Regulation of Arachidonic Acid Release by Calcium Influx in Human Endothelial Cells

In response to stimuli, endothelial cells release arachidonic acid, a lipid precursor of various vasoactive substances. We have investigated the relationships between cytosolic Ca²⁺ movements and arachidonic acid release in human umbilical vein endothelial cells. Histamine, a receptor-dependent agonist, and thapsigargin, a specific inhibitor of sarco-/endoplasmic Ca²⁺ pumps, time- and dose-dependently increased the release of [1-¹⁴C]-arachidonic acid. This release was inhibited by AACOCF₃, a selective inhibitor of cytosolic phospholipase A₂ (PLA₂). In the absence of Ca²⁺ influx, arachidonic acid release was suppressed in both histamine- and thapsigargin-stimulated cells, despite marked elevations of cytosolic Ca²⁺ concentration ([Ca²⁺]_i). In the presence of Ca²⁺ influx, arachidonic acid release was reduced in cells treated with BAPTA, an intracellular Ca²⁺ buffer, or with SK&F 96365, a receptor-operated Ca²⁺ channel blocker. Arachidonic acid release was analyzed as a function of the two successive phases of Ca²⁺ response to stimulation: Ca²⁺ peak and plateau phase, reflecting Ca²⁺ mobilization from internal stores and Ca²⁺ influx, respectively. The amount of arachidonic acid released was directly related to [Ca²⁺]_i values measured at the influx phase with a 80 n<i>M</i> [Ca²⁺]_i threshold, similar to that reported for PLA₂ translocation. This suggests that Ca²⁺ entry from the extracellular space is essential for activating cytosolic PLA₂ in human endothelial cells.