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     

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.     

Up-regulation of endothelin (ETA and ETB) receptors and down-regulation of nitric oxide synthase in the detrusor of a rabbit model of partial bladder outlet obstruction

Bladder outlet obstruction (BOO) is associated with altered bladder structure and function. Endothelin-1 (ET-1) has mitogenic and potent contractile properties. There are two ET receptors: ET_A and ET_B. Nitric oxide synthase (NOS) is the enzyme responsible for the synthesis of nitric oxide (NO) which is involved in smooth muscle relaxation. We investigated whether there are any changes in the density of ET-receptors and NOS in the detrusor and bladder neck in a rabbit model of BOO. Partial BOO was induced in adult male New Zealand White rabbits. Sham operated age-matched rabbits acted as controls. After six weeks the urinary bladders were excised and detrusor and bladder neck sections incubated with radioligands for ET-1, ET_A and ET_B receptors and with [³H]–l-NOARG (a ligand for NOS). NADPH histochemistry was also performed. BOO bladder weights were significantly increased (P=0.002). ET-1 binding and ET_A receptor binding sites were significantly increased in the BOO detrusor smooth muscle (P=0.04, P=0.03 respectively) and urothelium (P=0.002, P=0.02 respectively). ET_B receptor binding sites were also significantly increased in the BOO detrusor smooth muscle (P=0.04). However, there was no change in the BOO bladder neck. NOS was significantly decreased in the detrusor smooth muscle (P=0.003) and urothelium (P=0.0002). In the bladder neck NOS was also significantly reduced in the urothelium (P=0.003). NADPH staining was decreased in the detrusor and bladder neck. The up-regulation of ET receptors along with the down-regulation of NOS in the detrusor may contribute to the symptoms associated with BOO. Since ET-1 has a mitogenic role, especially via its ET_A receptors, the increase in ET_A receptors may also be involved in detrusor hyperplasia and hypertrophy in BOO. ET antagonists may therefore have a role in the treatment of patients with BOO. Received: 24 March 1999 / Accepted: 25 June 1999     

Autoradiographic localisation and contractile properties of prostatic endothelin receptors in patients with bladder outlet obstruction

OBJECTIVES: Previous studies have used endothelin (ET) receptor agonists and antagonists to localise ET receptor subtypes in prostatic tissue. We have utilised high affinity ET(A) ([(125)I]PD151242) and ET(B) ([(125)I]BQ3020) receptor-specific radioligands to determine the density and distribution of ET receptor subtypes in prostatic tissues obtained from patients with symptomatic benign prostatic hyperplasia (BPH). The contractile properties of the ET receptor subtypes as well as the effect of ET-1 on alpha(1)-adrenergic receptor-mediated prostatic smooth muscle contraction were assessed. PATIENTS AND METHODS: Saturation binding and quantitative autoradiographic studies were performed using specific radioligands for ET(A) and ET(B) receptors on prostate sections obtained from patients with bladder outflow obstruction secondary to BPH. In vitro isometric tension studies were carried out to characterise the ET receptor subtypes in prostatic smooth muscle strips from the same group of patients. In addition, the effect of ET-1 on alpha(1)-adrenergic receptor-induced prostatic smooth muscle contraction was also investigated. RESULTS: There were dense ET(A) and ET(B) receptor-binding sites in the prostatic stroma. ET(A) receptor-binding sites were also prominent on the prostatic epithelium. ET-1 and sarafotoxin 6 c (ET(B) receptor agonist) elicited prostatic smooth muscle contraction (-log EC(50) 8.31+/-0.15 and 8.22+/-0.22 M, respectively). Both BQ123 (ET(A) antagonist) and BQ788 (ET(B) antagonist) significantly inhibited ET-1- and S6c-mediated prostatic smooth muscle contractile responses, respectively. ET-1 at sub-threshold concentrations significantly enhanced alpha(1)-adrenergic receptor-mediated prostatic smooth muscle contractile responses. CONCLUSIONS: ET(A) receptor-binding sites are prominent in both prostatic stroma and epithelium, whereas ET(B) receptor-binding sites were predominantly seen in the prostatic stroma in symptomatic BPH. Both ET(A) and ET(B) receptors mediate prostatic smooth muscle contraction. ET-1 enhances alpha(1)-adrenergic receptor-mediated contractile responses, suggesting that ET may play a pathophysiological role in bladder outlet obstruction associated with BPH.     

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.     

内皮素1及其受体拮抗剂对HSC-T6细胞内皮素受体mRNA表达的影响

目的探讨内皮素1(ET-1)及其受体拮抗剂对HSC-T6细胞内皮素受体mRNA表达的作用及其机制,进一步了解缩血管物质在门静脉高压症发病机制中的作用。方法培养的肝星状细胞HSC-T6细胞系分为7组,分别为空白对照组,ET-1组(培养瓶中加入10 nmol/L的ET-1),BQ-123组[加入1μmol/L的选择性内皮素A型受体(ETRA)拮抗剂BQ-123],BQ-788组[加入1μmol/L的选择性内皮素B型受体(ETRB)拮抗剂BQ-788],ET-1+BQ-123组(加入10 nmol/L的ET-1和1μmol/L的BQ-123),ET-1+BQ-788组(加入10 nmol/L的ET-1和1μmol/L的BQ-788),ET-1+BQ-123+BQ-788组(加入10 nmol/L的ET-1、1μmol/L的BQ-123和1μmol/L的BQ-788)。采用逆转录聚合酶链反应(RT-PCR)检测HSC-T6细胞内皮素受体mRNA的表达。结果ET-1+BQ123+BQ788组ETRA mRNA的表达与空白对照组相比差异具有统计学意义(分别为0.292±0.023和0.440±0.030,P<0.05),其余各组与之相比无明显差异(P>0.05);与ET-1组相比,ET-1+BQ788组和ET-1+BQ123+BQ788组ETRA mRNA表达低,差异具有统计学意义(分别为0.329±0.044,0.292±0.023和0.487±0.039,P<0.05];与空白对照组相比,ET-1组ETRB mRNA表达上调,但差异无统计学意义(分别为0.499±0.136和0.289±0.047,P=0.134];与ET-1组相比,ET-1+BQ788组ETRB mRNA表达明显低,差异具有统计学意义(分别为0.153±0.071和0.499±0.136,P<0.05)。结论ET-1对ETRA mRNA的表达无明显作用;ET-1本身可能会上调HSC-T6 ETRB mRNA的表达,ET-1作用于ETRA时则会抑制ETRB mRNA的表达。     

Changes of bladder activity and glycine levels in the lumbosacral cord after partial bladder outlet obstruction in rats

Objectives: We investigated the time course of changes in bladder activity as well as in spinal and serum levels of glutamate and glycine after partial bladder outlet obstruction (BOO) in rats. Methods: A total of 36 female rats were divided into six groups: sham operation (control); 3 days, 14 days, and 28 days after BOO; 3 days and 28 days after relief of BOO. Under urethane anesthesia, isovolumetric cystometry was carried out in each group. Then, spinal and serum levels of glutamate and glycine were measured. Results: The interval between bladder contractions was shorter in all of the groups compared with the control group. The amplitude and duration of bladder contractions was decreased at 3 days, 14 days, and 28 days after BOO, and at 3 days after relief of BOO. Spinal and serum glutamate levels showed no changes. However, the spinal glycine level was decreased at 14 days and 28 days after BOO, and at 28 days after relief of BOO. Serum glycine level was also decreased at 28 days after BOO and 28 days after relief of BOO. Conclusions: Detrusor overactivity during the chronic phase of partial BOO is partly caused by a decrease of glycinergic neuronal activity in the lumbosacral cord. A 3‐day period of BOO produces detrusor overactivity, which might be due to an irreversible decrease of spinal glycinergic neuronal activity.     

Reduced endothelin-1- and nitric oxide-mediated arteriolar tone in hypertensive renal transplant recipients

BACKGROUND: The prevalence of hypertension is high in renal transplant recipients. It has been suggested that calcineurin inhibitors (CI) contribute to the development of post-transplant hypertension by stimulating endothelin (ET-1)-mediated and/or reducing nitric oxide (NO)-mediated vascular tone. METHODS: We tested this hypothesis using 2 groups of renal transplant recipients [normotensive patients without a need for antihypertensive medication (Normo-Tx), and hypertensive patients requiring antihypertensives (Hyper-Tx)] in the presence of CI-based immunosuppression. In addition, we studied matched control subjects (C). BQ 123 (ET-A receptor antagonist), BQ123 + BQ788 (ET-A/B-receptor antagonist), ET-1, L-NMMA (NO-synthase inhibitor), acetylcholine (ACH; endothelium-dependent vasodilator), glyceroltrinitrate (GTN, NO donor), and norepinephrine (NE, endothelium-independent vasoconstrictor) were infused into the brachial artery. Forearm blood flow (FBF) was measured by venous occlusion plethysmography. RESULTS: Endothelium-independent vasomotion in response to GTN and NE was similar in all groups. Vascular responses to selective and combined blockade of ET receptors in both Normo-Tx and Hyper-Tx did not exceed those of C. In fact, we observed a significantly lower increase in FBF after BQ 123 (P= 0.03), as well as after BQ 123/788 (P= 0.03) in Hyper-Tx compared with Normo-Tx. This was associated with an increased vascular sensitivity to exogenous ET-1 in Hyper-Tx compared with Normo-Tx (P= 0.04). Vasoconstriction after L-NMMA was reduced in Hyper-Tx compared with Normo-Tx (P= 0.015), while the response to ACH was reduced in both groups of Tx patients to a similar degree (P= 0.005 vs. C). CONCLUSION: Our results do not support a major role for the vascular endothelin system in the hypertension of renal transplant recipients, whereas deficient baseline NO production may be a contributing factor.     

Regional differences in the functional and biochemical properties of endothelin receptor subtypes in the rabbit prostatic urethra

OBJECTIVE: To examine the regional differences in the functional (pharmacological) and biochemical properties of endothelin (ET) receptors in the rabbit prostatic urethra. MATERIALS AND METHODS: The properties of ET receptors in 6-month-old male rabbit prostatic urethras were examined using isolated muscle-bath and radioligand receptor-binding techniques. Using plasma membrane suspensions, saturation and inhibition experiments with [(125)I]ET-1 and unlabelled agonists and antagonists (ET(A)-selective antagonist BQ123, and ET(B)-selective agonist sarafotoxin 6c, STX6c) were done to determine the ET receptor densities and their subtype specificities in the different regions of the urethra. RESULTS: The ETs (ET-1 and ET-3) produced significant concentration-dependent contractile responses in the smooth muscle strips from the different regions of the urethra. Although the maximum contractile responses induced by ET-1 were similar in the different regions, the maximum contractile responses induced by ET-3 were greater in the distal region than in the proximal or middle regions, suggesting that the contractile response to ET-1 is more potent than that to ET-3 in all regions, and that there are region-specific differences in the responses to ET-3 but not ET-1. Moreover, the ET-3-induced contractile response was suppressed by BQ788 (a selective antagonist of the ET(B) receptor) suggesting that the ET(B) receptor subtype contributes to the contractile responses mediated by ET-3. The ET receptors were expressed in higher concentrations in the distal than in the proximal or middle regions. BQ123 and STX6c inhibited [(125)I]ET-1 binding in all regions with high and low affinity constants, indicating the presence of both ET(A) and ET(B) receptor subtypes. The proportions of high-affinity binding sites for BQ123, representing ET(A) receptors, were approximately 68%, 63% and 42% in the proximal, middle and distal regions, respectively. By contrast, the proportions of high-affinity binding sites for STX6c, representing ET(B) receptors, were approximately 27%, 35% and 52% in the proximal, middle, and distal regions, respectively. These data indicate the presence of regional differences in the densities and subtype specificities of ET receptor subtypes, and the existence of regional differences in the rabbit prostatic urethra. CONCLUSION: The results suggest regional differences in ET(B) receptor subtypes that mediate contractile responses to ET-3, reflecting differences in the densities and specificities of the ET receptor subtypes in the rabbit prostatic urethra.     

Endothelin contributes to basal vascular tone and endothelial dysfunction in human obesity and type 2 diabetes

Endothelium-dependent vasodilation is impaired in clinical states of insulin resistance such as obesity and type 2 diabetes. Individuals who have hyperinsulinemic insulin resistance have relatively elevated circulating levels of endothelin (ET)-1, suggesting that ET-1 may be important in the endothelial dysfunction and alterations of vascular tone in these conditions. In 8 lean subjects, 12 nondiabetic obese subjects, and 8 subjects with type 2 diabetes, we measured basal and methacholine-stimulated rates of leg blood flow (LBF) and total serum nitrates (NOx) before and after the intrafemoral arterial administration of BQ123, a specific blocker of ET(A) receptors. BQ123 produced significant vasodilation in the obese and type 2 diabetic subjects (leg vascular resistance = mean arterial pressure/LBF fell by 34 and 36%; P < 0.005) but not in the lean subjects (13%; P = NS, P = 0.018 comparing all groups). ET(A) blockade did not change basal NOx flux (NOx*LBF). This suggests increased basal ET-1 constrictor tone among obese and type 2 diabetic subjects. BQ123 corrected the baseline defect in endothelium-dependent vasodilation seen in obese and type 2 diabetic subjects, suggesting an important contribution of ET-1 to endothelial dysfunction in these subjects. In contrast to basal conditions, stimulated NOx flux was augmented by BQ123 in obese and type 2 diabetic subjects but not in L subjects (P = 0.04), suggesting a combined effect of ET(A) blockade to reduce constrictor tone and augment dilator tone. Endothelin seems to contribute to endothelial dysfunction and the regulation of vascular tone in human obesity and type 2 diabetes.     

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.     

Increase in detrusor wall thickness indicates bladder outlet obstruction (BOO) in men

Detrusor wall thickness decreases continuously while the bladder fills to 50% of its capacity and then remains constant until 100%. Therefore, detrusor wall measurements were performed in patients when the bladder was filled to maximum capacity only. Mean detrusor wall thickness for unobstructed (n = 14), equivocal (n=23) and obstructed patients (n=33) were 1.33, 1.62 and 2.4 mm, respectively (P <0.001). With increasing CHESS letters and CHESS numbers, the thickness of the detrusor wall increased as well (P< 0.001). The positive predictive value of detrusor wall measurement (95.5% for a cut-off value greater than or equal to 2 mm) was superior to all other predictors investigated. The thickness of the detrusor wall increases depending on the extent of BOO. Both constrictive and compressive BOO lead to an increase in detrusor wall thickness. BOO is found in 95.5% of men with a detrusor wall thickness greater than or equal to 2 mm. Measuring the thickness of the detrusor wall can be used as a screening test to detect BOO.     

良性前列腺增生患者膀胱内前列腺突入测定的临床意义

目的探讨良性前列腺增生（BPH）患者前列腺突入膀胱内的程度对膀胱出口梗阻及逼尿肌功能的预测与评价。方法以经腹超声证实前列腺突入膀胱内的BPH患者为研究组,无突入的患者为对照组,分析两组间临床资料及尿动力学检查结果的关系。结果研究组临床资料中,前列腺体积、残余尿量、急性尿潴留及膀胱小梁化的比率与对照组相比差异有统计学意义（p〈0．05）,膀胱内前列腺突入程度与前列腺体积、残余尿量呈正相关（r分别为0．401,0．342,p值分别为0．013,0．0231）;在尿动力学结果中,研究组排尿期最大尿流率（Qmax）、逼尿肌不稳定及低顺应性膀胱的比率与对照组相比差异显著（p〈0．01）,排尿期最大逼尿肌压力（Pdet.max）及梗阻指数显著高于对照组（p〈0．05）,膀胱内前列腺突入程度与Qmax呈负相关（r=-0．284,p=0．045）,与Pdet．max及膀胱出口梗阻指数（BOOI）呈正相关（r分别为0．252,0．456,p值分别为0．041,0．032）。结论前列腺突入膀胱的BPH患者膀胱出口梗阻及膀胱功能受损的程度明显高于无突入患者;经B超测定膀胱内前列腺突入的程度,可以预测及评价膀胱出口梗阻的程度和膀胱功能的改变。     

女性膀胱流出道梗阻的影像尿动力学检查特点分析

目的 探讨女性膀胱出口梗阻(BOO)患者影像尿动力学检查特点及意义.方法 女性BOO患者42例,根据梗阻部位分为膀胱颈梗阻(7例)、中段尿道梗阻(13例)、远端尿道梗阻(15例)、尿道外口梗阻(3例)、盆腔器官重度脱垂(4例)5组.患者术前均行影像尿动力学检查,比较5组病例Qmax、最大膀胱容量、Pdet atQmax、残余尿、逼尿肌无抑制收缩、双侧肾积水等指标.结果 42例患者中以尿频、尿急等储尿症状为主者17例(40.5%),以排尿困难等为主者4例(9.5%),混合症状者21例(50.0%).42例Qmax(10.9±5.6)ml/s、最大膀胱容量(253±140.7)ml、Pdet atQmax(53.3±25.7)cm H2O、残余尿量(76.2±70.3)ml,逼尿肌无抑制收缩者21例(50.0%);5组患者比较:最大膀胱容量差异无统计学意义;膀胱颈梗阻组Pdet atQmax最高、残余尿量最多、Qmax最低、肾积水比例最高,与其他各组比较差异有统计学意义(P<0.05);逼尿肌无抑制收缩在外括约肌部梗阻患者中10例(76.9%),与其他各组比较差异有统计学意义(P<0.05).结论 影像尿动力学检查能有效评估女性BOO下尿路功能,提示梗阻部位并指导临床治疗.女性BOO患者中膀胱颈梗阻程度是影响上尿路损害的主要因素.     

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.     

New approaches in the modulation of bladder smooth muscle cells on viable detrusor constructs

Identification of biochemical and mechanical stimuli in order to modulate the function of bladder smooth muscle cells (SMC) in viable detrusor constructs. Human bladder detrusor cells were seeded on bladder acellular matrix and cultured under different conditions. Cell viability and proliferation were assessed by fluorescent microscopic analyses. Histological, immunohistochemical and flow cytometric analyses were performed to compare growth characteristics and differentiation of SMC. The combination of medium conditioned with proliferative urothelium and mechanical stretch resulted in a more densely populated membrane. In this culture system, the expression of α-smooth muscle actin (α-SMA) and desmin were clearly induced after serum elimination. SMC-phenotype can be modulated in viable detrusor constructs by applying selected combinations of urothelial-conditioned media and mechanical stimulation under stepwise reduction and elimination of serum.     

女性膀胱颈纤维化导致膀胱出口梗阻的诊治（附22例报告）

目的报告女性膀胱颈纤维化导致膀胱出口梗阻（BOO）的诊断治疗体会。方法膀胱颈纤维化导致BOO女性病例22例，经尿流动力学及膀胱镜检确诊，均行经尿道膀胱颈部分切除术，并联合药物治疗。结果随访2～15个月，排尿情况均有改善，最大尿流率〉15mL／s，国际前列腺症状评分（IPSS）5．2±1．6。病理报告显示所有病例膀胱颈组织纤维增生，其中合并慢性炎症改变10例。结论根据临床症状、尿流动力学检查及膀胱镜检可以明确诊断膀胱颈纤维化导致的BOO，经尿道膀胱颈部分切除术可以取得良好的治疗效果。     

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.     

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.     

膀胱出口梗阻大鼠膀胱过度活动的研究

目的:建立膀胱出口梗阻大鼠模型,诱发逼尿肌不稳定(DI),研究膀胱出口梗阻伴发膀胱过度活动的病理生理学特征。方法:选择38只成年SD雌性大鼠,随机分为模型组和对照组,结扎膀胱颈部建立膀胱出口梗阻模型。建模后3、6、9、12周采用BL-410生物机能实验系统测定膀胱压,以充盈期出现DI作为膀胱过度活动存在的标准,记录并计算DI阳性率和频率、最大排尿压(MVP)、最大膀胱容量(MCC)、膀胱顺应性(BC)和剩余尿量(PVR)。用光镜观察建模不同时期膀胱组织的病理学改变。结果:模型组大鼠3、6、9、12周DI阳性率分别为37.50%、75.00%、75.00%、62.50%。MVP、MCC、BC、PVR和DI频率较对照组增高(P<0.01),第9周大鼠PVR、MVP、MCC高于第3、6和12周。不同时期病理学改变呈现出膀胱容量增加、肌层逐渐增厚和纤维化的过程。结论:膀胱出口梗阻与逼尿肌不稳定的发生具有潜在的相关性,其病理学改变和尿流动力学参数反映了膀胱的病理生理学特点。