Urothelium-specific antibody and lectin surface mapping of bladder urothelium

Summary Coupled ligand-colloidal gold complexes were found to provide a convenient approach for the localization by scanning electron microscopy of cell surface membrane antigens and lectin-binding sites on bladder urothelium and for the immunocytochemical identification of urothelial cell populations at different stages of differentiation. The ligands used to probe the membrane were a urothelium-specific rabbit antibody raised to a urothelial membrane-associated antigen (UMA), and two lectins: Concanavalin A (Con A) and peanut agglutinin (PNA). A complex luminal surface distribution pattern was demonstrated by the UMA antigen related to the stage of urothelial cell maturation and differentiation. UMA could be detected on the surface of immature and early differentiating intermediate cells, but was absent from the late differentiation stage, becoming re-expressed as the cells matured and was found in greatest abundance on the terminally differentiated superficial cells. It was absent on cells in benign hyperplasia of the urothelium. Cellular and regional differences in lectin binding to the urothelial cell surface was suggested with Con A receptors localized uniformly over the superficial cells, and PNA receptors confined to linear arrays or occasional clusters over the apical surface but evenly dispersed over the lateral surface of these cells.     

Cell cycle of normal bladder urothelium in developing and adult mice

The present research has employed a novel, nonradioactive technique to quantitatively study normal urothelial proliferation in foetal, neonatal, juvenile and adult mouse bladder. Using whole mount histological preparations, the total number of urothelial nuclei per mouse bladder, and per given urothelial cell layer, have been assessed to provide data of the (unstimulated) kinetic behaviour of basal urothelial cells (the proliferative population), to analyse characteristics of the normal urothelial cell cycle. The urothelial cell cycle time increases from 30.6 h (foetal) to 40 weeks (adult), the duration of mitosis from 0.23 h (foetal) to 2.71 h (adult) and the duration of DNA synthesis from 2.52 h (neonatal) to 10.83 h (adult). These are average values for the urothelial cell cycle, which do not preclude the possible existence of proliferative units. The ratio of superficial nuclei to basal and intermediate nuclei, possibly indicative of a urothelial proliferative unit, declines to reach a plateau (1:40) in adult mice. These findings indicate that rapid urothelial proliferation during early murine development was likely to be a) biologically useful, since intrauterine foetal metabolic activity may require a functional bladder urothelium at an early stage, b) kinetically similar to acutely regenerating adult urothelial cells after cytotoxic insult. During murine life, the range of durations of mitosis and DNA synthesis is much less than the range of cell cycle times. Normal unstimulated urothelium of adult mice was confirmed to proliferate slowly.     

Ultrastructure of cell surface coat (glycocalyx) in rat urinary bladder epithelium

Earlier statements to the contrary, the present study demonstrates the presence of a cell surface coat (glycocalyx) on the luminal plasma membrane of the superficial transitional epithelial cells lining the urinary bladder of male Buffalo rats. This coat was demonstrated with ruthenium red, an electron dense stain, which revealed a surface layer, 60-80 A thick, separated from the outer leaflet of the plasma membrane by an electron lucent layer, approximately 30 A thick. The structure of the glycocalyx was not affected by 12 weeks of treatment with dibutylnitrosamine, a known bladder carcinogen.     

Differentiation-dependent localisation of tissue-type plasminogen activator in human bladder urothelium

Human bladder urothelium is able to secrete tissue-type plasminogen activator (tPA). The aim of our study was to analyse localisation of tPA antigen in comparison to differentiation state of cells in samples of histologically normal urothelium and non-invasive tumours of the human urinary bladder. Twenty-five samples of normal urothelium and 31 non-invasive papillary tumours from 36 patients were examined. The presence of tPA antigen was evaluated immunohistochemically. Differentiation of superficial cells was assessed by the presence of urothelial cell differentiation markers, uroplakins (UPs; immunohistochemistry) and cell's apical surface architecture (scanning electron microscopy). All tissue samples stained anti-tPA positive. In normal urothelium, the intensity of anti-tPA staining was the strongest in superficial cells, which were well-differentiated. In tumours, all cell layers stained anti-tPA positive. The intensity of anti-tPA positive reaction in the upper cell layer correlated with the percentage of anti-UP positive superficial cells. Superficial cells showed various differentiation states. The localisation of tPA antigen in human in vivo tissue is not confined to the well-differentiated superficial cells. Our results suggest a positive correlation between tPA secretion and cell differentiation.     

Non-neuronal acetylcholine and urinary bladder urothelium

Non-neuronal release of acetylcholine (ACh) has been proposed to play a role in urinary bladder function. These studies investigated the expression and function of the non-neuronal cholinergic system in cultured urothelial cells isolated from the rat urinary bladder. Our findings have revealed that urothelial cells express the high-affinity choline transporter (CHT1) and acetylcholine-synthesizing enzymes, choline acetyltransferase (ChAT) and carnitine acetyltransferase (CarAT). In contrast to neurons, urothelial cells do not express the vesicular acetylcholine transporter (VAChT) but do express OCT3, a subtype of polyspecific organic cation transporter (OCT) that is thought to be involved in the release of acetylcholine from non-neuronal cells. Following exposure of cultured urothelial cells to (3)H-choline, radioactivity was detected in the cells and increased release of radioactivity into the eternal media was evoked by mechanical stimulation (exposure of the cells to 50% hypotonic Krebs) or chemical stimulation of purinergic receptors by 100 muM ATP. The present experiments did not establish if the evoked release of radioactivity (termed (3)H-ACh release in this paper) was due to release of acetylcholine or choline. (3)H-ACh release was not evoked by application of acetylcholine alone, however pretreatment with the non-selective muscarinic receptor antagonist atropine prior to application of acetylcholine facilitated (3)H-ACh release, suggesting that the acetylcholine released from urothelial cells may participate in a negative feedback mechanism by acting on muscarinic receptors to inhibit its own release in the urothelium. Brefeldin, an agent which disrupts vesicular exocytosis, did not block hypotonic-evoked (3)H-ACh release. These observations indicate that acetylcholine release from urothelial cells is mediated by different mechanisms than those such as vesicular storage and exocytosis that underlie the release of neurotransmitters from nerves.     

Enhanced ATP release from rat bladder urothelium during chronic bladder inflammation: effect of botulinum toxin A

The effects of mechanoreceptor stimulation and subsequent ATP release in cyclophosphamide evoked chronic bladder inflammation was examined to demonstrate: (1) whether inflammation modulates ATP release from bladder urothelium and (2) whether intravesical botulinum toxin A administration inhibits urothelial ATP release, a measure of sensory nerve activation. ATP release was measured from rat bladders in a Ussing chamber, an apparatus that allows one to separately measure resting and mechanoreceptor evoked (e.g. hypoosmotic stimulation) ATP release from urothelial and serosal sides of the bladder. Cystometry was utilized to correlate changes in ATP release with alterations in the frequency of voiding and non-voiding bladder contractions, in vivo measures of bladder afferent activity. The resting urothelial release of ATP was not significantly affected by either cyclophosphamide or botulinum toxin A treatment. However, evoked ATP release following hypoosmotic stimulation was significantly increased (i.e. 94%) in chronic cyclophosphamide treated bladder urothelium compared to control bladders. In addition, botulinum toxin A treatment significantly reduced hypoosmotic shock induced ATP release in cyclophosphamide treated animals by 69%. Cystometry revealed that cyclophosphamide and botulinum toxin A treatments altered non-voiding (i.e. cyclophosphamide increased, botulinum toxin A decreased) but not voiding contraction frequency suggesting that alterations in urothelial ATP release selectively diminished underlying bladder C-fiber nerve activity. Finally, intravesical instillation of botulinum toxin A did not affect ATP release from the serosal side implying that its effects were confined to the urothelial side of the bladder preparation.     

Selective binding of lectins to normal and neoplastic urothelium in rat and mouse bladder carcinogenesis models

Bladder cancer adjuvant intravesical therapy could be optimized by more selective targeting of neoplastic tissue via specific binding of lectins to plasma membrane carbohydrates. Our aim was to establish rat and mouse models of bladder carcinogenesis to investigate in vivo and ex vivo binding of selected lectins to the luminal surface of normal and neoplastic urothelium. Male rats and mice were treated with 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water and used for ex vivo and in vivo lectin binding experiments. Urinary bladder samples were also used for paraffin embedding, scanning electron microscopy and immunofluorescence labelling of uroplakins. During carcinogenesis, the structure of the urinary bladder luminal surface changed from microridges to microvilli and ropy ridges and the expression of urothelial-specific glycoproteins uroplakins was decreased. Ex vivo and in vivo lectin binding experiments gave comparable results. Jacalin (lectin from Artocarpus integrifolia) exhibited the highest selectivity for neoplastic compared to normal urothelium of rats and mice. The binding of lectin from Amaranthus caudatus decreased in rat model and increased in mouse carcinogenesis model, indicating interspecies variations of plasma membrane glycosylation. Lectin from Datura stramonium showed higher affinity for neoplastic urothelium compared to the normal in rat and mouse model. The BBN-induced animal models of bladder carcinogenesis offer a promising approach for lectin binding experiments and further lectin-mediated targeted drug delivery research. Moreover, in vivo lectin binding experiments are comparable to ex vivo experiments, which should be considered when planning and optimizing future research.     

Postnatal restoration of the mouse urinary bladder urothelium

Mouse urothelium is disrupted just before birth, followed by a postnatal restoration process which includes cell proliferation, death and differentiation. We assessed urothelial proliferation by the expression of proliferating cell nuclear antigen (PCNA), desquamation by electron microscopy, and apoptosis by TUNEL staining and urothelial differentiation by the expression of uroplakins and cytokeratin 20 (CK20) as well as the apical plasma membrane maturation. Our results indicated that urothelial proliferation was high from birth until about the 14th postnatal day. A majority of basal cells and even occasional superficial cells were PCNA positive during the first 5 postnatal days. Cell death occurred during the first 9 postnatal days. Between birth and day 5, single cells underwent apoptosis, whereas between days 6 and 9 cells mainly desquamated. CK20 and uroplakins were expressed in all superficial cells in postnatal urothelium. Their subcellular distribution characteristically changed in accordance with the progressive differentiation of superficial cells. During the urothelial postnatal development, proliferation activity slowly decreases to the proliferatively quiescent urothelium of the adult animal. Apoptosis is present in the first 9 postnatal days and within a few days of this period it appears simultaneously with desquamation. Superficial urothelial cells gradually differentiate, which is reflected in the changeable morphology of the apical plasma membrane.     

Cell surface proteins of rat myoblasts

During the course of cell fusion of a rat myoblast cell line, L6, the relative amount of a cell surface protein of molecular weight (MW) 200 000-250 000 (L200), detected on the two-dimensional gel electrophoretogram, increases to one of a few major cell surface proteins in the older culture in which myotubes are being formed. In the non-fusing variant (M3A) of L6, such a spot is not detectable. Instead, one protein of MW 90 000-100 000 (M100) is found in large quantities in M3A, but in a small amount in L6. These results suggest the possibility that these cell surface proteins, particularly L200, may play a key role in the development of muscle cells.     

Use of lymphocytic DNA by the urothelium of bladder tumors

In 35 rats tumors of the urinary bladder were induced by nitrosomethylurea. A rubber loop was established on the neck of the urinary bladder and tightened for 1.5 h. This allowed one to completely isolate the tumor from the circulation. Shortly after the tightening of the loop 3H-thymidine was injected intraperitoneally. The rats were sacrificed at different times after the loop was removed. The tumors were examined by histoautoradiography. Lymphocytes were the first labeled cells that appeared in the stroma of the tumors 9 h after operation. Following 36 hours the radioisotope was detectable in the epitheliocytes. That meant that the label was reutilized by the tumor urothelium. It was shown that the only donor of the isotope could be a live labeled lymphocyte transmitting its own DNA in the course of a direct contact with the tumor cell. That was a manifestation of the trophic function of lymphocytes. It is concluded that lymphocytes support the proliferative activity of the tumorous tissue.     

Cell surface human alpha-L-fucosidase.

The acid alpha-L-fucosidase is usually found as a soluble component of lysosomes where fucoglycoconjugates are degraded. In the present investigation, we have demonstrated the existence of a cell surface protein with enzymatic alpha-L-fucosidase activity that crossreacts specifically with a rabbit anti-(alpha-L-fucosidase) Ig. By different approaches, this alpha-L-fucosidase, which represents 10-20% of the total cellular fucosidase activity, was detected in all the tested human cells (hemopoietic, epithelial, mesenchymal). Two bands of approximately 43-49 kDa were observed, although theoretical data support the possibility of having the same genetic origin that the known 50 to 55-kDa Mr alpha-L-fucosidase. We speculate about an alternative traffic pathway for the plasma membrane alpha-L-fucosidase to work on the rapid turnover of glycoproteins.     

Demonstration of the fuzzy surface coat of rat intestinal microvilli by freeze-etching

In freeze-etch replicas of epithelial cells of rat rectum, the fuzzy surface coat is composed of discrete filaments which are aligned in parallel with each other and with the long axes of the microvilli.     

Epithelial cells transdifferentiation into bladder urothelium in experiments in vivo

Atoplastic surgery using intestinal tissue has been used for the reconstructive therapy of the urinary tract since the mid-20th century; however, cell mechanisms of the urothelium engraftment are still unclear. Intestinal stem cells possess plasticity and, after autoplastic surgery, are presumably able to transdifferentiate into mature cells of the urinary tract. Using the preliminarily developed model for evaluating of the transdifferentiaion of somatic cells into urothelium in vivo, we found that, in syngeneic C57BL mice, epithelial Gfp-producing intestinal cells transdifferentiate into the cryoinjured bladder urothelium. Gfp was detected in the bladder tissue of recipient mice using reverse polymerase chain reaction, fluorescence and immunofluorescence. Colocalization of Her-4 protein revealed by common urothelium expression pattern and Gfp was demonstrated in few urothelial cells by double immunohistochemical staining of the bladder tissue with specific antibodies. The results obtained suggest that epithelial intestinal cells are able to transdifferentiate into bladder urothelium; however, the level of transdifferentiation is low and, presumably, cannot ensure the full functional urothelium engraftment in the case of autoplastic bladder surgery using intestinal tissue.     

Trypanostatic activity of rat IgG purified from the surface coat of Trypanosoma lewisi

Host IgG is a component of the surface coat of Trypanosoma lewisi; it is specifically acquired during infection in the rat, concomitant with a rise in titer of trypanostatic (ablastic) activity of host serum. Host IgG was eluted from trypomastigotes at 7 to 9 days postinfection with a high salt-low pH buffer. Surface coats and trypanosome ultrastructure were not notably altered by the elution procedure, as determined by electron microscopy. Rat IgG was removed and purified from the trypanosome eluates on an immunoadsorbent column made with the IgG fraction of anti-rat IgG serum coupled to Sepharose beads. Concentrated column eluates, by comparison with a standard, were shown to be rat IgG by immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. As a control, IgG from normal rat serum was purified by the same technique. IgG-negative trypanosomes harvested from immunosuppressed rats bound IgG purified from surface coats of trypanosomes, but not IgG purified from normal rat serum, as demonstrated by subsequent labelling with FITC-conjugated, rabbit anti-rat IgG. The IgG purified from surface coats inhibited the reproduction of T. lewisi in an in vitro assay, but purified, normal IgG did not. These data show that antigen-specific host IgG, adsorbed to the surface of T. lewisi, is ablastic antibody.     

Cell surface antigens on rat islet tumors

To examine the antigenic properties of the rat pancreatic beta cell tumor, RIN, we used a cell surface enzyme-linked immunosorbent assay (CELISA). Monoclonal antibodies of known specificity are used to validate the assay, and the results show that antibodies directed at glycoproteins and glycolipids are detected by this technique. The principal glycolipid targets are found in the ganglio and lacto series of glycosphingolipids, and not in the globo series. When the CELISA was used to study sera from type I diabetics, no differences in binding of control and diabetic samples were detected at low dilutions of serum. However, at higher serum dilutions (1/30 to 1/120) binding of IgG antibodies from type I diabetics was greater than that observed with normal sera. Similar reactivity was not detected in these sera when rat hepatocytes were used as targets in the CELISA. Prolonged culture of RIN cells, however, resulted in the loss of reactivity with sera from type I subjects, and is associated with a corresponding decrease in expression of cell surface gangliosides. Accordingly, subclones selected for ganglioside expression were used to compare anti-RIN binding in type I diabetics with that of normal controls and unaffected siblings. The results indicate that some rat islet tumors express antigenic determinants recognized by anti-islet antibodies associated with type I diabetes. Both nonspecific interaction at low serum dilutions and variable expression of cell surface antigens may explain the difficulties encountered when these cells are used for diagnostic purposes. An objective assay such as the CELISA may help to avoid these problems.     

Temporal and spatial dimensions of postnatal growth of the mouse urinary bladder urothelium

Postnatal growth and renewal of mouse urothelium start on the day of birth. In the present study, temporal and spatial dimensions of urothelial growth were studied during the first two postnatal weeks. Quantitative analysis showed that the rate of urothelial cell proliferation is significantly higher during all 14 postnatal days than in adult mice. Three peaks of proliferative and mitotic activity were revealed: on the day of birth and postnatal day 1, on days 6 and 7, and on day 14. The high proliferation rate around the day of birth and at postnatal days 6 and 7 coincides with cell death in the urothelium. Semiquantitative analysis showed that during all 14 postnatal days, the urothelial proliferative response is mostly confined to the basal cell layer. Urothelial cells divide predominantly in parallel to the plain of the urothelium on all chosen postnatal days. Increased portions of urothelial cells, dividing perpendicularly to the urothelium were observed only on the day of birth and on postnatal day 7. Our results suggest that postnatal growth of mouse urothelium is particularly the result of an increasing number of cells in individual cell layers and not the result of an increasing number of cell layers.