Pyrimidinoceptor potentiation of macrophage PGE2 release involved in the induction of nitric oxide synthase

1 We have previously demonstrated that Ca2+/calmodulin-dependent protein kinase (CaMK) mediates pyrimidinoceptor potentiation of LPS-elicited inducible nitric oxide synthase (iNOS) induction in murine 5774 macrophages. In the present paper, we have explored the role of cyclooxygenase (COX)-dependent prostaglandin E-2 (PGE(2)) formation in this event. 2 In J774 macrophages predominantly expressing P2Y(6) receptors, the simultaneous addition of UTP and lipopolysaccharide (LPS) resulted in potentiated increase in PGE(2) release. 3 UTP-induced increased PGE(2) release was demonstrated by a concomitant increase in COX-2 protein expression, and was decreased by inhibitors specific for phosphatidylinositide-phospholipase C (PI-PLC), CaMK, protein kinase C (PKC), nuclear factor-kappa B (NF-kappa B) or COX-2. 4 NS-398 (a selective COX-2 inhibitor) reduced LPS plus UTP-elicited iNOS induction and nitrite accumulation, supporting for the positive regulation of iNOS gene expression by endogenous PGE(2). 5 Moreover, the cyclic AMP/PKA-dependent up-regulation of iNOS expression mediated by PGE(2) was drawn from the inhibitory effects of 2',5'-dideoxyadenosine, KT5720 and H-89. Exogenous PGE(2) induced NF-kappa B activation and potentiated nitrite accumulation in response to LPS. 6 In addition to COX-2 induction, arachidonic acid (AA) release and steady-state mRNA levels of type V secretory phospholipase A(2) (sPLA(2)) and Ca2+-independent PLA(2) (iPLA(2)) were also increased in the presence of LPS and UTP; the LPS-induced increase in iPLA(2) activity was also potentiated by UTP. 7 Taken together, we conclude that UTP-mediated COX-2 and iPLA(2) potentiation and PGE(2) formation contribute to the iNOS induction, and that CaMK activation is the primary step in the UTP enhancement of COX-2 induction.