NO: an Inhibitor of Cell Death

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Limin Liu1 and Jonathan S Stamler*,1,2 process in particular, are among NO's major biological functions. 1 Howard Hughes Medical Institute, Duke University Medical Center, Durham Inhibition of apoptosis by NO was first demonstrated in B NC, 27710, USA lymphocytes5,6 (although a protective effect of NO had 2 Department of Medicine and Cell Biology, Duke University Medical Center, been reported in rat liver7 and rat neurons8). Anti-apoptotic Durham NC, 27710, USA NO effects were subsequently shown in a variety of cells, * corresponding author: JS Stamler, Duke University Medical Center, MSRB Room 321 (Box 2612), Durham N.C. 27710, USA. including T cells, hepatocytes, endothelial cells, neurons, tel: 919-684-6933; fax: 919-684-6998; ovarian follicle cells, eosinophils, thymocytes and embryo- e-mail: [email protected] nic kidney cells (see Table 1). A remarkable feature of these studies is that NO inhibition of cell death is largely independent of the apoptotic stimulus: protection is seen Received 28.6.99; accepted 15.7.99 against anti-Fas antibody, tumor necrosis factor a (TNFa) Edited by G Melino and ceramide on the one hand, and to ultraviolet radiation, growth factor withdrawal and NO synthase blockade on the other hand. Moreover, these salutary effects are broadly Abstract conferred by multiple sources of NO-related activity, Nitric oxide (NO) or related molecules of both endogenous and including constitutive and cytokine-inducible NO exogenous origin inhibit programmed cell death in a variety of synthases, overexpression of NOS and several classes of cells and tissues. This general protective function is largely NO donors. This review will concentrate on the antiapopto- independent of the apoptotic stimulus. S-nitrosylation of the tic effects of endogenous NOS activity. catalytic-site cysteine of caspases is a well-established and possibly widespread mechanism of enzyme inhibition that Anti-apoptotic effects of NO protects from cell death. However, NO may inhibit apoptosis by additional mechanisms. The physiological and pathologi- Lymphocytes cal significance of NO's anti-apoptotic activity remains to be Epstein-Barr virus (EBV)-infected as well as EBV negative determined in most cases. Burkitt's lymphoma cell lines express iNOS (`inducible' NOS) constitutively.6 Inhibition of such iNOS activity with NG- Keywords: nitric oxide; apoptosis; caspase S-nitrosylation monomethyl-L-arginine (L-NMMA) or by substrate depletion resulted in more than threefold increases in the rate of Abbreviations: ActD, actinomycin D; Casp, caspase; Cyt, spontaneous apoptosis, which in these cells was *3% over 6 cytochrome; DETA/NO, (Z)-1-[N-(2-aminoethyl)-N-(2-ami- several days. This increase in apoptosis was partially noethyl)diazen-1-ium-1,2-diolate; FADD, Fas-associating protein blocked by excess L- but not D-arginine and was abrogated with death domain; GSNO, S-nitrosoglutathione; L-NMMA, NG- by concomitant application of the NO donor S-nitroso- monomethyl-L-arginine; NGF, nerve growth factor; NOR3, (+)-(E)- penicillamine. Thus, constitutive low level NOS activity 4-ethyl-2-[(E)-hydroxyimino]-5-nitroso-3-hexenamide; NTR, neu- protects human lymphocyte cell lines from spontaneous rotrophin receptor; SIN-1, 3-morpholinosydnonimine; SNAP, S- apoptosis. Constitutive expression of iNOS also blocked nitroso-N-acetyl-penicillamine; SNP, sodium nitroprusside apoptosis in primary B cell cultures from patients with B-cell chronic lymphocytic leukemia (B-CLL),9 a disorder which results principally from decreased cell death rather than increased cell proliferation.10 In particular, application of the Introduction NOS inhibitor L-NG-nitroarginine methyl ester (L-NAME), Apoptosis is a highly organized process that is characterized either alone or in combination with anti-Fas antibody, by caspase activation, chromatin condensation and DNA substantially increased apoptotic DNA fragmentation in B- fragmentation.1 It functions to remove excess, autoreactive, CLL cells. Thus, endogenous NO is probably anti-apoptotic in damaged or infected cells in development, immune reactions vivo, and might contribute to the extended survival of B-CLL and many other biological processes.2 Apoptosis is tightly cells that is characteristic of the disease. It is not regulated; too little may cause cancer and autoimmune inconceivable that the anti-apoptotic activity of NOS may diseases while too much can manifest in fulminant hepatitis, contribute to the abnormal survival or pathologic proliferation heart failure or neurodegenerative disease.3,4 The inhibition of of cells and, more generally, to accumulation of mutations and such programmed cell death by nitric oxide (NO) or related development of tumors. Indeed, NOS is often highly molecules is a common and widespread phenomenon that expressed in malignant cells.11,12 has attracted considerable interest in the last few years. NOS not only protects B cells against spontaneous Regulation of apoptosis in general, and attenuation of the apoptosis but also from apoptosis induced by Fas NO: an inhibitor of cell death LLiuandJSStamler 938

activation.9,13 The same apparently holds true for T cells antibody, overexpression of FADD or caspase-8 and by and monocytic cell lines.13,14 However, the NO synthases various alternative stimuli (see Table 1).6,16,17 The involved and the mechanism of NO action may be quite biological relevance of this antiapoptotic effect, however, different in each case (Table 1). For instance, constitutive is not known. iNOS expression was detected in the B cells, monocytic cells and two T cell lines,9,13 whereas nNOS was co- expressed with iNOS in Jurkat T cells15 and only eNOS Hepatocytes expression was detected in allogeneically activated gdT The protective effects of NO in the liver were discovered 9 cells.14 years ago.7 Mice were injected with Corynebacterium parvum NO production was not detected in splenic B lympho- and then lipopolysaccharide (LPS)7 in a model of acute cytes from wild-type mice,5 and addition of a NOS inhibitor hepatic necrosis. The LPS resulted in a substantial increase in

(nitromethylarginine) to the culture medium did not change NOx production, which clearly protected from liver damage. the apoptotic response. Nevertheless, exogenous NO or Specifically, suppression of NO production with L-NMMA NO donors significantly delayed and inhibited both markedly exacerbated hepatic necrosis and other markers of spontaneous and antigen-induced apoptosis in these injury. It has been more recently reported that L-NMMA or L- cells.5 Moreover, exogenous NO substantially repressed NAME but not the iNOS-specific inhibitor L-N6-(1-imi- apoptosis in various T cells or B cells activated by anti-Fas noethyl)lysine (L-NIL), potentiate LPS-induced necrosis in

Table 1 Inhibition of apoptosis by NO Cell type NO source Apoptosis induced by Target or pathway* References Lymphocytes B lymphoma cell lines iNOS; SNAP L-NMMA; A23187; Fas+ (7) Casp S-nitrosylation; 6, 13, 15 L-NMMA cGMP-independent B cell chronic lymphocytic iNOS L-NMMA+Fas Unknown 9 leukemia cells Mouse splenic B cells SIN-1; NO; isosorbide dinitrate spontaneous; soluble MHC-I (+) cGMP; (7) decline in 5 Bcl-2 T cell lines iNOS; SNAP; SNP; GSNO Fas+L-NMMA; FADD (7) Casp S-nitrosylation; 13, 15, 16, 17 or Casp8 over expression cGMP-independent Human gdT cells Allogenetically activated eNOS Fas (+) cGMP; (+) ceramide 14 Rodent hepatocytes Cytokine-induced or Spontaneous; TNFa (Fas)+ (7) Casp S-nitrosylation; 20, 23 and liver overexpressed iNOS; SNAP; ActD; TNFa+galactosamine; (+) cGMP; (7) Bcl-2 7, 18, 58 V-PYRRO/NO LPS; hyperthermia cleavage; (7) Cyt c 54, 55 release; (+) HSP70 Endothelial cells Human umbilical vein Shear stress-stimulated NOS; TNFa; OxLDL; oxidative (7) Casp S-nitrosylation; 29, 33, 34 SNP; SNAP; PAPA NONOate stress; angiotensin II; cGMP-independent 27, 28, 35 caspase-3 overexpression Rat aorta Cytokine-induced iNOS; UVA (+) Bcl-2; (7) Bax 38 DETA/NO Sheep pulmonary artery Overexpressed iNOS LPS (7) Casp 30, 31 Porcine aorta DETA/NO; SNAP LPS then arsenite cGMP-independent 32 Neurons Rat embryonic motor eNOS L-NMMA (+) cGMP 40 neuron Differentiated PC12 cell NOS; NO donor NOS inhibitor; NGF withdrawal Unknown 41 Neuroblastoma NB-39-nu SNAP; NOR3 vincristine (7) Casp; cGMP- 44, 59 independent Neuroblastoma SK-N-BE SNAP P75NTR overexpression; cGMP-independent 43 ceramide Human eosinophils Activated U937 cells; SNAP; Fas; spontaneous (+) cGMP 50, 51 ceramide; azide; hydroxylamine U937 monocytic cell line iNOS; GSNO; SIN-1 Fas+L-NMMA (7) Casp; cGMP- 13, 60 independent Rat thymocyte GSNO (2 mM) Dexamethasone Unknown 64 Rat ovarian follicle NOS induced by IL-1b; SNP Spontaneous; L-NMMA Probably cGMP-related 49 Human kidney KEK-293 cell nNOS overexpression Caspase-1 overexpression (7) Casp 57 MCF-7 breast cancer cell SNAP TNFa+ActD (7) Casp; (7) Bcl-2 56 cleavage; (7) Cyt c

Abbreviations: ActD, actinomycin D; Casp; caspase; Cyt, cytochrome; DETA/NO, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)diazen-1-ium-1,2-diolate; GSNO, S- nitrosoglutathione; L-NMMA, NG-monomethyl-L-arginine; NGF, nerve growth factor; NOR3, (+)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitroso-3-hexenamide; NTR, neurotrophin receptor; SIN-1, 3-morpholinosydnonimine; SNAP, S-nitroso-N-acteyl-penicillamine; SNP, sodium nitroprusside, UVA, ultraviolet light A. *(+), increase in level; (7), inhibition involving/via. NO: an inhibitor of cell death LLiuandJSStamler 939 rat liver.18 This finding has been interpreted in terms of a endothelial survival. It was also shown that cutaneous protective role of eNOS but not iNOS in inflammation-induced iNOS expression and NO production were activated by UV necrosis. In contrast, both L-NAME and L-NIL increased irradiation.39 Whether this UV-induced NO production levels of apoptosis in LPS-treated livers. LPS, L-NAME and L- protects against the apoptotic effects of UV irradiation in NIL do not induce apoptosis when given alone. Taken vivo remains to be determined, but the data certainly together, these data suggest that either iNOS or both iNOS support this possibility. and eNOS prevent apoptosis in rat liver, whereas eNOS but not iNOS protects against inflammation-induced necrosis. iNOS is widely expressed in rodent liver cells exposed Neurons to cytokines and LPS.19±22 This NO-related activity NO is produced from both eNOS and nNOS in rat prevented hepatocytes of rats and mice from undergoing embryonic motor neurons. Only eNOS is constitutively spontaneous apoptosis and increased cell viability after expressed.40 Application of L-NAME alone had no effect, prolonged culture.20 Such cytokine-mediated protection but resulted in apoptosis of motor neurons cultured in the was not seen in hepatocytes of iNOS-knockout mice.20 presence of brain-derived neurotrophic factor (BDNF). Additionally, S-nitroso-N-acetyl-penicillamine (SNAP) or Treatment with DETA/NO blocked such L-NAME-induced the liver-selective NO donor V-PYRRO/NO suppressed apoptosis. Inhibition of NOS activity reportedly led to both spontaneous hepatocyte apoptosis and that induced massive apoptosis in fully differentiated PC12 cells that by a combination of actinomycin D (ActD) and TNFa or had been treated with nerve growth factor.41 NO donors anti-Fas antibody in vitro.20,23,24 When constantly infused have also been reported to inhibit apoptosis in both human into rat livers, V-PYRRO/NO almost completely blocked neuroblastoma and rat PC12 cell lines (see Table 1 for the massive liver damage and apoptosis induced by details).17,41 ± 44 Thus, NO from eNOS is likely a survival injection of TNFa and the transcription inhibitor galacto- factor for motor neurons exposed to BDNF, and eNOS, samine.24 Thus NO is an obligatory survival factor for nNOS or iNOS may be obligatory for maintenance of other hepatocytes in vivo, and has therapeutic potential. It is neuronal cells. unclear, however, whether this protective NO function is nNOS is induced by trophic factor deprivation in motor the sole purview of the hepatocyte or if NO derived from neuron cells.45 In this case, L-NAME or L-nitro-arginine other cells in the liver can protect hepatocytes under actually inhibited apoptosis and supported survival of the inflammatory conditions. motor neurons deprived of BDNF. L-NAME protection was abolished by excess L-arginine. Therefore, NO from nNOS contributed to motor neuron apoptosis induced by Endothelial cells neurotrophic factor deprivation. More typically, nNOS has Endothelial integrity and NO production are major been associated with N-methyl-D-aspartate-induced deterrents against atherosclerosis. Shear stress is the neuronal injury,46,47 against which it may also protect principle physiological stimulus for eNOS in vivo.In through feedback down-regulation of channel activity48 cultured endothelial cells, laminar shear stress not only via S-nitrosylation.8 stimulated NO production but also eNOS expression.25,26 Shear stress protected human umbilical venous endothelial cells from apoptosis induced by TNFa,27,28 oxidative Ovarian follicles stress28,29 or oxidized LDL28 in culture, an effect that Interleukin-1b (IL-1b) treatment increased NO production and was partially blocked by L-NMMA. NO donors SNAP and repressed apoptotic DNA fragmentation in rat preovulatory sodium nitroprusside (SNP) further inhibited both TNFa and follicles that had been cultured in vitro.49 L-NMMA decreased

H2O2-induced apoptosis in these studies. Moreover, NO the protective effect of IL-1b whereas sodium nitroprusside from either NO donors or transfected iNOS protected inhibited DNA fragmentation in the follicles. Thus, NO may endothelial cells from apoptosis induced by a variety of play an anti-apoptotic role in preovulatory follicles. The treatments (see Table 1), including LPS,30,31 LPS plus identify of the NO synthase isoform involved, however, is arsenite,32 angiotensin II,33 caspase-3 overexpression34 not known. and TNFa.35 Thus, endogenous NO or related molecules probably play a protective role in endothelial cells, in keeping with their anti-atherogenic and generally salutary Eosinophils functions36 (see the article by Dimmeler and Zeiher in this Hebestreit et al50 reported that NO derived from activated issue). U937 monocyte-like cells protected eosinophils from Fas- Rat aortic endothelial cells upregulate iNOS when mediated apoptosis when the two cells were co-cultured. The exposed to cytokines.37,38 Such iNOS expression and NO generating compounds SNAP, azide and hydroxylamine NO-related activity were positively correlated with cytokine- also inhibited Fas-mediated apoptosis in eosinophils. In induced protection against apoptosis mediated by UVA addition, Beauvais et al51 reported that both azide and irradiation.38 Specifically, the iNOS inhibitor L-N5-imi- hydroxylamine inhibited spontaneous apoptosis of eosino- noethyl ornithine (L-NIO) suppressed NO production and phils that were cultured for more than 48 h in vitro. abolished the anti-apoptotic effect of cytokine treatment, Eosinophils constitutively express iNOS and produce NO,52 whereas pretreatment of the cells with NO donor DETA/ but it remains unclear whether NO from iNOS plays any role in NO protected from UVA-induced apoptosis and increased regulation of apoptosis. NO: an inhibitor of cell death LLiuandJSStamler 940

Mechanism of NO inhibition of apoptosis in TNFa-stimulated hepatocytes was inhibited by either iNOS overexpression or SNAP.55 Overexpression of NOS Cyclic GMP (cGMP) also inhibited caspase activity in endothelial cells,31 kidney Multiple mechanisms or pathways have been identified by cells57 and hepatocytes.55,58 which NO may inhibit apoptosis. cGMP has been implicated in By far the best-established mechanism of caspase some instances but its overall contribution to the NO effect is inhibition is the S-nitrosylation of the catalytic-site generally unclear and its mechanism of action is very poorly cysteine. This has been well demonstrated for several understood. In an excellent study40 and case in point, eNOS classes of NO donors and is also carried out by eNOS and protection of rat motor neurons was shown to be dependent iNOS. For example, SNAP efficiently S-nitrosylated on soluble guanylate cyclase: ODQ (1H-[1, 2, 4] oxadiazolo recombinant caspase-3,20 caspase-1 and caspase-816 in [4, 3-a] quinoxalin-1 one), an inhibitor of soluble guanylate vitro and inhibited purified caspases20,27,54,56,59 and cyclase, resulted in apoptosis, which was reversed by caspase activity in cell lysates.27,59,60 These effects were addition of cell permeable analogs of cGMP. Additionally, reversed by treatment with dithiothreitol, an agent known to cGMP analogs prevented apoptosis that resulted from break down nitrosothiol modifications in proteins.20,54,56,60 inhibition of NO synthesis by L-NAME. However, ODQ (or a Rossig et al34 further demonstrated with electron spin metabolite) can also inhibit NOS and other heme-containing resonance techniques that the overexpressed P17 subunit enzymes.53 Thus while cGMP is almost certainly involved, it of caspase-3 was S-nitrosylated in COS-7 cells exposed to remains unclear to what extent it mediates NOS protection or S-nitrosocysteine or SNP. In contrast, S-nitrosylation of if additional mechanisms of resistance are involved. That said, transfected protein was not observed when the active site a similar case for cGMP-mediated protection has been made cysteine was replaced by alanine. Most significantly, in sympathetic neurons, PC12 cells,42 eosinophils,50,51 endogenous S-nitrosylation of the catalytic-site cysteine of hepatocytes,20,24 and gdT lymphocytes.14 In addition, cGMP native caspase-3 zymogen was recently demonstrated in might be used by ovarian follicle cells49 and splenic B cells.5 unstimulated human lymphocyte cell lines.15 Fas activation The effect of cGMP on apoptosis may be mediated through of these cells resulted in denitrosylation of the catalytic cGMP-dependent kinase,20 but how cGMP-dependent kinase cysteine as well as proteolytic cleavage of caspase-3. It achieves this effect is not known. appears, therefore, that caspase S-nitrosylation helps to NO protection against apoptosis is likely independent of protect cells from unwanted apoptosis by maintaining the cGMP in the Burkitt's lymphoma B cell lines Akata and zymogen in an inactive state, at least in lymphocyte cell 10C9.6,13 Specifically, addition of the cGMP analog 8- lines. It is not clear, however, how widespread or how bromo-cGMP failed to rescue the B cells in which NO critical S-nitrosylation of caspases is in the general synthesis was blocked by L-NMMA. Likewise, 8-bromo- phenomenon of NO protection against apoptosis or cGMP did not mimic the anti-apoptotic effects of NO donors whether this effect should be viewed as salutary or in endothelial cells,32,33 Jurkat T cells16 and SK-N-BE tumorgenic. neuroblastoma cells.43 Moreover, inhibition of soluble guanylyl cyclase by LY83583 or methylene blue did not interfere with the protective effects of SNAP in neuroblas- Bcl-2 and Bax toma NB-39-nu cells.44 Therefore, NO protection is Both the mRNA and protein content of the anti-apoptotic probably independent of cGMP in these cases. protein Bcl-2 was increased by cytokine-induced iNOS or NO donors in rat aortic endothelial cells.38 NO from either source additionally suppressed the upregulation of the pro-apoptotic Caspases Bax protein induced by UVA radiation. NO donors inhibited NO inhibition of caspase activity has been observed in a the cleavage of Bcl-2 protein and the release of cytochrome c number of cells. In particular, L-NMMA increased Fas-induced in both MCF-7 human breast carcinoma cells56 and rat caspase-3-like activity15 and poly (ADP-ribose) polymerase hepatocytes,55 and prevented the decrease of Bcl-2 upon (PARP) cleavage13 in both B and T cells, and reversed the induction of apoptosis in splenic B lymphocytes.5 On the other inhibition of caspase-3-like activity mediated by cytokines in hand, Ceneviva et al30 reported that NO from either SNAP or hepatocytes.20 L-NMMA also inhibited shear stress-induced overexpressed iNOS had no effect on Bcl-2 levels in sheep NO production in human umbilical vein endothelial cells and pulmonary artery endothelial cells. It is not clear how activated caspase-3-like enzymes in cells that were concur- widespread or how important these changes on Bcl-2 and 29 rently treated with H2O2. It is thus clear that endogenous NO Bax are in the case of NO protection against apoptosis. inhibited caspase-3-like activity in these cells. NO donors have also been shown to inhibit stimulus- mediated activation of caspase-3-like activities in lympho- Inducible heat shock protein 70 (HSP70) cyte cell lines,15 ± 17 endothelial cells,27,33,34 hepato- NO-related activity from cytokine-induced iNOS or SNAP cytes,20,54,55 neuroblastoma cells44 and breast carcinoma induced the expression of HSP70 in rat hepatocytes.23 cells.56 More generally, they appear to prevent proteolytic Antisense oligonucleotide to HSP70 blocked its induction by cleavage of caspase-3, whether induced by Fas activation SNAP, and abolished SNAP protection from TNFa and in Jurkat T cells,16 angiotensin II activation in endothelial ActD.23 It is not clear how important HSP70 upregulation is cells33 or TNFa stimulation in hepatocytes.55 In addition, in the anti-apoptotic function of endogenous NO in general or the cleavage of multiple caspases, in particular caspase-8, what the relationship is between HSP70 upregulation and the NO: an inhibitor of cell death LLiuandJSStamler 941

S-nitrosylation of caspases20 that also serve to protect from 9. Zhao H, Dugas N, Mathiot C, Delmer A, Dugas B, Sigaux F and Kolb JP (1998) B- apoptosis in hepatocytes. HSP70 expression was not induced cell chronic lymphocytic leukemia cells express a functional inducible nitric oxide by transfected iNOS, and is unlikely to be involved in NO synthase displaying anti-apoptotic activity. Blood 92: 1031 ± 1043 30 10. Jurlander J (1998) The cellular biology of B-cell chronic lymphocytic leukemia. protection in sheep pulmonary artery endothelial cells. Crit. Rev. Oncol. Hematol. 27: 29 ± 52 11. Gal A and Wogan GN (1996) Mutagenesis associated with nitric oxide production in transgenic SJL mice. Proc. Natl. Acad. Sci. USA 93: 15102 ± 15107 Ceramide 12. Ambs S, Merriam WG, Bennett WP, Felley-Bosco E, Ogunfusika MO, Oser SM, Sciorati et al14 reported that SNAP inhibited the `intermediate' Klein S, Shields PG, Billiar TR and Harris CC (1998) Frequent nitric oxide (within minutes) and reversible accumulation of ceramide that synthase-2 expression in human colon adenomas: implication for tumor angiogenesis and colon cancer progression. Cancer Res. 58: 334 ± 341 resulted from Fas activation in human gdT lymphocytes. While 13. Mannick JB, Miao XQ and Stamler JS (1997) Nitric oxide inhibits Fas-induced prolonged (over hours) and persistent elevation of cellular apoptosis. J. Biol. Chem. 272: 24125 ± 24128 61 ceramide levels may lead to apoptosis, it is not clear how 14. Sciorati C, Rovere P, Ferrarini M, Heltai S, Manfredi AA and Clementi E (1997) important the inhibition of `intermediate' ceramide is in Autocrine nitric oxide modulates CD95-induced apoptosis in gammadelta T protection against Fas-induced apoptosis. In fact, recent lymphocytes. J. Biol. Chem. 272: 23211 ± 23215 evidence suggests that Fas-induced apoptosis in T cells is 15. Mannick JB, Hausladen A, Liu L, Hess DT, Zeng M, Miao QX, Kane LS, Gow AJ independent of ceramide.62,63 On the other hand, SNAP and Stamler JS(1999)Fas-inducedcaspasedenitrosylation.Science284;651 ± 654 inhibited cell death that resulted from addition of exogenous 16. Dimmeler S, Haendeler J, Sause A and Zeiher AM (1998) Nitric oxide inhibits 14 50 ceramides to lymphocytes, eosinophils and neuroblasto- APO-1/Fas-mediated cell death. Cell Growth Differ. 9: 415 ± 422 ma cells.43 17. Melino G, Bernassola F, Knight RA, Corasaniti MT, Nistico G and Finazzi-Agro A (1997) S-nitrosylation regulates apoptosis [letter]. Nature 388: 432 ± 433 18. Ou J, Carlos TM, Watkins SC, Saavedra JE, Keefer LK, Kim YM, Harbrecht BG Conclusion and Billiar TR (1997) Differential effects of nonselective nitric oxide synthase (NOS) and selective inducible NOS inhibition on hepatic necrosis, apoptosis, The anti-apoptotic activity of NO and related molecules has ICAM-1 expression, and neutrophil accumulation during endotoxemia. Nitric been well established, and all evidence points to it playing Oxide1:404±416 a key function in biology. Whether NO ultimately inhibits or 19. Geller DA, Nussler AK, Di Silvio M, Lowenstein CJ, Shapiro RA, Wang SC, promotes (see the review by Brune in this issue) apoptosis Simmons RL and Billiar TR (1993) Cytokines, endotoxin, and glucocorticoids likely depends on the cell, the signal, the source, the regulate the expression of inducible nitric oxide synthase in hepatocytes. Proc. molecule, the amount, and the presence or absence of co- Natl. Acad. Sci. USA 90: 522 ± 526 20. Kim YM, Talanian RV and Billiar TR (1997b) Nitric oxide inhibits apoptosis by reactants. However, inhibition of cell death would appear to preventing increases in caspase-3-like activity via two distinct mechanisms. J. be the more significant NO function. S-nitrosylation of Biol. Chem. 272: 31138 ± 31148 caspase-3 is a well-characterized and possibly conserved 21. Helyar L, Bundschuh DS, Laskin JD and Laskin DL (1994) Induction of hepatic Ito mechanism of inhibition that explains the widespread cell nitric oxide production after acute endotoxemia. Hepatology 20: 1509 ± 1515 protective effect, but additional targets and signal transduc- 22. Laskin DL, Heck DE, Gardner CR, Feder LS and Laskin JD (1994) Distinct tion pathways are being actively identified. NO protects the patterns of nitric oxide production in hepatic macrophages and endothelial cells liver from inflammatory injury and the vessel wall from following acute exposure of rats to endotoxin. J. Leukoc. Biol. 56: 751 ± 758 23. Kim YM, de Vera ME, Watkins SC and Billiar TR (1997a) Nitric oxide protects atherogenesis. More generally, however, the physiological cultured rat hepatocytes from tumor necrosis factor-alpha-induced apoptosis by and pathological significance of NO's anti-apoptotic action inducing heat shock protein 70 expression. J. Biol. Chem. 272: 1402 ± 1411 remains to be determined. 24. Saavedra JE, Billiar TR, Williams DL, Kim YM, Watkins SC and Keefer LK (1997) Targeting nitric oxide (NO) delivery in vivo. Design of a liver-selective NO donor prodrugthatblockstumornecrosisfactor-alpha-inducedapoptosisandtoxicityin the liver. J. Med. Chem. 40: 1947 ± 1954 References 25. Forstermann U, Boissel JP and Kleinert H (1998) Expressional control of the `constitutive' isoforms of nitric oxide synthase (NOS I and NOS III). Faseb J. 12: 1. Thornberry NA and Lazebnik Y (1998) Caspases: enemies within. Science 281: 773 ± 790 1312 ± 1316 26. Noris M, Morigi M, Donadelli R, Aiello S, Foppolo M, Todeschini M, Orisio S, 2. Jacobson MD, Weil M and Raff MC (1997) Programmed cell death in animal Remuzzi G and Remuzzi A (1995) Nitric oxide synthesis by cultured endothelial development. Cell 88: 347 ± 354 cells is modulated by flow conditions. Circ. Res. 76: 536 ± 543 3. Nagata S (1997) Apoptosis by death factor. Cell 88: 355 ± 365 27. Dimmeler S, Haendeler J, Nehls M and Zeiher AM (1997a) Suppression of 4. Evan G and Littlewood T (1998) A matter of life and cell death. Science 281: apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme 1317 ± 1322 (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J. Exp. Med. 5. Genaro AM, Hortelano S, Alvarez A, Martinez C and Bosca L (1995) Splenic B 185: 601 ± 607 lymphocyte programmed cell death is prevented by nitric oxide release through 28. Dimmeler S, Hermann C, Galle J and Zeiher AM (1999) Upregulation of mechanisms involving sustained Bcl-2 levels. J. Clin. Invest. 95: 1884 ± 1890 superoxide dismutase and nitric oxide synthase mediates the apoptosis- 6. Mannick JB, Asano K, Izumi K, Kieff E and Stamler JS (1994) Nitric oxide suppressive effects of shear stress on endothelial cells. Arterioscl. Thromb. produced by human B lymphocytes inhibits apoptosis and Epstein-Barr virus Vasc. Biol. 19: 656 ± 664 reactivation. Cell 79: 1137 ± 1146 29. Hermann C, Zeiher AM and Dimmeler S (1997) Shear stress inhibits H2O2- 7. Billiar TR, Curran RD, Harbrecht BG, Stuehr DJ, Demetris AJ and Simmons RL induced apoptosis of human endothelial cells by modulation of the glutathione (1990) Modulation of nitrogen oxide synthesis in vivo: NG-monomethyl-L- redox cycle and nitric oxide synthase. Arterioscler. Thromb. Vasc. Biol. 17: arginine inhibits endotoxin-induced nitrate/nitrite biosynthesis while promoting 3588 ± 3592 hepatic damage. J. Leukoc. Biol. 48: 565 ± 569 30. Ceneviva GD, Tzeng E, Hoyt DG, Yee E, Gallagher A, Engelhardt JF, Kim YM, 8. Lipton SA, Choi YB, Pan ZH, Lei SZ, Chen HS, Sucher NJ, Loscalzo J, Singel DJ Billiar TR, Watkins SA and Pitt BR (1998) Nitric oxide inhibits lipopolysaccharide- and Stamler JS (1993) A redox-based mechanism for the neuroprotective and induced apoptosis in pulmonary artery endothelial cells. Am. J. Physiol. 275: neurodestructive effects of nitric oxide and related nitroso-compounds [see L717 ± 728 comments]. Nature 364: 626 ± 632 NO: an inhibitor of cell death LLiuandJSStamler 942

31. Tzeng E, Kim YM, Pitt BR, Lizonova A, Kovesdi I and Billiar TR (1997) Adenoviral 47. Sattler R, Xiong Z, Lu WY, Hafner M, MacDonald JF and Tymianski M (1999) transfer of the inducible nitric oxide synthase gene blocks endothelial cell Specific Coupling of NMDA Receptor Activation to Nitric Oxide Neurotoxicity by apoptosis. Surgery 122: 255 ± 263 PSD-95 Protein. Science 284: 1845 ± 1848 32. DeMeester SL, Qiu Y,BuchmanTG,HotchkissRS,DunniganK,KarlIEandCobb 48. Manzoni O, Prezeau L, Marin P, Deshager S, Bockaert J and Fagni L (1992) Nitric JP (1998) Nitric oxide inhibits stress-induced endothelial cell apoptosis [see oxide-induced blockade of NMDA receptors. Neuron 8: 653 ± 662 comments]. Crit. Care Med. 26: 1500 ± 1509 49. Chun SY, Eisenhauer KM, Kubo M and Hsueh AJ (1995) Interleukin-1 beta 33. Dimmeler S, Rippmann V, Weiland U, Haendeler J and Zeiher AM (1997b) suppressesapoptosisinratovarianfolliclesbyincreasingnitricoxideproduction. Angiotensin II induces apoptosis of human endothelial cells. Protective effect of Endocrinology 136: 3120 ± 3127 nitric oxide. Circ. Res. 81: 970 ± 976 50. Hebestreit H, Dibbert B, Balatti I, Braun D, Schapowal A, Blaser K and Simon HU 34. Rossig L, Fichtlscherer B, Breitschopf K, Haendeler J, Zeiher AM, Mulsch A and (1998) Disruption of fas receptor signaling by nitric oxide in eosinophils. J. Exp. Dimmeler S (1999) Nitric oxide inhibits caspase-3 by S-nitrosation in vivo. J. Biol. Med. 187: 415 ± 425 Chem. 274: 6823 ± 6826 51. Beauvais F, Michel L and Dubertret L (1995) The nitric oxide donors, azide and 35. Shen YH, Wang XL and Wilcken DE (1998) Nitric oxide induces and inhibits hydroxylamine, inhibit the programmed cell death of cytokine-deprived human apoptosis through different pathways. FEBS Lett. 433: 125 ± 131 eosinophils. FEBS Lett. 361: 229 ± 232 36. Moroi M, Zhang L, Yasuda T, Virmani R, Gold HK, Fishman MC and Huang PL 52. del Pozo V, de Arruda-Chaves E, de Andres B, Cardaba B, Lopez-Farre A, (1998) Interaction of genetic deficiency of endothelial nitric oxide, gender, and Gallardo S, Cortegano I, Vidarte L, Jurado A, Sastre J, Palomino P and Lahoz C pregnancyin vascularresponseto injury in mice. J. Clin. Invest.101: 1225 ± 1232 (1997) Eosinophils transcribe and translate messenger RNA for inducible nitric 37. Suschek C, Rothe H, Fehsel K, Enczmann J and Kolb-Bachofen V (1993) oxide synthase. J. Immunol. 158: 859 ± 864 Induction of a macrophage-like nitric oxide synthase in cultured rat aortic 53. Feelisch M, Kotsonis P, Siebe J, Clement B and Schmidt HHHW (1999) The endothelial cells. IL-1 beta-mediated induction regulated by tumor necrosis soluble guanylyl cyclase inhibitor, ODQ, is a non-selective heme protein inhibitor factor-alpha and IFN-gamma. J. Immunol. 151: 3283 ± 3291 of NO synthase and other cytochrome P450 enzymes involved in NO donor 38. Suschek CV, Krischel V, Bruch-Gerharz D, Berendji D, Krutmann J, Kroncke KD bioactivation. Mol. Pharmacol. 56: 243 ± 253 and Kolb-Bachofen V (1999) Nitric oxide fully protects against UVA-induced 54. Li J, Billiar TR, Talanian RV and Kim YM (1997) Nitric oxide reversibly inhibits apoptosis in tight correlation with Bcl-2 up-regulation. J. Biol. Chem. 274: 6130 ± seven members of the caspase family via S-nitrosylation. Biochem. Biophys. 6137 Res. Commun. 240: 419 ± 424 39. Kuhn A, Fehsel K, Lehmann P, Krutmann J, Ruzicka T and Kolb-Bachofen V 55. Li J, Bombeck CA, Yang S, Kim YM and Billiar TR (1999) Nitric oxide suppresses (1998) Aberrant timing in epidermal expression of inducible nitric oxide synthase apoptosis via interrupting caspase activation and mitochondrial dysfunction in after UV irradiation in cutaneous lupus erythematosus. J. Invest. Dermatol. 111: cultured hepatocytes. J. Biol. Chem. 274: 17325 ± 17333 149 ± 153 56. Kim YM, Kim TH, Seol DW, Talanian RV and Billiar TR (1998) Nitric oxide 40. Estevez AG, Spear N, Thompson JA, Cornwell TL, Radi R, Barbeito L and suppression of apoptosis occurs in association with an inhibition of Bcl-2 Beckman JS (1998b) Nitric oxide-dependent production of cGMP supports the cleavage and cytochrome c release. J. Biol. Chem. 273: 31437 ± 31441 survival of rat embryonic motor neurons cultured with brain-derived neurotrophic 57. Tenneti L, DM DE and Lipton SA (1997) Suppression of neuronal apoptosis by S- factor. J. Neurosci. 18: 3708 ± 3714 nitrosylation of caspases. Neurosci. Lett. 236: 139 ± 142 41. Peunova N and Enikolopov G (1998) Nitric oxide mediates differentiation and 58. Tzeng E, Billiar TR, Williams DL, Li J, Lizonova A, Kovesdi I and Kim YM (1998) survival of neuronal cells. In: Nitric oxide and the cell (eds. S Moncada, G Nistico, Adenovirus-mediated inducible nitric oxide synthase gene transfer inhibits G Bagetta, EA Higgs), pp 171 ± 178. Portland Press Ltd, London hepatocyte apoptosis. Surgery 124: 278 ± 283 42. Farinelli SE, Park DS and Greene LA (1996) Nitric oxide delays the death of 59. Ogura T, Tatemichi M and Esumi H (1979) Nitric oxide inhibits CPP32-like activity trophic factor-deprived PC12 cells and sympathetic neurons by a cGMP- under redox regulation. Biochem. Biophys. Res. Commun. 236: 365 ± 369 mediated mechanism. J. Neurosci. 16: 2325 ± 2334 60. Mohr S, Zech B, Lapetina EG and Brune B (1997) Inhibition of caspase-3 by S- 43. Lievremont JP, Sciorati C, Morandi E, Paolucci C, Bunone G, Della Valle G, nitrosation and oxidation caused by nitric oxide. Biochem. Biophys. Res. Meldolesi J and Clementi E (1999) The p75(NTR)-induced apoptotic program Commun. 238: 387 ± 391 develops through a ceramide-caspase pathway negatively regulated by nitric 61. Hannun YA (1996) Functions of ceramide in coordinating cellular responses to oxide [In Process Citation]. J. Biol. Chem. 274: 15466 ± 15472 stress. Science 274: 1855 ± 1859 44. Ogura T, DeGeorge T, Tatemichi M and Esumi H (1998) Suppression of anti- 62. Hsu SC, Wu CC, Luh TY, Chou CK, Han SH and Lai MZ (1998) Apoptotic signal of microtubule agent-induced apoptosis by nitric oxide: possible mechanism of a Fas is not mediated by ceramide. Blood 91: 2658 ± 2663 new drug resistance. Jpn. J. Cancer Res. 89: 199 ± 205 63. Watts JD, Gu M, Polverino AJ, Patterson SD and Aebersold R (1997) Fas- 45. Estevez AG, Spear N, Manuel SM, Radi R, Henderson CE, Barbeito L and induced apoptosis of T cells occurs independently of ceramide generation. Proc. Beckman JS (1998a) Nitric oxide and superoxide contribute to motor neuron Natl. Acad. Sci. USA 94: 7292 ± 7296 apoptosis induced by trophic factor deprivation. J. Neurosci. 18: 923 ± 931 64. Sandau K and Brune B (1996) The dual role of S-nitrosoglutathione (GSNO) 46. Gonzalez-Zulueta M, Ensz LM, Mukhina G, Lebovitz RM, Zwacka RM, during thymocyte apoptosis. Cell Signal 8: 173 ± 177 Engelhardt JF, Oberley LW, Dawson VL and Dawson TM (1998) Manganese superoxide dismutase protects nNOS neurons from NMDA and nitric oxide- mediated neurotoxicity. J. Neurosci. 18: 2040 ± 2055