Mammalian Urinary Bladder Research Articles

Identifying unique subtypes of spinal afferent nerve endings within the urinary bladder of mice

Spinal afferent neurons are responsible for the transduction and transmission of noxious (painful) stimuli and innocuous stimuli that do not reach conscious sensations from visceral organs to the central nervous system. Although the location of the nerve cell bodies of spinal afferents is well known to reside in dorsal root ganglia (DRG), the morphology and location of peripheral nerve endings of spinal afferents that transduce sensory stimuli into action potentials is poorly understood. The individual nerve endings of spinal afferents that innervate the urinary bladder have never been unequivocally identified in any species. We used an anterograde tracing technique developed in our laboratory to selectively label only spinal afferents. Mice were anesthetized and unilateral injections of dextran-amine made into lumbosacral DRGs (L5-S2). Seven to nine days postsurgery, mice were euthanized, the urinary bladder removed, then fresh-fixed and stained for immunoreactivity to calcitonin-gene-related-peptide (CGRP). Four distinct morphological types of spinal afferent ending in the bladder were identified. Three types existed in the detrusor muscle and one major type in the sub-urothelium and urothelium. Most nerve endings were located in detrusor muscle where the three types could be identified as having: "branching", "simple", or "complex" morphology. The majority of spinal afferent nerve endings were CGRP-immunoreactive. Single spinal afferent axons bifurcated many times upon entering the bladder and developed varicosities along their axon terminal endings. We present the first morphological identification of spinal afferent nerve endings in the mammalian urinary bladder.

The influence of intravesical administration of resiniferatoxin (RTX) on the chemical coding of sympathetic chain ganglia (SChG) neurons supplying the porcine urinary bladder.

Resiniferatoxin (RTX) is used as an experimental drug in therapy of neurogenic urinary bladder disorders. The present study investigated the chemical coding of sympathetic chain ganglia (SChG) neurons supplying porcine urinary bladder after intravesical RTX instillation. The SChG neurons were visualized with retrograde tracing method and their chemical profile was disclosed with double-labeling immunohistochemistry using antibodies against dopamine β-hydroxylase (DβH; marker of noradrenergic neurons), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin, Leu(5)-enkephalin and neuronal nitric oxide synthase (nNOS). It was found that in both the control (n = 5) and RTX-treated pigs (n = 5), the vast majority (90.4 ± 2.8 and 89.7 ± 2.3%, respectively) of FB-positive (FB+) nerve cells were DβH+. RTX instillation caused a decrease in the number of FB+/DβH+ neurons immunopositive to NPY (71.1 ± 12.1 vs 43.2 ± 6.7%), VIP (21.3 ± 10.7 vs 5.3 ± 4.3%) or SOM (16.5 ± 4.6 vs 2.3 ± 2.6%) and a distinct increase in the number of FB+/DβH+ neurons immunoreactive to nNOS (0.8 ± 1 vs 5.3 ± 1.9 %). The present study for the first time has provided some information that therapeutic effects of RTX on the mammalian urinary bladder can be partly mediated by SChG neurons.

Changes in chemical coding of sympathetic chain ganglia (SChG) neurons supplying porcine urinary bladder after botulinum toxin (BTX) treatment.

Botulinum toxin (BTX) is a neurotoxin used in medicine as an effective drug in experimental therapy of neurogenic urinary bladder disorders. We have investigated the influence of BTX on the chemical coding of sympathetic chain ganglia (SChG) neurons supplying the porcine urinary bladder. The toxin was injected into the wall of the bladder. SChG neurons were visualized by a retrograde tracing method with fluorescent tracer fast blue (FB) and their chemical coding was investigated by double-labelling immunohistochemistry with antibodies against dopamine β-hydroxylase (DβH; a marker of noradrenergic neurons), neuropeptide Y (NPY), vasoactive intestinal polypeptide (VIP), somatostatin (SOM), galanin (GAL), Leu5-enkephalin (L-ENK) and neuronal nitric oxide synthase (nNOS). In both the control (n = 5) and BTX-treated pigs (n = 5), the vast majority (91 ± 2.3 % and 89.8 ± 2.5 %, respectively) of FB-positive (FB+) nerve cells were DβH+. BTX injections caused a decrease in the number of FB+/DβH+ neurons that were immunopositive to NPY (39.5 ± 4.5 % vs 74.5 ± 11.9 %), VIP (8.9 ± 5.3 % vs 22.3 ± 8.8 %), SOM (5.8 ± 2.3 % vs 17.4 ± 3.7 %) or GAL (0.9 ± 1.2 % vs 5.4 ± 4.4 %) and a distinct increase in the number of FB+/DβH+ neurons that were immunoreactive to L-ENK (3.7 ± 2.9 % vs 1.1 % ± 0.8 %) or nNOS (7.7 ± 3.5 % vs 0.8 ± 0.6 %). Our study provides novel evidence that the therapeutic effects of BTX on the mammalian urinary bladder are partly mediated by SChG neurons.

Active urea transport in lower vertebrates and mammals.

Some unicellular organisms can take up urea from the surrounding fluids by an uphill pumping mechanism. Several active (energy-dependent) urea transporters (AUTs) have been cloned in these organisms. Functional studies show that active urea transport also occurs in elasmobranchs, amphibians, and mammals. In the two former groups, active urea transport may serve to conserve urea in body fluids in order to balance external high ambient osmolarity or prevent desiccation. In mammals, active urea transport may be associated with the need to either store and/or reuse nitrogen in the case of low nitrogen supply, or to excrete nitrogen efficiently in the case of excess nitrogen intake. There are probably two different families of AUTs, one with a high capacity able to establish only a relatively modest transepithelial concentration difference (renal tubule of some frogs, pars recta of the mammalian kidney, early inner medullary collecting duct in some mammals eating protein-poor diets) and others with a low capacity but able to maintain a high transepithelial concentration difference that has been created by another mechanism or in another organ (elasmobranch gills, ventral skin of some toads, and maybe mammalian urinary bladder). Functional characterization of these transporters shows that some are coupled to sodium (symports or antiports) while others are sodium-independent. In humans, only one genetic anomaly, with a mild phenotype (familial azotemia), is suspected to concern one of these transporters. In spite of abundant functional evidence for such transporters in higher organisms, none have been molecularly identified yet.

Abstract SY05-02: Hedgehog/wnt feedback supports regenerative proliferation of epithelial stem cells in bladder

Abstract Epithelial integrity in metazoan organs is maintained through the regulated proliferation and differentiation of organ-specific stem and progenitor cells. Although the epithelia of organs such as the intestine regenerate constantly and thus remain continuously proliferative, other organs, such as the mammalian urinary bladder, shift from near-quiescence to a highly proliferative state in response to epithelial injury caused by bacterial infection or chemical agents. The cellular and molecular mechanisms underlying this injury-induced mode of regenerative response are poorly defined. Here we show that proliferation within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. We demonstrate that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal, Sonic hedgehog (Shh). Upon injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Our findings provide a model for injury-induced epithelial regeneration in endodermal organs, and may provide a basis for understanding the roles of signaling pathways in cancer growth and metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY05-02. doi:10.1158/1538-7445.AM2011-SY05-02

Expression and Distribution of Ectonucleotidases in Mouse Urinary Bladder

BackgroundNormal urinary bladder function requires bidirectional molecular communication between urothelium, detrusor smooth muscle and sensory neurons and one of the key mediators involved in this intercellular signaling is ATP. Ectonucleotidases dephosphorylate nucleotides and thus regulate ligand exposure to P2X and P2Y purinergic receptors. Little is known about the role of these enzymes in mammalian bladder despite substantial literature linking bladder diseases to aberrant purinergic signaling. We therefore examined the expression and distribution of ectonucleotidases in the mouse bladder since mice offer the advantage of straightforward genetic modification for future studies.Principal FindingsRT-PCR demonstrated that eight members of the ectonucleoside triphosphate diphosphohydrolase (NTPD) family, as well as 5′-nucleotidase (NT5E) are expressed in mouse bladder. NTPD1, NTPD2, NTPD3, NTPD8 and NT5E all catalyze extracellular nucleotide dephosphorylation and in concert achieve stepwise conversion of extracellular ATP to adenosine. Immunofluorescent localization with confocal microscopy revealed NTPD1 in endothelium of blood vessels in the lamina propria and in detrusor smooth muscle cells, while NTPD2 was expressed in cells localized to a region of the lamina propria adjacent to detrusor and surrounding muscle bundles in the detrusor. NTPD3 was urothelial-specific, occurring on membranes of intermediate and basal epithelial cells but did not appear to be present in umbrella cells. Immunoblotting confirmed NTPD8 protein in bladder and immunofluorescence suggested a primary localization to the urothelium. NT5E was present exclusively in detrusor smooth muscle in a pattern complementary with that of NTPD1 suggesting a mechanism for providing adenosine to P1 receptors on the surface of myocytes.ConclusionsEctonucleotidases exhibit highly cell-specific expression patterns in bladder and therefore likely act in a coordinated manner to regulate ligand availability to purinergic receptors. This is the first study to determine the expression and location of ectonucleotidases within the mammalian urinary bladder.

Hedgehog/Wnt feedback supports regenerative proliferation of epithelial stem cells in bladder.

Epithelial integrity in metazoan organs is maintained through the regulated proliferation and differentiation of organ-specific stem and progenitor cells. Although the epithelia of organs such as the intestine regenerate constantly and thus remain continuously proliferative, other organs, such as the mammalian urinary bladder, shift from near-quiescence to a highly proliferative state in response to epithelial injury. The cellular and molecular mechanisms underlying this injury-induced mode of regenerative response are poorly defined. Here we show in mice that the proliferative response to bacterial infection or chemical injury within the bladder is regulated by signal feedback between basal cells of the urothelium and the stromal cells that underlie them. We demonstrate that these basal cells include stem cells capable of regenerating all cell types within the urothelium, and are marked by expression of the secreted protein signal Sonic hedgehog (Shh). On injury, Shh expression in these basal cells increases and elicits increased stromal expression of Wnt protein signals, which in turn stimulate the proliferation of both urothelial and stromal cells. The heightened activity of this signal feedback circuit and the associated increase in cell proliferation appear to be required for restoration of urothelial function and, in the case of bacterial injury, may help clear and prevent further spread of infection. Our findings provide a conceptual framework for injury-induced epithelial regeneration in endodermal organs, and may provide a basis for understanding the roles of signalling pathways in cancer growth and metastasis.

Interstitial cells in the urinary bladder—localization and function

This review summarizes the currently available literature on the localization and proposed functions of a novel group of cells in the urinary bladder known as interstitial cells or interstitial cells of Cajal (ICC). On-line searches of "Pubmed" for bladder, c-Kit, ICC, interstitial cell and myofibroblast were performed to identify relevant studies for the review. The literature contains substantial data that several sub-populations of ICC are present in the wall of the mammalian urinary bladder. These are located in the lamina propria and within the detrusor with distinctive cell shapes and morphological arrangements. Bladder ICC are identified with transmission electron microscopy or by immunohistochemical labeling using antibodies to the Kit receptor which is an established ICC marker. Lamina propria-ICC form a loose network connected via Cx43 gap junctions and are associated with mucosal nerves. Detrusor ICC track the smooth muscle bundles and make frequent contacts with intramural nerves. Both groups of ICC exhibit spontaneous electrical and Ca2+-signalling and also respond to application of neurotransmitter substances including ATP and carbachol. There is emerging evidence that the expression of ICC is upregulated in pathophysiological conditions including the overactive bladder. There is now a convincing body of evidence that specialized ICC are present in the urinary bladder making important associations with other cells that make up the bladder wall and possessing physiological properties consistent with a role of bladder activity modulation.

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Absorption through the bladder wall is dependent on 2 variables, the molecular weight of the agent, and the integrity of the asymmetric unit membrane (AUM) of the epithelial lining. 1 Waltz T. Haner M. Wu X.R. et al. Towards the molecular architecture of the asymmetric unit membrane of the mammalian urinary bladder epithelium: a closed “twisted ribbon” structure. J Mol Biol. 1995; 248: 887-900 Crossref PubMed Scopus (93) Google Scholar With regard to gemcitabine, what we observe in systemic toxicity, namely, the side effects referred to in the above comment, is dependent also on the expression of cytidine deaminase (CDA), which is known to be affected by variation. In previous studies, we observed occasional systemic toxicity also with the drug administered to intact bladders, 2 Maffezzini M. Campodonico F. Canepa G. et al. Short-schedule intravesical gemcitabine with ablative intent in recurrent Ta-T1, G1-2, low- or intermediate Risk transitional cell carcinoma of the bladder. Eur Urol. 2007; 51: 956-961 Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar and enhancement of absorption through tumor tissue, as compared with normal urothelium, was observed by others. 3 Paroni R. Salonia A. Lev A. et al. Effect of local hyperthermia to the bladder on mitomycin C pharmacokinetics during intravesical chemotherapy for the treatment of superficial transitional cell carcinoma. Br J Clin Pharmacol. 2001; 52: 273-278 Crossref PubMed Scopus (74) Google Scholar Systemic Absorption and Pharmacokinetics of Single-dose Intravesical Gemcitabine After Transurethral Resection of the Bladder in Non-muscle-invasive Bladder CancerUrologyVol. 74Issue 5PreviewTo evaluate the systemic quantitative absorption, pharmacokinetics, and toxicities of gemcitabine administered intravesically at the recommended, high-concentration dose of 40 mg/mL, immediately after transurethral resection. Full-Text PDF Editorial CommentUrologyVol. 74Issue 5PreviewThe authors demonstrate the limited absorption of gemcitabine in the post-TURB setting, and confirm that it is safe to use not only immediately after TURB, but also after a large resection. The toxicities of gemcitabine are dose-dependent and are primarily related to marrow suppression, with myelosuppression, anemia, and thrombocytopenia being the most common. At the low systemic doses absorbed and with rapid clearance, none of the patients studied reached an exposure near intravenous administration levels. Full-Text PDF

Urine Derived Cells are a Potential Source for Urological Tissue Reconstruction

Contemporary approaches to tissue engineering and cell therapy for urinary tract reconstruction require invasive tissue biopsies to obtain autologous cells. However, these procedures are associated with potential complications. We determined whether the cells present in urine have characteristics of normal bladder cells and investigated their potential uses for urological reconstructive procedures. A total of 55 urine samples were collected from 15 healthy individuals and 8 patients with vesicoureteral reflux. Urine derived cells were isolated, expanded and tested for progenitor and differentiated cell specific markers using flow cytometry, immunofluorescence and Western immunoblotting. The chromosomal stability of cultured urine derived cells was determined by karyotype analysis. Clones were successfully established from primary cultures of urine derived cells. Isolated cells showed 3 phenotypes, including fully differentiated, differentiating and progenitor-like cells. Some urine derived cells stained positive for the surface markers c-Kit, SSEA4, CD105, CD73, CD91, CD133 and CD44. Two to 7 cells per 100 ml urine were multipoint progenitors that could expand extensively in culture. Single progenitor cells had the ability to differentiate into the cell lineages expressing urothelial, smooth muscle, endothelial and interstitial cell markers. The expression of lineage markers was characterized by Western blot and immunofluorescence analysis. Urine derived cells also maintained a normal karyotype after serial culture. A subpopulation of cells isolated from urine had progenitor cell features and the potential to differentiate into several bladder cell lineages. Urine derived cells could serve as an alternative cell source for urinary tract tissue engineering and reconstruction.

Integrity of all four transmembrane domains of the tetraspanin uroplakin Ib is required for its exit from the ER

The surface of the mammalian urinary bladder is covered by a crystalline, asymmetric unit membrane (AUM) structure that contains the four major uroplakins (UPs): Ia, Ib, II and IIIa. UPIa and UPIb belong to the family of tetraspanins. Although UPIa and UPIb are structurally conserved, only UPIb could exit from the endoplasmic reticulum (ER) and reach the cell surface when expressed alone in 293T cells. Modifications of the large extracellular loop of UPIb, such as mutation of the N-glycosylation site or the cysteines involved in the formation of three disulfide bridges, or exchanging the large luminal loop of UPIb with that of UPIa did not affect the ability of UPIb to reach the cell surface. However, modifications of any of the four transmembrane domains of UPIb led to ER retention, suggesting that the proper formation of helical bundles consisting of the tetraspanin transmembrane domains is a prerequisite for UPIb to exit from the ER. Results of sedimentation analysis suggested that aggregate formation is a mechanism for ER retention.

The role of Ca2+ influx and intracellular Ca2+ release in the muscarinic‐mediated contraction of mammalian urinary bladder smooth muscle

OBJECTIVE To study the involvement of extracellular Ca2+ and the properties of the intracellular Ca2+ ([Ca2+]i) stores on the carbachol-induced contraction of mammalian urinary bladder smooth muscle strips under polarized and depolarized conditions. Strips of bladder were suspended between platinum ring electrodes in a cylindrical organ bath (0.2 mL) and continuously superfused with Krebs' solution at 1 mL/min. The effect of nifedipine, cyclopiazonic acid (CPA), thapsigargin, procaine, ryanodine and caffeine before and during a 10-s application of 100 microm carbachol under polarized conditions were studied. The effect of these drugs was also assessed under depolarized conditions using a protocol that allowed a more detailed assessment of the role of [Ca2+]i stores, consisting of emptying the stores by exposure to Ca2+-free solution, rapidly refilling them by a 10-s application of 81.5 mm Ca2+ (priming), returning to the Ca2+-free solution for 3 min and then applying 100 microm carbachol (10 s) in Ca2+-free solution (store release). Under polarized conditions, nifedipine and Ca2+ removal almost completely inhibited the carbachol-induced contractions. CPA increased the amplitude and duration of both carbachol- and electrical field stimulation-induced contractions. Although ryanodine had no inhibitory effect, caffeine and procaine significantly inhibited the carbachol-induced contraction. Under depolarized conditions nifedipine blocked both priming and store release contractions. CPA, thapsigargin, procaine and ryanodine significantly increased the priming and inhibited the store release contractions. However, caffeine virtually abolished both priming and store release contractions. These results suggest that in guinea-pig urinary bladder smooth muscle the Ca2+ necessary for contraction enters the cell through voltage-dependent dihydropyridine-sensitive Ca2+ channels and is pumped into an intracellular store that is released by carbachol. Under polarized conditions, the blockade of sarco-endoplasmic reticulum calcium ATP-ase (SERCA) with CPA increases [Ca2+]i and carbachol-induced contractions. The effects of caffeine and procaine suggest that store release involves ryanodine receptors and calcium-induced calcium release. Under depolarized conditions, Ca2+ entry is blocked by nifedipine and the stores diminish. Stored Ca2+ is also greatly reduced by the blockade of SERCA with either CPA or thapsigargin. Procaine, ryanodine and caffeine blocked the store release contractions, suggesting that this involves ryanodine receptors and calcium-induced calcium release.

Developmental regulation of nerve and receptor mediated contractions of mammalian urinary bladder smooth muscle

The development of nerve-induced activation, receptor properties and cellular signalling was examined by comparing the urinary bladder of new-born (0–2 days) and adult mice. Tissue strips were isolated and the effects of different neuromuscular agents on force were investigated during electrical field stimulation. The nerve-induced contractions of the urinary bladders from new-born mice were less influenced by desensitisation with α, β-methylene ATP and more sensitive to scopolamine compared with those of the adult bladder. There were no differences in α, β-methylene ATP or ATP responsiveness between adult and new-born tissue, showing that the lower purinergic component of the nerve-induced responses in the new-born mice was due to properties of the transmitter release rather than to a change in receptor function. Dose–response curves for carbachol revealed a lower peak response in new-born bladders compared with adults. The phasic component of the cholinergic contractions was pronounced and initiated at low carbachol concentrations in the new-born tissue. The carbachol contractions of both new-born and adult urinary bladder tissue were inhibited by the Rho kinase inhibitor Y27632 and by the protein kinase G activator 8-Br-cGMP. However, the sustained phase of carbachol contraction was significantly less sensitive to Y27632 and 8-Br-cGMP in new-born tissue. These results suggest that the receptor mediated calcium sensitisation mechanism is less prominent in new-born compared with adult mice and that the contractions of new-born bladders are less influenced by the nitric oxide (NO)-cGMP inhibitory pathway.

Effect of anoxia‐glucopenia and re‐superfusion on intrinsic nerves of mammalian detrusor smooth muscle: Importance of glucose metabolism

To investigate the effect of anoxia/glucopenia and re-superfusion on intrinsic nerves in the mammalian urinary bladder. Strips of detrusor smooth muscle were dissected from monkey and human urinary bladder and mounted for tension recording in organ baths superfused with Krebs solution. Human, monkey, and guinea-pig urinary bladders were treated to evaluate glycogen contents by a biochemical method. Detrusor strips from both monkeys and humans had to be exposed to anoxia-glucopenia for up to 2-2.5 hr to observe a progressive decline in the response to electrical field stimulation (EFS) of the intrinsic nerves, at variance with guinea-pig detrusor strips. In contrast, the response to direct activation of the smooth muscle with carbachol remained almost unaltered. Incubation of human and monkey detrusor strips with 2-deoxyglucose (2-DG) during 1 hr anoxia-glucopenia, however, caused a marked damage to the intrinsic nerves. The glycogen contents of both human detrusor specimens and monkey urinary bladders were 2.0- and 1.4-fold higher, respectively, than that found in guinea-pig urinary bladder; furthermore, untreated monkey detrusor sections showed a greater number of glycogen granules as compared to those subjected to anoxia-glucopenia and re-superfusion. In guinea-pig and in monkey detrusor sections glycogen granules were found in smooth muscle cells but not in neurons of intramural ganglia. A higher susceptibility of guinea-pig as compared to monkey and human nerves has been demonstrated; it is suggested that anaerobic glucose metabolism during anoxia-glucopenia is crucial for the functional recovery of detrusor intrinsic nerves from damage caused by anoxia-glucopenia and re-superfusion.

Colistin interactions with the mammalian urothelium.

Here we describe the effect of colistin on the barrier function of the mammalian urinary bladder epithelium. Addition of colistin to the mucosal solution of the rabbit urinary bladder epithelium (urothelium) resulted in an increase in the transepithelial conductance. The magnitude of the increase in transepithelial conductance was dependent on the membrane voltage, concentration of colistin, and presence of divalent cations in the bath solution. The initial site of action of colistin was at the apical membrane. Colistin increased the membrane conductance only when the apical membrane potential was cell interior negative. The more negative the membrane potential, the larger the conductance increase. The concentration dependence of the conductance increase saturated, suggesting a membrane binding site. Divalent cations decreased the magnitude of the conductance increase. This divalent cation action occurred at two sites: one in competition with colistin for a membrane binding site, and the other by rapidly blocking the induced conductance. At short exposure times, the increase in conductance was reversed by either removing colistin from the bath or changing the voltage so that the apical membrane was cell interior positive. At long exposure times, the increase was only partially reversible by voltage or removal from the bath. This finding suggests that at long exposure times, there is a toxic effect of colistin on the urothelium.

Analysis of hydrostatic pressure-induced changes in umbrella cell surface area

All cells experience and respond to external mechanical stimuli including shear stress, compression, and hydrostatic pressure. Cellular responses can include changes in exocytic and endocytic traffic. An excellent system to study how extracellular forces govern membrane trafficking events is the bladder umbrella cell, which lines the inner surface of the mammalian urinary bladder. It is hypothesized that umbrella cells modulate their apical plasma membrane surface area in response to hydrostatic pressure. Understanding the mechanics of this process is hampered by the lack of a suitable model system. We describe a pressure chamber that allows one to increase hydrostatic pressure in a physiological manner while using capacitance to monitor real-time changes in the apical surface area of the umbrella cell. It is demonstrated that application of hydrostatic pressure results in an increase in umbrella cell apical surface area and a change in the morphology of umbrella cells from roughly cuboidal to squamous. This process is dependent on increases in cytoplasmic Ca 2+. This system will be useful in further dissecting the mechanotransduction pathways involved in cell shape change and regulation of exocytic and endocytic traffic in umbrella cells.

In Vitro Biocompatibility Evaluation Of Naturally Derived And Synthetic Biomaterials Using Normal Human Bladder Smooth Muscle Cells

Tissue engineering of the urinary tract often requires the use of various biomaterials. Adequate biomaterial biocompatibility is necessary for successful urinary reconstruction. In this study using a primary normal human bladder smooth muscle cell culture system we evaluated the in vitro biocompatibility of a number of naturally derived biomaterials, including bladder submucosa, small intestinal submucosa, collagen and alginate, and polymeric biomaterials, including polyglycolic acid, poly(L-lactic acid) and poly(lactic-co-glycolic acid, which have been used for urinary reconstruction experimentally or clinically. To determine the cytotoxic and bioactive effects of bladder submucosa, small intestinal submucosa, collagen, alginate, polyglycolic acid, poly(L-lactic acid) and poly(lactic-co-glycolic acid) we measured cell viability, metabolic activity, apoptotic properties and DNA synthesis activity with 4 types of assays, namely Neutral Red (Sigma Chemical Co., St. Louis, Missouri), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma Chemical Co.), apoptotic activity and tritiated thymidine incorporation (Dupont NEN, Boston, Massachusetts) assays. Normal human bladder smooth muscle cells were cultured with the extracts of the biomaterials or cultured in direct contact with the biomaterials. All naturally derived and synthetic biomaterials tested in this study except alginate exhibited nontoxic and bioactive effects on human bladder smooth muscle cells (HBSMCs) in vitro, as indicated by the 4 types of biocompatibility assays using the extract and direct contact methods. Cell viability, apoptotic properties, metabolic activity and DNA synthesis activity of HBSMCs cultured with the extracts of the biomaterials or cultured in direct contact with the biomaterials were not significantly different from those of negative controls (fresh medium with no extracts or tissue culture plates without biomaterials). All naturally derived and synthetic biomaterials tested in this study except alginate exhibited nontoxic and bioactive effects on HBSMCs in vitro. This normal primary human bladder smooth muscle cell culture model is suitable for in vitro biocompatibility assessment. It provides information on cell-biomaterial interactions and on the ability of biomaterials to support bioactive cell functions.

Structure of the major membrane protein complex from urinary bladder epithelial cells by cryo-electron crystallography

Numerous protein plaques cover the apical surface of mammalian urinary bladder epithelial cells. These plaques contain four integral membrane proteins, called uroplakins, which form a well-ordered array of hexameric complexes. The 3D structure of these naturally occurring 2D crystals was studied by cryo-electron-crystallographic methods using a slow-scan charged-coupled device (CCD) camera to record the electron micrographs. A 1.2 nm projection map calculated from untilted crystals shows that each hexamer comprises a ring of six inner and six outer domains at a radius of 5.7 nm and 9.2 nm respectively. The 3D structure shows that the mass is distributed strongly asymmetrically with respect to the membrane, with most of the mass protruding from the luminal face. Both domains in the asymmetric unit traverse the membrane and protrude from the membrane on the cytoplasmic side. On the luminal side, the two domains are bridged forming a stretched arc. The total thickness of the complex is about 13.2 nm. A model of the urothelial plaque reveals that contacts between the hexamers are much less extended than within the hexamers.

COCULTURE OF BLADDER UROTHELIAL AND SMOOTH MUSCLE CELLS ON SMALL INTESTINAL SUBMUCOSA: POTENTIAL APPLICATIONS FOR TISSUE ENGINEERING TECHNOLOGY

COCULTURE OF BLADDER UROTHELIAL AND SMOOTH MUSCLE CELLS ON SMALL INTESTINAL SUBMUCOSA: POTENTIAL APPLICATIONS FOR TISSUE ENGINEERING TECHNOLOGY

REGENERATION OF FUNCTIONAL BLADDER SUBSTITUTES USING LARGE SEGMENT ACELLULAR MATRIX ALLOGRAFTS IN A PORCINE MODEL

REGENERATION OF FUNCTIONAL BLADDER SUBSTITUTES USING LARGE SEGMENT ACELLULAR MATRIX ALLOGRAFTS IN A PORCINE MODEL