Effects of phosphoramidon in endothelial cell cultures on the endogenous synthesis of endothelin-1 and on conversion of exogenous big endothelin-1 to endothelin-1.

Several studies have shown that phosphoramidon (PHOS) reduces the release of endothelin-1 (ET-1) from cultured endothelial cells. Moreover, the main endothelin-converting enzyme (ECE) activity in these cells is a membrane-bound metallopeptidase that is also inhibited by PHOS. We have investigated further the role of the PHOS-sensitive ECE in the conversion of big ET-1 to ET-1. ET-1 was measured using a radioimmunoassay specific for the C-terminal ET[16-21] sequence. The effect of PHOS on the production of ET-1 from endogenous precursors was determined using cultured bovine aortic endothelial cells (BAECs) and the human endothelial cell line (EA.hy 926). The concentrations of ET-1 accumulating in the medium over 24 h from BAECs were lowered by PHOS (-27% 10 microM, -76% 100 microM). In contrast, with EA.hy 926 cells, the same concentrations of PHOS increased by five- to sixfold the amount of ET-1 present in the medium after 24-h incubation. In other experiments, incubation of big ET-1 (1 microM) with intact BAECs or EA.hy 926 cells resulted in the generation of ET-1, and with both cell types this was inhibited by PHOS (IC50: BAECs = 6.4 microM; EA.hy 926 = 0.26 microM). These results are consistent with both cell types having a PHOS-sensitive ECE that is readily accessible to exogenous big ET-1 and is therefore probably located on the plasma membrane. Furthermore, another intracellular ECE may play a part in the endogenous intracellular formation of ET-1 in EA.hy 926 cells.