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Vol. 294, Issue 3, 997-1008, September 2000
Departments of Internal Medicine (Division of Digestive Diseases),
Pharmacology, and Molecular Biophysics and Physiology, Rush University
Medical Center, Chicago, Illinois
Loss of gastrointestinal (GI) barrier integrity has been implicated in
a wide range of inflammatory illnesses, including alcoholic cirrhosis.
Using monolayers of Caco-2 (intestinal) cells as a model, we showed
that the ability of ethanol (EtOH) to disrupt intestinal barrier
integrity depends on damage to the microtubule (MT) cytoskeleton,
especially oxidative injury. One drug that prevented both the MT damage
and barrier disruption was
L-N6-1-iminoethyl-lysine, a
selective inhibitor of the inducible form of nitric-oxide synthase
(iNOS). Because of this finding and because overproduction of nitric
oxide (NO) and generation of peroxynitrite (ONOO
) have
been proposed to be responsible for mucosal injury in other GI
disorders, we sought to determine whether NO overproduction and
ONOO formation mediates EtOH-induced MT damage and loss
of intestinal barrier function. To this end, Caco-2 monolayers were
exposed to EtOH or to authentic ONOO or
ONOO generators with or without pretreatment with iNOS
inhibitors or antioxidants. We found that EtOH caused 1) iNOS
activation, 2) NO overproduction, 3) increases in oxidative stress and
superoxide anion production (superoxide dismutase quenchable
fluorescence of dichlorofluorescein), 4) nitration and oxidation of
tubulin (immunoblotting), 5) decreased levels of stable polymerized
tubulin, and 6) increased levels of disassembled tubulin. EtOH also 7) extensively damaged the MT cytoskeleton and 8) disrupted barrier function. Authentic ONOO or ONOO donors had
similar effects. Pretreatment with a selective iNOS inhibitor,
L-N6-1-iminoethyl-lysine, or
with antioxidants (ONOO scavengers urate or
L-cysteine; superoxide anion scavenger superoxide dismutase) attenuated damage due to EtOH or to ONOO
generators. We conclude that EtOH-induced 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-induced GI disorders that inhibit this mechanism.
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