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 Ca 2+ movements and arachidonic acid release in human umbilical vein endothelial cells. Histamine, a receptor-dependent agonist, and thapsigargin, a specific inhibitor of sarco-/endoplasmic Ca 2+ pumps, time- and dose-dependently increased the release of [1- 14 C]-arachidonic acid. This release was inhibited by AACOCF 3 , a selective inhibitor of cytosolic phospholipase A 2 (PLA 2 ). In the absence of Ca 2+ influx, arachidonic acid release was suppressed in both histamine- and thapsigargin-stimulated cells, despite marked elevations of cytosolic Ca 2+ concentration ([Ca 2+ ] i ). In the presence of Ca 2+ influx, arachidonic acid release was reduced in cells treated with BAPTA, an intracellular Ca 2+ buffer, or with SK&F 96365, a receptor-operated Ca 2+ channel blocker. Arachidonic acid release was analyzed as a function of the two successive phases of Ca 2+ response to stimulation: Ca 2+ peak and plateau phase, reflecting Ca 2+ mobilization from internal stores and Ca 2+ influx, respectively. The amount of arachidonic acid released was directly related to [Ca 2+ ] i values measured at the influx phase with a 80 nM [Ca 2+ ] i threshold, similar to that reported for PLA 2 translocation. This suggests that Ca 2+ entry from the extracellular space is essential for activating cytosolic PLA 2 in human endothelial cells.