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某种机制起作用 独立于蛋白质偶联阿片受体，与 葡萄糖刺激胰腺β细胞分泌胰岛素 朗格汉斯岛。 将检验以下假设。 强啡肽通过以下方式使β细胞对葡萄糖诱导的胰岛素分泌敏感 激活（或延长激活）N-甲基-D-天冬氨酸 (NMDA)-选择性兴奋性离子型谷氨酸受体。 强啡肽 A 是阿片肽家族的成员，其作用似乎是 主要通过与 G 蛋白偶联相互作用作为神经调节剂 受体（GPCR）； mu 和 kappa 以及 NMDA 受体是 一类配体门控离子通道，激活后会增加 细胞表面膜对 Ca2（以及 Na 和 K ）的渗透性和 从而提高细胞质游离Ca 2 浓度。 海拔高度 细胞质游离 C2 反过来会使细胞对刺激敏感 葡萄糖和耦合刺激胰岛素分泌。 将追求的具体目标是： 1) 确定是否 强啡肽和相关肽与葡萄糖刺激协同作用 通过 NMDA 受体发出信号来分泌胰岛素。 PI 将雇用 小鼠胰岛素瘤细胞系 MIN6，用于研究结合和信号传导 胰岛素内源性表达的 NMDA 受体的特征 分泌细胞并将这些发现与观察结果进行比较 人胚肾细胞（HEK 293细胞）和猴肾COS-1 表达 NMDA 受体的细胞由特定基因亚基组成 转移。 2) 确定哪些亚基形成强啡肽结合 NMDA 受体以便开始描绘亚基上的结构域 直接结合强啡肽和相关肽。 实验 涉及 NMDA 受体亚基表达的实验将在 转染HEK 293细胞和COS-1细胞，其中受体可以 表达到高水平。 3) 确定药效基团 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(1-17) 的特性。这些实验将在 MIN6、HEK 293 和 COS-1 细胞。 如果强啡肽-NMDA受体-钙 研究表明该途径可使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是开发 可用于治疗糖尿病的非肽类口服活性药物 人类中的梅利图斯。

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.