Inhibition of the permeability response to vasopressin and oxytocin in the toad bladder: Effects of bradykinin,kallidin, eledoisin,and physalaemin

Summary It has been shown by means of Bentley'sin vitro preparation of the isolated urinary bladder of the toad,Bufo marinus paracnemis Lutz, that bradykinin reversibly inhibited the increase brought about by vasopressin on the permeability to water of the toad bladder. The increased hydro-osmotic response of the bladder to oxytocin was also inhibited by the kinin. The effect on water permeability was observed when bradykinin was added either to the serosal Ringer's solution or to the mucosal solution. The addition of bradykinin alone did not alter the basal osmotic water transfer across the bladder. In this context, bradykinin acted as a competitive antagonist of vasopressin (and oxytocin). Although lacking intrinsic activity, bradykinin exhibited affinity for receptor sites that are also common to the neurohypophysial hormones, causing a parallel shift of the log-dose/response curve for vasopressin without changing the maximal responses. The effects of other kinins (namely kallidin, eledoisin and physalaemin) on the toad bladder were also tested. Each of these drugs alone did not change the basal water flux across the bladder wall. Like bradykinin, these peptides inhibited the increase in water permeability evoked by vasopressin and oxytocin in the bladder. In view of the importance of neurohypophysial hormones and their target tissues to the osmotic homeostasis of amphibians, and the observation of antagonism between the kinins and the pituitary hormones coupled to the abundance of kinins in the amphibian organism, particularly in the skin and urinary bladder, teleological reasoning predicts a physiological role for the kinins, possibly functioning to dampen excesses and oscillations in membrane permeability that could occur in face of a constant and variable secretion of neurohypophysial hormone, thus adding to the homeostatic response of the amphibian organism.     

Vasopressin and water permeability of artificial lipid membranes

Vasopressin markedly stimulated the water permeability of bilayer lipid membranes: a two-fold increase was measured at 25° in presence of 1.7·10⁻⁹ M (50 μunits/ml) vasopressin. Oxytocin and a mixture of the amino acids comprising the vasopressin molecule could not substitute for vasopressin at comparable concentration. The experimental activation energy of water transport was reduced in the presence of vasopressin from 14 to 4 kcal/mole, in agreement with the effect of the hormone on water permeability of toad bladder.     

The State of Water in the Isolated Toad Bladder in the Presence and Absence of Vasopressin

An attempt has been made to assess the validity of applying the frictional and viscous coefficients of bulk water to the movement of water and solutes through the urinary bladder of the toad. The temperature dependence of diffusion of THO, C¹⁴-urea, C¹⁴-thiourea, and net water transfer across the bladder was determined in the presence and absence of vasopressin. The activation energy for diffusion of THO was 9.8 kcal per mole in the absence of vasopressin and 4.1 kcal per mole with the hormone present. Activation energies simultaneously determined following vasopressin for diffusion and net transfers of water were similar, and in the same range as known activation energies for diffusion and viscous flow in water. Urea had activation energies for diffusion of 4.1 and 3.9 kcal per mole in the absence and presence of vasopressin, respectively. Thiourea had a high activation energy for diffusion of 6.3 kcal per mole, which was unchanged, 6.6 kcal per mole, following hormone. These findings suggest that in its rate-limiting permeability barrier, water is present in a structured state, offering a high resistance to penetration by water. Vasopressin enlarges the aqueous channels so that the core of water they contain possesses the physical properties of ordinary bulk water. Urea penetrates the tissue via these aqueous channels while thiourea is limited by some other permeability barrier.     

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.     

The role of microtubules and microfilaments in the hydrosmotic response to antidiuretic hormone

To test the effects of colchicine and cytochalasin B on the ADH-induced response, unidirectional and net water fluxes were measured at one or two minutes intervals in frog urinary bladder. The action of these agents on the appearance of intramembrane particles aggregates in the luminal membrane of target cells under oxytocin stimulation and the changes in the tissue ultrastructure induced by cytochalasin B were also studied. It was observed that: the time-course of the response to oxytocin was strongly slowed by colchicine while the washout was not affected; the time-course of the 'on and off' of the response to oxytocin was not modified by cytochalasin B; cytochalasin B pretreatment proportionally reduced unidirectional and net water fluxes measured after glutaraldehyde fixation; the combined action of colchicine and cytochalasin B proportionally reduced the net water flux and the number of intramembrane particles aggregates, observed in freeze-fracture studies; after cytochalasin B action the dilation of intercellular spaces classically observed under oxytocin stimulation is strongly reduced. It is concluded that: microtubules probably play an important role in the water channels plug-in, but not in their removal; microfilaments integrity is necessary for the mechanisms inducing intercellular space dilation and the observed results confirm that water permeability is controlled by the number of permeation units present in the luminal border of granular cells and probably represented by the intramembrane particle aggregates.     

Effect of cytochalasins on the surface topography of neoplastic cells in suspension

The effect of cytochalasins B or D on the surface topography of mouse neoplastic fibroblasts of L line (detached from glass by trypsin-EDTA solution) or of its LS subline (adapted to the growth in suspension in vitro), as well as on that of Ehrlich's ascites tumor cells was investigated by screening electron microscopy. Incubation of suspended cells with cytochalasin B (2 micrograms/ml) or cytochalasin D (0.2 microgram/ml) for 30-180 min led to the following changes: (I) progressive decrease of the proportion of the cells with a microvillous surface relief and simultaneous increase in the percentage of the cells with a blebbed microrelief; (2) shortening of the microvilli and decrease of their density on the cell surface; (3) appearance of surface areas with a rough folded relief; (4) formation of very large blebs on the LS or L cell surfaces; (5) unusual "polar" distribution of blebs on Ehrlich's tumor and L cells: the blebs were concentrated in one locus on the cell surface. The data show that normal organization of the actin microfilament system in the cell cortex is necessary for formation of the microvilli but not for the blebs.     

The morphology of the toad urinary bladder: A stereoscopic and transmission electron microscopical study

Summary The appearance of the mucosal cell layer of the isolated urinary bladder of the toadBufo marinus, has been examined using stereoscopic and conventional transmission electron microscopy.Three cell types can be identified in surface view, these are granular cells, mitochondriarich cells and goblet cells. Cell boundaries between granular cells are clearly defined by membranous folds along their margins. Although no changes are seen in the stereoscopic electron micrographs when the granular cells are made permeable to water by vasopressin, the changes observed on transmission electron micrographs include swelling of cell bodies and nuclei, filling of intercellular channels with water, and the appearance beneath the mucosal cell membrane surface of electron dense granules.Differences between the appearance of the bladder mucosal cells by the two methods of electron microscopical examination are due largely to water loss when the tissue is freeze dried prior to stereoscopic examination.Grateful thanks are due to Dr. W. D. E. Thomas and Miss Elizabeth Hull of the Long Ashton Research Station for use of the Stereoscan microscope, and also to Dr. John Clamp and Mr. P. J. Summers for help and advice.In receipt of a personal research grant from the Medical Research Council, London.