Attenuation of inducible nitric oxide synthase gene expression by delta 9-tetrahydrocannabinol is mediated through the inhibition of nuclear factor- kappa B/Rel activation.

delta 9-Tetrahydrocannabinol (delta 9-THC) a prototypic compound belonging to the family of agents known as cannabinoids, produces a wide variety of biological effects, including inhibition of immune function. The putative mechanism for cannabinoid biological action involves binding to cannabinoid receptor types 1 and 2 (CB1 and CB2) to negatively regulate adenylate cyclase and inhibit intracellular signaling via the cAMP cascade. In the current study, we show that delta 9-THC produces a marked inhibition of inducible nitric oxide synthase (iNOS) transcription and nitric oxide production by the macrophage line RAW 264.7 in response to lipopolysaccharide (LPS). Analysis of RAW 264.7 cell RNA demonstrated transcripts for CB2 but not CB1. Treatment of RAW 264.7 with delta 9-THC inhibited forskolin-stimulated cAMP production in a dose-related manner, verifying the expression of functional cannabinoid receptors by this cell line. iNOS transcription, which is regulated in part by the nuclear factor-kappa B/Rel (NF-kappa B/Rel) family of transcription factors, has been shown to be under the control of the cAMP signaling cascade. We demonstrate that delta 9-THC inhibits the activation and binding of NF-kappa B/Rel proteins to their cognate DNA site, kappa B, in response to LPS stimulation. LPS treatment of RAW 264.7 cells also induced the activation of the cAMP cascade, as indicated by an increase in binding of nuclear factors to the cAMP response element. Activation of CRE binding proteins was inhibited by delta 9-THC. Forskolin treatment of RAW 264.7 cells induced both kappa B and cAMP response element binding activity and was likewise inhibited by delta 9-THC. Collectively, this series of experiments indicates that NF-kappa B/Rel is positively regulated by the cAMP cascade to help initiate iNOS gene expression in response to LPS stimulation of macrophages. This activation of iNOS is attenuated by delta 9-THC through the inhibition of cAMP signaling.