Endothelial cell plasma membrane obtained by chemically induced vesiculation.
A method using low concentrations of formaldehyde and dithiothreitol was applied to obtain 'right-side out' luminal plasmalemma-derived vesicles from bovine aortic endothelial cells (EC) in culture, and from human umbilical vein and bovine or porcine aortas perfused ex vivo with the vesiculation solution. Vesicle formation and shedding were examined by phase-contrast microscopy and by transmission (TEM) and scanning electron microscopy (SEM). Vesicles showed the characteristic trilaminar pattern of the unit membrane and did not contain cellular organelles. As detected in freeze-fracture preparations, vesicle membrane displayed intramembrane particles and filipin-detectable cholesterol. Like EC plasmalemma, vesicle surface was heavily stained by Ruthenium Red and bound under a normal pattern cationized ferritin and ferritin hydrazide. As indicated by lectin agglutination assays and by ultrastructural cytochemistry, vesicles maintained on their ectodomains glycoconjugates bearing monosaccharides such as N-acetyl-neuraminic acid, beta-N-acetylglucosamine and beta-D-galactose, and expressed 5'-nucleotidase activity. The electrophoretic profiles of externally disposed 125I-labelled polypeptides of vesicles were found to be similar to those of intact EC. Chemically-induced vesiculation appears as a suitable method to obtain EC plasmalemma for studying its composition and functions in various vascular beds.
D000818:Animals; D002417:Cattle; D002458:Cell Fractionation / Q000379:methods*; D002462:Cell Membrane / Q000032:analysis / Q000187:drug effects / Q000201:enzymology / Q000648:ultrastructure*; D002478:Cells, Cultured; D004229:Dithiothreitol / Q000494:pharmacology; D004727:Endothelium / Q000648:ultrastructure*; D005557:Formaldehyde / Q000494:pharmacology; D005614:Freeze Fracturing; D006801:Humans; D008565:Membrane Proteins / Q000032:analysis; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D008970:Molecular Weight; D013552:Swine
D008565:Membrane Proteins; D005557:Formaldehyde; D004229:Dithiothreitol