EVIDENCE FOR VESICLES THAT TRANSPORT ENDOTHELIN-1 IN BOVINE AORTIC ENDOTHELIAL-CELLS

To facilitate studies of the intracellular processing of endothelin-1 (ET-1) in endothelial cells we developed an enrichment procedure for the isolation of transport or secretory vesicles containing ET-1. Cultured bovine aortic endothelial cells (BAECs) were disrupted using a tight-fitting Dounce homogenizer, and subcellular fractions were isolated by sucrose-density-gradient ultracentrifugation. ET-1 immunoreactivity (ET-IR) in the fractions was measured with a specific ET[16-21] radioimmunoassay (RIA). The major peak of ET-IR was consistently localized at the 1.0/1.2 M sucrose interface (10.96 fmol/10(6) cells) in each preparation of subcellular fractions. The mean level of ET-IR at this interface, expressed as a percentage of the total, was 47.9 +/- 3.5% (n = 6). This finding provides strong evidence for the existence of a vesicle that transports ET-1 within BAECs, which may be an important site for endogenous ET-1 processing. A number of studies have reported that the final processing step in ET-1 biosynthesis by the hypothetical endothelin-converting enzyme (ECE) is inhibited by phosphoramidon (PHOS). Therefore, levels of PHOS-sensitive ECE activity in each fraction were assessed in parallel with ET-IR levels. The ECE activity associated with the peak of ET-IR (2.67 pmol ET-1/h/10(6) cells) was insensitive to 100 muM PHOS. However, the peak of ECE-like activity (10.4 pmol ET-1/h/10(6) cells) localized in the 0.8 M sucrose band was inhibited by 79% in the presence of 100 muM PHOS. Angiotensin-converting enzyme activity, a marker for plasma membranes, was also assayed in these fractions and localized to the same fractions as the PHOS-sensitive ECE activity. This finding suggests that the abundant peak of ECE-like activity that is present in subcellular fractions obtained from BAECs, but separated from the ET-rich fractions, is associated with the plasma membrane of endothelial cells and therefore not involved in intravesicle processing.