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Novel bioactive endothelins (1-31) produced by chymase and their physiological functions

食糜酶产生的新型生物活性内皮素（1-31）及其生理功能

基本信息

批准号：
09670130
负责人：
KIDO Hiroshi
金额：
$ 2.05万
依托单位：
The University of Tokushima
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1998
项目状态：
已结题

项目摘要

We report the novel role of human chymase in the production of bioactive 31-amino acid cngth endothelins (ETs), which may play a role in allergies and vascular diseases. In the bronchi of asthmatic patients, the vascular tissue in atherosclerosis, and the heart muscle in cardiac hypertrophy, both ET-like immunoreactivity and the accumulation of mast cells significantly increase. Chymase from human mast cells selectively cleaves big ET-1, -2 and -3 at their Tyr^<31> -Gly^<32> bonds, and produces novel bioactive 31-amino acid length ETs, ETs(1-31), without any further degradation products. However, chymases from other species, human cathepsin G, and porcine alpha-chymotrypsin, degrade big Ets, ETs(1-31) at concentrations between 10^<-9>M and 10^<-7>M exhibited various contractile potencies in rat tracheae and porcine coronary arteries in a dose-dependent manner. Furthermore, ET-1(1-31) at concentrations between 10^<-14>M and 10^<-10>M caused a significant increase in the intracellular free Ca^<2+> concentration. The contractile activity of ETs(1-31) may not be the consequence of conversion to the corresponding ETs(1-21) by phosphoramidon-sensitive ET concerting enzyme(s) or other chymotrypsin-type proteases and metallo-endopeptidases, because the contractile activity was not significantly inhibited on treatment with inhibitors of these proteases prior to the addition of ET-1(1-31). To determine the levels of ETs(1-31), were established highly sensitive and pecific sandwich-enzyme immunoassays (ELAs) for ETs(1-31), which showed no cross reactivity with 21 -amino acid-length endothelins [ETs(1-21)] and big ETs. The ELAs for ET-1 -2, and 3(1-31) could detect as little as 0.3 pg/well for ET- 1(1-31), 0.8 pg/well for ET-2(1-31) and 0.3 pg/well for ET-3(1 -31), respectively.

我们报道了人糜酶在产生生物活性的31-氨基酸cngth内皮素（cngth endothelins，cngth endothelins）中的新作用，cngth endothelins可能在过敏和血管疾病中发挥作用。在哮喘患者支气管、动脉粥样硬化血管组织和心肌肥厚心肌中，ET样免疫反应性和肥大细胞的积聚均显著增加。来自人肥大细胞的糜蛋白酶选择性地在其Tyr^ -Gly^键处切割大ET-1、-2和-3<31><32>，并产生新的生物活性的31-氨基酸长度的β，β（1-31），而没有任何进一步的降解产物。然而，来自其他物种的糜酶，人组织蛋白酶G和猪α-糜蛋白酶，在10 μ M和10 μ M之间的浓度下降解大Ets，Ets（1-31）<-9><-7>，在大鼠气管和猪冠状动脉中以剂量依赖性方式表现出各种收缩效力。此外，浓度在10 μ M和10 μ M之间的ET-1（1-31）<-14><-10>引起细胞内游离Ca^2+浓度的显著增加。ET（1-31）的收缩活性可能不是通过磷酰胺敏感性ET结合酶或其他胰凝乳蛋白酶型蛋白酶和金属内肽酶转化为相应的ET（1-21）的结果，因为在加入ET-1（1-31）之前，用这些蛋白酶的抑制剂处理时，收缩活性没有受到显著抑制。为了测定β-内酰胺酶（1-31）的水平，建立了β-内酰胺酶（1-31）的高灵敏度和特异性β-内酰胺酶免疫测定法（ELA），其显示与21 -氨基酸长度的内皮素[β-内酰胺酶（1-21）]和大β-内酰胺酶无交叉反应。ET-1 - 2和3（1-31）的ELA可分别检测低至0.3pg/孔的ET- 1（1-31）、0.8pg/孔的ET-2（1-31）和0.3pg/孔的ET-3（1 - 31）。