The permeability of rat transitional epithelium. Kertinization and the barrier to water

Permeability barriers must exist in transitional epithelium to prevent the free flow of water from underlying blood capillaries through the epithelium into the hypertonic urine, and such a barrier has now been demonstrated in isolated bladders. This barrier is passive in function and can be destroyed by damaging the luminal surface of the transitional epithelium with sodium hydroxide and 8 M urea solutions, by digesting it with trypsin, lecithinase C, and lecithinase D, or by treating it with lipid solvents such as Triton x 100 and saponin. From this it is concluded that the barrier depends on the integrity of lipoprotein cell membranes. The barrier function is also destroyed by sodium thioglycollate solutions, and electron microscope investigations show that sodium thioglycollate damages the thick asymmetric membrane which limits the luminal face of the superficial squamous cell. Cytochemical staining shows the epithelium to contain disulfide and thiol groups and to have a concentration of these groups at the luminal margin of the superficial cells. It thus appears that the permeability barrier also depends on the presence of disulfide bridges in the epithelium, and it is presumed that these links are located in keratin. Because of the effect of thioglycollates, both on the barrier function and on the morphology of the membrane, it is suggested that keratin may be incorporated in the thick barrier membrane. It is proposed that the cells lining the urinary bladder and ureters should be regarded as a keratinizing epitheluim.     

The maintenance of the permeability barrier of bladder facet cells requires a continuous fusion of discoid vesicles with the apical plasma membrane

The luminal surface of the bladder epithelium is continuously exposed to urine that differs from blood in its ionic composition and osmolality. The apical plasma membrane of facet or umbrella cells, facing the urine, is covered with rigid-looking plaques consisting of hexagonal uroplakin particles. Together with tight junctions these plaques form a specialized membrane compartment that represents one of the tightest and most impermeable barriers in the body. Plaques also occur in the membrane of cytoplasmic discoid vesicles. Here it is shown shown that synaptobrevin, SNAP23 and syntaxin are perfectly colocalized with uroplakin III at the apical plasma membrane as well as with membranes of discoid vesicles. Such a distribution suggests that discoid vesicles in facet cells may gain access to the apical plasma membrane probably by combination of homotypic and heterotypic fusion events. Furthermore, we detected uroplakin III-containing membranes of different sizes in the urine of healthy humans and rats. Probably facet cells maintain their permeability barrier by a process of continuous membrane regeneration that includes the cutting off of areas of the apical membrane and its replacement by newly fused discoid vesicles.     

Alkaline phosphatase, 5'-nucleotidase and magnesium-dependent adenosine triphosphatase activities in the transitional epithelium of the rat urinary bladder.

The cerium-based method was used to demonstrate cytochemically the ultrastructural localization of alkaline phosphatase (ALPase), 5'-nucleotidase (5'-Nase) and magnesium-dependent adenosine triphosphatase (Mg-ATPase) on the transitional epithelium of the rat urinary bladder. The reaction product for ALPase was found on the plasma membrane of all epithelial cells, except the luminal surface of superficial cells. The activity of 5'-Nase appeared on the plasma membrane of all bladder transitional epithelial cells, including the free surface of superficial cells. The Mg-ATPase reaction product was seen on the plasma membrane of superficial, intermediate and basal cells, but never on the luminal surface of superficial cells and it was only occasionally seen on the basal surface. The possible functions of these phosphatases have been discussed, and it was emphasized that the 5'-Nase activity present on the luminal surface of superficial cells may play a special role in the membrane movement of these cells in the transitional epithelium.     

Hypercompliant Apical Membranes of Bladder Umbrella Cells

Urinary bladder undergoes dramatic volume changes during filling and voiding cycles. In the bladder the luminal surface of terminally differentiated urothelial umbrella cells is almost completely covered by plaques. These plaques (500 to 1000 nm) are made of a family of proteins called uroplakins that are known to form a tight barrier to prevent leakage of water and solutes. Electron micrographs from previous studies show these plaques to be interconnected by hinge regions to form structures that appear rigid, but these same structures must accommodate large changes in cell shape during voiding and filling cycles. To resolve this paradox, we measured the stiffness of the intact, living urothelial apical membrane and found it to be highly deformable, even more so than the red blood cell membrane. The intermediate cells underlying the umbrella cells do not have uroplakins but their membranes are an order of magnitude stiffer. Using uroplakin knockout mouse models we show that cell compliance is conferred by uroplakins. This hypercompliance may be essential for the maintenance of barrier function under dramatic cell deformation during filling and voiding of the bladder.     

Hypercompliant Apical Membranes of Bladder Umbrella Cells

Urinary bladder undergoes dramatic volume changes during filling and voiding cycles. In the bladder the luminal surface of terminally differentiated urothelial umbrella cells is almost completely covered by plaques. These plaques (500 to 1000 nm) are made of a family of proteins called uroplakins that are known to form a tight barrier to prevent leakage of water and solutes. Electron micrographs from previous studies show these plaques to be interconnected by hinge regions to form structures that appear rigid, but these same structures must accommodate large changes in cell shape during voiding and filling cycles. To resolve this paradox, we measured the stiffness of the intact, living urothelial apical membrane and found it to be highly deformable, even more so than the red blood cell membrane. The intermediate cells underlying the umbrella cells do not have uroplakins but their membranes are an order of magnitude stiffer. Using uroplakin knockout mouse models we show that cell compliance is conferred by uroplakins. This hypercompliance may be essential for the maintenance of barrier function under dramatic cell deformation during filling and voiding of the bladder.     

The isolation and analysis of the luminal plasma membrane of calf urinary bladder epithelium.

The luminal plasma membrane of calf urinary bladder epithelium (urothelium) has been isolated by a method designed to preserve enzymic activity as well as structural integrity. The yield was about 80 micrograms per calf bladder. Low levels of 5' nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase activities were found in the luminal membrane fraction. Cerebroside was the major lipid present and dodecyl sulphate gel electrophoresis revealed a complex protein and glycoprotein composition in the whole membrane. A membrane fraction consisting of only the plaque areas was shown to have a simpler protein composition with major polypeptides of apparent Mr 12 000 and 22 000. These may associate to form a 30 000 apparent Mr complex which could represent the individual 'particles' of the dodecameric subunits seen by electron microscopy in the plaque regions.     

Ultrastructural organization of the hamster renal pelvis.

The renal pelvis of the hamster has been studied by light microscopy (epoxy resin sections), transmission electron microscopy, and morphometric analysis of electron micrographs. Three morphologically distinct epithelia line the pelvis, and each covers a different zone of the kidney. A thin epithelium covering the outer medulla (OM) consists of two cell types: (1) granular cells are most numerous and have apically positioned granules which stain intensely with toluidine blue, are membrane-bound, and contain a fine particulate matter that stains light grey to black in electron micrographs. (2) Basal cells do not have granules, are confined to the basal lamina region, and do not reach the mucosal epithelial surface. The inner medulla (IM) is covered by a pelvic epithelium morphologically similar to collecting duct epithelium of IM. Some cells in this portion of the pelvic epithelium (IM) stain intensely dark with toluidine blue, osmium tetroxide, lead, and uranyl acetate. Transitional epithelium, which separates cortex (C) from pelvic urine, has an asymmetric luminal plasma membrane and discoid vesicles, each of which is similar to those previously observed in mammalian ureter and urinary bladder epithelia. Based on morphological comparisons with other epithelia, the IM and OM pelvic epithelia would appear permeable to solutes and/or water, while the transitional epithelium covering the C appears relatively impermeable. It would also appear that the exchange of solutes and water between pelvic urine and OM would involve capillaries, primarily, since morphometric analysis showed that both fenestrated and continuous capillaries of the OM were extremely abundant (greater than 60% of OM pelvic surface area) just under the thin pelvic epithelium.     

The superficial cells of the transitional epithelium in the expanded and unexpanded rat urinary bladder. Transmission and scanning electron-microscopic study.

The superficial epithelial layer in the urinary bladder of adult rats was examined, in various states, using the transmission and scanning electron microscopes. A good agreement was obtained between the results of the two methods. When the urinary bladder is unexpanded, the superficial cells show marked bulges into the bladder lumen and the contacts between cells (mainly desmosomes) are displaced deep into the epithelium. The luminal surface is bizarrely bent and large parts of the membrane intrude into the cytoplasm, where they give the appearance of discoid and fusiform vesicles. Between neighboring cells, deep interdigitations are observed. In the scanning electron microscope, the surface of the epithelium appears cauliflower-like and has deep grooves, gullys and folds. When the bladder is expanded, the surface becomes smoother and the contacts between cells move to the surface. The stretched cells are angular in form (5-, 6- or 7-sided) and show great variations in surface area (150-500 mum2). The luminal cell membrane consists of an alternation of asymmetrical areas (120 A thick and 0.2-0.4 mum in length) with normal sections which are 80 A thick. In the scanning electron microscope, these thick areas appear as 4-, 5- or 6-sided plaques with a maximal diameter of 0.4 mum. The borders of the plaques are formed of portions of cell membrane which have a normal thickness and extrude as microcristae into the lumen. This produces a honeycomb appearance on the cell surface.     

Freeze-fracture study of the pavement cell in the lamprey gill epithelium. Analogy of membrane structure with the granular cell in the amphibian urinary bladder

Freeze-fracture studies of the lamprey gill epithelium reveal structural differences of the luminal and basolateral plasma membrane of the pavement cells. The luminal membrane is characterized by only a few intramembrane particles on the P face and numerous large (10-13 nm) particles on the E face, whereas the basolateral membrane shows the majority of intramembrane particles (6-8 nm) on the P face. The structural specialization of the luminal membrane and the differences between the luminal and basolateral membranes of the pavement cell are similar to those previously demonstrated for the unstimulated granular cell of the amphibian urinary bladder. Because of this similarity, it is suggested that the 2 cell types are analogous and that the luminal membrane of the pavement cell in the lamprey gill epithelium is functionally characterized by a low water permeability. A possible role of sodium uptake by the pavement cells from freshwater and putative differences of osmoregulatory mechanisms in the gills of lampreys and teleosts in freshwater environments are discussed.     

Structure of the urinary bladder in the Pacific cod

The urinary bladder of the Pacific cod,Gadus macrocephalus was investigated. A pair of ureters was found to unite and form a sac-like protrusion on the posteroventral aspect of the gonad. This protrusion was confirmed as the urinary bladder based on its histology and the presence of ammonia and urea in the fluid within this structure. The urinary bladder consisted of a central lumen and two lateral expansions found on the right and left sections. The luminal epithelial cells of the Pacific cod urinary bladder, unlike those of other animals, were characterized by microvilli on the free surface, and the presence of a number of vesicles and mitochondria across the apical portion on the cells. This suggests that the luminal epithelium of the urinary bladder might be actively engaged in transportation of water or other materials from the urine. The Pacific cod urinary bladder may therefore be closely associated with osmoregulation by reabsorbing water from the urine. Ammonia-N and urea-N levels of 65–213 μg/dl and 1.5–3.5 mg/dl were measured in the fluid which filled the urinary bladder.     

THE FINE STRUCTURE OF THE TRANSITIONAL EPITHELIUM OF RAT URETER

The fine structure of the transitional epithelium of rat ureter has been studied in thin sections with the electron microscope, including some stained cytochemically to show nucleoside triphosphatase activity. The epithelium is three to four cells deep with cuboidal or columnar basal cells, intermediate cells, and superficial squamous cells. The basal cells are attached by half desmosomes, or attachment plates, on their basal membranes to a basement membrane which separates the epithelium from the lamina propria. Fine extracellular fibres, ca. 100 A in diameter, are to be found in the connective tissue layer immediately below the basement membrane of this epithelium. The plasma membranes of the basal and intermediate cells and the lateral and basal membranes of the squamous cells are deeply interdigitated, and nucleoside triphosphatase activity is associated with them. All the cells have a dense feltwork of tonofilaments which ramify throughout the cytoplasm. The existence of junctional complexes, comprising a zonula occludens, zonula adhaerens, and macula adhaerens or desmosome, between the lateral borders of the squamous cells is reported. It is suggested that this complex is the major obstacle to the free flow of water from the extracellular spaces into the hypertonic urine. The free luminal surface of the squamous cells and many cytoplasmic vesicles in these cells are bounded by an unusually thick plasma membrane. The three leaflets of this unit membrane are asymmetric, with the outer one about twice as thick as the innermost one. The vesicles and the plasma membrane maintain angular conformations which suggest the membrane to be unusually rigid. No nucleoside triphosphatase activity is associated with this membrane. Arguments are presented to support a suggestion that this thick plasma membrane is the morphological site of a passive permeability barrier to water flow across the cells, and that keratin may be included in the membrane structure. The possible origin of the thick plasma membrane in the Golgi complex is discussed. Bodies with heterogeneous contents, including characteristic hexagonally packed stacks of thick membranes, are described. It is suggested that these are "disposal units" for old or surplus thick membrane. A cell type is described, which forms only 0.1 to 0.5 per cent of the total cell population and contains bundles of tubular fibres or crystallites. Their origin and function are not known.     

The uroepithelium: not just a passive barrier

The uroepithelium lines the inner surface of the renal pelvis, the ureters, and the urinary bladder, where it forms a tight barrier that allows for retention of urine, while preventing the unregulated movement of ions, solutes, and toxic metabolites across the epithelial barrier. In the case of the bladder, the permeability barrier must be maintained even as the organ undergoes cyclical changes in pressure as it fills and empties. Beyond furthering our understanding of barrier function, new analysis of the uroepithelium is providing information about how detergent-insoluble membrane/protein domains called plaques are formed at the apical plasma membrane of the surface umbrella cells, how mechanical stimuli such as pressure alter exocytic and endocytic traffic in epithelial cells such as umbrella cells, and how changes in pressure are communicated to the underlying nervous system.     

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)     

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.     

Membrane structural specialization of the toad urinary bladder revealed by the freeze-fracture technique

Summary Examination of the toad urinary bladder by freeze-fracture electron microscopy reveals that the mitochondria-rich cells of the epithelium possess distinctive and characteristic membrane structural specialization. Unique rod-shaped intramembrane particles are found in luminal and basal membranes as well as certain intracellular vesicles of this cell type. The consistent finding of two discrete patterns of luminal membrane structural organization supports the possibility that two morphological forms of mitochondria-rich cell exist within the toad bladder epithelium.     

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.     

A spin label study of the urinary bladder luminal membrane

Spin label experiments have been carried out on the urinary bladder luminal membrane of the bovine transitional epithelium employing the 5-, 7-, 12-, and 16-doxyl substituted stearic acid methyl esters, and compared for reference to similarly labeled bovine erythrocytes. The bladder membranes are significantly different from the bovine red blood cell membranes and show a lower order and polarity near the membrane surface. This fact and the general similarity of results for the bladder and isolated plaque membranes suggests that the highly organized proteins of the bladder membrane may act as a coat on the lipid bilayer and, while intrinsic in nature, do not significantly perturb the hydrophobic core of the lipid bilayer.     

Stretch-regulated exocytosis/endocytosis in bladder umbrella cells

The epithelium of the urinary bladder must maintain a highly impermeable barrier despite large variations in urine volume during bladder filling and voiding. To study how the epithelium accommodates these volume changes, we mounted bladder tissue in modified Ussing chambers and subjected the tissue to mechanical stretch. Stretching the tissue for 5 h resulted in a 50% increase in lumenal surface area (from approximately 2900 to 4300 microm(2)), exocytosis of a population of discoidal vesicles located in the apical cytoplasm of the superficial umbrella cells, and release of secretory proteins. Surprisingly, stretch also induced endocytosis of apical membrane and 100% of biotin-labeled membrane was internalized within 5 min after stretch. The endocytosed membrane was delivered to lysosomes and degraded by a leupeptin-sensitive pathway. Last, we show that the exocytic events were mediated, in part, by a cyclic adenosine monophosphate, protein kinase A-dependent process. Our results indicate that stretch modulates mucosal surface area by coordinating both exocytosis and endocytosis at the apical membrane of umbrella cells and provide insight into the mechanism of how mechanical forces regulate membrane traffic in non-excitable cells.     

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.     

Cyclic AMP-regulated exocytosis of Escherichia coli from infected bladder epithelial cells

The superficial bladder epithelium is a powerful barrier to urine and also serves as a regulator of bladder volume, which is achieved by apical exocytosis of specialized fusiform vesicles during distension of the bladder. We report that type 1 fimbriated uropathogenic Escherichia coli (UPEC) circumvents the bladder barrier by harboring in these Rab27b/CD63-positive and cAMP-regulatable fusiform vesicles within bladder epithelial cells (BECs). Incorporation of UPEC into BEC fusiform compartments enabled bacteria to escape elimination during voiding and to re-emerge in the urine as the bladder distended. Notably, treatment of UPEC-infected mice with a drug that increases intracellular cAMP and induces exocytosis of fusiform vesicles reduced the number of intracellular E. coli.