Role of sodium in intracellular calcium elevation and leukotriene B 4 formation by receptor-mediated activation of human neutrophils

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±0.9pmol/10⁶ neutrophils to 1.7±1.0pmol/ 10⁶ neutrophils at 0.3μM fMLP). Isotonic substitution of Na ⁺ with NMDG⁺ also induced a significant inhibition of fMLP-induced rise in cytosolic Ca²⁺ concentration ([Ca ²⁺]_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.