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Nitrosative stress
[38]
NITROSATIVESTRESS
[38] By A L F R E D
389
Nitrosative Stress
H A U S L A D E N a n d J O N A T H A N S. S T A M L E R
Introduction Nitric oxide (NO) and its aerobic reaction products have long been recognized as cytotoxic, mutagenic, and carcinogenic. The discovery of endogenous NO biosynthesis and a multitude of NO signaling functions has greatly increased interest in identifying the targets involved and the nature of the interactions. Nitros(yl)ation of transition metals or of basic amino acids, in particular cysteines, form the basis of NO-related activityI : The best-characterized mechanism of signal transduction is the activation of guanylate cyclase by nitrosyl-heme formationI ; other mechanisms include the activation of the bacterial transcription factors soxR2 and oxyR3 by nitrosylation of an iron-sulfur cluster and a cysteine, respectively, and the activation of G proteins by nitrosylation of a conserved cysteine conforming to a consensus motif.4 However, nitros(yl)ation--either when excessive or when happening in less than regulated fashion--can lead to a loss of cellular function. The molecular correlates of nitrosative injury include enzyme inactivation and damage to DNA. We have termed such impairment of metabolism "nitrosative stress," 3 and the causal species nitrosants, emphasizing the relationship to--and analogy with--oxidative stress and oxidants. Here we focus on the differences, the parallels, and the synergism between nitrosative stress and oxidative stress with emphasis on mechanism of cytotoxicity and the protective response, in particular, the activation of antinitrosative defenses that lead to the acquisition of resistance. An approach to assessment of nitrosative stress is also provided. Interactions between Oxidants and Nitrosants Nitric oxide does not react with most cellular constituents and is usually quite selective in the choice of cysteine or heme-containing target protein, with which it reacts in the presence of molecular oxygen. However, in combination with reactive oxygen species or with increases in the NO level, 1 j. S. Stamler, Cell 78, 931 (1994). 2 T. Nunoshiba, T. DeRojas-Walker, J. S. Wishnok, S. R. T a n n e n b a u m , and B. Demple, Proc. Natl. Acad. Sci. USA 90, 9993 (1993). 3 A. Hausladen, C. T. Privalle, T. Keng, J. D e A n g e l o , and J. S. Stamler, Cell 86, 719 (1996). 4 j. S. Stamler, E. J. Toone, S. A. Lipton, and N. J. Sucher, Neuron 18, 691 (1997).
METHODS IN ENZYMOLOGY,VOL. 300
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nitrosation and oxidation are more readily observed. This may extend the signaling repertoire of reactive nitrogen species (e.g., beyond activation of guanylate cyclase or inhibition of caspases) on the one hand, 5'6 but predisposes to toxic and mutagenic effects on the other hand. Subtle changes in the molecular mechanism of nitrosation may account for such a loss of selectivity toward targets during stress. For instance, NO may be directly responsible for S-nitrosylation in certain regulated functions, in which case the reaction is first-order in NO and critically dependent on thiol pK] or S-nitrosylation may be enzymatically controlled--as exemplified in hemoglobin catalysis--and thus highly selective for substrate. 8 On the other hand, N2039-11 probably acts as the principal nitrosant when the NO level is >10 lxM. ~2 Nitrosation and deamination of D N A bases and mutagenicity are much more likely in this case because N 2 0 3 donates reactive NO +. Other examples of powerful nitrosating and oxidizing species are the NO-superoxide product peroxynitrite, 13'14the poorly characterized derivatives that form when NO and 02- are generated in unequal amounts, and the species with NO* and hydroxyl radical character that form in transition metal-catalyzed reactions between hydrogen peroxide and N O . 15'16 Indeed, the connection between oxidative and nitrosative stress is well exemplified by, but not limited to, reactions of NO/O2 , which can nitrate, a7-22nitrosate, 5 j. Haendeler, U. Weiland, A. M. Zeiher, and S. Dimmeler, Nitric Oxide 1, 282 (1997). 6 j. B. Mannick, X. Q. Miao, and J. S. Stamler, J. Biol. Chem. 272, 24125 (1997). 7 A. J. Gow, D. G. Buerk, and H. Ischiropoulos, J. Biol. Chem. 272, 2841 (1997). 8 A. J. Gow and J. S. Stamler, Nature 391, 169 (1998). 9 V. G. Kharitonov, A. R. Sundquist, and V. S. Sharma, J. Biol. Chem. 270, 28158 (1995). 10D. A. Wink, J. A. Cook, S. Y. Kim, Y. Vodovotz, R. Pacelli, M. C. Krishna, A. Russo, J. B. Mitchell, D. Jourd'heuil, A. M. Miles, and M. B. Grisham, J. Biol. Chem. 272, 11147 (1997). 11 N. Hogg, R. J. Singh, and B. Kalyanaraman, F E B S Lett. 382, 223 (1996). 12 D. A. Wink, K. S. Kasprazak, C. M. Maragos, R. K. Elespuru, M. Misra, T. M. Dunams, T. A. Cebula, W. H. Koch, A. W. Andrews, J. S. Allen, and L. K. Keefer, Science 254, 1001 (1991). 13j. S. Beckman, Chem. Res. Toxicol. 9, 836 (1996). 14W. H. Koppenol, J. J. Moreno, W. A. Pryor, H. Ischiropoulos, and J. S. Beckman, Chem. Res. Toxicol. 5, 834 (1992). 15 R. Pacelli, D. A. Wink, J. A. Cook, M. C. Krishna, W. DeGraff, N. Friedman, M. Tsokos, A. Samuni, and J. B. Mitchell, J. Exp. Med. 182, 1469 (1995). 16 R. Farias-Eisner, G. Chaudhuri, E. Aeberhard, and J. M. Fukuto, J. Biol. Chem. 271, 6144 (1996). 17A. Gow, D. Duran, S. R. Thom, and H. Ischiropoulos, Arch, Biochem. Biophys. 3339 42 (1996). 18H. Ischiropoulos, M. F. Beers, S. T. Ohnishi, D. Fisher, S. E. Garner, and S. R. Thorn, J. Clin. Invest. 97, 2260 (1996). 19A. J. Gow, D. Duran, S. Malcolm, and H. Ischiropoulos, FEBS Lett. 385, 63 (1996). 20 I. Y. Haddad, S. Zhu, H. Ischiropoulos, and S. Matalon, Am. J. Physiol. 270, L281 (1996). 21 j. p. Crow and H. Ischiropoulos Methods Enzymol. 269, 185 (1996).
[38]
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a n d o x i d i z e 23-26 biological molecules. Thus, NO by itself has little inhibitory
effect on bacteria or cultured cells, while N 2 0 3 , N O / O 2 - , S N O s , and peroxynitrite can be highly c y t o t o x i c . 15'16'27'28 Manifestations of Nitrosative Stress The targets of nitrosative stress--protein, DNA, lipids--are in many cases the same as those damaged by oxidative stress, and exposure to high levels of nitrosants can result in thiol oxidation u,zg,3° or induce an oxidative stress through the depletion of antioxidants. However, while a change in redox state is the sine qua n o n of an oxidative stress, it is not a prerequisite for a nitrosative stress. This state is alternatively characterized by (excessive) covalent attachments (e.g., NO group attachment) or additions to target nucleophiles. At the molecular level, for example, proteins experience nitrosative modifications that impair function. 31 Such nitros(yl)ation can occur in the absence of oxidation. The following examples are cases in point: RSH + NOz- + H + ~ RSNO + H20 RSH + N 2 0 3 ---->RSNO + N O 2 - + H +
(1) (2)
Acidified nitrite is equivalent, through dehydration, to NO +, and N 2 0 3 to [NO + N O 2 - ] , that is, the reactions involve substitution of H + for NO + rather than a change in oxidation state of either N or S. Inasmuch as the assignment of electrons to S (RS- NO +) depends on the nature of the R group, oxidation may occur in some instances. However, many RSNOs transfer NO +, preserving the reduced thiol, which suggests that the electron assignment is appropriate as written. 22 j. S. Beckman, H. Ischiropoulos, L. Zhu, M. van der Woerd, C. Smith, J. Chen, J. Harrison, J. C. Martin, and M. Tsai, Arch. Biochem. Biophys. 298, 438 (1992). 23 M. G. Salgo and W. A. Pryor, Arch. Biochem. Biophys. 333, 482 (1996). 24 R. M. Uppu, G. L. Squadrito, and W. A. Pryor, Arch. Biochem. Biophys. 327, 335 (1996). 25 M. G. Salgo, E. Bermudez, G. L. Squadrito, and W. A. Pryor, Arch. Biochem. Biophys. 322, 500 (1995). 26 R. Radi, J. S. Beckman, K. M. Bush, and B. A. Freeman, J. Biol. Chem. 266, 4244 (1991). 27 I. Ioannidis, M. B~itz, M. Kirscb, H. G. Korth, R. Sustmann, and H. De Groot, Biochem. J. 329, 425 (1998). 28 M. A. De Groote, D. Granger, Y. Xu, G. Campbell, R. Prince, and F. C. Fang, Proc. Natl. Acad. Sci. USA 92, 6399 (1995). 29 E. G. DeMaster, B. J. Quast, B. Redfern, and H. T. Nagasawa, Biochemistry 34, 11494 (1995). 3o S. P. Singh, J. S. Wishnok, M. Keshive, W. M. Deen, and S. R. Tannenbaum, Proc. Natl. Acad. Sci. USA 93, 14428 (1996). 31 D. I. Simon, M, E. Mullins, L. Jia, B. Gaston, D. J. Singel, and J. S. Stamler, Proc. Natl. Acad. Sci. USA 93, 4736 (1996).
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Nitrosylation can also have effects distinct from oxidation. This is best exemplified in regulation of the cardiac calcium release channel (ryanodine receptor). Poly-S-nitrosylation reversibly activates the calcium channel, whereas thiol oxidation leads to irreversible channel activation and a loss of control. 32 In other words, channel regulation is not the result of a general change in redox state. Rather, the channel can sense and distinguish between the nitrosative and oxidative modifications. Likewise, the activation of the bacterial transcription factor oxyR by S-nitrosylation seems to be more readily reversible than activation by oxidation 3. Taken together, the data suggest the possibility of differential control by nitrosative and oxidative signals. Moreover, SNO modifications may protect thiols from irreversible oxidation.
Resistance to Nitrosative Stress The first line of defense against production of nitrosants is glutathione (GSH). The high intracellular concentrations make it a primary target for oxidants and nitrosants alike. S-Nitrosoglutathione, however, does not accumulate to any significant degree, suggesting the presence of SNO metabolizing activity? Such "SNOase" candidates include the enzymes thioredoxin and thioredoxin reductase, which cleave S-nitrosoglutathione more readily than glutathione disulfide. Ubiquitous reductants like ascorbate, moreover, reduce SNO to N O . 4'33-35 Protein thiol may also serve to buffer nitrosants; proteins readily modified without loss of function are cases in point. 31,32'36But ultimately, SNO accumulation in proteins will be associated with untoward effects3 and glutathione depletion is likely to be a contributing factor. Indeed, several enzymes involved in maintaining the GSH pool, including y-glutamylcysteine synthetase (glutamate-cysteine ligase), glutathione reductase, and glutathione p e r o x i d a s e 37-39 a r e themselves inhibited by S-nitrosylation or NO-mediated oxidation. Additional regulatory pro32 L. Xu, J. P. Eu, G. Meissner, and J. S. Stamler, Science 279, 234 (1998). 33 D. Nikitovic and A. Holmgren, J. Biol. Chem. 271, 19180 (1996). 34 G. Scorza, D. Pietraforte, and M. Minetti, Free Radic. Biol. Med. 22, 633 (1997). 35 M. Kashiba-Iwatsuki, M. Yamaguchi, and M. Inoue, F E B S Lett. 389, 149 (1996). 36j. S. Stamler, O. Jaraki, J. Osborne, D. I. Simon, J. Keaney, J. Vita, D. Singel, C. R. Valeri, and J. Losealzo, Proc. Natl. Acad. Sci. USA 89, 7674 (1992). 37 M. A. Keese, M. Bose, A. Mulsch, R. H. Schirmer, and K. Beeker, Biochem. Pharmacol. 54, 1307 (1997). 38 M. Asahi, J. Fujii, T. Takao, T. Kuzuya, M. Hori, Y. Shimonishi, and N. Tanigucbi, J. Biol. Chem. 272, 19152 (1997). 39j. Han, J. S. Stamler, H.-L. Li, and O. Grifith, in "Biology of Nitric Oxide (IV)" (J. S. Stamler, S. Gross, S. Moncada, and A. Higgs, Eds.), Portland Press, London (1995).
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teins, lipids, and D N A 4°'41 a r e subject to nitrosative damage as well. Nitrosative stress might increase mutations, inhibit growth, promote cell d e a t h , 3,28'4°-46 o r contribute to the pathophysiological processes of atherosclerosis, chronic inflammation, and sepsis. 47 Accumulating evidence suggests that cells possess inducible mechanisms to counter the toxic effects of nitrosants in much the same way as oxidative stress is controlled by an elaborate system of inducible antioxidative defenses. Demple and co-workers first showed that the soxRS regulon in Escherichia coli, which controls the expression of antioxidative genes in response to increased fluxes of superoxide, is also induced by NO and provides resistance to its untoward effects.2,4s We have shown that the hydrogen peroxide responsive trancription factor oxyR, which controls the expression of peroxide scavenging genes, also provides resistance to the cytostatic effects of S N O s . 3 Molecular recognition of NO by soxRS and SNO by OxR is performed by an iron-sulfur cluster and cysteine, respectively. Thus, metals and thiols in proteins are not only the sites most prone to the damaging effects of NO/SNO exposure, but also serve to alert the cell to their presence. None of the genes controlled by soxRS or oxyR are known to detoxify nitrosants, so the protective effect has been explained by virtue of (elimination of) harmful interactions with oxidants. There is, however, evidence that E. coli metabolize nitrosants and recovery from SNO-induced growth inhibition correlates well with SNO decomposition. 3 Resistance to SNOs has also been shown in Salmonella typhimurium, where mutation of a dipeptide transporter or homocysteine-producing gene provide protection. 28,44 Rat hepatocytes are protected from SNO toxicity by pretreatment with a low dose of SNO, indicating an inducible mechanism of SNO resistance, 49 and macrophages became resistant to NO inducedgrowth inhibition by continuous stimulation of NO biosynthesis. 5°,51 NO 40 T. Nguyen, D. Brunson, C. L. Crespi, B. W. Penman, J. S. Wishnok, and S. R. Tannenbaum, Proc. Natl. Acad. Sci. USA 89, 3030 (1992). 41 A. Gal and G. N. Wogan, Proc. Natl. Acad. Sci. USA 93, 15102 (1996). 42 M. N. Routledge, D. A. Wink, L. K. Keefer, and A. Dipple, Carcinogenesis 14, 1251 (1993). 43 S. Tamir, S. Burney, and S. R. Tannenbaum, Chem. Res. Toxicol. 9, 821 (1996). 44 M. A. De Groote, T. Testerman, Y. Xu, G. Stauffer, and F. C. Fang, Science 272, 414 (1996). 45 j. C. Zhuang and G. N. Wogan, Proc. Natl. Acad. Sci. USA 94, 11875 (1997). 46 F. C. Fang, J. Clin. Invest. 99, 2818 (1997). 47 j. F. Kerwin, Jr., J. R. Lancaster, Jr., and P. L. Feldman, J. Med. Chem. 38, 4343 (1995). 48 T. Nunoshiba, T. DeRojas-Walker, S. R. Tannenbaum, and B. Demple, Infect. Immun. 63, 794 (1995). 49 y. M. Kim, H. Bergonia, and J. R. Lancaster, Jr., FEBS Lett. 374, 228 (1995). 50 B. Briine, C. G6tz, U. K. Mebmer, K. Sandau, M.-H. Hirvonen, and E. G. Lapatina, J. Biol. Chem. 272, 7253 (1997). 51 M. R. Hirvonen, B. Brune, and E. G. Lapetina, Biochem. J. 315, 845 (1996).
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TABLE I DETECTION OF NITROSANTSAND PRODUCTSOF NITROSATIVESTRESS Nitrosant or product
NO2SNO
Nitrosamines Metal nitrosyls Exhaled NO Deaminated DNA NO. + O2"-/NO ÷ + H202
Method Capillary electrophoresis HPLC Griess reaction Photolysis/chemiluminescence Saville assay HPLC/postcolumn reactor HPLC/mass spectrometry Capillary electrophoresis GC/chemiluminescence Fluorescence Electron paramagnetic resonance GC/mass spectrometry Mutagenesis Tyrosine nitration, thiol nitrosation
Ref. 57 57 58 59 59 60 61 62 63, 57 65, 64 67, 21,
64 66 68 59-62
scavenging by superoxide, 5° metallothionein, 52 or heat shock protein 53 has been implicated as a resistance mechanism. In addition, a n u m b e r of enzymes have been implicated in N O or SNO resistance, but their physiological significance remains to be e s t a b l i s h e d . 33,54-56 A s s e s s m e n t of Nitrosative S t r e s s The assessment of a nitrosative stress is an important objective. One way is to measure products of N O metabolism that can serve as nitrosants. These include nitrite (which has the potential to serve as an N O + equivalent), S N O s , n i t r o s a m i n e s ( o r d e r i v a t i v e s t h e r e o f ) a n d m e t a l n i t r o s y l s , p a r ticularly low mass species. The formation of a fluorescent triazole following n i t r o s a t i o n o f 2 , 3 - d i a m i n o n a p h l a l e n e at p h y s i o l o g i c a l p H 57 o r a n i n c r e a s i n g r a t i o o f n i t r i t e t o n i t r a t e a r e u s e f u l m e a s u r e s o f n i t r o s a t i v e stress. A s e c o n d a p p r o a c h is t o m e a s u r e t h e b i o l o g i c a l e n d p r o d u c t s o f n i t r o s a t i o n , in p a r t i c u 52 M. A. Schwarz, J. S. Lazo, J. C. Yalowich, W. P. Allen, M. Whitmore, H. A. Bergonia, E. Tzeng, T. R. Billiar, P. D. Robbins, J. R. Lancaster, Jr., and B. R. Pitt, Proc. Natl. Acad. Sci. USA 92, 4452 (1995). 53 K. Bellmann, M. Jaattela, D. Wissing, V. Burkart, and H. Kolb, F E B S Lett. 391, 185 (1996). 54 M. P. Gordge, J. S. Hothersall, G. H. Neild, and A. A. Dutra, Br. J. Pharmacol. 119, 533 (1996). 55 N. Hogg, R. J. Singh, E. Konorev, J. Joseph, and B. Kalyanaraman, Biochem. J. 323, 477 (1997). 56V. Borutait6 and G. C. Brown, Biochem. J. 315, 295 (1996). 57 p. j. Andrew, M. Auer, I. J. Lindley, H. F. Kauffmann, and A. J. Kungl, F E B S Lett 408, 319 (1997).
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OXIDANTS/ANTIOXIDANTS AND CELL-CELL ADHESION
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lar, nitrosylated proteins and deaminated D N A bases. Table I provides a list of nitrosants and nitrosated products as well as references to some chemical, electrophoretic, spectroscopic, and chemiluminescent methods used for their detection. 57 A. M. Leone and M. Kelm, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 499, Wiley, Chichester, UK (1996). 58 H. H. H. W. Schmidt and M. Kelm, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 491. Wiley, Chichester, UK (1996). 59j. S. Stamler and M. Feelisch, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 521. Wiley, Chichester, UK (1996). 60 T. Akaike, K. Inoue, T. Okamoto, H. Nishino, M. Otagiri, S. Fujii, and H. Maeda, J. Biochem. (Tokyo) 122, 459 (1997). 61 I. Kluge, U. Gutteck-Amsler, M. Zollinger, and K. Q. Do, J. Neurochem. 69, 2599 (1997). 62 j. S. Stamler and J. Loscalzo, Anal Chem. 64, 779 (1992). 63 t . K. Keefer and D. L. H. Williams, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 509. Wiley, Chichester, UK (1996). 64 A. M. Leone, L. E. Gustafsson, P. L. Francis, M. G. Persson, N. P. Wiklund, and S. Moncada, Biochem. Biophys. Res. Commun. 201, 883 (1994). 65 D. J. Singel and J. R. Lancaster, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 341. Wiley, Chichester, UK (1996). 66 y. m. Henry and D. J. Singel, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 357. Wiley, Chichester, UK (1996). 67 S. Christen, P. Gee, and B. N. Ames, Methods Enzymol. 269, 267 (1996). 68 S. Tamir, T. deRojas-Walker, J. S. Wishnok, and S. R. Tannenbaum, Methods Enzymol. 269, 230 (1996).
[39] D e t e r m i n a t i o n o f C e l l - C e l l A d h e s i o n i n R e s p o n s e Oxidants and Antioxidants
to
By SASHWATIROY, CHANDANK. SEN,and LESTER PACKER Introduction Adhesion of leukocytes to endothelial cells is the earliest step in immune recognition process and is mediated by cell adhesion molecules (CAM). I In the early phases of cell adhesion, leukocytes transiently adhere to the vessel wall in a process termed "rolling." Rolling of leukocytes is mediated by a family of adhesive molecules called selectins, expressed both on the leukocyte and endothelial cell surface. 2 After "rolling," leukocytes firmly 1 M. P. Bevilacqua, Annu. Rev. lmmunol. U , 767 (1993). 2 C. W. Smith, Semin. Hematol. 30, 45 (1993).
METHODS IN ENZYMOLOGY, VOL. 300
Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. 0076-6879/99 $30,00
NITROSATIVESTRESS
[38] By A L F R E D
389
Nitrosative Stress
H A U S L A D E N a n d J O N A T H A N S. S T A M L E R
Introduction Nitric oxide (NO) and its aerobic reaction products have long been recognized as cytotoxic, mutagenic, and carcinogenic. The discovery of endogenous NO biosynthesis and a multitude of NO signaling functions has greatly increased interest in identifying the targets involved and the nature of the interactions. Nitros(yl)ation of transition metals or of basic amino acids, in particular cysteines, form the basis of NO-related activityI : The best-characterized mechanism of signal transduction is the activation of guanylate cyclase by nitrosyl-heme formationI ; other mechanisms include the activation of the bacterial transcription factors soxR2 and oxyR3 by nitrosylation of an iron-sulfur cluster and a cysteine, respectively, and the activation of G proteins by nitrosylation of a conserved cysteine conforming to a consensus motif.4 However, nitros(yl)ation--either when excessive or when happening in less than regulated fashion--can lead to a loss of cellular function. The molecular correlates of nitrosative injury include enzyme inactivation and damage to DNA. We have termed such impairment of metabolism "nitrosative stress," 3 and the causal species nitrosants, emphasizing the relationship to--and analogy with--oxidative stress and oxidants. Here we focus on the differences, the parallels, and the synergism between nitrosative stress and oxidative stress with emphasis on mechanism of cytotoxicity and the protective response, in particular, the activation of antinitrosative defenses that lead to the acquisition of resistance. An approach to assessment of nitrosative stress is also provided. Interactions between Oxidants and Nitrosants Nitric oxide does not react with most cellular constituents and is usually quite selective in the choice of cysteine or heme-containing target protein, with which it reacts in the presence of molecular oxygen. However, in combination with reactive oxygen species or with increases in the NO level, 1 j. S. Stamler, Cell 78, 931 (1994). 2 T. Nunoshiba, T. DeRojas-Walker, J. S. Wishnok, S. R. T a n n e n b a u m , and B. Demple, Proc. Natl. Acad. Sci. USA 90, 9993 (1993). 3 A. Hausladen, C. T. Privalle, T. Keng, J. D e A n g e l o , and J. S. Stamler, Cell 86, 719 (1996). 4 j. S. Stamler, E. J. Toone, S. A. Lipton, and N. J. Sucher, Neuron 18, 691 (1997).
METHODS IN ENZYMOLOGY,VOL. 300
Copyright © 1999by AcademicPress All rights of reproduction in any form reserved. 0076-6879/99$30.00
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nitrosation and oxidation are more readily observed. This may extend the signaling repertoire of reactive nitrogen species (e.g., beyond activation of guanylate cyclase or inhibition of caspases) on the one hand, 5'6 but predisposes to toxic and mutagenic effects on the other hand. Subtle changes in the molecular mechanism of nitrosation may account for such a loss of selectivity toward targets during stress. For instance, NO may be directly responsible for S-nitrosylation in certain regulated functions, in which case the reaction is first-order in NO and critically dependent on thiol pK] or S-nitrosylation may be enzymatically controlled--as exemplified in hemoglobin catalysis--and thus highly selective for substrate. 8 On the other hand, N2039-11 probably acts as the principal nitrosant when the NO level is >10 lxM. ~2 Nitrosation and deamination of D N A bases and mutagenicity are much more likely in this case because N 2 0 3 donates reactive NO +. Other examples of powerful nitrosating and oxidizing species are the NO-superoxide product peroxynitrite, 13'14the poorly characterized derivatives that form when NO and 02- are generated in unequal amounts, and the species with NO* and hydroxyl radical character that form in transition metal-catalyzed reactions between hydrogen peroxide and N O . 15'16 Indeed, the connection between oxidative and nitrosative stress is well exemplified by, but not limited to, reactions of NO/O2 , which can nitrate, a7-22nitrosate, 5 j. Haendeler, U. Weiland, A. M. Zeiher, and S. Dimmeler, Nitric Oxide 1, 282 (1997). 6 j. B. Mannick, X. Q. Miao, and J. S. Stamler, J. Biol. Chem. 272, 24125 (1997). 7 A. J. Gow, D. G. Buerk, and H. Ischiropoulos, J. Biol. Chem. 272, 2841 (1997). 8 A. J. Gow and J. S. Stamler, Nature 391, 169 (1998). 9 V. G. Kharitonov, A. R. Sundquist, and V. S. Sharma, J. Biol. Chem. 270, 28158 (1995). 10D. A. Wink, J. A. Cook, S. Y. Kim, Y. Vodovotz, R. Pacelli, M. C. Krishna, A. Russo, J. B. Mitchell, D. Jourd'heuil, A. M. Miles, and M. B. Grisham, J. Biol. Chem. 272, 11147 (1997). 11 N. Hogg, R. J. Singh, and B. Kalyanaraman, F E B S Lett. 382, 223 (1996). 12 D. A. Wink, K. S. Kasprazak, C. M. Maragos, R. K. Elespuru, M. Misra, T. M. Dunams, T. A. Cebula, W. H. Koch, A. W. Andrews, J. S. Allen, and L. K. Keefer, Science 254, 1001 (1991). 13j. S. Beckman, Chem. Res. Toxicol. 9, 836 (1996). 14W. H. Koppenol, J. J. Moreno, W. A. Pryor, H. Ischiropoulos, and J. S. Beckman, Chem. Res. Toxicol. 5, 834 (1992). 15 R. Pacelli, D. A. Wink, J. A. Cook, M. C. Krishna, W. DeGraff, N. Friedman, M. Tsokos, A. Samuni, and J. B. Mitchell, J. Exp. Med. 182, 1469 (1995). 16 R. Farias-Eisner, G. Chaudhuri, E. Aeberhard, and J. M. Fukuto, J. Biol. Chem. 271, 6144 (1996). 17A. Gow, D. Duran, S. R. Thom, and H. Ischiropoulos, Arch, Biochem. Biophys. 3339 42 (1996). 18H. Ischiropoulos, M. F. Beers, S. T. Ohnishi, D. Fisher, S. E. Garner, and S. R. Thorn, J. Clin. Invest. 97, 2260 (1996). 19A. J. Gow, D. Duran, S. Malcolm, and H. Ischiropoulos, FEBS Lett. 385, 63 (1996). 20 I. Y. Haddad, S. Zhu, H. Ischiropoulos, and S. Matalon, Am. J. Physiol. 270, L281 (1996). 21 j. p. Crow and H. Ischiropoulos Methods Enzymol. 269, 185 (1996).
[38]
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a n d o x i d i z e 23-26 biological molecules. Thus, NO by itself has little inhibitory
effect on bacteria or cultured cells, while N 2 0 3 , N O / O 2 - , S N O s , and peroxynitrite can be highly c y t o t o x i c . 15'16'27'28 Manifestations of Nitrosative Stress The targets of nitrosative stress--protein, DNA, lipids--are in many cases the same as those damaged by oxidative stress, and exposure to high levels of nitrosants can result in thiol oxidation u,zg,3° or induce an oxidative stress through the depletion of antioxidants. However, while a change in redox state is the sine qua n o n of an oxidative stress, it is not a prerequisite for a nitrosative stress. This state is alternatively characterized by (excessive) covalent attachments (e.g., NO group attachment) or additions to target nucleophiles. At the molecular level, for example, proteins experience nitrosative modifications that impair function. 31 Such nitros(yl)ation can occur in the absence of oxidation. The following examples are cases in point: RSH + NOz- + H + ~ RSNO + H20 RSH + N 2 0 3 ---->RSNO + N O 2 - + H +
(1) (2)
Acidified nitrite is equivalent, through dehydration, to NO +, and N 2 0 3 to [NO + N O 2 - ] , that is, the reactions involve substitution of H + for NO + rather than a change in oxidation state of either N or S. Inasmuch as the assignment of electrons to S (RS- NO +) depends on the nature of the R group, oxidation may occur in some instances. However, many RSNOs transfer NO +, preserving the reduced thiol, which suggests that the electron assignment is appropriate as written. 22 j. S. Beckman, H. Ischiropoulos, L. Zhu, M. van der Woerd, C. Smith, J. Chen, J. Harrison, J. C. Martin, and M. Tsai, Arch. Biochem. Biophys. 298, 438 (1992). 23 M. G. Salgo and W. A. Pryor, Arch. Biochem. Biophys. 333, 482 (1996). 24 R. M. Uppu, G. L. Squadrito, and W. A. Pryor, Arch. Biochem. Biophys. 327, 335 (1996). 25 M. G. Salgo, E. Bermudez, G. L. Squadrito, and W. A. Pryor, Arch. Biochem. Biophys. 322, 500 (1995). 26 R. Radi, J. S. Beckman, K. M. Bush, and B. A. Freeman, J. Biol. Chem. 266, 4244 (1991). 27 I. Ioannidis, M. B~itz, M. Kirscb, H. G. Korth, R. Sustmann, and H. De Groot, Biochem. J. 329, 425 (1998). 28 M. A. De Groote, D. Granger, Y. Xu, G. Campbell, R. Prince, and F. C. Fang, Proc. Natl. Acad. Sci. USA 92, 6399 (1995). 29 E. G. DeMaster, B. J. Quast, B. Redfern, and H. T. Nagasawa, Biochemistry 34, 11494 (1995). 3o S. P. Singh, J. S. Wishnok, M. Keshive, W. M. Deen, and S. R. Tannenbaum, Proc. Natl. Acad. Sci. USA 93, 14428 (1996). 31 D. I. Simon, M, E. Mullins, L. Jia, B. Gaston, D. J. Singel, and J. S. Stamler, Proc. Natl. Acad. Sci. USA 93, 4736 (1996).
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SIGNAL TRANSDUCTION AND GENE EXPRESSION
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Nitrosylation can also have effects distinct from oxidation. This is best exemplified in regulation of the cardiac calcium release channel (ryanodine receptor). Poly-S-nitrosylation reversibly activates the calcium channel, whereas thiol oxidation leads to irreversible channel activation and a loss of control. 32 In other words, channel regulation is not the result of a general change in redox state. Rather, the channel can sense and distinguish between the nitrosative and oxidative modifications. Likewise, the activation of the bacterial transcription factor oxyR by S-nitrosylation seems to be more readily reversible than activation by oxidation 3. Taken together, the data suggest the possibility of differential control by nitrosative and oxidative signals. Moreover, SNO modifications may protect thiols from irreversible oxidation.
Resistance to Nitrosative Stress The first line of defense against production of nitrosants is glutathione (GSH). The high intracellular concentrations make it a primary target for oxidants and nitrosants alike. S-Nitrosoglutathione, however, does not accumulate to any significant degree, suggesting the presence of SNO metabolizing activity? Such "SNOase" candidates include the enzymes thioredoxin and thioredoxin reductase, which cleave S-nitrosoglutathione more readily than glutathione disulfide. Ubiquitous reductants like ascorbate, moreover, reduce SNO to N O . 4'33-35 Protein thiol may also serve to buffer nitrosants; proteins readily modified without loss of function are cases in point. 31,32'36But ultimately, SNO accumulation in proteins will be associated with untoward effects3 and glutathione depletion is likely to be a contributing factor. Indeed, several enzymes involved in maintaining the GSH pool, including y-glutamylcysteine synthetase (glutamate-cysteine ligase), glutathione reductase, and glutathione p e r o x i d a s e 37-39 a r e themselves inhibited by S-nitrosylation or NO-mediated oxidation. Additional regulatory pro32 L. Xu, J. P. Eu, G. Meissner, and J. S. Stamler, Science 279, 234 (1998). 33 D. Nikitovic and A. Holmgren, J. Biol. Chem. 271, 19180 (1996). 34 G. Scorza, D. Pietraforte, and M. Minetti, Free Radic. Biol. Med. 22, 633 (1997). 35 M. Kashiba-Iwatsuki, M. Yamaguchi, and M. Inoue, F E B S Lett. 389, 149 (1996). 36j. S. Stamler, O. Jaraki, J. Osborne, D. I. Simon, J. Keaney, J. Vita, D. Singel, C. R. Valeri, and J. Losealzo, Proc. Natl. Acad. Sci. USA 89, 7674 (1992). 37 M. A. Keese, M. Bose, A. Mulsch, R. H. Schirmer, and K. Beeker, Biochem. Pharmacol. 54, 1307 (1997). 38 M. Asahi, J. Fujii, T. Takao, T. Kuzuya, M. Hori, Y. Shimonishi, and N. Tanigucbi, J. Biol. Chem. 272, 19152 (1997). 39j. Han, J. S. Stamler, H.-L. Li, and O. Grifith, in "Biology of Nitric Oxide (IV)" (J. S. Stamler, S. Gross, S. Moncada, and A. Higgs, Eds.), Portland Press, London (1995).
[38]
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teins, lipids, and D N A 4°'41 a r e subject to nitrosative damage as well. Nitrosative stress might increase mutations, inhibit growth, promote cell d e a t h , 3,28'4°-46 o r contribute to the pathophysiological processes of atherosclerosis, chronic inflammation, and sepsis. 47 Accumulating evidence suggests that cells possess inducible mechanisms to counter the toxic effects of nitrosants in much the same way as oxidative stress is controlled by an elaborate system of inducible antioxidative defenses. Demple and co-workers first showed that the soxRS regulon in Escherichia coli, which controls the expression of antioxidative genes in response to increased fluxes of superoxide, is also induced by NO and provides resistance to its untoward effects.2,4s We have shown that the hydrogen peroxide responsive trancription factor oxyR, which controls the expression of peroxide scavenging genes, also provides resistance to the cytostatic effects of S N O s . 3 Molecular recognition of NO by soxRS and SNO by OxR is performed by an iron-sulfur cluster and cysteine, respectively. Thus, metals and thiols in proteins are not only the sites most prone to the damaging effects of NO/SNO exposure, but also serve to alert the cell to their presence. None of the genes controlled by soxRS or oxyR are known to detoxify nitrosants, so the protective effect has been explained by virtue of (elimination of) harmful interactions with oxidants. There is, however, evidence that E. coli metabolize nitrosants and recovery from SNO-induced growth inhibition correlates well with SNO decomposition. 3 Resistance to SNOs has also been shown in Salmonella typhimurium, where mutation of a dipeptide transporter or homocysteine-producing gene provide protection. 28,44 Rat hepatocytes are protected from SNO toxicity by pretreatment with a low dose of SNO, indicating an inducible mechanism of SNO resistance, 49 and macrophages became resistant to NO inducedgrowth inhibition by continuous stimulation of NO biosynthesis. 5°,51 NO 40 T. Nguyen, D. Brunson, C. L. Crespi, B. W. Penman, J. S. Wishnok, and S. R. Tannenbaum, Proc. Natl. Acad. Sci. USA 89, 3030 (1992). 41 A. Gal and G. N. Wogan, Proc. Natl. Acad. Sci. USA 93, 15102 (1996). 42 M. N. Routledge, D. A. Wink, L. K. Keefer, and A. Dipple, Carcinogenesis 14, 1251 (1993). 43 S. Tamir, S. Burney, and S. R. Tannenbaum, Chem. Res. Toxicol. 9, 821 (1996). 44 M. A. De Groote, T. Testerman, Y. Xu, G. Stauffer, and F. C. Fang, Science 272, 414 (1996). 45 j. C. Zhuang and G. N. Wogan, Proc. Natl. Acad. Sci. USA 94, 11875 (1997). 46 F. C. Fang, J. Clin. Invest. 99, 2818 (1997). 47 j. F. Kerwin, Jr., J. R. Lancaster, Jr., and P. L. Feldman, J. Med. Chem. 38, 4343 (1995). 48 T. Nunoshiba, T. DeRojas-Walker, S. R. Tannenbaum, and B. Demple, Infect. Immun. 63, 794 (1995). 49 y. M. Kim, H. Bergonia, and J. R. Lancaster, Jr., FEBS Lett. 374, 228 (1995). 50 B. Briine, C. G6tz, U. K. Mebmer, K. Sandau, M.-H. Hirvonen, and E. G. Lapatina, J. Biol. Chem. 272, 7253 (1997). 51 M. R. Hirvonen, B. Brune, and E. G. Lapetina, Biochem. J. 315, 845 (1996).
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TABLE I DETECTION OF NITROSANTSAND PRODUCTSOF NITROSATIVESTRESS Nitrosant or product
NO2SNO
Nitrosamines Metal nitrosyls Exhaled NO Deaminated DNA NO. + O2"-/NO ÷ + H202
Method Capillary electrophoresis HPLC Griess reaction Photolysis/chemiluminescence Saville assay HPLC/postcolumn reactor HPLC/mass spectrometry Capillary electrophoresis GC/chemiluminescence Fluorescence Electron paramagnetic resonance GC/mass spectrometry Mutagenesis Tyrosine nitration, thiol nitrosation
Ref. 57 57 58 59 59 60 61 62 63, 57 65, 64 67, 21,
64 66 68 59-62
scavenging by superoxide, 5° metallothionein, 52 or heat shock protein 53 has been implicated as a resistance mechanism. In addition, a n u m b e r of enzymes have been implicated in N O or SNO resistance, but their physiological significance remains to be e s t a b l i s h e d . 33,54-56 A s s e s s m e n t of Nitrosative S t r e s s The assessment of a nitrosative stress is an important objective. One way is to measure products of N O metabolism that can serve as nitrosants. These include nitrite (which has the potential to serve as an N O + equivalent), S N O s , n i t r o s a m i n e s ( o r d e r i v a t i v e s t h e r e o f ) a n d m e t a l n i t r o s y l s , p a r ticularly low mass species. The formation of a fluorescent triazole following n i t r o s a t i o n o f 2 , 3 - d i a m i n o n a p h l a l e n e at p h y s i o l o g i c a l p H 57 o r a n i n c r e a s i n g r a t i o o f n i t r i t e t o n i t r a t e a r e u s e f u l m e a s u r e s o f n i t r o s a t i v e stress. A s e c o n d a p p r o a c h is t o m e a s u r e t h e b i o l o g i c a l e n d p r o d u c t s o f n i t r o s a t i o n , in p a r t i c u 52 M. A. Schwarz, J. S. Lazo, J. C. Yalowich, W. P. Allen, M. Whitmore, H. A. Bergonia, E. Tzeng, T. R. Billiar, P. D. Robbins, J. R. Lancaster, Jr., and B. R. Pitt, Proc. Natl. Acad. Sci. USA 92, 4452 (1995). 53 K. Bellmann, M. Jaattela, D. Wissing, V. Burkart, and H. Kolb, F E B S Lett. 391, 185 (1996). 54 M. P. Gordge, J. S. Hothersall, G. H. Neild, and A. A. Dutra, Br. J. Pharmacol. 119, 533 (1996). 55 N. Hogg, R. J. Singh, E. Konorev, J. Joseph, and B. Kalyanaraman, Biochem. J. 323, 477 (1997). 56V. Borutait6 and G. C. Brown, Biochem. J. 315, 295 (1996). 57 p. j. Andrew, M. Auer, I. J. Lindley, H. F. Kauffmann, and A. J. Kungl, F E B S Lett 408, 319 (1997).
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lar, nitrosylated proteins and deaminated D N A bases. Table I provides a list of nitrosants and nitrosated products as well as references to some chemical, electrophoretic, spectroscopic, and chemiluminescent methods used for their detection. 57 A. M. Leone and M. Kelm, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 499, Wiley, Chichester, UK (1996). 58 H. H. H. W. Schmidt and M. Kelm, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 491. Wiley, Chichester, UK (1996). 59j. S. Stamler and M. Feelisch, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 521. Wiley, Chichester, UK (1996). 60 T. Akaike, K. Inoue, T. Okamoto, H. Nishino, M. Otagiri, S. Fujii, and H. Maeda, J. Biochem. (Tokyo) 122, 459 (1997). 61 I. Kluge, U. Gutteck-Amsler, M. Zollinger, and K. Q. Do, J. Neurochem. 69, 2599 (1997). 62 j. S. Stamler and J. Loscalzo, Anal Chem. 64, 779 (1992). 63 t . K. Keefer and D. L. H. Williams, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 509. Wiley, Chichester, UK (1996). 64 A. M. Leone, L. E. Gustafsson, P. L. Francis, M. G. Persson, N. P. Wiklund, and S. Moncada, Biochem. Biophys. Res. Commun. 201, 883 (1994). 65 D. J. Singel and J. R. Lancaster, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 341. Wiley, Chichester, UK (1996). 66 y. m. Henry and D. J. Singel, in "Methods in Nitric Oxide Research" (M. Feelisch and J. S. Stamler, Eds.), p. 357. Wiley, Chichester, UK (1996). 67 S. Christen, P. Gee, and B. N. Ames, Methods Enzymol. 269, 267 (1996). 68 S. Tamir, T. deRojas-Walker, J. S. Wishnok, and S. R. Tannenbaum, Methods Enzymol. 269, 230 (1996).
[39] D e t e r m i n a t i o n o f C e l l - C e l l A d h e s i o n i n R e s p o n s e Oxidants and Antioxidants
to
By SASHWATIROY, CHANDANK. SEN,and LESTER PACKER Introduction Adhesion of leukocytes to endothelial cells is the earliest step in immune recognition process and is mediated by cell adhesion molecules (CAM). I In the early phases of cell adhesion, leukocytes transiently adhere to the vessel wall in a process termed "rolling." Rolling of leukocytes is mediated by a family of adhesive molecules called selectins, expressed both on the leukocyte and endothelial cell surface. 2 After "rolling," leukocytes firmly 1 M. P. Bevilacqua, Annu. Rev. lmmunol. U , 767 (1993). 2 C. W. Smith, Semin. Hematol. 30, 45 (1993).
METHODS IN ENZYMOLOGY, VOL. 300
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