Grimminger F, Sibelius U, Aktories K, Suttorp N, Seeger W
Department of Internal Medicine, Justus Liebig University, Giessen, FRG.
Mol Pharmacol. 1991 Oct;40(4):563-71.
Botulinum C2 toxin, a binary toxin that ADP-ribosylates nonmuscle G-actin, was used as a selective tool to evaluate the role of actin-dependent cytoskeletal rearrangement in ligand-evoked lipid mediator generation. Human neutrophils (PMN) were preincubated with varying concentrations of the toxin for 30 min. Lipoxygenase products of arachidonic acid were measured by chromatographic techniques in the presence of exogenous arachidonic acid to probe PMN 5-lipoxygenase activity. Formation of platelet-activating factor (PAF) was assayed by the bioincorporation of [3H]acetate. Stimulation was performed with the soluble chemotactic ligands formyl-methionyl-leucyl-phenylalanine (FMLP) and PAF, as well as opsonized zymosan. PMN pretreatment with C2 toxin in the range between 200/400 and 800/1600 ng/ml C2I/II caused a dose-dependent suppression of the basal F-actin content and of stimulus-induced actin assembly. Phosphoinositide hydrolysis (measured as liberated inositol phosphates) and PAF generation in response to FMLP and exogenous PAF were markedly increased at these toxin doses. Minor C2 toxin concentrations (range, approximately 25/50 to 200/400 ng/ml C2I/II) were sufficient to amplify stimulus-induced formation of leukotriene B4 and its omega-oxidation products, nonenzymatic hydrolysis products of leukotriene A4, and 5-hydroxyeicosatetraenoic acid (5-HETE). With increasing toxin doses, leukotriene generation declined and 5-HETE became the predominant metabolite. In contrast to the soluble ligands, the zymosan-effected generation of PAF and leukotrienes was dose-dependently inhibited by C2 toxin concentrations of greater than 200/400 ng/ml, paralleled by a loss of motile and phagocytotic functions in these cells. We conclude that selective inhibition of actin assembly amplifies PAF and 5-lipoxygenase product formation in response to soluble chemoattractants with distinct dose dependences. The augmentation of PAF generation may be linked to amplified second messenger levels at higher doses of C2 toxin, whereas the sensitivity of the 5-lipoxygenase metabolism to low concentrations may indicate toxin effect on a small, functionally specified, actin pool. The present data support an important role of cytoskeletal rearrangement in temporal and/or spatial limitation of chemoattractant-evoked PMN activation.
肉毒杆菌C2毒素是一种可使非肌肉G-肌动蛋白发生ADP核糖基化的二元毒素，被用作一种选择性工具，以评估肌动蛋白依赖性细胞骨架重排在配体诱发的脂质介质生成中的作用。将人中性粒细胞（PMN）与不同浓度的毒素预孵育30分钟。在存在外源性花生四烯酸的情况下，通过色谱技术测量花生四烯酸的脂氧合酶产物，以检测PMN的5-脂氧合酶活性。通过[3H]乙酸的生物掺入来测定血小板活化因子（PAF）的形成。用可溶性趋化配体甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）和PAF以及调理酵母聚糖进行刺激。用200/400至800/1600 ng/ml C2I/II范围内的C2毒素预处理PMN会导致基础F-肌动蛋白含量和刺激诱导的肌动蛋白组装呈剂量依赖性抑制。在这些毒素剂量下，响应FMLP和外源性PAF的磷酸肌醇水解（以释放的肌醇磷酸测量）和PAF生成显著增加。低浓度的C2毒素（范围约为25/50至200/400 ng/ml C2I/II）足以放大刺激诱导的白三烯B4及其ω-氧化产物、白三烯A4的非酶水解产物和5-羟基二十碳四烯酸（5-HETE）的形成。随着毒素剂量增加，白三烯生成减少，5-HETE成为主要代谢产物。与可溶性配体相反，大于200/400 ng/ml的C2毒素浓度可剂量依赖性抑制酵母聚糖诱导的PAF和白三烯生成，同时这些细胞的运动和吞噬功能丧失。我们得出结论，对肌动蛋白组装的选择性抑制可放大对可溶性趋化剂的PAF和5-脂氧合酶产物形成，且具有不同的剂量依赖性。PAF生成的增加可能与较高剂量C2毒素时第二信使水平的放大有关。而5-脂氧合酶代谢对低浓度的敏感性可能表明毒素对一个小的、功能特定的肌动蛋白池有作用。目前的数据支持细胞骨架重排在趋化剂诱发的PMN激活的时间和/或空间限制中起重要作用。
