LEUKOTRIENE SYNTHESIS IN CALCIUM-DEPLETED HUMAN NEUTROPHILS - ARACHIDONIC-ACID RELEASE CORRELATES WITH CALCIUM INFLUX

The relationship between intracellular calcium concentration ([Ca2+](i)), the release of arachidonic acid and the synthesis of leukotriene B-4 (LTB(4)) was investigated using Ca2+-depleted human polymorphonuclear leucocytes (PMNs) in which [Ca2+](l) can be manipulated by varying the concentration of exogenous Ca2+ added with agonists. In this model, Ca2+, platelet-activating factor (PAF) and N-formyl-Met-Leu-Phe (FMLP), added alone, were unable to induce arachidonic acid release or LTB(4) synthesis, as assessed by measurements of the products by MS and HPLC, respectively. However, the simultaneous addition of Ca2+ and either PAF or FMLP to these Ca2+-depleted PMNs resulted in an influx of Ca2+ proportional to the extracellular concentration of Ca2+ and caused a substantial release of arachidonic acid and synthesis of LTB(4). The [Ca2+](i) values for threshold and maximal arachidonic acid release were found to be 150 nM and 350 nM respectively, suggesting the involvement of cytosolic phospholipase A(2) (cPLA(2)). Under stimulatory conditions resulting in similar [Ca2+](i). Ca2+-depleted PMNs released significant amounts of arachidonic acid but normal (Ca2+-repleted) PMNs did not, indicating that Ca2+ depletion of PMNs altered the normal regulation of arachidonic acid release and facilitated the release of the fatty acid upon stimulation with agonists. cPLA(2) and mitogen-activated protein kinase (MAP kinase) phosphorylation, as assessed by changes of electrophoretic mobility, occurred in both Ca2+-repleted and Ca2+- depleted PMNs upon addition of agonist. These data demonstrate that in Ca2+-depleted PMNs stimulated with agonists, arachidonic acid release and LTB(4) synthesis correlated with extracellular Ca2+ influx.