Scrivens Michelle, Dickenson John M
School of Biomedical and Natural Sciences, Nottingham Trent University, Clifton Lane, Nottingham, NG11 8NS, UK.
Eur J Pharmacol. 2006 Aug 14;543(1-3):166-73. doi: 10.1016/j.ejphar.2006.05.037. Epub 2006 Jun 7.
Previous studies using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis have shown that the P2Y(14) receptor is expressed at high levels in human neutrophils. Therefore the primary aim of this study was to determine whether the P2Y(14) receptor is functionally expressed in human neutrophils. In agreement with previous studies RT-PCR analysis detected the expression of P2Y(14) receptor mRNA in human neutrophils. UDP-glucose (IC(50)=1 microM) induced a small but significant inhibition (circa 30%) of forskolin-stimulated cAMP accumulation suggesting functional coupling of endogenously expressed P2Y(14) receptors to the inhibition of adenylyl cyclase activity in human neutrophils. In contrast, the other putative P2Y(14) receptor agonists UDP-galactose and UDP-glucuronic acid (at concentrations up to 100 microM) had no significant effect, whereas 100 microM UDP-N-acetylglucosamine-induced a small but significant inhibition of forskolin-stimulated cAMP accumulation (20% inhibition). UDP-galactose, UDP-glucuronic acid and UDP-N-acetylglucosamine behaved as partial agonists by blocking UDP-glucose mediated inhibition of forskolin-induced cAMP accumulation. Treatment of neutrophils with pertussis toxin (G(i/o) blocker) abolished the inhibitory effects of UDP-glucose on forskolin-stimulated cAMP accumulation. UDP-glucose (100 microM) also induced a modest increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, whereas the other sugar nucleotides had no effect on ERK1/2 activation. Finally, UDP-glucose and related sugar nucleotides had no significant effect on N-formyl-methionyl-leucyl-phenylalanine-induced elastase release from neutrophils. In summary, although we have shown that the P2Y(14) receptor is functionally expressed in human neutrophils (coupling to inhibition of forskolin-induced cAMP and ERK1/2 activation) it does not modulate neutrophil degranulation (assessed by monitoring elastase release). Clearly further studies are required in order to establish the functional role of the P2Y(14) receptor expressed in human neutrophils.
此前使用定量逆转录聚合酶链反应（RT-PCR）分析的研究表明，P2Y(14)受体在人中性粒细胞中高水平表达。因此，本研究的主要目的是确定P2Y(14)受体在人中性粒细胞中是否有功能表达。与先前的研究一致，RT-PCR分析检测到人中性粒细胞中P2Y(14)受体mRNA的表达。UDP-葡萄糖（IC(50)=1微摩尔）对福斯可林刺激的环磷酸腺苷（cAMP）积累有轻微但显著的抑制作用（约30%），提示内源性表达的P2Y(14)受体与抑制人中性粒细胞中的腺苷酸环化酶活性存在功能偶联。相比之下，其他假定的P2Y(14)受体激动剂UDP-半乳糖和UDP-葡萄糖醛酸（浓度高达100微摩尔）无显著作用，而100微摩尔UDP-N-乙酰葡糖胺对福斯可林刺激的cAMP积累有轻微但显著的抑制作用（20%抑制）。UDP-半乳糖、UDP-葡萄糖醛酸和UDP-N-乙酰葡糖胺通过阻断UDP-葡萄糖介导的对福斯可林诱导的cAMP积累的抑制作用而表现为部分激动剂。用百日咳毒素（G(i/o)阻断剂）处理中性粒细胞消除了UDP-葡萄糖对福斯可林刺激的cAMP积累的抑制作用。UDP-葡萄糖（100微摩尔）也使细胞外信号调节激酶1/2（ERK1/2）磷酸化适度增加，而其他糖核苷酸对ERK1/2激活无作用。最后，UDP-葡萄糖和相关糖核苷酸对N-甲酰甲硫氨酰-亮氨酰-苯丙氨酸诱导的中性粒细胞弹性蛋白酶释放无显著作用。总之，虽然我们已表明P2Y(14)受体在人中性粒细胞中有功能表达（与抑制福斯可林诱导的cAMP和ERK1/2激活偶联），但它不调节中性粒细胞脱颗粒（通过监测弹性蛋白酶释放评估）。显然，为确定人中性粒细胞中表达的P2Y(14)受体的功能作用，还需要进一步研究。
