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J. Biol. Chem., Vol. 277, Issue 43, 40666-40674, October 25, 2002

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Induction of Heme Oxygenase 1 by Nitrosative Stress

A ROLE FOR NITROXYL ANION^*

Patrick Naughton, Roberta Foresti, Sandip K. Bains, Martha Hoque, Colin J. Green, and Roberto Motterlini

From the Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex HA1 3UJ, United Kingdom

Received for publication, April 22, 2002, and in revised form, August 11, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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 (t_1/2 = 2.3 min). 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Heme oxygenase, the rate-limiting step in heme degradation to CO and bilirubin, exists in inducible (HO-1)¹ and constitutive (HO-2 and HO-3) isoforms, the synthesis and activities of which are differentially regulated in mammalian tissues (1-3). The common conception that these enzymes are merely components of a catabolic pathway that facilitates the elimination of toxic products from the organism has been disputed by strong evidence demonstrating that endogenously generated CO and bilirubin act as crucial effector molecules in the mitigation of vascular and cellular dysfunction (4-12). Disparate conditions and a number of pathological states including hypoxia, endotoxic shock, atherosclerosis, and inflammation have been found to promote overexpression of the HO-1 gene and increased heme oxygenase activity (13-18). 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 (15, 19-21). 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 (3, 22).

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. 22 and 23; Refs. 15 and 24). 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 (25). 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 (26-28). Subsequent reports have confirmed these findings (24, 29-31), and more recent works have established that NO-related species (such as peroxynitrite and S-nitrosoglutathione) as well as endogenously generated NO and S-nitrosothiols are also capable of HO-1 activation (15, 32). In light of the rather complex and diverse chemistry of the NO group, which enables it to exist in a variety of interrelated redox-activated forms, investigations are now required to explore which additional NO congeners might trigger HO-1 expression. 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),² respectively, the one-electron oxidation and reduction products of NO (33). 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 (33, 34). 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).

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- Sodium trioxodinitrate (Na₂N₂O₃) or AS, diethylamine-NO (DEA/NO), DETA NONOate (DETA/NO), and 2-(4-carboxylphenyl)-4,4,5,5-tetramethylimidazolin-1-oxyl 3-oxide (C-PTIO) were purchased from Alexis Corp. (Bingham, UK). Stock solutions of AS, DETA/NO, and DEA/NO were freshly prepared on the day of the experiments by solubilizing the drugs in 0.1 M NaOH, maintained on ice, and used within 1 h of preparation. C-PTIO was prepared in ethanol at a final concentration of 50 mM. N-Acetyl-L-cysteine (NAC), CuSO₄, buthionine sulfoximine (BSO), and all of the other chemicals were obtained from Sigma unless otherwise specified.

Cell Culture-- Rat H9c2 cells were purchased from the American Tissue Culture Collection (Manassas, VA) and cultured as described previously (35) in complete medium consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 4 mM L-glutamine, 100 units/ml penicillin, and 0.1 mg/ml streptomycin. The cultures were maintained at 37 °C in a 5% CO₂ humidified atmosphere, and the experiments were conducted on confluent cells.

Experimental Protocols-- H9c2 cells were exposed to increasing concentrations of AS (0.25-2 mM) added as a bolus to complete medium. Lower concentrations (50 and 100 µM) of AS were also added to cells at 1-h intervals over a period of 6 h (total addition of 250 and 500 µM, respectively). At the end of the incubations, the cells were harvested and analyzed for heme oxygenase activity, HO-1 protein, and mRNA expression (15). A time course (0-24 h) for heme oxygenase activity and HO-1 protein was also determined in cells incubated with 0.75 mM AS. To establish whether induction of the HO-1 gene occurs at transcriptional levels, the cells were treated with AS (0.75 mM) in the presence of a transcriptional inhibitor, actinomycin D (0.5-1 µg/ml). To examine a possible effect of culture medium components on NO-mediated heme oxygenase activation, the cells were pretreated for 15 min with AS (0.75 mM) in Dulbecco's phosphate-buffered saline (DPBS) followed by incubation in complete medium (DMEM) for 6 h. Notably, the half-life of AS at 37 °C (pH 7.4) is 2.3 min, and, thus, the selected preincubation time allowed the majority of NO to be released and exert its effect prior to the 6-h incubation period with DMEM. In another set of experiments, H9c2 cells were incubated with AS in the presence of increasing concentrations of NAC (1-2 mM) or the glutathione synthesis inhibitor, BSO (0.5 or 1 mM), to assess the effect of thiol groups on NO-mediated induction of heme oxygenase activity and HO-1 protein expression. These treatments were performed both in complete medium for 6 h and in DPBS for 15 min followed by incubation in DMEM for 6 h. Experiments were also conducted to ascertain the direct contribution of NO/NO on heme oxygenase activation. For this purpose, the cells were treated with AS in the presence of CuSO₄ (10-200 µM), which converts NO to NO (36) or C-PTIO (25 µM), a scavenger of NO. To compare the effect of NO with the one elicited by NO on heme oxygenase activity, we performed experiments in which cells were treated with two NO-releasing agents that possess different rates of NO release. The cells were exposed to increasing concentrations (0-2 mM) of DETA/NO (t_1/2 = 20 h at 37 °C) or DEA/NO (t_1/2 = 2 min at 37 °C) in complete medium, and both heme oxygenase activity and HO-1 protein expression were assessed after 6 h of incubation. Cell metabolism and apoptosis were also determined in cells exposed for 6 h to various concentrations of AS, DETA/NO, or DEA/NO. Finally, a direct measurement of NO in solutions containing either AS or the NO-releasing agents was assessed using a Clark-type NO electrode to examine a possible correlation between the amount of NO released and heme oxygenase activation.

Assay for Heme Oxygenase Activity-- Heme oxygenase activity was determined at the end of each treatment using a modification of a method described previously by our group (15, 24, 28, 35). Briefly, the harvested cells were subjected to three cycles of freeze-thawing and sonicated before addition to a reaction mixture consisting of phosphate buffer (1 ml of final volume, pH 7.4) containing magnesium chloride (2 mM), NADPH (0.8 mM), glucose 6-phosphate (2 mM), glucose-6-phosphate dehydrogenase (0.2 unit), 3 mg of rat liver cytosol as a source of biliverdin reductase, and the substrate hemin (20 µM). The reaction was conducted at 37 °C in the dark for 1 h and terminated by the addition of 1 ml of chloroform, and the extracted bilirubin was measured by the difference in absorbance between 464 and 530 nm ( = 40 mM¹ cm¹). Heme oxygenase activity was expressed as picomol of bilirubin/mg protein/h. The total protein content of confluent cells was determined using a Bio-Rad DC protein assay by comparison with a standard curve obtained with bovine serum albumin.

Western Blot Analysis-- Cells treated with the various agents were also analyzed by Western immunoblot technique as described previously by our group (15, 24, 35). Briefly, 30 µg of protein was separated by SDS-polyacrylamide gel electrophoresis and transferred overnight to nitrocellulose membranes, and the nonspecific binding of antibodies was blocked with 3% nonfat dried milk in phosphate-buffered saline. The membranes were then probed with a polyclonal rabbit anti-HO-1 antibody (Stressgen, Victoria, Canada) (1:1000 dilution in Tris-buffered saline, pH 7.4) for 2 h at room temperature. After three washes with phosphate-buffered saline containing 0.05% (v/v) Tween 20, the blots were visualized using an amplified alkaline phosphatase kit from Sigma (Extra-3A), and the relative density of bands was analyzed by an imaging densitometer (model GS-700; Bio-Rad).

RNA Extraction, Northern Blot, and RT-PCR Analyses-- Total RNA was extracted by phenol chloroform using the method described by Chomczynski and Sacchi (37). Total RNA was run on a 1.3% denaturing agarose gel containing 2.2 M formaldehyde and transferred onto a nylon membrane. The membrane was hybridized using [-³²P]dCTP-labeled cDNA probes to rat HO-1 gene as described previously (35, 38), whereas staining of the 18 S rRNA band was used to confirm integrity and equal loading of RNA. The hybridized membrane was exposed to radiographic film, and the bands were analyzed using an imaging densitometer. For RT-PCR analysis, 40 ng of RNA was reverse-transcribed (RT-PCR beads; Amersham Biosciences), and cDNA was amplified using rat primers for HO-1 and -actin as described previously by our group (8).

NO Detection-- NO was measured amperometrically using a NO-sensitive electrode (ISO-NO Mark II, World Precision Instruments, Sarasota, FL). The experiments were performed by immersing the electrode in 10 ml of either DPBS or DMEM followed by addition of the agents under analysis. The NO concentration was recorded continuously under constant stirring at room temperature. Calibration of the electrode was performed daily by measuring known amounts of NO in solution according to the manufacturer's instructions. This consisted of generating NO by cumulative additions of KNO₂ to an acidic solution (0.1 M H₂SO₄) containing 0.1 M KI.

Cell Metabolism and Apoptosis Assay-- Cell metabolism was performed by an Alamar Blue assay according to the manufacturer's instructions (Serotec, UK) as previously reported by us (12, 39). The assay is based on detection of metabolic activity of living cells using a redox indicator that changes from the oxidized (blue) to the reduced (red) form. The intensity of the red color is proportional to the metabolism of cells, which is calculated as the difference in absorbance between 570 and 600 nm and expressed as a percentage of control. Apoptosis was detected using an Annexin V-FITC kit (Clontech, Palo Alto, CA); the percentage of annexin V-positive cells was determined by measuring the fluorescence using a FacsScan flow cytometer (Becton Dickinson) as described previously by our group (23).

Statistical Analysis-- Differences in the data among the groups were analyzed by using one-way analysis of variance combined with the Bonferroni test. The values were expressed as the means ± S.E., and differences between groups were considered to be significant at p < 0.05 or p < 0.01.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Angeli's Salt Increases Heme Oxygenase Activity, HO-1 Protein, and mRNA Expression in Rat H9c2 Cells-- A concentration-dependent increase in heme oxygenase activity was observed in cells exposed for 6 h to 0.25-1 mM AS; higher concentrations of the drug (1.5 and 2 mM) did not further enhance heme oxygenase activity but rather promoted a reduction in the activation of the enzyme (Fig. 1A). These results were reflected by a concentration-dependent increase in HO-1 protein expression, which was maximal at 1 mM AS and gradually declined at 1.5 and 2 mM (Fig. 1B). In contrast, HO-1 mRNA transcript constantly increased from 0.25 to 2 mM AS, indicating that any mechanism interfering with elevation of heme oxygenase activity at higher concentrations of AS (1.5 and 2 mM) is likely to occur at post-transcriptional levels (Fig. 1C). A time course of heme oxygenase activity and HO-1 protein expression in H9c2 cells treated with 0.75 mM AS revealed a maximal activation at 6 h followed by a gradual decline at 12 and 18 h (Fig. 2, A and B, respectively). After 24 h of treatment, heme oxygenase activity and HO-1 protein levels were comparable with control values. Incubation of H9c2 cells with the transcriptional inhibitor actinomycin D (0.5-1 µg/ml) completely blocked AS-mediated increase in both HO-1 mRNA and heme oxygenase activity (Fig. 3). Interestingly, multiple additions of AS at lower concentrations (50 or 100 µM) over a period of 6 h still resulted in a significant (p < 0.05) increase in heme oxygenase activity (Fig. 4).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   AS increases heme oxygenase activity, HO-1 protein expression, and mRNA transcript in rat H9c2 cells. A, effect of various concentrations of AS on heme oxygenase activity. The enzymatic activity was measured spectrophotometrically after exposure of cells to AS (0-2 mM) for 6 h as described under "Experimental Procedures." Each bar represents the mean (± S.E.) of five or six experiments performed independently. *, p < 0.05 versus control (0 mM AS). B, Western blot analysis showing HO-1 protein expression in H9c2 cells exposed for 6 h to various concentrations of AS. The samples were processed and probed with HO-1 antibodies as described under "Experimental Procedures." The blot is representative of three independent experiments. C, Northern blot analysis showing HO-1 mRNA induction in cells exposed for 6 h to various concentrations of AS. The samples were processed as described under "Experimental Procedures." The 18 S band is shown to confirm integrity and equal loading of RNA. The positive control (+ve CON) represents cells treated for 6 h with hemin (50 µM).

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Time course of heme oxygenase activity and HO-1 protein expression in H9c2 cells exposed to AS. A, heme oxygenase activity was determined at different times (0-24 h) after exposure to 0.75 mM AS. Each bar represents the mean (± S.E.) of 5-6 experiments performed independently. *, p < 0.05 versus control (0 h). B, Western blot analysis showing HO-1 protein expression in H9c2 cells exposed to 0.75 mM AS for different times. The blot is representative of three independent experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   AS-mediated induction of HO-1 mRNA and increase in heme oxygenase activity is prevented by actinomycin D. H9c2 cells were exposed to AS (0.75 mM) with or without the transcriptional inhibitor actinomycin D (ActD, 0.5 or 1 µg/ml). After 6 h, HO-1 mRNA expression was determined by RT-PCR (upper panel) using rat HO-1 primers as described under "Experimental Procedures," and -actin was used as an internal control. Similarly, heme oxygenase activity (lower panel) was measured in cells treated for 6 h with AS in the presence or absence of actinomycin D (0.5 µg/ml). Each bar represents the mean (± S.E.) of four experiments performed independently. *, p < 0.05 versus control (CON); , p < 0.05 versus AS.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Effect of multiple additions of AS on heme oxygenase activity in H9c2 cells. AS (50 µM or 100 µM) was added to cells at 1-h intervals over a period of 6 h. AS**, total addition of 250 and 500 µM AS, respectively. Heme oxygenase activity was determined as reported under "Experimental Procedures." Each bar represents the mean (± S.E.) of four experiments performed independently. *, p < 0.01 versus control (CON).

Influence of Medium Components on AS-mediated Increase in Heme Oxygenase Activity-- The NO is a short-lived species in solution and reacts with a variety of targets present in the cellular milieu (40, 41). Because of the complexity of NO chemistry in cell culture media and being aware of a possible interconversion between NO, NO, and NO⁺, we investigated the effect of AS on heme oxygenase activity using DPBS instead of complete medium. For this purpose, the cells were pretreated with AS (0.75 mM) in DPBS for 15 min followed by incubation for 6 h in complete medium. As shown in Fig. 5, AS in DPBS was much less effective in increasing heme oxygenase activity compared with the same treatment in complete medium (1209 ± 134 versus 725 ± 54 pmol bilirubin/mg protein/h, respectively) (p < 0.05). Given that AS has a relatively short half-life (2.3 min at 37 °C), we also wanted to verify whether inactivation of this drug would prevent heme oxygenase activity. For this purpose, "decomposed" AS was prepared by preincubating the NO generator in complete medium (or DPBS) for 1 h at 37 °C. The cells were then exposed to decomposed AS, and heme oxygenase activity was measured after 6 h of treatment. As shown in Fig. 5, no significant increase in heme oxygenase activity was detected in either medium- or DPBS-treated cells containing decomposed AS. These data reveal that NO promotes an increase in heme oxygenase activity in H9c2 cells and are indicative of an interaction between NO and the culture medium components in amplifying heme oxygenase activation. The possibility that the rate of decomposition of AS in culture media is accelerated compared with DPBS cannot be excluded a priori (see below).

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Influence of medium components on AS-mediated increase in heme oxygenase activity. H9c2 cells were exposed to 0.75 mM AS either in complete DMEM or DPBS as described under "Experimental Procedures." Decomposed AS was prepared by preincubating the NO generator in DMEM or DPBS for 1 h at 37 °C prior to cell treatment. Each bar represents the mean (± S.E.) of five or six experiments performed independently. *, p < 0.05 versus control (CON); , p < 0.05 versus AS.

Dissecting the Effects of NO and NO in AS-mediated Increase in Heme Oxygenase Activity-- The interconversion between NO and its redox-activated forms has been reported to occur within the biological systems (33). The extent of these reactions depends upon the composition of the intracellular milieu; in this respect, thiols are known to play a major role because they are preferential sites for the NO groups (33). In particular, NO seems to react more readily than NO with sulfydryl moieties (33, 41, 42). In addition, it is known that certain transition metals, such as Cu²⁺ and Fe³⁺, can catalyze the conversion of NO to NO in vitro and in vivo (36, 43). Therefore, the use of these compounds may help to assess the different bioactivity of NO and NO and their contribution as effector molecules in a specific experimental setting. To verify further that NO generated from AS is directly involved in the activation of the heme oxygenase pathway, we utilized the thiol donor NAC as an exogenous source of thiols and tested its ability to modulate NO-mediated changes in heme oxygenase activity and HO-1 protein expression. As shown in Fig. 6, 1 mM NAC considerably attenuated the increase in heme oxygenase activity caused by 0.75 mM AS (p < 0.05), irrespective of whether cells were incubated in DMEM (Fig. 6A) or DPBS (Fig. 6B). When 2 mM NAC was combined with AS in culture medium, heme oxygenase activation was diminished even further, reaching levels comparable with control. The inhibitory effect of NAC on AS-mediated induction in heme oxygenase activity was associated with a marked reduction in HO-1 protein expression (Fig. 6C), indicating that activation of the HO-1 pathway by NO may proceed via redox reactions with target sulfydryl groups. The cells were also exposed to AS in the presence of the glutathione synthesis inhibitor, BSO. Under these conditions, the increase in heme oxygenase activity elicited by AS was less pronounced (Fig. 7), suggesting that intracellular thiols are important in preserving the bioactivity and signal transduction properties of NO.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   NAC prevents AS-mediated increase in heme oxygenase activity and HO-1 protein levels. Heme oxygenase activity was measured in H9c2 cells 6 h after exposure to 0.75 mM AS with or without NAC (1 and 2 mM). The experiments were carried out either in DMEM (A) or DPBS (B) as described under "Experimental Procedures." Each bar represents the mean (± S.E.) of five or six experiments performed independently. *, p < 0.05 versus control (CON); , p < 0.05 versus AS. C, HO-1 protein expression in H9c2 cells 6 h after exposure to 0.75, 1, or 2 mM AS (in DMEM) with or without an equal concentration of NAC. The blot is representative of three independent experiments.

View larger version (15K): [in this window] [in a new window]   Fig. 7.   Effect of BSO on AS-mediated increase in heme oxygenase activity. Heme oxygenase activity was measured in cardiomyocytes 6 h after exposure to 0.75 mM AS with or without BSO (0.5 and 1 mM). Each bar represents the mean (± S.E.) of four experiments performed independently. *, p < 0.01 versus control (CON); , p < 0.05 versus AS.

To exclude the involvement of NO eventually formed during decomposition of AS in the culture medium, additional experiments were conducted with C-PTIO (25 µM), a NO-trapping agent. As shown in Fig. 8A, the increase in heme oxygenase activity in H9c2 cells exposed to AS was completely unaffected by the presence of the NO scavenger, suggesting that AS-derived NO does not contribute to the observed effect. Interestingly, conversion of NO to NO with CuSO₄ potentiated the AS-mediated increase in heme oxygenase activity (in both DMEM and DPBS) and HO-1 protein expression (Fig. 8, B and C, respectively). Despite the fact that CuSO₄ per se stimulated an increase in HO-1 protein levels at concentrations equal or above 50 µM, 10 µM CuSO₄ was without effect but significantly amplified the increase in heme oxygenase activity mediated by AS. Collectively, these data suggest that, although NO originating from AS does not appear to be an effective HO-1 inducer in our system, the extent and rate of NO oxidation to generate NO might be important determinants for the regulation of HO-1 transcription (23). The results presented below on the effect of DEA/NO and DETA/NO on heme oxygenase activity highlight this intriguing aspect (see "Comparison of the Effects of NO and NO on Heme Oxygenase Activity, Cell Metabolism, and Apoptosis").

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Dissecting the role of NO and NO in AS-mediated increase in heme oxygenase activity. A, effect of C-PTIO on AS-mediated increase in heme oxygenase activity. H9c2 cells were exposed for 6 h to 0.75 mM AS with or without the NO scavenger, C-PTIO (25 µM). Each bar represents the mean (± S.E.) of five or six experiments performed independently. *, p < 0.05 versus control (CON). B, effect of cupric sulfate (CuSO4) on AS-mediated increase in heme oxygenase activity. H9c2 cells were exposed for 6 h to 0.75 mM AS (either in DMEM or DPBS) in the presence of 10 µM CuSO4 and compared with cells treated with AS or CuSO4 alone. Each bar represents the mean (± S.E.) of five or six experiments performed independently. *, p < 0.05 versus control (CON); , p < 0.05 versus DMEM; , p < 0.05 versus AS. C, HO-1 protein expression in H9c2 cells exposed for 6 h to AS (0.75 mM) with or without increasing concentrations of CuSO4 (10-200 µM).

Measurements of NO released from AS in DPBS and culture medium were also performed using a Clark-type NO electrode and compared with the amount of NO released from DEA/NO and DETA/NO, two commonly used NO releasing agents (see table incorporated in Fig. 9). It was found that: 1) 0.5 mM AS in DMEM generates much less NO (0.31 ± 0.01 µM × s) compared with DEA/NO (10.1 ± 0.78 µM × s, p < 0.05) but significantly more than DETA/NO (0.03 ± 0.01 µM × s, p < 0.05); 2) the amount of NO generated from AS in culture medium (0.31 ± 0.01 µM × s) is significantly (p < 0.05) higher than the one measured in DPBS (0.078 ± 0.01 µM × s), clearly indicating that conversion of NO to NO is indeed a feasible process in the cell culture environment; and 3) at a concentration of 20 µM DEA/NO, which releases the same amount of NO over time as 0.5 mM AS in the cell culture medium (0.31 ± 0.01 µM × s and 0.32 ± 0.01 µM × s, respectively), heme oxygenase activity remained unchanged and comparable with control levels over a 6-h period (Fig. 9). Altogether, these data confirm that NO liberated from AS can directly stimulate the expression of the HO-1 pathway and suggest that NO does not contribute to this effect unless its rate of production from NO, and therefore the extent of NO oxidation to NO, is significantly augmented.

View larger version (18K): [in this window] [in a new window]   Fig. 9.   Direct involvement of AS-derived NO in increasing heme oxygenase activity. The table inserted in this figure reports data on the amounts of NO released over time from various agents measured amperometrically using a NO-sensitive electrode. The experiments were performed by immersing the electrode in 10 ml of either DPBS or DMEM followed by the addition of AS, DEA/NO, or DETA/NO as reported under "Experimental Procedures." Each value represents the mean (± S.E.) of four experiments performed independently. *, p < 0.05 versus DPBS; , p < 0.05 versus AS. The figure reports data on heme oxygenase activity measured 6 h after treatment of H9c2 cells with DMEM supplemented with AS (0.5 mM) or DEA/NO (20 µM). As shown by the table, the amounts of NO released from 0.5 mM AS and 20 µM DEA/NO in DMEM are in the same order of magnitude. Each bar represents the mean (± S.E.) of five or six experiments performed independently. *, p < 0.05 versus control (CON).

Comparison of the Effects of NO and NO on Heme Oxygenase Activity, Cell Metabolism, and Apoptosis-- Despite the fact that the small amounts of NO liberated during the decomposition of AS are not responsible for an increase in heme oxygenase activity, specific NO-releasing agents have the ability to stimulate HO-1 induction at appropriate concentrations. DETA/NO, which slowly releases NO (t_1/2 = 20 h at 37 °C), caused a concentration-dependent (0.25-1.5 mM) increase in heme oxygenase activity that was associated with a significant elevation in HO-1 protein (Fig. 10A). After 6 h of treatment, heme oxygenase activity was maximal at 1.5 mM DETA/NO and started to decline at 2 mM, whereas protein expression increased constantly with increasing concentrations of the NO releasing agent. This pattern is very similar to the one observed with AS (Fig. 1). In contrast, the results obtained with DEA/NO indicate that liberation of NO over a short period of time (DEA/NO t_1/2 = 2 min at 37 °C) causes a transient loss in cell metabolism. Although maximal activation of heme oxygenase by DEA/NO after 6 h of treatment occurs at lower concentrations (0.5 mM) compared with DETA/NO (1.5 mM) or AS (1 mM), stimulation of heme oxygenase activity and HO-1 protein expression are significantly impaired at 1.5 and 2 mM (Fig. 10B). Fig. 11 shows that although no adverse effects were detected with AS within the concentration range used, both DETA/NO and DEA/NO caused a reduction in cell metabolism at high concentrations (1-2 mM). It has to be noted that 1.5 and 2 mol of NO dissociate, respectively, from 1 mol of DEA/NO and DETA/NO, whereas 1 mol of NO is released per mol of AS. At the highest concentration used (2 mM), the metabolism of cells treated with DETA/NO and DEA/NO was 79.4 ± 2 and 63.5 ± 1% of control, respectively (p < 0.001). Despite a significant reduction in cell metabolism after treatment with DEA/NO and DETA/NO, no increase in apoptosis was detected even when cells were exposed to 2 mM of the NO releasing agents (see table incorporated in Fig. 11). Taken together, these data typify the intriguing dual aspect of NO in its capacity to induce adaptation to stress and cell injury depending on its rate of generation and the interconversion between NO and its redox-activated forms.

View larger version (26K): [in this window] [in a new window]   Fig. 10.   Effects of DETA/NO and DEA/NO on heme oxygenase activity and HO-1 protein levels. Heme oxygenase activity and HO-1 protein expression were measured in H9c2 cells after treatment with various concentrations (0-2 mM) of DETA/NO (A) or DEA/NO (B) for 6 h according to "Experimental Procedures." Each bar represents the mean (± S.E.) of five or six experiments performed independently. *, p < 0.05 versus control (0 mM DETA/NO or DEA/NO).

View larger version (28K): [in this window] [in a new window]   Fig. 11.   Comparison between the effects of NO and NO on cell metabolism and apoptosis. H9c2 cells were treated with various concentrations (0-2 mM) of AS, DETA/NO, or DEA/NO and both cell metabolism (see bar graph) and apoptosis (see table) were determined after 6 h as described under "Experimental Procedures" using Alamar Blue and Annexin V-FITC kits, respectively. Each bar represents the mean (± S.E.) of six experiments performed independently. *, p < 0.05 versus control (0 mM).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Numerous 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 (33). 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).

(Eq. 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 (33). 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 (44, 45). On the other hand, excessive or uncontrolled nitrosylation can lead to impaired NO metabolism resulting in nitrosative stress and development of disease states (46-48). Considerable attention has also been directed toward the nitrosative chemistry of NO, although its contribution in modulating specific biological activities remains controversial (40). Different proteins and enzymes including mammalian and bacterial hemoglobins (49, 50), ferrocytochrome c (51), superoxide dismutase (52), and NO synthase (53), as well as decomposition of S-nitrosothiols (34), may give rise to NO. The fate of NO within the cellular system is unknown, but this unstable species has been reported to react readily with thiols (41, 42), and in the presence of molecular oxygen or other oxidants, it can generate other RNS with specific reactivity toward cellular targets (54, 55). 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 (56). Recent reports using donors of nitroxyl revealed opposite effects by showing either NO-mediated exacerbation of post-ischemic myocardial injury (43) or cytoprotection against neuronal damage (57). In another study, the positive inotropic effects of NO and its beneficial cardiovascular activities have been reported (58). 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 (8, 24, 32, 38, 59), 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 (15, 22, 23, 28). 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 generated in 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 (15, 24, 59). 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 (23). 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 (t_1/2 = 2.3 min for AS and t_1/2 = 2 min 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 (60) and may account for the pronounced overexpression of HO-1 observed in this and our previous studies (23). 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 (55, 61) and has been shown to increase endothelial HO-1 protein and heme oxygenase activity in vitro (24, 32). 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 (62). 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) (63), and exacerbation of cellular damage was observed only when AS was combined with hydrogen peroxide (56). Of interest, and in agreement with results using NO donors (62), 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 (22, 28) or nitrosating agents (NO⁺ donors) such as S-nitrosoglutathione and S-nitroso-N-acetyl penicillamine (15, 24, 59), 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.

	ACKNOWLEDGEMENT

We are grateful to Prof. Martin Hughes for stimulating discussion.

	FOOTNOTES

^* This work was supported by British Heart Foundation Grants PG/1999-005 and PG/2001-037 (to R. M.) and PG/2000-047 (to R. F.) and by funds from the Dunhill Medical Trust. The National Heart Research Fund and the Wellcome Trust provided travel grants to present this work to international meetings.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, HA1 3UJ, UK. Tel.: 44-20-88693181; Fax: 44-20-88693270; E-mail: r.motterlini@ic.ac.uk.

Published, JBC Papers in Press, August 22, 2002, DOI 10.1074/jbc.M203863200

² After the submission of this manuscript, a report by Shafirovich and Lymar (61) 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.

	ABBREVIATIONS

The 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.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES