DIFFERENTIAL REGULATION OF VON-WILLEBRAND-FACTOR EXOCYTOSIS AND PROSTACYCLIN SYNTHESIS IN ELECTROPERMEABILIZED ENDOTHELIAL-CELL MONOLAYERS

We have developed a system to permeabilize human umbilical vein endoth elial cells in monolayer culture by application of a high-voltage elec tric field. The permeabilized preparation allows access of small molec ules (M(r) < 1000) without loss of large cytosolic proteins. Electrope rmeabilized cells exocytose highly multimeric von Willebrand factor fr om secretory granules in response to added Ca2+ (EC(50) = 0.8+/-0.02 m u M), with levels comparable with those observed on stimulation of int act endothelial cells by physiological agonists. MgATP(2-) potentiates Ca2+-driven von Willebrand factor secretion. Other nucleoside triphos phates, but not non-hydrolysable analogues, can replace ATP. Electrope rmeabilized cells also synthesize and release prostacyclin in response to added Ca2+ (EC(50)=0.3+/-0.08 mu M), but nucleoside triphosphates markedly inhibit, whereas non-hydrolysable GTP analogues increase, Ca2 +-driven prostacyclin synthesis. We conclude that elevation of the int racellular [Ca2+] is sufficient to cause efficient exocytosis of von W illebrand factor from permeabilized cells, despite evidence that addit ional second messengers are needed in intact cells. We find no evidenc e in endothelial cells for a guanine nucleotide-binding protein promot ing exocytosis, although one is clearly involved in stimulating Ca2+-d riven prostacyclin synthesis.