Abstract
Purpose: :
Unlike its constitutive isoforms, inducible nitric oxide synthase (iNOS) is generated for the pathologic cause by the immune system activator in inflammatory diseases. In this study, transgenic mice with iNOS overexpression in photoreceptors were generated to evaluate the effect of iNOS itself, devoid of its associated immune response contributions. Such an endeavor to pinpoint the exact role of iNOS using well-defined transgenic mice has not previously been attempted.
Methods: :
Opsin/iNOS transgene for pronuclei injection was generated by cloning 4.4 kb Acc65I/Xhol mouse opsin promoter into the Acc65I/Sall sites of pCMVSport6-iNOS. A line of homozygotes and two lines of heterozygotes were generated. Presence of iNOS was confirmed and the iNOS-derived oxidative and nitrosative damages were detected by protein nitration using Western and confocal localization and by apoptosis using TUNEL.
Results: :
The iNOS insertion was demonstrated by a single 130 kDa iNOS band in Western analysis and was also found to localize specifically to the inner segments (IS) and outer plexiform layer (OPL). Under basal conditions, the cellular toxicity of sustained iNOS expression was detected by the formation of nitrosative product, namely, tyrosine-nitrated proteins, the most significant of all was nitrated mitochondrial cytochrome c in IS, OPL and retinal pigment epithelium. Marked apoptotic cells were detected in the outer nuclear layer.
Conclusions: :
This study generated a pathologic phenotype with sustained iNOS expression in photoreceptors delivering high output of nitric oxide and peroxynitrite. The endogenous iNOS without any immunological stimulation caused marked nitration/release of mitochondrial cytochrome c and apoptosis in photoreceptors. Thus, manipulation of pathways leading to iNOS upregulation, or an effective neutralization of iNOS can be of significant therapeutic benefit in photoreceptor degeneration mediated by mitochondrial oxidative stress.
Keywords: nitric oxide • mitochondria • transgenics/knock-outs