The rate-limiting step in hydrosmotic response of frog urinary bladder

Summary The ADH-induced water fluxes and the associated appearance of intramembranous particle aggregates in the luminal membrane of frog urinary bladders have been correlated in a time course study. Plots of the onset and reversal of the oxytocin-induced hydrosmotic response were sigmoidal in shape, symmetrical and slowed by low temperature to the same degree. Parallel freezefracture studies showed that the mean size distribution of the aggregates was constant at different temperatures and at different times during hormonal stimulation and washout. No qualitatively different picture of aggregate formation was detected at low temperature: this suggests that the insertion and removal of individual aggregates into or from the apical plasma membrane is a rather rapid process, both at 20 and at 6.5° C. As in the case of water permeability, both aggregate appearance and disappearance were similarly slowed by lowering the temperature.A similar time-course study of the inhibition of the hydrosmotic response by acidification of the medium was also made. In this case, lowering the incubation temperature induced a clear dissociation between net water flow and the surface area occupied by the aggregates. For the first time, a low water permeability was found associated with a high aggregate surface area in the apical membrane, indicating that cellular acidification induces an impairment of aggregate function rather than a reduction of surface area.J.C. is a career investigator at the Institut National de la Santé et de la Recherche Médicale, INSERM V.48     

A time course study of water permeability and morphological alterations induced by mucosal hyperosmolarity in frog urinary bladder

Summary The role of the tight junction in the hydrosmotic response of the frog urinary bladder has been analysed by comparative kinetic studies and freeze etching examination. The comparison of the time course of the variations in transepithelial water net flux and of the alterations of tight junction ultrastructure in bladders exposed to mucosal hyperosmolar solutions shows that blisters are present in the tight junction before any increase in transepithelial water net flux. This indicates that the two phenomena are dissociated.In the same experimental conditions, freeze etching examination shows the presence in the tight junction of large areas of smooth and apparently stretched membrane where the typical network structure has disappeared. These alterations are reduced by further treatment with oxytocin and are probably not involved in the physiological hydrosomotic response.This work was supported in part by a grant from the Medical Research Council of Canada (J.H.).     

Giant vacuoles arising during ADH-induced transcellular bulk water flow across the epithelium of the frog urinary bladder

Structural changes of the cytoplasm of urinary bladder granular cells after an antidiuretic hormone (ADH) stimulation of water transport were studied using standard and cryogenic methods of electron microscopy. Numerous changes occurred in these cells, the cytoplasm of the granular cells becoming swollen, and the intercellular spaces enlarged. Most granules become fused with the apical membrane. Under maximal ADH action, giant vacuoles appear in the cytoplasm of granular cells, in association with microfilaments and microtubules. Analysis of ultrastructure of the granular cells has established the origin of giant vacuoles from the cis -cisterna of the Golgi complex. A hypothesis based on the morphofunctional homology of giant vacuoles in granular cells with the contractile vacuoles of Protozoa is proposed in which the giant vacuoles ('contractile-like' vacuoles) are seen as operating a osmoregulatory role in these cells. It is also proposed that microtubules and microfilaments participate in giant vacuole migration through the cytoplasm.     

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.     

A vascular network closely linked to the epithelium of the urinary bladder of the rat

Summary Scanning electron microscopy was used on the mucosa of the rat urinary bladder after digestion with strong alkali and microdissection. The underside of the epithelium (and the plane of the epithelium-tunica propria interface) is not smooth but is scored by grooves-10 m wide and 3–4 m deep—connected into a fine mesh. A net of blood capillaries located in the uppermost part of the tunica propria occupies these grooves. They measure 3–9 m in diameter, are separated from the epithelium by a gap of 0.3 m, often show fenestrations, and are accompanied by numerous and extensive pericytes and by some fibroblasts. We discuss these observations in the light of current knowledge of blood flow in the bladder, contraction and distension of the bladder wall and formation of mucosal folds, transport of solutes through the epithelium, and plasma extravasation from mucosal blood vessels in neurogenic inflammation.     

Cellular pH and water permeability control in frog urinary bladder a possible action on the water pathway

Mucosal acidification (from pH 8.1 to 6.0) reversibly inhibited the hydroosmotic responses to oxytocin, cyclic AMP and 8-bromo-cyclic AMP in frog urinary bladder. These inhibitory effects were only observed in the presence of a permeant buffer in the apical medium and could also be elicited by CO₂ bubbling, even when the mucosal pH was clamped at 8.1. Acid pH reduced the oxytocin-induced net water flux faster than norepinephrine or oxytocin removal and the difference was especially important at low temperature. The time course of recovery from acid pH inhibition was, at 20°C, similar to that of the hormonal action, but when the medium temperature was reduced to 6–7°C, the recovery from acid pH inhibition paradoxically became faster while the oxytocin action was markedly slowed down ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of changes in net water fluxes (expressed in min): oxytocin addition at 20°C, $\text{6.2 ± 0.9}$; at 6°C, $\text{24 ± 3}$; oxytocin removal at 20°C, $\text{4.7 ± 0.8}$; at 6°C, $\text{22 ± 3}$; pH inhibition at 20°C, $\text{2.6 ± 0.2}$; at 6°C $\text{2.5 ± 0.2}$; recovery from pH 6 at 20°C, $\text{6.5 ± 0.9}$; at 6°C, $\text{2.7 ± 0.3}$). These results can be explained by accepting two main loci sensitive to medium acidification: (1) the cyclase system and (2) an intracellular, temperature-independent, post-cyclic AMP site. The fact that the intramembranous particle aggregates associated with the oxytocin-induced water permeability increase did not disappear after the flow inhibition by acid pH at low temperature suggests that the second effect could be located at the water channel itself.     

Osmotic permeabilities across corneal endothelium and antidiuretic hormone-stimulated toad urinary bladder structures

Osmotic permeabilities of several epithelial structures have been determined with novel optical procedures based on specular microscopy. The osmotic permeabilities of several tissue layers were determined by continuously monitoring the position of the apical tissue borders while an osmotic flow was imposed across those layers. The values found were (in μm/s; mean ± SE): corneal epithelium, 137 ± 30 (n = 5); antidiuretic hormone stimulated toad bladder, 429 ± 64 (n = 6); and corneal endothelium, 711 ± 34 (n = 7). In addition, the osmotically-induced transient change in thickness of the corneal endothelial cells was determined with the help of a computer, and the apparent osmotic permeability measured for the apical membrane was 1420 ± 160 μm/s (n = 5). It is concluded that the osmotic permeability across the endothelial layer is sizably larger than had been previously detected and that osmotic flows across such layer largely traverse the cellular membranes. With osmotic permeability values (per unit of cell membrane area) as large as presently reported, isotonic fluid transport by epithelia can be explained simply on the basis of local osmotic gradients.     

Nocodazole inhibition of the vasopressin-induced water permeability increase in toad urinary bladder

Nocodazole is a synthetic antitumor drug that binds rapidly to tubulin. When this drug is applied to toad bladder prior to vasopressin stimulation it inhibits the vasopressin response. A maximum inhibition (68%) is reached with a dose level of 10 μ/ml applied one-half hour prior to vasopressin stimulation (20 mU/ml). This compares with an inhibition of 50% seen with a 3-h exposure of the tissue to colchicine (0.1 mM) prior to stimulation with vasopressin. Application of nocodazole (1 μ/ml) 3 min after hormonal stimulation shows no inhibition of the response at one-half hour past stimulation. These data support the view that microtubules are involved in the vasopressin-induced increase in water permeability in toad bladder and also indicate that this involvement is limited to the period prior to or directly after stimulation.     

Immunocytochemical studies on translocation of phosphorylated aquaporin-h2 protein in granular cells of the frog urinary bladder before and after stimulation with vasotocin

We have generated a specific antibody against phosphorylated aquaporin-h2 (pAQP-h2) protein to investigate the role of phosphorylation in the translocation of AQP-h2 protein within the granule cells of the urinary bladder of the frog (Hyla japonica). The antibody was generated against a synthetic peptide (ST-160) corresponding to amino acids 255–268, with a phosphorylated Ser-262, a residue that is putatively phosphorylated by protein A kinase. Using this antibody, we found, by Western blot analysis, that phosphorylation of the AQP-h2 protein rapidly increased within 2 min after vasotocin (AVT) stimulation and remained at a higher than normal level for 15 min. Moreover, quantitative immunoelectron microscopy indicated that the location of the AQP-h2 protein dramatically changed after AVT stimulation. Before stimulation, pAQP-h2 protein was localized in only a small number of intracellular vesicles near the nucleus of the granular cells, whereas the labeling density of the intracellular vesicles and the apical membrane rapidly increased after stimulation. This finding was also confirmed by the results of an immunofluorescence study. Thus, phosphorylation of AQP-h2 protein seems to be essential for translocation of the protein from the cytoplasmic pool to the apical plasma membrane of the granular cells in frog urinary bladder. This work was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan to S.T.     

Fine structure of intramembranous particle aggregates in ADH-treated frog urinary bladder and skin: Influence of glutaraldehyde and N-ethyl maleimide

Summary The fine structure of ADH-induced intramembrane particle aggregates has been studied in different tissues and under different experimental conditions. Particle aggregates similar to those previously observed in the amphibian urinary bladder and in the mammalian collecting duct were also found in the frog skin, another ADH target tissue. In the frog urinary bladder, typical aggregates were observed in the absence of glutaraldehyde fixation. Two experimental approaches were used a) the absence of both fixative and cryoprotectant treatments and b) the absence of only glutaraldehyde treatment. In the latter case the reversal of hydrosmotic action was prevented by exposing the preparations to N-ethyl maleimide. In specimens of frog urinary bladder conventionally fixed with glutaraldehyde, two fracture levels could be observed in the aggregates, suggesting that the aggregated particles span an appreciable part of the membrane thickness.J. Chevalier is a career investigator from the Institut National de la Santé et de la Recherche Médicale, INSERM U 48, France     

Stem cell applications for pathologies of the urinary bladder

New stem cell based therapies are undergoing intense research and are widely investigated in clinical fields including the urinary system. The urinary bladder performs critical complex functions that rely on its highly coordinated anatomical composition and multiplex of regulatory mechanisms. Bladder pathologies resulting in severe dysfunction are common clinical encounter and often cause significant impairment of patient’s quality of life. Current surgical and medical interventions to correct urinary dysfunction or to replace an absent or defective bladder are sub-optimal and are associated with notable complications. As a result, stem cell based therapies for the urinary bladder are hoped to offer new venues that could make up for limitations of existing therapies. In this article, we review research efforts that describe the use of different types of stem cells in bladder reconstruction, urinary incontinence and retention disorders. In particular, stress urinary incontinence has been a popular target for stem cell based therapies in reported clinical trials. Furthermore, we discuss the relevance of the cancer stem cell hypothesis to the development of bladder cancer. A key subject that should not be overlooked is the safety and quality of stem cell based therapies introduced to human subjects either in a research or a clinical context.     

Study of NO-synthase activity and detection of NO-dependent antimicrobial effects in primary culture of frog urinary bladder epithelial cells

We have shown previously that endogenous nitric oxide (NO) in frog urinary bladders modulates the influence of arginine-vasotocin on the increase in the osmotic water permeability of the bladder epithelium. The goal of the present work was to obtain a primary culture of epithelial cells from a frog urinary bladder in order to study in vitro the cellular activity of NO-synthase (NOS) and its regulation. The best conditions for cultivating the cells turned out to be with the use of a modified L-15 medium containing 10% fetal bovine serum and gentamicin (40 μg/ml) at room temperature. Under these conditions, at least 50% of the cells preserved their viability for 8 days. The NOS activity was estimated from the accumulation of nitrites (NO ₂ ^? ) in the cultivation medium; the amount of NO ₂ ^? in the presence of nitro-L-arginine methyl ester (L-NAME), an inhibitor of all types of NOS, was considered unspecific and was subtracted from each value. The NO ₂ ^? accumulation was linear in time for three days of cultivation and was inhibited by 1400W, an inducible NOS (iNOS) inhibitor, or by 7-nitroindazole, an inhibitor of constitutive NOSs, at concentrations of 5–50 and 10–200 μM, respectively. One-day incubation of the cells in the medium with low doses of gentamicin (1 or 2 μg/ml) led to a marked increase in the amount of bacteria in the cultivation m5 mM edium identified as E. coliand Acinetobacter sp. An addition of 5 mM L-NAME to the cultivation medium increased the amount of the bacteria by 1.5-and 2.5-fold in the presence of 2 and 1 μg/ml gentamicin/ml, respectively. Thus, the epithelial cells of frog urinary bladders have the NO-dependent antibacterial effect, which seems to be the result of enhanced iNOS expression. The obtained data indicate that the primary culture of the epithelial cells from frog urinary bladders is a prospective model for studying the role of NO and the regulation of NOS activity, which is of particular interest for studying protective NO effects in epithelial tissues.     

Non-electrolyte diffusion across the frog gastric mucosa under osmotic gradients

Serosal-to-mucosal and mucosal-to-serosal diffusion of ¹⁴C-labelled inulin, sucrose, erythritol and propionamide was compared with ³HHO diffusion in mucosae incubated with isosmotic solutions at both surfaces, as well as isosmotic solution at serosal surface and hyperosmotic solution at the mucosal surface. The use of a hyperosmotic solution at the mucosal surface significantly increases unidirectional diffusion fluxes of inulin and of sucrose. To a nonsignificant extent, it affects the fluxes of erythritol and propionamide and significantly reduces the ³HHO diffusion. A size increment of the diffusion path utilized by the larger molecules is proposed.     

Arginase activity in frog urinary bladder epithelial cells and its involvement in regulation of nitric oxide production

The activity of arginase converting arginine into ornithine and urea is of particular interest among many factors regulating NO production in the cells. It is known that by competing with NO-synthase for common substrate (arginine), arginase can affect NO synthesis. In the present work, properties of arginase from the common frog Rana temporaria L. urinary bladder epithelial cells containing the NO-synthase were characterized, and possible contribution of arginase to regulation of NO production by epithelial cells was studied. It has been shown that the enzyme has temperature optimum in the range of 55–60°C, K _M for arginine 23 mM, and V _max about 10 nmole urea/mg of protein/min, and its activity was efficiently inhibited by (S)-(2-boronoethyl)-L-cysteine (BEC), an inhibitor of arginase, at concentrations from 10^?6 to 10^?4 M. The comparison of arginase activity in various frog tissues revealed the following pattern: liver > kidney > brain > urinary bladder (epithelium) > heart > testis. The arginase activity in isolated urinary bladder epithelial cells was 3 times higher that in the intact urinary bladder wall. To evaluate the role of arginase in regulation of NO production, the epithelial cells were cultivated in the media L-15 or 199 containing different amounts of arginine; the concentration of NO₂ ^?, the stable NO metabolites, was de-termined in the cultural fluid after 18–20 h of cell incubation. The vast majority of the produced nitrites are associated with NOS activity, as L-NAME, the NO inhibitor, decreased their accumulation by 77.1% in the L-15 medium and by 80% in the 199 medium. BEC (10^?4 M) increased nitrite production by 18.0% ± 2.7% in the L-15 medium and by 24.4% ± 3.5% in the 199 medium (p < 0.05). The obtained data indicate a relatively high activity of arginase in the frog urinary bladder epithelium and its involvement in regulation of NO production.     

Reversal of muscle hypertrophy in the rat urinary bladder after removal of urethral obstruction

We studied the ultrastructure of the bladder musculature after first inducing hypertrophy by means of urethral obstruction and subsequently removing the obstruction. With hypertrophy the bladder musculature increases ten-fold or more in volume; after de-obstruction approximately 4/5 of the hypertrophic muscle weight and volume is lost within six weeks. In spite of this very large decrease in muscle mass there is no degeneration of muscle cells or nerve endings or of other cell types in the de-obstructed bladder either at 5 days or at 6 weeks. The individual muscle cells are smaller in size than in the hypertrophic bladder but still larger than control muscle cells. The decrease in muscle cell size is more substantial than the decrease in muscle cell surface. There are no lysosomes or other signs of intracellular degradation in any cells of the muscle layer. The musculature contains a very large amount of intercellular material, mainly collagen. This study documents the great plasticity of the musculature in the reduction of muscle mass after de-obstruction. However, some of the fine structural features are almost as different from the controls as in the hypertrophic muscle.     

The distribution of noradrenergic nerves and small,intensely fluorescent (SIF) cells in the cat urinary bladder

Summary Fluorescence and electron microscopy have been used to study the distribution of noradrenergic nerves in the smooth muscle of the cat urinary bladder. Using the former technique, relatively few fluorescent noradrenergic nerves were observed in the body and fundus, while a rich plexus occurred adjacent to muscle cells of the bladder neck. The trigone could not be distinguished neuromorphologically from detrusor muscle in this region. Electron microscopy showed that the majority of noradrenergic terminals in the body and fundus were associated with presumptive cholinergic axons, while in the bladder neck noradrenergic terminals formed typical neuroeffector relationships with individual smooth muscle cells.Numerous ganglia occurred both in the adventitia and among the smooth muscle bundles, particularly in the bladder neck. The majority of the nerve cell bodies were non-fluorescent, although many contained bright orange autofluorescent granules, believed to be lysosomes. A small minority of ganglion cells were associated with fluorescent noradrenergic nerve terminals, thereby providing structural evidence for limited intraganglionic inhibition. In addition, occasional groups of small intensely fluorescent (SIF) cells were observed in some intramural ganglia and these were subsequently identified in the electron microscope. The possibility that these cells may provide a second inhibitory influence on bladder activity was considered.     

Ultrastructural correlates of the antidiuretic hormone-dependent and antidiuretic hormone-independent increase of osmotic water permeability in the frog urinary bladder epithelium

Electron and confocal microscopy, using immunocytochemical methods, was employed to assess osmotic water permeability of the frog (Rana temporaria) urinary bladder during transcellular water transport, induced by antidiuretic hormone (ADH) or by wash-out of autacoids from serosal, ADH-free Ringer solution. The increase of osmotic water permeability of the urinary bladder was accompanied by relevant ultrastructural changes, the most remarkable being: (1) the appearance of aggregates of intramembranous particles in the apical membrane of granular cells, and the extent of the membrane area covered by the aggregates proportional to that of the water flow; (2) redistribution of actin filaments in the cytoplasm of granular cells; judging from the anti-actin label density, the number of actin filaments in the apical region of cytoplasm was reduced by 2.5–4 times compared with normal; (3) a decrease in the total electron density of the cytoplasm due to the increased water content of granular cells.