EFFECTS OF PHOSPHORAMIDON AND PEPSTATIN-A ON THE SECRETION OF ENDOTHELIN-1 AND BIG ENDOTHELIN-1 BY HUMAN UMBILICAL VEIN ENDOTHELIAL-CELLS -MEASUREMENT BY 2-SITE ENZYME-LINKED IMMUNOSORBENT ASSAYS

1. Two-site enzyme-linked immunosorbent assays have been developed for the rapid, sensitive and nonisotopic measurement of endothelin-l and big endothelin-1. The sensitivities of detection were 0.5 and 03 fmol/ well, with ED(50) values of 13 and 12 fmol/well for the endothelin-1 a nd big endothelin-1 assays, respectively. Each assay is highly selecti ve for its corresponding antigen. The ET-1 assay showed no detectable cross-reactivity with ET-1-(1-20), indicating that the assay only reco gnizes the 21-amino acid biologically active peptide. 2. The two assay s were used to measure the effects of two classes of protease inhibito r on the basal release of enothelin-1 and big endothelin-1 from cultur ed first-passage human umbilical vein endothelial cells. 3. The secret ion of both peptides was time-dependent over 12 h. The metalloprotease inhibitor phosphoramidon (1 x 10(-4) mol/l) significantly reduced the amount of endothelin-1 secreted into the medium (P<0.05), with a conc omitant increase in the secreted levels of big endothelin-1 (P<0.01). The aspartyl protease inhibitor, pepstatin A, also caused a significan t decrease in the secretion of endothelin (P<0.05). However, unlike ph osphoramidon, there was no increase in the levels of big ET-1 compared with the controls. At these concentrations, neither inhibitor affecte d the viability of the cells as indicated by Trypan Blue exclusion. 4. The two assays permit the direct measurement of endothelin-1 and its precursor, and will be of use in the elucidation of the putative human endothelin-converting enzyme(s). In addition to a phosphoramidon-sens itive enzyme, the results also suggest the existence of a pepstatin A- sensitive enzyme in human endothelial cells. Furthermore, pepstatin A also appears to limit the generation of big endothelin-1. Thus pepstat in A-sensitive enzymes may be better targets for inhibitors to reduce the harmful vasoconstrictive effects of endothelin-1 in cardiovascular diseases.