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 microM) 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 microM) pretreatment mimicked the effect of Ca2+ removal. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) prevented 10 microM fMLP from increasing [Ca2+]i; whereas 1 microM thapsigargin still significantly increased [Ca2+]i after pretreatment with 10 microM fMLP. Addition of 3 mM Ca2+ induced a concentration-dependent [Ca2+]i increase after pretreatment with fMLP in Ca(2+)-free medium. This Ca2+ entry was partly inhibited by econazole (25 microM), SKF96365 (50 microM), and a phospholipase A2 inhibitor (aristolochic acid; 20 microM). The fMLP (10 microM)-induced Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 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 microM 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.
Chen
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LW
Department of Surgery, Kaohsiung Veterans General Hospital, 386 Ta Chung 1st Rd., Kaohsiung 813, Taiwan.
Jan
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D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D016257:Fura-2; D006801:Humans; D066298:In Vitro Techniques; D007811:Lanthanum / Q000494:pharmacology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D017382:Reactive Oxygen Species; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D017382:Reactive Oxygen Species; D009240:N-Formylmethionine Leucyl-Phenylalanine; D007811:Lanthanum; D011493:Protein Kinase C; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2