Agonist-stimulated divalent cation entry into single cultured human umbilical vein endothelial cells.
1. The free cytoplasmic Ca2+ concentration ([Ca2+]i) can be measured using Fura-2 in superfused single human umbilical vein endothelial cells. When an endothelial cell is stimulated by a maximal dose of histamine (100 microM), [Ca2+]i rises to a peak and then falls back to a maintained plateau which is due to a stimulated Ca2+ influx. 2. If extracellular Ca2+ is replaced by 50 microM-Mn2+ then 100 microM-histamine causes a rise in [Ca2+]i accompanied by a fluorescence quench that signals the stimulated entry of Mn2+ into the cytoplasm. 3. If in Ca2(+)-free solution a cell is stimulated by 100 microM-histamine for 120 s to discharge the internal Ca2+ store, and then exposed to 50 microM-Mn2+ after removal of the histamine, a similar stimulated Mn2+ entry is seen. This quench is unaffected by readdition of histamine and is not seen if the store is refilled by exposure to 1 mM-extracellular Ca2+ for 180 s before exposure to the Mn2+. 4. The refilling of the internal store by exposure to 1 mM-Ca2+ and the stimulated entry of Mn2+ are both blocked by 2 mM-Ni2+. 5. If [Ca2+]i is stimulated to produce repetitive spikes by a low dose of histamine (0.3-1 microM) in nominally Ca2(+)-free solution containing Mn2+, then the stimulated quench is uniform and is not modulated by the [Ca2+]i spiking. 6. If the internal store is discharged by exposure to histamine in Ca2(+)-free solution and then refilled for a short period then the cell is in a state where the internal store is partly full to an extent that depends on the duration of the refilling. In such an experiment, the rate of Mn2+ influx may be estimated by measuring the rate of quench during a short exposure to 50 microM-Mn2+. The rate of Mn2+ entry varies inversely with the degree of fullness of the internal Ca2+ store. 7. If a similar experiment is repeated but with the fullness of the internal store being varied by varying the period of the initial exposure to 100 microM-histamine, with no refilling, the same inverse relationship between Mn2+ influx and fullness of the internal store is obtained. 8. These experiments show that Mn2+ enters human umbilical vein endothelial cells following agonist stimulation by a pathway that is controlled by the degree of fullness of the internal store; it does not, however, enter the cytoplasm by exactly the same route as Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)
Jacob
R
R
Smith Kline & French Research Ltd, Welwyn, Herts.
D000200:Action Potentials / Q000187:drug effects; D001572:Benzofurans; D002118:Calcium / Q000378:metabolism / Q000493:pharmacokinetics*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D005456:Fluorescent Dyes; D016257:Fura-2; D006632:Histamine / Q000494:pharmacology*; D006801:Humans; D008345:Manganese / Q000493:pharmacokinetics*; D009532:Nickel / Q000494:pharmacology; D013268:Stimulation, Chemical; D014471:Umbilical Veins / Q000378:metabolism*
D001572:Benzofurans; D005456:Fluorescent Dyes; D008345:Manganese; D009532:Nickel; D006632:Histamine; D002118:Calcium; D016257:Fura-2