Inhibition of diabetic bladder smooth muscle cell proliferation by endothelin receptor antagonists

Urinary bladder hypertrophy and hyperplasia are well recognised in diabetic cystopathy. The urinary bladder is known to synthesise endothelin-1 (ET-1), a potent vasoconstrictor peptide with mitogenic properties. Using diabetic New Zealand White (NZW) rabbits, we investigated the potential role of ET receptor subtypes (ET_A and ET_B) on the proliferation of bladder smooth muscle cells (SMC). Diabetes mellitus was induced in adult male NZW rabbits. After 6 months, control (n=6) and diabetic (n=6) bladders were removed and SMC from the dome and bladder neck were grown using standard explant methodology. At passage two, the cells were made quiescent and then further incubated in foetal calf serum (FCS), control age-matched rabbit serum (CRS) or diabetic rabbit serum (DRS) in the presence or absence of ET_A-antagonist (BQ123) or ET_B-antagonist (BQ788). SMC proliferation was then measured with 5-bromo-2′deoxy-uracil 24 h later and by cell counting (using a haemocytometer) at 48 h. Neither BQ123 nor BQ788 influenced detrusor or bladder neck SMC proliferation in FCS or CRS. However, in the presence of DRS, BQ123 and BQ788 significantly inhibited diabetic detrusor and bladder neck SMC proliferation at 30 and 100 nmol/l (P < 0.03 and P < 0.01, respectively). Cell counts were also significantly reduced from the diabetic detrusor and bladder neck (P < 0.01 and P < 0.03 with BQ123 and BQ788, respectively). These results suggest that ET may play a pathophysiological role in the bladder SMC hyperplasia associated with diabetes mellitus. Received: 24 November 1999 / Accepted: 21 March 2000     

Down-regulation of endothelin-B receptor sites in cavernosal tissue of a rabbit model of partial bladder outlet obstruction: potential clinical relevance

Erectile dysfunction (ED) is a common problem that significantly affects quality of life and psychological well-being. Benign prostatic hyperplasia (BPH) is the commonest known benign proliferative disorder. Recently there has been growing evidence to suggest that patients with high BPH symptom scores have an increased incidence of ED. Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is thought to play an important role as a modulator of erectile physiology and dysfunction. We investigated whether there were any changes in the penile histology and in the density and distribution of ET-1 and its receptor subtypes in the corpora cavernosa of a rabbit model of partial bladder outflow obstruction (BOO). BOO was induced in 12 adult New Zealand White rabbits; 12 sham-operated rabbits acted as controls. Penises were excised after 3 and 6 weeks (n=6 each for control and BOO). Low- and high-resolution autoradiography was performed using radioligands for ET-1, ET_A and ET_B receptors and the results were analysed densitometrically. Ultrastructural evaluation of the corpus cavernosum (CC) was also performed. ET-1, ET_A and ET_B receptor-binding sites were primarily localised to the smooth-muscle cells (SMC) of the CC and to the endothelium lining the cavernosal space. ET_B receptor-binding sites were significantly decreased (P=0.04) in the 6-week BOO cavernosal tissue. These receptor changes were accompanied by ultrastructural changes in the CC. ET-1 may play a role in the pathophysiology of ED associated with BPH. This may partly be due to enhanced vasoconstrictor actions and SMC proliferation secondary to a reduction in ET_B receptors. Further work is needed to evaluate this possibility.     

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.     

Muscarinic and purinergic receptor expression in the urothelium of rats with detrusor overactivity induced by bladder outlet obstruction

OBJECTIVE

To investigate the expression of muscarinic and purinergic receptors in rat urothelium, and changes in their distribution and expression following detrusor overactivity induced by bladder outlet obstruction (BOO).

MATERIALS AND METHODS

Thirty Sprague‐Dawley rats were divided into control (10) and BOO groups (20). Partial BOO was induced for 3 weeks and the rats assessed by cystometrography. A portion of the bladder was stained using immunofluorescence for M₂ and M₃ muscarinic receptors, and P2X₃ purinergic receptors. The remainder was dissected into bladder urothelium and the smooth muscle layer, and the expression of the receptor proteins analysed by Western blotting.

RESULTS

Cystometrography showed a significant decrease in contraction interval and increase in contraction pressure in the BOO group. On immunofluorescence staining, muscarinic and purinergic receptors were localized in both the urothelium and the muscle layer. Immunoreactivity of M₂ and M₃ muscarinic receptors was greater in the urothelium of the BOO group than in the control group; there was a smaller increase in P2X₃ immunoreactivity. On Western blotting, the expression of M₂, M₃ and P2X₃ receptors was increased in the urothelium of the BOO group, and there was increased M₃ receptor expression in the muscle layer of the BOO group.

CONCLUSIONS

There were detectable changes in muscarinic and purinergic receptors with bladder overactivity induced by BOO. Our results suggest that changes in urothelium receptor expression could have a role in mediating the afferent sensory responses in the urinary bladder.     

Increased expression of endothelin B receptors in the diabetic rabbit urinary bladder: functional relevance

OBJECTIVES: To determine the effect of diabetes mellitus on the density and distribution of endothelin A (ETA ) and endothelin B (ETB ) receptor subtypes in the rabbit urinary bladder, and to assess the in vitro functional properties of endothelin-1 (ET-1) receptors in bladder smooth muscle strips from diabetic and healthy rabbits. MATERIALS AND METHODS: Diabetes mellitus was induced in six male New Zealand White rabbits with alloxan and their urinary bladders excised 6 months after the induction of diabetes. On serial detrusor and bladder neck sections, low- and high-resolution autoradiography was performed using radioligands for ET-1, ETA and ETB receptors; these sections were then analysed densitometrically. The results were compared with those from six age-matched healthy control rabbits. Functional responses were investigated using isometric tension studies. RESULTS: ETA and ETB receptor binding sites were localized to both the urothelium and smooth muscle of the detrusor and bladder neck. There were significantly more ETB receptor binding sites in the diabetic detrusor and bladder neck sections than in controls. ET-1 smooth muscle contractile responses were ETA receptor-mediated. The smooth muscle contractile responses to ET-1 were unaltered in the detrusor, but significantly impaired in the bladder neck of diabetic animals compared with controls. CONCLUSION: Alteration in the expression of ETB receptors and in vitro contractile smooth muscle responses to ET-1 in the diabetic rabbit urinary bladder neck may play a role in the pathophysiology of diabetic cystopathy.     

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.     

Endothelin-1, endothelin-3 and their receptors (endothelinA and endothelinB) in chronic renal transplant rejection in rats1

Endothelins (ET) are a family of vasoactive peptides that play an important role in several disorders affecting kidneys. In this study we investigated the expressions of ET-1, ET-3, and their receptors, ET_A and ET_B, in a rat chronic renal transplant rejection model. Renal allografts were performed (F344 → Lewis) with bilateral nephrectomy in recipients. For isograft control, lewis → lewis transplantations were performed. All recipients were sacrificed 140 days after transplantation and the grafts were analyzed histologically. ET-1 and ET-3 protein expression in grafts was measured by immunohistochemistry and ELISA. Semiquantitative RT-PCR methods were used for mRNA levels of ET-1, ET-3, ET_A and ET_B. No evidence of chronical rejection was manifested in isografts. The allografted rats showed proteinuria and increased serum creatinine levels. Histologically, renal allografts showed atrophy and sclerosis of the glomeruli, cortical scarring and vascular intimal thickening. Immunohistochemically, ET-1 and ET-3 were localized in the convoluted tubules, collecting ducts, endothelium and smooth muscle cells of the large blood vessels. Significantly increased staining for ET-1 and ET-3 were found in allografts compared to isografts. Simultaneously, ELISA for ET-1 and ET-3 showed elevated protein concentrations in allografts compared to isografts. Allografts showed significantly increased ET-1- and ET-3 mRNA compared to isografts. On the other hand, a significant down regulation of the ET_A mRNA was noted, and the ET_B mRNA remained unchanged. The data from the present study suggest that alteration of ET system may be of importance in the pathogenesis of chronic renal transplant rejection. Received: 19 January 1999 Revised: 17 December 1999 Accepted: 28 January 2000     

Up-regulation of endothelin-B (ETB) receptors and ETB receptor-mediated rabbit detrusor contraction in partial bladder outlet obstruction.

OBJECTIVE: To investigate, in a rabbit model of bladder outlet obstruction (BOO), whether ETB receptors initiate any contractile activity, and to assess the density of these receptors. MATERIALS AND METHODS: Partial BOO was produced in male New Zealand White rabbits, with age-matched sham-operated rabbits acting as controls. One and 3 weeks later, the detrusor and bladder neck strips were incubated in organ baths with either BQ788 (an ETB antagonist), BQ123 (an ETA antagonist) or vehicle. Concentration-response curves were constructed using IRL-1620 (a selective ETB agonist). Low-resolution autoradiography was performed on serial detrusor and bladder neck sections from control and partial BOO (3-week) rabbits using radioligands for ETA and ETB. RESULTS: In strips from controls and after 1 week of partial BOO, IRL-1620 induced no contractions, but after 3 weeks of BOO, IRL-1620 induced significant concentration-dependent detrusor contractions (producing 12%, 25% and 70% of the KCl response at 10-8, 10-7 and 10-6 mol/L, respectively). The ETA antagonist had no effect on IRL-1620-mediated contractions. In contrast, the ETB antagonist completely abolished these contractions. Autoradiography showed the presence of ETA and ETB receptors in the detrusor and bladder neck of normal and obstructed animals, and a significant up-regulation of ETA and ETB receptors only in the obstructed detrusor smooth muscle. CONCLUSIONS: In BOO, ETB receptors initiate detrusor contractile activity. This is a time-dependent process that may depend on the up-regulation of ETB receptors in the detrusor. Therefore, ETB receptors may play a role in the pathophysiology of partial BOO.     

Evaluation of the Effect of Endothelin-1 and Characterization of the Selective Endothelin A Receptor Antagonist PD155080 in the Prostate

Purpose

To evaluate the contractile effect of endothelin-1 (ET-1) on prostatic urethral pressure and to characterize the effect of the selective ET_A receptor antagonist PD155080 on ET-1 mediated prostatic urethral pressure.

Materials and Methods

The effect of intravenous ET-1 administration on canine urethral pressure was determined in the presence and absence of PD155080. The affinity of PD155080 for endothelin mediated contraction was determined using antagonist dissociation studies. Saturation and competition binding studies were performed using [(¹²⁵) I] ET-1 in both human and canine prostate.

Results

ET-1 bolus injection elicited shallow and prolonged increases the prostatic urethral pressure. Pretreatment with PD155080 totally abolished the urethral contractile response to ET-1. Specific [(¹²⁵) I] ET-1 binding was saturable and of high affinity. Two ET receptor subtypes (ET_A receptor, ET_B receptor) have been identified in human prostate. The ratio of ET_A to ET_B receptors was approximately 1.5:1 in both human and canine prostates. Isometric tension studies revealed that PD155080 shifted the ET-1 dose-response curves to the right and exhibited no effect on the ET_B receptor selective agonist sarafotoxin dose-response curves.

Conclusion

ET-1 mediates prostate smooth muscle tone and may play a role in the pathophysiology and treatment of benign prostatic hyperplasia (BPH).     

Endothelin antagonists in hypertension and kidney disease

The endothelin (ET) system seems to play a pivotal role in hypertension and in proteinuric kidney disease, including the micro- and macro-vascular complications of diabetes. Endothelin-1 (ET-1) is a multifunctional peptide that primarily acts as a potent vasoconstrictor with direct effects on systemic vasculature and the kidney. ET-1 and ET receptors are expressed in the vascular smooth muscle cells, endothelial cells, fibroblasts and macrophages in systemic vasculature and arterioles of the kidney, and are associated with collagen accumulation, inflammation, extracellular matrix remodeling, and renal fibrosis. Experimental evidence and recent clinical studies suggest that endothelin receptor blockade, in particular selective ET_AR blockade, holds promise in the treatment of hypertension, proteinuria, and diabetes. Concomitant blockade of the ET_B receptor is not usually beneficial and may lead to vasoconstriction and salt and water retention. The side-effect profile of ET receptor antagonists and relatively poor antagonist selectivity for ET_A receptor are limitations that need to be addressed. This review will discuss what is currently known about the endothelin system, the role of ET-1 in the pathogenesis of hypertension and kidney disease, and summarize literature on the therapeutic potential of endothelin system antagonism.     

Time-dependent up-regulation of neuronal 5-hydroxytryptamine binding sites in the detrusor of a rabbit model of partial bladder outlet obstruction

Serotonin (5-hydroxytryptamine; 5-HT), a vasoactive bioamine with potent contractile activity, is thought to act indirectly in the urinary bladder by the stimulation of its presynaptic receptors. This results in the release of acetylcholine (ACh), which then acts on muscarinic receptors to produce bladder contractility. Bladder outlet obstruction (BOO) can lead to detrusor instability associated with denervation supersensitivity to ACh. Using a rabbit model of partial BOO, we investigated whether there were any associated changes in the neuronal 5-HT binding sites. Partial BOO was induced in adult male New Zealand White rabbits. Sham-operated age-matched rabbits acted as controls. After 1, 3 and 6 weeks the urinary bladders were excised. Detrusor sections were incubated with [³H]-5-HT. Autoradiographs were generated and analysed densitometrically. The presence of nerves was detected using immunohistochemistry with NF200. Autoradiography demonstrated a time-dependent, significant (P < 0.0001) up-regulation of [³H]-5-HT binding sites in the detrusor smooth muscle after the induction of BOO. Immunohistochemistry confirmed that the [³H]-5-HT binding sites were neuronal. In the rabbit model of partial BOO there was a significant time-dependent up-regulation of neuronal [³H]-5-HT binding sites in the detrusor. This change may influence 5-HT-mediated ACh release, resulting in increased bladder contractility. This, in turn, may play a role in detrusor instability associated with denervation post-junctional supersensitivity. These results provide a possible rationale for further investigation into the use of 5-HT antagonists in the treatment of detrusor instability associated with BOO.     

Expression of transient receptor potential vanilloid 4 and effects of ruthenium red on detrusor overactivity associated with bladder outlet obstruction in rats

Purpose

To investigate transient receptor potential vanilloid 4 (TRPV4) expression and the effects of ruthenium red (RR)—TRPV antagonist—on detrusor overactivity (DO) associated with bladder outlet obstruction (BOO).

Methods

Rats were randomly assigned to 3 groups. The control group (n = 10) included sham-operated rats. The BOO-group without RR (n = 15) and BOO-group with RR (n = 15) underwent partial BOO surgery. Three weeks postoperatively, cystometrography was performed in all rats. After confirming DO, RR was instilled intravesically in the BOO-group with RR. Urodynamic parameters were investigated, including contraction interval (CI) and contraction pressure (CP). TRPV4 expression was evaluated through immunofluorescence staining and western blotting.

Results

The BOO-group without RR had significantly shorter CI and significantly higher CP compared to the control. In the BOO-group with RR, CI was significantly longer compared to the BOO-group without RR. However, change in CP between BOO-group without and with RR was not significantly different. Immunofluorescence staining showed that TRPV4 was localized in the urothelium and detrusor muscles. TRPV4 immunofluorescence signals were increased in the urothelium and detrusor muscle in BOO-group without RR, compared with the control. In western blot analysis, immunoreactive bands indicating expression of TRPV4 were detected in the urothelium and detrusor muscle, and those were significantly increased in the BOO-group without RR compared with the control in the urothelium and detrusor muscle.

Conclusions

TRPV4 plays an important role in the pathophysiology of DO, and RR has a beneficial effect on DO associated with BOO.     

In vitro effect of cyclosporine-A on angiotensins secretion by glomerular cells

Aim: Cyclosporine‐A (CyA) is used to control transplant rejections and to treat autoimmune diseases. We investigated the possibility that changes induced by CyA on endothelin 1 (ET), angiotensin I (AI) and angiotensin II (AII) concentrations recognize a common pathway through which different mechanisms operate. Methods: We measured ET, AI and AII concentrations, before and after either ET or CyA addition to the incubation medium of glomeruli of pig kidneys, isolated in vitro. The measurements were carried out with or without selective (ET_A and ET_B) or unselective ET_A‐ET_B receptor inhibitors. Results: In the presence of CyA, AI and ET are positively correlated either when ET_B receptors are blocked, or when both receptors are free, while this correlation becomes negative when ET_A receptors alone are blocked. Adding ET to the medium, the correlations between AI and ET are negative when either ET_A, or ET_B or both are blocked. The effects of CyA and ET are significant only during the first 2 h of incubation. Conclusion: Cyclosporine‐A recruits angiotensins and ET through ET_A receptors, a mechanism possibly responsible of glomerular damage. This stimulation is time‐dependent. Prevention of the renal damage from CyA should require selective ET_A receptor blockade.     

Time-dependent up-regulation of endothelin-A receptors and down-regulation of endothelin-B receptors and nitric oxide synthase binding sites in the renal medulla of a rabbit model of partial bladder outlet obstruction: potential clinical relevance

OBJECTIVE: To assess the density of endothelin (ET) receptors (ET-1 is a potent vasoconstrictor peptide acting on two known receptors, ETA and ETB ) and nitric oxide synthase (NOS) binding sites in the kidney of a rabbit model of bladder outlet obstruction (BOO). MATERIALS AND METHODS: Partial BOO was created in adult New Zealand White rabbits; after 1, 3, 4 and 6 weeks of BOO, kidney sections were incubated with radioligands for ET-1, ETA, ETB receptors and with [3H]-NOARG (a ligand for NOS). Autoradiographs were generated and analysed densitometrically. Sections were also assessed by NADPH histochemistry. Plasma creatinine, urea and electrolyte levels were regularly monitored. The control and 6-week BOO kidneys were also evaluated ultrastructurally by electron microscopy. RESULTS: There was no significant change in plasma creatinine, urea and electrolyte levels. ETA and ETB receptor density was significantly greater in the medulla than in the cortex (P<0.001) in all animals. There was an up-regulation of ETA receptors (P=0.03) and down-regulation of ETB receptors (P=0.03) and NOS binding sites (P<0.001), as well as decreased NADPH staining in the medulla of 6-week partial BOO kidneys. Electron microscopy detected glomerular disruption of the obstructed kidneys. CONCLUSION: The time-dependent changes in ETA and ETB receptors, NOS binding sites and NADPH staining in the renal medulla, as well as ultrastructural changes, occur despite normal renal function. These changes appear to be an early event and may play a role in the development of renal failure. Hence, the use of ETA receptor antagonists at this early stage may prevent the development of renal failure/impairment in BOO.     

Endothelin‐1 Acutely Reduces the Permeability of Visceral Sheep Peritoneum In Vitro Through Both Endothelin‐A and Endothelin‐B Receptors

Mesothelium is an important part of the peritoneal barrier for water and ion transport, essential for effective peritoneal dialysis (PD). Peritoneal fibrosis has been associated with PD treatment failure. Endothelin‐1 (ET‐1) is a potent vasoactive peptide, involved in pathologic fibrotic processes. Its action is mediated mainly by endothelin type A (ET_A) and type B (ET_B) receptors. The aim of this study was to investigate, by Ussing chamber experiments, the effect of ET‐1 on the transmesothelial electrical resistance (R_TM) of the isolated visceral sheep peritoneum. Intact sheets of visceral peritoneum were obtained from 40 adult sheep and mounted in Ussing‐type chambers. ET‐1 (10^?7 M), BQ‐123 (ET_A receptor antagonist; 10^?6 M), BQ‐788 (ET_B receptor antagonist; 10^?6 M), and their combinations were added on the apical and the basolateral side of the peritoneum. R_TM was measured before and serially after addition of the substances, and changes were registered as percentage (ΔR_TM %). R_TM increased within 1 min after addition of ET‐1 apically (ΔR_TM 65.03 ± 15.87%; P < 0.05) or basolaterally (ΔR_TM 85.5 ± 20.86%; P < 0.05). BQ‐123 and BQ‐788 and their combination significantly reduced (P < 0.05) the effect of ET‐1 to a similar degree in all cases. These results clearly indicate that ET‐1 reduces ionic permeability of the visceral sheep peritoneum in vitro. Additionally, it is obvious that this inhibitory effect is mediated through both ET_A and ET_B receptors.     

Endothelin-A-receptor antagonist LU 302146 inhibits electrostimulation-induced bladder contractions in vivo

OBJECTIVES: Endothelin (ET) is a strong constrictor of smooth muscle structures. The relevance of Endothelin-A receptors in the bladder was demonstrated in several in vitro studies. The aim of this functional study was to evaluate the acute effect of the selective ET-A-antagonist LU 302146 (LU) on neurostimulation-induced bladder contractions in vivo. METHODS: Eight male mini pigs were anesthesized. The bladder was exposed and a double lumen catheter was inserted to perform intravesical pressure (pves) measurements. Laminectomy was performed for sacral anterior root stimulation (SARS) of S2. Four animals received the selective ET-A-antagonist LU, three atropine and one animal was treated with vehicle. Pves was recorded before and after drug administration as well as before and during neurostimulation. At the end of each LU trial, a supplementary application of 4 mg atropine was administered followed by a final SARS. RESULTS: In all experiments reproducible pves values were elicited during electrostimulation before administration of the test substance. The selective ET-A-antagonist reduced stimulation-induced bladder contraction by a mean of 57%. Additional administration of atropine inhibited the detrusor contraction almost completely during SARS. The vehicle had no effect on bladder contraction. CONCLUSIONS: In the presented animal model, ET-1 inhibition with the selective ET receptor-A-antagonist LU 302146 decreases stimulation-induced bladder contraction in vivo. The results suggest that the selective ET-A antagonist LU acts on the atropine-resistant component of efferent detrusor activation since additional administration of atropine almost completely abolish detrusor contraction. This observation in addition to the involvement of ET-1 in bladder smooth muscle proliferation, raises the possibility that ET-receptor antagonists might be beneficial in patients with neurogenic bladder dysfunction or in patients with functional or anatomical BOO.     

Immunohistochemical estimation of hypoxia in human obstructed bladder and correlation with clinical variables

OBJECTIVE

To investigate the tissue distribution of ischaemia in human detrusor in patients with bladder outlet obstruction (BOO) and to correlate the results with clinical variables, as clinical BOO is a common problem in ageing men and ischaemia might be important in detrusor dysfunction.

PATIENTS AND METHODS

From September 2004 to October 2006, 70 patients were recruited, comprising 60 scheduled for surgery to treat benign prostatic hyperplasia (the study group) and 10 as controls. Detrusor tissue was retrieved and stained for hypoxia‐inducible factor (HIF)‐1α, a cellular marker of hypoxia.

RESULTS

The mean (sd ) total number of cells immunoreactive to HIF‐1α in the study group was 93.3 (48.09), and in the specimens from the control group only few rare cells showed weak immunoreactivity to HIF‐1α (0–2). Positive cells were in different proportions between muscle bundles and submucosa, expressed mainly in stromal cells. The urothelium and detrusor muscle showed no immunoreactivity to HIF‐1α. There was strong immunoreactivity in patients with prolonged BOO (<10 years), declining thereafter, and in those patients with urinary retention.

CONCLUSIONS

The urothelium and detrusor seem to be more resistant to hypoxic stress, while stromal cells perceive low oxygen tension. The bladder response to chronic hypoxia through HIF‐1α expression is limited in time and might depend on the functional status of the detrusor.     

Angiotensin II type 1 (AT-1) receptor inhibition partially prevents the urodynamic and detrusor changes associated with bladder outlet obstruction: a mouse model

Study Type – Therapy (case control) Level of Evidence 3b What's known on the subject? and What does the study add? Angiotensin II is the main effector peptide in the bladder local renin‐angiotensin system. This experiment demonstrates the role of this local renin‐angiotensin system with respect to bladder outlet obstruction.

OBJECTIVE

• ? To determine if treatment with an angiotensin II type 1 (AT‐1) receptor antagonist, losartan, can prevent the structural and functional changes that occur in a mouse model of bladder outlet obstruction (BOO).

MATERIALS AND METHODS

• ? Twenty‐four Balb/CAN mice underwent partial urethral obstruction for 6 weeks.
• ? Twelve mice were given oral losartan (10 mg/kg/day), and 12 were not. Six mice served as unobstructed controls, and six unobstructed mice were given oral losartan (10 mg/kg/day) to determine the effect of angiotensin II inhibition on the normal bladder.
• ? Bladder capacity (C), detrusor pressure during voiding (Pdet) and volume at first non‐voiding contraction (NVC1) as a percentage of C were recorded after 6 weeks.
• ? Bladders were stained with haematoxylin and eosin for measurement of detrusor muscular thickness, and graded as 1 = atrophy (<100 µm thick), 2 = normal (100–200 µm thick), 3 = hypertrophy (>200 µm thick) compared with controls.

RESULTS

• ? Compared with controls, BOO mice had greater C (153.5 ± 20.9 vs 57.5 ± 7.4 µl, P < 0.01), higher Pdet (28.8 ± 2.1 vs 12.1 ± 2.1 mm Hg), lower NVC1 (median = 24% vs 54% P= 0.03). BOO mice manifested greater bladder weight (93.2 ± 11.7 mg vs 26.8 ± 2.40 mg, P < 0.01) and greater detrusor muscle thickness (median 3 vs 2, P= 0.02).
• ? Compared with untreated BOO mice, mice treated with losartan had greater mean C (248.8 ± 28.6 vs 153.5 ± 20.9 µL, P= 0.01), no significant change in mean Pdet (24.7 ± 1.6 vs 28.8 ± 2.1 mm Hg, P= 0.2) and a higher mean NVC1 (47% vs 24%, P= 0.02).
• ? Treatment with losartan mediated an insignificant reduction in mean bladder weight (68.1 ± 9.1 mg vs 93.2 ± 11.7 mg, P= 0.10), but a significant reduction in detrusor muscle thickness (median 2 vs 3, P= 0.02). Losartan did not mediate any significant structural or functional changes in the unobstructed mouse bladder.

CONCLUSION

• ? In a mouse model of BOO, treatment with an AT‐1 antagonist partially prevented the urodynamic and structural changes that otherwise occur with BOO.

     

Modulating effect of a selective endothelin A receptor antagonist on pulmonary endothelin system protein expression in experimental diaphragmatic hernia

Background/Purpose

Previously, we reported that perinatal administration of atrasentan, a selective endothelin A receptor (ET_A) antagonist, provided a beneficial effect on the cardiopulmonary profile under short-term conditions in newborn lambs with surgically induced congenital diaphragmatic hernia (CDH). We hypothesized that changes in the hemodynamic profile that we observed at birth in treated animals could be influenced by pulmonary modulation of the endothelin (ET) system.

Methods

The effect of atrasentan on protein expression levels of ETs and ET receptors (ET_A and ET_B receptor) was investigated by immunohistochemistry in lung tissues of untreated control (n = 3), treated control (n = 6), untreated CDH (n = 6), and treated CDH newborn lambs (n = 8).

Results

Right lung tissue of treated control lambs showed significantly higher ET_A protein expression levels in both vascular adventitia and airway epithelia when compared with that of untreated control lambs (P < .05). In contrast, protein expression levels of ET_A and ET _B receptor were significantly lower in the vascular smooth muscle cells among other tissue subcompartments of the right lung of treated CDH newborn lambs vs CDH lambs (P < .02 and P = .005, respectively).

Conclusions

We speculate that rapid pulmonary modulation of ET system protein expression levels by atrasentan results from an indirect effect possibly dependent on ventilation and/or perfusion. In CDH groups, this could contribute to the beneficial effect of the treatment.