Korean J Ophthalmol.  2007 Mar;21(1):21-27. 10.3341/kjo.2007.21.1.21.

Direct Detection of Reactive Nitrogen Species in Experimental Autoimmune Uveitis

• Bae SR
• Wu GS
• Sevanian A
• Schultz BE
• Zamir E
• Rao NA

• KMID: 1105026
• DOI: http://doi.org/10.3341/kjo.2007.21.1.21

Abstract

PURPOSE: Demonstrate unequivocally the generation of nitric oxide in experimental autoimmune uveoretinitis by electron spin resonance spectroscopy (ESR) using ferrous iron complex of N-methyl-D-glucamine dithiocarbamate, (MGD)2-Fe2+, as a spin trap. METHODS: Experimental autoimmune uveitis was induced in Lewis rats, and at the peak of the intraocular inflammation, the animals received intravitreous injections of the spin trap. The retina and choroid dissected from the enucleated globes were subjected to ESR. Similarly, the retina and choroid obtained at the peak of experimental autoimmune uveo-retinitis (EAU) were placed in a vial containing luminal, and chemiluminescence was counted on a Packard liquid scintillation analyzer. RESULTS: The ESR three-line spectrum (g=2.04; a(N)=12.5 G) obtained was characteristic of the adduct [(MGD)2-Fe2+-NO]. The majority of this signal was eliminated by the inducible nitric oxide synthase (iNOS) specific inhibitor aminoguanidine injected inflamed retina was detected when compared with that of the non inflamed controls. The chemiluminescent activity was further increased two-fold by the addition of bicarbonate to the inflamed retina; the phenomenon is attributable only to the presence of a high steady-state concentration of peroxynitrite. CONCLUSIONS: The study shows an unequivocal presence of nitric oxide in EAU retina and choroid and the generation of peroxynitrite. High levels of these reactive nitrogen species generated in the inflamed retina and choroids are certain to cause irreversible tissue damage, especially at the susceptible sites such as photoreceptors.

Keyword

Electron spin resonance spectroscopy; Luminol chemiluminescence; Nitric oxide; Peroxynitrite; Spin trapping

MeSH Terms

Uveitis/immunology/*metabolism
Thiocarbamates
Spin Trapping
Spin Labels
Sorbitol/analogs & derivatives
Retina/metabolism
Reactive Nitrogen Species/*metabolism
Rats, Inbred Lew
Rats
Peptide Fragments/immunology
Humans
Electron Spin Resonance Spectroscopy
Choroid/metabolism
Autoimmune Diseases/immunology/*metabolism
Arrestin/immunology
Animals

Figure

• 

  Fig. 1 ESR spin trapping of nitric oxide in the retina of EAU rats. ESR spectra were recorded from two inflamed retinas from Lewis rats 12 days post-immunization. Ninety minutes before sacrifice, the eyes were injected intravitreally with the spin trap, (MGD)2-Fe2+; after isolation, the retinas were incubated with (MGD)2-Fe2+ for 30 minutes before ESR recording. Each experiment was repeated three times, using a total of six eyes from different animals, and a representative spectrum is shown. normal control: non-immunized retina without spin trap; EAU control: EAU retina without spin trap; normal + (MGD)2-Fe2+: non-immunized retina with spin trap; EAU + (MGD)2-Fe2+: EAU retina with spin trap; (MGD)2-Fe2+-NO: nitric oxide generated from SNAP in vitro reacted with spin trap.

• 

  Fig. 2 Effect of AG on the ESR spin trapping of nitric oxide in the EAU retina. Lewis rats with EAU were injected intravitreally with either AG or PBS (as control) two hours prior to the injection of the spin trap, (MGD)2-Fe2+. Following enucleation, the retina was incubated with the spin trap for 30 minutes before ESR measurement. Each experiment was repeated three times using a total of six eyes. Representative ESR spectra are shown. EAU+(MGD)2-Fe2+: EAU retina with spin trap; EAU+(MGD)2-Fe2++ AG: EAU retina with spin trap plus AG.

• 

  Fig. 3 Luminol chemiluminescence of EAU and control retinas. The retina and choroid from Lewis rats were collected at the peak of inflammation, 12 days post-immunization. Luminol (0.2 µ g/ml) was added to the tissues, and the chemiluminescence measurement commenced immediately with a Packard liquid scintillation analyzer set at an out-of coincidence mode for single photon counting. Six sets of retina/choroid were used for each sample, and each measurement was repeated three times, using a total of 18 eyes for each experimental category. Chemiluminescence is expressed as mean±standard error for each category (n=3). An ANOVA single-parameter comparison between maximal chemiluminescent count in EAU vs. that in non-immunized control eyes indicated significance at p<0.0001.

• 

  Fig. 4 Effects of bicarbonate, SOD, L-NAME, AG, and AG + SOD on luminol chemiluminescence of EAU retina/choroids. The enhancement by the bicarbonate (50 mM, n=3) addition was first determined by counting six sets of retina/choroid with and without bicarbonate. The countings from the bicarbonate/luminol/retina system were then used as a background level for evaluating the inhibition displayed by SOD (5270 units/sample; n=3), L-NAME (1 mM, n=3), AG (5 mM, n=3), and SOD (5270 units) + AG (5 mM) (n=3). The counts without the inhibitor were established first, and then the inhibitor was added. The counting was continued until it became stabilized (60 minutes). A set of six retinas and choroids were used for one measurement. Measurements were repeated three times. All eyes used were 12 days post-immunization. Single-variable ANOVA was used to compare counts with the inhibitor groups to bicarbonate/luminal/chemiluminescence without inhibitors. *p<0.005; **p<0.005; ***p<0.0001. Control: non-immunized retina; EAU: EAU retina; HCO3-: EAU retina + bicarbonate; L-NAME: EAU retina + bicarbonate + L-NAME; AG: EAU retina + bicarbonate + AG; SOD: EAU retina + bicarbonate + SOD; AG + SOD: EAU retina + bicarbonate + SOD + AG.

Reference

1. Rao NA. Role of oxygen free radicals in retinal damage associated with experimental uveitis. Trans Am Ophthalmol Soc. 1990. 88:797–850.

2. Rao NA, Wu GS. Free radical mediated photoreceptor damage in uveitis. Progress in Retina and Eye Res. 2000. 19:41–68.

3. Zhang J, Wu GS, Rao NA. Role of nitric oxide (NO) in experimental autoimmune uveitis (EAU). Invest Ophthalmol Vis Sci. 1993. 1000:abstr.

4. Nathan C, Xie Q-W. Nitric oxide synthases: roles, tolls, and controls. Cell. 1994. 78:915–918.

5. Zhang J, Wu LY, Wu GS, et al. Differential expression of nitric oxide synthase in experimental uveoretinitis. Invest Ophthalmol Vis Sci. 1999. 40:1899–1905.

6. Jacquemin E, de Kozak Y, Thillaye B, et al. Expression of inducible nitric oxide synthase in the eye from endotoxin-induced uveitis rats. Invest Ophthalmol Vis Sci. 1996. 37:1187–1196.

7. Schmidt HHHW, Hofmann H, Schindler U, et al. No ˙NO from NO synthase. Proc Natl Acad Sci USA. 1996. 93:14492–14497.

8. Komarov AM, Wink DA, Feelisch M, et al. Electron paramagnetic resonance spectroscopy using N-metyl-D-glucamine dithiocarbamate iron cannot discriminate between nitric oxide synthase. Free Radic Biol Med. 2000. 28:739–742.

9. Lai CS, Komarov AM. Spin trapping of nitric oxide produced in vivo in septic-shock mice. FEBSL Lett. 1994. 345:120–124.

10. Zweier JL, Wang P, Samouilov A, et al. Enzyme-independent formation of nitric oxide in biological tissues. Nature Med. 1995. 1:804–809.

11. Komarov AM, Kramer JH, Mak IT, et al. EPR detection of endogenous nitric oxide in postischemic heart using lipid and aqueous-soluble dithiocarbamate-iron complexes. Mol Cell Biochem. 1997. 175:91–97.

12. Obolenskaya M, Vanin AF, et al. EPR evidence of nitric oxide production by the regenerating rat liver. Biochem Biophys Res Commun. 1994. 571–576.

13. Komarov AM, Lai CS. Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy. Biochim Biophys Acta. 1995. 1272:29–36.

14. Bune AJ, Shergill JK, Cammack R, et al. L-arginine depletion by arginase reduces nitric oxide production in endotoxemic shock: an electron paramagnetic resonance study. FEBS Lett. 1995. 366:127–130.

15. Allen RC. Phagocytic leukocyte oxygeneration activities and chemiluminescence: a kinetic approach to analysis. Methods Enzymol. 1986. 133:449–493.

16. Merenyi G, Lind J, Eriksen TE. The reactivity of superoxide (O₂^-)and its ability to induce chemiluminescence with luminol. Photochem Photobiol. 1985. 41:203–208.

17. Radi R, Rubbo H, Thomson L, et al. Luminol chemiluminescence using xanthine and hypoxanthine as xanthine oxidase substrates. J Free Rad Biol Med. 1990. 8:121–126.

18. Archer SL, Nelson DP, Weir EK. Simultaneous measurement of O₂ radicals and pulmonary vascular reactivity in rat lung. J Appl Physiol. 1989. 67:1903–1911.

19. Dowling EJ, Symons AM, Parke DV. Free radical production at the site of an acute inflammatory reaction as measured by chemiluminescence. Agents Actions. 1986. 19:203–207.

20. Beckman JS, Beckman TW, Chen J, et al. Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc Natl Acad Sci USA. 1990. 87:1620–1624.

21. Radi R, Cosgrove TP, Beckman JS, et al. Peroxynitrite-induced luminol chemiluminescence. Biochem J. 1993. 290:51–57.

22. Wang P, Zweier JL. Measurement of nitric oxide and peroxynitrite generation in the postischemic heart. Evidence for peroxynitrite-mediated reperfusion injury. J Biol Chem. 1996. 271:29223–29230.

23. Zweier JL, Wang P, Kuppusamy P. Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy. J Biol Chem. 1995. 270:304–307.

24. Xia Y, Zweier JL. Direct measurement of nitric oxide generation from nitric oxide synthase. Proc Natl Acad Sci USA. 1997. 94:12705–12710.

25. Mulsch A, Vanin A, Mordvintcev P, et al. NO accounts completely for the oxyenated nitrogen species generated by enzymic L-arginine oxygenation. Biochem J. 1992. 288:597–603.

26. Kubrina LN, Mikoyan VD, Mordvintcev PI, et al. Iron potentiates bacterial lipopolysaccharide-induced nitric oxide formation in animal organs. Biochim Biophys Acta. 1993. 1176:240–244.

27. Corbett JA, McDaniel ML. Selective inhibition of inducible nitric oxide synthase by aminoguanidine. Methods Enzymol. 1996. 268:398–408.

28. Shinobu LA, Jones SG, Jones MM. Sodium N-methyl-D-glucamine dithiocarbamate and cadmium intoxication. Acta Pharmacol Toxicol (Copenh). 1984. 54:189–194.

29. Denicola A, Freeman BA, Trujillo M, et al. Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations. Arch Biochem Biophys. 1996. 333:49–58.

30. Gow A, Duran D, Thom SR, et al. Carbon dioxide enhancement of peroxynitrite-mediated tyrosine nitration. Arch Biochem Biophys. 1996. 333:42–48.

31. Zhang H, Joseph J, Felix C, et al. Bicarbonate enhances the hydroxylation, nitration, and peroxidation reactions catalyzed by copper, zinc superoxide dismutase. Intermediacy of carbonate anion radical. J Biol Chem. 2000. 275:14038–14045.