ACTH RELEASE BY DYN A--ROLE OF NMDA RECEPTORS

描述(申请人摘要)： 这项建议的长期目标是提供对 非阿片类药物刺激前额叶的细胞机制 促肾上腺皮质激素分泌强啡肽(Dyn)释放ACTH。具体的 目的是：1)确定Dyn是否作用于垂体前叶细胞，在 缺乏下丘脑释放因子，通过非阿片类药物释放ACTRh 机械装置。这将通过检测ACTH分泌来实现。 小鼠垂体前叶肿瘤细胞ATT-20对Dyn等的反应 Mu-、Delta-和kapPay-阿片受体多肽，并测定 这些药物对纳洛酮的敏感性。2)确定 促肾上腺皮质激素分泌中的NMDAR和钙流通量 来自ATT-20细胞。功能和结合研究，以及蛋白质印迹 将被用来确定垂体细胞上是否存在NMDAR。这个 Zeta1 NMDAR亚基将与epsilon 1，epsilon 2， Epsilon3、epsilon4均瞬时进入COS-1细胞，并稳定进入HEK 293细胞，以形成以下NMDA亚单位的组合：Zeta1 1，zeta1，zeta1-epsilon4。这些构造 将表征为由NMDAR配体和Dyn结合，并在 刺激下的细胞内钙离子。3)从分子和分子水平确定 结构水平上Dyn与NMDAR、嵌合NMDAR的结合部位 将使用保守的分段交换来构造子单元 方法，其中包括交换选定的化学相似的 NMDAR亚基胞外区的化学残基 在另一个NMDAR亚基上有类似的残基。这些嵌合体将被分析 DYN和DYN对细胞内钙离子水平的影响 NMDAR配体。对Dyn在ACTH中调控的认识 分泌及其与NMDAR相互作用的机制很重要，因为 Dyn在兴奋性氨基酸和受体上的作用可能是 治疗上有效的神经内分泌疾病的治疗 或者可能会对…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，并消除发展致敏对 可卡因。这些作用的机制尚不清楚，但它们都在 至少部分是非阿片类药物。因此，这些研究可能会为 Dyn在治疗可卡因和阿片类药物滥用中的作用机制。

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.