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.     

Growth of bone marrow stromal cells on small intestinal submucosa: an alternative cell source for tissue engineered bladder

OBJECTIVE: To assess the potential use of bone marrow stromal cell (BMSC)-seeded biodegradable scaffold for bladder regeneration in a canine model, by characterizing BMSCs and comparing them to bladder smooth muscle cells (SMCs) by immunohistochemistry, growth capability, and contractility. MATERIALS AND METHODS: Bone marrow was taken by direct needle aspiration from the femurs of five beagle dogs for the in vitro study. Mononuclear cells were isolated by Ficoll-Paque density gradient centrifugation and cultivated in medium 199 with 10% fetal bovine serum. BMSCs were characterized by cell proliferation, in vitro contractility, immunohistochemical analysis, and the growth pattern on small intestinal submucosa (SIS) scaffolds compared to bladder SMC cultures from the same dogs. Another six dogs had a hemicystectomy and bladder augmentation with BMSC-seeded (two), bladder cells including urothelial cells plus SMC-seeded SIS (two) and unseeded SIS scaffolds (two). The six dogs were followed for 10 weeks after augmentation. RESULTS: In vitro BMSCs had a significant contractile response to calcium-ionophore, with a mean (sem) 36 (2)%, relative contraction (P < 0.01), which was similar to bladder SMCs but markedly different from fibroblasts. BMSCs also expressed alpha-smooth muscle actin by immunohistochemical staining and Western blotting, but did not express desmin or myosin. In vivo, both BMSC-seeded and bladder cell-seeded SIS grafts had solid smooth-muscle bundle formation throughout the graft. CONCLUSIONS: BMSCs had a similar cell proliferation, histological appearance and contractile phenotype as primary cultured bladder SMCs. SIS supported three-dimensional growth of BMSCs in vitro, and BMSC-seeded SIS scaffold promoted bladder regeneration in a canine model. BMSCs may serve as an alternative cell source in urological tissue engineering.     

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.     

Heterogeneous expression of nonmuscle myosin heavy chain-B in mesangial cells of patients with Gitelman's syndrome

AIMS: It has been suggested that angiotensin II (Ang II) promotes hypertrophy and hyperplasia of mesangial cells. Nonmuscle myosin heavy chain-B (NMHC-B) and alpha-smooth muscle (SM) actin are considered to be molecular markers for phenotypic change ofproliferative mesangial cells. One of the clinical characteristics in Gitelman's syndrome (GS) is the elevation of plasma Ang II. However, little is known about the relation between Ang II and phenotypic change of mesangial cells in patients with GS. In this report, we examined the expression of NMHC-B and alpha-SM actin in mesangial cells of two GS patients. MATERIALS AND METHODS: Plasma renin activity, and the concentrations of Ang II, 6-keto-prostaglandin F1alpha (6-keto-PGF1alpha), urinary kallikrein, and 6-keto-PGF1alpha were measured. Immunohistochemical staining of NMHC-B and alpha-SM actin in mesangial cells of GS patients was also performed. RESULTS: Both cases of GS showed normal glomerular function, few histological abnormalities, and higher than normal plasma concentrations of renin and Ang II. Furthermore, one case showed a high urinary concentration of kallikrein and the expression of both NMHC-B and alpha-SM actin in mesangial cells. The other case showed a high urinary concentration of 6-keto-PGF1alpha but not kallikrein and without the expression of NMHC-B and alpha-SM actin. CONCLUSION: Not only plasma kinin-kallikrein and prostaglandins, but the renal expression of NMHC-B and alpha-SM actin may be variable in different patients with GS.     

Microarray analysis of exstrophic human bladder smooth muscle

OBJECTIVE

To compare the genetic profiles of ‘healthy’ bladder smooth muscle cells (SMCs) and exstrophic SMCs (ESMCs) to identify genes that are over‐ and under‐expressed in ESMCs, thus providing a molecular evaluation of the quality and therapeutic potential of ESMC tissue.

PATIENTS, MATERIAL AND METHODS

Classical bladder exstrophy is a rare disorder, occurring in 1 in 30 000 live births. Studies have shown that exstrophic bladders are developmentally immature at birth. After surgical closure, the bladder typically undergoes abnormal remodelling (such as over‐expression of collagen III) throughout early development. We hypothesized that the predominant genetic differences between normal SMCs and ESMCs are in the developmental genes. This hypothesis was tested by the use of microarray analysis. Bladder SM biopsies were taken from ‘healthy’ subjects undergoing bladder surgeries for other conditions (for example, urinary reflux) and patients with bladder exstrophy. Cells were expanded in vitro, and total RNA was isolated and hybridized to the Affymetrix U133A GeneChip® (Affymetrix Inc., Santa Clara, CA, USA) by the Wake Forest University School of Medicine Affymetrix core facility, using standard protocols.

RESULTS

We created a genetic signature consisting of 961 genes that are over‐expressed and 432 genes that are under‐expressed in ESMCs. Analysis of these signatures identified an over‐expression of inflammatory genes and an under‐expression of developmental genes.

CONCLUSION

Our data is in concordance with previous studies and histological data showing that ESMCs are developmentally immature relative to healthy bladder SM. The clinical implication of the ESMC genetic signature is that it provides a list of targets that can be (i) manipulated ex vivo and/or in vivo to induce differentiation (the completion of development) and (ii) used as biomarkers to explain the variability of the clinical symptoms after surgical closure.     

Denervation and outlet obstruction induce a net synthesis of contractile and cytoskeletal proteins in the urinary bladder of the male rat

The concentrations of the contractile proteins actin and myosin and the cytoskeletal protein desmin were determined in urinary bladders from normal rats, and from rats with bladder outlet obstruction or denervation. Ten days of obstruction or total denervation by bilateral removal of the pelvic ganglia resulted in an almost fourfold increase in bladder weight. Actin and myosin concentrations did not change significantly. The total amount of actin was 1624±235 g in the control bladder. In the obstructed and denervated bladders it increased significantly to 6277±648 g and 7671±835 g, respectively. The desmin/actin ratio was 0.237±0.012 in the control bladders, and increased significantly to 0.369±0.015 in the obstructed and 0.343±0.022 in the denervated bladders. Partial denervation by removal of the pelvic ganglion on one side only increased bladder weight by 52%, but did not increase the desmin/actin ratio. The content of actin in such bladders increased by 82%. Both obstruction (which increases the functional load of the detrusor muscle cells) and denervation (which produces bladder paralysis) are known to induce hypertrophy of the detrusor smooth muscle cells. The study shows that the desmin/actin ratio and the total amount of contractile proteins increase in response to the hypertrophy as such, and not to the work performed by the smooth muscle cells, and that the nerves have no trophic influence on the growth response. Also, even a limited lesion of the bladder innervation is associated with growth and a net increase in the amount of contractile proteins.     

Smooth-muscle myosin heavy-chain isoform expression in bladder-outlet obstruction

Summary Bladder-outlet obstruction leads to detrusor smooth-muscle hypertrophy/hyperplasia. Despite this overall increase in muscle mass, smooth-muscle contractility decreases and bladder emptying is impaired. The goal of this study was to determine whether smooth-muscle myosin heavy-chain (MHC) alterations occur in conjunction with partial obstruction of the rabbit bladder. Total MHC and MHC-isoform protein concentrations were determined by quantitative gel electrophoresis in rabbit bladders partially obstructed for 1–4 weeks. MHC gene expression was assessed by Northern and nuclease protection assays. Two MHC isoforms (SM1/SM2) were identified in the normal rabbit bladder. After 2 weeks of obstruction the ratio of SM1/SM2 changed from 0.4:0.6 to 0.5:0.5 (P<0.01). As compared with sham-operated values, the level of MHC mRNA decreased significantly as of 1 day after obstruction. Quantitation of MHC-isoform mRNA levels revealed a nearly 3-fold increase in the SM1/SM2 ratio. In this animal model of bladder-outlet obstruction, early changes in MHC isoforms as well as an overall decrease of MHC mRNA expression were demonstrated, suggesting that obstruction induces significant alterations in myofilament gene expression.     

Morphology,phenotype and ultrastructure of fibroblastic cells from normal and neuropathic human detrusor: absence of myofibroblast characteristics

PURPOSE: Fibroblasts are functionally diverse and fibroblastic cells with smooth muscle-like characteristics (myofibroblasts) regulate smooth muscle activity in certain tissues. The presence of myofibroblasts has been reported in the bladder with important implications for normal function and detrusor overactivity. We assessed fibroblastic cell characteristics to discern features suggesting a myofibroblast phenotype in normal or neuropathic human detrusor. MATERIALS AND METHODS: A total of 25 control samples were obtained from cadaveric organ donors or patients with a mean age of 42.3 years investigated for hematuria and compared with samples from 18 patients with a mean age of 37.4 years with neurogenic detrusor overactivity. Morphology, phenotypic expression of various markers and the ultrastructure of each fibroblastic cell visible in multiple sections from each specimen was evaluated by 2 independent assessors. RESULTS: Fibroblastic cells were observed throughout the smooth muscle and connective tissue. They were located peripherally on muscle fascicles and had a polar stellate appearance with processes ramifying in interfascicular planes and muscle. They possessed vimentin-like immunoreactivity and weak c-kit-like immunoreactivity but not desmin or alpha-smooth muscle actin-like immunoreactivity. Ultrastructurally they showed dilated rough endoplasmic reticulum with a moderately electron dense amorphous content and prominent golgi complexes. Nuclei had clumped peripheral heterochromatin. There were extensive flattened processes that lacked basal laminae. There was no specific contact with nerve fibers or smooth muscle. Neuropathic bladder samples did not differ overtly from those of controls. CONCLUSIONS: The detrusor possesses an extensive network of fibroblastic cells and processes. No evidence of myofibroblast differentiation was discerned in normal or neuropathic detrusor, although a minority subpopulation or regional variability in cellular phenotype could not be excluded.     

Smooth muscle myosin heavy chains are developmentally regulated in the rabbit bladder

PURPOSE: In smooth muscle (SM), myosin heavy chain (MHC) is expressed predominantly as two isoforms, SM1 and SM2, which are encoded by a single gene and expressed by alternative splicing mechanisms. Although functional differences of these isoforms are unknown, changes in SM1/SM2 ratio have been reported in various pathophysiologic conditions. We analyzed MHC composition of bladder detrusor SM from rabbits of different ages to determine whether SM1 and SM2 isoform expressions are developmentally regulated. MATERIALS AND METHODS: Rabbit bladders on the -11, -4, 1, 7, 14, 21, and 90th days of life were analyzed for SM MHC isoform expression at protein and mRNA levels. Porous sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), S1 protection assay, and histological analysis were employed. RESULTS: The predominant MHC isoform in fetal and neonatal bladders was SM1. In the third postnatal week, the SM1/SM2 ratio decreased from 2.3 to 1.0. A stable SM1/SM2 ratio of 0.6 was observed in the adult animal. Although expression of SM1 mRNA was 2.6-fold greater than that of SM2 in the fetus, the relative amount of SM2 mRNA increased rapidly after birth and remained the predominant isoform throughout adult life. Developmental changes in relative amounts of SM1 and SM2 protein in bladder tissues were virtually identical to those of SM1 and SM2 mRNA. SM cell growth and disappearance of primitive mesenchyme from the bladder occurred concomitantly with the MHC isoform shift. CONCLUSIONS: The parallel temporal course of MHC mRNA and protein isoform levels suggests detrusor SM MHC expression may be developmentally regulated at the mRNA level.     

CHARACTERIZATION OF CULTURED BLADDER SMOOTH MUSCLE CELLS: ASSESSMENT OF IN VITRO CONTRACTILITY

PURPOSE: The contractile properties of in vitro cultured bladder smooth muscle cells (SMC) are unknown. This study characterized the in vitro contractile response of human and rat bladder SMC to several pharmacological agonists known to induce in vivo contraction of intact bladder muscle. MATERIALS AND METHODS: Human and rat bladder SMC were seeded separately within attached collagen lattices. Contractility of SMC was analyzed by measuring alterations in lattice diameter after exposure and release to the following contractile agonists: carbachol (10(-7)-10(-3) microM), calcium-ionophore (10 microM), lysophosphatidic acid (LPA) (1 microM), endothelin (0.1 microM), KCl (3.33 mmicroM) angiotensin II (10 microM), and serotonin (100 microM). Results were recorded as a mean reduction of the lattice diameter. In addition, immunohistochemical analysis for phenotypic markers of smooth muscle cell differentiation was performed on bladder SMC cultured within collagen lattices. Human palmar fascia fibroblasts, which have been previously well characterized by in vitro contractility and immunohistochemistry, were tested in parallel and used as controls for all the above experiments. RESULTS: Human SMC had significant contractile responses to calcium-ionophore (31% +/- 4 relative percent contraction, p <0.05), LPA (34% +/- 4, p <0.05), and endothelin (37 +/- 5%, p <05). There was no significant contraction in response to carbachol, angiotensin II, KCl, or serotonin. Rat bladder SMC had a similar contractile response but did not contract in response to endothelin. In contrast to human and rat bladder SMC, fibroblasts did not contract to calcium-ionophore. CONCLUSIONS: In vitro cultured bladder SMC demonstrate loss of contractile response to normal in vivo pharmacologic agonists. Both human and rat bladder SMC can be distinguished in vitro from fibroblasts based upon their lack of contractile response to calcium- ionophore. These results demonstrate the ability to further characterize cultured bladder SMC with in vitro contractility. Further characterization is essential if we are to advance our understanding of the clinical applicability of in vitro studies utilizing cultured bladder SMC.     

PHENOXYBENZAMINE IN THE MANAGEMENT OF NEUROPATHIC BLADDER FOLLOWING SPINAL CORD INJURY

Background : The present study aims to show the clinical and urodynamic effects of phenoxybenzamine on the neuropathic bladder of spinal cord-injured patients who failed to be free of catheter by attaining satisfactory voiding function, despite initial bladder training. Methods : Forty-six spinal cord-injured patients were subjected to pharmacological manipulation with phenoxybenzamine. It was used as an adjunct in the management of neuropathic bladder dysfunction that caused failure of the bladder to empty, by tapping or crede to achieve satisfactory residual urine volume of < 100 mL. Phenoxybenzamine was started with a dose of 10 mg daily, increased by 10 mg every 3 days to a dose of 30 mg daily; this was maintained from 3 weeks to 6 months (mean: 39 days). The pre-treatment residual urine volume ranged between 100 and 1050 mL (mean: 360 mL). Follow-up periods ranged between 12 and 36 months (mean: 16 months). Results : Five patients (11%) were excluded due to either inadequate treatment or inadequate follow-up. Nineteen patients (41%) with reflex (upper motor neurone) bladders showed improvement of bladder evacuation. There was a reduction of the maximum urethral closure pressure, which ranged between 10 and 32 cm of water (mean: 22 cm). Twenty-two patients (48%) did not respond, requiring other measures to be taken which included transurethral surgery (n = 19). Nine of the failures involved areflex (lower motor neurone) bladders, and seven failures involved reflex bladders with an extremely tight outlet and urethral closure pressure of > 50 cm of water. Six failures involved reflex bladders that were lacking strong enough detrusor contractions to attain a balanced bladder responsive to abdominal tapping; response was achieved by administration of a parasympatheticomimetic drug. Neuropathic bladders with uninhibited detrusor contractions responded well to phenoxybenzamine. Conclusions : Phenoxybenzamine proved useful in reducing bladder outlet resistance after spinal cord injury, provided that detrusor bladder contractions were present. It is useful in controlling detrusor–sphincter dyssynergia and autonomic hyperreflexia. It was not useful in areflex bladders, perhaps due to the development of spasticity of the striated muscle component of the external sphincter. The presence of bladder neck (internal sphincter) dysfunction may modify or abolish its effect.     

Focal hypoxia of the obstructed rabbit bladder wall correlates with intermediate decompensation

AIMS: We showed that partial obstruction of the rabbit bladder outlet caused decreases in detrusor blood flow that were directly proportional to the level of decompensation present. Bladder decompensation is characterized by decreases in detrusor contractility, mitochondrial function, and sarco/endoplasmic reticulum calcium ATPase (SERCA) activity in obstructed rabbits. The current study was designed to create bladder decompensation and to relate its characteristic dysfunctions to the presence or absence of hypoxia in the obstructed rabbit bladder wall. Tissue hypoxia was visualized immunohistochemically after administration of a hypoxia probe in vivo. METHODS: Twelve New Zealand White rabbits were separated into two groups. The rabbits in group 1 received sham operations; the rabbits in group 2 received partial outlet obstructions by standard methods. Four weeks after surgery, each rabbit received an intraperitoneal injection of aqueous Hypoxyprobe-1, which forms protein adducts in cells having O(2) concentrations less than 14 microM. Two hours after injection, the rabbit was anesthetized and the bladder exposed through a midline incision. One full-thickness bladder strip was cut and immediately placed in fixative for immunohistochemical recognition and visualization of Hypoxyprobe-1-protein adducts. The remaining bladder was then excised, and three additional strips were cut for contractility studies. The remainder of the bladder was frozen for biochemical and slot-blot analyses. RESULTS: Bladder weight was increased fourfold after obstruction, and significant contractile and biochemical dysfunctions were observed that indicated an intermediate level of decompensation. Immunohistochemical visualization revealed focal areas of moderate to severe hypoxia in the detrusor smooth muscle (SM) and subserosal regions of these bladders. No hypoxia was observed in the obstructed bladder mucosa, consistent with the absence of biochemical dysfunction in this compartment, or in unobstructed bladders. Slot-blot analyses confirmed the presence of significant Hypoxyprobe-1-protein adducts in the detrusor of the obstructed bladder, whereas none were present in the control bladder detrusors. CONCLUSIONS: Partial outlet obstruction of rabbit bladders resulted in focal areas of moderate to severe hypoxia in the detrusor SM and subserosal regions concomitant with increased bladder mass, decreased contractile function, and selective metabolic dysfunctions of the SM consistent with an intermediate stage of decompensation. The metabolic characteristics of the normoxic mucosa were normal a were those of unobstructed bladders.     

Structural basis of neurogenic bladder dysfunction. II. Myogenic basis of detrusor hyperreflexia

PURPOSE: We describe the ultrastructure of detrusor smooth muscle in long-standing neurogenic bladder dysfunction in the human. MATERIALS AND METHODS: Detrusor biopsies were obtained from (15 female and 31 male) patients 7 to 96 years old with neurogenic bladder dysfunction for less than 1 to 43 years. Of the patients 9 had meningomyelocele, 25 spinal cord injury and 12 brain disorder. Urodynamically, all patients had detrusor hyperreflexia (neurogenic detrusor overactivity) in addition to bladder outlet obstruction in 4, impaired detrusor contractility in 19, decreased bladder compliance in 4, and detrusor-sphincter dyssynergia in 24. Ultrastructural changes in detrusor, including those associated with detrusor overactivity, impaired detrusor contractility and bladder outlet obstruction, were evaluated qualitatively and quantitatively. RESULTS: Intermediate junctions of muscle cells were absent or reduced in 45 biopsies, which instead had dominant intimate cell appositions with much narrower junctional gaps. A greater than 2 intimate cell apposition-to-intermediate junction ratio was present in 45 biopsies (98%), and intimate cell apposition linked chains of 5 muscle cells or greater in all biopsies (100%). Muscle cell degeneration was observed in 34 biopsies from 20 of 27 patients (74%) with normal contractility and 14 of 19 (74%) with impaired detrusor contractility. No particular changes were associated with functional bladder outlet obstruction due to detrusor-sphincter dyssynergia. CONCLUSIONS: The ultrastructural complete dysjunction pattern is a feature of hyperreflexia as well as nonneuropathic detrusor overactivity of various etiology. A greater than 2 intimate cell apposition-to-intermediate junction ratio had 98% sensitivity but its specificity remains to be determined. The lack of relationship between muscle cell degeneration and detrusor contractility probably reflects limitations of urodynamic measurement of contractility in patients with spinal cord injury and meningomyelocele.     

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     

A prospective controlled quantitative study of ultrastructural changes in the underactive detrusor

PURPOSE: We quantify and compare ultrastructural changes seen at high power on electron microscopic examination of detrusor biopsies from underactive and control bladders. MATERIALS AND METHODS: The detrusor underactivity group consisted of 14 patients (mean age 64.0 years) with post-micturition residuals greater than 300 ml., impaired contractility and no obstruction on urodynamics. A total of 17 patients who voided normally voiders with residual volume less than 50 ml. served as the control group. Bladder biopsies were obtained endoscopically using cold cup technique and processed using standard methods. All specimens were randomized and studied at high power (x12,000 to x24,000) by an examiner who was blinded to the groups. All complete cells within each random grid field were counted up to a total of 500. The number of disrupted cells per 500 cells was noted. RESULTS: Disruptive cell profiles were found in all biopsies. Median cell count was 20 cells/500 in 14 controls and 96.5 cells/500 in the underactive detrusor group (p <0.001). The number of disruptive cell profiles did not correlate with age in the control group (r = 0.34, p = 0.18). CONCLUSIONS: There were distinct morphological changes in the detrusor associated with bladder dysfunction with approximately 4 times more disruptive cells in patients with an underactive bladder. However, there was no correlation between age and number of disruptive cells, suggesting that this is not a process of aging per se. Ultrastructure studies may have a role as an adjunct to urodynamics in the diagnosis of bladder dysfunction.     

Regeneration of detrusor muscle after subtotal cystectomy in the rat: effects on contractile proteins and bladder mechanics.

The aim of the present study was to determine to what extent adult rats can produce new contracting bladder muscle and to see if such newly formed bladder tissue possesses characteristic mechanical properties or whether the ability to recover mechanically is so pronounced that the prehistory of the bladder is unimportant. Subtotal cystectomy was performed in adult female rats, leading to a pronounced decrease in total bladder weight. At 10 weeks, bladder weight had normalized. The histological appearance of such bladders was similar to that of the controls. Active and passive length-tension relations for the detrusor muscle were determined in controls and up to 10 weeks after surgery. Immediately after surgery active and passive forces showed a leftward shift and maximum active force decreased markedly. With time the length-tension curves shifted back to normal, but a decreased active force still remained at 10 weeks. Detrusor actin concentration and detrusor myosin/actin ratio were unaffected by the subtotal cystectomy. Intermediate filament protein/actin ratio showed a significant but transitory increase. We conclude that there is a remarkable recovery of detrusor muscle function after subtotal cystectomy, leading to a normalization of optimum length for active force and a net synthesis of contractile and cytoskeletal proteins. The ability to produce active force does, however, not fully recover.     

Adenovirus mediated gelsolin gene therapy for orthotopic human bladder cancer in nude mice

PURPOSE: Gelsolin is an actin regulatory protein that is undetectable or reduced in human bladder tumors compared with normal epithelial cells. Whether the over expression of gelsolin could inhibit tumor growth was investigated in an orthotopic bladder cancer nude mouse model using recombinant adenovirus encoding wild-type gelsolin (Ad-GSN). MATERIALS AND METHODS: The 2 human bladder cancer cell lines KU-7 and UMUC-2 were transduced with Ad-GSN in vitro. Flow cytometric analysis was done to examine the cell cycle after transducing the adenovirus. Cell growth was compared with control groups of these cells transduced with adenovirus containing the Escherichia coli beta-galactosidase gene Ad-betagal. In vivo KU-7 cells were introduced into the bladder of nude mice (day 0), followed by 3 injections into the urethra (days 2 to 4) with Ad-GSN or Ad-betagal (1 x 10 pfu). At 8 days after initial adenovirus exposure (day 10) each bladder was sectioned and stained, and the mass of the tumor was digitally determined. RESULTS: Bladder cancer cell growth (KU-7 and UMUC-2) was inhibited after these cells were transduced with Ad-GSN in vitro. Based on flow cytometric analysis over expression of gelsolin may cause these cells to arrest or delay at the G2/M phase of the cell cycle. In the orthotopic bladder cancer model the mass of the tumor was approximately 90% less in Ad-GSN treated animals than in controls. CONCLUSIONS: Ad-GSN provides a significant tumor suppressive effect on human bladder cancer cells in this orthotopic nude mouse model. Adenovirus mediated over expression of gelsolin may be useful therapy for human bladder cancer.