Anderson Ronald, Theron Annette J, Gravett Cornelia M, Steel Helen C, Tintinger Gregory R, Feldman Charles
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa.
Br J Pharmacol. 2009 Jan;156(1):105-15. doi: 10.1111/j.1476-5381.2008.00012.x. Epub 2008 Dec 6.
The objective of this study was to characterize the effects of the cysteinyl leukotriene receptor antagonist, montelukast (0.1-2 micromol x L(-1)), on Ca(2+)-dependent pro-inflammatory activities, cytosolic Ca(2+) fluxes and intracellular cAMP in isolated human neutrophils activated with the chemoattractants, N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 micromol x L(-1)) and platelet-activating factor (200 nmol x L(-1)).
Generation of reactive oxygen species was measured by lucigenin- and luminol-enhanced chemiluminescence, elastase release by a colourimetric assay, leukotriene B(4) and cAMP by competitive binding ELISA procedures, and Ca(2+) fluxes by fura-2/AM-based spectrofluorimetric and radiometric ((45)Ca(2+)) procedures.
Pre-incubation of neutrophils with montelukast resulted in dose-related inhibition of the generation of reactive oxygen species and leukotriene B(4) by chemoattractant-activated neutrophils, as well as release of elastase, all of which were maximal at 2 micromol x L(-1) (mean percentages of the control values of 30 +/- 1, 12 +/- 3 and 21 +/- 3 respectively; P < 0.05). From a mechanistic perspective, treatment of chemoattractant-activated neutrophils with montelukast resulted in significant reductions in both post-peak cytosolic Ca(2+) concentrations and store-operated Ca(2+) influx. These montelukast-mediated alterations in Ca(2+) handling by the cells were associated with a significant elevation in basal cAMP levels, which resulted from inhibition of cyclic nucleotide phosphodiesterases.
Montelukast, primarily a cysteinyl leukotriene (CysLT(1)) receptor antagonist, exhibited previously undocumented, secondary, neutrophil-directed anti-inflammatory properties, which appeared to be cAMP-dependent.
本研究的目的是描述半胱氨酰白三烯受体拮抗剂孟鲁司特（0.1 - 2微摩尔×升⁻¹）对用趋化因子N-甲酰-L-甲硫氨酰-L-亮氨酰-L-苯丙氨酸（1微摩尔×升⁻¹）和血小板活化因子（200纳摩尔×升⁻¹）激活的分离人中性粒细胞中钙依赖的促炎活性、胞质钙通量和细胞内cAMP的影响。
通过光泽精和鲁米诺增强的化学发光法测量活性氧的产生，通过比色法测定弹性蛋白酶释放，通过竞争性结合酶联免疫吸附测定法测定白三烯B₄和cAMP，通过基于fura-2/AM的光谱荧光法和放射性测量法（⁴⁵Ca²⁺）测定钙通量。
中性粒细胞与孟鲁司特预孵育导致趋化因子激活的中性粒细胞产生活性氧和白三烯B₄以及弹性蛋白酶释放受到剂量相关的抑制，所有这些在2微摩尔×升⁻¹时达到最大抑制（分别为对照值的平均百分比30±1、12±3和21±3；P<0.05）。从机制角度来看，用孟鲁司特处理趋化因子激活的中性粒细胞导致峰后胞质钙浓度和储存-操作性钙内流均显著降低。细胞中这些孟鲁司特介导的钙处理改变与基础cAMP水平显著升高相关，这是由于环核苷酸磷酸二酯酶受到抑制所致。
孟鲁司特主要作为半胱氨酰白三烯（CysLT₁）受体拮抗剂，展现出此前未被记录的、针对中性粒细胞的继发性抗炎特性，这些特性似乎依赖于cAMP。
