Expression of Inducible Nitric Oxide Synthase and Cationic Amino Acid Transporter in Endotoxin-Induced Uveitis
Date Issued
2012
Date
2012
Author(s)
Lee, Yi-An
Abstract
Purpose：To examine the expression of cationic amino acid transporter (CAT) and inducible nitric oxide synthase (iNOS) in endotoxin-induced uveitis (EIU).
Methods：EIU was induced by a footpad injection of 200 μg LPS in Lewis rats. Proteasome inhibitor bortezomib (valcade) was injected intraperitoneally 30 minutes before the LPS administration. The rats were sacrificed 24 hours later and the eyes were enucleated. The expression of iNOS and CAT mRNA and protein in the iris and ciliary body was determined by RT-PCR and Western blotting. The production of NO in aqueous was measured by ELISA. Immunochemical staining of the iris and ciliary body was performed to evaluate the inhibitory effect of valcade. The activity of NF-κB was assessed by electrophoretic mobility shift assay (EMSA). In addition to animal model, we also used RAW264.7 cells as an animal model to investigate our hypothesis.
Results： Proteasome inhibitor valcade abrogated the inflammation of EIU as demonstrated by immunochemical stain and the decreased NO production in aqueous. The expression of iNOS and CAT mRNA in EIU mouse eyes were suppressed by valcade. Supershift assay revealed that NF-κB activation was responsible for iNOS and CAT induction. Similar results were found in cellular model study.
Conclusions：We confirmed the existence of CAT in intraocular tissue. NF-κB is an essential transcription factor not only for the induction of iNOS, but also for the up-regulation of CAT-2. The simultaneous up-regulation of CAT-2 and iNOS may play an important role in the pathogenesis of EIU.
Subjects
uveitis
inducible nitric oxide synthase
cationic amino acid transporter-1
cationic amino acid transporter-2
nitric oxide
Type
thesis
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