Regional differences in bladder enlargement and in vitro contractility after outlet obstruction in the rabbit

PURPOSE: Bladder outlet obstruction leads to bladder enlargement and subsequent decreases in contractile function in vivo and in vitro. We determined whether there were regional differences in bladder wall properties and in vitro contractile responses after 2 weeks of bladder outlet obstruction. MATERIALS AND METHODS: Male rabbits underwent cystometry. The bladder was then filled to 40 ml. and the surface was marked with 2-zero silk knots placed approximately 1 cm. apart. The distance between the knots was measured at 20, 40 and 80 ml. The animals then underwent the creation of surgical obstruction. After 2 weeks the obstruction was removed. Cystometry and measurements were repeated and strips were obtained from defined dorsal and ventral areas. Contractile responses to electrical field stimulation, adenosine triphosphate, carbachol and KCl were determined and compared with strips from unobstructed controls. RESULTS: In vivo expansion during bladder filling occurred evenly throughout the bladder wall in controls and the contractile response to all stimuli was similar in ventral and dorsal strips. After 2 weeks of bladder outlet obstruction the upper dome expanded to a significantly higher degree than the lower bladder body. The response to all stimuli was significantly reduced after bladder outlet obstruction and there was a significantly decreased response to all stimuli in dorsal compared with ventral strips. Strips from the dorsal midline showed a relaxation response to electrical field stimulation at low frequencies, whereas all ventral strips contracted. CONCLUSIONS: Functional remodeling after bladder outlet obstruction is a process that does not occur to the same extent throughout the bladder. The obstructed bladder is an inhomogeneous organ with significant regional differences in mechanical and pharmacological properties.     

Lipid signaling changes in smooth muscle remodeling associated with partial urinary bladder outlet obstruction

AIMS: Hypertrophy of the urinary bladder smooth muscle (detrusor) is associated with partial bladder outlet obstruction (PBOO). Hypertrophied detrusor smooth muscle (DSM) reveals altered contractile characteristics. In this study, we analyzed the lipid-dependent signaling system that includes phospholipase A2 in PBOO-induced DSM remodeling and hypertrophy to determine whether the release of arachidonic acid (AA) from phospholipid is altered in the detrusor. METHODS: Partial bladder outlet obstruction (PBOO) was produced by partial ligation of the urethra in New Zealand white rabbits. Two weeks after the surgery, the bladder function was studied by keeping the rabbits in metabolic cages for 24 hr. Bladders were removed from rabbits that had bladder dysfunction (increased urinary frequency and decreased void volume) and the DSM separated from mucosa and serosa. The isolated smooth muscle was incubated with [3H] AA to equilibrate the cytoplasmic AA. The level of AA release was compared with the level obtained with 2-week sham-operated rabbits. RESULTS: The rate of AA release was high in DSM from bladders with PBOO-induced hypertrophy. Carbachol stimulated AA release in control DSM but DSM from obstructed rabbits revealed no further increase from the elevated basal AA release. The half-maximal concentration of carbachol that was required to stimulate AA release from control samples of detrusor was 35 microM. CONCLUSIONS: The increased levels of AA release that are observed in this tissue after PBOO indicate the activation of phospholipase A2. The finding that carbachol could induce contraction, but not an increase in AA, indicates that the carbachol-induced contraction in the obstructed bladders is independent of lipid signaling pathways that involve AA. It is possible that the increased rate of arachidonic acid release from obstructed bladders correlates with the enhanced rates of prostaglandin production reported by other investigators from the same tissue.     

Contractile protein expression in bladder smooth muscle is a marker of phenotypic modulation after outlet obstruction in the rabbit model

PURPOSE: We determined changes in contractile protein expression before and after the relief of partial bladder outlet obstruction in the rabbit model and assessed their potential role as predictors of recovery. MATERIALS AND METHODS: We examined the ratio of the smooth muscle myosin heavy chain isoforms SM2-to-SM1, caldesmon isoform expression and bladder function in obstructed and unobstructed adult rabbit bladders. Cystometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis were done to determine changes in bladder function and contractile protein expression. RESULTS: Overall we observed significant correlation of bladder weight with the SM2-to-SM1 ratio (p <0.05). Regardless of the duration of obstruction (up to 10 weeks) the ratio appeared to stabilize around a value comparable to that in fetal rabbit smooth muscle cells, suggesting a reversal of SM2 and SM1 expression to a level similar to that at the fetal stage. The pattern of h and l-caldesmon isoform expression showed an increase in l-caldesmon expression in obstructed bladders. Except for decreased leak point pressure in the obstructed group we noted no statistically significant urodynamic changes in bladder capacity or compliance. CONCLUSIONS: There is significant correlation of bladder weight, which is the best known marker of obstruction, with the SM2-to-SM1 ratio. The myosin heavy chain isoform expression ratio appears to be an indicator of phenotypic modulation in bladder smooth muscle before and after the relief of bladder outlet obstruction. Thus, it may be useful as a marker of bladder dysfunction and predictor of functional recovery. Regression to a fetal pattern of protein expression may suggest irreversible damage to smooth muscle cells, possibly limiting recovery.     

Effect of bladder outlet obstruction on detrusor smooth muscle cell: an in vitro study

BACKGROUND: Although relieving obstruction is generally curative on bladder outlet obstruction (BOO), bladder dysfunction persists in some patients. Repetitive stretch and relaxation applied to cultured bladder smooth muscle (SM) cells in vitro have been used to mimic increases in urodynamic load experienced by the detrusor muscle under conditions of BOO. We first clarified the relationship between phenotype transformation and biomechanical properties of detrusor smooth muscle cell (DSMC) subjected to the cyclic mechanical stretch. MATERIALS AND METHODS: Cultured rat DSMC were grown on collagen-coated silicone membranes and subjected to continuous cycles of stretch-relaxation. All experiments were performed on cells between passages 2 and 4. Each cycle consists of 5 seconds of stretch and 5 seconds of relaxation. The computer controlled vacuum induced 10% (1), 20% (2), and 30% (3) maximum elongation of the plate membrane at different designed pressures. The deoxyribonucleic acid synthesis rate was assessed by performing tritiated thymidine incorporation assay. The expression of SM-alpha-actin and proliferation of DSMC were analyzed by immunofluorescent assay and flow cytometry. The image analysis and micropipet aspiration systems were used to investigate the single cell contraction and viscoelasticity. Using the 3-element standard linear solid model, the elastic modulus K(1), K(2), and viscoelastic coefficient mu were determined, which show the passive deformation ability of detrusor cells. RESULTS: As the basic structural changes to mechanical stretch, DSMC undergo phenotypic modulation from their normal contractile phenotype to a "synthetic" phenotype: the DSMC become more proliferative and the actin less organized along the cell's long axis. The cell proliferation index of control and stretched group (10%, 20%, 30% elongation) are 0.24, 0.43, 0.58, and 0.65, respectively. The actin filaments in unstimulated cells were evident and orientated along the major axis of the cell. After mechanical stretch, the well-spread filaments changed their orientation. The function, such as contraction, and viscoelasticity of a single DSMC subjected to stretch both decreased significantly compared with control. The maximum contractile velocity and maximum cell length shortening rate of group 3 (30% elongation) showed significant decreases compared with unstretched control (P < 0.01). K(1) and K(2) were decreased with the increase of mechanical overload. However, there was no statistic difference between groups 2 and 3. CONCLUSIONS: Functional abnormalitie of BOO have the structural basis: phenotype transformation (i.e., remodeling) of the detrusor cells. Cyclic stretch and relaxation applied to DSMC in vitro can be used to model increases in urodynamic load experienced by the bladder detrusor muscle under conditions of BOO. Phenotype transformation is the structural basis of functional changes of DSMC subjected to periodic overload mechanical stretch.     

The relationship of detrusor instability and symptoms with objective parameters used for diagnosing bladder outlet obstruction: a prospective study

PURPOSE: Detrusor instability is a common urodynamic finding in patients with prostatic obstruction. In prospective fashion we evaluated detrusor instability in patients with lower urinary tract symptoms attributable to benign prostatic hyperplasia and determined its possible association with the degree of obstruction. MATERIALS AND METHODS: A total of 459 men with a mean age plus or minus standard deviation of 60.4 +/- 9.4 years who were investigated for lower urinary tract symptoms at our facility answered an Arabic standardized version of International Prostate Symptom Score and underwent simple uroflowmetry, outpatient cystoscopy and transrectal ultrasound. Invasive urodynamics, including filling and voiding cystometry, was done with pressure flow analysis according to the Sch?fer nomogram. Statistical significance was tested by the Mann-Whitney U and Wilcoxon rank sum tests. RESULTS: Of the 459 patients 108 (23.5%) had detrusor instability. Instability significantly affected patient symptom score and conception of quality of life. Moreover, instability significantly affected the degree of obstruction, as measured by the maximum flow rate, post-void residual urine, prostate volume and Sch?fer grade of obstruction. CONCLUSIONS: Detrusor instability affects patient symptoms and quality of life. It also signifies a more severe degree of obstruction in male patients with lower urinary tract symptoms and bladder outlet obstruction due to benign prostatic hyperplasia.     

Effect of oral Tadenan treatment on rabbit bladder structure and function after partial outlet obstruction

PURPOSE: There is increasing evidence that the progressive dysfunction induced by partial outlet obstruction is mediated by ischemia-reperfusion, and bladder decompensation results from ischemia-reperfusion induced damage to the cellular and subcellular organelle membranes of nerve and smooth muscle, mitochondria and sarcoplasmic reticulum. Tadenan, an extract of Pygeum africanum, is a therapeutic prescribed in Europe to relieve symptoms of obstructive bladder dysfunction secondary to benign prostatic hyperplasia. There is excellent experimental evidence that Tadenan treatment of obstructed rabbits reduces and reverses the progression of bladder decompensation. We determined whether Tadenan therapy can reverse the morphological damage associated with obstructive dysfunction. MATERIAL AND METHODS: A total of 36 male New Zealand White rabbits were separated into 6 groups of 6 each. Rabbits in groups 1 and 2 underwent sham operation. For 3 weeks beginning 2 weeks after sham operation group 1 was treated with vehicle and group 2 was treated with 30 mg./kg. Tadenan daily. Rabbits in groups 3 to 6 underwent partial outlet obstruction surgery. Two weeks after obstruction each rabbit was treated for 3 weeks with vehicle in group 3, and with 1, 10 and 30 mg./kg. Tadenan in groups 4, 5 and 6, respectively. After the completion of treatment cystometry was performed on each rabbit and isolated bladder strips were evaluated for contractile responses to field stimulation, adenosine triphosphate, carbachol and KCl. Separate strips were fixed for electron microscopy to determine the location and severity of cellular and subcellular membrane damage. RESULTS: Partial outlet obstruction resulted in reduced compliance, decreased responses of bladder strips to all forms of stimulation tested, and significant and extensive damage to cellular and subcellular organelle membranes consistent with an ischemia-reperfusion etiology. Daily 1 and 10 mg./kg. Tadenan treatments had little effect on the obstruction induced increase in bladder weight or the deleterious changes in bladder function and structure. However, treating obstructed rabbits with 30 mg./kg. Tadenan daily resulted in reduced bladder hypertrophy, improved compliance, improved contractile responses to nearly normal levels of isolated bladder strips to all stimuli tested and reversal of obstruction induced structural damage to cellular and subcellular organelle membranes. CONCLUSION: Tadenan treatment of obstructed rabbits resulted in a dose dependent improvement in bladder ultrastructure in parallel with improved bladder compliance and contractile responses of isolated strips to stimulation, providing support for the hypothesis that damage to cellular and subcellular organelle membranes mediates the contractile dysfunction induced by partial outlet obstruction.     

Characterization of neuropathic bladder smooth muscle cells in culture

PURPOSE: Clinically bladder cells used in tissue engineering techniques will come from neuropathic bladders and not normal bladders. We determined if neuropathic bladder smooth muscle (SM) cells (SMCs) retain functional differences when cultured in vitro. MATERIALS AND METHODS: Primary cultures of SMCs were established from patients with a neuropathic bladder (5) and a normal bladder (5). Expression of alpha-SM actin and SM myosin heavy chain was determined using immunocytochemical staining and Western blot analysis. Baseline cell proliferation and the mitogenic response to angiotensin II was assessed by cell counting and cell viability assays. Cell contractility was determined for normal and neuropathic SMCs using an in vitro collagen lattice assay. Cell adherence was measured assessed using partial and complete trypsinization assays. RESULTS: Normal and neuropathic SMCs showed similar morphology in culture, and similar patterns of alpha-SM actin and SM myosin expression. Following 10 days of plating under optimal growth conditions the number of neuropathic SMCs was 170% more than normal SMCs. In response to angiotensin II neuropathic SMCs reached 54% of maximal growth capacity as opposed to 30% for normal SMCs (p <0.01). Neuropathic SMCs contracted significantly less in 10% serum and calcium ionophore (p <0.05), as determined by in vitro contractility assays. Neuropathic SMCs had 19% and 30% less adherent cells than normal SMCs (p <0.01) following isotonic solution washes and trypsinization, respectively. CONCLUSIONS: These results demonstrate that cultured neuropathic bladder SMCs possess and maintain different characteristics than normal SMCs in vitro. The potential clinical implications of using these cells in conjunction with tissue engineering techniques for the promotion of bladder regeneration requires further investigation.     

Changes in the smooth muscle of the corpora cavernosum related to reversal of partial bladder outlet obstruction in rabbits

Previous studies have demonstrated that partial bladder outlet obstruction (PBOO) in the rabbit induces an increase in corpus cavernosum smooth muscle (CCSM) tone, which may make it difficult for the CCSM to relax. Thus, to determine whether the corpus cavernosum restores relaxation after reversal of PBOO, we investigated the physiologic, histologic, and cell biology in penises obtained from rabbits 4 weeks and 8 weeks after reversal of PBOO. CCSM from bladder outlet-obstructed and obstruction-reversed rabbits showed significant decreases in the contractile responses to phenylephrine. The relaxation responses to electrical field stimulation (EFS), ATP, acetylcholine, and sodium nitroprusside (SNP) were decreased in obstructed and reversed for 4 weeks groups. By 8 weeks of reversal, the relaxation of CCSM was increased gradually in response to EFS, SNP, and acetylcholine. However, the response to ATP did not result in the relaxation of CCSM to control levels. The ratio of SM to collagen decreased after obstruction and remained low after reversal. Expression of both isoforms of Rho kinase (ROK) was increased in obstruction groups. At 4 weeks of reversal, the expression of ROK alpha remained at obstruction level, whereas ROK beta expression decreased in comparison with the obstruction group. By 8 weeks of reversal, expression of both ROK alpha and beta significantly decreased when compared with the obstruction group. These results suggested that the poor relaxation response at reversal of 4 weeks was associated with incomplete decreased expression of both isoforms of ROK, whereas the incomplete recovery of the CCSM relaxation response at reversal of 8 weeks may be associated with structural alterations in the CC and irreversible damage from PBOO.     

Effects of neferine on cytosolic free calcium concentration in corpus cavernosum smooth muscle cells of rabbits

To study the relaxation mechanisms of neferine (Nef) on the corpus cavernosum smooth muscle (CCSM), the CCSM cells from New Zealand White rabbits were cultured in vitro. [Ca(2+)](i) was measured by fluorescence ion digital imaging system (FIDIS), using Fluo-2/AM as a Ca(2+)-sensitive fluorescent indicator. Nef (0.1, 1 and 10 micromol l(-1)) had no effect on the resting [Ca(2+)](i) (P > 0.05). In the presence of extracellular Ca(2+) (2.5 mmol l(-1)), Nef (0.1, 1 and 10 micromol l(-1)) inhibited [Ca(2+)](i) elevation induced by high K(+) and phenylephrine (PE) in a concentration-dependent manner (P < 0.05). In calcium free solution containing egtaic acid (EGTA), Nef (0.1 micromol l(-1)) had no inhibitory effects on [Ca(2+)](i) elevation induced by PE (P > 0.05). However, Nef (1 and 10 micromol l(-1)) inhibited [Ca(2+)](i) elevation induced by PE (P < 0.05). These data suggest that Nef inhibited [Ca(2+)](i) in CCSM cells via blocking voltage-dependent Ca(2+) channel, alpha(1)-adrenoceptor-operated Ca(2+) channel and Ca(2+) release from intracellular Ca(2+) pool. This inhibitory action on [Ca(2+)](i) might be one of the relaxation mechanisms of Nef on the CCSM.     

Construction of cavernosum smooth muscle using umbilical artery smooth muscle cells seeded on acellular corporal collagen matrices

The objective of this study was to investigate the feasibility of tissue engineering of corpus cavernosal smooth muscle. Acellular corporal collagen matrices (ACCMs) were obtained from the penis of adult rabbits by a cell removal procedure. ACCMs were implanted into the back muscles of allogenic rabbits to investigate the resulting immunological reaction. Human umbilical artery smooth muscle cells (HUASMCs) were isolated from human umbilical arteries through explant techniques and expanded in vitro . Subsequently, third and fifth passage HUASMCs were seeded to ACCMs at a concentration of 30 × 10⁶ cells/mL. Then, seeded ACCMs were implanted subcutaneously in athymic mice. The implants were retrieved at 10, 20 and 40 days after implantation. Histochemistry, immunohistochemistry and scanning electron microscopy were performed to analyse the morphological characteristics of the engineered tissues. Additionally, organ bath studies were performed to address the contractility of the engineered tissues. The decellularization process successfully extracted all cellular components while preserving the original collagen fibers. The immunological reaction to ACCMs consisted of only a transient nonspecific inflammatory response. Light and scanning electron microscopy demonstrated that HUASMCs extended onto the three-dimensional ACCMs scaffolds in vitro . Histological analyses of the explants from all time points demonstrated a progressive regeneration of smooth muscle, with structures very similar to native corpus cavernosum smooth muscle. The maximum contraction force induced by phenylephrine and electrical stimulation were 3.64 ± 0.18 g/100 mg and 2.50 ± 0.21 g/100 mg, respectively. Our study demonstrates that HUASMCs can be seeded on three-dimensional ACCM scaffolds and will develop tissues similar to that of the native corpus cavernosum smooth muscle.     

人阴茎海绵体平滑肌细胞三维培养模型的建立

目的 观察人阴茎海绵体平滑肌细胞(hCCMCs)在三维培养系统中的生物学特性.方法 体外分离hCCMCs,并用差速贴壁法进行纯化.免疫组化的方法检测α-平滑肌肌动蛋白(α-SMA)的表达,进行hCCMCs的鉴定.在单层贴壁培养的基础上,将hCCMCs在胶原凝胶中培养,形成三维培养系统,并对hCCMCs的形态结构、增殖情况进行研究.结果 原代培养的hCCMCs细胞形态不均一,经过差速贴壁法纯化后,细胞形态均一,免疫组化显示大多数细胞α-SMA阳性表达.三维培养系统中,hCCMCs在不同层面上呈三维生长.hCCMCs在三维培养系统中培养10d后,细胞数量无明显增殖(P>O.05),明显低于贴壁培养条件下细胞增殖速率(P<0.01).结论 hCCMCs三维培养系统可以观察hCCMCs在三维条件下的生长、增殖状况,研究hCCMCs与微环境的相互作用关系,有望为相关研究找到一种更为简单、可控性更强的体内实验替代方法.     

兔阴茎海绵体平滑肌细胞的快速分离及鉴定

目的探讨兔阴茎海绵体平滑肌细胞快速分离方法,为应用膜片钳技术研究阴茎勃起机制提供实验材料.方法采用木瓜蛋白酶和胶原酶两步酶解消化法,快速分离出新西兰大白兔阴茎海绵体平滑肌细胞并应用免疫组化鉴定.结果分离的细胞成活率较高,贴壁呈长梭形,胞膜光滑完整,胞浆均匀,可用于膜片钳记录.免疫组化鉴定为兔阴茎海绵体平滑肌细胞.结论酶解消化快速分离兔阴茎海綿体平滑肌细胞为全细胞膜片钳技术研究阴茎勃起功能障碍的电生理机制提供了较好的实验材料.     

低氧对SD大鼠阴茎海绵体平滑肌纤维化的影响

目的:探讨低氧对SD大鼠阴茎海绵体平滑肌纤维化的影响。方法:体外培养阴茎海绵体平滑肌细胞,免疫组化鉴定细胞;常规氧浓度(21%O2浓度)分别培养12、24、48、72h作为对照,低氧(1%O2浓度)干预12、24、48、72h,RT-PCR分别测定各组TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量。结果:体外培养的阴茎海绵体平滑肌细胞生长良好,抗平滑肌α-肌动蛋白单克隆抗体免疫组化染色阳性;RT-PCR结果提示TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量在48h内与低氧时间成正相关,时间进一步延长不能增加其相对表达量。结论:在低氧环境下,SD大鼠阴茎海绵体平滑肌细胞的TGF-β1、Ⅰ型胶原、Ⅲ型胶原的相对表达量随时间的延长逐渐增加,48h达到最大值。低氧可导致SD大鼠阴茎海绵体平滑肌纤维化。     

Rabbit urinary bladder blood flow changes during the initial stage of partial outlet obstruction

PURPOSE: The rabbit urinary bladder's early response to partial outlet obstruction includes bladder wall remodeling with marked urothelial and fibroblast hyperplasia (1 day) and smooth muscle hypertrophy (3-5 days) resulting in a 4-5 fold increase in bladder mass within 7 days. In this study, we examined the effect of partial outlet obstruction on bladder blood flow during the initial period of rapid growth (1-7 days). MATERIALS AND METHODS: Each New Zealand White rabbit was partially obstructed by tying a 2-0 silk ligature loosely around the vesical outlet. After 0 (unoperated), 4 hours, 1, 3, or 7 days of obstruction, 5 rabbits per group were anesthetized and the carotid and femoral arteries cannulated with polyethylene tubing. Additional rabbits receiving sham surgeries were treated like obstructed animals at 4 hours and 1 day post-obstruction (5/group). Using standard methods, fluorescent microspheres were infused through the right carotid artery. Bladder and right kidney were rapidly removed upon completion of sphere infusion; bladder mucosa and muscle were separated. Sphere densities in detrusor, mucosa, and kidney were measured by Interactive Medical Technologies, Ltd. A section of each detrusor tissue was fixed in formalin and immunostained for smooth muscle alpha-actin. RESULTS: Mucosal blood flow (0.20 +/- 0.03 ml./min./gm.) was approximately 4-fold greater than that of detrusor (0.05 +/- 0.01 ml./min./gm.). Sham surgery caused a significant increase in bladder blood flow at 4 hours post-obstruction that returned to control levels by 1 day. Both mucosal and muscle blood flows were slightly higher in rabbit bladders obstructed for 4 hours than in sham-operated rabbits, and substantially greater in those obstructed for 1 day: 0.68 +/- 0.13 ml./min./gm. (mucosa) and 0.26 +/- 0.04 ml./min./gm. (muscle). Blood flows returned to control values by 3 days post-obstruction and remained constant through 7 days. Kidney blood flow was unchanged. Although bladder weight increased 4-fold after 7 days of obstruction, the volume fraction of smooth muscle (transverse section) remained constant at approximately 40%. CONCLUSIONS: Blood flow was approximately 4-fold greater in bladder mucosa than in muscle, which may relate to the significantly higher metabolic rate and lower high energy phosphate concentration of mucosa than muscle. Partial outlet obstruction resulted in a significant increase in blood flow at 1 day post-obstruction, which coincides temporally with the early cellular hyperplasia and hypertrophy of obstructed rabbit bladder. This increase in blood flow may be an essential factor for the initial increase in bladder mass. By three days, the blood flow per gram of tissue returned to control levels. The mechanisms relating to the changes in blood flow induced by partial outlet obstruction are currently under investigation.     

Microarray analysis of bladder smooth muscle from patients with myelomeningocele

OBJECTIVE

To examine whether gene profiles can provide a molecular evaluation of the quality and therapeutic potential in patients with myelomeningocele (MM), by comparing genetic profiles of smooth muscle cells (SMCs) from healthy bladders and bladders from patients, to identify genes that are over‐ and under‐expressed in MM bladder SMCs.

MATERIAL AND METHODS

Bladder SM biopsies were obtained from ‘healthy’ subjects undergoing bladder surgery for vesico‐ureteric reflux and from patients with a neurogenic bladder secondary to MM. Bladder SMCs were expanded in vitro and total RNA was isolated and hybridized to gene chips to evaluate the differential expression levels of 22 283 genes. Differentially expressed genes were identified by two methods. In the first analysis, we directly compared raw data sets of healthy SMCs to those derived from patients with MM. In the second analysis, we indirectly compared healthy SMCs and MM SMCs to a reference file, to create a genetic signature of genes that are over‐ and under‐expressed in MM SMCs.

RESULTS

The direct analysis identified 240 genes that were over‐expressed and 104 that were under‐expressed in MM SMCs. Gene ontology classifications were used to identify biological themes and pathways. Genes that were over‐expressed in MM SMCs were involved in development: mesenchyme homeobox 2 (‐fold change, 9.3); bone morphogenic protein 6 (4.0); fibroblast growth factor 2 (4.8); inhibin A (4.2), cartilage oliogomeric matrix protein (9.97); collagen 11A (6); collagen 5A2 (3) and collagen 1A1 (2.18). The indirect analysis identified 665 genes that were over‐expressed and 1343 that were under‐expressed in MM SMCs. Pathway‐based analysis of these genetic signatures showed an over‐expression of genes involved in muscle development and focal adhesion/extracellular matrix interactions. Genes that were under‐expressed in MM SMCs were mapped to muscle contraction, transmission of nerve impulses, and cell‐cell adhesion pathways.

CONCLUSION

Our results are consistent with previous studies showing that MM bladders have an excess of extracellular matrix deposition, improper contraction, and are developmentally immature relatively to healthy SMCs. The clinical implication of microarray analysis of MM SMCs is that it provides potential targets that could induce muscle differentiation and inhibit extracellular matrix production.     

Partial bladder outlet obstruction induces urethral smooth muscle hypertrophy and decreased force generation

PURPOSE: PBOO leads to increased urinary frequency, decreased void volume, hypertrophy of the detrusor SM, and alterations in contractile and regulatory proteins. This study was done to determine whether PBOO induced increases in urinary frequency and detrusor SM hypertrophy are associated with an alteration in the contractility and expression of myosin isoforms in urethral SM. MATERIALS AND METHODS: PBOO was surgically induced in male New Zealand White rabbits, and sham operated rabbits served as controls. After surgery, rabbits were kept 12 days, and prior to sacrifice, urine output and voiding frequency were monitored by keeping the animals in metabolic cages for 24 hours. Animals with increased urinary frequency (mean +/- SEM 43 +/- 12 voids per 24 hours) and sham operated rabbits (6 +/- 3 voids per 24 hours) were used for this study. Morphology of the urethra was studied using light and immunofluorescence microscopy. The expression of myosin isoforms was analyzed at the mRNA and protein levels by RT-PCR and Western blotting. RESULTS: The urethral wall and SM of PBOO rabbits showed hypertrophy. The force produced by the longitudinal muscle strips of PBOO animals in response to phenylephrine, KCl, or electrical field stimulation was decreased 50%, 37% and 40%, respectively. Immunofluorescence microscopy revealed a decrease in nerve density. RT-PCR and Western blotting showed a decrease in the expression of myosin isoform SM-B with a concomitant increase in SM-A at the mRNA and protein levels. CONCLUSIONS: Our data show hypertrophy of the urethral wall and SM, and alterations in contraction, innervation, and myosin isoforms in PBOO induced detrusor hypertrophy.     

粉防己碱(Tet)对兔子阴茎海绵体平滑肌细胞质中自由钙离子浓度的影响(英文)

目的:探讨粉防己碱(tetrandrine,Tet)松弛阴茎海绵体平滑肌的作用机制。方法:体外培养新西兰白兔阴茎海绵体平滑肌细胞,经钙荧光指示剂 Fluo-2/AM 负载后,用荧光离子数字成像系统观察 Tet 对平滑肌细胞内[Ca~(2 )]_i 的影响。结果:Tet(1,10,100μmol/L)对平滑肌细胞内静息[Ca~(2 )]_i无明显影响(P>0.05)。当细胞外钙离子浓度为2.5 mmol/L 时,Tet(1μmol/L,10 μmol/L,100 μmol/L)抑制了高钾和去氧肾上腺素(PE)导致的细胞内[Ca~(2 )]_i 升高(P<0.05),这种抑制作用具有浓度依赖性。在无细胞外钙时,1 μmol/L和10μmol/L Tet 对 PE 引起的细胞内[Ca~(2 )]_i 升高无明显影响(P>0.05);而100 μmol/L Tet 能明显抑制 PE 引起的细胞内[Ca~(2 )]_i 升高(P<0.05)。结论:Tet 通过阻滞电压依赖性钙通道、α_1受体依赖性钙通道和抑制细胞内钙库释放,降低阴茎海绵体平滑肌细胞内[Ca~(2 )]_i 水平,这是 Tet 松弛阴茎海绵体平滑肌的作用机制之一。