SK-AND-F-96365 INHIBITS INTRACELLULAR CA2-OXIDE AND VON-WILLEBRAND-FACTOR( PUMPS AND RAISES CYTOSOLIC CA2+ CONCENTRATION WITHOUT PRODUCTIONOF NITRIC)

The effects of the imidazole compound SK&F 96365 on Ca2+ movements and production of nitric oxide (NO) and von Willebrand factor (vWF) have been investigated in human endothelial cells. Changes in cytosolic Ca2 + concentration ([Ca2+](i)) were measured with Fura-2. Real-time produ ction of NO was monitored with a porphyrinic microsensor and the relea se of vWF with an enzyme-linked immunosorbent assay. Irrespective of t he transmembrane Ca2+ gradient, 30 mu M SK&F 96365 doubled [Ca2+](i) s uggesting a Ca(2+) release from intracellular stores. The SK&F 96365-i nduced [Ca2+](i) rise was not accompanied by detectable NO and vWF pro duction, while 1 mu M thapsigargin enhanced [Ca2+](i) 2.5 times, doubl ed the secretion of vWF and increased the NO production to 10 +/- 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from inc reasing [Ca2+](i), NO production and vWF secretion. To investigate the mechanism by which SK&F 96365 released Ca2+ from internal pools, its effect and that of thapsigargin on the ATP-dependent Ca-45(2+) uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigar gin, dose-dependently reduced the initial rate of Ca-45(2+) uptake. In conclusion, we demonstrate that, in the absence of Ca2+ entry from th e extracellular space, the [Ca2+](i) increase elicited by SK&F 96365 o r thapsigargin is not sufficient to initiate NO synthesis and vWF secr etion. This confirms the important role of Ca2+ influx in endothelial secretion processes.