PHOSPHORAMIDON INHIBITION OF THE IN-VIVO CONVERSION OF BIG ENDOTHELIN-1 TO ENDOTHELIN-1 IN THE HUMAN FOREARM

1 The vasoconstrictor peptide, endothelin-l (ET-I) and a biologically inactive C-terminal fragment (CTF) are generated from an intermediate big ET-1 by a putative ET converting enzyme, sensitive to phosphoramidon. We have developed a procedure using selective solid-phase extraction and specific radioimmunoassays to measure the levels of immunoreactive (IR) big ET-I and the products of conversion (ET-I and CTF) in human plasma. These techniques have been used to determine the levels of the three peptides in venous plasma following local infusions of ET-1 and big ET-1, both alone and together with phosphoramidon. 2 Infusion of ET-1 into the brachial artery (5 pmol min(-1)) significantly increased (P<0.05) IR ET levels from a basal level of 2.3 pM to 55.2 pM in plasma from the infused arm after 60 min of infusion. This corresponded with a marked decrease in forearm blood flow from a basal level of 2.6 mi dl(-1) min(-1) to 1.7 ml dl(-1) min(-1) The levels of IR big ET-1 and CTF were unchanged. Co-infusion of phosphoramidon (30 nmol min(-1)) with ET-1 had no significant effect on the plasma IR levels of ET, big ET-I, CTF, or blood flow. 3 Big ET-I (50 pmol min(-1)) significantly increased (P<0.05) venous concentrations of all three IR peptides after 60 min compared to basal (ET: from 2.2 to 7.7 pM, big ET-I: from 0 to 386.0 pM, CTF: from 0.2 to 37.0 pM). Forearm blood flow decreased significantly (P<0.05) from a basal level of 3.0 mi dl(-1) min(-1) to 1.6 mi dl(-1) min(-1) 4 When phosphoramidon was co-infused with big ET-1, both the rise in IR ET and associated vasoconstriction were abolished. However, IR CTF was still detected, suggesting that either some conversion by phosphoramidon-insensitive enzyme(s) was occurring, and/or that CTF was being protected from further degradation by phosphoramidon. 5 These data show that in the human forearm the activity of a phosphoramidon-sensitive ET converting enzyme is at least in part responsible for the vasoconstrictor properties of exogenous big ET-1. Furthermore, because measurable levels of newly synthesized ET-1 are likely to be rapidly reduced in the blood/plasma through receptor binding, assay of IR big ET-I and CTF may be a more sensitive measure of ET-1 generation in disease.