First 11 pages of thesis. - OPUS - Universität Würzburg

vessels with the iNOS specific inhibitor 1400W significantly reduced superoxide
levels, indicating that iNOS itself contributes to superoxide production in
atherosclerosis. To the best of our knowledge this is the first evidence
supporting superoxide generation by iNOS in atherosclerosis. In contrast, ample
evidence supports eNOS “uncoupling” and subsequent superoxide, instead of
nitric oxide production 151, 206 . The mechanism of superoxide generation by iNOS
differs from eNOS and nNOS. The latter two enzymes produce superoxide from
the heme centers of their oxygenase domains 35, 117 , while iNOS produces
superoxide from the reductase domain 136 . While 100 µM of L-arginine was
sufficient to block superoxide generation from nNOS 117 , there was no effect of
100 µM L-arginine on iNOS mediated superoxide generation 136 . Higher
concentrations of L-arginine (1mM–5mM) only reduced, but did not abolish
superoxide production, indicating that superoxide generation by iNOS may even
occur when substrate is not limited 136 . Xia et al. reported that induction of iNOS
in macrophages reduced L-arginine concentrations, indicating high substrate
demand 37 . These authors hypothesized that iNOS may catalyze nitric oxide
from the oxygenase- and superoxide from the reductase domain
simultaneously, which could directly lead to peroxynitrite formation.
Quantification of tissue 3-nitrotyrosine, considered a footprint of
peroxynitrite mediated protein damage showed reduced levels of 3-nitrotyrosine
in apoE/iNOS dko vessels, compared to apoE ko vessels. Peroxynitrite oxidizes
BH4, a cofactor of NOS 207 which in its oxidized form causes uncoupling of the
constitutive NOS enzymes 103 . Further, peroxynitrite can directly cause
irreversible uncoupling and inactivation of nNOS 208 . iNOS mediated
peroxynitrite formation and its high substrate demand may thus cause
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