Regulation of the soluble epoxide hydrolase by peroxynitrite

Abstract

Inhibition of the soluble epoxide hydrolase (sEH), which for example converts epoxyeicosatrienoic acids to dihydroxyeicosatrienoic acids (DHETs), has anti‐inflammatory as well as anti‐hypertensive effects. As the enzymatic core of the sEH hydrolase domain contains two tyrosine residues (Tyr383 & 466), we addressed the hypothesis that the activity of the sEH may be affected by oxidative stress, in particular by nitration of Tyr383 & 466.sEH activity was measured in human and murine endothelial cells by LC‐MS/MS and was sensitive to the specific sEH inhibitor 1‐adamantyl‐3‐cyclohexylurea. The importance of the tyrosine residues was demonstrated in that mutation of Tyr383 & 466 to phenylalanine resulted in a complete loss of DHET production. sEH activity in both cell types was significantly (80±6%, P<0.001) attenuated by preincubation with the peroxynitrite (ONOO−) generator 3‐morpholino‐sydnonimine (SIN‐1, 0.5 mmol/L). The latter compound and authentic ONOO− also elicited the tyrosine nitration of the sEH (immunoprecipitation and Western blotting) an effect that was not be triggered by hydrogen peroxide (1 mmol/L). Moreover, in an in vivo murine model, oxidative stress induced by bacterial lipopolysaccharide significantly (20±9%, P<0.01) attenuated lung sEH activity. These data indicate that the activity of the sEH and thus cellular EET levels can be regulated by the tyrosine nitration of the protein.