The hydrosmotic effect of vasopressin: a scanning electrom-microscope study.

Scanning electron-microscopy (SEM) was used to investigate the hydrosmotic effect of vasopressin on the apical surface of urinary bladders of toads Bufo marinus. Bladders were mounted on glass chambers and water fluxes were monitored with an optical method. Tissues were fixed in 2% glutaraldehyde and processed for SEM. Three types of cells were seen on the surface of control bladders:large polygonal (granular) cells, with blunt microvilli; smaller (mitochondria-rich) cells, with longer microvilli; goblet cells. Neither exposure of the bladders to a large osmotic gradient nor exposure to vasopressin in the absence of a gradient altered appreciably the epithelial surface. In contrast, the combination of vasopressin and an osmotic gradient resulted ina conspicuous diminution of the blunt microvilli. However, the small cells with longer microvilli remained unchanged. Identical results were seen with cAMP or theophylline in the presence of an osmotic gradient. These findings suggest that the hydrosmotic effect of vasopressin is mainly exerted on the granular cells of toad bladder and confirm observations made by others with the electron-microscope.     

The effects cytochalasin B on the surface morphology of the toad urinary bladder epithelium: a scanning electron microscopic study.

Cytochalasin B depresses the hydroosmotic response of the toad urinary bladder to vasopressin without affecting basal (bulk flow) permeability, diffusional permeability, or the hormone induced increase in short circuit current. Fine structural studies demonstrated that this macrolide fungal metabolite, in the presence of both an osmotic gradient and vasopressin, induces the formation of large intracellular vacuoles or 'lakes' in epitelial cells lining the bladder mucosa. Some surface changes (shortening and irregularity of microvilli, clumping of the glycocalyx, etc.) were reported by transmission electron microscopy. Scanning electron microscopy demonstrates that cytochalasin B drastically alters the mucosal surface morphology of the hormone stimulated bladder. Lesser changes were seen in the absence of vasopressin. In the presence of arginine vasopressin, excessive cellular swelling and possible rupturing, as well as surface membrane infolding and rippling, were seen in the cytochalasin treated tissues, The specific entity most affected by this treatment is the granular cell.     

The cellular specificity of the effect of vasopressin on toad urinary bladder

Summary Phase and electron micrographs of toad bladders were obtained following dilution of bathing media in the presence and absence of vasopressin. Dilution of the mucosal medium alone resulted in no morphologic changes. Subsequent addition of vasopressin produced an increase in the cell volume of the granular cells, manifested by some or all of the following changes: increased area of granular cell profiles as observed in sections, rounding of the cell nucleus, displacement of the two components of the nuclear envelope, loss of nuclear heterochromatin, sacculation of the endoplasmic reticulum and the Golgi apparatus, and reduction in the electron density of the cell cytoplasm. No such morphologic changes were noted in the other cell types comprising the mucosal epithelium — the mitochondria-rich, the goblet, and the basal cells. On the other hand, dilution of the serosal bathing medium in the absence of vasopressin caused a marked increase in the cell volume of all these cell types. The results demonstrate that the action of vasopressin to enhance bulk water flow across toad bladder is exerted specifically on the apical surface of the granular cells. It is suggested that the hormonal effect on sodium transport may also be limited to the granular cells. The route of osmotic water flow and the possible role of the other mucosal epithelial cells is discussed.     

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.     

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.     

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.     

Study of enzymes regulating vasopressin-stimulated cyclic AMP metabolism in separated mitochondria-rich and granular epithelial cells of toad urinary bladder

Summary The epithelial cells of the toad urinary bladder are morphologically heterogeneous. In order to relate the effect of vasopressin on cyclic AMP metabolism to cell type, the epithelial cells were separated by the density gradient technique of Scott, Sapirstein and Yoder (Science 184: 797, 1974). The separation was verified by electron-microscopy and by observing that the band of cells enriched in mitochondria-rich cells was enriched in carbonic anhydrase activity compared to the band of granular cells. A large portion of the cells collected from the gradient was considered to be nonviable, precluding further study of their function as intact cells. Vasopressin-stimulated adenylate cyclase activity in homogenates of granular cells was similar to that in homogenates of mitochondria-rich cells. Cyclic nucleotide phosphodiesterase activity was also similar in the two types of cell. Thus, the enzymes known to be involved in cyclic AMP metabolism in response to vasopressin appear to be located in both major cell types.     

THE ANATOMIC SITE OF THE TRANSEPITHELIAL PERMEABILITY BARRIERS OF TOAD BLADDER

An examination of the mucosal epithelium of the urinary bladder of the toad reveals that the two major cell types which abut on the urinary surface, the granular and mitochondria-rich cells, also contact the basement membrane. Thus, the epithelium functions as a single cell layer. Although basal cells are interpolated between the granular cells and the basement membrane over a large portion of the epithelium, they do not constitute an additional continuous cell layer. This finding is consistent with extensive physiological data which had assumed that the major permeability barriers of this epithelium were the apical and basal-lateral plasma membranes of a single layer of cells.     

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     

Intracellular solute gradients during osmotic water flow: An electron-microprobe analysis

Summary In an attempt to quantify possible intracellular water activity gradients during ADH-induced osmotic water flow, we employed energy dispersive X-ray microanalysis to thin, freezedried cryosections obtained from fresh, shock-frozen tissue of the toad urinary bladder. The sum of all detectable small ions (Na + K + Cl) in the cellular water space was taken as an index of the intracellular osmolarity. Presuming that all ions are osmotically active, they comprise about 90% of the cellular solutes. When the cells were exposed to dilute serosal medium, the reduction in the sum of the ions agreed well with the expected reduction in osmolarity. After inducing water flow by addition of ADH and dilution of the mucosal medium, all epithelial cells showed a fall in osmolarity. The change was more pronounced in granular cells than in basal or mitochondria-rich cells, consistent with the notion that granular cells represent the main transport pathway. Most significantly, intracellular osmolarity gradients, largely caused by an uneven distribution of K and Na, were detectable in granular cells. The gradients were not observed after ADH or mucosal dilution alone, or when the direction of transepithelial water flow was reversed. We conclude from these results that there is a significant cytoplasmic resistance to water flow which may lead to intracellular gradients of water activity. Concentration gradients of diffusible cations can be explained by a flow-induced Donnan-type distribution of fixed negative charges. With regard to transepithelial Na transport, the data suggest that ADH stimulates transport by increasing the Na permeability of the apical membranes of granular cells specifically.     

Regulation of membrane permeability by vasopressin; activation of the water permeability pathway in toad urinary bladder by N-ethyl-maleimide

1. Vasopressin induces a rapid increase in water permeability and stimulates net sodium transport in responsive epithelia through the mediation of cAMP. 2. In amphibian urinary bladder, the increase in water permeability is dependent on an intact cytoskeleton and is associated with the exocytotic insertion of tubular vesicles containing particle aggregates (the putative water channels) into the apical membrane of the granular epithelial cells. 3. In the toad bladder, mucosal addition of NEM, 0.1 mM, elicits a slow and irreversible increase in transepithelial water flow, whilst decreasing net sodium transport. 4. The hydrosmotic response to mucosal NEM is inhibited by cellular acidification, by pretreatment with cytoskeleton-disruptive drugs, and by agents that increase cytosolic calcium. 5. Mucosal NEM potentiates the hydrosmotic response to a submaximal, but not a maximal, dose of vasopressin. 6. Mucosal NEM, like vasopressin, induces both vesicle fusion and the appearance of particle aggregates at the granular cell apical surface. 7. NEM, unlike vasopressin, does not increase cellular cAMP content. 8. Mucosal NEM appears to increase transcellular water flow by activating cellular processes normally triggered by vasopressin, at a step beyond cAMP.     

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)     

Hyaluronate-hydrolases system and hydroosmotic effect of vasopressin]

Involvement of enzymes catabolizing hyaluronic acid (hyaluronidase, beta-glucuronidase, N-acetyl-beta-D-hexosaminidase) in the hydroosmotic action of vasopressin on the amphibian urinary bladder Rana Ridibunda was studied. It was found that vasopressin (50 nM), agonist of V2 receptors dDAVP (1.5 mcM) and forscolin (30 mcM) induce an activation of enzymes and its release into the Ringer solution at the mucosal surface simultaneously with the increase in the osmotic water flow. Maximal effect was observed 10 min later than hydroosmotic response. Release of enzymes under vasopressin effect was found in the absence of osmotic gradient and water flow through the epithelium. The repeated substitution of the outer Ringer solution for the fresh one resulted in the increase in the both the water permeability and the release of enzymes through the mucosal surface. We suggested that involvement of hyaluronate-hydrolases in the vasopressin effect is mediated by the cAMP-dependent mechanism. It is supposed that this effect creates conditions for the increase in the permeability of glycosaminoglycan structures covering adjacent to the apical cell surface.     

Effect of colchicine on the osmotic water flow across the toad urinary bladder.

Osmotic water movement across the toad urinary bladder in response to both vasopressin and cyclic AMP was inhibited by 10(-5) to 10(-4) M colchicine on the serosal but not on the mucosal side. This inhibitory effect was found to be time- and dose-dependent. Colchicine alone did not change basal osmotic flow and a baseline of the short-circuit current (Isc) and also did not affect a vasopressin-induced rise of the Isc. The inhibitory effect was not prevented by the addition of pyruvate. The osmotic water movement produced by 360 mM Urea (mucosal), 360 mM mannitol (serosal) or 2 mug/ml amphotericin B (mucosal), was not affected by 10(-4) M colchicine. These results suggest that colchicine inhibits some biological process subsequent to the formation of cyclic AMP except a directional cytoplasmic streaming process where microtubules may be involved.     

Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.     

Effect of monensin on osmotic water flow across the toad bladder and its stimulation by vasopressin and cyclic AMP

The effects of the sodium ionophore monensin on osmotic water flow across the urinary bladder of the toad Bufo marinus were studied. Monensin alone did not alter osmotic water flow; however, the ionophore inhibited the hydrosmotic response to vasopressin and cyclic AMP in a dose-dependent manner. The inhibitory effects of monensin were apparent when the ionophore was added to th serosal bathing solution but not when it was added to the mucosal bathing solution. The inhibitory effect of serosal monensin required the presence of sodium in the serosal bathing solution but not the presence of calcium in the bathing solutions. Thus, it appears that intracellular sodium concentration is a regulator of the magnitude of the hydrosmotic response to vasopressin and cyclic AMP.     

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.     

Stimulative effect of guanylylimidodiphosphate on the vasopressin-induced increment of osmotic water flow of the toad bladder.

The effect of guanylylimidodiphosphate [Gpp(NH)p] on vasopressin-induced osmotic water flow across the bladder of the toad, $\text{Bufo}\text{bufo}\text{japonicus}$ was examined. Gpp(NH)p significantly enhanced vasopressin-induced osmotic water flow of the bladder at a concentration of 1 × 10^?5M, while it showed no effect on the water flow without vasopressin. Gpp(NH)p alone could not enhance cyclic AMP-induced osmotic water flow of the toad bladder. Adenylylimidodiphosphate [App(NH)p] could not enhance vasopressin-induced osmotic water flow of the bladder at a concentration of 1 × 10^?5M. The results suggest that Gpp(NH)p can enhance the physiological effect of vasopressin by stimulating vasopressin activation of adenylate cyclase during substrate and hormone depletion of the toad bladder.     

Barriers to water flow in vasopressin-treated toad urinary bladder

Summary Unstirred layers of water complicate the measurement of water permeability across epithelia. In the toad urinary bladder, the hormone vasopressin increases the osmotic water permeability of the granular epithelial cell's luminal membrane, and also leads to the appearance of aggregates of particles within this membrane. The aggregates appear to be markers for luminal membrane osmotic water permeability. This report analyzes the relationship between transbladder osmotic water flow and aggregate frequency, and demonstrates that flow across the bladder is significantly attenuated by unstirred layers of water or by structural barriers other than the luminal membrane when the luminal membrane is made permeable by vasopressin. This analysis in addition yields unique values for the permeabilities of both the luminal membrane and the barriers to water flow which lie in series with it.     

Electron microscopic study of colonic epithelial cells from the grass frog Rana temporaria under different intensity of water absorption

Three cell types have been revealed in the epithelium of the frog large intestine: granular, mitochondria-rich, and mucosal cells. Under a low water permeability (0.12 +/- 0.10 mkl/(min.cm2)) the distribution of intramembrane particles (IMP) in the apical cell membrane was the same as in the most cell plasma membranes studied with freeze-fracture method. Under rising osmotic permeability and water absorption (0.43 +/- 0.05 mkl/(min.cm2)) the IMP distribution did not change. In these conditions, the quantity of fusion sites between granule membranes and the apical membrane increased, and the intercellular spaces in basolateral epithelial region were diluted. A a low water permeability, in addition to usual microtubules, bundles of noncentrosomal microtubules with associated osmiophilic globules were revealed. A comparative analysis has been made of the present evidence and previously obtained data on the frog urinary bladder epithelium.