Induction of Heme Oxygenase 1 by Nitrosative Stress
2002; Elsevier BV; Volume: 277; Issue: 43 Linguagem: Inglês
10.1074/jbc.m203863200
ISSN1083-351X
AutoresPatrick Naughton, Roberta Foresti, Sandip K. Bains, Martha Hoque, Colin J. Green, Roberto Motterlini,
Tópico(s)Neuroscience of respiration and sleep
ResumoNitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO+), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997)J. Biol. Chem. 272, 18411–18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613–13620). Here, we report that Angeli's salt (AS) (0.25–2 mm), a compound that releases nitroxyl anion (NO−) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO−-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 °C (t12=2.3min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 μm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO−, not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO− oxidation.N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO−. We conclude that NO−, in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress. Nitric oxide and S-nitrosothiols modulate a variety of important physiological activities. In vascular cells, agents that release NO and donate nitrosonium cation (NO+), such as S-nitrosoglutathione, are potent inducers of the antioxidant protein heme oxygenase 1 (HO-1) (Foresti, R., Clark, J. E., Green, C. J., and Motterlini, R. (1997)J. Biol. Chem. 272, 18411–18417; Motterlini, R., Foresti, R., Bassi, R., Calabrese, V., Clark, J. E., and Green, C. J. (2000) J. Biol. Chem. 275, 13613–13620). Here, we report that Angeli's salt (AS) (0.25–2 mm), a compound that releases nitroxyl anion (NO−) at physiological pH, induces HO-1 mRNA and protein expression in a concentration- and time-dependent manner, resulting in increased heme oxygenase activity in rat H9c2 cells. A time course analysis revealed that NO−-mediated HO-1 expression is transient and gradually disappears within 24 h, in accordance with the short half-life of AS at 37 °C (t12=2.3min). Interestingly, multiple additions of AS at lower concentrations (50 or 100 μm) over a period of time still promoted a significant increase in heme oxygenase activity. Experiments performed using a NO scavenger and the NO electrode confirmed that NO−, not NO, is the species involved in HO-1 induction by AS; however, the effect on heme oxygenase activity can be amplified by accelerating the rate of NO− oxidation.N-Acetylcysteine almost completely abolished AS-mediated induction of HO-1, whereas a glutathione synthesis inhibitor (buthionine sulfoximine) significantly decreased heme oxygenase activation by AS, indicating that sulfydryl groups are crucial targets in the regulation of HO-1 expression by NO−. We conclude that NO−, in analogy with other reactive nitrogen species, is a potent inducer of heme oxygenase activity and HO-1 protein expression. These findings indicate that heme oxygenase can act both as a sensor to and target of redox-based mechanisms involving NO and extend our knowledge on the biological function of HO-1 in response to nitrosative stress. Heme oxygenase, the rate-limiting step in heme degradation to CO and bilirubin, exists in inducible (HO-1) 1The abbreviations used are: HO-1, heme oxygenase 1; RNS, reactive nitrogen species; NO−, nitroxyl anion; NO+, nitrosonium cation; AS, Angeli's salt; DEA/NO, diethylamine-NO; DETA/NO, DETA NONOate; C-PTIO, 2-(4-carboxylphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide; NAC, N-acetyl-l-cysteine; BSO, buthionine sulfoximine; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; RT, reverse transcriptase. 1The abbreviations used are: HO-1, heme oxygenase 1; RNS, reactive nitrogen species; NO−, nitroxyl anion; NO+, nitrosonium cation; AS, Angeli's salt; DEA/NO, diethylamine-NO; DETA/NO, DETA NONOate; C-PTIO, 2-(4-carboxylphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide; NAC, N-acetyl-l-cysteine; BSO, buthionine sulfoximine; DMEM, Dulbecco's modified Eagle's medium; DPBS, Dulbecco's phosphate-buffered saline; RT, reverse transcriptase. and constitutive (HO-2 and HO-3) isoforms, the synthesis and activities of which are differentially regulated in mammalian tissues (1Maines M.D. Trakshel G.M. 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Jiang B.H. Chin B.Y. Iyer N.V. Alam J. Semenza G.L. Choi A.M.K. J. Biol. Chem. 1997; 272: 5375-5381Google Scholar, 15Motterlini R. Foresti R. Bassi R. Calabrese V. Clark J.E. Green C.J. J. Biol. Chem. 2000; 275: 13613-13620Google Scholar, 16Yet S.F. Pellacani A. Patterson C. Tan L. Folta S.C. Foster L. Lee W.S. Hsieh C.M. Perrella M.A. J. Biol. Chem. 1997; 272: 4295-4301Google Scholar, 17Ishikawa K. Navab M. Leitinger N. Fogelman A.M. Lusis A.J. J. Clin. Invest. 1997; 100: 1209-1216Google Scholar, 18Willis D. Moore A.R. Frederick R. Willoughby D.A. Nat. Med. 1996; 2: 87-90Google Scholar). Although the molecular mechanism(s) leading to HO-1 induction by these and other conditions remains to be fully elucidated, a common denominator that characterizes the prompt stimulation of HO-1 under most circumstances is the transient decrease in cellular glutathione levels and a drastic change in the redox status of the intracellular milieu (15Motterlini R. Foresti R. Bassi R. Calabrese V. Clark J.E. Green C.J. J. Biol. Chem. 2000; 275: 13613-13620Google Scholar, 19Lautier D. Luscher P. Tyrrell R.M. Carcinogenesis. 1992; 13: 227-232Google Scholar, 20Ewing J.F. Maines M.D. J. Neurochem. 1993; 60: 1512-1519Google Scholar, 21Choi A.M.K. Alam J. Am. J. Respir. Cell Mol. Biol. 1996; 15: 9-19Google Scholar). It is not surprising, therefore, that conditions associated with increased production of reactive oxygen species and reactive nitrogen species (RNS) favor the activation of the HO-1/CO/bilirubin pathway, which is now regarded as an important cellular stratagem to counteract and resist different stress insults (3Maines M.D. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 517-554Google Scholar, 22Foresti R. Motterlini R. Free Radical Res. 1999; 31: 459-475Google Scholar).In the context of redox reactions and signal transduction events that elicit the expression of HO-1 in vascular tissue, the gaseous molecule NO has recently been highlighted as an important biological modulator (see reviews in Refs. 22Foresti R. Motterlini R. Free Radical Res. 1999; 31: 459-475Google Scholar and 23Motterlini R. Green C.J. Foresti R. Antiox. Redox Signal. 2002; 4: 615-624Google Scholar; Refs. 15Motterlini R. Foresti R. Bassi R. Calabrese V. Clark J.E. Green C.J. J. Biol. Chem. 2000; 275: 13613-13620Google Scholar and 24Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Google Scholar). NO has been implicated in a wide range of processes critical to normal functions in the cardiovascular, nervous, and immune systems; the cytotoxic nature of NO has also been extensively emphasized when excessive production of this gas is triggered under certain pathological conditions (25Darley-Usmar V. Wiseman H. Halliwell B. FEBS Lett. 1995; 369: 131-135Google Scholar). The conception that HO-1 might function to counteract the potential toxic effects evoked by NO first emerged from the discovery that certain NO-releasing agents can stimulate an increase in HO-1 transcript and heme oxygenase activity, resulting in protection against oxidative stress (26Kim Y.M. Bergonia H.A. Muller C. Pitt B.R. Watkins W.D. Lancaster Jr., J.R. J. Biol. Chem. 1995; 270: 5710-5713Google Scholar, 27Kim Y.M. Bergonia H. Lancaster Jr., J.R. FEBS Lett. 1995; 374: 228-232Google Scholar, 28Motterlini R. Foresti R. Intaglietta M. Winslow R.M. Am. J. Physiol. 1996; 270: H107-H114Google Scholar). Subsequent reports have confirmed these findings (24Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Google Scholar,29Hara E. Takahashi K. Tominaga T. Kumabe T. Kayama T. Suzuki H. Fujita H. Yoshimoto T. Shirato K. Shibahara S. Biochem. Biophys. Res. 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In fact, the biological response(s) mediated by NO cannot be confined solely to the ability of this free radical to interact with important intracellular targets but must be extended to the reactivity of the nitrosonium cation (NO+) and the nitroxyl anion (NO−), 2After the submission of this manuscript, a report by Shafirovich and Lymar (61Shafirovich V. Lymar S.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7340-7345Google Scholar) revealed that the species generated by Angeli's salt in aqueous solutions at neutral pH is HNO rather than NO−. Although we are aware of these new findings, throughout the text we will refer to the species released by Angeli's salt still as NO− to simplify the terminology of the distinct redox-activated forms of NO. 2After the submission of this manuscript, a report by Shafirovich and Lymar (61Shafirovich V. Lymar S.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7340-7345Google Scholar) revealed that the species generated by Angeli's salt in aqueous solutions at neutral pH is HNO rather than NO−. Although we are aware of these new findings, throughout the text we will refer to the species released by Angeli's salt still as NO− to simplify the terminology of the distinct redox-activated forms of NO.respectively, the one-electron oxidation and reduction products of NO (33Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar). Each of these redox forms can evoke a variety of biological responses depending upon their concentration and location, the presence of thiols, and the composition of the cellular microenvironment (33Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar,34Arnelle D.R. Stamler J.S. Arch. Biochem. Biophys. 1995; 318: 279-285Google Scholar). In the present study, we utilized rat H9c2 cells to examine the effect of a NO− generator (Angeli's salt) on HO-1 protein expression as well as heme oxygenase activity in an attempt to discern the contribution of NO and its redox forms in the cellular adaptation to the stress inflicted by nitrosative reactions (i.e.nitrosative stress).DISCUSSIONNumerous reports point to a crucial role for the free radical NO in regulating physiological processes; however, scientists are becoming aware that the effects elicited by the NO group can be better appreciated by recognizing the complexity of NO chemistry when applied to biological systems. The reactivity of the NO group is dictated by the oxidation state of the nitrogen atom, which enables this diatomic molecule to exist in different redox-activated forms (33Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar). Therefore, in contrast to NO, which contains one unpaired electron in the outer orbital, NO+ and NO− are charged molecules being, respectively, the one-electron oxidation and reduction products of NO (Equation 1). NO+←−e−NO→+e−NO−Equation 1 These chemical congeners of NO can be generated either exogenously or endogenously and may provoke different effects depending on their concentrations and composition of the surrounding cellular microenvironment (33Stamler J.S. Singel D.J. Loscalzo J. Science. 1992; 258: 1898-1902Google Scholar). For instance, persuasive evidence demonstrated that post-translational modifications by reversible transfer of NO+ to critical sulfhydryl residues (S-nitrosylation) is a ubiquitous signaling mechanism in the control of protein activity, underlying the importance of redox-based nitrosative reactions in cellular function (44Stamler J.S. Cell. 1994; 78: 931-936Google Scholar, 45Stamler J.S. Lamas S. Fang F.C. Cell. 2001; 106: 675-683Google Scholar). On the other hand, excessive or uncontrolled nitrosylation can lead to impaired NO metabolism resulting in nitrosative stress and development of disease states (46Hausladen A. Privalle C.T. Keng T. Deangelo J. Stamler J.S. Cell. 1996; 86: 719-729Google Scholar, 47Eu J.P. Liu L.M. Zeng M. Stamler J.S. Biochemistry. 2000; 39: 1040-1047Google Scholar, 48Patel R.P. Moellering D. Murphy-Ullrich J., Jo, H. Beckman J.S. Darley-Usmar V.M. Free Radical Biol. Med. 2000; 28: 1780-1794Google Scholar). Considerable attention has also been directed toward the nitrosative chemistry of NO−, although its contribution in modulating specific biological activities remains controversial (40Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Google Scholar). Different proteins and enzymes including mammalian and bacterial hemoglobins (49Gow A.J. Stamler J.S. Nature. 1998; 391: 169-173Google Scholar, 50Hausladen A. Gow A. Stamler J.S. Proc. Natl. Acad. Sci. U. S. 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Alcohol. 2000; 20: 55-59Google Scholar), and in the presence of molecular oxygen or other oxidants, it can generate other RNS with specific reactivity toward cellular targets (54Patel R.P. McAndrew J. Sellak H. White C.R., Jo, H.J. Freeman B.A. Darley-Usmar V.M. Biochim. Biophys. Acta. 1999; 1411: 385-400Google Scholar, 55Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Google Scholar). In the presence of hydrogen peroxide and transition metals, NO− but not NO appears to cause loss of cell viability by site-specific DNA damage (56Chazotte-Aubert L. Oikawa S. Gilibert I. Bianchini F. Kawanishi S. Ohshima H. J. Biol. Chem. 1999; 274: 20909-20915Google Scholar). Recent reports using donors of nitroxyl revealed opposite effects by showing either NO−-mediated exacerbation of post-ischemic myocardial injury (43Ma X.L. Cao F. Liu G.L. Lopez B.L. Christopher T.A. Fukuto J.M. Wink D.A. Feelisch M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14617-14622Google Scholar) or cytoprotection against neuronal damage (57Kim W.K. Choi Y.B. Rayudu P.V. Das P. Asaad W. Arnelle D.R. Stamler J.S. Lipton S.A. Neuron. 1999; 24: 461-469Google Scholar). In another study, the positive inotropic effects of NO− and its beneficial cardiovascular activities have been reported (58Paolocci N. Saavedra W.F. Miranda K.M. Martignani C. Isoda T. Hare J.M. Espey M.G. Fukuto J.M. Feelisch M. Wink D.A. Kass D.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10463-10468Google Scholar). Although the mechanism by which nitroxyl anion manifests these divergent effects is currently under intense investigation, the possible involvement of this reduced form of NO in the expression of stress inducible genes has not been previously examined. Here, we show for the first time that NO−, generated from the spontaneous degradation of AS, directly promotes an induction of the HO-1 pathway leading to a marked increase in heme oxygenase activity in H9c2 cells. These findings, together with our previous reports on the high inducibility of vascular HO-1 by NO and NO-related species (8Sammut I.A. Foresti R. Clark J.E. Exon D.J. Vesely M.J.J. Sarathchandra P. Green C.J. Motterlini R. Br. J. Pharmacol. 1998; 125: 1437-1444Google Scholar, 24Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Google Scholar, 32Foresti R. Sarathchandra P. Clark J.E. Green C.J. Motterlini R. Biochem. J. 1999; 339: 729-736Google Scholar, 38Vesely M.J.J. Exon D.J. Clark J.E. Foresti R. Green C.J. Motterlini R. Am. J. Physiol. 1998; 275: C1087-C1094Google Scholar, 59Sawle P. Foresti R. Green C.J. Motterlini R. FEBS Lett. 2001; 508: 403-406Google Scholar), extend our knowledge on the versatile biochemical features of HO-1 as a redox-sensitive protein and reinforce our view on a possible role for the heme oxygenase pathway in the cellular adaptation to nitrosative chemistry (15Motterlini R. Foresti R. Bassi R. Calabrese V. Clark J.E. Green C.J. J. Biol. Chem. 2000; 275: 13613-13620Google Scholar, 22Foresti R. Motterlini R. Free Radical Res. 1999; 31: 459-475Google Scholar, 23Motterlini R. Green C.J. Foresti R. Antiox. Redox Signal. 2002; 4: 615-624Google Scholar, 28Motterlini R. Foresti R. Intaglietta M. Winslow R.M. Am. J. Physiol. 1996; 270: H107-H114Google Scholar). Our study emphasizes that the rate of NO production and the conversion of NO to its redox-activated forms by cellular components might be important factors in determining how the expression of cytoprotective enzymes, including HO-1, is regulated and to what extent these defense systems could counteract the damaging effects mediated by RNS.Of particular interest are the findings that multiple additions of AS at concentrations of 50 and 100 μm over a period of time still induced high heme oxygenase activity levels. This suggests that a limiting factor in HO-1 induction by AS is the short half-life of the NO− generator and that, by mimicking more closely a physiological/pathophysiological scenario of continuous NO− production, it is possible to maintain and prolong the signaling activities involved in AS-mediated heme oxygenase activation. At this stage we do not know whether the concentrations of AS used in our experiments are biologically relevant because sensitive methodologies for the measurements of NO− generatedin vivo still need to be developed. That NO−, not AS, is the chemical entity promoting this response is corroborated by the observations that: 1) decomposed AS does not affect the basal levels of heme oxygenase in H9c2 cells and 2) the thiol donor, NAC, significantly prevents AS-mediated increase in heme oxygenase activity. This is in keeping with the notion that thiols, possibly through the formation of endogenous S-nitrosothiols, are important modulators of HO-1 expression by NO and NO-related species (15Motterlini R. Foresti R. Bassi R. Calabrese V. Clark J.E. Green C.J. J. Biol. Chem. 2000; 275: 13613-13620Google Scholar, 24Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Google Scholar,59Sawle P. Foresti R. Green C.J. Motterlini R. FEBS Lett. 2001; 508: 403-406Google Scholar). This concept is further emphasized by the fact that BSO, a glutathione synthesis inhibitor, reduced heme oxygenase activation by AS, implicating endogenous glutathione in the preservation of NO− bioactivity and signaling properties. Moreover, our results are indicative of a direct involvement of redox NO species in the transcriptional activation of the HO-1 gene (23Motterlini R. Green C.J. Foresti R. Antiox. Redox Signal. 2002; 4: 615-624Google Scholar). Notably, the effect of NO− appears to be independent of NO but can be amplified by accelerating the rate of NO− oxidation. The discrimination between these two species was possible by comparing the effect of AS with the one elicited by DEA/NO as these agents release either NO− or NO, respectively, with very similar half-lives ( t12=2.3min for AS and t12=2min for DEA/NO). By using a sensitive NO electrode, we verified that in DMEM, 0.5 mm AS generates the same amount of NO as 20 μm DEA/NO; however, at these concentrations, only AS was capable of significantly increasing heme oxygenase activity. Accordingly, the NO scavenger, C-PTIO, did not affect heme oxygenase activation by AS. Of interest is the finding that AS in DPBS releases considerably less NO (4-fold) compared with AS in complete medium; because the increase in heme oxygenase activity mediated by AS is more pronounced in cells cultured with DMEM than those cultured with DPBS, collectively these data indicate that NO− can directly stimulate HO-1 induction, but at the same time transformation of NO− to NO (and eventually other RNS) by cell culture components can amplify the increase in heme oxygenase activity. This is confirmed by the observation that CuSO4, which catalyzes the oxidation of NO− to NO, potentiates HO-1 expression in the presence of AS. Although the exact mechanism of NO−-mediated increase in heme oxygenase activity needs to be fully elucidated, it appears to involve transcriptional activation of the HO-1 gene because both increases in HO-1 mRNA expression and heme oxygenase activity are completely suppressed by actinomycin D. It is plausible that nitrosation of selective targets (possibly thiol groups) localized in transcription proteins are responsible for activation of inducible genes (60Marshall H.E. Merchant K. Stamler J.S. FASEB J. 2000; 14: 1889-1900Google Scholar) and may account for the pronounced overexpression of HO-1 observed in this and our previous studies (23Motterlini R. Green C.J. Foresti R. Antiox. Redox Signal. 2002; 4: 615-624Google Scholar). It cannot be excluded that formation of other NO intermediates such as peroxynitrite may contribute to HO-1 transcriptional activation because this powerful oxidant can be generated from NO− at physiological pH (55Miranda K.M. Espey M.G. Yamada K. Krishna M. Ludwick N. Kim S. Jourd'heuil D. Grisham M.B. Feelisch M. Fukuto J.M. Wink D.A. J. Biol. Chem. 2001; 276: 1720-1727Google Scholar, 61Shafirovich V. Lymar S.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7340-7345Google Scholar) and has been shown to increase endothelial HO-1 protein and heme oxygenase activity in vitro (24Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Google Scholar, 32Foresti R. Sarathchandra P. Clark J.E. Green C.J. Motterlini R. Biochem. J. 1999; 339: 729-736Google Scholar). The specific transcription factor(s) sensitive to nitrosative reactions that regulate HO-1 induction remains, however, to be identified.When comparing the degree and duration of heme oxygenase activation by NO− with the effect elicited by NO, it is important to assess the way cells adapt to the stress inflicted by these nitrosative species. The use of specific NO-releasing compounds that have the ability to liberate NO at different rates allows us to examine the relative potency of NO in causing the nitrosative stress response. Our data show that DETA/NO, which releases NO at a slow rate generating an amount of ∼0.03 μm × s (see table in Fig. 9), is significantly less effective than DEA/NO (amount of NO release = 10.1 μm × s) at stimulating an early increase HO-1 expression and heme oxygenase activity. This is in line with and partially explained by previous findings showing that, compared with treatment with slow NO releasers, a burst of NO considerably extends the half-life of HO-1 mRNA in human fibroblasts, suggesting that translation-independent mRNA stability could be an important mechanism by which cells sense the NO challenge (62Bouton C. Demple B. J. Biol. Chem. 2000; 275: 32688-32693Google Scholar). Notably, our data seem to indicate that NO− is less harmful than NO because exposure of cells to AS did not result in any detectable reduction in cell metabolism, whereas DEA/NO, which has a half-life similar to that of AS, caused a significant decline in this parameter. Despite this observation, no increase in apoptosis was detected in cells treated with AS or DEA/NO, indicating that the reduction in cell metabolism by NO could be a reversible process. Although these findings appear to be in contrast with previous reports showing cytotoxicity for NO−, it needs to be pointed out that in those studies higher concentrations of AS were used (2–5 mm) (63Wink D.A. Feelisch N. Fukuto J. Chistodoulou D. Jourdheuil D. Grisham M.B. Vodovotz Y. Cook J.A. Krishna M. DeGraff W.G. Kim S. Gamson J. Mitchell J.B. Arch. Biochem. Biophys. 1998; 351: 66-74Google Scholar), and exacerbation of cellular damage was observed only when AS was combined with hydrogen peroxide (56Chazotte-Aubert L. Oikawa S. Gilibert I. Bianchini F. Kawanishi S. Ohshima H. J. Biol. Chem. 1999; 274: 20909-20915Google Scholar). Of interest, and in agreement with results using NO donors (62Bouton C. Demple B. J. Biol. Chem. 2000; 275: 32688-32693Google Scholar), we also found that NO−-mediated HO-1 expression is transient and gradually disappears once the spontaneous generation of NO− ceases. This may have important implications in the design of agents that are capable of amplifying the induction of anti-nitrosative systems without causing a major threat to the cellular components.In analogy with previous reports showing increased heme oxygenase activity in vascular cells challenged with NO releasers (22Foresti R. Motterlini R. Free Radical Res. 1999; 31: 459-475Google Scholar, 28Motterlini R. Foresti R. Intaglietta M. Winslow R.M. Am. J. Physiol. 1996; 270: H107-H114Google Scholar) or nitrosating agents (NO+ donors) such asS-nitrosoglutathione andS-nitroso-N-acetyl penicillamine (15Motterlini R. Foresti R. Bassi R. Calabrese V. Clark J.E. Green C.J. J. Biol. Chem. 2000; 275: 13613-13620Google Scholar, 24Foresti R. Clark J.E. Green C.J. Motterlini R. J. Biol. Chem. 1997; 272: 18411-18417Google Scholar, 59Sawle P. Foresti R. Green C.J. Motterlini R. FEBS Lett. 2001; 508: 403-406Google Scholar), we demonstrate in the present study that NO− induces HO-1 mRNA/protein expression and enhances heme oxygenase activity in H9c2 cells. Our findings corroborate the concept that HO-1 is not only highly sensitive to oxidant challenges but can be finely modulated by redox reactions involving nitrosative chemistry. Heme oxygenase, the rate-limiting step in heme degradation to CO and bilirubin, exists in inducible (HO-1) 1The abbreviations used are: HO-1, heme oxygenase 1; RNS, reactive nitrogen species; NO−, nitroxyl anion; NO+, nitrosonium cation; AS, Ang
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