SK&F 96365 inhibits intracellular Ca2+ pumps and raises cytosolic Ca2+ concentration without production of nitric oxide and von Willebrand factor

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 production of NO was monitored with a porphyrinic microsensor and the release of vWF with an enzyme-linked immunosorbent assay. Irrespective of the transmembrane Ca2+ gradient, 30 mu M SK&F 96365 doubled [Ca2+](i) suggesting a Ca(2+) release from intracellular stores. The SK&F 96365-induced [Ca2+](i) rise was not accompanied by detectable NO and vWF production, while 1 mu M thapsigargin enhanced [Ca2+](i) 2.5 times, doubled the secretion of vWF and increased the NO production to 10 +/- 4 nM (n = 5). Pretreatment with SK&F 96365 prevented thapsigargin from increasing [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 thapsigargin, dose-dependently reduced the initial rate of Ca-45(2+) uptake. In conclusion, we demonstrate that, in the absence of Ca2+ entry from the extracellular space, the [Ca2+](i) increase elicited by SK&F 96365 or thapsigargin is not sufficient to initiate NO synthesis and vWF secretion. This confirms the important role of Ca2+ influx in endothelial secretion processes.