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 microM SK&F 96365 doubled [Ca2+]i suggesting a Ca2+ release from intracellular stores. The SK&F 96365-induced [Ca2+]i rise was not accompanied by detectable NO and vWF production, while 1 microM 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 45Ca2+ uptake into platelet membrane vesicles were compared. SK&F 96365 as thapsigargin, dose-dependently reduced the initial rate of 45Ca2+ 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.
Iouzalen
L
L
Department of Pharmacology, URA CNRS 1482, Necker University School of Medicine, Paris, France.
Lantoine
F
F
Pernollet
M G
MG
Millanvoye-Van Brussel
E
E
Devynck
M A
MA
David-Dufilho
M
M
D000255:Adenosine Triphosphate / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D001792:Blood Platelets / Q000201:enzymology / Q000648:ultrastructure; D002118:Calcium / Q000378:metabolism* / Q000493:pharmacokinetics; D002121:Calcium Channel Blockers / Q000494:pharmacology*; D002132:Calcium Radioisotopes / Q000493:pharmacokinetics; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002478:Cells, Cultured / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007425:Intracellular Membranes / Q000378:metabolism; D009569:Nitric Oxide / Q000096:biosynthesis*; D019284:Thapsigargin / Q000494:pharmacology; D014471:Umbilical Veins / Q000166:cytology; D014841:von Willebrand Factor / Q000096:biosynthesis* / Q000378:metabolism
D002121:Calcium Channel Blockers; D002132:Calcium Radioisotopes; D004791:Enzyme Inhibitors; D007093:Imidazoles; D014841:von Willebrand Factor; D009569:Nitric Oxide; D019284:Thapsigargin; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; C063159:1-(2-(3-(4-methoxyphenyl)propoxy)-4-methoxyphenylethyl)-1H-imidazole; D002118:Calcium