Regulated Exocytosis in Vascular Endothelial Cells Can be Triggered by Intracellular Guanine Nucleotides and Requires a Hydrophobic, Thiol-Sensitive Component. Studies of Regulated von Willebrand Factor Secretion from Digitonin Permeabilized Endothelial Cells

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Abstract
To study the intracellular events leading to regulated exocytosis in human umbilical vein endothelial cells (HUVEC) the plasma membrane of HUVEC was selectively permeabilized with digitonin while retaining secretory function. Fusion of Weibel-Palade bodies, the secretory organelle of HUVEC, with the plasma membrane was detected by assaying the media for von Willebrand factor (vWF). The secretion from permeabilized cells faithfully reflects that in intact cells by a number of criteria. First, in the presence of calcium, permeabilized HUVEC secreted vWF with the same kinetics and to the same extent as intact cells stimulated with secretagogue. In addition, the vWF secreted by permeabilized cells after stimulus was exclusively the processed mature form found in Weibel-Palade bodies. Release required micromolar levels of calcium. In addition, GTPgammaS could also stimulate release by a parallel pathway. Both calcium- and GTPgammaS-stimulated secretion required a thiol-sensitive component. The hydrophobic thiol alkylating agent U73122 inhibited calcium-dependent and GTPgammaS-stimulated secretion. Surprisingly, N-ethylmaleimide, a hydrophilic alkylating agent, did not inhibit secretion. The N-ethylmaleimide-sensitive fusion protein (NSF), a protein implicated in a variety of vesicle fusion events, did not appear to be the target of U73122. These data strongly suggests the participation of a non-NSF, membrane-associated protein in regulated secretion in endothelial cells. Further, there appear to be two parallel pathways leading to secretion in HUVEC, one stimulated by elevated levels of calcium and the other mediated by a GTP-binding protein.