Inhibition of P2Y6 receptor-mediated phospholipase C activation and Ca2+ signalling by prostaglandin E2 in J774 murine macrophages

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 Ca2+ concentration ([Ca2+-]i) stimulated by P2X(4), P2Y(2), and P2Y(6) receptors in 1774 murine macrophages. In this study, we examined the mechanism of PGE2 inhibitory effects on P2Y(6) receptor-mediated function in 1774 cells. The P2Y(6) receptor agonist UDP induced a sustained elevation of [Ca2+-]i by stimulating the phospholipase C (PLC) signalling pathway. PGE(2) inhibited [Ca2+-]i elevation and phosphatidylinositol (Pl) hydrolysis in a concentration-dependent manner.1774 cells highly expressed the E-type prostanoid 2 (EP2) receptor subtype, a Gscoupled receptor. PGE(2) and a selective EP2 receptor agonist caused cyclic AMP (cAMP) accumulation in 1774 cells. The inhibitory effects of PGE(2) on P2Y(6) receptor-mediated responses were mimicked by the selective EP2 receptor agonist. Although EP2 receptor is linked to adenylyl cyclase activation, PGE(2)-induced inhibition of Ca2+ response and Pl hydrolysis could not be mimicked by a lipophilic cAMP derivative, clibutyryl cAMP, or an adenylyl cyclase activator, forskolin. The inhibition of UDP-induced PLC activation by PGE(2) was not affected by down-regulation of protein kinase C by phorbol-12-myristate-13-acetate treatment. PGE(2) inhibited PLC activation induced by aluminium fluoride, but not by the Ca2+-ionophore, ionomycin. Finally, the inhibition of UDP-induced PLC activation by PGE(2) was impaired by Gs knockdown usi(n)g siRNA. These results suggest that EP2 receptor activation in macrophages negatively controls the G(q/11)-PLC signalling through a Gs-mediated, but cAMP-independent signalling mechanism. (C) 2015 Elsevier B.V. All rights reserved,