ROLE OF NITRIC OXIDE (NO) IN FUNCTION OF LOWER URINARY TRACT

[研究目的]探讨一氧化氮（nitric oxide，NO）在离体兔尿道条和尿道平滑肌细胞中的生理作用。我们还研究了PTIO对慢性脊髓损伤家兔膀胱尿道功能的影响。在这个项目中，我们使用了一类新的NO清除剂，即，合成了一系列2-苯基-4，4，5，5-四甲基咪唑啉-3-氧化物（PTIO）的衍生物，它们与NO发生自由基-自由基反应。[方法] 1）功能实验：成年雌性家兔尿道分离。将肌条转移到装有Krebs-Henseleit溶液的器官浴槽中，每条肌条的末端连接到力位移传感器，记录等长力。用1 μ M苯肾上腺素收缩肌条，并施加电场刺激（EFS）。为了测量肌条的N 0 ^-_/N 0 ^-_（NOx）释放，将微透析探针插入尿道条，并用Krebs-Henseleit灌注。 ...更多信息 解决方案将BPS期间获得的透析液级分注入基于Griess反应的自动NOx分析系统中。2)尿道平滑肌细胞培养实验：去除尿道上皮后，将尿道平滑肌切成小块，用含胶原酶的PBS分离。将细胞转移至MEM培养液中，并在CO2培养箱中孵育。将建立好的尿道平滑肌细胞培养液转移到EFS电极的小浴槽中，用显微镜下的视频测微计测量细胞长度的变化。3)脊髓动物实验：成年雌性家兔在T8-T9胸段行椎板切除，横断脊髓。术后8周行双腔导尿管膀胱内留置术，并行尿动力学检查。评价PTLO或L-NNA（NOS抑制剂）对各参数的影响。[结果] EFS引起离体兔尿道条频率依赖性舒张反应。用L-NNA预处理几乎完全抑制舒张反应，PTIO抑制舒张45%。在FES过程中，尿道条和培养细胞中的NOx增加。L-NNA和PTIO均能抑制NO的增加，L-NNA的抑制作用明显大于PTIO，慢性脊髓损伤家兔最大膀胱容量降低，最大排尿压和排尿反射次数明显增加。尿道闭合压明显下降。静脉注射PTIO或L-NNA对膀胱功能无明显影响，但可使尿道闭合压明显升高。[结论]尿道舒张反应是由自由基NO和NO生物产物共同引起的，PTIO是治疗神经源性尿失禁的有效药物。少

[Aimes of study] We have investigated the physiological role of nitric oxide (NO) in isolated rabbit urethral strips or urethral smooth muscle cells. We also investigated the effects of PTIO on vesico-urethral function in the chronic spinal rabbits. In this project, we used a new class of NO scavengers, i.e., a series of derivatives of 2-phenyl-4, 4, 5, 5, -tetramethylimidazoline-oxyl 3-oxide (PTIO), which showed a radical-radical reaction with NO.[Methods] 1) Functional experiments : Urethra was isolated from adult female rabbits. The muscle strip was transferred to organ bath filled with Krebs-Henseleit solution, and end of each strip was connected to force displacement transduser, and isometric force was recorded. The muscle strip was contracted with 1 muM phenylephrine, and electrical field stimulation (EFS) was applied For measurement of N0^-_/N0^-_ (NOx) release from muscle strips, a microdialysis probe was inserted into the urethral strip, and was perfused with Krebs-Henseleit s … More olution. The dialyzate fraction obtained during BPS was infused into an automated NOx analyzing system based on the Griess reaction. 2) Experiments in caltured urethral smooth muscle cells : After removal of urethral epithelium, the urethral smooth muscle was cut into small pieces, and isolated by PBS with collagenase. The cell was transffered to MEM culture solution and incubated CO2 incubator. The establised cultured urethral smooth muscle cell was transffered to small bath with electrode of EFS, and the change in the length of the cell was evaluated by videomicrometer using microscope. 3) Experiments in spinal rabbits : Laminectomy was performed and spinal cord was transected at thracic level T8-T9 in adult femal rabbits. Eight weeks after operation, double lumen catheter was placed into bladder, and urodynamic studies were performed. The effects of PTLO or L-NNA (NOS inhibitor) on each parameter was evaluated.[Results] EFS caused frequency-dependent relaxation responses in isoleted rabbit urethral strips. Pretreatment with L-NNA almost completely inhibited the relaxation responses, and PTIO inhibited the relaxation by 45 %. During FES, NOx increased in the urethral strip and cultured cell. The increased NOx was inhibited by both L-NNA and PTIO.The inhibition was significantly larger in L-NNA than in PTIO.In the chronic spinal rabbits, the maximun bladder capacity was decreased and the maximum voiding pressure and the number of micturition reflax was significantly increased. The urethral closing pressure was significantly decresed. The intravenous injection on PTIO or L-NNA did not change the bladder function, but caused a significant incraese in the urethral closing pressure.[Conclusion] The data suggest that relaxation response in urethra was caused by both radical NO and NO bioaducts, and that PTIO was useful drug for urunary incontinece in the neurogenic condition. Less