Open AccessIdentification of endothelin 1 and big endothelin 1 in secretory vesicles isolated from bovine aortic endothelial cells.
Author(s) -
Vanessa J. Harrison,
Kathleen Barnes,
Anthony J. Turner,
Elizabeth G. Wood,
Roger Corder,
John R. Vane
Publication year - 1995
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.92.14.6344
Subject(s) - vesicle , differential centrifugation , cytoplasm , staining , polyclonal antibodies , biology , immunoelectron microscopy , immunostaining , centrifugation , immunogold labelling , cell fractionation , biochemistry , immunofluorescence , ultracentrifuge , microbiology and biotechnology , negative stain , chemistry , electron microscope , ultrastructure , membrane , antibody , anatomy , immunohistochemistry , genetics , physics , optics , immunology
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 approximately 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.