Mechanisms and modulation of formyl-methionyl-leucyl-phenylalanine (fMLP)-induced Ca²⁺ mobilization in human neutrophils

The effect of fMLP (N-formyl-methionyl-leucyl-phenylalanine), a neutrophil-stimulating bacterial peptide, on Ca²⁺ mobilization in human neutrophils was examined using fura-2 as a Ca²⁺ indicator. fMLP (10 nM-10 μM) increased [Ca²⁺]_i concentration-dependently. The [Ca²⁺]_i signal comprised an initial rise followed by a gradual decay and a sustained phase. External Ca²⁺ removal partly decreased the signal. La³⁺ (50 μM) pretreatment mimicked the effect of Ca²⁺ removal. In Ca²⁺-free medium, pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) prevented 10 μM fMLP from increasing [Ca²⁺]_i; whereas 1 μM thapsigargin still significantly increased [Ca²⁺]_i after pretreatment with 10 μM fMLP. Addition of 3 mM Ca²⁺ induced a concentration-dependent [Ca²⁺]_i increase after pretreatment with fMLP in Ca²⁺-free medium. This Ca²⁺ entry was partly inhibited by econazole (25 μM), SKF96365 (50 μM), and a phospholipase A₂ inhibitor (aristolochic acid; 20 μM). The fMLP (10 μM)-induced Ca²⁺ release was abolished by inhibiting phospholipase C with 2 μM U73122. The fMLP-induced [Ca²⁺]_i increase was inhibited by 25% by pretreatment with 10 nM phorbol ester to activate protein kinase C but was augmented by 27% by pretreatment with 2 μM GF 109203X to inactivate protein kinase C. We found that fMLP increase reactive oxygen intermediate (ROI) production in neutrophils, which can be suppressed by U73122 pretreatment. Collectively, this study shows that in human neutrophils, fMLP increased [Ca²⁺]_i concentration-dependently by releasing Ca²⁺ from phospholipase C-coupled, thapsigargin-sensitive stores, accompanied by Ca²⁺ entry. The fMLP-induced [Ca²⁺]_i rise was modulated by protein kinase C, and the fMLP-induced Ca²⁺ entry was abolished by La³⁺, and was reduced by econazole, SKF96365 and inhibition of phospholipase A₂.