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

To study the intracellular events leading to regulated exocytosis in h uman umbilical vein endothelial cells (HUVEC) the plasma membrane of H UVEC was selectively permeabilized with digitonin while retaining secr etory function. Fusion of Weibel-Palade bodies, the secretory organell e of HUVEC, with the plasma membrane was detected by assaying the medi a for von Willebrand factor (VWF). The secretion from permeabilized ce lls faithfully reflects that in intact cells by a number of criteria. First, in the presence of calcium, permeabilized HUVEC secreted vWF wi th the same kinetics and to the same extent as intact cells stimulated with secretagogue. In addition, the vWF secreted by permeabilized cel ls after stimulus was exclusively the processed mature form found in W eibel-Palade bodies. Release required micromolar levels of calcium. In addition, GTP gamma S could also stimulate release by a parallel path way. Both calcium-and GTP gamma S-stimulated secretion required a thio l-sensitive component. The hydrophobic thiol alkylating agent U73122 i nhibited calcium-dependent and GTP gamma S-stimulated secretion. Surpr isingly, N-ethylmaleimide, a hydrophilic alkylating agent, did not inh ibit secretion. The N-ethylmaleimide-sensitive fusion protein (NSF), a protein implicated in a variety of vesicle fusion events, did not app ear to be the target of U73122. These data strongly suggests the parti cipation of a non-NSF, membrane-associated protein in regulated secret ion in endothelial cells. Further, there appear to be two parallel pat hways leading to secretion in HUVEC, one stimulated by elevated levels of calcium and the other mediated by a GTP-binding protein.