Agonist‐stimulated divalent cation entry into single cultured human umbilical vein endothelial cells.

Abstract

1. The free cytoplasmic Ca2+ concentration ([Ca2+]1) 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 *mM), [Ca2+]i rises to a peak and then falls back to a maintained plateau which is due to stimulated Ca2+ influx. 2. If extracellular Ca2+ is replaced by 50 .mu.M-Mn2+ then 100 .mu.M-histamine causes a rise in [Ca2+]i accompanied by a fluroscence quench that signals the stimulated entry of Mn2+ into the cytoplasm. 3. If in Ca2+-free solution a cell is stimulated by 100 .mu.M-histamine for 120 s to discharge the internal Ca2+ store, and then exposed to 50 .mu.M-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 1mM-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 .MU.) in nominally Ca2+-free solutions 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 .mu.M-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 the exposure to 100 .mu.M-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 forming 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+. It is proposed that Mn2+ enters by a pathway that is in part the same as the pathway by which Ca2+ refills into the internal store. The common element of these two pathways contains the point at which divalent cation entry is controlled by the degree of fullness of the internal store.