Pyrimidinoceptor potentiation of macrophage PGE(2) release involved in the induction of nitric oxide synthase.
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 cyclo-oxygenase (COX)-dependent prostaglandin E(2) (PGE(2)) formation in this event. In J774 macrophages predominantly expressing P2Y(6) receptors, the simultaneous addition of UTP and lipopolysaccharide (LPS) resulted in potentiated increase in PGE(2) release. 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-kappaB) or COX-2. 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). 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-kappaB activation and potentiated nitrite accumulation in response to LPS. 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. 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.
Chen
B C
BC
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Lin
W W
WW
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D051546:Cyclooxygenase 2; D015232:Dinoprostone / Q000378:metabolism* / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D054501:Group II Phospholipases A2; D007211:Indoles / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D010716:Phosphatidylinositols / Q000378:metabolism; D010741:Phospholipases A / Q000235:genetics; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011759:Pyrrolidines / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000235:genetics; D058473:Receptors, Purinergic P2Y2; D013859:Thiocarbamates / Q000494:pharmacology; D014316:Tritium; D014544:Uridine Triphosphate / Q000494:pharmacology
D007211:Indoles; D007527:Isoenzymes; D008070:Lipopolysaccharides; D016328:NF-kappa B; C109734:P2RY11 protein, human; C546385:P2RY2 protein, human; D010716:Phosphatidylinositols; D011759:Pyrrolidines; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; D013859:Thiocarbamates; C099845:purinoceptor P2Y6; C074644:uridine triphosphate receptors; D014316:Tritium; C020972:pyrrolidine dithiocarbamic acid; D016718:Arachidonic Acid; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D011493:Protein Kinase C; D010741:Phospholipases A; D054501:Group II Phospholipases A2; D015232:Dinoprostone; D002118:Calcium; D014544:Uridine Triphosphate; C064758:Ro 31-8220