2型糖尿病大鼠逼尿肌舒张功能及β受体的变化

目的探讨2型糖尿病大鼠随着病程发展,膀胱舒张功能及逼尿肌细胞β受体的变化。方法①运用离体逼尿肌肌条机械牵张刺激实验,以β受体激动剂异丙肾上腺素(ISO),在不同药物浓度(1×10-9～3×10-5)刺激逼尿肌肌条,观察糖尿病大鼠与正常大鼠在不同时期的舒张力变化。②在不同时间段,消化逼尿肌细胞,用流式细胞学方法检测逼尿肌细胞膜上β3受体的变化。③制备膀胱匀浆,用蛋白印迹法方法检测G蛋白亚型在各时间段的变化。结果①不同浓度ISO刺激逼尿肌肌条的机械性牵张刺激实验研究发现:8周时,半数计量有效浓度(IC50)糖尿病组比对照组敏感(P<0.05),对照组最大舒张力Emax比糖尿病组小,差异有统计学意义(P<0.01)。16周时,IC50对照组比实验组敏感(P<0.01),糖尿病组最大舒张力Emax降低(P<0.05);24周时,IC50对照组比糖尿病组敏感(P<0.01),糖尿病组Emax明显降低(P<0.01)。②采用流式细胞学方法检测逼尿肌细胞上β3受体密度发现,8周时糖尿病组与对照组相比增加,差异有统计学意义(P<0.05);16周时,糖尿病组β3受体平均荧光强度比正常对照组降低(P<0.05);24周时,糖尿病组与对照组β3受体平均荧光强度相比显著下降(P<0.01)。结论糖尿病大鼠逼尿肌舒张功能在病程前期表现为舒张力增强,对ISO敏感增强,相比较同期β3受体含量糖尿病组增高。     

2型糖尿病大鼠不同时期膀胱收缩力动态变化的实验研究

目的观察2型糖尿病(T2DM)大鼠不同时期膀胱收缩力的动态变化情况,以探讨糖尿病膀胱病变的病理生理学机制。方法制备T2DM大鼠模型,以同龄正常大鼠为对照,在成模后6、12和20周,应用M受体激动剂卡巴胆碱,在不同药物浓度水平下进行离体逼尿肌环收缩刺激实验。结果 6周时实验组的最大收缩力(1.12±0.16)g/mg,比对照组(0.99±0.06)g/mg增高;半数有效剂量浓度(EC50)实验组(1.7±0.4)×10-7mol/L比对照组(2.7±0.3)×10-7mol/L降低;12周时实验组的最大收缩力(1.20±0.12)g/mg与对照组(1.21±0.08)g/mg差异无统计学意义;EC50实验组(2.2±0.4)×10-7mol/L与对照组(2.3±0.4)×10-7mol/L差异无统计学意义;20周时实验组的最大收缩力(1.01±0.05)g/mg比对照组(1.90±0.09)g/mg降低;EC50实验组(2.7±0.4)×10-7mol/L比对照组(2.0±0.4)×10-7mol/L增高。结论不同时期的糖尿病大鼠膀胱收缩力表现不同,随着病程的进展,膀胱逼尿肌收缩力呈现先增高后降低的趋势,对卡巴胆碱的敏感性也呈现先增高后降低的变化趋势。     

SREBP-1在1型糖尿病大鼠肾脏的表达和胰岛素的干预性研究

目的探讨1型糖尿病大鼠肾脏脂质沉积和固醇调节元件结合蛋白-1(sterol regulatory element binding protein-1,SREBP-1)的表达以及胰岛素处理的影响。方法以Wistar大鼠建立链脲佐菌素1型糖尿病模型并随机分为正常对照组,糖尿病模型组和胰岛素处理组。喂养2 wk后处死,测定肾皮质组织甘油三酯含量,油红O染色检测脂质沉积的部位;免疫组织化学、Western blot和原位杂交检测肾脏SREBP-1表达。结果与正常对照组相比,1型糖尿病大鼠肾脏甘油三酯含量明显升高,油红O检测示脂质沉积定位于肾近曲小管上皮细胞,肾小球内未见着色。胰岛素处理明显降低了甘油三酯含量,和糖尿病模型组相比差异有统计学意义。免疫组织化学检测SREBP-1定位于大鼠肾脏近曲小管上皮细胞胞质,糖尿病组表达量明显高于正常组和胰岛素处理组。Western blot证实了SREBP-1蛋白前体片段和成熟片段在糖尿病组的高表达,前体片段积分光密度比值为0.673±0.027,成熟片段为0.670±0.028,分别是正常组的1.86倍和1.77倍;胰岛素处理后SREBP-1蛋白前体片段表达下降了52.8%,成熟片段表达量下降了30.9%。原位杂交结果证实SREBP-1 mRNA定位于近曲小管上皮细胞胞质,糖尿病组表达明显升高,与正常对照组相比差异有统计学意义(P<0.01);胰岛素处理后其表达明显下降。结论1型糖尿病大鼠肾近曲小管上皮细胞SREBP-1 mRNA和蛋白表达升高可能参与了肾脏脂质沉积,胰岛素处理可有效降低SREBP-1mRNA和蛋白表达。     

罗格列酮对糖尿病大鼠心肌纤维化及核因子κB、结缔组织生长因子表达的影响

目的探讨罗格列酮对糖尿病大鼠心肌纤维化及心肌核因子κB(NF-κB)、结缔组织生长因子(CTGF)表达的影响。方法链脲佐菌素(STZ)诱导糖尿病大鼠模型。30只大鼠分为3组:正常对照组(n=8)、模型组(n=11)和罗格列酮组(n=11)。罗格列酮组每日予罗格列酮5 mg·kg~(-1)灌胃,其余2组予同体积生理盐水,共12 wk。采用Masson染色检测左室心肌胶原含量、免疫组化法检测心肌NF-κB的表达、Western印迹技术检测心肌CTGF蛋白质表达。结果与正常对照组相比,模型组大鼠左室心肌组织胶原含量明显升高(10±s 3)%vs(16.2±2.4)%,P<0.0l;NF-κB、CTGF蛋白质表达水平亦明显升高,P<0.01。与模型组比较,罗格列酮组大鼠左室心肌组织胶原含量有所下降(16.2±2.4)%vs (12±3)%,P<0.05;NF-κB、CTGF蛋白质表达水平明显减少,P<0.01。结论罗格列酮可抑制糖尿病大鼠心肌纤维化及心肌NF-κB、CTGF的蛋白表达。     

早期糖尿病大鼠背根神经节以及主动脉中COX-2 mRNA和血中PGE_2的变化

目的研究早期糖尿病大鼠背根神经节(DRG)以及主动脉中COX-2mRNA及血中PGE2的变化,探讨COX-2mRNA和血中PGE2之间的关系。方法单次注射链佐霉素60mg·kg-1制作大鼠糖尿病模型,随机分成5组:分别为糖尿病模型组1W,3W,4W,8W和对照组。用RT-PCR技术半定量测定DRG中COX-2mRNA,用放射免疫法检测血浆PGE2的浓度。结果DRG中COX-2mRNA与血浆PGE2的变化趋势相同:1W开始增加,3W达峰值,患病后4W内降至基础水平。对照组血浆PGE2未检出,模型组1W增加至453.3±172.8pg·mL-1,3W达到峰值2500±592.14pg.mL-1,到4W时降至380±112.3pg·mL-1,8W后又增至740±173.2pg·mL-1。DRG中COX-2mRNA1W开始上升,3W达到峰值,4W内降至基础水平。主动脉中COX-2mRNA:对照组最高,1W开始下降,8W恢复至正常水平。结论糖尿病大鼠早期血浆PGE2浓度可能部分受DRG中COX-2mRNA水平的调节。     

血管紧张素Ⅱ在内皮素诱导的大鼠高血压中的作用

目的探讨血管紧张素Ⅱ在内皮素-1诱导的大鼠高血压中的作用。方法 5～6周雄性SD大鼠随机分为3组:①对照组(1%氯化钠慢性灌注,n=7);②内皮素-1慢性灌注组[2.5pmol/(kg.min),n=7];③内皮素-1+奥美沙坦治疗组(0.01%食物中混入,n=7)。Western blotting检测血浆血管紧张素原和主动脉ACE与AT1受体表达。放射免疫法检测血浆肾素活性、血管紧张素Ⅰ和血管紧张素Ⅱ水平。结果与对照组相比,内皮素-1慢性灌注组大鼠血压明显升高[(121±2)vs(141±2)mmHg,P<0.05)],血浆肾素活性明显增加[(3.1±0.6)vs(8.1±0.8)AngI/(mL.h),P<0.05)],血管紧张素Ⅰ[(45±8)vs(122±28)fmol/mL]及血管紧张素Ⅱ水平[(47±7)vs(94±13)fmol/mL]水平明显升高(P<0.05)。内皮素-1灌注未影响大鼠血浆血管紧张素原以及主动脉ACE和AT1受体表达。AT1受体拮抗剂奥美沙坦降低了内皮素-1慢性灌注引起的大鼠高血压。结论内皮素-1诱导的大鼠高血压与血管紧张素Ⅱ水平增高相关。内皮素-1与血管紧张素Ⅱ共同作用,促进了内皮素-1慢性灌注诱导的大鼠高血压进展。     

无创性肢体缺血预适应的早期及延迟效应对中年大鼠心肌缺血再灌注损伤的保护作用和差异

目的研究无创性肢体缺血预适应的早期及延迟效应对中年大鼠心肌缺血再灌注(I/R)损伤的保护作用和差异。方法对预适应组中年大鼠实施1d或连续3d无创性后肢缺血预适应后,分别对其立即实施心脏I/R处理,或24h后再实施心脏I/R处理,与对照组(单纯实施心脏I/R)相比较,观察无创性肢体缺血预适应的早期和延迟效应对中年大鼠心脏I/R后心脏生理学指标[心率(HR)、平均动脉压(MAP)、ST段]、血清学指标、心律失常Lambeth评分、各组I/R后的心肌梗死面积的影响。结果无创性后肢缺血预适应的早期效应组保护效应作用明显。1E,3E组心脏梗死面积(IS/AAR)减小,与对照组IS/AAR[(50±9)%]比较,1E[(15±5)%]和3E[(35±10)%]组IS/AAR显著降低(P<0.05);心律失常Lambeth评分降低,1E[(2.6±0.9)分]、3E[(2.6±1.1)]分组与对照组[(4.2±0.8)分]比较,差异有统计学意义(P<0.05);血清中丙二醛(MDA)的含量减少,血浆中MDA的含量在1E[(7.4±1.2)nmol/ml]、3E[(6.8±0.9)nmol/ml]组中较对照组大鼠[(9.4±1.0)]nmol/ml均显著降低(P<0.05);超氧化物歧化酶(SOD)酶活性增加,血浆中SOD酶活性在1E[(295±30)U/ml]、3E[(345±22)U/ml]组中较对照组大鼠[(257.4±21.0)U/ml]均显著升高(P<0.05,P<0.01);谷胱甘肽过氧化物酶(GSH-PX)酶活性增加,与对照组[(1196±127)U/L]相比,早期预适应组1E[(1547±193)U/L],3E[(1624±69)U/L]组血清中GSH-PX的活性均显著增高(P<0.05)。结论无创性后肢缺血预适应的早期效应对中年大鼠心脏I/R损伤具有保护作用,且其作用大于其延迟效应。     

2型糖尿病大鼠脂联素受体基因表达的相关性研究

目的研究2型糖尿病大鼠骨骼肌和肝脏脂联素受体的表达变化。方法将20只健康雄性SD大鼠随机分为糖尿病组(DM组)和对照组(NC组),每组10只,分别饲以高脂饲料和普通饲料。第13周末DM组大鼠一次性腹腔注射2%链脲佐菌素(STZ)溶液(STZ 25mg/kg,以0.1mol/L柠檬酸-柠檬酸钠缓冲液配制,pH=4.4),NC组腹腔注射等体积的无菌柠檬酸-柠檬酸钠缓冲液。第14周末进行葡萄糖耐量试验(OGTT)筛选成模大鼠,第25周末处死所有大鼠,取血检测空腹血糖、甘油三酯、胆固醇、脂联素、胰岛素水平,并计算胰岛素敏感指数(ISI),使用半定量反转录聚合酶链反应(RT-PCR)的方法分别检测2组大鼠骨骼肌和肝脏组织中脂联素受体(AR)mRNA表达水平。结果 DM组大鼠空腹血糖、甘油三酯、胆固醇、胰岛素水平高于NC组,脂联素水平和ISI低于NC组(P<0.05);DM组大鼠骨骼肌中AR1mRNA和肝脏组织中AR2mRNA的表达水平低于NC组(0.64±0.25与1.36±0.30,0.93±0.18与1.52±0.21;P<0.05),AR1和AR2表达水平分别与空腹血糖、甘油三酯、胆固醇、胰岛素、脂联素、ISI相关。结论 2型糖尿病大鼠AR mRNA的表达水平显著降低,与胰岛素敏感性密切相关。     

雄性大鼠盆神经损伤神经源性膀胱动物模型的建立

目的建立盆神经损伤后神经源性膀胱的大鼠模型,研究引起神经源性膀胱的相关因素。方法将 16只雄性 SD大鼠采用随机数字表法分为神经损伤组和假手术组,每组 8只,神经损伤组损伤双侧盆神经,假手术组仅显露神经但不做处理。 4周后行膀胱功能检测,并留取膀胱组织行 Masson染色及 VAChT阳性神经纤维免疫荧光染色。结果术后 4周,神经损伤组大鼠膀胱质量( 123.6±16.8)mg较假手术组( 87.2±11.2)mg显著增加( P＜0.05)。神经损伤组大鼠膀胱最大容量( 3.45±0.45)mL较假手术组( 1.46±0.11)mL显著增加( P＜0.05)。神经损伤组大鼠膀胱逼尿肌收缩力明显下降,最大排尿压峰值为( 12.1±6.5) mmHg,明显低于假手术组( 42.9±10.5)mmHg(P＜0.05)。 Masson染色示神经损伤组大鼠膀胱壁组织中平滑肌含量减少,胶原纤维增生明显,其胶原占比( 19.75±5.46)%显著高于假手术组( 9.95±3.25)%,(P＜0.05)。免疫荧光结果显示神经损伤组膀胱壁内 VAChT阳性神经纤维占比( 7.46±1.68)%较假手术组( 12.67±3.24)%显著减少( P＜0.05)。结论损伤大鼠双侧盆神经可成功建立神经源性膀胱动物模型,表现为膀胱逼尿肌纤维化, VAChT阳性神经纤维减少及排尿功能受损。     

糖尿病大鼠膀胱逼尿肌兴奋性与收缩性实验研究

目的观察糖尿病膀胱病变逼尿肌兴奋性与收缩性变化,并探讨其发病机制。方法建立糖尿病大鼠模型,正常大鼠作对照,测定不同糖尿病病程离体逼尿肌肌条机械性牵张及胆碱能药物刺激后逼尿肌的兴奋性、收缩性变化。结果糖尿病组大鼠膀胱逼尿肌机械性牵张及胆碱能药物刺激后最大收缩力、平均收缩力、收缩频率均较对照组下降。结论糖尿病膀胱病变逼尿肌收缩功能受损,且随病程延长损害程度逐渐加重。逼尿肌对早期胆碱能药物干预反应敏感。     

Alteration of M(3) subtype muscarinic receptors in the diabetic rat urinary bladder

The M(3) receptor (M(3)-mAChR) is the major muscarinic subtype in the animal bladder responsible for detrusor contraction. The alterations in its protein quantity and biosynthesis during diabetic cystopathy were investigated. 3-month-old male Wistar rats were divided into two groups: (1) 2-week diabetic rats and (2) normoglycemic control rats. Diabetes was induced by a single intravenous injection of 60 mg/kg streptozotocin. The amount of M(3) receptor protein in the rat bladder body tissue was measured by Western immunoblotting using monoclonal antibodies. For determination of M(3) muscarinic receptor mRNA in the bladder tissue, the method of Northern blotting was employed. The results of the Western immunoblotting showed that the amount of M(3)-mAChR protein in the diabetic bladder was significantly increased by about 70.2 +/- 8.5% when compared to the control bladder (p < 0.05, n = 8). Northern blotting demonstrated a 54.7 +/- 6.0% increase of M(3)-mAChR mRNA in the diabetic bladder (p < 0.05, n = 8). The findings of the present study demonstrated an upregulation of M(3)-mAChR biosynthesis in the diabetic urinary bladder. This phenomenon offers an explanation of the increased contractility after muscarinic stimulation of the detrusor muscle of diabetic animals.     

Assessment of muscarinic receptor subtypes in human and rat lower urinary tract by tissue segment binding assay

Muscarinic receptors in the human and rat lower urinary tract (urinary bladder detrusor muscle and mucosa, and prostate) were identified by intact tissue segment binding assays with two radioligands, and the effects of prolonged receptor activation in vitro on muscarinic receptors were examined. Hydrophilic [(3)H]-NMS and hydrophobic [(3)H]-QNB bound to the detrusor muscle segments with the same density, suggesting that the muscarinic receptors were localized at the plasma membrane. While the density of muscarinic receptor was higher in detrusor muscle than in the bladder mucosa and prostate, there was no species-specific difference either in density or in subtype distribution (M(1), M(2), and M(3) subtypes in detrusor; M(2) and M(3) subtypes in bladder mucosa; and M(1) and M(2) subtypes in prostate). Incubation of detrusor strips with carbachol decreased [(3)H]-NMS binding sites within 20 min, followed by a reduction of [(3)H]-QNB binding sites after a 60-min lag phase. The loss of the binding sites over 3 h after carbachol treatment was the same (approximately 40%) for both radioligands. The present intact tissue segment binding assay reveals tissue-specific and plasma membrane distribution of distinct muscarinic receptor subtypes and their dynamic changes (internalization and down-regulation) in lower urinary tract of humans and rats.     

Ability of cyclohexenonic long-chain fatty alcohol to ameliorate diabetes-induced cystopathy in the rat

We investigated the pharmacological effects of N-hexacosanol on diabetic rat detrusor. Eight-week-old male Sprague-Dawley rats were randomly divided into 4 groups: diabetic rats induced by 50 mg/kg intraperitoneally of streptozotocin treated with N-hexacosanol (0, 2 or 8 mg/kg, subcutaneously every day) and control rats. Bladder function was estimated by functional studies using carbachol and KCl. Contractile response curves to increasing concentrations of carbachol were constructed in the absence and presence of various concentrations of subtype-selective muscarinic antagonists, that is, atropine, pirenzepine, methoctramine and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP). The participation levels of muscarinic M(2) and M(3) receptor mRNAs in detrusor were investigated by real-time polymerase chain reaction. Treatment with N-hexacosanol did not alter diabetic status of the rats, but significantly improved the diabetes-induced hypercontractility of the rat bladder. Estimations of the pA(2) values for atropine, pirenzepine, methoctramine and 4-DAMP indicate that the carbachol-induced contractile response is mediated through the M(3) receptor subtype in all groups. Furthermore, N-hexacosanol ameliorated the diabetes-induced upregulation of muscarinic M(2) receptor mRNAs in streptozotocin-diabetic rat detrusor. Our data indicate that N-hexacosanol has therapeutic effects on hypercontractility in the diabetic bladder by ameliorating overexpression of muscarinic M(2) and M(3) receptor mRNAs without significant alternations of pharmacological profiles.     

Long-term nitric oxide deficiency causes muscarinic supersensitivity and reduces beta(3)-adrenoceptor-mediated relaxation, causing rat detrusor overactivity

BACKGROUND AND PURPOSE: Overactive bladder is a complex and widely prevalent condition, but little is known about its physiopathology. We have carried out morphological, biochemical and functional assays to investigate the effects of long-term nitric oxide (NO) deficiency on muscarinic receptor and beta-adrenoceptor modulation leading to overactivity of rat detrusor muscle. EXPERIMENTAL APPROACH:Male Wistar rats received N(omega)-nitro-L-arginine methyl ester (L-NAME) in drinking water for 7-30 days. Functional responses to muscarinic and beta-adrenoceptor agonists were measured in detrusor smooth muscle (DSM) strips in Krebs-Henseleit solution. Measurements of [(3)H]inositol phosphate, NO synthase (NOS) activity, [(3)H]quinuclidinyl benzilate ([(3)H]QNB) binding and bladder morphology were also performed. KEY RESULTS:Long-term L-NAME treatment significantly increased carbachol-induced DSM contractile responses after 15 and 30 days; relaxing responses to the beta(3)-adrenoceptor agonist BRL 37-344 were significantly reduced at 30 days. Constitutive NOS activity in bladder was reduced by 86% after 7 days and maintained up to 30 days of L-NAME treatment. Carbachol increased sixfold the [(3)H]inositol phosphate in bladder tissue from rats treated with L-NAME. [(3)H]QNB was bound with an apparent K(D) twofold higher in bladder membranes after L-NAME treatment compared with that in control. No morphological alterations in DSM were found. CONCLUSIONS AND IMPLICATIONS:Long-term NO deficiency increased rat DSM contractile responses to a muscarinic agonist, accompanied by significantly enhanced K(D) values for muscarinic receptors and [(3)H]inositol phosphate accumulation in bladder. This supersensitivity for muscarinic agonists along with reductions of beta(3)-adrenoceptor-mediated relaxations indicated that overactive DSM resulted from chronic NO deficiency.     

Regulation of bladder muscarinic receptor subtypes by experimental pathologies

1 The M3 muscarinic receptor subtype is widely accepted as the receptor on smooth muscle cells that mediates cholinergic contraction of the normal urinary bladder and other smooth muscle tissues, however, we have found that the M2 receptor participates in contraction under certain abnormal conditions. The aim of this study was to determine the effects of various experimental pathologies on the muscarinic receptor subtype mediating urinary bladder contraction. 2 Experimental pathologies resulting in bladder hypertrophy (denervation and outlet obstruction) result in an up-regulation of bladder M2 receptors and a change in the receptor subtype mediating contraction from M3 towards M2. Preventing the denervation-induced bladder hypertrophy by urinary diversion prevents this shift in contractile phenotype indicating that hypertrophy is responsible as opposed to denervation per se. 3 The hypertrophy-induced increase in M2 receptor density and contractile response is accompanied by an increase in the tissue concentrations of mRNA coding for the M2 receptor subtype, however, M3 receptor protein density does not correlate with changes in M3 receptor tissue mRNA concentrations across different experimental pathologies. 4 This shift in contractile phenotype from M3 towards M2 subtype is also observed in aged male Sprague-Dawley rats but not females or either sex of the Fisher344 strain of rats. 5 Four repeated, sequential agonist concentration response curves also cause this shift in contractile phenotype in normal rat bladder strips in vitro, as evidenced by a decrease in the affinity of the M3 selective antagonist p-fluoro-hexahydro-sila-diphenidol (p-F-HHSiD). 6 A similar decrease in the contractile affinity of M3 selective antagonists (darifenacin and p-F-HHSiD) is also observed in bladder specimens from patients with neurogenic bladder as well as certain organ transplant donors. 7 It is concluded that although the M3 receptor subtype predominantly mediates contraction under normal circumstances, the M2 receptor subtype can take over a contractile role when the M3 subtype becomes inactivated by, for example, repeated agonist exposures or bladder hypertrophy. This finding has substantial implications for the clinical treatment of abnormal bladder contractions.     

Synthesis and pharmacological evaluation of enantiomerically pure 4-deoxy-4-fluoromuscarines.

Four isomers of [(4-fluoro-5-methyl-tetrahydrofuran-2-yl)methyl]trimethylammonium iodide (4-deoxy-4-fluoro-muscarines) were prepared in enantiomerically and diastereomerically pure form from (S)-(-)-methyl 4-methylphenyl sulfoxide, ethyl fluoroacetate, and allyl bromide. Their absolute configurations were assigned by 1H NMR analyses. The four optically pure compounds were tested in vitro on guinea pig and their muscarinic potency was evaluated at M3 (ileum and bladder) and M2 (heart) muscarinic receptor subtypes. Compound 1a, the most potent isomer of the series, was also tested in vivo on pithed rat and its muscarinic activity at the M1 receptor subtype was compared with that of muscarine. Moreover, affinity and relative efficacy were calculated in vitro for this compound at M2 (heart force and rate) and M3 (ileum and bladder) receptors in order to investigate muscarinic receptor heterogeneity. The 4-deoxy-4-fluoromuscarines display a similar trend of potency as the corresponding muscarines and compound 1a shows differences in the affinity constants among the studied tissues. Replacement of a hydroxyl group for a fluorine atom in the 4 position of muscarine produces 1 order of magnitude increase in affinity for cardiac M2 muscarinic receptors controlling rate, while the affinity at cardiac M2 muscarinic receptors controlling force is unchanged, opening the possibility of a further classification of cardiac muscarinic receptors.     

The Forefront for Novel Therapeutic Agents Based on the Pathophysiology of Lower Urinary Tract Dysfunction: Bladder Selectivity Based on In Vivo Drug–Receptor Binding Characteristics of Antimuscarinic Agents for Treatment of Overactive Bladder

We have reviewed the binding of antimuscarinic agents, used to treat urinary dysfunction in patients with overactive bladder, to muscarinic receptors in target and non-target tissues in vivo. Transdermal administration of oxybutynin in rats led to significant binding in the bladder without long-term binding in the submaxillary gland and the abolishment of salivation evoked by oral oxybutynin. Oral solifenacin showed significant and long-lasting binding to muscarinic receptors in mouse tissues expressing the M₃ subtype. Oral tolterodine bound more selectively to muscarinic receptors in the bladder than in the submaxillary gland in mice. The muscarinic receptor binding activity of oral darifenacin in mice was shown to be pronounced and long-lasting in the bladder, submaxillary gland, and lung. In vivo quantitative autoradiography using (+)N-[¹¹C] methyl-3-piperidyl benzilate in rats showed significant occupancy of brain muscarinic receptors on intravenous injection of oxybutynin, propiverine, solifenacin, and tolterodine. The estimated in vivo bladder selectivity compared to brain was significantly greater for solifenacin and tolterodine than oxybutynin. Darifenacin occupied few brain muscarinic receptors. Similar findings were also observed with positron emission tomography in conscious rhesus monkeys. The newer generation of antimuscarinic agents may be advantageous in the bladder selectivity after systemic administration.     

Direct labeling of rat M3-muscarinic receptors by [3H]4DAMP

The muscarinic receptors of rat submaxillary gland, rat heart and rat cortex were directly labeled using the ligand [3H]4-diphenylacetoxy-N-methyl-piperidine methiodide [( 3H]4DAMP). In the rat submaxillary gland, [3H]4DAMP predominantly bound with high affinity (Kd = 0.2 nM) to a population of binding sites that displayed the pharmacology of the M3 muscarinic receptor subtype. In rat heart, [3H]4DAMP labeled the M2 muscarinic receptor with low affinity (Kd = 4 nM). In rat cortex [3H]4DAMP predominantly bound to a population of sites with high affinity (Kd = 0.2 nM). The pharmacology of these sites was consistent with [3H]4DAMP labeling both M1 and M3 muscarinic receptors present in rat cortex with high affinity. These data indicate that [3H]4DAMP represents a useful ligand for selectively labeling the M1 and M3 muscarinic receptor subtypes.     

In vivo characterization of muscarinic receptors in peripheral tissues: evaluation of bladder selectivity of anticholinergic agents to treat overactive bladder

The present study was undertaken to characterize in vivo muscarinic receptors in peripheral tissues (urinary bladder, submaxillary gland, colon, stomach, heart) of mice, and further to evaluate bladder-selectivity of anticholinergic agents to treat overactive bladder. Following i.v. injection of [(3)H]QNB in mice, the radioactivity in peripheral tissues was exclusively detected as the unchanged form. The in vivo specific [(3)H]QNB binding in particulate fraction of tissue homogenates of mice showed a pharmacological specificity which characterized muscarinic receptors. Binding parameters (K (d) and B (max)) for in vivo specific [(3)H]QNB binding differed between mouse tissues. Oral administration of oxybutynin attenuated significantly in vivo specific [(3)H]QNB binding in all tissues of mice. From ratios of AUC(urinary bladder)/AUC(other tissues) of time-dependent muscarinic receptor occupancy, oral oxybutynin has been shown to exert little urinary bladder selectivity. Following oral administration of propiverine, there was a significant reduction of in vivo specific [(3)H]QNB binding in the urinary bladder, colon and submaxillary gland, but not in the stomach and heart. From the ratios of AUC(urinary bladder) to AUC(submaxillary gland) or AUC (heart), it has been shown that oral propiverine exerts higher selectivity to muscarinic receptors in the urinary bladder than in the submaxillary gland and heart. Similarly, tolterodine displayed high selectivity to muscarinic receptors in the urinary bladder than in the submaxillary gland. Thus, the present study has demonstrated that [(3)H]QNB may be a useful ligand for in vivo characterization of muscarinic receptor binding of anticholinergic agents to treat overactive bladder. Propiverine and tolterodine have exhibited in vivo selectivity of muscarinic receptor in the mouse urinary bladder rather than in the submaxillary gland, and such receptor binding specificity may be the reason of lower incidence of dry mouth.