Non-opioid Actions of Dynorphin A in Anterior Pituitary

描述：（申请摘要）下丘脑中k-阿片受体的高密度以及下丘脑和垂体中Dyn肽的高浓度提示Dyn可能参与神经内分泌调节。最近在绵羊胎儿中的研究使我们提出Dyn是促ACTH的重要促激素，并且Dyn可能通过非阿片和阿片机制起作用。具体来说，我们假设Dyn通过两种不同但互补的途径释放ACTH。Dyn可作用于下丘脑的k-阿片受体，释放CRH和AVP。此外，假设Dyn与AVP和CRH共同从下丘脑释放，然后这个Dyn池可以作用于垂体前叶的非阿片受体，释放ACTH和b-内啡肽（bEnd）。本项目的目的是了解Dyn在ACTH和bEnd释放过程中所涉及的阿片和非阿片通路的调控作用，我们的具体目标是：1)了解Dyn在垂体前叶中非阿片作用的机制。我们假设Dyn和/或其代谢物可能通过NMDA受体或通过垂体前叶一种新的未知受体起作用。为了深入了解这种受体的结构特异性，我们计划比较一些dyn相关肽的活性；2)确定反复暴露于Dyn后，是否对Dyn的阿片和非阿片成分均产生耐受性。我们假设耐受性只会发展到下丘脑阿片成分，NMDA在调解这一过程中起作用；3)确定皮质醇是否在下调Dyn的阿片和非阿片成分的作用中起负反馈作用。我们假设只有下丘脑的阿片成分会受到皮质醇的负反馈控制。将使用体内（长期仪器化的羊胎儿制备）和体外（羊胎儿垂体前叶细胞的原代培养）技术的结合。这些研究将显著提高我们对Dyn在神经内分泌功能中的作用及其复杂作用机制的调控的认识。除了共享Dyn A的非阿片类作用的共同主题外，该组件将与所有其他组件共享主动交互。李博士将提供dyn相关肽进行结构-活性研究；Hook博士将建议我们进行ACTH释放测试的相关内源性dyn相关肽；我们的数据将提供给Weinstein博士进行分子建模。

DESCRIPTION: (Applicant's Abstract) The high density of k-opioid receptors in the hypothalamus and the high concentration of Dyn peptides in both hypothalamus and pituitary suggest a possible involvement of Dyn in neuroendocrine regulation. Recent studies in the ovine fetus have led us to propose that Dyn is an important secretagogue of ACTH, and that Dyn may act via non-opioid as well as opioid mechanisms. Specifically, we hypothesize that Dyn releases ACTH via two different, but complementary, pathways. Dyn can act on k-opioid receptors in the hypothalamus to release CRH and AVP. In addition, it is hypothesized that Dyn is co-released with AVP and CRH from the hypothalamus, and this Dyn pool can then act on non-opioid receptors in the anterior pituitary to release ACTH and b-endorphin (bEnd). The goal of this project is to understand the regulatory control of both opioid and non-opioid pathways involved in the release of ACTH and bEnd by Dyn. Our specific aims are: 1) To understand the mechanisms behind the non-opioid actions of Dyn in the anterior pituitary. We hypothesize that Dyn and/or its metabolite(s) may act via the NMDA receptor or via a novel unidentified receptor in the anterior pituitary. To gain some insight into the structural specificity of this receptor, we plan to compare the activity of a number of Dyn-related peptides; 2) To determine if tolerance develops to both the opioid and non-opioid component of Dyn's action with repeated Dyn exposure. We hypothesize that tolerance will only develop to the hypothalamic opioid component and that NMDA plays a role in mediating this process; 3) To determine if cortisol plays a negative feedback role in down-regulating the opioid and non-opioid component of Dyn's action. We hypothesize that only the hypothalamic opioid component will be subjected to negative feedback control by cortisol. A combination of in vivo (chronically-instrumented ovine fetus preparation) and in vitro (primary cultures of ovine fetal anterior pituitary cells) techniques will be used. The proposed studies will significantly enhance our understanding of the role of Dyn in neuroendocrine function and the regulatory control of its complex mechanisms of action. Besides sharing the common theme of non-opioid actions of Dyn A, this component will share active interaction with all other components. Dr. Lee will provide the Dyn-related peptides for structure-activity studies; Dr. Hook will advise us on the relevant endogenous Dyn-related peptides to be tested for ACTH release; our data will be provided to Dr. Weinstein for molecular modeling.