Phosphoramidon inhibition of the in vivo conversion of big endothelin-1 to endothelin-1 in the human forearm.

1. The vasoconstrictor peptide, endothelin-1 (ET-1) 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-1 and the products of conversion (ET-1 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 ml 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-1, CTF, or blood flow. 3. Big ET-1 (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-1; 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 ml dl-1 min-1 to 1.6 ml 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-1 and CTF may be a more sensitive measure of ET-1 generation in disease.