Role of the cyclic AMP-protein kinase A pathway in lipopolysaccharide-induced nitric oxide synthase expression in RAW 264.7 macrophages. Involvement of cyclooxygenase-2.
The signaling pathway for lipopolysaccharide (LPS)-induced nitric oxide (NO) release in RAW 264.7 macrophages involves the protein kinase C and p38 activation pathways (Chen, C. C., Wang, J. K., and Lin, S. B. (1998) J. Immunol. 161, 6206-6214; Chen, C. C., and Wang, J. K. (1999) Mol. Pharmacol. 55, 481-488). In this study, the role of the cAMP-dependent protein kinase A (PKA) pathway was investigated. The PKA inhibitors, KT-5720 and H8, reduced LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression. The direct PKA activator, Bt(2)cAMP, caused concentration-dependent NO release and iNOS expression, as confirmed by immunofluorescence studies. The intracellular cAMP concentration did not increase until after 6 h of LPS treatment. Two cAMP-elevating agents, forskolin and cholera toxin, potentiated the LPS-induced NO release and iNOS expression. Stimulation of cells with LPS or Bt(2)cAMP for periods of 10 min to 24 h caused nuclear factor-kappaB (NF-kappaB) activation in the nuclei, as shown by detection of NF-kappaB-specific DNA-protein binding. The PKA inhibitor, H8, inhibited the NF-kappaB activation induced by 6- or 12-h treatment with LPS but not that induced after 1, 3, or 24 h. The cyclooxygenase-2 (COX-2) inhibitors, NS-398 and indomethacin, attenuated LPS-induced NO release, iNOS expression, and NF-kappaB DNA-protein complex formation. LPS induced COX-2 expression in a time-dependent manner, and prostaglandin E(2) production was induced in parallel. These results suggest that 6 h of treatment with LPS increases intracellular cAMP levels via COX-2 induction and prostaglandin E(2) production, resulting in PKA activation, NF-kappaB activation, iNOS expression, and NO production.
Chen
C C
CC
Department of Pharmacology, College of Medicine, National Taiwan University, Taipei 10018, Taiwan.
Chiu
K T
KT
Sun
Y T
YT
Chen
W C
WC
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002451:Cell Compartmentation; D002460:Cell Line; D002772:Cholera Toxin / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051546:Cyclooxygenase 2; D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000276:immunology*; D008264:Macrophages / Q000201:enzymology*; D051379:Mice; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D052247:Nitric Oxide Synthase Type II; D009573:Nitrites / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D015398:Signal Transduction
D052246:Cyclooxygenase 2 Inhibitors; D016861:Cyclooxygenase Inhibitors; D007527:Isoenzymes; D007546:Isoquinolines; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009573:Nitrites; D005576:Colforsin; D003994:Bucladesine; C053177:N-(2-(methylamino)ethyl)-5-isoquinolinesulfonamide; D002772:Cholera Toxin; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse; D051546:Cyclooxygenase 2; D011451:Prostaglandin-Endoperoxide Synthases; D054746:Cyclic AMP-Dependent Protein Kinase Type II; D017868:Cyclic AMP-Dependent Protein Kinases