Signalling Pathways Regulating Human Neutrophil Migration Induced By Secretory Phospholipases A2

This study was designed to elucidate the signalling pathways by which secretory phospholipases A2 (sPLA2s) induce in vitro neutrophil migration. The cell migration assays were performed with Naja mocambique venom PLA2 (sPLA2 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 (30-300nM) and the selective protein kinase C (PKC) inhibitor 1-(5-isoquinolinesulfonyl)-2- methylpyperazine (H7; 50-200 μM) as well as the Gi inactivator pertussis toxin (30-300nM) caused a concentration-dependent inhibition of the neutrophil migration induced by either N. mocambique venom PLA2 (100 μg/ml) or bothropstoxin-I (100 μg/ml). Pertussis toxin nearly abolished PAF-induced migration, while staurosporine and H7 partly (but significantly) inhibited the chemotactic responses to PAF. The dual inhibitor of cytosolic PLA2 and Ca2+-independent PLA2 (iPLA2), arachidonil-trifluoromethyl-ketone (ATK; 0.2-20 μM), or the specific iPLA2 inhibitor bromoenol lactone (1-30 μM) caused a concentration-dependent inhibition of the migration induced by either sPLA 2s. 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 sPLA2, 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 PLA2 through activation of GTP-binding protein and PKC is the main mechanism by which exogenous sPLA2s cause neutrophil migration. © 2004 Elsevier Ltd. 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The Amino Acid Sequence Of Bothropstoxin-ii, An Asp-49 Myotoxin From Bothrops Jararacussu (jararacucu) Venom With Low Phospholipase A2 Activity

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