Vagal Action of CCK in Mediating GI Function

描述（由申请人提供）：迷走神经CCK-A受体以高和低亲和力状态存在。在上一个资助期间，我们证明了生理水平的CCK作用于迷走神经高亲和力CCK-A受体，以介导胰腺分泌。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，可能由低亲和力CCK-A受体介导。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，以调节短期的食物摄入，这可能会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。一组结状神经元含有高亲和力CCK-A受体，其通过迷走-迷走胆碱能反射介导胰腺分泌。第二组结状神经节神经元含有低亲和力CCK和瘦素受体; CCK通过Src激酶放大STAT 3的信号传导来增强瘦素信号转导途径。这些神经元投射到NTS，然后投射到下丘脑，以控制短期的进食行为。该建议的特点，同时含有CCK和瘦素受体的神经元，并调查CCK放大瘦素信号转导通路的细胞内机制。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，然后表明预处理利用体外分离的胃迷走神经制备增强特定组胃迷走神经传入纤维的瘦素反应。这些纤维的电生理学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响应的纤维进行对比。然后，我们将通过细胞内记录和标记技术来表征两组结状神经节神经元所使用的化学编码。CCK增强瘦素信号转导通路的机制将通过膜片钳研究，使用各种Src和PI 3拮抗剂从结状神经节分离的神经元进行检查。本研究将利用CCK-A受体和Src显性负性基因转染的神经母细胞瘤SY 5 Y细胞系，研究Src和STAT 3之间的相互作用，以放大电激发。最后，将进行化学研究以检查CCK增强瘦素诱发的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表征，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。该神经通路的故障可能导致导致肥胖的摄食过多。

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.