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The role of Na+ and Na+ exchangers in intracellular Ca2+ elevation and leukotriene B4 (LTBs) formation was investigated in granulocyte macrophage colony-stimulating factor (GM-CSF)-primed, fMLP-stimulated human neutrophils. Isotonic substitution of extracellular Na+ with N-methyl-d-glucamine+ (NMDG+) resulted in over 85% inhibition of the LTBs generation observed (from 14.1±0.9 pmol/106 neutrophils to 1.7±1.0 pmol/106 neutrophils at 0.3 μM fMLP). Isotonic substitution of Na+ with NMDG+ also induced a significant inhibition of fMLP-induced rise in cytosolic Ca2+ concentration ([Ca2+]i) (from 2.17- to 0.78-fold increase over basal levels). Pretreatment with an inhibitor of the Na+/Ca2+ exchanger (benzamil) did not inhibit either [Ca2+]i rise or LTBs production, indicating that the observed effects of extracellular Na+-deprivation were unrelated to the Na+/Ca2+ exchanger in receptor-mediated Ca2+ influx, as previously hypothesized. LTBs production by thapsigargin-activated neutrophils was not affected by Na+ depletion, but was totally abolished in the presence of EGTA, suggesting that store depletion-driven extracellular Ca2+ influx is required for leukotriene synthesis and that this process is independent of Na+-deprivation. Exposure to Na+-free medium for the time of GM-CSF priming led to a significant decrease of intracellular pH values, suggesting a role of the Na+/H+ exchanger in intracellular Na+ depletion. Reducing the time of Na+-deprivation totally reversed the observed effect on LTBs production, resulting in enhanced, rather than inhibited, formation of LTBs. These results indicate that LTBs generation and [Ca2+]i rise in human neutrophils primed by GM-CSF and stimulated with fMLP is dependent on intracellular Na+ concentration, and, at variance with previously published results, unrelated to the Ca2+ influx through the Na+/Ca2+ exchanger.