Changes in cytosolic Ca2+ associated with von Willebrand factor release in human endothelial cells exposed to histamine. Study of microcarrier cell monolayers using the fluorescent probe indo-1.
A method for measuring fluorescence in anchored monolayers of human endothelial cells is described and used to demonstrate changes in the cytosolic free-calcium concentration ([Ca2+]c) in these cells exposed to histamine and thrombin; some endothelial responses to both agonists (e.g., mitogenesis) have been suggested to be Ca2+-mediated. Umbilical vein endothelial cells were cultured on microcarriers and loaded with the Ca2+ indicator, indo-1. Enzymatic cell detachment was avoided by monitoring the indo-1 fluorescence ratio (400/480 nm) of a stirred suspension of cell-covered microcarriers. Basal [Ca2+]c was estimated to be 70-80 nM. Thrombin induced a transient dose-dependent increase in [Ca2+]c, which was active-site dependent. Histamine stimulated a dose-dependent increase in [Ca2+]c, which was reversed by removal of histamine and inhibited competitively by the H1-receptor antagonist pyrilamine, but not by the H2-receptor antagonist cimetidine. Furthermore, histamine induced a dose-dependent secretion of von Willebrand factor, which paralleled the rise in [Ca2+]c and was similarly blocked by the H1-receptor antagonist, and which may contribute to platelet deposition at sites of inflammation.
D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003600:Cytosol / Q000378:metabolism; D004727:Endothelium / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D007211:Indoles; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000502:physiology; D014471:Umbilical Veins; D014841:von Willebrand Factor / Q000378:metabolism*
D005456:Fluorescent Dyes; D007211:Indoles; D014841:von Willebrand Factor; D006632:Histamine; D013917:Thrombin; C048960:indo-1; D002118:Calcium