Detrusor Muscle Cells Research Articles

MORPHOFUNCTIONAL AND CLINICAL SIGNIFICANCE OF THE INTERSTITIAL CELLS OF CAJAL IN THE URINARY BLADDER (REVIEW ARTICLE)

The therapy of symptoms of the lower urinary tract that in most cases are caused by benign prostatic hyperplasia is one of challenges for modern medicine. Knowing the origin of bladder muscle hyperactivity will promote the treatment in this group of patients. The studies of interstitial cells of Cajal in the urinary bladder open up new opportunities to widen our understanding of bladder physiology. The purpose of this study is to conduct an analytical review of the literature on the role of interstitial cells of Cajal in the physiology of the urinary bladder. We investigated the current literature highlighting the morphological and physiological significance of interstitial cells of Cajal in the urinary bladder. Interstitial cells of Cajal are in close proximity to muscle cells, vegetative nerve endings and urothelial cells. There is increasing evidence that interstitial cells of Cajal play a role in the development of lower urinary tract symptoms. Interstitial cells of Cajal may be responsible for generating electrical potentials and inducing detrusor muscle contractions. New pathomechanisms of the development of bladder hyperactivity were put forward, namely: impairment of spontaneous contractility caused by altered signal transduction of interstitial Cajal cells between nerves and detrusor muscle cells; change in signal transmission through suburothelial interstitial cells of Cajal. The c–kit receptor is not only a marker for identifying these cells, but may also play a critical role in controlling bladder function. Interstitial cells of Cajal, discovered more than 100 years ago, are still remaining a poorly studied object. Having long processes, interstitial cells of Cajal form multiple contacts with smooth muscle and nerve cells, building up a specific network. Current knowledge about interstitial cells of Cajal in the urinary bladder suggest that these cells and c–kit receptors may become a new “target” for the pharmaceutical therapy of lower urinary tract symptoms and overactive bladder.

Decreased autophagic activity of detrusor cells is involved in the inflammatory response of interstitial cystitis/bladder pain syndrome.

Genome-wide association studies suggest that autophagy plays an important regulatory role in inflammatory and autoimmune diseases. Inflammation and immune regulation disorders are involved in the occurrence and development of interstitial cystitis/bladder pain syndrome (IC/BPS). However, the changes and roles of autophagy in IC/BPS have not been reported. Therefore, this study aimed to investigate bladder autophagy and inflammation changes in patients with IC/BPS. Bladder specimens (n = 5) from patients with cystectomy due to end-stage IC/BPS were collected. The bladder samples of the control group (n = 5) were derived from the normal area bladder tissue after radical cystectomy. H&E and toluidine blue staining were used for histological evaluation. The co-location of LC3, alpha-smooth muscle actin (α-SMA), and autophagosome was investigated with double-labeled immunofluorescence and transmission electron microscopy (TEM). The expression of IL-6, TNF-α, Bax, caspase-3, and BCL-2 in the detrusor layer was analyzed using immunohistochemistry (IHC) and Western blot (WB). Compared with the control group, bladder tissue from IC/BPS patients revealed thinner and edematous epithelium with many mast cells (P < 0.05) infiltrating into the muscle layer. By using TEM (P < 0.05), double-labeled immunofluorescence (P < 0.05), and Western blot (P < 0.05) in IC/BPS patients, autophagy was also found and was significantly increased in detrusor myocytes. IHC and WB indicate the expression of BCL-2 (P < 0.05) was decreased, while IL-6, TNF-α, Bax, and caspase-3 expression was elevated (P < 0.05). The number of autophagosomes in detrusor cells was increased in IC/BPS. However, autophagy of detrusor muscle cells may not have sufficient phagocytic activity to effectively remove damaged proteins and mitochondria, which may lead to the continued deterioration of IC/BPS inflammation and apoptosis.

Human amniotic fluid stem cells can alleviate detrusor dysfunction caused by bladder outlet obstruction in rats

The present study examined whether bladder detrusor dysfunction due to partial bladder outlet obstruction (pBOO) could be improved after the treatment of human amniotic fluid stem cells (hAFSCs). 72 female rats were grouped into sham operation, pBOO, and pBOO with hAFSCs treatment (pBOO + hAFSCs) for in vitro and in vivo studies. Bladder weight, bladder wall thickness, the ratio of collagen to smooth muscle and the levels of positive CD11b/c and HIS48 cells was significantly increased after pBOO but improved after hAFSCs treatment. Cystometries showed impaired bladder function after pBOO. Protein and mRNA levels of hypoxia inducible factor-1α, CCL2, interleukin-1β, transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), α-smooth muscle actin, collagen I and collagen III were increased at 2 and/or 6 weeks, but proteins and mRNA expressions of protein gene product 9.5 were decreased at 2 and 6 weeks after pBOO. These abnormalities were improved after hAFSCs treatment. The expressions of TGF-β1 and CTGF in cultured detrusor cells of pBOO rats were increased but were improved after hAFSCs treatment. The present results showed hAFSCs treatment could improve bladder detrusor dysfunction in pBOO rats, which may be related to the reduction of inflammatory and pro-fibrotic markers in detrusor muscle cells.

Expression and localization of pannexin-1 and CALHM1 in porcine bladder and their involvement in modulating ATP release.

ATP release from urinary bladder is vital for afferent signaling. The aims of this study were to localize calcium homeostasis modulator 1 (CALHM1) and pannexin-1 expression and to determine their involvement in mediating ATP release in the bladder. To determine gene expression and cellular distribution, PCR and immunohistochemistry were performed, respectively, in the porcine bladder. CALHM1 and pannexin-1-mediated ATP release in response to hypotonic solution (0.45% NaCl)-induced stretch, and extracellular Ca2+ depletion ([Ca2+]0) was measured in isolated urothelial, suburothelial, and detrusor muscle cells. CALHM1 and pannexin-1 mRNA and immunoreactivity were detected in urothelial, suburothelial, and detrusor muscle layers, with the highest expression on urothelium. Hypotonic stretch caused a 2.7-fold rise in ATP release from all three cell populations (P < 0.01), which was significantly attenuated by the pannexin-1 inhibitor, 10Panx1, and by the CALHM1 antibody. Brefeldin A, a vesicular transport inhibitor, and ruthenium red, a nonselective CALHM1 channel blocker, also significantly inhibited stretch-mediated ATP release from urothelial cells. [Ca2+]0 caused a marked, but transient, elevation of extracellular ATP level in all three cell populations. CALHM1 antibody and ruthenium red inhibited [Ca2+]0-induced ATP release from urothelial cells, but their effects on suburothelial and detrusor cells were insignificant. 10Panx1 showed no significant inhibition of [Ca2+]0-induced ATP release in any types of cells. The results presented here provide compelling evidence that pannexin-1 and CALHM1, which are densely expressed in the porcine bladder, function as ATP release channels in response to bladder distension. Modulation of extracellular Ca2+ may also regulate ATP release in the porcine bladder through voltage-gated CALHM1 ion channels.

Effect of Naftopidil on Bladder Microcirculation in a Rat Model of Bladder Outlet Obstruction.

To evaluate the effect of naftopidil on bladder capillary blood flow using bladder outlet obstruction model rats. Female Sprague-Dawley rats were divided into three groups: control group, bladder-outlet-obstruction group, and bladder-outlet-obstruction + naftopidil group. Bladder-outlet-obstruction surgery was performed in the bladder-outlet-obstruction and bladder-outlet-obstruction + naftopidil groups. The control group received sham-operation. The bladder-outlet-obstruction + naftopidil group were treated with naftopidil (30 mg/kg) for 14 days after bladder-outlet-obstruction operation, while the control and bladder-outlet-obstruction groups were treated with vehicle. Continuous cystometry was performed 14 days after the surgery. Bladder blood flow was measured after 14 days using a pencil lens charge-coupled device microscopy system. The bladder was then harvested for histology and measuring 8-hydroxy-2'-deoxyguanosine tissue level by enzyme-linked immunosorbent assay. In cystometry, the bladder-outlet-obstruction rats showed bladder overactivity, while naftopidil treatment improved the cystometric pattern. The blood flow through the submucosal capillaries of the bladder base in the bladder-outlet-obstruction group was lesser than that in the control, whereas the bladder-outlet-obstruction + naftopidil group showed significantly greater blood flow than the bladder-outlet-obstruction group. The bladder tissue in the bladder-outlet-obstruction group showed a tendency to contain more hypertrophic detrusor muscle and inflammatory cells compared to those in the control group, while naftopidil treatment suppressed these histological changes. The 8-hydroxy-2'-deoxyguanosine levels in the bladder tissue significantly differed among the three groups (the bladder-outlet-obstruction group > the bladder-outlet-obstruction + naftopidil group > the control group). Naftopidil improved bladder overactivity as well as the impaired bladder blood flow.caused by bladder-outlet-obstruction.

Laser-capture microdissection for analysis of cell type-specific gene expression of muscarinic receptor subtypes in the rat bladder with cyclophosphamide-induced cystitis.

This study examined whether the laser-capture microdissection (LCM) method can achieve separation of urothelial cells from detrusor cells or superficial urothelial cells from intermediate/basal urothelial cells, using α-smooth muscle actin (SMA) and cytokeratin 20 (CK20). In addition, we investigated the changes in expression of muscarinic receptors in laser-captured urothelial and detrusor cells in rats with chronic cystitis. Female SD rats were injected with cyclophosphamide (75 mg/kg) intraperitoneally at day 1, 4, 7 and 10 to induce chronic cystitis. Saline was injected in the same protocol for controls. Bladder specimens were cut at 8 μm thickness, fixed in 70% ethanol and lightly stained by hematoxylin and eosin, and then superficial urothelium, intermediate/basal urothelium and detrusor muscles were laser-captured separately. Real-time PCR was performed to examine expressions of α-SMA, CK20, muscarinic 2 receptors (M2R) and muscarinic 3 receptors (M3R). The expression of α-SMA mRNA in detrusor muscle cells was 200 times higher than that in urothelial cells in controls. CK20 mRNA expression in apical urothelial cells was 55 times more than that in detrusor muscle and four times more than that in intermediate/basal urothelial cells. Expressions of M2R and M3R mRNA were increased in urothelial cells and decreased in detrusor muscles following chronic cystitis. The LCM could be useful for tissue collection of detrusor muscle and different layers of urothelial cells with minimal contamination of other cell types, and cell type-specific changes in molecular expression could accurately be analyzed. Increased expression of urothelial MR might enhance urothelial-afferent interactions to induce bladder overactivity/pain conditions associated with bladder inflammation.

High fat diet-induced molecular and physiological dysfunction of the urinary bladder

Obesity with its multiple comorbidities has become a global pandemia. We here report on the pathophysiological aspects of obesity-associated urinary bladder dysfunctions. Our results are based on multiple in vitro and in vivo studies including a high fat diet (HFD) rat animal model of which the details are given in the cited publications. In cultured human detrusor muscle cells, obesity-related pathophysiological mechanisms were directly induced by the saturated free fatty acid palmitate. In HFD animals, we found serious fibrosis of the bladder wall and downregulation of the muscarinic M3-receptor leading to diminished contractility of the urinary bladder. Bariatric surgical intervention (sleeve gastrectomy) reversed the fibrosis. Our results support the relevance of obesity for urological bladder dysfunction. The epidemic dimension of obesity with its steadily growing number of cases requires a re-evaluation of this pathological condition in the urological context.

Pathophysiology of idiopathic overactive bladder and the success of treatment: a systematic review from ICI-RS 2013.

To investigate the frequency of phenotype profiling of patients with idiopathic overactive bladder (OAB) syndrome, and to determine the effectiveness of treatment among individuals with different pathophysiologic profiles. The electronic databases MEDLINE, EMBASE, Cochrane CENTRAL, Cochrane Database of Systematic Reviews, and CINAHL were searched from January 1, 1980 to August 12, 2013 for interventional randomized controlled treatment trials (RCTs) of idiopathic OAB. Phenotying for pathophysiologies originating in the urothelial/mucosal layer of the bladder, the detrusor muscle cell layer, and the central nervous system were sought. Articles that analyzed urgency outcomes based on pathophysiologic profiling were selected. Due to the heterogeneity of the included interventions and outcome assessment measures, meta-analysis was not appropriate and a qualitative synthesis was undertaken. Of 239 original RCTs of idiopathic OAB, 48 (20%) profiled participants on underlying pathophysiology. Less than half of these (n = 20) reported treatment efficacy for urgency symptoms by pathophysiological sub-type. One examined the effect of botulinum A toxin on interstitial cell protein expression. Four compared treatment efficacy in OAB patients with and without involuntary detrusor contractions. Fifteen compared the effect of treatment on urgency reduction in patients with detrusor overactivity. There were no consistent trends in treatment efficacy according to pathophysiologic sub-type. No studies examined urothelial dysfunction or abnormal central processing of bladder afferent signaling in response to treatment. In order to advance the field of idiopathic OAB, more trials are needed that profile and test urgency outcomes in participants according to suspected underlying pathophysiology. Neurourol. Urodynam. 33:611-617, 2014. © 2014 Wiley Periodicals, Inc.

Adhesion molecules of detrusor muscle cells are influenced by a hypercholesterolemic diet or bladder outlet obstruction in a wistar rat model

BackgroundCell adhesion molecules (CAMs) are essential for maintaining tissue integrity by regulating intercellular and cell to extracellular matrix interactions. Cadherins and catenins are CAMs that are located on the cell membrane and are important for adherens junction (AJ) function. This study aims to verify if hypercholesterolemic diet (HCD) or bladder outlet obstruction (BOO) promotes structural bladder wall modifications specific to alterations in the expression of cadherins and catenins in detrusor muscle cells.MethodsForty-five 4-week-old female Wistar rats were divided into the following three groups: group 1 was a control group that was fed a normal diet (ND); group 2 was the BOO model and was fed a ND; and group 3 was a control group that was fed a HCD (1.25% cholesterol). Initially, serum cholesterol, LDL cholesterol and body weight were determined. Four weeks later, groups 1 and 3 underwent a sham operation; whereas group 2 underwent a partial BOO procedure that included a suture tied around the urethra. Six weeks later, all rats had their bladders removed, and previous exams were repeated. The expression levels of N-, P-, and E-cadherin, cadherin-11 and alpha-, beta- and gamma-catenins were evaluated by immunohistochemistry with a semiquantitative analysis.ResultsWistar rats fed a HCD (group 3) exhibited a significant increase in LDL cholesterol levels (p=0.041) and body weight (p=0.017) when compared to both groups that were fed a normal diet in a ten-week period. We found higher β- and γ-catenin expression in groups 2 and 3 when compared to group 1 (p = 0.042 and p = 0.044, respectively). We also observed Cadherin-11 overexpression in group 3 when compared to groups 1 and 2 (p = 0.002).ConclusionsA HCD in Wistar rats promoted, in addition to higher body weight gain and increased serum LDL cholesterol levels, overexpression of β- and γ-catenin in the detrusor muscle cells. Similar finding was observed in the BOO group. Higher Cadherin-11 expression was observed only in the HCD-treated rats. These findings may be associated with bladder dysfunctions that occur under such situations.

Changes in the Function and Expression of T-Type and N-Type Calcium Channels in the Rat Bladder after Bladder Outlet Obstruction

We evaluated possible changes in the function and expression of T-type and N-type Ca(2+) channels in the bladder of rats with bladder outlet obstruction. Female Sprague Dawley® rats were divided into a group with bladder outlet obstruction created by partial urethral ligation and a sham operated group. Six weeks postoperatively we determined the mRNA expression of T-type and N-type Ca(2+) channels in the bladder, dorsal root ganglion and spinal cord. We also cystometrically investigated expression by intravenous administration of the T-Ca blocker RQ-00311610 or the N-type Ca(2+) channel blocker ω-conotoxin GVIA. We then performed in vitro functional studies of detrusor strips using these blockers. mRNA expression of T-type Ca(2+) channels in the bladder detrusor and mucosa layers, and the spinal cord dorsal horn, and N-type Ca(2+) channels in the whole bladder and detrusor layer, and the spinal cord dorsal horn was greater in the obstructed group than the sham operated group. In obstructed rats bladder capacity and voided volume increased after RQ-00311610 administration but the number of nonvoiding contractions decreased after ω-conotoxin GVIA administration. Detrusor strips from obstructed rats showed weaker contractile responses to electrical field stimulation, particularly in regard to the purinergic component. ω-Conotoxin GVIA suppressed electrical field stimulation induced contractions only in the detrusor of obstructed rats, especially the cholinergic component. Blocking T-type Ca(2+) channels increased bladder capacity while N-type Ca(2+) channel blockade inhibited nonvoiding contractions in rats with bladder outlet obstruction. Decreased bladder efferent neurotransmission occurred after bladder outlet obstruction, predominantly in its purinergic component and detrusor contractions via cholinergic neurotransmission were activated in a compensatory manner, probably via N-type Ca(2+) channel up-regulation.

Expression and function of vascular endothelial growth inhibitor in aged porcine bladder detrusor muscle cells

Aging of the bladder detrusor muscle plays an important role in lower urinary tract symptoms in elderly people. Our previous work demonstrated that elderly patients have increased levels of vascular endothelial growth inhibitor (VEGI) in bladder tissue. Therefore, we hypothesized that VEGI may play a role in aging of the bladder detrusor muscle cells. This study aims to develop and characterize primary cultures of aged porcine bladder detrusor muscle cells in order to explore the expression and function of VEGI. Bladder samples from female pigs were divided into two groups: the aged group (Model) and the young group (Control). We confirmed β-galactosidase expression, a marker for senescence, in aged muscle cells (identified by α-smooth muscle actin (α-SMA) staining), but not in the young group. mRNA levels of VEGI-251 and death receptor 3 (DR3) were up-regulated (P<0.05) and total cell protein levels of VEGI-251, DR3 and nuclear factor-kappa B [NF-κB (p65)], membrane protein levels of DR3, and nuclear protein levels of NF-κB (p65) were significantly higher (P<0.01) in the Model cells compared to Control cells. In conclusion, we have established a method to culture aged detrusor muscle cells derived from porcine bladder. Higher levels of VEGI-251, DR3 and NF-κB (p65) were observed in the aged cells. VEGI-251 may function by increasing DR3 on cellular membranes and promoting the transfer of NF-κB into the nucleus. This suggests that VEGI may be a target for reversing the aging process of bladder detrusor muscle cells.

Urothelial signaling.

The urothelium, which lines the inner surface of the renal pelvis, the ureters, and the urinary bladder, not only forms a high-resistance barrier to ion, solute and water flux, and pathogens, but also functions as an integral part of a sensory web which receives, amplifies, and transmits information about its external milieu. Urothelial cells have the ability to sense changes in their extracellular environment, and respond to chemical, mechanical and thermal stimuli by releasing various factors such as ATP, nitric oxide, and acetylcholine. They express a variety of receptors and ion channels, including P2X3 purinergic receptors, nicotinic and muscarinic receptors, and TRP channels, which all have been implicated in urothelial-neuronal interactions, and involved in signals that via components in the underlying lamina propria, such as interstitial cells, can be amplified and conveyed to nerves, detrusor muscle cells, and ultimately the central nervous system. The specialized anatomy of the urothelium and underlying structures, and the possible communication mechanisms from urothelial cells to various cell types within the bladder wall are described. Changes in the urothelium/lamina propria ("mucosa") produced by different bladder disorders are discussed, as well as the mucosa as a target for therapeutic interventions.

Cajal-like interstitial cells as a novel target in detrusor overactivity treatment: true or myth?

IntroductionThe Cajal–like intestitial cells (ICCs) act as a pacemaker and are responsible for generating smooth muscle activity in the gastrointestinal tract (GI). Interstitial cells that resemble ICCs in the GI have been identified in the urinary bladder.Materials and methodsThis review is based on a systemic literature research. The medline/pubmed, scopus, embase, and Web of Science databases were browsed in order to identify original and review articles, as well as editorials relating to cajal–like cells, urinary bladder, detrusor overactivity, overactive bladder, glivec, etc. The controlled vocabulary of the Medical Subject Headings (MeSH) database was used to ensure the sensitivity of the searches. 40 papers met the criteria and were used for this review.ResultsCajal cells lie in close proximity to the muscle cells, autonomic nerve endings, and urothelial cells. There is increasing evidence that ICCs play role in urinary tract dysfunction development (e.g. detrusor overactivity, primary obstructive megaureter, congenital ureteropelvic junction obstruction, etc.). ICCs may be responsible for generating electrical potentials and induction of detrusor muscle contractions. Novel pathomechanisms of detrusor overactivity development have been postulated, as follows: 1) the disturbance of spontaneous contractility caused by altered signal transduction of ICCs between nerves and detrusor muscle cells, and 2). the alteration in signal transduction between urothelium and afferent nerve endings via suburothelial ICCs. The c–kit receptor is not only a detection marker of these cells, but may also play a crucial role in the control of bladder function.ConclusionsCajal cells in urinary bladder suggest that the c–kit receptor may provide a novel target for treating detrusor overactivity. The review presents the current knowledge of ICCs, its role in urinary bladder function, and potential novel therapeutic strategy.

Examination of the Presence and Differential Expression of Connexin Isoforms in the Urinary Bladder of the Male Rat During Postnatal Period

The coordinated action of the detrusor muscle cells in the urinary bladder is governed by cell-cell communication through gap junction, consisted of connexin (Cx) molecules. Even though a number of researches have been mostly focused on expressional changes of a few Cx isoforms in clinically dysfunctional condition of the bladder, less attention has been paid for investigation of Cx isoforms present in the bladder. Using real-time PCR analysis, the present study examined Cx isoforms expressing in the male rat bladder during postnatal period. Also, expressional patterns of Cx isoforms were evaluated in the bladder at different postnatal ages. Of a total of 13 Cx isoforms tested in the present study, we were able to detect mRNAs of 6 Cx isoforms in the rat urinary bladder, including Cxs 31, 31.1, 32, 37, 40, and 45. The transcript levels of Cxs 31, 31.1, 37, 40, and 45 were gradually increased from 1 week of age until 25 days of age, followed by transient decreases at 45 days of age. However, abundance of Cx32 transcript was drastically increased at 15 days of age, followed by a sharp drop at 45 days of age. These results indicate that differential expression of Cx isoforms in the bladder during postnatal development would be necessary for maintaining proper function of the bladder. A question remains to be answered if significant decreases of transcript levels of some Cx isoforms at the elderly are associated with age-dependent dysfunction of the bladder.

Acetylcholine Induces Human Detrusor Muscle Cell Proliferation: Molecular and Pharmacological Characterization

The purpose of the study is to understand whether the cholinergic stimulation is important, not only in inducing contraction of the detrusor muscle, but also in modulating the proliferation of smooth muscle cells. These results could help to better understand the role of antimuscarinic drugs, which are currently used for the treatment of many urological diseases. Primary cultures were prepared from biopsies of human detrusor muscle of subjects >65 years. From the cell culture set-up for each patient, mRNA was extracted and both the gene expression and the influence of increasing passages on the expression of muscarinic receptor subtypes were evaluated by semi-quantitative and quantitative PCR (RT-PCR and Q-RT-PCR). The rate of cell proliferation induced by cholinergic drugs was assessed by the evaluation of the [3H]-thymidine incorporation. The gene expression analysis demonstrated that the range of expression of muscarinic subtypes in human detrusor smooth muscle cells (HDSMCs) is M2 > M3 > M1 > M4 >> M5. The exposure to the cholinergic agonist carbachol induced a concentration-dependent increase in cell proliferation rate. The pharmacological characterization indicated that this effect was mainly mediated by the receptor subtypes M3 and M2. The cholinergic stimulation led to an increase in HDSMC proliferation, suggesting that this phenomenon might be involved in the pathogenic mechanism through which the cervico-urethral obstruction causes a detrusor hypertrophy, followed by a loss of function. These results could then provide an indication of the use of antimuscarinic drugs in the treatment of lower urinary tract disorders.

Calcineurin and Akt expression in hypertrophied bladder in STZ-induced diabetic rat

Diabetes causes significant increases in bladder weight but the natural history and underlying mechanisms are not known. In this study, we observed the temporal changes of detrusor muscle cells (DMC) and the calcineurin (Cn) and Akt expressions in detrusor muscle in the diabetic rat. Male Sprague–Dawley rats were divided into 3 groups: streptozotocin-induced diabetics, 5% sucrose-induced diuretics, and age-matched controls. The bladders were removed 1, 2, or 9weeks after disease induction and the extent of hypertrophy was examined by bladder weights and cross sectional area of DMC. Cn and Akt expression were evaluated by immunoblotting. Both diabetes and diuresis caused significant increases in bladder weight. The mean cross sectional areas of DMC were increased in both diabetic and diuretic animals 1, 2, or 9weeks after disease induction. The expression levels of both the catalytic A (CnA) and regulatory B (CnB) subunits of Cn were increased at 1 and 2weeks, but not at 9weeks. Expression of Akt was similar among control, diabetic, and diuretic rat bladder at all time points. In conclusion, diabetes and diuresis induce similar hypertrophy of detrusor muscle during the first 9weeks, indicating that bladder hypertrophy in the early stage of diabetes is in response to the presence of increased urine output in diabetes. Our results suggest that the Cn, but not the Akt signaling pathway may be involved in the development of bladder hypertrophy.

Porcine Bladder Urothelial, Myofibroblast, and Detrusor Muscle Cells: Characterization and ATP Release

ATP is released from the bladder mucosa in response to stretch, but the cell types responsible are unclear. Our aim was to isolate and characterize individual populations of urothelial, myofibroblast, and detrusor muscle cells in culture, and to examine agonist-stimulated ATP release. Using female pig bladders, urothelial cells were isolated from bladder mucosa following trypsin-digestion of the luminal surface. The underlying myofibroblast layer was dissected, minced, digested, and cultured until confluent (10–14 days). A similar protocol was used for muscle cells. Cultures were used for immunocytochemical staining and/or ATP release investigations. In urothelial cultures, immunoreactivity was present for the cytokeratin marker AE1/AE3 but not the contractile protein α-smooth muscle actin (α-SMA) or the cytoskeletal filament vimentin. Neither myofibroblast nor muscle cell cultures stained for AE1/AE3. Myofibroblast cultures partially stained for α-SMA, whereas muscle cultures were 100% stained. Both myofibroblast and muscle stained for vimentin, however, they were morphologically distinct. Ultrastructural studies verified that the suburothelial layer of pig bladder contained abundant myofibroblasts, characterized by high densities of rough endoplasmic reticulum. Baseline ATP release was higher in urothelial and myofibroblast cultures, compared with muscle. ATP release was significantly stimulated by stretch in all three cell populations. Only urothelial cells released ATP in response to acid, and only muscle cells were stimulated by capsaicin. Tachykinins had no effect on ATP release. In conclusion, we have established a method for culture of three cell populations from porcine bladder, a well-known human bladder model, and shown that these are distinct morphologically, immunologically, and pharmacologically.

Sex- and Age-Related Changes in Connexin 43 Expression in Normal Rat Bladder

PurposeGap junctions are intercellular channels to facilitate electrical and metabolic communication between adjacent cells. Connexin 43 is the most predominant type of connexin expressed on rat detrusor muscle cells. We investigated the connexin 43 expressions in various age groups of either sex in normal rats.MethodsEighty Sprague-Dawley rats were used for analysis. Each group was quantified by 8 rats at 1 week, 2 weeks, 1 month, 3 months, and 6 months of age in either sex. In each animal, bladder was removed without any kind of intervention and fresh-frozen in liquid nitrogen. Total RNA extraction was done with easy-BLUE total RNA extraction kit. Reverse transcription polymerase chain reaction was done for connexin 43 and glyceraldehyde-3-phosphate dehydrogenase as an internal control using ImProm-II Reverse Transcription System.ResultsIn female rats, no age-related change was detected in connexin 43 expressions. In male rats, connexin expression at 3 months of age showed significant decrease compared with 1 week, 2 weeks, and 6 months of age (P<0.05). When connexin expression at the same age in male and female were compared, only 3 months group in male showed significant decrease than the same age group in female.ConclusionsOur data suggest that the expressions of connexin 43 mRNA in normal detrusor muscle cell showed age-related changes especially in male rats. Although it is difficult to interpret these findings at this stage, age should be considered as a possible compounding factor affecting connexin 43 expressions in male rats.

Partial urethral obstruction: ATF3 and p-c-Jun are involved in the growth of the detrusor muscle and its motor innervation

Infravesical obstruction leads to growth of urinary bladder smooth-muscle cells. The ganglion cells innervating the bladder muscle also increase in size. Stretch of detrusor muscle cells rapidly activates c-Jun NH₂-terminal kinase (JNK), which phosphorylates the transcription factor c-Jun, and stimulates the synthesis of the cotranscription factor ATF3. The aim of the study was to determine whether ATF3 and p-c-Jun were involved in growth of bladder smooth-muscle and ganglion cells. The urethra was partially obstructed in female rats. After 3 days or 10 weeks bladders were weighed, fixated and cut for immunohistochemistry to demonstrate ATF3 and p-c-Jun. Ganglia were processed separately. Unoperated and sham-operated rats were used as controls. There was no ATF3 or p-c-Jun in control detrusor muscle. After 3 days of obstruction bladder weight had nearly doubled. Almost all nuclei in the detrusor showed immunofluorescence for ATF3 and p-c-Jun. After 10 weeks bladder weight had increased 10-fold. Almost all detrusor nuclei still showed p-c-Jun, but few had ATF3 activity. In control ganglia there was no ATF3 and only faint nuclear p-c-Jun activity. After 3 days of obstruction the ganglion cells had increased in size and many nuclei showed intense immunofluorescence for ATF3 and p-c-Jun. After 10 weeks the ganglion cell size had increased further. There was no ATF3 activity and no more p-c-Jun than in control ganglia. ATF3 and p-c-Jun seem to be involved in the growth of the detrusor muscle and its motor innervation following infravesical outlet obstruction.

Effect of ischemia/reperfusion on bladder nerve and detrusor cell damage

Ischemia, reperfusion, and subsequent free radical damage have been implicated in many voiding disorders. Our goal was to investigate further the mechanisms of these disorders, with particular emphasis on nerve and mitochondrial function and on detrusor smooth-muscle cells. The effects on contractile responses to various stimulations, citrate synthase, choline acetyltransferase activities, and vesicular acetylcholine transporter were evaluated after ischemia alone and ischemia/reperfusion 2 h, 7 days, and 14 days. Nerve density and detrusor cell apoptosis were also measured. The contractile responses were significantly decreased at both 7 and 14 days reperfusion, although at 14 days some recovery was observed. Similar patterns were seen for the intramural nerves, both nerve cell cytoskeletal structures and cholinergic neurotransmitters. Citrate synthase activity was also depressed by ischemia and 2 h reperfusion, but the activity recovered by 7 days. Detrusor cell apoptosis was not significantly affected by ischemia and 2 h reperfusion; but showed an approximately 14-fold increase at both 7 and 14 days reperfusion. Reperfusion following ischemia resulted in worsening intramural bladder nerve dysfunction, nerve fiber injury, mitochondrial injury, and damaged detrusor muscle cells. However, at 14 days reperfusion, nerve and mitochondrial regeneration occurred and resulted in partial recovery of contractile function.