Microfilament-rich cells in the toad bladder epithelium

Summary Basal cells of the bladder epithelium ofBufo marinus have been found heterogenous and consist of microfilament-rich cells (MFR-cell) and undifferentiated cells (Un-cell). The MFR-cell, which represents approximately 20% of the epithelial cell population, lies between the epithelial layer lining the urinary space and the basement membrane; it extends under several epithelial cells by processes of varying widths and lengths which contact, via desmosomes, other MFR-cells, as well as cells in the superficial layer, i.e., granular and mitochondria-rich cells. The cytoplasm of MFR-cell is filled with intermediate filaments arranged in bundles which run parallel to the plane of the epithelium and no dense granules, typical of granular cells, have been detected. Strong immunofluorescence for actin is associated with cells which occupy the same basal position as MFR-cells. Undifferentiated cells have no contact via desmosomes with adjacent cells and their cytoplasm is filled with free ribosomes; they lack bundles of intermediate filaments and posses no specialized organelles.After a 4-hr pulse of³H-thymidine, 1.5% of epithelial cells incorporate thymidine into nuclear DNA, out of which 3/4 are basally 1/4 are apically located. Identification of cell types by electron microscopy reveals that 10% of undifferentiated basal cells are labeled, whereas less than 0.1% of granular cells and no MFR-cells incorporate³H-thymidine into DNA. When dissociated from the epithelium and separated by isopycnic centrifugation, MFR-cells possess a mean buoyant density of approximately 1.025, cosediment with mitochondria-rich cells and exhibit a strong immunofluorescence for actin. The function of MFR-cells remains unknown; however, they may play a role in cell coupling and responses to hormonal and physical factors.     

The presence of G1 and G2 populations in normal epithelium of rat urinary bladder

The cell population kinetics of transitional epithelium of the rat urinary bladder was analysed by (3H) thymidine autoradiography and pararosanilin Feulgen DNA cytofluorometry. By flash and 72 h continuous DNA labelling, the generative cells of the transitional epithelium were found to be well localized in the basal layer, and it was postulated that che cells produced by cell proliferation in the basal layer would migrate towards the surface, maintaining direct attachment to the basement membrane by anchorage of a cellular process. Analyses of normal and wounded transitional epithelium revealed that 58.8% of all basal cells are G0 cells in G1 phase (G1-population), and 59.0% of the remaining basal cells reside in prolonged (75.1-108.0 h) G2 phase, preserving the ability to divide (G2-population). The cell cycle time of the generative basal cells including the long G2 phase was calculated as 129.1-162 h. All the cells existing in upper layers were found to be also G0 cells in G1 phase, with the DNA amounts of 2C class. No polyploid cells could be detected except for 2C-2C binucleated cells in the superficial layer. The existence of a G2-population may serve for the urgent need of cell incrementation to repair cell loss as the cells in G2 phase can divide without the time-delay needed for DNA synthesis. The rat transitional epithelium, which is composed exclusively of proliferating and potentially proliferative cells, will have much greater capability to repair damage than stratified squamous epithelia.     

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.     

The fine structure of the gall bladder epithelium of the sheep

Summary The electron microscopy of the gall bladder epithelium in the sheep shows that the cells are secretory. They have an extensive Golgi apparatus and sparse endoplasmic reticulum with secretory droplets localised in the apical region and have been referred to the group known as mucoid cells. The intercellular spaces which are elaborately developed in those species whose gall bladder is primarily absorptive are poorly developed. Pinocytosis is not a prominent feature. It is believed that the features noted are correlated with the reported absence of absorption in the ungulate gall bladder.     

Options for histological study of the structure and ultrastructure of human urinary bladder epithelium

The urothelium lines all urinary passages, with exception of the distal portions of the urethra. For the first time the structure of the human bladder was described by Leonardo Da Vinci in 15^th century, however, the exact ultrastructure and function of the bladder’s epithelium have not been fully understood. The aim of our study was to investigate the structure of normal human urinary bladder epithelium with methods of classical histology, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). We obtained biopsies from non-tumor areas from the human urinary bladder of tumor-bearing patients during transurethral resections of these tumours in general or spinal anaesthesia. Totally we investigated biopsies from 20 patients, 16 males and 4 females. The mean age of this group of patients was averaged 66.5 years. The urothelium is comprised of three cell types including polyhedral basal cells, piriform intermediate cells, and superficial umbrella cells. In human urinary bladder epithelium we found a direct connection between intermediate cells and the basement membrane. These thin cytoplasmic projections are detectable not only on slides for light microscopy (semi-thin sections), but also in transmission electron-micrographs. In semi-thin sections we found also direct connections between superficial umbrella cells and basement membrane. These connections we were not able to verify via transmission electron-microscopy. Nevertheless our results show that the human urinary bladder urothelium is a special type of pseudostratified epithelium and each cell has a thin cytoplasmic projection with a direct contact with basement membrane.     

Urinary bladder epithelium antigen induces CD8+ T cell tolerance, activation, and autoimmune response

The effort to explore the specific autoimmune mechanisms of urinary bladder has long been hindered due to a lack of proper animal models. To better elucidate this issue, we developed a novel line of transgenic (Tg) mice, designated as URO-OVA mice, that express the model Ag OVA as a "self"-Ag on the bladder epithelium. URO-OVA mice are naturally tolerant to OVA and show no response to OVA stimulation. Adoptive transfer of naive OVA-specific T cells showed cell proliferation, activation, and infiltration but no bladder histopathology. In contrast, adoptive transfer of activated OVA-specific T cells induced OVA-mediated histological bladder inflammation. Increased mast cells and up-regulated mRNA expressions of TNF-alpha, nerve growth factor, and substance P precursor were also observed in the inflamed bladder. To further facilitate bladder autoimmunity study, we crossbred URO-OVA mice with OVA-specific CD8(+) TCR Tg mice (OT-I mice) to generate a dual Tg line URO-OVA/OT-I mice. The latter mice naturally acquire clonal deletion for autoreactive OT-I CD8(+) T cells (partial deletion in the thymus and severe deletion in the periphery). Despite this clonal deletion, URO-OVA/OT-I mice spontaneously develop autoimmune cystitis at 10 wk of age. Further studies demonstrated that the inflamed bladder contained infiltrating OT-I CD8(+) T cells that had escaped clonal deletion and gained effector functions before developing histological bladder inflammation. Taken together, we demonstrate for the first time that the bladder epithelium actively presents self-Ag to the immune system and induces CD8(+) T cell tolerance, activation, and autoimmune response.     

Mechanisms of spontaneous and induced heteroploidization and polyploidization.

Mechanisms of heteroploidization and polyploidization were studied in tissue cultures, using various methods and cell lines. In untreated populations cells with different chromosome number are formed by mitotic non-disjunction. Mononuclear polyploid cells are formed by repeated DNA synthesis (endoreduplication) and polynuclear giant cells by postmitotic fusion of the daughter cells. These phenomena occur more frequently in cell populations treated with cystostatic drugs. Most polyploid cells are not viable.     

The structure of the gas bladder of the spotted gar,Lepisosteus oculatus

We report here on the macroscopic, light microscopic, and electron microscopic structure of the gas bladder (GB) of the spotted gar, Lepisosteus oculatus. The GB opens into the pharynx, dorsal to the opening of the oesophagus, through a longitudinal slit bordered by two glottal ridges. Caudal to the ridges, the GB is an elongated sac divided into a central duct and right and left lobes. The lobes are formed by a cranio‐caudal sequence of large air spaces that open into the central duct. The structure of the GB is that of a membranous sac supported by a system of septa arising from the walls of a central duct. The septa contain variable amounts of striated and smooth muscle might function to maintain the bladder shape and in providing contractile capabilities. The presence of muscle cells, nerves, and neuroepithelial cells in the wall of the GB strongly suggests that GB function is tightly regulated. The central duct and the apical surface of the thickest septa are covered by mucociliated epithelium. Most of the rest of the inner bladder surface is covered by a respiratory epithelium which contains goblet cells and a single type of pneumocyte. These two cell types produce surfactant. The respiratory barrier contains thick areas with fibrillar material and cell prolongations, and thin areas that only contain basement membrane material between the capillary wall and the respiratory epithelium. Lungs and GBs share many anatomical and histological features. There appears to be no clear criterion for structural distinction between these two types of respiratory organs. J. Morphol. 276:90–101, 2015. © 2014 Wiley Periodicals, Inc.     

Isolation of radio-iodinated apical and basal-lateral plasma membranes of toad bladder epithelium

Summary The apical and basal-lateral plasma membranes of toad bladder epithelium were radio-iodinated with the glucose-glucose oxidase-lactoperoxidase system. The covalently bound radio-iodine was used as a marker during subcellular fractionation and membrane isolation. Homogenization conditions that ensured rupture of more than 80% of the cells without substantial nuclear damage were defined by Nomarski optics. The nuclei were separated by differential centrifugation and the apical and basal-lateral components were resolved by differential and sucrose density gradient centrifugation. The apical components yielded two radioactive bands that were identified as glycocalyx and plasma membrane labeled with¹²⁵I. The basal-lateral components yielded a heterodisperse pattern made up of at least 3 radioactive bands, but the bulk of the activity of ouabain-sensitive ATPase comigrated with only one of these bands. The mitochondia, identified by assays for cytochrome oxidase and NADH cytochromec reductase activities, were separated from the radio-iodine labeled components by centrifugation in sucrose density gradients under isokinetic conditions. The labeled glycocalyx and the slowly migrating components of basal-lateral labeling were separated from the radio-iodinated membranes by centrifugation at 100,000 × g × 1 hr after removal of the mitochondria by the isokinetic method. The labeled membranes were then subjected to ultracentrifugation in sucrose density gradients under isopycnic conditions; the basal-lateral membranes containing ouabain-sensitive ATP-ase were well resolved from the apical membranes by this method. These results provide a relatively rapid method of attaining partial purification of the apical and basal-lateral plasma membranes of toad bladder epithelium.     

Bacterial penetration of bladder epithelium through lipid rafts

Type 1 fimbriated Escherichia coli represents the most common human uropathogen, owing much of its virulence to invasion of the uroepithelium, which is highly impermeable due to the preponderance of uroplakins and highly ordered lipid components. We sought to elucidate the molecular basis for E. coli invasion of the bladder epithelium by employing human 5637 bladder epithelial cells, and we found the following: (i) intracellular E. coli associated with caveolae and lipid raft components; (ii) RNA(i) reduction of caveolin-1 expression inhibited bacterial invasion; (iii) a signaling molecule required for E. coli invasion was located in lipid rafts and physically associated with caveolin-1; (iv) bacterial invasion was inhibited by lipid raft disrupting/usurping agents. In the mouse bladder, the E. coli type 1 fimbrial receptor, uroplakin Ia, was located in lipid rafts, and lipid raft disruptors inhibited E. coli invasion. Cumulatively, E. coli uroepithelial invasion occurs through lipid rafts, which, paradoxically, contribute to bladder impermeability.     

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.     

The conjunctival epithelium in domestic ruminants. II. Electronmicroscopic investigations

The basal cells of all types of epithelia in the domestic ruminants have a smooth surface in their proximal part or are furnished with branched cytoplasmic projections. The cytoplasm of many basal cells contains a few or many micropinocytotic vesicles, which can be arranged in a moniliform manner. The intermediate cells as well as the cells of the stratum superficiale - with the exception of the uppermost layers of the stratified squamous epithelium - are characterized by the presence of a great number of membrane-bound granules. Microvilli which project over the uppermost epithelial cells only occasionally contain a filamentous internal structure. The apical closure of the epithelium is preferentially formed by zonulae occludentes (tight junctions) with maximally 2-3 tight junction seals, they are followed proximally by 1-2 desmosomes. Zonulae adhaerentes can only rarely be observed. The nervous supply of the conjunctival epithelium which is best developed in the bulbusfacing side of the nictitating membrane is brought about by sub- and intraepithelial nerve-endings.     

Rodlet cell distribution in the gall bladder epithelium of Fundulus heteroclitus

Rodlet cells within the epithelial mucosa of the gall bladder of the estuarine killifish Fundulus heteroclitus (L.) obtained from a highly contaminated creek adjacent to a landfill, were arranged within the constraints of the epithelium. Furthermore, the rodlet cells established a close intimate association with electron dense epithelioid cells. A comparison with fish from a non impacted estuary revealed a significantly greater number of rodlet cells in the 'contaminated' group. The abundance of rodlet cells within the gall bladder of the fish exposed to contaminants further strengthens the hypothesis that these cells participate in the fish's immune system.     

12-O-tetradecanoylphorbol-13-acetate induces selective desquamation of superficial cells in rat urinary bladder epithelium

Summary 12-O-tetradecanoylphorbol-13-acetate (TPA) is known to affect the proliferation and/or differentiation of several types of cells. We injected TPA directly into the lumen of rat bladder to determine, using scanning and transmission electron microscopy, its effects on the bladder epithelium in vivo. At 1 h after TPA injection (1g/ml), the superficial cells of the epithelium had changed their morphology, and large spherical vacuoles occupied their cytoplasm. In some areas, the underlying intermediate cells were exposed by the desquamation of the superficial cells. During the next few hours, TPA was excreted from the bladder lumen by voluntary micturition, but the desquamation of the superficial cells proceeded further. All the superficial cells were lost from the luminal surface by 24 h after TPA injection. The changes noted were specific for the superficial cells and were not observed in the intermediate or basal cells. After 24h, part of the epithelium had a three-layer structure, indicating that regeneration was taking place. These results demonstrate that TPA selectively affects and desquamates superficial cells in a short period of time. This experimental system may be useful for studying in vivo cell proliferation and/or differentiation.     

Modal DNA values of normal and malignant urothelial cells of the bladder in relation to nuclear size

In a study of the correlation between mean nuclear size and DNA content in urinary bladder carcinoma, the modal DNA values of cell suspensions from 125 biopsies, obtained from 86 patients with malignant or normal urinary bladder epithelium, were analyzed by flow cytometry (FCM). Light microscopic measurements of nuclear size were carried out on smears from the same material. The results were correlated to the histopathologic stage and grade. The mean nuclear volumes were significantly larger in diploid tumor cells than in cells of normal epithelium. Aneuploid tumors showed significantly larger nuclei than did diploid tumors. Although there was a significant correlation between increases in the nuclear volume and in the DNA content, there was some overlapping between various grades of malignancy: mean nuclear volumes in aneuploid grade 2 tumors did not differ from those in aneuploid grade 3 tumors. A combination of FCM and morphometry discriminated all but 16% of the tumors from the normal cases. It is concluded that FCM and morphometry are complementary and can be used for the objective characterization of urinary bladder carcinomas.     

An epidermal proliferative unit-like structure in the epithelium of mouse bladder observed by backscattered electron imaging

Summary The simultaneous use of a silver-staining technique, backscattered electron imaging and stereo-tilts has made it possible to visualize the spatial distribution of cell nuclei in the stretched epithelium of the bladder of mice. This study has led to the observation that a structural organization resembling the epidermal proliferative unit, previously found in the skin exists also in bladder epithelium. However, the proliferative unit in the bladder was different in that it contained a higher number of cells per unit, and an absence of columns of inactive squamous cells. These findings may indicate that epidermal proliferative unit-like structures represent a common form of organization in some epithelia.     

Cell kinetics of mouse urinary bladder epithelium. V. Changes in the proportion of cells with various nuclear DNA content after repeated doses of cyclophosphamide.

A dose of 2 mg cyclophosphamide (Sendoxan, "Pharamcia", Sweden) dissolved in 0.2 ml distilled water was injected intraperitoneally once a week to 12 hairless mice for three months. Four animals were killed at 1, 2 and 3 months and micro-flow fluormetric histograms of the bladder epithelial cells were made. The proportion of cells in diploid S phase remained normal at 1 and 2 months, but increased at 3 months. The proportion of tetraploid S-phase cells fell rapidly and markedly and there were almost no cells in this phase at 1, 2 and 3 months. The proportion of diploid cells increased, the proportion of tetraploids was significantly reduced and the octoploid cells disappeared after 2 months. The changes are similar to those seen after repeated injections of the bladder carcinogen dibutylnitrosamine, but less pronounced. Since cyclophosphamide is a strong alkylating agent it is possible that, in the doses used, it is also a weak carcinogen for hte bladder epithelium. This must be tested in direct, long-term treatment experiments. Bladder cancers in humans receiving cyclophosphamide therapy have been described.     

Genetic factors influencing bystander signaling in murine bladder epithelium after low-dose irradiation in vivo

Radiation-induced bystander effects occur in cells that are not directly hit by radiation tracks but that receive signals from hit cells. They are well-documented in vitro consequences of low-dose exposure, but their relevance to in vivo radiobiology is not established. To investigate the in vivo production of bystander signals, bladder explants were established from two strains of mice known to differ significantly in both short-term and long-term radiation responses. These were investigated for the ability of 0.5 Gy total-body irradiation in vivo to induce production of bystander signals in bladder epithelium. The studies demonstrate that irradiated C57BL/6 mice, but not CBA/Ca mice, produce bystander signals that induce apoptosis and reduce clonogenic survival in reporter HPV-G-transfected keratinocytes. Transfer of medium from explants established from irradiated animals to explants established from unirradiated animals confirmed these differences in bladder epithelium. The responses to the in vivo-generated bystander signal exhibit genotypic differences in calcium signaling and also in signaling pathways indicative of a major role for the balance of pro-apoptosis and anti-apoptosis proteins in determining the overall response. The results clearly demonstrate the in vivo induction of bystander signals that are strongly influenced by genetic factors and have implications for radiation protection, medical imaging, and radiotherapy.     

Eosinophil peroxidase increases membrane permeability in mammalian urinary bladder epithelium

Eosinophilperoxidase (EPO), a cationic protein found in eosinophils, has beenreported to be cytotoxic independent of its peroxidase activity. Thisstudy investigated with electrophysiological methods whether EPO istoxic to mammalian urinary bladder epithelium. Results indicate thatEPO, when added to the mucosal solution, increases apical membraneconductance of urinary bladder epithelium only when the apical membranepotential is cell interior negative. The EPO-induced conductance wasconcentration dependent, with a maximumconductance of 411 µS/cm² and aMichaelis-Menten constant of 113 nM. The EPO-induced conductance wasnonselective for K⁺ andCl. The conductance waspartially reversed using voltage but not by removal of EPO from thebulk solution. Mucosal Ca²⁺reversed the EPO-induced conductance by a mechanism involving reversible block of the conductance. Prolonged exposure (up to 1 h) toEPO was toxic to the urinary bladder epithelium, as indicated by anirreversible increase in transepithelial conductance. These resultssuggest that EPO is indeed toxic to urinary bladder epithelium via amechanism that involves an increase in membrane permeability.     

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.