Determination of specific receptor sites for platelet activating factor in bovine neutrophils
Determination of specific receptor sites for platelet activating factor in bovine neutrophils
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DOI：
10.2460/ajvr.2004.65.628
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发表时间：
2004-05-01
影响因子：
1
通讯作者：
Hancke, JL
中科院分区：
文献类型：
--
作者：
Burgos, RA;Hidalgo, MA;Hancke, JL
目的：通过放射配基结合、逆转录-聚合酶链式反应(RT-PCR)和免疫印迹分析，鉴定和鉴定牛中性粒细胞中的血小板活化因子(PAF)受体。用合适的人引物进行RT-PCR检测，用从牛PAF受体多肽中获得的多克隆抗体进行蛋白质印迹分析。结果：动力学结合数据分析支持单一类PAF受体。H-3-PAF与膜制剂的结合被PAF和一个非水解性的三磷酸鸟嘌呤类似物(GPP[NH]p)和Lyso-PAF(一种生物活性较低的PAF类似物)选择性地取代。在PAF受体拮抗剂中，14-脱氧穿心莲内酯和WEB 2086抑制中性粒细胞膜H-3-PAF结合作用最强，木脂素、五味子甲素和五味子甲素也有效，而银杏内酯(BN52020和BN52021)仅轻度抑制H-3-PAF结合。RT-PCR检测和免疫印迹分析证实中性粒细胞存在PAF受体。结论和临床相关性研究结果表明，牛中性粒细胞只表达一种PAF受体，该受体可能参与炎症反应。最有效的PAF拮抗剂是14-脱氧穿心莲内酯和WEB 2086；这些PAF拮抗剂可能在治疗牛的炎症过程中潜在有用。
Objective - To identity and characterize a platelet activating factor (PAF) receptor in bovine neutrophils by use of radioligand binding, reverse transcription-polymerase chain reaction (RT-PCR) assay, and western blot analysis.Animals - 4 healthy adult cows.Procedure - Bovine neutrophil membranes were isolated for association, dissociation, and saturation binding experiments with PAF labeled with hydrogen 3 (H-3-PAF). The RT-PCR assay was performed with appropriate human primers, and western blot analysis was developed with a polyclonal antibody obtained from a peptide of bovine PAF receptor.Results - Analysis of kinetic binding data supported a single class of PAF receptor. Binding of H-3-PAF to membrane preparations was selectively displaced by PAF and a nonhydrolyzable analogue of guanine triphosphate (Gpp[NH]p) and by lyso-PAF (a biologically inactive analogue of PAF) to a lesser extent. Among other PAF receptor antagonists, 14-deoxyandrographolide and WEB 2086 were the most effective in inhibiting H-3-PAF binding sites in neutrophil membranes; 2 lignans, schisandrin-A and gamma-schisandrin were also effective, but 2 gingkolides (BN52020 and BN52021) only mildly inhibited H-3-PAF binding. Results of RT-PCR assay and western blot analysis of neutrophil crude membranes confirmed the presence of a PAF receptor.Conclusions and Clinical Relevance - Results indicated that bovine neutrophils express only 1 type of PAF receptor, and it is likely that this receptor is involved in inflammatory responses. The most effective PAF antagonists were 14-deoxyandrographolide and WEB 2086; these PAF antagonists may be potentially useful in the treatment of inflammatory processes in cattle.