Nitric oxide and its metabolites mediate ethanol-induced microtubule disruption and intestinal barrier dysfunction

Loss of gastrointestinal (GI) barrier integrity has been implicated in a wi de range of inflammatory illnesses, including alcoholic cirrhosis. Using mo nolayers of Caco-2 (intestinal) cells as a model, we showed that the abilit y of ethanol (EtOH) to disrupt intestinal barrier integrity depends on dama ge to the microtubule (MT) cytoskeleton, especially oxidative injury. One d rug that prevented both the MT damage and barrier disruption was L-N-6-1-im inoethyl-lysine, a selective inhibitor of the inducible form of nitric-oxid e synthase (iNOS). Because of this finding acid because overproduction of n itric oxide (NO) and generation of peroxynitrite (ONOO-) have been proposed to be responsible for mucosal injury in other GI disorders, we sought to d etermine whether NO overproduction and ONOO- formation mediates EtOH-induce d MT damage and loss of intestinal barrier function, To this end, Caco-2 mo nolayers were exposed to EtOH or to authentic ONOO- or ONOO- generators wit h or without pretreatment with iNOS inhibitors or antioxidants. We found th at EtOH caused 1) iNOS activation, 2) NO overproduction, 3) increases in ox idative stress and superoxide anion production (superoxide dismutase quench able fluorescence of dichlorofluorescein), 4) nitration and oxidation of tu bulin (immunoblotting), 5) decreased levels of stable polymerized tubulin, and 6) increased levels of disassembled tubulin. EtOH also 7) extensively d amaged the MT cytoskeleton and 8) disrupted barrier function. Authentic ONO O- or ONOO- donors had similar effects. Pretreatment with a selective iNOS inhibitor, L-N-6-1-iminoethyl-lysine, or with antioxidants (ONOO- scavenger s urate or L-cysteine; superoxide anion scavenger superoxide dismutase) att enuated damage due to EtOH or to ONOO- generators. We conclude that EtOH-in duced MT damage and intestinal barrier dysfunction require iNOS activation followed by NO overproduction and ONOO- formation. These findings provide a rationale for the development of novel therapeutic agents for alcohol-indu ced GI disorders that inhibit this mechanism.