pThe relationship between intracellular calcium concentration ([Ca2+]i), the release of arachidonic acid and the synthesis of leukotriene B4 (LTB4) was investigated using Ca(2+)-depleted human polymorphonuclear leucocytes (PMNs) in which [Ca2+]i 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 LTB4 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 Ca(2+)-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 LTB4. 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 A2 (cPLA2). Under stimulatory conditions resulting in similar [Ca2+]i, Ca(2+)-depleted PMNs released significant amounts of arachidonic acid but normal (Ca(2+)-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. cPLA2 and mitogen-activated protein kinase (MAP kinase) phosphorylation, as assessed by changes of electrophoretic mobility, occurred in both Ca(2+)-depleted and Ca(2+)-depleted PMNs upon addition of agonist. These data demonstrate that in Ca(2+)-depleted PMNs stimulated with agonists, arachidonic acid release and LTB4 synthesis correlated with extracellular Ca2+ influx./p
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