MECHANISMS FOR ALTERED DETRUSOR CONTRACTION IN DIABETES

说明(改编自应用程序) 人类糖尿病与一系列排尿功能障碍有关 特点是膀胱感觉受损，排尿后残留物增加 逼尿肌可能进展的体积和逼尿肌收缩能力降低 反射不全和尿流减少。动物模型研究， 尤其是链脲佐菌素(STZ)诱导的糖尿病大鼠和 遗传性尿崩症(di/di)Brattleboro(BB)大鼠，显示 与人类糖尿病相似的变化，例如较大的膀胱 排尿功能受损的容量。拟议研究的数据 将提供有关糖尿病对健康影响的基本信息 逼尿肌收缩力和膀胱功能，并测试潜在的 假设糖尿病对分子机制有影响 规范部队的生成和维护，以清空 膀胱。为了解决这一假设，我们建议使用逼尿肌 糖尿病大鼠(STZ诱导的糖尿病BB大鼠)的平滑肌，胰岛素 治疗和对照大鼠(非糖尿病渗透性利尿，正常)和 确定调控机制如何(通过蛋白质)控制 蛋白质相互作用或酶作用)由这些蛋白质改变。 具体地说。我们将确定：(1)膀胱内是否缺乏 糖尿病患者的膀胱压力和过度扩张是由低水平引起的。 肌球蛋白轻链(MLC)磷酸化的改变或 静息状态下逼尿肌中MLC的磷酸化 语气或在部队发展期间；(2)活动和 MLC激酶和/或蛋白C激酶(PCK)亚型的表达， 在糖尿病患者的逼尿肌中，与信号通路有关的信号通路发生了改变； (3)平滑肌特异性钙蛋白的表达是否发生变化 (h-caldesmon)，调节肌动蛋白-肌球蛋白的细丝成分 糖尿病患者逼尿肌中的相互作用是导致不能 对于糖尿病患者的肌肉无力维持力量；(4)是否 肌球蛋白亚型表达的改变 对糖尿病的反应；以及(5)肌动蛋白/激活的ATPase活性 在糖尿病患者的膀胱中，逼尿肌中肌球蛋白的含量发生了变化。我们提议 使用来自糖尿病和非糖尿病渗透性膀胱和尿路的肌肉 利尿剂和正常大鼠。肌球蛋白的表达将用 定量逆转录聚合酶链式反应 比较聚合酶链式反应、Northern和Western印迹分析。的功能 肌球蛋白亚型将通过测量肌球蛋白ATPase的作用来分析 肌球蛋白头上方肌动蛋白细丝的活动和运动 体外动力测定。逼尿肌收缩力将用力分析 完整肌条的测量。收缩功能的改变 收缩的装置和调节，不依赖于膜和 肌浆网，将通过化学“剥皮”来确定。 肌肉条。对分子事件的理解 糖尿病患者的收缩功能障碍对于靶向 药物治疗药物开发的分子步骤 糖尿病膀胱病。

DESCRIPTION (adapted from application) Diabetes in humans is associated with a spectrum of voiding dysfunctions characterized by impaired bladder sensation, increased postvoidal residual volume and decreased detrusor contractility that may progress in detrusor areflexia and diminished urinary flow. Studies on animal models, particularly the streptotzotozin (STZ) induced diabetic rats and Brattleboro (BB) rats with hereditary diabetic insipidus (di/di), show changes similar to those in human diabetes, e.g., a greater bladder capacity with impaired voiding function. Data from the proposed studies will provide the basic information regard the effect of diabetes on detrusor contractility and bladder function, and test the underlying hypothesis that diabetes has an effect on the molecular mechanisms that regulate force generation and maintenance that are required to empty the urinary bladder. To address this hypothesis, we propose to use detrusor smooth muscle from diabetic (STZ induced diabetes BB rats), insulin treated, and control rats (non diabetic osmotic diuresis, and normal) and to determine how the regulatory mechanisms controlled (through protein protein interaction or enzymatically) by these proteins are altered. Specifically. we will determine: (1) whether the lack of intravesical pressure and over distension of the diabetic bladder is due to a low level of myosin light chain (MLC) phosphorylation or a change in the site of phosphorylation of the MLC in the detrusor smooth muscle at the resting tone or during force development; (2) whether the activities and expression of MLC kinases and/or the protein C kinase (PCK) isoforms, implicated in signaling pathways, are altered in the detrusor in diabetes; (3) whether changes in the expression of smooth muscle specific caldesmon (h-caldesmon), the thin filament component that regulates actin-myosin interactions, in the detrusor in diabetes is responsible for the inability for the inability of diabetic smooth muscle to maintain force; (4) whether thre is a change in the expression of smooth muscle myosin isoforms in response to diabetes; and (5) whether the actin/activated ATPase activity of myosin in the detrusor is altered in diabetic bladders. We proposed to use muscles from bladders and urethras of diabetic, non diabetic osmotic diuretic and normal rats. The expression of myosin will be studied using reverse transcribed polymerase chain reaction (PCR), quantitative comparative PCR, and Northern and Western blot analyses. The function of myosin isoforms will be analyzed by measuring the action-myosin ATPase activity and the movement of actin filaments over myosin heads in the in vitro motility assays. Detrusor contractility will be analyzed by force measurements of intact muscle strips. Alterations in the contractile apparatus and regulation of contraction, independent of the membranes and sarcoplasmic reticulum, will be determined using chemically "skinned" muscle strips. An understanding of the molecular events that lead to contractile dysfunctions in diabetes is crucial in order to target the molecular steps for the development for pharmacological gents to treat diabetic cystopathy.