EET displays anti-inflammatory effects in TNF-α-stimulated human bronchi

The aim of the present study was to investigate the anti-inflammatory effects of 14,15-epoxyeicosatrienoic acid (EET) on reactivity and Ca2+ sensitivity in TNF-alpha-stimulated human bronchi. Tension measurements performed on either control, TNF-alpha-, or TNF-alpha + EET-pretreated bronchi revealed that 100 nM 14,15-EET pretreatments significantly reduced the reactivity of TNF-alpha-p retreated tissues to contractile agonists. EET also normalized the relaxing response to isoproterenol in TNF-alpha-treated bronchi. Pretreatment with 100 nM 14,15-EET prevented TNF-alpha-induced I kappa B alpha degradation, as demonstrated by an increase in I kappa B alpha protein levels on Western blot analysis. The anti-inflammatory properties of EET were mediated by the inhibition of I kappa B alpha degradation, suggesting a lower activation of NF-kappa B. The Ca2+ sensitivity of TNF-alpha-stimulated bronchi was also evaluated on beta-escin-permeabilized preparations. Observed mean responses demonstrated that EET pretreatments abolished Ca2+ hypersensitivity developed by TNF-alpha-stimulated bronchial explants. Moreover, 14,15-EET significantly reduced PDBu-induced Ca2+ sensitivity in TNF-alpha-stimulated bronchi. Western blot and RT-PCR analyses revealed that CPI-17 protein and transcript levels were increased in TNF-alpha-treated bronchi, as opposed to being decreased in the presence of 14,15-EET. This eicosanoid also reduced U-46619-induced Ca2+ sensitivity, which is related to the activation of Rhokinase pathway. These results were also correlated with an increase in protein staining and transcription level of p116(Rip), a RhoA inhibitory-binding protein. Altogether, these data demonstrate that 14,15-EET is a potent modulator of the hyperreactivity triggered by TNF-alpha in human airway smooth muscle cells.