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ACTH RELEASE BY DYN A--ROLE OF NMDA RECEPTORS

DYN A 释放 ACTH——NMDA 受体的作用

基本信息

批准号：
6150437
负责人：
PETER Y CHENG
金额：
$ 0.39万
依托单位：
WEILL MEDICAL COLL OF CORNELL UNIV
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-02-01 至 2000-04-07
项目状态：
已结题

项目摘要

DESCRIPTION (Applicant's Abstract): The long term objective of this proposal is to provide an understanding of the cellular mechanisms underlying the non-opioid stimulation of anterior pituitary corticotrophs by dynorphin (Dyn) to release ACTH. The specific aims are: 1) to determine if Dyn acts on anterior pituitary cells, in the absence of hypothalamic releasing factors, to release ACTRh, via non-opioid mechanisms. This will be accomplished by determing ACTH secretion from mouse anterior pituitary tumor cells, AtT-20, in response to Dyn and other mu-, delta-, and kappaid-opioid receptor peptides, and determing the sensitivity of those repsonses to naloxone. 2) to determine the role of NMDA receptors (NMDAR) and Ca2+ flux in Dyn-stimulation of ACTH secretion from AtT-20 cells. Functional and binding studies, as well as Western Blots will be used to determine the presence of NMDAR on pituitary cells. The zeta1 NMDAR subunit will be co-transfected with epsilon1, epsilon2, epsilon3, epsilon4 both transiently, into COS-1 cells, and stably into HEK 293 cells, to make the following combinations of NMDA subunits: Zeta1 epsilon1, zeta1 epsilon2, zeta1 epsilon3, zeta1-epsilon4. These constructs will be characterized for binding by NMDAR ligand and Dyn, and changes in intracellular Ca2+ upon stimulation. 3) to determine at the molecular and structural level the binding site of Dyn on the NMDAR, chimeric NMDAR subunits will be constructed, employing the conservative segement exchange approach, which involves the exchange of selected chemically similar residues on the extracellular domain of one NMDAR subunit with chemically similar residues on another NMDAR subunit. These chimeras will be analyzed for binding and change of intracellular Ca2+ levels in response to Dyn and NMDAR ligands. An understanding of the regulatory control of Dyn in ACTH secretion, and its mechanism of interaction with NMDAR is important since the actions of Dyn at excitatory amino acid and receptors may be therapeutically useful in the treatment of neuroendocrine disorders of alternatively may have the potential for producing adverse effects on neuroendocrine function. In addition, Dyn has been shown to inhibit the development of tolerance to opiates, restore spinal/supraspinal synergism in morphine tolerant mice, and to eliminate the development of sensitization to cocaine. The mechanism of these actions are not known but they are all, at least in part, non-opioid. Thus, the studies may shed new light into the mechanisms behind the role of Dyn for treatment of cocaine and opioid abuse.

描述（申请人摘要）：本提案的长期目标是提供对以下内容的了解：非阿片类物质刺激前额叶皮层的细胞机制垂体促肾上腺皮质激素细胞通过强啡肽（Dyn）释放ACTH。具体目的是：1）确定Dyn是否作用于垂体前叶细胞，缺乏下丘脑释放因子，通过非阿片样物质释放ACTRh 机制等这将通过测定促肾上腺皮质激素分泌来完成，小鼠垂体前叶肿瘤细胞AtT-20对强啡肽等药物的反应 μ-、δ-和κ-阿片样受体肽，并测定这些反应对纳洛酮的敏感性。 2)以确定强啡肽刺激促肾上腺皮质激素分泌中NMDA受体和Ca ~（2+）流的研究 AtT-20细胞功能和结合研究，以及蛋白质印迹将用于确定垂体细胞上NMDAR的存在。的 zeta 1 NMDAR亚基将与ε 1，ε 2， ε 3、ε 4均瞬时进入COS-1细胞，并稳定进入HEK 293细胞，以制备以下NMDA亚基组合： ε 1，zeta 1 ε 2，zeta 1 ε 3，zeta 1-ε 4。这些构建体将表征NMDAR配体和Dyn的结合，以及细胞内Ca 2+。 3)以确定在分子和在结构水平上，Dyn在NMDAR、嵌合NMDAR上的结合位点亚基将被构建，采用保守的节段交换方法，其中涉及交换选定的化学类似物，在一个NMDAR亚基的胞外结构域上的残基与化学修饰结合，在另一个NMDAR亚基上的类似残基。这些嵌合体将被分析结合和细胞内Ca 2+水平的变化，以响应Dyn和 NMDAR配体。对Dyn在ACTH中调节作用的认识分泌，其与NMDAR相互作用的机制是重要的，因为强啡肽对兴奋性氨基酸和受体的作用可能是在治疗上可用于治疗神经内分泌病症，也可能对环境产生不利影响，神经内分泌功能此外，Dyn已被证明可以抑制发展对阿片类药物的耐受性，恢复脊髓/脊髓上的协同作用，吗啡耐受小鼠，并消除对可卡因这些作用的机制尚不清楚，但它们都是，在至少部分是非阿片类药物。因此，这些研究可能会为 Dyn治疗可卡因和阿片类药物滥用的作用机制。