Role of the Lipid Peroxidation Product, 4-Hydroxynonenal, in the

Article pubs.acs.org/crt

Role of the Lipid Peroxidation Product, 4‑Hydroxynonenal, in the Development of Nitrate Tolerance Yohan D’Souza,† Toshihiro Kawamoto,‡ and Brian M. Bennett*,† †

Department of Biomedical & Molecular Sciences, Faculty of Health Sciences, Queen’s University, Kingston, Ontario, Canada K7L 3N6 ‡ Department of Environmental Health, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahatanishi-ku, Kitakyushu 807-8555, Japan ABSTRACT: Tolerance to nitrates such as nitroglycerin (GTN) is associated with oxidative stress, inactivation of aldehyde dehydrogenase 2 (ALDH2), and decreased GTNinduced cGMP accumulation and vasodilation. We hypothesized that GTN-induced inactivation of ALDH2 results in increased 4-hydroxy-2-nonenal (HNE) adduct formation of key proteins involved in GTN bioactivation, and, consequently, an attenuated vasodilator response to GTN (i.e., tolerance). We used an in vivo GTN tolerance model, a cell culture model of nitrate action, and Aldh2−/− mice to assess whether GTN exposure resulted in HNE adduct formation, and whether exogenous HNE affected GTN-induced relaxation and cGMP accumulation. Immunoblot analysis indicated a marked increase in HNE adduct formation in GTN-tolerant porcine kidney epithelial cells (PK1) and in aortae from GTN-tolerant rats and untreated Aldh2−/− mice. Preincubation of PK1 cells with HNE resulted in a dose-dependent decrease in GTN-induced cGMP accumulation, and pretreatment of isolated rat aorta with HNE resulted in dose-dependent decreases in the vasodilator response to GTN, thus mimicking GTN-tolerance. Pretreatment of aortae from Aldh2−/− mice with 10 μM HNE resulted in a desensitized vasodilator response to GTN. In the in vivo rat tolerance model, changes in HNE adduct formation correlated well with the onset of GTN tolerance and tolerance reversal. Furthermore, coadministration of an HNE scavenger during the tolerance induction protocol completely prevented HNE adduct formation and GTN tolerance but did not prevent the inactivation of ALDH2. The data are consistent with a novel mechanism of GTN tolerance suggesting a primary role of HNE adduct formation in the development of GTN tolerance.

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Furthermore, in the original hypothesis of Chen et al.,1 NO2− was proposed as the obligate intermediate in the formation of NO from GTN and that this further biotransformation occurs in mitochondria. However, we have argued that although NO2− is the predominant N,O-containing species formed during the vascular biotransformation of GTN, due to the low vasodilator potency of NO2− and the high endogenous levels of NO2− relative to those that could be derived from pharmacologically relevant concentrations of GTN, it is unlikely that NO2− is the pharmacological activator of sGC or an intermediate in the formation of an activator of sGC.9 Oxidative stress has long been associated with nitrate tolerance, both in humans10 and experimental animals,11 and the oxidative stress hypothesis suggests that chronic GTN treatment results in increased vascular superoxide production secondary to angiotensin II-induced up-regulation of a vascular NADH/NADPH oxidase, resulting in the quenching of NO by reaction with superoxide. However, other studies have shown dissociation between superoxide production and nitrate

INTRODUCTION The clinical utility of antianginal nitrate drugs such as nitroglycerin (glyceryl trinitrate, GTN) is limited by the onset of tolerance, which results in a desensitized vasodilator response. GTN is a prodrug that requires bioactivation to yield nitric oxide (NO) or an NO-like species (termed NO bioactivity) resulting in the activation of soluble guanylyl cyclase (sGC), increased cGMP production, and vascular smooth muscle relaxation. Among the numerous proposals to explain nitrate tolerance, decreased bioactivation to the proximal activator of sGC is considered a primary mechanism. Aldehyde dehydrogenase 2 (ALDH2) has been proposed as a major bioactivating enzyme of GTN, based on the findings that ALDH activity is decreased during GTN tolerance and that ALDH2 inhibitors (albeit nonspecific) attenuate the vasodilator response to GTN and inhibit GTN-induced cGMP formation.1 Other studies have shown that Aldh2 null mice exhibit a desensitized response to GTN2 and that ALDH2 activity and expression are decreased in GTN tolerant tissues.3−6 In contrast, a number of studies have questioned the role of ALDH2 in nitrate action, suggesting that ALDH2 may not be directly involved in GTN bioactivation,3,7−9 let alone tolerance. © 2014 American Chemical Society

Received: December 31, 2013 Published: February 20, 2014 663

dx.doi.org/10.1021/tx4004787 | Chem. Res. Toxicol. 2014, 27, 663−673

Chemical Research in Toxicology

Article

Animal Care Committee. Animals were maintained under a 12 h light/ dark cycle, with free access to food and water. Rats (male Sprague− Dawley, 300−400 g, Charles River Laboratories, Montreal, QC, Canada) were randomly assigned into GTN-treated or sham groups with each group consisting of 3−4 animals. In some experiments, rats in GTN-treated or sham groups were administered AG-01 (20 mg/kg, intraperitoneally) or the vehicle just prior to the implantation of GTNcontaining or drug-free patches and then a second dose 24 h later. Wild type male C57BL/6 mice (20−30 g) were obtained from Jackson Laboratory, (Bar Harbor ME) and matched with respect to age to Aldh2−/− mice, kindly provided by Dr. T. Kawamoto (University of Occupational and Environmental Health, Kitakyushu, Japan). The Aldh2−/− mice were generated as previously described20 and have a C57BL/6 background, backcrossed for at least 10 generations. Cell Culture. LLC-PK1 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cells were inoculated at a density of 3.0 × 105 cells per well in 6-well plates in 2.0 mL of DMEM/Ham’s F12 medium (1:1) supplemented with 10% fetal bovine serum, 5 μg/mL insulin, 2 mM glutamine, 10 mM HEPES (pH 6.9), 50 U/mL penicillin, and 50 μg/mL streptomycin, and grown at 37 °C in an atmosphere of 5% CO2 in air until confluent. In some experiments, cells were preincubated with 10 μM GTN for 2 h prior to assessment of HNE adduct formation, in order to simulate GTN tolerance.8 Induction of GTN Tolerance. GTN tolerance was induced by exposing rats or mice to a continuous source of GTN via the subdermal implantation of two 0.2 mg·h−1 (1/4 patch for mice) transdermal GTN patches for 48 h as previously described.21 Briefly, under isoflurane anesthesia, a 1-cm transverse incision was made in the upper dorsal region of the animal and the skin separated from the underlying fascia by blunt dissection. Two transdermal patches were inserted back to back into the resulting subdermal space. The site was sutured closed and disinfected with 10% providone−iodine solution. Animals were administered buprenorphine (0.05 mg·kg−1 s.c.) preoperatively. The site was reopened after 24 h, and both patches were replaced. Animals in the sham groups received identical treatments; however, sham (drug-free) patches were used instead. Animals were then sacrificed, and the aorta was removed for functional or biochemical analysis. Ex Vivo Aorta Studies. Rats were sacrificed, and the thoracic aortae were removed, cleaned, and cut into 4 equal pieces and placed in 6-well plates with 2.0 mL of DMEM/Ham’s F12 medium (1:1) supplemented with 10% fetal bovine serum, 5 μg/mL insulin, 2 mM glutamine, 10 mM HEPES (pH 6.9), 50 U/mL penicillin, and 50 μg/ mL streptomycin, and incubated at 37 °C in an atmosphere of 5% CO2 in air. Aortic segments were allowed to equilibrate for 1 h before exposure to vehicle (saline), GTN (1 μM), or HNE (10 μM) for 24 h. Immunoblot and Dot Blot Analyses. Aortae were homogenized in lysis buffer (25 mM HEPES at pH 7.0, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.1% SDS, and protease inhibitors (Roche Diagnostics, Mannheim, Germany)) and centrifuged at 480g for 10 min. Proteins in the supernatant fraction were separated by SDS− PAGE on 10% gels and transferred electrophoretically to PVDF membranes. Blots were probed with a rabbit polyclonal antibody to HNE or ALDH2 and immunoreactive bands visualized by enhanced chemiluminescence. Membranes were then stripped and reprobed with a mouse monoclonal antibody to β-actin to ensure equal loading. Immunoreactive bands were quantified by optical densitometry using ImageJ software (version 1.43). For dot blot analysis, 20 μg of aortic protein was spotted directly onto a PVDF membrane, dried, and incubated with 3% bovine serum albumin for 1 h at room temperature and then probed with anti-HNE antibody. Immunoprecipitation. Whole cell homogenates from aortae or liver (200 μg protein) were incubated with 2 μg of anti-HNE antibody overnight at 4 °C with gentle rotation, followed by incubation with 15 μL of protein A/G PLUS agarose beads for an additional 2 h. The beads were pelleted and washed 4 times with phosphate buffered saline (pH 7.4), and bound proteins were eluted by boiling in Laemmli sample buffer for 5 min followed by centrifugation at 1000g for 1 min. Immunoprecipitated proteins were separated by SDS−PAGE on 7.5%

tolerance, and have suggested that the initiating step in tolerance involves GTN-induced S-oxidation of multiple cysteine-containing proteins.12 A hallmark of oxidative stress is lipid peroxidation, which is considered to be important in the pathogenesis of proliferative and neurodegenerative diseases.13,14 Lipid peroxidation results in the formation of several reactive aldehydes, such as acrolein, malondialdehyde, and 4-hydroxy-2-nonenal (HNE).15,16 Among these, HNE is one of the most abundant and cytotoxic aldehydes and forms DNA and protein adducts, resulting in altered cell function. Under physiological conditions, the concentration of HNE can vary between 0.1 to 3 μM. However, under periods of oxidative stress, the concentration of HNE can reach up to 5 mM.15,16 Three detoxification pathways for HNE have been described: conjugation with glutathione by glutathione transferases, reduction by several aldo-keto reductases, and oxidation by ALDH2. Of the three, ALDH2 has the highest catalytic efficiency for HNE oxidation and is the most sensitive to irreversible inactivation by HNE.17 These findings raise the possibility that ALDH2 is inactivated by HNE formed as a consequence of oxidative stress induced by GTN tolerance. Alternatively, enzyme inactivation via oxidation of critical sulfhydryl groups in the active site of ALDH2 by GTN5,18 could result in impaired catabolism of HNE by ALDH2. In both cases, inactivation of ALDH2 could result in decreased clearance of HNE and would be expected to result in increased HNE protein adduct formation. If certain of these proteins are involved in GTN bioactivation, the consequence would be an attenuated vasodilator response to GTN. In the present study, we used an in vivo GTN tolerance model and a cell culture model of nitrate action to assess the role of HNE in the development of nitrate tolerance. More specifically, we determined whether GTN tolerance results in increased HNE adduct formation, whether HNE alters GTNinduced cGMP formation, or GTN-induced vascular relaxation, and whether the HNE scavenger L-histidyl hydrazide (AG-01) prevents GTN tolerance.

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EXPERIMENTAL PROCEDURES

Drugs and Solutions. Transdermal GTN patches were obtained as Transderm-Nitro (0.2 mg/h) from Novartis Pharmaceuticals (Dorval, QC, Canada). Drug-free patches were produced by soaking patches for at least 2 days in 95% ethanol (patches were allowed to airdry for at least 15 min before implantation). Removal of GTN from the patches by this procedure was confirmed by the absence of GTN or GTN metabolites in the plasma of rats implanted with these sham patches.19 GTN was obtained as a solution (TRIDIL, 5 mg/mL) in ethanol, propylene glycol, and water (1:1:1.33) from Sabex Inc. (Boucherville, QC, Canada). Isoflurane for inhalational anesthesia was obtained from Halocarbon Laboratories (River Edge, NJ). Chemiluminescence reagents were from Kirkegaard and Perry Laboratories (Gaithersburg, MA). The rabbit antihuman ALDH2 antiserum was a gift from Dr. V. Vasiliou (University of Colorado Health Science Center, Denver, CO), a rabbit polyclonal anti4-HNE antibody (HNE11-S) was obtained from Alpha Diagnostics International (cat #HNE11-S; San Antonio, TX), and a mouse monoclonal anti-β-actin antibody was obtained from Sigma (St. Louis, MO, USA). HNE was purchased from Cayman Chemicals (Ann Arbor, MI). Protein A/GAgarose Plus was purchased from Santa Cruz Biotechnology (sc-2003; Santa Cruz, CA, USA). All other chemicals were of reagent grade and were obtained from a variety of commercial sources. Animals. All procedures for animal experimentation were undertaken in accordance with the principles and guidelines of the Canadian Council on Animal Care and were approved by the Queen’s University 664



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Figure 1. HNE adduct formation in GTN-tolerant PK1 cells and aortae from GTN-tolerant rats and naı̈ve Aldh2−/− mice. Representative immunoblot of (A) PK1 cells exposed to 10 μM GTN for 2 h, (B) aortae from control and GTN-tolerant rats, and (C) aortae from wild type and Aldh2−/− mice. Thirty micrograms of whole-cell homogenates were resolved on a 10% SDS−PAGE gel, transferred to a PVDF membrane and probed with antibody to HNE or β actin as indicated. (D) Immunoreactive bands were quantitated by densitometry. Data are presented as the mean ± SD (n = 4) and were analyzed by a Student’s t-test for unpaired data. Asterisks (*) were used to indicated significant differences from the control or wild type (***p < 0.001). substrate (0.05 or 5 mM propionaldehyde).24,25 ALDH2 activity is reflected by changes in A340 at low substrate concentration. Data Analysis. Relaxation responses to GTN were measured as the percentage decrease in phenylephrine-induced tone. EC50 values for relaxation were determined from the concentration−response curves using a sigmoidal dose−response curve-fitting algorithm. All data are expressed as the mean ± SD and were analyzed by two-way analysis of variance with a Bonferroni posthoc test or Student’s t test for unpaired data, as indicated. A p-value of 0.05 or less was considered statistically significant.

gels and probed with a rabbit polyclonal antibody to ALDH2. Cell lysate and protein A/G Plus agarose beads without the addition of anti-HNE antibody were used as a negative control. Additional control experiments using HNE-conjugated BSA and unconjugated BSA confirmed the specificity of the anti-HNE antibody. GTN-Induced cGMP Accumulation. Cells grown to confluence in 6-well plates were washed twice with phosphate-buffered saline and incubated in serum-free media for 30 min with HNE (1−100 μM). Cells were then incubated in serum-free medium containing 0.5 mM isobutylmethylxanthine for 3 min at 37 °C with GTN (1 μM) or 1,1diethyl-2-hydroxy-2-nitrosohydrazine (DEA/NO) (10 μM). After incubation, the medium was aspirated, and the cells were immediately treated with 6% ice-cold trichloroacetic acid and cGMP determined by radioimmunoassay.22 Isolated Blood Vessel Relaxation Responses. Isolated rings of aorta (2−3 mm) from rats or mice were prepared for isometric tension measurements and were equilibrated for 1 h at an optimal resting tension of 9.8 mN for rat aorta 23 and 5 mN for mouse aorta. Vascular preparations were contracted submaximally with phenylephrine (0.2− 5 μM), and after the induced tone had stabilized, cumulative concentration−response curves were obtained for GTN (0.1 nM−30 μM) or DEA/NO (0.1 nM−10 μM). In some experiments, aortic rings were preincubated with HNE (1−100 μM) for 30 min followed by washout, prior to obtaining relaxation responses to GTN or DEA/ NO. Quantification of Hepatic Mitochondrial ALDH Activity. Rat livers were homogenized in 0.25 M sucrose, 5 mM Tris-HCl, and 0.5 mM EDTA, pH 7.2, and centrifuged at 480g; the supernatant was centrifuged at 4,800g to obtain the mitochondrial fraction. Mitochondria were solubilized with deoxycholate (2.5 mg/mg protein), and ALDH activity was measured as the change in A340 during incubation with 1 mM NAD + in 50 mM sodium pyrophosphate, pH 8.8, containing 2 μM rotenone (to inhibit NADH consumption by complex I of the electron transfer chain), 1 mM 4-methylpyrazone (to inhibit alcohol dehydrogenase), and

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RESULTS

Detection of HNE Adducts in GTN Tolerance. Exposure of PK1 cells to 10 μM GTN for 2 h resulted in a marked increase in HNE adduct formation (Figure 1A). We detected an increase in the number of immunoreactive bands as well as an increase in the density of HNE bands (p < 0.001). Together, this resulted in a 2.5-fold increase in HNE adduct formation after the exposure of PK1 cells to GTN (Figure 1D). GTN tolerance induction in rats also resulted in marked increases in aortic HNE adduct formation (Figure 1B). Similar to adduct formation in PK1 cells, there was a significant increase in the number and density of adducts formed, resulting in a 2-fold increase in HNE adduct formation compared to aortae from sham-treated rats (Figure 1D). We also assessed HNE adduct formation in aortae from Aldh2−/− mice and observed a 4-fold increase in HNE adduct formation compared to that from aortae from wild type mice (Figure 1D), suggesting a marked increase in oxidative stress in these animals. Similar to the findings in GTN-tolerant PK1 cells and GTN-tolerant rat aorta, an increase in the density and number of HNE immunoreactive bands was observed (Figure 1C). 665



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HNE Inhibits GTN-Induced cGMP Accumulation in PK1 Cells. Cellular cGMP levels were measured in response to 1 μM GTN in the presence or absence of HNE. The preincubation of PK1 cells with HNE resulted in a concentration-dependent decrease in GTN-induced cGMP accumulation (Figure 2A). A 30 min preincubation with 10

Figure 2. Effect of HNE incubation on GTN-induced and DEA/NOinduced cGMP formation in PK1 cells. PK1 cells were incubated for 30 min with the concentrations of HNE indicated and then exposed to 1.0 μM GTN (A) or 10 μM DEA/NO (B) for 3 min in the presence of 0.5 mM isobutylmethylxanthine, after which cGMP accumulation was quantitated by a radioimmunoassay. Data are presented as the mean ± SD (n = 4) and were analyzed by a Student’s t-test for unpaired data. * indicates significant difference from the control (*p < 0.05; ***p < 0.001).

Figure 3. HNE exposure results in decreased vasodilator responses to GTN in rat aorta. Isolated aortic rings from rats were incubated with 0.1, 1, and 10 μM HNE for 30 min, followed by washout. Aortic rings were then contracted submaximally with phenylephrine, and cumulative concentration−response curves for (A) GTN or (B) DEA/NO were obtained. Data are presented as the mean ± SD (n = 3).

μM HNE resulted in a 30% decrease in the cGMP response to 1 μM GTN, whereas incubation with 30 μM and 100 μM HNE resulted in an over 60% and 90% reduction in GTN-induced cGMP formation, respectively. In order to determine if HNE impaired the cell’s ability to respond to NO, we used the spontaneous NO-releasing compound, DEA/NO. Preincubation with various concentrations of HNE had no effect on the cGMP response to 10 μM DEA/NO (Figure 2B) indicating that the ability of sGC to respond to NO was unaltered by HNE exposure and suggesting that HNE specifically inhibits GTN bioactivation. HNE Inhibits GTN-Induced Vascular Relaxation. A 30 min preincubation of rat aorta with HNE resulted in concentration-dependent rightward shifts in the concentration−response curve to GTN (Figure 3A). The EC50 value for GTN-induced relaxation of control rat aorta was 12 ± 6 nM. A 0.1 μM HNE treatment resulted in a 2.5-fold decrease in sensitivity to GTN with a corresponding EC50 value of 29 ± 6 nM (p < 0.01). Similarly, preincubation with 1 μM and 10 μM HNE resulted in significant decreases in GTN potency and

EC50 values for relaxation of 79 ± 15 nM and 190 ± 67 nM (p < 0.001). No significant differences were observed in the maximum contractile responses to phenylephrine or the maximum relaxation responses to GTN. Similar to the cGMP responses to DEA/NO in PK1 cells after HNE treatment, the treatment of blood vessels with HNE did not alter the vasodilator activity of DEA/NO, indicating that the responsiveness of aortic sGC to activation by NO was not affected by HNE treatment (Figure 3B), again suggesting that HNE specifically inhibits GTN bioactivation. Using an in vivo GTN tolerance protocol in which mice were continuously exposed to GTN for 48 h, we observed a 4-fold rightward shift in the concentration−response curves for GTN in aortae from Aldh2−/− mice (EC50 values of 340 ± 52 nM in control vs 1.5 ± 0.95 μM in tolerant (p < 0.01)), and a 6-fold rightward shift in aortae from wild type mice (EC50 values of 61 ± 31 nM in control vs 390 ± 52 nM in tolerant mice (p < 0.01)) (Figure 4A). Pretreatment of Aldh2−/− mouse aorta for 30 min with 0.1 μM or 1 μM HNE did not significantly alter 666



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in rats exposed to GTN for 24 and 48 h, respectively, with corresponding EC50 values of 31 ± 5 nM and 78 ± 26 nM vs the control value of 9 ± 1 nM (p < 0.001) (Figure 5E). A 1-day nitrate-free period after 48 h of GTN exposure resulted in complete restoration of the relaxation response to GTN, consistent with the return of HNE adduct formation to control levels. HNE Scavenger AG-01 Prevents GTN Tolerance. Previous studies have reported that the histidine analogue, AG-01, is a highly effective HNE scavenger and significantly protects against HNE-induced oxidative damage.26,27 Accordingly, we determined whether AG-01 inhibited GTN-induced HNE adduct formation, protected against GTN-mediated inhibition of ALDH2 activity, or prevented the development of GTN tolerance. Dot blot analysis revealed that AG-01 significantly reduced HNE adduct formation in both control and GTN tolerant rat aorta (Figure 5B and D). Furthermore, AG-01 administration completely reversed the impaired vasodilator response to GTN resulting from GTN treatment (Figure 5F). GTN Tolerance Results in HNE Adduct Formation with ALDH2. To determine whether HNE adducts of ALDH2 are formed in GTN-tolerant tissues, we performed immunoprecipitation/immunoblot (IP/IB) analysis on GTN-tolerant rat aorta. IP/IB analysis indicated the presence of HNE adducts on ALDH2 after a 48 h exposure to GTN (Figure 6A) suggesting that a significant interaction between HNE and ALDH2 occurs during GTN tolerance development. The administration of AG-01 significantly reduced HNE adduct formation during GTN tolerance (Figure 5B and D), and consistent with these observations, the administration of AG-01 prevented the formation of HNE adducts on ALDH2 during GTN tolerance (Figure 6B). ALDH2 Inactivation Occurs in the Absence of HNE Adduct Formation in GTN Tolerance. To determine whether prevention of HNE adducts of ALDH2 by AG-01 affects ALDH2 enzyme activity, we performed IP/IB analysis and measured ALDH2 and total ALDH activity in liver mitochondria from control and GTN-tolerant rats. We used hepatic ALDH2 activity as a surrogate marker for vascular ALDH2 activity due to the paucity of vascular ALDH2 and, consequently, the large amount of tissue required for vascular ALDH2 activity measurements.9 The administration of AG-01 prevented the formation of HNE adducts on hepatic ALDH2 during GTN treatment (Figure 7A) and is consistent with the findings observed in GTN-tolerant aortae (Figure 6B). However, the administration of AG-01 did not prevent GTNinduced inhibition of ALDH2 activity, and an 8.5-fold reduction in ALDH2 activity was observed. The specific activity values for ALDH2 of 1.3 ± 0.2 and 11.1 ± 1.6 nmol/min/mg for GTN-treated and control rat liver, respectively (p < 0.001, Figure 7B), are very similar to those obtained previously but in the absence of AG-01 treatment.9 Assuming that AG-01 inhibits HNE adduct formation in liver and blood vessels to the same degree (as suggested by comparison of Figures 6B and 7A), these data suggest that a direct inhibitory effect of GTN on the enzyme is sufficient to cause ALDH2 inactivation. Downregulation of ALDH2 by HNE and GTN. Exposure of rat aortic segments ex vivo to 1 μM GTN or 10 μM HNE for 24 h resulted in approximately a 2.5-fold increase in HNE adduct formation (p < 0.001) (Figure 8A and B), concomitant with a 30% and 40% decrease in ALDH2 expression after

Figure 4. GTN-induced tolerance in wild type and Aldh2−/− mice, and the effect of HNE on GTN-induced relaxation of aortae from Aldh2−/− mice. (A) Isolated aortic rings from the GTN-tolerant wild type or Aldh2−/− mice were contracted submaximally with phenylephrine, and cumulative concentration−response curves for GTN were obtained. (B) Aortae from Aldh2−/− mice were incubated with 0.1, 1, or 10 μM HNE for 30 min followed by washout. Aortic rings were then contracted submaximally with phenylephrine and cumulative concentration−response curves for GTN obtained. Data are presented as the mean ± SD (n = 5−8).

GTN-induced relaxation, with EC50 values of 370 ± 170 nM and 860 ± 170 nM, respectively (Figure 4B). However, incubation with 10 μM HNE resulted in a 4-fold rightward shift in the concentration−response curve for GTN with a corresponding EC50 value of 1.4 ± 0.7 μM (p < 0.01 vs untreated). This 4-fold rightward shift was similar in magnitude to the shift observed in aortae from GTN-tolerant Aldh2−/− mice. Time Course of HNE Adduct Formation during GTN Tolerance Development and Reversal. An approximate 1.5-fold and 3-fold increase in HNE adduct formation was observed in aortae taken from rats exposed to GTN for 24 and 48 h, respectively (p < 0.001) (Figure 5A and C). However, a 48 h exposure to GTN followed by a 1-day nitrate-free period resulted in no significant differences in HNE adduct formation compared to that of the control. The increases in HNE adduct formation observed after a 24 and 48 h exposure to GTN correlated well with the decreased functional responses to GTN in rats exposed to GTN; 3.5-fold and 8.5-fold rightward shifts in the concentration−response curve for GTN were observed 667



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Figure 5. HNE adduct formation correlates with vascular GTN tolerance and reversal, and AG-01 prevents GTN-induced HNE adduct formation and vascular GTN tolerance. Dot blot analysis of (A) aortae from rats treated with vehicle (control), GTN for 24 or 48 h, or GTN for 48 h followed by a 1-day nitrate free interval (1 day off) and (B) aortae from vehicle or 48 h GTN-treated rats coadministered AG-01 (20 mg/kg i.p.). Twenty micrograms of whole-cell homogenates were spotted onto a PVDF membrane and probed with an HNE antibody. Immunoreactive dots from A and B were quantitated by densitometry in panels C and D, respectively. Data are presented as the mean ± SD (n = 4) and were analyzed by a one-way ANOVA with a Newman-Keuls posthoc test. Asterisks (*) were used to indicated significant differences from the control (***p < 0.001, **p < 0.01, and *p < 0.05). Isolated aortic rings from rats treated as in A and B were contracted submaximally with phenylephrine, and cumulative concentration−response curves for GTN were obtained (panels E and F, respectively). Data are presented as the mean ± SD (n = 4).

homogenates from GTN-treated animals coadministered AG01 indicated no loss of ALDH2 protein (Figure 9).

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DISCUSSION

Our study shows that GTN-tolerant cells and blood vessels exhibit a marked increase in HNE adduct formation and that naı̈ve blood vessels exposed to HNE exhibit a decreased vasodilator response to GTN, mimicking GTN tolerance. Furthermore, there was a marked increase in HNE adduct formation in aortae from Aldh2−/− mice compared to that in wild type mice and also a desensitized response to GTN after exposure of Aldh2−/− mouse aorta to HNE, which mimicked GTN tolerance in these mice. Additionally, the HNE scavenger, AG-01, prevented both GTN-induced HNE adduct formation and the development of GTN tolerance but did not prevent the inhibition of ALDH2 enzyme activity. Taken together, these data suggest a significant role for HNE adduct formation in the development of GTN tolerance. Clinically, the utility of GTN is limited by the onset of tolerance to its hemodynamic and antianginal effects, which occur after continuous exposure to GTN. Following the initial report proposing ALDH2 as the principal enzyme that bioactivates GTN resulting in vascular smooth muscle

Figure 6. Formation of HNE adducts of ALDH2 after induction of GTN tolerance and prevention by AG-01. IP/IB analysis of (A) aortae from GTN-tolerant and control rats (n = 3) and (B) aortae from GTN-tolerant and control rats coadministered AG-01 (20 mg/kg i.p.) (n = 3). Two hundred micrograms of aortic whole-cell homogenates were immunoprecipitated using an HNE antibody, and immunoprecipitates were subjected to immunoblot analysis using an antibody to ALDH2. No immunoreaction was observed in incubations of cell lysates and protein A/G beads without the HNE antibody. Right-hand lane, purified ALDH2 standard (50 ng).

incubation with GTN and HNE, respectively (Figure 8C and D). In contrast, immunoblot analysis of ALDH2 in aortic 668



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Figure 7. Absence of HNE adducts of ALDH2 after AG-01 treatment does not prevent the decrease in ALDH2 activity in rat hepatic mitochondria from GTN-tolerant rats. (A) IP/IB analysis of liver from control rats (n = 3) and GTN-tolerant rats coadministered AG-01 (20 mg/kg i.p.)(n = 3). Two hundred micrograms of hepatic whole-cell homogenates were immunoprecipitated using an HNE antibody, and immunoprecipitates were subjected to immunoblot analysis using an antibody to ALDH2. Right-hand lane, purified ALDH2 standard (50 ng). (B) ALDH activity was measured at low- and high-substrate concentrations in hepatic mitochondria prepared from control rats and GTN-tolerant rats coadministered AG-01. Data are presented as the mean ± SD (n = 4) and were analyzed by a Student’s t-test for unpaired data. Asterisks (*) were used to indicated significant differences from the control + AG-01 (***p < 0.001).

Figure 9. Co-administration of AG-01 during the induction of GTN tolerance prevents down-regulation of aortic ALDH2. Immunoblot analysis of aortae from control rats (n = 4) and from GTN-tolerant rats (n = 4) coadministered AG-01 (20 mg/kg i.p.). Thirty micrograms of whole-cell homogenates were resolved on a 10% SDS−PAGE gel, transferred to a PVDF membrane, probed with antibody to ALDH2 or β actin as indicated (A), and immunoreactive bands quantitated by densitometry (B). Data are presented as the mean ± SD (n = 4) and were analyzed by a Student’s t-test for unpaired data (p > 0.05).

ALDH2 is the basis for GTN tolerance. The ALDH2 hypothesis has not gone without challenge; one clinical study investigating the effects of the ALDH2*2 mutation, which renders ALDH2 almost completely inactive, found no differences in the maximal vasodilator response to GTN compared to individuals with the wild type enzyme, although there was a delay in the time to attain maximal vasodilation; the authors concluded that enzymes other than ALDH2 are involved in GTN bioactivation.7 Recent studies from our laboratory have shown that the reduced ALDH activity and ALDH2 expression in nitrate-tolerant blood vessels is dissociated from the duration of the impaired vasodilator and biotransformation responses to GTN and that the relatively specific ALDH2 inhibitor, daidzin, does not inhibit GTN biotransformation or GTN-induced relaxation of the rat aorta.3 Furthermore, in our PK1 cell culture model of nitrate tolerance, overexpression of ALDH2 resulted in increases in ALDH2-mediated GTN biotransformation but had no effect on GTN-induced cGMP formation. siRNA depletion of endogenous ALDH2 also had no effect on the cGMP response to GTN in these cells, indicating that GTN bioactivation is unaltered in either the presence or absence of ALDH2. Although these studies suggested that ALDH2 is not directly involved in GTN bioactivation, the desensitized GTN response in Aldh2−/− mice suggests some sort of role for ALDH2 in nitrate action. In an attempt to reconcile these apparently disparate findings, we investigated a potential link between oxidative stress and ALDH2 inactivation hypotheses of tolerance by examining the role of HNE, a byproduct of oxidative stress and an inhibitor of ALDH2. We detected 2−3-fold increases in HNE adduct formation in GTN-tolerant aorta and in tolerant PK1 cells (Figure 1). This finding is consistent with other studies showing the presence of

Figure 8. Ex vivo incubation of rat aorta with GTN and HNE results in decreased ALDH2 expression. Rat aortic segments were incubated in serum-free media with GTN (1 μM) or HNE (10 μM) for 24 h. Thirty micrograms of whole-cell homogenates were resolved on a 10% SDS−PAGE gel, transferred to a PVDF membrane, probed with antibody to HNE, ALDH2, or β actin as indicated (A), and immunoreactive bands for HNE (B) and ALDH2 (C) were quantitated by densitometry. Data are presented as the mean ± SD (n = 4) and were analyzed by a Student’s t-test for unpaired data. Asterisks (*) were used to indicated significant differences from the control (***p < 0.001; **p < 0.01).

relaxation,1 a number of studies have linked ALDH2 to GTN bioactivation,2,28−30 including studies showing that Aldh2−/− mice exhibit both reduced cGMP and vasodilator responses to GTN.2 This led to the hypothesis that the inactivation of 669



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Figure 10. Proposed model of the mechanisms of GTN bioactivation and tolerance in the vasculature. The box on the left depicts the bioactivation mechanism of GTN in GTN-sensitive blood vessels. The box on the right depicts the mechanism of GTN tolerance. Bold arrows indicate prominent pathways, whereas dotted arrows indicate pathways that are diminished. See text for details. Abbreviations: ALDH2, aldehyde dehydrogenase 2; HNE, 4-hydroxynonenal; NO2−, nitrite; NO, nitric oxide; ROS, reactive oxygen species.

oxidative stress during GTN tolerance. The findings of increased superoxide levels in GTN-tolerant blood vessels11 and evidence for peroxynitrite formation31 led to the oxidative stress hypothesis of GTN tolerance, which proposes that GTN tolerance is due to the formation of reactive oxygen species such as superoxide, which reacts with GTN-derived NO, resulting in decreased NO bioavailability and altered NO signaling. The formation of HNE adducts observed in GTNtolerant aorta as well as those found in aortae from Aldh2−/− mice links the presence of oxidative stress with a desensitized response to GTN. Further, our data shows that HNE specifically affects the GTN bioactivation process since the vasodilator and cGMP responses to the spontaneous NO donor, DEA/NO, were unaltered after exposure to HNE (Figures 2B and 3B). The increase in HNE formation that occurs during GTN tolerance is of interest given the complex relationship between ALDH2 and HNE. In addition to being an ALDH2 substrate, at low concentrations, HNE is a reversible inhibitor of ALDH2,17,32 and at high concentrations, it results in irreversible covalent modification of the enzyme.17,33 HNE irreversibly inactivates ALDH2 through covalent modification of Cys302 in the active site, resulting in almost complete inhibition of enzymatic activity.34 In simile, Chen et al. proposed that during ALDH2-mediated biotransformation of GTN to NO2− and glyceryl dinitrate metabolites, critical sulfhydryl groups at the active site are oxidized, inactivating the enzyme in the process.1 Several studies have attempted to use reducing agents such as dihydrolipoic acid and dithiothreitol to restore ALDH2 activity or prevent ALDH2 inactivation by GTN.4,18 However, these agents only partially restored ALDH2 activity, and the authors concluded that significant irreversible inactivation of ALDH2 occurs during GTN tolerance. In the present study, we observed significant HNE adduct formation on ALDH2 during GTN tolerance (Figure 6A). This finding suggests that the binding of HNE to ALDH2 in GTN-tolerant aortae could

contribute to the inhibition of ALDH2 activity observed during GTN tolerance. Although HNE is one of the more reactive and better studied cytotoxic aldehydes, many other lipid peroxidation-derived aldehydes are formed under conditions of oxidative stress43 that cannot be excluded from participating in the inactivation both of ALDH2 and of enzymes responsible for GTN bioactivation. The administration of the HNE scavenger AG-01 prevented GTN tolerance, vascular HNE adduct formation (Figure 5), and HNE adduct formation on ALDH2 (Figure 6B), but it did not prevent ALDH2 inactivation during GTN treatment, as indicated by the marked decrease in hepatic mitochondrial ALDH2 activity despite the absence of HNE adducts on hepatic ALDH2 in GTN-tolerant rats treated with AG-01 (Figure 7). This suggests that a direct inhibitory effect on the enzyme by GTN or by some unknown species is sufficient to cause ALDH2 inactivation in GTN tolerance and that inactivation of ALDH2 by HNE adduct formation is not required for the observed decrease in enzyme activity. However, this does not preclude the formation of HNE adducts of ALDH2 in the absence of AG-01 treatment that could contribute to decreased enzyme activity. Regardless of the precise mechanism of ALDH2 inactivation during tolerance development, the prevention of GTN tolerance by AG-01, concomitant with marked ALDH2 inactivation, does not support a role for ALDH2 in mediating GTN bioactivation. A number of studies have shown that ALDH2 protein levels are decreased during GTN tolerance.3−6 Although this is at least partly due to effects at the transcriptional level,6 the reduction in ALDH2 protein is also likely due to the irreversible inactivation of ALDH2 that occurs during chronic GTN exposure. In the current study, we observed a similar effect with HNE in rat aorta; a 24-h incubation of the aorta with 10 μM HNE resulted in a 40% reduction in ALDH2 protein levels (Figure 8). Given that HNE forms covalent adducts with ALDH2,17,33 this could target the protein for proteasomal 670



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that show that in addition to forming adducts with ALDH2,17,39 HNE inhibits and forms adducts with a number of GTN biotransformation enzymes, including glutathione transferases, xanthine oxidoreductases, and cytochromes P450.40−42 Additionally, we observed marked increases in HNE adduct formation in aortae from GTN-treated animals and the increases in HNE adducts correlated with rightward shifts in the concentration−response curves to GTN (Figure 5). Furthermore, a one-day nitrate-free period was sufficient to resensitize responsiveness of rat aorta to GTN, and this resensitization was accompanied by a marked reduction in HNE adduct formation, with HNE adduct levels similar to the levels observed in control rat aorta. In summary, we have provided evidence for the role of HNE in the development of GTN tolerance. HNE adducts are present in GTN-tolerant rat aorta and in Aldh2−/− mice, and exogenous HNE inhibits both GTN-induced cGMP formation and GTN-induced relaxation. Furthermore, both GTN tolerance and GTN-induced HNE adduct formation are prevented by an HNE scavenger. We propose that inactivation of ALDH2 during GTN tolerance, either by HNE adduct formation or by a direct action of GTN, results in decreased HNE catabolism by ALDH2 and HNE adduct formation and inactivation of enzymes critical for the bioactivation of GTN.

degradation, as has been found with other proteins that form HNE adducts.35 Consistent with this idea, coadministration of AG-01 during GTN treatment prevented both ALDH2-HNE adduct formation (Figure 6) and down-regulation of ALDH2 protein (Figure 9) in rat aorta. Aldh2−/− mice are less responsive to GTN than wild type mice (a finding used to support the ALDH2 hypothesis of tolerance2). However, Aldh2−/− mice continuously administered GTN for 48 h exhibit further decreases in responsiveness to GTN. The GTN tolerance protocol resulted in a 4-fold rightward shift in the concentration−response curve for GTN in Aldh2−/− mice compared to a 6-fold rightward shift in wild type mice (Figure 4A). We suspect that the lesser shift seen in Aldh2−/− mice is due to the increased presence of HNE adducts observed in naı̈ve Aldh2−/− mice (Figure 1C and D), prior to GTN exposure. The demonstration of GTN tolerance in Aldh2−/− mice observed in this study is significant and provides additional evidence against the ALDH2 inactivation hypothesis of GTN tolerance (since there is no Aldh2 in these mice to be inactivated by GTN treatment). The observation that HNE adducts are 4-fold higher in Aldh2−/− mouse aorta compared to wild type aorta (Figure 1) suggests that Aldh2−/− mice are under persistent oxidative stress due to the absence of ALDH2. This is consistent with other mouse models associating the lack of ALDH2 activity with increased oxidative stress and HNE formation.36,37 We propose that the increase in endogenous HNE may be the basis for the desensitized GTN response in Aldh2−/− mice relative to wild type mice, via HNE adduct formation of GTN bioactivation enzymes, and may also be the reason we did not observe a rightward shift in the concentration−response curve to GTN after treatment with low concentrations of HNE (Figure 4B). However, treatment of Aldh2−/− mouse aorta with a higher HNE concentration resulted in a 4-fold rightward shift in the concentration−response curve to GTN, mimicking the rightward shift observed in GTN tolerance in these mice. These data suggest that in Aldh2−/− mice, exposure to exogenous HNE further targets GTN bioactivation enzymes responsible for GTN-induced vasodilation, resulting in GTN tolerance. On the basis of the findings herein, we propose a new unifying model of GTN tolerance (Figure 10) that accommodates not only the oxidative stress and ALDH2 hypotheses of tolerance but also the critical sulfhydryl hypothesis proposed by Needleman and Johnson some 40 years ago.38 We hypothesize that GTN is biotransformed by a number of enzymes, resulting in either the formation of NO2− in the absence of a vasodilator response (clearance-based biotransformation) or the formation of NO bioactivity resulting in a vasodilator response (mechanism-based biotransformation). During periods of GTN sensitivity, GTN biotransformation leads to the formation of NO bioactivity and vasodilation. During this period, endogenous HNE formed from physiological processes in the cell is catabolized by ALDH2, which serves to limit the accumulation of HNE and prevent HNEinduced inactivation of GTN-bioactivation enzymes. However, in GTN tolerance, ALDH2 is inactivated either by HNE adduct formation or by GTN-mediated oxidation of critical SH groups in the active site of ALDH2 and is unable to detoxify HNE, resulting in increased HNE adduct accumulation and the inactivation of the GTN biotransformation enzyme pool. This inactivation reduces the mechanism-based biotransformation of GTN resulting in reduced NO bioactivity and decreased vasodilation. Support for this hypothesis stems from studies

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AUTHOR INFORMATION

Corresponding Author

*Tel: 613-533-6473. Fax: 613-533-6412. E-mail: bennett@ queensu.ca. Funding

This work was supported by a grant from the Canadian Institute of Health Research [MOP 81175]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Mrs. Diane Anderson for technical assistance. ABBREVIATIONS AG-01, L-histidyl hydrazide; ALDH, aldehyde dehydrogenase; DEA/NO, 1,1-diethyl-2-hydroxy-2-nitrosohydrazine; HNE, 4hydroxy-2-nonenal; GTN, glyceryl trinitrate; GDN, glyceryl dinitrate; PK1, porcine kidney epithelial cells; sGC, soluble guanylyl cyclase

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REFERENCES

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Role of the Lipid Peroxidation Product, 4-Hydroxynonenal, in the

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