Phosphoramidon blocks the pressor activity of porcine big endothelin-1-(1-39) in vivo and conversion of big endothelin-1-(1-39) to endothelin-1-(1-21) in vitro.
In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate called "big endothelin-1" (big ET-1) by a putative ET-converting enzyme (ECE) that cleaves the 39-mer at the bond between Trp-21 and Val-22. Since big ET-1 has only 1/100-1/150th the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of ET-1. Big ET-1 injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET-1 into ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET-1 in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were all ineffective at blocking the pressor response to big ET-1. However, the metalloprotease inhibitors phosphoramidon and thiorphan dose-dependently inhibited the pressor response to big ET-1, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on mean arterial pressure when administered alone. In a rabbit lung membrane preparation, ECE activity was identified that was blocked by the metalloprotease inhibitors phosphoramidon and 1,10-phenanthroline in a concentration-dependent manner. This enzyme converted big ET-1 to a species of ET that comigrated on HPLC with ET-1 and produced an ET-like contraction in isolated rat aortic rings. Our results suggest that the physiologically relevant ECE is a metalloprotease.
McMahon
E G
EG
Searle Research and Development, GD Searle & Co., St. Louis, MO.
Palomo
M A
MA
Moore
W M
WM
McDonald
J F
JF
Stern
M K
MK
D000818:Animals; D001011:Aorta / Q000187:drug effects / Q000502:physiology*; D019332:Endothelin-1; D016232:Endothelins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D006020:Glycopeptides / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology*; D010618:Phenanthrolines / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013552:Swine; D014661:Vasoconstriction / Q000187:drug effects
D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D010618:Phenanthrolines; D011480:Protease Inhibitors; D011498:Protein Precursors; C008890:phosphoramidon; C025205:1,10-phenanthroline