IDENTIFICATION OF ENDOTHELIN-1 AND BIG ENDOTHELIN-1 IN SECRETORY VESICLES ISOLATED FROM BOVINE AORTIC ENDOTHELIAL-CELLS

Vesicles containing endothelin 1 (ET-1) were isolated from bovine aortic endothelial cells (BAECs) by fractionation of homogenates on sucrose density gradients by ultracentrifugation. The vesicles were localized at the 1.0/1.2 M sucrose interface using a specific anti-ET-1-(16-21) RIA, Identification of ET-1 and big ET-1 in this fraction was confirmed by HPLC analysis combined with RIA, Morphological examination of the ET-1-enriched fraction by electron microscopy identified clusters of vesicles approximate to 100 nm in diameter, Immunostaining of ultrathin cryosections prepared from the vesicle fraction for ET-1 or big ET-1 showed clusters of 15-nm gold particles attached to or within vesicles, Immunofluorescence staining of whole BAECs using a specific ET-1(16-21) IgG purified by affinity chromatography revealed punctate granulation of the cell cytoplasm viewed under light microscopy, This distinct pattern of staining was shown by confocal light microscopy to be intracellular. Immunofluorescence staining of whole cells with a polyclonal antiserum for big ET-1-(22-39) showed a defined perinuclear localization of precursor molecule. Hence, several different approaches have demonstrated that ET-1 and big ET-1 are localized within intracellular vesicles in BAECs, suggesting that these subcellular compartments are an important site for processing of big ET-1 by endothelin-converting enzyme.