Ito Masaaki, Matsuoka Isao
Laboratory of Pharmacology, Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki-shi, Gunma 370-0033, Japan.
Eur J Pharmacol. 2015 Feb 15;749:124-32. doi: 10.1016/j.ejphar.2014.12.024. Epub 2015 Jan 19.
Extracellular nucleotides act as inflammatory mediators through activation of multiple purinoceptors. Under inflammatory conditions, the purinergic signalling is affected by various inflammatory mediators. We previously showed that prostaglandin (PG) E2 suppressed the elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) stimulated by P2X4, P2Y2, and P2Y6 receptors in J774 murine macrophages. In this study, we examined the mechanism of PGE2 inhibitory effects on P2Y6 receptor-mediated function in J774 cells. The P2Y6 receptor agonist UDP induced a sustained elevation of [Ca(2+)]i by stimulating the phospholipase C (PLC) signalling pathway. PGE2 inhibited [Ca(2+)]i elevation and phosphatidylinositol (PI) hydrolysis in a concentration-dependent manner. J774 cells highly expressed the E-type prostanoid 2 (EP2) receptor subtype, a Gs-coupled receptor. PGE2 and a selective EP2 receptor agonist caused cyclic AMP (cAMP) accumulation in J774 cells. The inhibitory effects of PGE2 on P2Y6 receptor-mediated responses were mimicked by the selective EP2 receptor agonist. Although EP2 receptor is linked to adenylyl cyclase activation, PGE2-induced inhibition of Ca(2+) response and PI hydrolysis could not be mimicked by a lipophilic cAMP derivative, dibutyryl cAMP, or an adenylyl cyclase activator, forskolin. The inhibition of UDP-induced PLC activation by PGE2 was not affected by down-regulation of protein kinase C by phorbol-12-myristate-13-acetate treatment. PGE2 inhibited PLC activation induced by aluminium fluoride, but not by the Ca(2+)-ionophore, ionomycin. Finally, the inhibition of UDP-induced PLC activation by PGE2 was impaired by Gs knockdown using siRNA. These results suggest that EP2 receptor activation in macrophages negatively controls the Gq/11-PLC signalling through a Gs-mediated, but cAMP-independent signalling mechanism.
细胞外核苷酸通过激活多种嘌呤受体作为炎症介质。在炎症条件下，嘌呤能信号传导受到多种炎症介质的影响。我们之前表明，前列腺素（PG）E2抑制J774鼠巨噬细胞中由P2X4、P2Y2和P2Y6受体刺激引起的细胞内Ca(2+)浓度（[Ca(2+)]i）升高。在本研究中，我们研究了PGE2对J774细胞中P2Y6受体介导功能的抑制作用机制。P2Y6受体激动剂UDP通过刺激磷脂酶C（PLC）信号通路诱导[Ca(2+)]i持续升高。PGE2以浓度依赖的方式抑制[Ca(2+)]i升高和磷脂酰肌醇（PI）水解。J774细胞高表达E型前列腺素2（EP2）受体亚型，一种与Gs偶联的受体。PGE2和选择性EP2受体激动剂导致J774细胞中环状AMP（cAMP）积累。选择性EP2受体激动剂模拟了PGE2对P2Y6受体介导反应的抑制作用。尽管EP2受体与腺苷酸环化酶激活有关，但PGE2诱导的对Ca(2+)反应和PI水解的抑制作用不能被亲脂性cAMP衍生物二丁酰cAMP或腺苷酸环化酶激活剂福斯可林模拟。PGE2对UDP诱导的PLC激活的抑制作用不受佛波醇-12-肉豆蔻酸酯-13-乙酸酯处理下调蛋白激酶C的影响。PGE2抑制氟化铝诱导的PLC激活，但不抑制Ca(2+)离子载体离子霉素诱导的激活。最后，使用小干扰RNA敲低Gs会损害PGE2对UDP诱导的PLC激活的抑制作用。这些结果表明，巨噬细胞中EP2受体的激活通过Gs介导但不依赖cAMP的信号机制对Gq/11-PLC信号传导进行负调控。
