Measurement of C-terminal fragment of big endothelin-1: a novel method for assessing the generation of endothelin-1 in humans.
We developed a procedure for individual measurement of big endothelin-1 (big ET-1) and the two products of big ET-1 conversion (ET-1 and C-terminal fragment (CTF) of big ET-1 (big ET-1(22-38)), using selective solid-phase extraction and specific radioimmunoassays. These techniques were used to measure the levels of these peptides in extracts of human plasma after brachial artery infusions of big ET-1. Infusion of big ET-1 (50 pmol/min, n = 6) caused a decrease in forearm blood flow from 3.03 to 1.55 ml/dl/min after 60 min (p < 0.05). In agreement, the levels of plasma immunoreactive (IR) big ET-1, ET, and CTF were significantly increased from basal levels in the infused arm (from undetectable to 386 pM, 2.2-7.5 pM, and 0.17-37 pM, respectively; p < 0.05). In the presence of the metalloprotease inhibitor phosphoramidon (30 nmol/min), the increase in IR-ET and the associated vasoconstriction were abolished. However, IR-CTF was still detected, suggesting that either some conversion by phosphoramidon-insensitive endothelin-converting enzyme (ECE) was occurring and/or that CTF was being preserved from further proteolysis by phosphoramidon. These data confirm that exogenous big ET-1 is locally converted to ET-1 and CTF in the human forearm, at least in part by a phosphoramidon-sensitive ECE. Furthermore, because measurable levels of newly synthesized ET-1 are probably rapidly reduced as a result of receptor binding, the assay of IR-CTF may be a more sensitive measure of the overexpression of ET-1 in disease.
Plumpton
C
C
Clinical Pharmacology Unit, University of Cambridge, Addenbrooke's Hospital, England.
Haynes
W G
WG
Webb
D J
DJ
Davenport
A P
AP
D000328:Adult; D016282:Aspartic Acid Endopeptidases / Q000378:metabolism; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000096:biosynthesis* / Q000097:blood*; D006020:Glycopeptides / Q000494:pharmacology; D006801:Humans; D008297:Male; D008666:Metalloendopeptidases; D010446:Peptide Fragments / Q000097:blood*; D011498:Protein Precursors / Q000097:blood*; D014661:Vasoconstriction
D019332:Endothelin-1; D016232:Endothelins; D006020:Glycopeptides; D010446:Peptide Fragments; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon