Anderson R, Visser S S, Ramafi G, Theron A J
Medical Research Council Unit for Inflammation and Immunity, Department of Immunology, Faculty of Medicine, University of Pretoria, South Africa.
Br J Pharmacol. 2000 Jun;130(4):717-24. doi: 10.1038/sj.bjp.0703344.
We have investigated the effects of the adenosine A(2A) receptor agonist CGS 21680 (0.01 - 1 microM) on reactive oxidant production by, and elastase release from FMLP-activated human neutrophils, as well as on cytosolic Ca(2+) fluxes and intracellular concentrations of cyclic AMP. Oxidant production, elastase release and cyclic AMP were assayed using lucigenin-enhanced chemiluminescence, colourimetric and radioimmunoassay procedures respectively, while cytosolic Ca(2+) fluxes were measured by fura-2 spectrofluorimetry in combination with radiometric procedures which distinguish between net efflux and influx of the cation. Treatment of neutrophils with CGS 21680 did not affect the FMLP-activated release of Ca(2+) from intracellular stores, but resulted in dose-related acceleration of the rate of decline in fura-2 fluorescence, as well as decreases in both efflux and store-operated influx of Ca(2+), compatible with enhancement of resequestration of the cation by the endo-membrane Ca(2+)-ATPase. These effects on neutrophil Ca(2+) handling were associated with increased intracellular cyclic AMP and with inhibition of oxidant production and release of elastase. In contrast, treatment of neutrophils with the selective A(2A) receptor antagonist, ZM 241385 (2.5 microM), prevented the transient increase in cyclic AMP in FMLP-activated neutrophils which was associated with delayed sequestration of incoming Ca(2+) during store-operated influx. The CGS 21680-mediated reduction of Ca(2+) efflux from FMLP-activated neutrophils was also antagonized by pretreatment of the cells with ZM 241385 (2.5 microM), as well as by thapsigargin (1 microM), an inhibitor of the endo-membrane Ca(2+)-ATPase. ZM 241385 also neutralized the cyclic AMP-elevating and anti-inflammatory interactions of CGS 21680 with neutrophils. We conclude that A(2A) receptors regulate the pro-inflammatory activities of human neutrophils by promoting cyclic AMP-dependent sequestration of cytosolic Ca(2+).
我们研究了腺苷A(2A)受体激动剂CGS 21680（0.01 - 1微摩尔）对FMLP激活的人中性粒细胞产生活性氧以及释放弹性蛋白酶的影响，同时也研究了其对胞质Ca(2+)通量和细胞内环磷酸腺苷浓度的影响。分别使用光泽精增强化学发光法、比色法和放射免疫分析法测定活性氧产生、弹性蛋白酶释放和环磷酸腺苷，而通过fura-2荧光光谱法结合区分阳离子净外流和内流的放射测量程序来测量胞质Ca(2+)通量。用CGS 21680处理中性粒细胞不会影响FMLP激活的细胞内储存Ca(2+)的释放，但会导致fura-2荧光下降速率呈剂量相关的加速，以及Ca(2+)外流和储存操纵性内流减少，这与内膜Ca(2+)-ATP酶增强阳离子再摄取相一致。这些对中性粒细胞Ca(2+)处理的影响与细胞内环磷酸腺苷增加以及活性氧产生和弹性蛋白酶释放受到抑制有关。相反，用选择性A(2A)受体拮抗剂ZM 241385（2.5微摩尔）处理中性粒细胞，可阻止FMLP激活的中性粒细胞中环磷酸腺苷的短暂增加，这与储存操纵性内流期间进入的Ca(2+)延迟螯合有关。用ZM 241385（2.5微摩尔）预处理细胞以及用内膜Ca(2+)-ATP酶抑制剂毒胡萝卜素（1微摩尔）预处理细胞，也可拮抗CGS 21680介导的FMLP激活的中性粒细胞中Ca(2+)外流的减少。ZM 241385还可中和CGS 21680与中性粒细胞的环磷酸腺苷升高和抗炎相互作用。我们得出结论，A(2A)受体通过促进环磷酸腺苷依赖性的胞质Ca(2+)螯合来调节人中性粒细胞的促炎活性。
