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

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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.