DYNORPHIN AND BETA CELL SENSITIZATION

在这个项目中，PI提出了一系列实验来确定 强啡肽和相关肽是否通过一种机制 独立于蛋白偶联阿片受体，协同作用， 葡萄糖刺激胰岛β细胞分泌胰岛素 朗格汉斯岛 将检验以下假设。 强啡肽使β细胞对葡萄糖诱导的胰岛素分泌敏感， 激活N-甲基-D-天冬氨酸（或延长其激活） （NMDA）-选择性兴奋性离子型谷氨酸受体。 强啡 A是阿片肽家族的一员， 主要作为神经调质，与G蛋白偶联 受体（GPCR）; μ和κ以及NMDA受体是一个 一类配体门控离子通道，激活时增加 细胞表面膜对Ca 2+（以及Na+和K+）的渗透性， 从而提高胞浆游离Ca 2+浓度。 升高 细胞质游离C2+将反过来使细胞对刺激敏感， 葡萄糖和偶联刺激胰岛素分泌。 具体目标是：（1）确定是否 强啡肽和相关肽协同葡萄糖刺激 胰岛素分泌通过NMDA受体信号传导。PI将使用 小鼠胰岛素瘤细胞系MIN 6，以研究结合和信号传导 胰岛素受体内源性表达的NMDA受体的特征- 分泌细胞，并将这些发现与 人胚肾细胞（HEK 293细胞）和猴肾COS-1 表达由基因特异性亚基组成的NMDA受体的细胞 转移2)确定哪些亚基形成强啡肽结合NMDA 从而开始描绘亚基上的结构域 直接与强啡肽和相关肽结合。 实验 涉及NMDA受体亚单位表达的研究将在 转染的HEK 293细胞和COS-1细胞，其中受体可以被 表达到高水平。3)为了确定药效团， 强啡肽 也就是说，确定最小的肽， 保留NMDA受体结合和胰岛素促分泌素 Dyn A的特征（1-17）。这些实验将在 MIN 6、HEK 293和COS-1细胞。 如果强啡肽NMDA受体钙 途径显示使β细胞对葡萄糖诱导的胰岛素敏感 分泌，这项研究的长期目标将是发展 可用于治疗糖尿病的非肽类口服活性药物 在人类中。

In this project, the PI proposes a series of experiments to determine whether dynorphin and related peptides, acting through a mechanism independent of the protein-coupled opioid receptors, synergize with glucose to stimulate insulin secretion from beta cells of the pancreatic islets of Langerhans. The following hypothesis will be tested. Dynorphin sensitizes beta cells to glucose-induced insulin secretion by activating (or prolonging the activation of) N-methyl-D-aspartate (NMDA)-selective excitatory ionotropic glutamate receptors. Dynorphin A is a member of the family of opioid peptides that appear to act primarily as neuromodulators by interacting with G protein-coupled receptors (GPCRs); mu and kappa and NMDA receptors are members of a class of ligand-gated ion channels that when activated increase the permeability of the cell surface membrane to Ca2+ (and Na+ and K+) and thereby elevate cytoplasmic free Ca 2+ concentration. Elevations in cytoplasmic free C2+ will in turn sensitize the cell to stimulation by glucose and couple stimulation to insulin secretion. The Specific Aims that will be pursued are: 1) To determine whether dynorphin and related peptides synergize with glucose stimulation of insulin secretion by signaling via NMDA receptors. The PI will employ a mouse insulinoma cell line, MIN6 to study binding and signaling characteristics of endogenously expressed NMDA receptors in insulin- secreting cells and compare those findings with observations made in human embryonic kidney cells (HEK 293 cells) and monkey kidney COS-1 cells expressing NMDA receptors comprised of specific subunits by gene transfer. 2) To determine which subunits form dynorphin-binding NMDA receptors so as to begin to delineate the domain(s) on the subunit(s) that directly bind dynorphin and related peptides. The experiments involving expression of NMDA receptor subunits will be performed in transfected HEK 293 cells and COS-1 cells in which the receptors can be expressed to high levels. 3) To determine the pharmacophore within the dynorphin peptide. That is, to determine the smallest peptide that retains the NMDA receptor-binding and insulin secretagogue characteristics of Dyn A(1-17). These experiments will be performed in MIN6, HEK 293 and COS-1 cells. If the dynorphin-NMDA receptor-calcium pathway were shown to sensitize beta cells to glucose-induced insulin secretion, a long-term goal of this research will be to develop nonpeptidic, orally active drugs that can be used to treat diabetes mellitus in humans.