Fiore S, Ryeom S W, Weller P F, Serhan C N
Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115.
J Biol Chem. 1992 Aug 15;267(23):16168-76.
Lipoxin A4 stimulates rapid lipid remodeling and a pertussis toxin-sensitive release of arachidonic acid in polymorphonuclear leukocytes (PMN) (Nigam, S., Fiore, S., Luscinskas, F.W., and Serhan, C.N. (1990) J. Cell. Physiol. 143, 512-523) and has been shown to inhibit leukocyte responses in several systems. To examine the basis underlying these actions, we have prepared [11,12-3H]lipoxin A4 (LXA4) and characterized its interactions with human PMN. Time course studies (0-90 min) with intact PMN demonstrated cell association of 3H label which was specific and reversible. PMN bound [3H]LXA4 with a Kd of 0.5 +/- 0.3 nM, representing approximately 1,830 sites/PMN, and the Hill plot value of 1.9 suggests cooperative binding. [3H]LXA4 binding was stereoselective since neither leukotriene B4 (LTB4), lipoxin B4 (LXB4), (6S)-LXA4, 11-trans-LXA4, nor SKF 104353 competed for [3H]LXA4-specific binding while LTD4 and LTC4 partially competed. Subcellular fractionation revealed that specific binding with [3H]LXA4 was associated with membrane (42.1%)-, granule (34.5%)-, and nuclear (23.3%)-enriched fractions, a distribution distinct from that of [14,15-3H] LTB4 binding. [11,12-3H]LXA4-specific binding was modulated by guanosine analogs, suggesting the involvement of G proteins. A fluorescent LXA4 derivative (methyl-7-methoxycoumarin-LXA4) competed with [3H]LXA4 binding to intact PMN and showed specific and reversible binding as monitored by flow cytometric analysis. These results indicate that PMN possess specific recognition sites for LXA4 which may mediate its actions.
脂氧素A4可刺激多形核白细胞（PMN）快速进行脂质重塑并释放花生四烯酸，且该释放对百日咳毒素敏感（尼甘姆，S.，菲奥雷，S.，卢辛斯卡，F.W.，和塞尔汉，C.N.（1990年）《细胞生理学杂志》143卷，512 - 523页），并且已证实在多个系统中它可抑制白细胞反应。为研究这些作用的潜在机制，我们制备了[11,12 - 3H]脂氧素A4（LXA4）并对其与人PMN的相互作用进行了表征。对完整PMN进行的时间进程研究（0 - 90分钟）表明3H标记与细胞的结合具有特异性且可逆。PMN结合[3H]LXA4的解离常数（Kd）为0.5±0.3 nM，相当于约1830个位点/PMN，希尔系数为1.9表明存在协同结合。[3H]LXA4的结合具有立体选择性，因为白三烯B4（LTB4）、脂氧素B4（LXB4）、（6S） - LXA4、11 - 反式 - LXA4以及SKF 104353均不与[3H]LXA4的特异性结合竞争，而LTD4和LTC4部分竞争。亚细胞分级分离显示，[3H]LXA4的特异性结合与富含膜（42.1%）、颗粒（34.5%）和细胞核（23.3%）的组分相关，这种分布与[14,15 - 3H]LTB4的结合不同。[11,12 - 3H]LXA4的特异性结合受鸟苷类似物调节，提示G蛋白参与其中。一种荧光LXA4衍生物（甲基 - 7 - 甲氧基香豆素 - LXA4）与[3H]LXA4竞争结合完整PMN，并通过流式细胞术分析显示出特异性且可逆的结合。这些结果表明PMN具有LXA4的特异性识别位点，这可能介导其作用。
