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

1. We have previously demonstrated that Ca 2+/calmodulin-dependent protein kinase (CaMK) mediates pyrimidinoceptor potentiation of LPS-elicited inducible nitric oxide synthase (iNOS) induction in murine J774 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-κB) 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-κB 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 Ca 2+-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.