Anandamide Inhibits Nuclear Factor-κB Activation through a Cannabinoid Receptor-Independent Pathway

Abstract

Anandamide (arachidonoylethanolamine, AEA), an endogenous agonist for both the cannabinoid CB 1 receptor and the vanilloid VR1 receptor, elicits neurobehavioral, anti-inflammatory, immunomodulatory, and proapoptotic effects. Because of the central role of nuclear factor-B (NF-B) in the inflammatory process and the immune response, we postulated that AEA might owe some of its effects to the suppression of NF-B. This study shows that AEA inhibits tumor necrosis factor-␣ (TNF␣)-induced NF-B activation by direct inhibition of the IB kinase (IKK)␤ and, to a lesser extent, the IKK␣ subunits of B inhibitor (IB) kinase complex, and that IKKs inhibition by AEA correlates with inhibition of IB␣ degradation, NF-B binding to DNA, and NF-B-dependent transcription in TNF␣-stimulated cells. AEA also prevents NF-B-dependent reporter gene expression induced by mitogen-activated protein kinase kinase kinase and NF-B-inducing kinase. The NF-B inhibitory activity of AEA was independent of CB 1 and CB 2 activation in TNF␣-stimulated 5.1 and A549 cell lines, which do not express vanilloid receptor 1, and was not mediated by hydrolytic products formed through the activity of the enzyme fatty acid amide hydrolase. Chemical modification markedly affected AEA inhibitory activity on NF-B, suggesting rather narrow structure-activity relationships and the specific interaction with a molecular target. Substitution of the alkyl moiety with less saturated fatty acids generally reduced or abolished activity. However, replacement of the ethanolamine "head" with a vanillyl group led to potent inhibition of TNF␣-induced NF-B-dependent transcription. These findings provide new mechanistic insights into the antiinflammatory and proapoptotic activities of AEA, and should foster the synthesis of improved analogs amenable to pharmaceutical development as anti-inflammatory agents.Endocannabinoids are a class of lipid mediators found in several tissues and structurally based on a polyunsaturated fatty acid amide or ester motifs . Anandamide (arachidonoylethanolamide, AEA) and 2-arachidonoylglicerol (2-AG) are the main endocannabinoids described to date. They act as mediators in the brain and in peripheral tissues mainly through the stimulation of brain (CB 1 ) and peripheral (CB 2 ) cannabinoid receptors. Although AEA preferentially binds to CB 1 , 2-AG is equipotent at both receptor subtypes. AEA is produced by neurons and other cell types from the hydrolysis of the phospholipid precursor N-arachydonoylphosphatidylethanolamide, catalyzed by a Ca 2ϩ

Cannabinoid Receptor Type 1 Protects Nigrostriatal Dopaminergic Neurons against MPTP Neurotoxicity by Inhibiting Microglial Activation

“…The CB 1 receptor regulates motor behavior in the basal ganglia via mediating both excitatory and inhibitory inputs to the SN reticulate and globus pallidus (53) as well as short-and long-term synaptic plasticity through suppressing the release of neurotransmitters, such as glutamate and GABA (54,55). Additionally, cannabinoids modulate inflammatory responses by regulating microglial function through both receptor-dependent and -independent mechanisms (39,(56)(57)(58).…”

Nitroglycerin increases serotonin transporter expression in rat spinal cord but anandamide modulated this effect

“…Literature data show that AEA regulates the expression of several genes and some of its effect is receptor (CB1) dependent, but receptor independent actions have also been reported (Correa et al, 2008;Mestre et al, 2011;Sancho et al, 2003). One can not exclude the possibility of some indirect action, because it is known that endocannabinoids are able to raise NO production by upregulating the nNOS activity (Carney et al, 2009).…”

The endocannabinoid system in pain and inflammation: Its relevance to rheumatic disease

“…Together, this helps slow the perpetuation of disease and alleviate associated arthritic pain primarily derived from localized inflammation (36). Further to this, elevated levels AEA and 2-AG are detected in the synovial fluid of RA and OA patients, but absent in healthy controls, suggest that local endocannabinoid secretion may assist in minimising inflammation in the arthritic joints (4,60). With both cannabinoid receptors and endogenous ligands present in inflamed human joints, targeting this system may hold therapeutic promise for both inflammatory, as well as degenerative arthritis (60).…”

“…Further to this, elevated levels AEA and 2-AG are detected in the synovial fluid of RA and OA patients, but absent in healthy controls, suggest that local endocannabinoid secretion may assist in minimising inflammation in the arthritic joints (4,60). With both cannabinoid receptors and endogenous ligands present in inflamed human joints, targeting this system may hold therapeutic promise for both inflammatory, as well as degenerative arthritis (60). Administration of cannabinoid agonists, WIN55212 and CP55940, have shown the ability to reduce inflammatory IL-6 and interleukin-8 (IL-8) cytokine production by fibroblast like synoviocytes cells, ameliorating acute inflammation and associated pain in arthritic joints (2,63).…”

“…In arthritis, persistent inflammation results in the infiltration of immune cells and the subsequent development of hypersensitivity. Synovial serum samples from patients with RA consistently express elevated cytokine levels such as TNF-α, interleukin-6 (IL-6), and IL-1β, which act directly to sensitize joint nociceptors and stimulate the release of prostaglandins (59,60). In an elegant study by Sancho et al it …”