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

The complete amino acid sequence of bothropstoxin-II (BthTX-II), a myotoxin isolated from Bothrops jararacussu snake venom, is reported. The results show that BthTX-II is an Asp-49 phospholipase A2 (PLA2)-like protein composed of a single polypeptide chain of 120 amino acid residues (Mr = 13,976), containing one methionine and 14 half-cystines. Despite a high degree of homology with other PLA2's and the presence of the strategic residues known to compose the Ca2+-binding loop, namely Tyr-28, Gly-30, Gly-32, and especially Asp-49, besides His-48, Tyr-52, and Asp-99, all of them directly or indirectly involved in catalysis, BthTX-II revealed a very low PLA2 activity when assayed on egg yolk phosphatidylcholine. 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No Role For Enzymatic Activity Or Dantrolene-sensitive Ca2+ Stores In The Muscular Effects Of Bothropstoxin, A Lys49 Phospholipase A2 Myotoxin

The role of low levels of phospholipase A2 (PLA2) activity and intracellular Ca2+ stores in the pharmacological action of bothropstoxin (BthTX), a myotoxic Lys49 PLA2 homologue isolated from the venom of Bothrops jararacussu, was investigated. We examined the muscular effects of BthTX in the mouse diaphragm and its PLA2 activity in radiolabeled human and rat primary cultures of skeletal muscle. Although it is a Lys49 PLA2 homologue, BthTX had a low, but easily detectable, level of enzymatic activity relative to two Asp49 PLA2 enzymes from Naja naja kaouthia and Naja naja atra venoms, and this activity was reduced by about 85% in the presence of Sr2+ (4.0 mM). However, the replacement of 1.8 mM Ca2+ by 4 mM Sr2+ did not alter the BthTX-induced contracture and blockade of the muscle twitch tension. In addition, Sr2+ decreased by 50% the time required to cause 50% paralysis, and evoked approximately a four-fold increase in the number of spontaneous spikes. In isolated sarcoplasmic reticulum preparations, BthTX opened the intracellular Ca2+ release channel (ryanodine receptor) and lowered the threshold of Ca2+-induced Ca2+ release by a second, as yet unidentified, mechanism. However, in intact muscle, dantrolene, an antagonist of some forms of intracellular Ca2+ release, had no effect on the actions of BthTX. These findings do not support any role for the low levels of PLA2 activity, or dantrolene-sensitive intracellular Ca2+ stores, in the action of BthTX. The mechanism whereby Sr2+ stimulates the pharmacological activity of BthTX remains to be clarified. © 1995.331114791489Cintra, Marangoni, Oliveira, Giglio, Bothropstoxin-I: amino acid sequence and function (1993) J. Prot. Chem., 12, pp. 57-64Condrea, Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom. 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