Evidence that agonists stimulate bivalent-cation influx into human endothelial cells.
Human umbilical-vein endothelial cells stimulated with thrombin or histamine show an increase in [Ca2+]i (cytoplasmic free calcium concn.) that is maintained well above the basal pre-stimulated value as long as agonist and a source of extracellular Ca2+ are present. These results provide circumstantial evidence that agonists stimulate influx of Ca2+ across the plasma membrane and into the cytoplasm. Here, we have used Mn2+ as the extracellular bivalent cation which can bind to the fluorescent Ca2+ indicator fura-2 to quench its fluorescence completely. Human umbilical-vein endothelial cells were loaded with fura-2 and, in the presence of extracellular Mn2+, thrombin and histamine were shown to cause quenching of the intracellular dye. This result demonstrates conclusively that agonists can stimulate the influx of bivalent cations. Stimulated discharge of Ca2+ from intracellular stores and influx of Mn2+ were temporally resolved in the same cells to show that release of Ca2+ from intracellular stores clearly precedes influx. Influx of Mn2+ was also demonstrated when extracellular Mn2+ was added after agonist at a time when [Ca2+]i had fallen back to the basal value, showing that influx is not dependent on elevated [Ca2+]i.
Hallam
T J
TJ
Department of Cellular Pharmacology, Smith Kline & French Research Ltd., Welwyn, Herts, U.K.
Jacob
R
R
Merritt
J E
JE
D002118:Calcium / Q000378:metabolism; D002712:Chlorides; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism*; D004987:Ethers / Q000494:pharmacology; D005260:Female; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D015759:Ionomycin; D008345:Manganese / Q000493:pharmacokinetics* / Q000494:pharmacology; D017895:Manganese Compounds; D009532:Nickel / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013917:Thrombin / Q000494:pharmacology*
D002712:Chlorides; D004987:Ethers; D017895:Manganese Compounds; D008345:Manganese; D015759:Ionomycin; C022838:nickel chloride; D009532:Nickel; D006632:Histamine; D013917:Thrombin; C025340:manganese chloride; D002118:Calcium