We have developed a system to permeabilize human umbilical vein endothelial cells in monolayer culture by application of a high-voltage electric field. The permeabilized preparation allows access of small molecules (M(r) < 1000) without loss of large cytosolic proteins. Electropermeabilized cells exocytose highly multimeric von Willebrand factor from secretory granules in response to added Ca2+ (EC50 = 0.8 +/- 0.02 microM), with levels comparable with those observed on stimulation of intact endothelial cells by physiological agonists. MgATP2- potentiates Ca(2+)-driven von Willebrand factor secretion. Other nucleoside triphosphates, but not non-hydrolysable analogues, can replace ATP. Electropermeabilized cells also synthesize and release prostacyclin in response to added Ca2+ (EC50 = 0.3 +/- 0.08 microM), but nucleoside triphosphates markedly inhibit, whereas nonhydrolysable GTP analogues increase, Ca(2+)-driven prostacyclin synthesis. We conclude that elevation of the intracellular [Ca2+] is sufficient to cause efficient exocytosis of von Willebrand factor from permeabilized cells, despite evidence that additional second messengers are needed in intact cells. We find no evidence in endothelial cells for a guanine nucleotide-binding protein promoting exocytosis, although one is clearly involved in stimulating Ca(2+)-driven prostacyclin synthesis.
Differential regulation of von Willebrand factor exocytosis and prostacyclin synthesis in electropermeabilized endothelial cell monolayers
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J A Frearson, P Harrison, M C Scrutton, J D Pearson; Differential regulation of von Willebrand factor exocytosis and prostacyclin synthesis in electropermeabilized endothelial cell monolayers. Biochem J 15 July 1995; 309 (2): 473–479. doi: https://doi.org/10.1042/bj3090473
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