Roles of Nitric Oxide and Superoxide in Cystitis

描述（由申请人提供）：骨盆区域照射可导致膀胱炎症和功能障碍。这种膀胱炎或其可能性也增加了膀胱癌的发病率，禁止对膀胱肿瘤进行放射治疗，并限制了治疗其他盆腔恶性肿瘤的允许辐射剂量。放射性膀胱炎的发病机制尚不清楚。它可能涉及伞细胞特有的线粒体一氧化氮合成酶（mtNOS）的激活，渗透性屏障的破坏和尿液渗入固有层。这反过来又会导致炎症和增加胶原III沉积在固有层。导致膀胱顺应性下降和功能障碍。我们已经开发了放射性膀胱炎的啮齿动物模型，其中辐照导致12小时内经皮细胞抗性降低，尿素和水的渗透性增加。6个月时，膀胱造影显示膀胱顺应性和收缩间期减少，而残余体积和基线压力增加，这是纤维化的特征。预先转染放射性保护剂锰超氧化物歧化酶（MnSOD）仅部分有效，很可能是由于过氧化物酶活性降低和过量过氧化氢形成。然而，在放疗期间使用NOS抑制剂的新型膀胱内治疗，或对无mtNOS的膀胱进行照射，提供了几乎完全的保护。抑制NO可阻止其与超氧化物（O2）反应生成过氧亚硝酸盐（ONO2-），破坏呼吸链复合物I和III，导致细胞凋亡/坏死死亡。特异性目的1将验证电离辐射激活mtNOS的假设，从而产生破坏尿路上皮通透性屏障的活性氮和活性氧（RNS和ROS）。我们已经开发了NO和ONO2微传感器，使我们能够同时实时测量完整小鼠膀胱和培养尿路上皮细胞中这些代谢物的变化水平。这些测量将与线粒体酶功能的测定变化相关联。特异性目的2将检验膀胱内给予NOS拮抗剂或具有过氧化物酶活性的MnSOD或SOD模拟物可保护膀胱免受放射性膀胱炎的假设。这些疗法在照射小鼠膀胱中的有效性将在1至6个月时通过渗透性测量、膀胱测量和胶原沉积的组织化学分析来评估。

DESCRIPTION (provided by applicant): Irradiation of the pelvic region can result in bladder inflammation and dysfunction. This cystitis or its likelihood also increases the incidence of bladder cancer, prohibits radiation treatment for bladder tumors, and limits the allowable radiation dose for treating other pelvic malignancies. The mechanism of radiation cystitis is unclear. It may involve activation of a mitochondria nitric oxide (NO) synthase (mtNOS) unique to the umbrella cells, disruption of the permeability barrier and infiltration of urine into the lamina propria. This in turn can lead to inflammation and increased collagen III deposition in the lamina propria. Decreased bladder compliance and dysfunction result. We have developed rodent models of radiation cystitis where irradiation results in decreased transepithelial resistance and increased urea and water permeabilities within 12 hours. At six months, cystometrograms show that bladder compliances and intercontractile intervals are decreased while residual volumes and baseline pressures are increased-features indicative of fibrosis. Prior transfection with the radioprotectant manganese superoxide dismutase (MnSOD) is only partially effective, most likely due to decreased peroxidase activity and excess hydrogen peroxide formation. However, novel intravesical therapy with a NOS inhibitor during irradiation, or irradiation of bladders devoid of mtNOS, offers almost complete protection. Inhibition of NO can prevent its reaction with superoxide (O2) to form peroxynitrite (ONO2-), which can damage complexes I and III of the respiratory chain and lead to apoptotic/necrotic cell death. Specific Aim 1 will test the hypothesis that ionizing radiation activates mtNOS, resulting in reactive nitrogen and oxygen species (RNS and ROS) which disrupt the urothelial permeability barrier. We have developed NO and ONO2- microsensors which allow us to simultaneously measure in real-time, the changing levels of these metabolites in intact mouse bladders and cultured urothelial cells. These measurements will be correlated with assayed changes in mitochondrial enzyme functions. Specific Aim 2 will test the hypothesis that the intravesical administration of NOS antagonists or MnSOD or SOD mimetics with peroxidase activity protect the bladder against radiation cystitis. The effectiveness of these therapies in irradiated mouse bladders will be assessed at 1 to 6 months by employing permeability measurements, cystometry and histochemical analyses for collagen deposition.