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William J. O’Brien, Tom Heimann, Li-Shih Tsao, Bruce T. Seet, Grant McFadden, Jerry L. Taylor; Regulation of Nitric Oxide Synthase 2 in Rabbit Corneal Cells. Invest. Ophthalmol. Vis. Sci. 2001;42(3):713-719.
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purpose. The purpose of these studies was to investigate the role of interferon-γ
(IFN-γ), tumor necrosis factor-α (TNF-α), interleukin 1β
(IL-1β), and transforming growth factor-β (TGF-β) in the
regulation of inducible nitric oxide synthase (NOS2) activity in rabbit
methods. Rabbit corneal epithelial, stromal, and endothelial cells were grown in
culture and treated with cytokines and growth factors, alone or in
combination. NOS activity was measured at times up to 72 hours after
treatment by assaying the culture medium for nitrite using the Griess
reaction. Cell lysates were analyzed by Western blot analysis for NOS2
protein. RNA was isolated and amplified with NOS1-, NOS2-, and
NOS3-specific primers by RT-PCR.
results. NOS2 expression was induced by combined cytokine treatment from
nondetectable levels to abundant levels in low passage (<4) stromal
cells and to low levels in corneal endothelial cells but not in corneal
epithelial cells. In the absence of IFN-γ, little or no nitrite
accumulation was induced by TNF-α, IL-1β, or lipopolysaccharide
(LPS) treatment. The inductive effects of IFN-γ were antagonized in a
dose-dependent manner by the myxoma virus rabbit IFN-γ receptor
homolog, M-T7. rRaIFN-γ, in combination with IL-1β and TNF-α,
induced the appearance of NOS2 mRNA within 24 hours but detectable
nitrite did not accumulate in large amounts (>10 μM) until after 24
hours postinduction. NOS2 was identified as a 130 kDa protein on
Western blot analysis using monoclonal antibody against murine NOS2.
TGF-β1 and β2 inhibited the accumulation of
cytokine-induced nitrite in a dose-dependent manner while not
significantly reducing the steady state level of NOS2 mRNA. The
activity of the induced NOS was inhibited by 1400W, a NOS2-selective
inhibitor, but not 7-nitroindazole, a NOS1-selective inhibitor.
conclusions. In cultured corneal stromal cells, NOS2 expression was upregulated by
IFN-γ in combination with IL-1β and TNF-α but not by any of these
cytokines alone, while TGF-β downregulated the activity. Cultures of
corneal epithelial cells could not be induced to express NOS2, yet
cultures of endothelial cells produced low amounts of NO in response to
cytokines. The NOS1 and NOS3 isoforms were not detected in any of these
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