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) Mel. Pharmacol. 55, 481-488). In this study, the role of the cAMP-dependent protein kinase A (PKA) pathway was investigated. The PEA inhibitors, KT-5720 and H8, reduced LPS-induced NO release and inducible nitric oxide synthase (iNOS) expression. The direct PHA 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-kappa B (NF-kappa B) activation in the nuclei, as shown by detection of NF-kappa B-specific DNA-protein binding The PKA inhibitor, H8, inhibited the NF-kappa B activation induced by 6- or 12-h treatment with LPS but not that induced after 1, 3, or 24 h. The cyclooxygenase-a (COX-2) inhibitors, NS-398 and indomethacin, attenuated LPS-induced NO release, iNOS expression, and NF-kappa B 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-kappa B activation, iNOS expression, and NO production.