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

Abstract

The role of Na + and Na + exchangers in intracellular Ca 2+ elevation and leukotriene B 4 (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/10 6 neutrophils to 1.7±1.0 pmol/10 6 neutrophils at 0.3 μM fMLP). Isotonic substitution of Na + with NMDG + also induced a significant inhibition of fMLP-induced rise in cytosolic Ca 2+ concentration ([Ca 2+] i) (from 2.17- to 0.78-fold increase over basal levels). Pretreatment with an inhibitor of the Na +/Ca 2+ exchanger (benzamil) did not inhibit either [Ca 2+] i rise or LTBs production, indicating that the observed effects of extracellular Na +-deprivation were unrelated to the Na +/Ca 2+ exchanger in receptor-mediated Ca 2+ 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 Ca 2+ 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 [Ca 2+] 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 Ca 2+ influx through the Na +/Ca 2+ exchanger.