Activation of the AMP-activated protein kinase (AMPK) in endothelial cells has been linked with anti-inflammatory effects but the events downstream of kinase activation are not well understood. We previously reported that nitric oxide (NO) is able to activate the AMPK in response to shear stress and as the shear stress- induced release of NO attenuates NFkappaB activation the aim of this study was to determine the relationship between AMPK and NFkappaB. In human umbilical vein and murine lung endothelial cells, the exogenous application of a NO donor (DETA-NO 100 mM, 30 minutes to 24 hours) resulted in the phosphorylation and activation of the AMPK and a significant attenuation of the TNFa-induced activation of NFkappaB (EMSA). The overexpression of a constitutively active AMPK mutant also attenuated the TNFa-induced activation of the transcription factor while a dominant negative mutant potentiated the response. Moreover the dominant negative AMPK prevented the NO-induced inhibition of NFkappaB. Consistent with these observations, the constitutively active AMPK mutant reduced the TNFa-induced expression of E-selectin expression and attenuated monocyte adhesion. In endothelial cells isolated from AMPKa2-/- mice the IL-1b- induced expression of E-selectin and VCAM-1 were significantly increased compared to wild-type endothelial cells. The acute activation of AMPK by pentobarbital resulted in a significant decrease in the TNFa-induced phosphorylation of the p65 subunit of NFkappaB as well as the inhibition of IkappaB phosphorylation. Moreover, the downregulation of the AMPK in human endothelial cells (siRNA) resulted in an increase in the TNFa-stimulated phosphorylation of the p65. Taken together, these results indicate that the anti-inflammatory effects associated with AMPK activation can be at least partially attributed to the inhibition of NFkappaB activation and that the activation of the AMPK by NO can explain its effects on E- selectin and VCAM-1 expression.