Vagal Action of CCK in Mediating GI Function

描述（由申请人提供）：迷走神经CCK-A受体存在高亲和力和低亲和力状态。在上一个资助期，我们证明了CCK在生理水平上通过迷走神经高亲和CCK- a受体介导胰腺分泌。迷走神经CCK受体似乎也在食物摄入的短期控制中发挥重要作用，可能是由低亲和力CCK- a受体介导的。然而，诱导饱腹感所需的CCK剂量远高于餐后水平。研究表明迷走神经CCK和瘦素受体之间的协同相互作用调节短期食物摄入，这可能降低诱导饱腹感所需的CCK剂量。我们假设CCK通过两种不同的迷走神经传入信号通路介导胰腺分泌和饱腹感。一组结节神经元含有高亲和力的CCK-A受体，通过迷走-迷走胆碱能反射介导胰腺分泌。第二组结节神经节神经元包含低亲和力CCK和瘦素受体；CCK通过Src激酶放大STAT 3信号通路，从而增强瘦素信号转导通路。这些神经元投射到NTS，然后是下丘脑来控制短期进食行为。本研究描述了含有CCK和瘦素受体的神经元的特征，并研究了CCK放大瘦素信号转导途径的细胞内机制。我们将证明瘦素受体与低亲和力但不高亲和力的CCK-A受体共定位，然后利用离体胃迷走神经制剂证明预处理增强了一组特定胃迷走神经传入纤维的瘦素反应性。这些纤维的电生理和生物学特性将被表征，并与仅对高亲和力CCK-A受体激动剂JMV 180有反应的纤维进行对比。然后，我们将通过细胞内记录和标记技术表征两组结节神经节神经元利用的化学编码。CCK增强瘦素信号转导途径的机制将通过膜片钳研究，使用来自结节神经节的分离神经元进行各种Src和PI3拮抗剂的研究。转染CCK-A受体和Src显性阴性基因的神经母细胞瘤SY5Y细胞系将被用来解剖Src与STAT 3之间的相互作用，以放大电刺激。最后，将进行化学研究，以检验CCK增强瘦素引起的STAT 3磷酸化的机制。这些研究将提供迷走神经低亲和力CCK受体如何介导短期饱腹感的详细特征，并描绘CCK与瘦素相互作用以增强STAT 3信号级联的细胞内机制。这种神经通路的失败可能导致嗜食导致肥胖。

DESCRIPTION (provided by applicant): Vagal CCK-A receptors exist in high and low affinity states. In the last funding period we demonstrated that CCK at physiological levels acts on vagal high affinity CCK-A receptors to mediate pancreatic secretion. Vagal CCK receptors also appear to play an important role in short term control of food intake, likely mediated by low affinity CCK-A receptors. The doses of CCK required to induce satiety however is much higher than postprandial levels. Studies indicate a synergistic interaction between vagal CCK and leptin receptors to regulate short term food intake, which may lower the dose of CCK required to induce satiety. We hypothesize CCK mediates pancreatic secretion and satiety using two different vagal afferent signaling pathways. One group of nodose neurons contain high affinity CCK-A receptors, which mediate pancreatic secretion via the vago-vagal cholinergic reflex. A second group of nodose ganglia neurons contain both low affinity CCK and leptin receptors; CCK enhances the leptin signal transduction pathway by amplifying the signaling of STAT 3 through Src kinase. These neurons project to the NTS and then the hypothalamus to control short term eating behavior. This proposal characterizes the neurons containing both CCK and leptin receptors and investigates the intracellular mechanisms by which CCK amplifies the leptin signal transduction pathway. We will demonstrate that leptin receptors co-localize with low but not high affinity CCK-A receptors, then show that pretreatment enhances leptin responsiveness of a specific group of gastric vagal afferent fibers utilizing an in vitro isolated stomach-vagus nerve preparation. Electrophysiological and biological characteristics of these fibers will be characterized and contrasted with fibers responding only to the high affinity CCK-A receptor agonist JMV 180. We will then characterize the chemical codings utilized by the two groups of nodose ganglia neurons by intracellular recording and labeling techniques. The mechanism by which CCK enhances the leptin signal transduction pathway will be examined by patch clamp studies using isolated neurons from nodose ganglia various Src and PI3 antagonists. Neuroblastoma SY5Y cell lines transfected with CCK-A receptor and Src dominant negative gene will be used to dissect the interaction between Src and STAT 3 for the amplification of electrical firings. Lastly, chemical studies to examine the mechanism by which CCK enhances STAT 3 phosphorylation evoked by leptin will be done. These studies will provide a detailed characterization of how vagal low affinity CCK receptors mediate short- term satiety and delineate the intracellular mechanism by which CCK interacts with leptin to enhance the STAT 3 signaling cascade. Failure of this neural pathway may result in hyperphagia resulting in obesity.