Signalling pathways regulating human neutrophil migration induced by secretory phospholipases A2

被引:22
作者
Gambero, A
Thomazzi, SM
Cintra, ACO
Landucci, ECT
De Nucci, G
Antunes, E
机构
[1] UNICAMP, Fac Med Sci, Dept Pharmacol, BR-13084970 Campinas, SP, Brazil
[2] USP, Dept Biochem, Ribeirao Preto, Brazil
基金
巴西圣保罗研究基金会;
关键词
phospholipase A(2); neutrophil migration; G-protein; protein kinase C; intracellular Ca2+;
D O I
10.1016/j.toxicon.2004.06.004
中图分类号
R9 [药学];
学科分类号
1007 ;
摘要
This study was designed to elucidate the signalling pathways by which secretory phospholipases A(2) (sPLA(2)s) induce in vitro neutrophil migration. The cell migration assays were performed with Naja mocambique venom PLA(2) (sPLA(2) with high catalytic activity), bothropstoxin-I (sPLA2 devoid of catalytic activity) and platelet-activating factor (PAF), using a 48-well microchemotaxis chamber. Both the non-selective protein kinase inhibitor staurosporine (30-300 nM) and the selective protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2-methylpyperazine (H7; 50-200 muM) as well as the Gi inactivator pertussis toxin (30-300 nM) caused a concentration-dependent inhibition of the neutrophil migration induced by either N. mocambique venom PLA(2) (100 mug/ml) or bothropstoxin-I (100 mug/ml). Pertussis toxin nearly abolished PAF-induced migration, while staurosporine and H7 partly (but significantly) inhibited the chemotactic responses to PAR The dual inhibitor of cytosolic PLA(2) and Ca2+-independent PLA(2) (iPLA(2)), arachidonil-trifluoromethyl-ketone (ATK; 0.2-20 muM), or the specific iPLA(2) inhibitor bromoenol lactone (1-30 muM) caused a concentration-dependent inhibition of the migration induced by either sPLA(2)s. At the maximal concentration used for each compound, the migration was almost suppressed. In contrast, both of these compounds caused only slight inhibitions of PAF-induced migration. No rise in intracellular Ca2+ Was observed in neutrophil-stimulated sPLA(2), as determined in cells preloaded with fura 2-AM. In the experimental condition used, pertussis toxin, staurosporine, H7, ATK or bromoenol lactone did not induce cytotoxic effects, according to MTT assay. Our results suggest that activation of an endogenous PLA(2) through activation of GTP-binding protein and PKC is the main mechanism by which exogenous sPLA(2)s cause neutrophil migration. (C) 2004 Elsevier Ltd. All rights reserved.
引用
收藏
页码:473 / 481
页数:9
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