The studies for expression and secretion of adrenomedullin and its signal transduction.

肾上腺髓质素 (AM) 是最近从人嗜铬细胞瘤组织中分离出来的一种有效的血管舒张肽，在结构上是 CGRP 家族的成员。 AM的mRNA在心脏、肺、肾、血管壁以及肾上腺髓质等多种组织中高表达。本项目对AM的表达、分泌及其信号转导进行了研究。为了研究心力衰竭中AM的产生和分泌位点，我们测定了心力衰竭大鼠血浆肾上腺髓质素的水平以及各组织中肾上腺髓质素的肽和mRNA的水平。我们还检查了心力衰竭患者的心脏是否确实将 AM 分泌到循环中。心力衰竭大鼠的血浆 AM 水平升高，与心室重量相关。心力衰竭大鼠的心脏和肺组织 AM 肽和 mRNA 水平增加，但肾脏或肾上腺没有增加。心力衰竭患者冠状窦的血浆 AM 水平高于主动脉。这些结果表明，心力衰竭中血浆肾上腺髓质素水平与其严重程度成比例增加，并且心力衰竭大鼠中心脏产生和分泌的肾上腺髓质素增加。肺可能是心力衰竭大鼠肾上腺髓质素产生增加的另一个部位。为了阐明人类AM基因表达的调控机制，利用荧光素酶载体在培养的人主动脉内皮细胞（HAEC）中检测了AM基因5'侧翼区的功能元件。 AM基因5'-侧翼DNA的启动子活性因失去-85至-93 bp处的NF-IL6位点、-33至-68 bp处的AP-2位点和-21至-26 bp处的TATA盒而降低。这些顺式作用元件被认为参与了EC中人类AM基因的转录调控。目前尚不清楚AM诱导的血管舒张是否是内皮依赖性的。主动脉环的内皮剥脱抑制了对 AM 的扩张反应。此外，用 cGMP 特异性磷酸二酯酶抑制剂 E-4021 对大鼠主动脉环和离体肾脏进行预处理，显着增强了 AM 诱导的血管舒张，表明 NO-cGMP 在 AM 的血管作用中发挥着重要作用。前肾上腺髓质素 N 末端 20 肽 (PAMP-20) 是一种强效降血压肽，由AM前驱体。我们已确定 PAMP-12 (PAMP[9-20]) 猪肾上腺髓质为 AM 前体的主要产物，并证明其具有有效的降血压作用。尽管 PAMP 特定受体的存在仍不清楚，但 PAMP-12 可能与 PAMP-20 具有相同的生物功能。

Adrenomedulin (AM) is a potent vasorelaxant peptide recently isolated from human pheochromocytoma tissue, and is structurally a member of CGRP family. mRNA of AM is highly expressed in several tissues including heart, lung, kindey and vascular wall as well as adrenal medulla. In this project, we have studied the expression and secretion of AM and its signal transduction.To investigate the sites of production and secretion of AM in heart failure, we measured plasma adrenomedullin levels and peptide and mRNA levels of adrenomedullin in various tissues in rats with heart failure. We also examined whether the heart actually secretes AM into the circulation in patients with heart failure. Plasma AM levels were increased in heart failure rats, correlating with ventricular weight. Tissue AM peptide and mRNA levels were increased in the heart and lungs, but not kidney or adrenals of rats with heart failure. Plasma AM levels were higher in the coronary sinus than in the aorta in patients with h … More eart failure. These results suggest that plasma adrenomedullin levels are increased in heart failure in proportion to its severity and that cardiac production and secretion of adrenomedullin is increased in heart failure rats. Lungs may be another site for increased production of adrenomedullin in heart failure rats.To elucidate the regulatory mechanism of human AM gene expression, functional elements of 5'-flanking region of AM gene were examined in cultured human aortic endothelial cells (HAEC) by using a luciferase vector. The promoter activity of AM gene 5'-flanking DNA was reduced by losing NF-IL6 site at -85 to -93 bp, AP-2 sites at -33 to -68 bp and TATA box at -21 to -26 bp. These cis-acting elements are supposed to participate in the transcriptional regulation of human AM gene in ECIt is still unclear whether AM-induced vasodilation is endothelium-dependent. Endothelium-denudation of the aortic ring suppressed the dilatory response to AM.Furthermore, pretreatments of the rat aortic rings and isolated kidney with E-4021, a cGMP specific phosphodiesterase inhibitor, significantly enhanced AM-induced vasodilation, suggesting an important role of NO-cGMP in the vascular action of AM.Proadrenomedullin N-terminal 20 peptide (PAMP-20) is a potent hypotensive peptide processed from the AM precursor. We have identified PAMP-12 (PAMP[9-20]) porcine adrenal medulla as a major product of the AM precursor, and demonstrated that it exhibits a potent hypotensive action. Although the existence of a specific receptors for PAMP remains obscure, PAMP-12 might share a biological functin with PAMP-20 Less