| Abstract |
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.
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