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

【研究目的】我们研究了一氧化氮（NO）在兔离体尿道条或尿道平滑肌细胞中的生理作用。我们还研究了PTIO对慢性脊髓兔膀胱尿道功能的影响。在本项目中，我们使用了一类新的NO清除剂，即一系列与NO发生自由基-自由基反应的2-苯基- 4,4,5,5，-四甲基咪唑-氧基3-氧化物（PTIO）衍生物。【方法】1)功能实验：从成年母兔分离尿道。将肌条转移到充满Krebs-Henseleit溶液的器官浴中，将每条肌条的末端连接到力位移传感器上，记录等长力。用1 μ m的苯肾上腺素收缩肌条，采用电场刺激（EFS）测定肌条的no ^-_/ no ^-_ （NOx）释放量，将微透析探针插入尿道条，并灌注Krebs-Henseleit溶液。在BPS过程中获得的透析产物馏分注入到基于Griess反应的自动NOx分析系统中。2)培养尿道平滑肌细胞实验：去除尿道上皮后，将尿道平滑肌切成小块，用PBS加胶原酶分离。将细胞转移至MEM培养液中，CO2培养箱孵育。将培养好的尿道平滑肌细胞用电刺激电极转移到小浴液中，用显微镜观察细胞长度的变化。3)脊髓兔实验：对成年母兔进行椎板切除术，并在椎体水平T8-T9处横切脊髓。术后8周，双腔导尿管置入膀胱，行尿动力学观察。评估PTLO或L-NNA （NOS抑制剂）对各参数的影响。【结果】电刺激引起兔离体尿道条的频率依赖性松弛反应。L-NNA预处理几乎完全抑制了松弛反应，PTIO抑制了45%的松弛反应。FES过程中，尿道条和培养细胞中NOx含量增加。L-NNA和PTIO均抑制了NOx的增加。L-NNA的抑制作用明显大于PTIO。慢性脊髓兔最大膀胱容量降低，最大排尿压力和排尿反次数明显增加。尿道闭合压力明显降低。静脉注射PTIO或L-NNA均未改变膀胱功能，但引起尿道闭口压力明显升高。【结论】本研究提示NO自由基和NO生物成人均可引起尿道松弛反应，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