Functioning of Nodose Ganglia in Diabetes

描述(申请人提供)：糖尿病控制不佳的患者经常出现胃肠功能异常。临床研究表明，这些异常大多可归因于迷走神经传入功能缺陷。我们的初步研究表明，链脲佐菌素诱导的糖尿病大鼠(STZ-D)的结状神经节(NG)神经元呈现超极化，导致兴奋性降低。这可能导致糖尿病状态下迷走神经功能的异常。我们假设慢性高血糖激活“背景”Tresk钾通道，导致NG超极化并降低兴奋性。这是一个分两步走的过程。最初，糖尿病神经元细胞内钙的增加会激活钙调神经磷酸酶。钙调神经磷酸酶结合到Tresk蛋白上的NFAT样的对接位置，导致丝氨酸276去磷酸化，导致通道激活，导致超极化。随着时间的推移，Tresk蛋白的上调不仅导致Tresk通道开放的频率增加，而且Tresk K通道的数量也增加。为了验证这一假设，我们有三个具体目标。目的1证明STZ-D的高血糖改变了NG神经元的基本电生理特性。膜片钳记录将被用来表征对照组和STZ-D大鼠NG神经节神经元的兴奋性。这些异常的生理学意义将通过体内电生理记录糖尿病大鼠的NG来评估，并研究其对CCK、瘦素和分泌素刺激的反应性。目的2研究慢性糖尿病状态下NG神经元的超极化是否由Tresk通道激活所介导。我们将通过电生理学研究以及蛋白质印迹和RT-PCR方法来确定NG中是否存在特定的TESK钾通道。Tresk通道的参与将通过使用基于病毒的系统传递siRNA来证明，以沉默Ng中Tresk通道的表达。为了评估Tresk在糖尿病NG超极化调节中的功能重要性，我们将通过Tresk siRNA对NG进行电穿孔，检测体内沉默Tresk通道表达后电生理和胃肠道异常的可逆性。目的3研究糖尿病大鼠NG神经元膜修饰的信号转导通路。使用信使特异性激活剂或抑制剂进行膜片钳记录和细胞内钙成像研究，以确定特定的细胞内级联元件在糖尿病动物NG兴奋性中的作用。在糖尿病中丝氨酸276去磷酸化的证明将涉及到使用磷酸蛋白质组方法。了解糖尿病状态下NG功能异常的细胞和分子机制，将为慢性糖尿病胃肠道功能异常的治疗提供重要靶点。 公共卫生相关性：控制不佳的糖尿病患者的胃肠功能经常异常，包括胃动力异常、胰腺和胃分泌减少以及饮食行为异常。这些功能中的许多是通过迷走神经控制的，而迷走神经在糖尿病患者中经常是异常的。本研究的目的是了解导致这一组感觉神经元异常的分子和细胞机制。这可能提供重要的治疗目标，并导致改善糖尿病患者的胃肠道并发症的管理。

DESCRIPTION (provided by applicant): Gastrointestinal function is frequently abnormal in patients with poorly controlled diabetes. Clinical investigations indicate that most of these abnormalities can be attributed to defective vagal afferent functioning. Our preliminary studies indicate that nodose ganglia (NG) neurons in streptozotocin induced diabetic rats (STZ- D) display hyperpolarization leading to decreased excitability. This may contribute to abnormal vagal function in the diabetic state. We hypothesize that chronic hyperglycemia activates "background" TRESK potassium channels, leading to hyperpolarization of the NG and decreased excitability. This is a two-step process. Initially increase in intracellular calcium in diabetic neurons activates calcineurin. Calcineurin binds to a NFAT- like docking site on the TRESK protein and causes dephosphorylation of serine 276, resulting in activation of the channel and leading to hyperpolarization. Over time upregulation of the TRESK protein occurs resulting in not only increased frequency of the opening of TRESK channel but an increase number of TRESK K+ channels. To test this hypothesis we have 3 specific aims. Aim 1 is to demonstrate that hyperglycemia in STZ- D modifies basic electrophysiological properties of NG neurons. Patch clamp recordings will be performed to characterize the excitability of NG ganglia neurons from control and STZ-D rats. Physiological implications of these abnormalities will be evaluated by in vivo electrophysiological recording of NG in diabetic rats and study its responsiveness to CCK, leptin and secretin stimulation. Aim 2 examines whether hyperpolarization of NG neurons in the chronic diabetic state is mediated by activation of TRESK channels. The presence of specific TESK potassium channels in NG will be identified using electrophysiological studies as well as western blot and RT-PCR methods. The participation of the TRESK channel will be demonstrated by the use of a virus based system for delivery of siRNA to silence the expression of the TRESK channel in NG. To evaluate the functional importance of TRESK in the mediation of hyperpolarization of NG in diabetes, we will examine reversibility of the electrophysiological and GI abnormalities following silencing TRESK channel expression in vivo through electroporation of the NG with TRESK siRNA. Aim 3 investigates the signal transduction cascades that mediate the membrane modifications of NG neurons in diabetes. Patch clamp recordings and intracellular calcium imaging studies will be performed with messenger specific activators or inhibitors to determine the role of specific intracellular cascade elements on NG excitability in diabetic animals. The demonstration of desphosphorylation of serine 276 in diabetes will involve the use of phosphoproteome method. Understanding the cellular and molecular mechanism responsible for abnormal functioning of the NG in the diabetic state will provide important therapeutic targets for the management of abnormal GI function in chronic diabetes. PUBLIC HEALTH RELEVANCE: Gastrointestinal functions are frequently abnormal in patients with poorly controlled diabetes, including abnormal stomach motility, diminished pancreatic and stomach secretions and abnormal eating behavior. Many of these functions are controlled through a vagus nervous system which is frequently abnormal in diabetic patients. The purpose of this study is to understand the molecular and cellular mechanisms responsible for the abnormalities of this group of sensory neurons. This could provide important therapeutic targets and lead to improved management of diabetic patients with GI complications.