Chau L Y, Peck K, Yen H H, Wang J Y
Division of Cardiovascular Research, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan, Republic of China.
Biochem J. 1994 Aug 1;301 ( Pt 3)(Pt 3):911-6. doi: 10.1042/bj3010911.
Prolonged exposure (8-24 h) of human promonocytic U937 cells to 100 nM 1-O-hexadecyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (carbarmyl-PAF), a non-metabolizable analogue of platelet-activating factor (PAF), reduced the numbers of PAF receptors by 50-75%, as determined by the radioligand-binding assay. To clarify whether the down-regulation of receptor numbers is due to decreased expression level of the PAF-receptor gene, the effect of carbamyl-PAF on the steady-state level of PAF-receptor mRNA was examined by a highly sensitive reverse-transcriptase PCR method. A 50% decline in the level of PAF-receptor mRNA was observed in U937 cells pretreated with 100 nM carbamyl-PAF for 24 h. The effect of carbamyl-PAF was dose-dependent, with an EC50 value around 10 nM. PAF-receptor antagonist, SRI-63675, was able to attenuate the effect of carbamyl-PAF. Furthermore lysoPAF, at 1 uM, was unable to induce a significant decrease in PAF-receptor mRNA after incubation for 24 h, indicating that the effect of carbamyl-PAF was specific. The half-life of the PAF-receptor mRNA measured in the presence of actinomycin D was unaffected by carbamyl-PAF treatment. In contrast, nuclear run-off experiments demonstrated that the transcription rate of the PAF-receptor gene in carbamyl-PAF-treated cells was about 65% of that in control cells. These results suggest that the PAF receptor in U937 cells is subject to down-regulation by agonist, at least partly, at the transcriptional level.
将人单核细胞白血病U937细胞长时间（8 - 24小时）暴露于100 nM 1 - O - 十六烷基 - 2 - N - 甲基氨基甲酰 - sn - 甘油 - 3 - 磷酸胆碱（氨基甲酰 - PAF），一种血小板活化因子（PAF）的不可代谢类似物，通过放射性配体结合试验测定，PAF受体数量减少了50 - 75%。为了阐明受体数量的下调是否是由于PAF受体基因表达水平降低所致，采用高灵敏度逆转录聚合酶链反应方法检测了氨基甲酰 - PAF对PAF受体mRNA稳态水平的影响。在用100 nM氨基甲酰 - PAF预处理24小时的U937细胞中，观察到PAF受体mRNA水平下降了50%。氨基甲酰 - PAF的作用呈剂量依赖性，EC50值约为10 nM。PAF受体拮抗剂SRI - 63675能够减弱氨基甲酰 - PAF的作用。此外，1 μM溶血PAF在孵育24小时后未能诱导PAF受体mRNA显著下降，表明氨基甲酰 - PAF的作用具有特异性。在放线菌素D存在下测量的PAF受体mRNA半衰期不受氨基甲酰 - PAF处理的影响。相反，核转录实验表明，氨基甲酰 - PAF处理细胞中PAF受体基因的转录速率约为对照细胞的65%。这些结果表明，U937细胞中的PAF受体至少部分地在转录水平上受到激动剂的下调作用。
