Endocytosis and exocytosis events regulate vesicle traffic in endothelial cells

We used water-soluble styryl pyridinium dyes that fluoresce at the membrane -water interface to study vesicle traffic in endothelial cells. Cultured en dothelial cells derived from bovine and human pulmonary microvessels were i ncubated in styryl probes, washed to remove dye from the plasmalemmal outer face, and observed by digital fluorescence microscopy. Vesicles that deriv ed from plasmalemma by endocytosis were filled with the styryl dye. These v esicles were distributed throughout the cytosol as numerous particles of he terogeneous diameter and brightness. Vesicle formation was activated 2-fold following addition of extracellular albumin whereas a control protein, imm unoglobulin G, had no effect. Dye uptake was abrogated by labeling at low t emperatures and inhibitors of phosphoinositide-3-kinase (PI 3-kinase). Tyro sine kinase inhibitors (genistein and herbimycin A) prevented the albumin-i nduced vesicle formation. Cytochalasin B prevented vesicle redistribution i ndicating involvement of actin filaments in translocation of endosomes away from sites of Vesicle formation. Styryl dye was lost from cells by exocyto sis as evident by the disappearance of discrete fluorescent particles. N-et hylmaleimide and botulinum toxin types A and B caused cells to accumulate i ncreased number of vesicles suggesting that exocytosis was regulated by NSF -dependent SNARE mechanism. The results suggest that phosphoinositide metab olism regulates endocytosis in endothelial cells and that extracellular alb umin activates endocytosis by a mechanism involving tyrosine phosphorylatio n, whereas exocytosis is a distinct process regulated by the SNARE machiner y. The results support the hypothesis that albumin regulates its internaliz ation and release in vascular endothelial cells via activation of specific endocytic and exocytic pathways.