Suppression of detrusor overactivity in rats with bladder outlet obstruction by a type 4 phosphodiesterase inhibitor

OBJECTIVE: To investigate the effects of a selective type 4 cyclic nucleotide phosphodiesterase (PDE4) inhibitor, IC486051, on bladder activity in normal rats and those with and bladder outlet obstruction (BOO), as inhibition of PDE4 leads to elevation of intracellular cAMP levels and relaxation of smooth muscle. MATERIALS AND METHODS: BOO was induced in female Sprague-Dawley rats by tying a silk ligature around the urethra. At 4 or 6 weeks after inducing BOO, conscious rats were assessed by cystometry with the urethral ligature intact. In unobstructed rats, blood pressure was also measured. RESULTS: In unobstructed rats, IC486051 (0.1 mg/kg intravenously) produced no significant changes in cystometric variables, while at a dose of 0.5 mg/kg maximum voiding pressure was reduced by 34%. At both doses, there was a small, transient increase in blood pressure. In both 4- and 6-week BOO rats IC486051 dose-dependently decreased the number and amplitude of non-voiding bladder contractions by up to 80%, relative to pre-treatment values. At doses of 0.1 and 0.5 mg/kg IC486051 had no significant effect on voiding variables. In the 4-week BOO rats, a dose of 1.0 mg/kg decreased bladder capacity, voided volume and residual volume by 21%, 32% and 18%, respectively. In 6-week BOO rats, a dose of 1.0 mg/kg decreased maximal voiding pressure by 17% and pressure threshold for voiding by 28%. In both groups of rats with BOO, voiding efficiency was unchanged. CONCLUSIONS: A selective PDE4 inhibitor can effectively suppress detrusor overactivity in rats with BOO, at doses that have no effect on voiding bladder contractions. Thus, selective PDE4 inhibitors should be considered for the treatment of overactive bladder in patients with BOO.     

Altered distribution of interstitial cells in the guinea pig bladder following bladder outlet obstruction

AIMS: We investigated the effects of bladder outlet obstruction (BOO) on the distribution of interstitial cells (ICs) in the guinea-pig bladder. METHODS: Bladder overactivity of BOO animals was validated with urodynamic studies. Immunohistochemical analyses for Kit and vimentin as markers for ICs were performed on both BOO and control bladders. Morphological and functional properties of detrusor smooth muscle (DSM) were examined with alpha-smooth muscle actin staining and intracellular recording, respectively. Electron microscopy was also carried out to characterize ultrastructural morphology of ICs. RESULTS: Two weeks after surgery, BOO animals showed an increased voiding frequency and a reduced voiding volume. Filling cystometry demonstrated a frequent incidence of non-voiding contractions, a reduced interval between voiding contractions and an increased voiding pressure in BOO bladders. In BOO bladders, the thickness of suburothelial and subserosal connective tissue layers was increased, whilst that of detrusor smooth muscle (DSM) layer was less affected. Population of Kit or vimentin immunoreactive ICs was increased in subserosal layers, and their distribution was altered in suburotherial layer in BOO bladders. Neither alpha-actin immunoreactivity nor spontaneous electrical activity of DSM was altered in BOO bladders. ICs were characterized by their numerous mitochondria and caveolae, and had a close contact with each other and with neighboring DSM or nerves. CONCLUSIONS: These results demonstrated the increased population of ICs in the BOO guinea-pig model for the first time, and suggest that the altered distribution of ICs may contribute to the pathophysiology of bladder overactivity.     

bFGF 、TGFβ-1在膀胱出口梗阻患者逼尿肌细胞中的表达

目的 观察膀胱出口梗阻（BOO）后膀胱平滑肌细胞碱性成纤维细胞生长因子（bFGF）、转化生长因子（TGFβ-1）表达的变化,探讨生长因子在BOO后膀胱平滑肌细胞继发改变中的作用。方法 应用逆转录－聚合酶链反应（RT－PCR）和免疫组织化学SP法在mRNA和蛋白水平检测bFGF、TGFβ-1在30例BOO及4例非BOO膀胱平滑肌组织中的表达。结果 BOO组与非BOO组膀胱平滑肌组织均有bFGF mRNA表达,BO组表达水平高于非BOO组,P＜0．01;TGFβ-1 mRNA在两组未见表达。结论 BOO 后磅胱平滑肌的继发改变与bFGF mRNA的表达有关。     

Possible role of endothelin-1 in the rabbit urinary bladder hyperplasia secondary to partial bladder outlet obstruction

OBJECTIVES: Urinary bladder hypertrophy and hyperplasia are common features of bladder outlet obstruction (BOO). The urinary bladder is known to synthesize endothelin-1 (ET-1), which is a potent vasoconstrictor peptide with mitogenic properties. Using an animal model of partial BOO, we investigated the potential role of ET-1 and its receptor subtypes (ET(A) and ET(B)) in bladder smooth muscle cell (SMC) proliferation. MATERIALS AND METHODS: Partial BOO was produced in adult male New Zealand White rabbits. After 3 weeks, the bladder was removed and SMCs from the dome and bladder neck were grown using standard explant methodology. At passage 2, the cells were made quiescent and then further incubated in foetal calf serum (FCS), control age-matched rabbit serum (CRS) or partial BOO serum (BRS) in the presence or absence of ET(A)-antagonist (BQ123) or ET(B)-antagonist (BQ788). SMC proliferation was then measured 24 h later with 5-bromo-2'deoxy-uracil and by cell counting using a haemocytometer at 48 h. Immunostaining for alpha-actin was performed on detrusor and bladder neck cells to confirm the presence of smooth muscle cells. RESULTS: BQ123 and BQ788 did not influence detrusor or bladder neck SMC proliferation in FCS or CRS. However, in the presence of BRS, BQ123 and BQ788 (100 nmol/L) significantly (p = 0.008) inhibited detrusor and bladder neck SMC proliferation. Cell counts were significantly reduced from the detrusor (p = 0.03, p = 0.01 with BQ123 and BQ788, respectively) and bladder neck (p = 0.01 for both BQ123 and BQ78). CONCLUSIONS: These results suggest that ET antagonists may have a role in preventing SMC hyperplasia associated with partial BOO.     

Change in acetylcholine release from rat bladder with partial outlet obstruction

OBJECTIVE

To investigate changes in acetylcholine release from the bladder of rats with partial bladder outlet obstruction (BOO), as partial BOO leads to hypertrophy and an alteration in the contractions of the detrusor smooth muscle, and acetylcholine plays an important role in urinary bladder contractions but there is little available information on acetylcholine release after BOO.

MATERIAL AND METHODS

Partial BOO was induced in adult female rats by ligating the proximal urethra over a 1 mm angiocatheter; sham‐operated rats served as controls. The rats were killed 2 weeks, 3 and 6 months after induction of BOO. We investigated the contractions induced by carbachol, KCl (80 mm ), ATP and electrical‐field stimulation (EFS, 2.5–40 Hz), and collected the dialysate obtained from a microdialysis probe inserted into the muscle strips during EFS, and measured the amount of acetylcholine in the dialysate fraction by high‐performance liquid chromatography with electro‐chemical detection. S‐100 immunohistochemical staining of the bladder preparations was used for histological examination in BOO and control rats.

RESULTS

The bladder weight gradually increased after BOO. There were no significant changes in KCl‐induced contractions throughout the experimental period in either group. There were no significant changes in carbachol‐induced contractions until 3 months after BOO but there was a significant reduction at 6 months. ATP‐induced contractions were significantly increased 2 weeks and 3 months after BOO. EFS‐induced contractions were gradually reduced after BOO. Acetylcholine release from the bladder strips was not significantly different between the groups until 2 weeks after BOO. However, acetylcholine release in BOO rats was significantly decreased 3–6 months after BOO, being significantly lower than that of the control rats. In the histological study, the number of nerve fibres in the BOO rats was significantly lower than in the control rats.

CONCLUSIONS

We suggest that the prolonged BOO caused a decrease in EFS‐induced acetylcholine release and the number of nerves in the rat urinary bladder, which might contribute to bladder underactivity in BOO.     

Effects of chronic partial outlet obstruction on blood flow and oxygenation of the rat bladder

PURPOSE: Experimental partial bladder outlet obstruction in rats and rabbits drives the bladder through 3 sequential responses, referred to as hypertrophy, compensation and decompensation. The hypertrophy phase, which is a period of rapid bladder growth, has previously been shown to be accompanied by a significant increase in bladder blood flow in rats and rabbits in a manner that likely supports the bladder cell growth process. However, chronic periods of obstruction in the rabbit have been shown to reduce significantly bladder blood flow, especially to the detrusor smooth muscle, corresponding with a loss of bladder contractile function or decompensation in these animals. We determined the effects of chronic 1 to 4-week partial outlet obstruction on rat bladder blood flow and directly correlated them with hypoxia in the rat bladder. MATERIALS AND METHODS: Rats underwent surgical partial bladder outlet obstruction under anesthesia. At weekly intervals after surgery relative blood flow to the bladder and spleen was measured by a fluorescent microsphere infusion technique. Sham operated rats were also studied 2 and 4 weeks following surgery. In a second experiment groups of similarly obstructed rats were treated with Hypoxyprobe-1 (Natural Pharmacia International, Inc., Research Triangle Park, North Carolina), a chemical probe for hypoxia, 3 days, 1 and 2 weeks after partial bladder outlet obstruction. The bladders were subsequently fixed and immunostained using a monoclonal antibody that detects Hypoxyprobe-1 adducts that are selectively formed in hypoxic cells. RESULTS: Neither bladder weight nor bladder relative blood flow was affected by sham surgery. Likewise, control and sham obstructed rat bladders were found to be free of Hypoxyprobe-1 reactive areas. In contrast, obstructed rats had significantly increased bladder weight at all time points. Relative weight of the obstructed rat bladders indicates the response to mild-moderate obstruction. Bladder relative blood flow in obstructed rats was significantly elevated 1 and 2 weeks after partial bladder outlet obstruction but it returned to almost control levels by 3 and 4 weeks. Hypoxyprobe-1 staining demonstrated a sequential transition of hypoxia from bladder mucosa and submucosal regions at 3 days to muscularis and serosal fibroblasts 1 week and finally to smooth muscle cells by 2 weeks after obstruction. CONCLUSIONS: In contrast to the rabbit model, global blood flow in the mild-moderate chronically obstructed rat bladder was found to be higher or nearly equivalent to blood flow in unobstructed control rat bladders. However, even in the presence of normal or above normal blood flow focal regions of hypoxia were still observed in obstructed rat bladders and these regions changed with time. These results provide a reason to understand better why rats are more resistant to the onset of bladder decompensation than rabbits and support the concept that hypoxia is involved in bladder remodeling as well as in progressive functional impairment of the bladder after partial bladder outlet obstruction.     

Modulation of insulin-like growth factor-I system of the bladder using a somatostatin analogue in chronic spinalized rats

PURPOSE: We have previously reported the possible role of the insulin-like growth factor-I (IGF-I) system of mitogens in the development of detrusor smooth muscle hyperplasia and hypertrophy after spinal cord injury. We evaluated the in vivo effects of the anti-growth factor somatostatin analogue octreotide on the IGF-I system as well as subsequent changes in bladder smooth muscle hypertrophy and function after spinal cord injury in rats. MATERIALS AND METHODS: Included in this study were 90 adult female Sprague-Dawley rats weighing 200 to 250 gm. Of the rats 18 served as sham operated controls, while the remaining 72 underwent were spinal cord transection at the level of the T10 vertebra. The spinalized animals were randomly divided into 4 equal groups of 18, of which 1 group served as paraplegic controls. The other 3 groups received octreotide (60 microgram. daily for 4 weeks) delivered via a subcutaneously implanted osmotic pump immediately, 2 and 4 weeks after spinal cord injury. At the end of the experiment (6 to 8 weeks) each group of animals was subdivided into 2 subgroups of 9. In the first group filling cystometrography was done, while in the second subgroup wet bladder weight was estimated and Northern blot analysis was performed. RESULTS: Mean wet bladder weight plus or minus standard deviation in sham operated and paraplegic controls was 0.11 +/- 0.01 and 0.64 +/- 0.33 gm., respectively (p <0.05). The increase in bladder weight in paraplegic controls was associated with over expression of the IGF-I gene and with marked suppression of IGF binding proteins-3 and 5 compared with sham operated controls. On the other hand, mean wet bladder weight in the animals that received octreotide immediately after spinal cord injury was 0.17 +/- 0.02 gm., which was associated with a dramatic decrease in IGF-I gene expression and increased expression of IGF binding proteins-3 and 5. Mean cystometric bladder capacity in paraplegic controls was 0.48 +/- 0.18 ml. with an associated voiding pressure of 71 +/- 13 cm. water. All paraplegic controls showed detrusor hyperreflexia. In animals that received octreotide immediately after spinal cord injury mean cystometric bladder capacity was 2.49 +/- 1.75 ml. with an associated voiding pressure of 32 +/- 7 cm. water. Detrusor hyperreflexia disappeared in 88.89% of the rats in this group. There were less marked changes in bladder weight (mean 0.24 and 0.29 +/- 0.3 gm.), IGF-I gene expression and its binding proteins and urodynamic parameters when the drug was given 2 and 4 weeks, respectively, after spinal cord injury. CONCLUSIONS: Modulating the IGF-I system of mitogens in detrusor smooth muscle with consequently decreased bladder hypertrophy and improved urodynamic behavior in spinal cord injured animals using somatostatin analogue could be a possible therapeutic modality in patients with spinal cord injury.     

The detrusor muscle cell in bladder outlet obstruction--ultrastructural and morphometric findings

OBJECTIVE: Lower urinary tract symptoms (LUTS) in elderly males are not solely caused by bladder outlet obstruction (BOO) and may be at least partly attributable to detrusor dysfunction. Urodynamically, patients may show instability, hypocontractility, BOO or combinations of these findings. These findings have been related to specific ultrastructural changes in detrusor smooth muscle cells; however, this relationship is controversial. The aim of this study was to correlate ultrastructural findings in patients with BOO with urodynamic parameters. MATERIAL AND METHODS: In 25 men with BOO verified by means of a full urodynamic evaluation, including a pressure-flow study, a detrusor biopsy was obtained. Six men without BOO served as controls. Biopsies for electron microscopy were analysed semiquantitatively and morphometrically to determine the presence of muscle cell hypertrophy, variation in intercellular distances, occurrence of abnormal cell junctions and configurations and intracellular changes. RESULTS: The only parameter which was found to relate to the degree of obstruction in BOO was an increase in intra- and interfascicular elastin, all other correlations not reaching significance. CONCLUSION: This study does not confirm a specific relationship between ultrastructural detrusor smooth muscle features and various types of BOO. Therefore ultrastructural investigation of detrusor smooth muscle cells cannot replace urodynamic evaluation in the classification of LUTS.     

Alterations in purinergic and cholinergic components of contractile responses of isolated detrusor contraction in a rat model of partial bladder outlet obstruction

OBJECTIVE: To study the effect of 3 weeks of partial bladder outlet obstruction (BOO), compared to a sham operation, on the cholinergic and purinergic components of detrusor contractile responses to agonists and to electrical field stimulation (EFS); the expression of P2X receptor subtypes was also examined. MATERIALS AND METHODS: Partial BOO was induced in female Sprague-Dawley rats by surgically applying a jeweller's silver 'jump' ring around the urethra, such that the urethra was constricted but not closed. Sham-operated female rats underwent an identical procedure without placement of a ring. RESULTS: After 3 weeks of partial BOO the rat bladders became significantly hypertrophied, doubling in weight. Spontaneous activity was markedly increased, but the contractile response to a single bolus of KCl (120 mM) was unaltered. The neurogenic-induced contractile responses of strips of detrusor from obstructed bladders were significantly greater than those from sham-operated bladders, and the responses of strips of detrusor from obstructed bladders to EFS showed a significantly greater atropine-sensitive component than sham-operated detrusor. However, the response of detrusor strips to EFS that was susceptible to desensitization by alpha,beta-methylene ATP was not significantly changed in obstructed bladders. The sensitivity of the strips from obstructed bladders to carbachol, ATP and beta,gamma-methylene ATP was less than in sham-operated detrusor. Immunohistochemical studies showed no difference in the P2X receptor subtypes expressed on detrusor smooth muscle from obstructed and sham-operated rats. CONCLUSION: In the rat, after moderate bladder hypertrophy, the atropine-sensitive component was significantly up-regulated, but the ATP-sensitive component was marginally reduced, although not significantly. These results suggest that up-regulation of the P2X component of bladder contraction seen in humans with bladder instability, and in other species models of BOO, is not mirrored in the rat, or occurs later in the pathological process of bladder hypertrophy.     

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.     

The effect of sildenafil citrate on bladder outlet obstruction: a mouse model

OBJECTIVE

To investigate if sildenafil citrate can inhibit the functional and structural changes of the detrusor in a murine model of bladder outlet obstruction (BOO). Phosphodiesterase type 5 (PDE‐5) inhibitors have recently been used for treating urinary symptoms associated with prostatic obstruction, but it is unclear whether PDE‐5 inhibition acts on the prostatic urethra or the bladder.

MATERIALS AND METHODS

In 18 male Balb/CAN mice, partial BOO was created and the mice allowed to survive for 6 weeks. Half of the mice (nine) were treated with oral sildenafil citrate daily (10 mg/kg) by oral lavage (BOO + V), and half (nine) were not (BOO). Six mice were used as sham‐operated controls and received no sildenafil. The mice were assessed by urodynamics at baseline and after 6 weeks, with a measurement of volume at first uninhibited non‐voiding contraction (V_DO1), bladder capacity (BC), and detrusor pressure during void (P_det). At 6 weeks, bladders were harvested, fixed and sectioned, and stained with haematoxylin and eosin (H&E) and trichrome stain. Detrusor muscle hypertrophy and fibrosis were evaluated on a scale of 1 (decreased) to 3 (increased), by two urologists and one pathologist unaware of the treatment group; the results were compared with those from normal controls.

RESULTS

BOO mice had a significantly greater BC than control mice, with a mean (sd ) of 153 (66) vs 58 (13) µL (P = 0.004). Treatment with sildenafil did not significantly alter BC. BOO caused an increase in Pdet compared to controls, with a mean (sd ) of 25 (7) vs 12 (5) cm H₂O. P_det was not significantly different after treatment with sildenafil. The median V_DO1 as a percentage of BC was significantly lower in BOO than in control mice (20% vs 53%, P > 0.03) and increased significantly after sildenafil treatment (20% vs 44%, P = 0.04). BOO was associated with a greater bladder weight than in control mice, with a mean (sd ) of 89 (32) vs 27 (6) mg (P = 0.001), which was decreased with sildenafil treatment, to 40 (14) vs 89 (32) mg (P = 0.013). BOO caused an increase in detrusor muscular hypertrophy vs control mice, with a median H&E score of 3 vs 2 (P = 0.01) and an increase in fibrosis vs control mice, with a median trichrome score of 3 vs 2 (P = 0.01). BOO + V mice had reduced muscular hypertrophy and fibrosis, with a median H&E score of 3 vs 2 (P = 0.01) and a median trichrome score of 3 vs 1 (P = 0.01).

CONCLUSIONS

BOO mediates both functional and structural changes in the mouse bladder. Six weeks of obstruction caused an increase in BC, detrusor overactivity and voiding pressure, and mediated an increase in bladder weight, detrusor muscle hypertrophy and collagen deposition in the lamina propria and smooth muscle. Treatment with 6 weeks of oral sildenafil beginning at the time of BOO prevented the increase in detrusor overactivity without affecting voiding pressures, and prevented the increase in detrusor muscle hypertrophy and collagen deposition that otherwise occurred with BOO. It appears therefore that sildenafil citrate acts on the bladder rather than on the outlet.     

Activity and expression of nitric oxide synthase in the hypertrophied rat bladder and the effect of nitric oxide on bladder smooth muscle growth

PURPOSE: We investigated the expression and activity of nitric oxide synthase (NOS) and the localization of cyclic guanosine monophosphate (cGMP) in hypertrophied rat bladder. We also examined whether nitric oxide (NO) has a growth inhibitory effect in bladder smooth muscle cells. MATERIALS AND METHODS: The urethra was partly ligated and the bladder was removed 3 days, 3 or 6 weeks after obstruction. NOS activity was determined as the conversion of L-[14C]citrulline from L-[14C]arginine (Amersham Life Science, Solna, Sweden). Neuronal NOS (nNOS) expression was studied with Western blot analysis and immunohistochemistry. The expression of inducible NOS (iNOS) and cGMP was evaluated by immunohistochemistry. The effect of NO on isolated bladder smooth muscle cell growth was assessed as protein and DNA synthesis by [3H]-leucine and [3H]-thymidine (NEN Life Science Products, Zaventem, Belgium) incorporation, respectively. RESULTS: Ca independent iNOS activity increased after short-term obstruction. Immunohistochemical studies in obstructed bladders demonstrated iNOS expression primarily in urothelial and inflammatory cells. Ca dependent nNOS activity decreased after obstruction, as confirmed by Western blot analysis. The cGMP immunoreactive cells were mainly found within the serosal layer of obstructed bladders. The NO donor DETA-NONOate (Alexis Biochemicals, Lausen, Switzerland) (300 microM.) reduced [3H]-leucine and [ H]-thymidine incorporation by a mean of 29% +/- 2% and 95% +/- 2%, respectively, in cultured bladder smooth muscle cells. CONCLUSIONS: Bladder obstruction caused a small increase in iNOS activity and a decrease in nNOS activity. NO was found to have a growth inhibitory effect in bladder smooth muscle cells, suggesting that changes in NOS activity may influence the progress of bladder hypertrophy.     

Partial outlet obstruction of the rat bladder induces a stimulatory response on proliferation of the bladder smooth muscle cells

Outlet obstruction of the ratbladder induces hypertrophy/hyperplasiacharacterized by increases in bladder mass,smooth muscle content, and collagen deposition.In order to understand the mechanism of theoutlet obstruction-induced hypertrophy andhyperplasia, we first determined the temporalpattern of changes in bladder mass afterinducing the outlet obstruction. Histologicalanalysis revealed that the smooth muscle cellswith hypertrophy and hyperplasia, fibroblastsand connective tissue were increased in atime-dependent manner, corresponding to thetemporal pattern observed in the changes inbladder mass, although the phase of changes inthese tissue components was somewhat different.In order to further determine whether anyproliferation-stimulatory factors were releasedfrom the bladder with obstruction incorrespondence with increased bladder mass,soluble fractions were prepared from thebladders with outlet obstruction for 3–30weeks, and their effects on proliferation ofsmooth muscle cells were examined. The solublefractions prepared from the bladders at 3 to 14weeks after obstruction slightly butsignificantly facilitated the proliferation ofcultured smooth muscle cells, while the solublefractions released after 20 weeks rathersuppressed the proliferation. These resultssuggest that the initial increase in bladdermass might be in part due to the facilitatedproliferation of smooth muscle cells of thebladder body induced by growth factors releasedinto the soluble fractions, and thathypertrophy might then play a role in theincreased bladder mass at later phases.     

Regulation of actomyosin and contraction in smooth muscle

Summary Unlike striated muscle cells, smooth muscle cells do not have an organized sarcomeric structure. However, all smooth muscle cells contain the contractile proteins, myosin, actin, and tropomyosin. Polymorphism of the myosin heavy chain exists in smooth muscle cells. Two myosin heavy chain (MHC) isoforms, SM1 (204 kDa) and SM2 (200 kDa), are present in smooth muscle cells; however, their ratios vary in smooth muscles from different sources. The hypertrophy of the urinary bladder induced by partial outlet obstruction in rabbits is associated with an alteration of the SM1-to-SM2 ratio from 1:3 to 1:1. Both heavy chains react with polyclonal antibody against smooth muscle myosin; however, antibody prepared against a peptide from the C-terminal region of the SM2 heavy chain cross-reacts only with the SM2 heavy chain. Removal of the obstruction reverses the bladder to normal mass with a concomitant change in the SM1-to-SM2 ratio back to 1:3. The expression of the SM1 mRNA is increased in response to obstruction-induced hypertrophy, and it also returns to normal upon removal of the obstruction. Urinary bladder smooth muscle contains predominantly -actin. Obstruction-induced hypertrophy of the bladder smooth muscle is associated with an increase in the -actin at both protein and mRNA levels. The -non-muscle actin is decreased and the -smooth muscle actin is unchanged in response to obstruction-induced bladder hypertrophy.Contraction of all smooth muscles involves similar mechanisms. This review describes our current understanding of the mechanisms regulating contraction of the smooth muscle of the urinary bladder.This work was supported in part by NIH grants HL 22246, DK 47514 and DK 39740     

Bladder outlet obstruction accelerates bladder carcinogenesis

OBJECTIVE

To examine the correlation between partial bladder outlet obstruction (PBOO) and bladder carcinogenesis.

MATERIALS AND METHODS

Female Wistar rats (6 weeks old) were divided into three groups of 10 each: group 1 was exposed to n‐butyl‐n‐butanol nitrosamine (BBN, a carcinogen) in drinking water for 8 weeks; group 2 had PBOO induced surgically after exposure to BBN for 8 weeks; group 3 had a sham operation and the rats drank normal water (control group). After 20 weeks, all of the rats were killed humanely and their bladders analysed.

RESULTS

There were no significant differences in body weight among the groups. The bladder weight of group 2 was significantly greater than either group 1 or group 3. Histopathologically, bladder smooth muscle hypertrophy was the major cause of the increased bladder weight for group 2. In group 2 there were increases in bladder wall thickness and many nipple‐shaped urothelial tumours. Basic fibroblast growth factor and hypoxia‐inducible factor‐1α expression were significantly greater in group 2 than in groups 1 and 3.

CONCLUSIONS

Exposure of the bladder to carcinogens during bladder hyperplasia and hypertrophy induced by PBOO results in a greater incidence of superficial bladder carcinoma.     

Processed lipoaspirate cells for tissue engineering of the lower urinary tract: implications for the treatment of stress urinary incontinence and bladder reconstruction

PURPOSE: We performed a pilot study to investigate the ability of human adipose derived, multipotent stem cells to be delivered to and survive within bladder and urethral smooth muscle. MATERIALS AND METHODS: Lipoaspirate was acquired from female patients undergoing liposuction. The lipoaspirate was processed to yield a pluripotent population of processed lipoaspirate (PLA) cells. For tissue delivery PLA cells were fluorescent labeled and suspended in Hanks' balanced salt solution (Sigma Chemical Co., St. Louis, Missouri). To assess PLA viability in multiple animal models 8 Rnu athymic rats (Charles River, Wilmington, Massachusetts) and 6 SCID mice (Taconic Farms, Oxnard, California) underwent laparotomy and injection of PLA cells into the bladder and urethra. An additional 8 rats underwent sham injection of Hanks' balanced salt solution alone. Experimental and control animals were sacrificed 2, 4, 8 and 12 weeks after injection, and the bladders and urethras were analyzed. RESULTS: Self-regenerating, pluripotent PLA cells were easily isolated from human adipose tissue. Evaluation 2, 4, 8 and 12 weeks after injection demonstrated PLA cell viability and incorporation into the recipient smooth muscle. Eight weeks following injection PLA cells demonstrated in vivo expression of alpha-smooth muscle actin, an early marker of smooth muscle differentiation. CONCLUSIONS: PLA cells are an easily accessible source of pluripotent cells, making them ideal for tissue regeneration. PLA cells remain viable up to 12 weeks in the lower urinary tract. Human PLA cells injected into the urinary tract show morphological and phenotypic evidence of smooth muscle incorporation and differentiation with time. PLA cells may provide a feasible and cost-effective cell source for urinary tract reconstruction.     

Inhibition of collagen deposit in obstructed rat bladder outlet by transplantation of superparamagnetic iron oxide-labeled human mesenchymal stem cells as monitored by molecular magnetic resonance imaging (MRI)

Bladder outlet obstruction (BOO) caused by collagen deposit is one of the most common problems in elderly males. The present study is to investigate if human mesenchymal stem cells (MSCs) are capable of inhibiting collagen deposition and improve cystometric parameters in bladder outlet obstruction in rats. Human MSCs were labeled with nanoparticles containing superparamagnetic iron oxide (SPION), and transplanted in rat BOO lesion site. Forty 6-week-old female Sprague-Dawley rats were divided into four groups (group 1: control, group 2: sham operation, group 3: BOO, and group 4: BOO rats receiving SPION-hMSCs). Two weeks after the onset of BOO, 1 × 10(6) SPION-hMSCs were injected into the bladder wall. Serial T2-weighted MR images were taken immediately after transplantation of SPION-labeled human MSCs and at 4 weeks posttransplantation. T2-weighted MR images showed a clear hypointense signal induced by the SPION-labeled MSCs. While the expression of collagen and TGF-β protein increased after BOO, the expression of both returned to the original levels after MSC transplantation. Expression of HGF and c-met protein also increased in the group with MSC transplantation. Maximal voiding pressure and residual urine volume increased after BOO but they recovered after MSC transplantation. Human MSCs transplanted in rat BOO models inhibited the bladder fibrosis and mediated recovery of bladder dysfunction. Transplantation of MSC-based cell therapy could be a novel therapeutic strategy against bladder fibrosis in patients with bladder outlet obstruction.     

Phenotypic changes in human bladder smooth muscle cell

Tissue specimens were resected from 15 patients (age 7 to 82, average 52.2 years old) with bladder diseases; i.e., 2 with neurogenic bladder, 6 recipients of kidney transplantation with defunctionalized bladder, 3 with benign prostatic hypertrophy, 3 with bladder cancer and 1 with vesicoureteral reflux. We investigated the phenotypic expression of the bladder smooth muscle cells with bladder diseases. The ratio of non-contractile to contractile phenotypes (nc/c ratio) showed a rising tendency with aging. The increase of nc/c ratio was especially notable with neurogenic bladder. Phenotypic expression was observed in human bladder smooth muscle cells as reported in vascular smooth muscle cells. Several bladder diseases cause a conversion of contractile smooth muscle cell phenotype from contractile type to non-contractile type, and this modulation of smooth muscle cell phenotype may play an important role in detrusor function.