Identification of the enzymatic mechanism of
`nitroglycerin bioactivation
`
`Zhiqiang Chen*†, Jian Zhang†, and Jonathan S. Stamler*†‡§
`
`*Howard Hughes Medical Institute, Departments of †Medicine and §Biochemistry, Duke University Medical Center, Durham, NC 27710
`
`Communicated by Irwin Fridovich, Duke University Medical Center, Durham, NC, April 15, 2002 (received for review March 26, 2002)
`
`Nitroglycerin (glyceryl trinitrate, GTN), originally manufactured by
`Alfred Nobel, has been used to treat angina and heart failure for
`over 130 years. However, the molecular mechanism of GTN bio-
`transformation has remained a mystery and it is not well under-
`stood why ‘‘tolerance’’ (i.e., loss of clinical efficacy) manifests over
`time. Here we purify a nitrate reductase that specifically catalyzes
`the formation of 1,2-glyceryl dinitrate and nitrite from GTN, lead-
`ing to production of cGMP and relaxation of vascular smooth
`muscle both in vitro and in vivo, and we identify it as mitochondrial
`aldehyde dehydrogenase (mtALDH). We also show that mtALDH is
`inhibited in blood vessels made tolerant by GTN. These results
`demonstrate that the biotransformation of GTN occurs predomi-
`nantly in mitochondria through a novel reductase action of
`mtALDH and suggest that nitrite is an obligate intermediate in
`generation of NO bioactivity. The data also indicate that attenu-
`ated biotransformation of GTN by mtALDH underlies the induction
`of nitrate tolerance. More generally, our studies provide new
`insights into subcellular processing of NO metabolites and suggest
`new approaches to generating NO bioactivity and overcoming
`nitrate tolerance.
`
`It is generally assumed that glyceryl trinitrate (GTN) is con-
`
`verted in vascular smooth muscle cells to NO or an NO
`congener (S-nitrosothiol, SNO), which activates guanylate cy-
`clase and thus relaxes vascular smooth muscle (1, 2). However,
`the molecular mechanism involved is not known, and there is no
`unifying explanation for the pharmacological profile of the drug
`(e.g., the preferential effects on capacitance vessels and sulfhy-
`dryl requirement for relaxation) or the phenomenon of nitrate
`tolerance (i.e., the loss of clinical efficacy over time), which
`leaves patients vulnerable to ischemia and may contribute to
`other deleterious consequences of long-term nitrate use (3, 4).
`GTN biotransformation is tissue and cell specific and dose
`dependent, and it yields 1,2-glyceryl dinitrate (1,2-GDN), 1,3-
`GDN, inorganic nitrite, and NO (or SNO) in differing amounts
`and ratios. In vascular smooth muscle cells, 1,2-GDN is the
`predominant dinitrate metabolite (2, 5, 6) and the vasorelax-
`ation-related formation of 1,2-GDN is diminished in GTN-
`tolerant blood vessels (7, 8). These data suggest that an enzyme
`with product specificity for 1,2-GDN (over 1,3-GDN) generates
`NO bioactivity from GTN and that loss of this activity at least
`partly accounts for tolerance. Several enzymatic mechanisms of
`vascular smooth muscle relaxation by GTN have been proposed
`(9 –15), and candidate enzymes include glutathione S-
`transferases, cytochrome P450 reductase, cytochrome P450,
`xanthine oxidoreductase, and an unidentified microsomal pro-
`tein. But none of these enzymes catalyzes the selective formation
`of 1,2-GDN (12, 15, 16) or is inhibited in tolerant vessels, and the
`roles of these enzymes in generating NO bioactivity remain
`controversial. Here we report on the purification of a GTN
`reductase that specifically generates 1,2-GDN, identify it as
`mitochondrial aldehyde dehydrogenase (mtALDH), and estab-
`lish its central role in GTN bioactivation, vasorelaxation, and
`tolerance.
`
`Materials Methods
`Chemicals and Drugs. GTN, 1,2-GDN, and 1,3-GDN standards
`were bought from Radian International (Austin, TX). GTN was
`
`synthesized according to ref. 17 and purified by TLC. 1,2-GDN
`and 1,3-GDN was prepared by hydrolysis of GTN and purified
`by TLC (18). [2-14C] GTN (55 mCi兾mmol) was obtained from
`American Radiolabeled Chemicals (St. Louis). Disulfiram, chlo-
`ral hydrate, cyanamide, acetaldehyde, phenylephrine, sodium
`nitroprusside (SNP), Q-Sepharose, DEAE-cellulose, and butyl-
`Sepharose were obtained from Sigma. Hydroxyapatite and
`protease inhibitor (mixture set III) were from CalBiochem.
`The cGMP assay (125I) kit was purchased from Amersham
`Pharmacia.
`
`Enzyme Purification. Mouse RAW264.7 cells (50-g pellet; ⬇1010
`cells) were disrupted by sonication in 30 mM phosphate buffer
`(KPi), pH 7.5 containing 1 mM DTT, 0.5 mM EDTA, and
`protease inhibitors (1:200 dilution). The 100,000-g supernatant
`was loaded onto a DEAE-cellulose column, and the flow-
`through fractions, which contained the enzyme activity, were
`pooled and concentrated by ultrafiltration (Amicon, 10-kDa
`cut-off membrane). After 3-fold dilution with cold water, the
`fractions were loaded onto a Q-Sepharose column, and the
`activity was eluted by a phosphate gradient (10 mM to 150 mM,
`pH 7.5); the active fractions were pooled and solid potassium
`chloride (KCl) was added to 1.25 M. The sample was then loaded
`onto a butyl-Sepharose column, and the enzyme activity was
`eluted by decreasing the salt concentration. The concentrated
`active fractions were then diluted and loaded onto a hydroxy-
`apatite column. After washing the column with 10 mM KPi兾0.4
`M KCl, the enzyme was eluted by a phosphate gradient from 10
`mM KPi to 150 mM KPi, pH 7.5. All steps were performed at
`4°C, and the elution buffers contained 1 mM DTT and 0.5 mM
`EDTA unless otherwise specified.
`
`GTN Biotransformation. 1,2-GDN and 1,3-GDN formation were
`measured by the TLC and liquid scintillation spectrometry
`method as described by Brien et al. (19) with a few modifications.
`During protein purification, the assay mixture (1 ml) contained:
`100 mM KPi (pH 7.5), 0.5 mM EDTA, 1 mM NADH, 1 mM
`NADPH, 0.1 or 1 ␮M GTN, and protein (with DTT) from
`column fractions. After incubation at 37°C for 10–30 min, the
`reaction was stopped (on dry ice or 4°C) and GTN and its
`metabolites were extracted with 3 ⫻ 4 ml ether and pooled, and
`the solvent was evaporated by a stream of nitrogen. The final
`volume was kept to less than 100 ␮l in ethanol for subsequent
`TLC separation and scintillation counting. For the activation of
`dialyzed enzyme samples and assay of the activity of purified
`enzyme, the assay mixture contained: 100 mM KPi (pH 7.5), 0.5
`mM EDTA, 0–1 mM DTT or other reductants (0.5 mM DTT for
`the bovine enzyme), and 1 ␮M GTN. For assays in tissues such
`as rabbit aorta, rings were blotted and weighed after sitting for
`1 h in Krebs solution (control) (composition described below).
`When used, inhibitors were added for 20 min before the addition
`
`Abbreviations: GTN, glyceryl trinitrate; GDN, glyceryl dinitrate; mtALDH, mitochondrial
`aldehyde dehydrogenase; SNO, S-nitrosothiol; SNP, sodium nitroprusside.
`
`See commentary on page 7816.
`‡To whom reprint requests should be addressed. E-mail: STAML001@mc.duke.edu.
`
`8306 – 8311 兩 PNAS 兩
`
`June 11, 2002 兩 vol. 99 兩 no. 12
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`www.pnas.org兾cgi兾doi兾10.1073兾pnas.122225199
`
`Page 1 of 6
`
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`CFAD v. Insys
`IPR2015-01800
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`Table 1. Purification of GTN reductase from mouse macrophage RAW264.7 cells
`
`Procedures
`
`100,000 g supernatant
`DEAE-cellulose
`Q-Sepharose
`Butyl-Sepharose
`Hydroxyapatite
`
`Total units,
`nmol兾h
`
`Total protein,
`mg
`
`Specific activity,
`nmol兾h per mg
`
`837.4
`756.0
`528.5
`205.4
`120.9
`
`2,265
`345
`123
`10.9
`3.6
`
`0.37
`2.19
`4.29
`18.81
`33.36
`
`Yield,
`%
`
`100
`90.3
`63.1
`24.5
`14.4
`
`Fold
`purification
`
`1.0
`5.9
`11.6
`50.8
`90.2
`
`Starting material: 50-g cell pellet.
`
`MEDICALSCIENCES
`
`of 1 ␮M GTN; the mixture (1 ml) was then kept at 37°C for 5 min.
`The extraction and TLC-liquid scintillation spectrometry anal-
`ysis were as described above. Buffer control (Krebs buffer plus
`GTN) and nonspecific biotransformation (heat-inactivated rings
`plus GTN) activities were also measured, and the results were
`corrected accordingly.
`
`Aortic Ring Bioassays. New Zealand White rabbits (2.5–3 kg) were
`killed by carbon dioxide inhalation. Thoracic aorta were re-
`moved, cleaned of fat and connecting tissue, and cut into 3-mm
`rings. The rings were mounted under 2 g of resting tension in
`tissue baths (25 ml) filled with Krebs solution (37°C) containing:
`118 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4,
`2.5 mM CaCl2, 25 mM NaHCO3, and 11 mM glucose, pH 7.4.
`The solution was bubbled with 20% O2, 5% CO2, and balance N2.
`Changes in isometric tension were recorded with Statham (Hato
`Rey, PR) transducers and a Grass Instruments (Quincy, MA)
`polygraph, and contractions were initiated with phenylephrine.
`Tolerance to GTN was induced by incubating rings with 0.3 mM
`GTN for 30 min. Rings were then washed several times with
`Krebs solution and left for 1 h before being further tested.
`
`Nitrite and Nitrate. Nitrite and nitrate concentrations (in the
`aqueous phase after ether extraction, see GTN biotransforma-
`tion) were determined in an NO analyzer (model 280, Sievers,
`Boulder, CO) according to the manufacturer’s instructions.
`
`cGMP Assay. Aortic rings were blotted and weighed before being
`incubated in aerated bioassay chambers for 1 h. Then, after
`treatment with different inhibitors, the rings were exposed to 1
`␮M GTN for 1 min and immediately frozen in liquid nitrogen
`and stored at ⫺80°C until the time of analysis. cGMP extraction
`and measurements were performed according to the manufac-
`turer’s protocol.
`
`ALDH Assay. Rabbit aortic rings were homogenized with a Kontes
`tissue grinder in 30 mM KPi buffer (deoxygenated with nitrogen
`gas), pH 7.5, and the homogenate was then sonicated and
`centrifuged at 10,000 g for 10 min. ALDH activity in the
`supernatant was monitored at room temperature by following
`NADH formation at 340 nm. The assay mixture (1 ml) con-
`tained: 100 mM Tris䡠HCl (pH 8.5), 1 mM NAD⫹, 1 mM
`propionaldehyde, and 1 mM 4-methylpyrazole.
`
`GTN Infusions inVivo. New Zealand White rabbits (2.5–3 kg) were
`anesthetized with isoflurane (1.5%). The carotid artery and
`jugular vein were isolated via cut down and cannulated. The
`blood pressure was monitored continuously with a Viggo Spec-
`tramed pressure transducer (AD Instruments, Grand Junction,
`CO) attached to a Gould (Cleveland) recorder. Drugs were
`infused via the jugular vein. mtALDH inhibitors (⬇5 mM) were
`infused 20 min before initiating studies with GTN. Cyanamide
`had no effect on mean arterial blood pressure, whereas chloral
`hydrate had a modest hypotensive effect. Similar studies were
`performed in Sprague–Dawley rats.
`
`Statistical Analysis. Results were statistically analyzed by the
`Student’s t test, and two sets of data were considered statistically
`different when P ⬍ 0.05.
`
`Results and Discussion
`We reasoned that the difficulty in isolating the enzyme respon-
`sible for GTN biotransformation from blood vessels may reflect
`a lack of starting material and兾or exposure to excessive concen-
`trations of GTN (under which conditions GTN metabolism may
`not represent biotransformation), and we thus screened alter-
`native animal tissues and cell lines by monitoring 1,2-GDN
`formation from physiological amounts of GTN (0.1 ␮M). We
`found that mouse macrophage RAW264.7 cells, which can be
`grown in large numbers, resemble vascular smooth muscle cells
`in generating mainly 1,2-GDN, and that after cellular disruption
`by sonication, the enzyme activity was present predominantly in
`the 100,000-g supernatant. We then developed a four-step
`purification procedure to isolate the enzyme from RAW264.7
`cells (Table 1). The enzyme activity passed freely through a
`DEAE-cellulose column and was preserved as a single peak after
`each of the following chromatographic steps:
`ion exchange
`(Q-Sepharose), hydrophobicity (butyl-Sepharose), and hydroxy-
`apatite column chromatography. At the end of the procedure the
`protein had been purified to near homogeneity (90-fold), with
`14.4% overall yield (Fig. 1a); its estimated subunit mass on SDS
`gel is about 53 kDa. N-terminal sequence of 30 aa residues
`(SAAATSAVPAPNHQPEVFXNQIFINNEWHD) was ob-
`tained by Edman degradation and matched exactly with mouse
`mtALDH (the unidentified residue 19 is a cysteine in the
`database). To confirm that this enzyme is located in mitochon-
`dria and responsible for the production of 1,2-GDN, we assayed
`purified preparations of mitochondria (20) that we had isolated
`from macrophage cells and found that they generated 1,2-GDN
`from GTN, whereas the mitochondria-free supernatant had no
`residual activity (data not shown). We then pretreated the
`macrophage cells with known ALDH inhibitors that either
`modify the active site residue (disulfiram or cyanamide) or
`compete for substrate binding (chloral hydrate) and found that
`all of the inhibitors, tested at concentrations from 50 ␮M to 1
`mM, blocked formation of 1,2-GDN (see below).
`In addition to the classical NAD⫹-dependent dehydrogena-
`tion activity, ALDH possesses an esterase activity (21), which is
`initiated by nucleophilic attack of the active site cysteine on
`substrate esters, such as p-nitrophenyl acetate, and is followed by
`hydrolysis to generate the acetic acid product. The analogous
`reaction with GTN would be expected to yield an E-S-NO2 active
`site intermediate that would then hydrolyze to yield nitrate
`⫺). However, we could not detect any nitrate formation.
`(NO3
`Rather, the purified enzyme catalyzed the stoichiometric for-
`⫺) from GTN. Specifically,
`mation of 1,2-GDN and nitrite (NO2
`the rates of 1,2-GDN formation were linear with physiological
`GTN concentrations in the range of 0.1 to 10 ␮M (data not
`shown), whereas 1,3-GDN was below detection limits, and nitrite
`was generated in stoichiometric amounts. The product ratio of
`1,2-GDN兾nitrite (0.83 ⫾ 0.14, n ⫽ 3) was similar to that of
`
`Chen et al.
`
`PNAS 兩
`
`June 11, 2002 兩 vol. 99 兩 no. 12 兩 8307
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`Page 2 of 6
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`

`
`Scheme 1.
`
`Km (acetaldehyde) and Ki (chloral hydrate) for dehydrogenase
`activity] reflect the absence of NAD⫹, which increases substrate
`binding (26). As expected, cyanamide did not have inhibitory
`effects on the purified enzyme because it first requires biocon-
`version in vivo.
`The classical sulfhydryl requirement for relaxation of vascular
`smooth muscle by GTN (2, 27) has never been satisfactorily
`explained. It has been suggested that thiols potentiate GTN
`activity through formation of bioactive SNOs and兾or protect
`against nitrate tolerance that results from depletion of intracel-
`lular thiols. However, the formation of SNO and depletion of
`intracellular thiol have not been confirmed (28), and alternative
`hypotheses, such as oxygen radical scavenging by thiol (29), have
`more recently found favor. We found that the purified mtALDH
`could catalyze 1,2-GDN formation only when DTT or 2-
`mercaptoethanol (2ME) were present (although these concen-
`trations of thiol had no effect on their own). Further, catalytic
`activity that was lost by dialysis could be fully restored by
`addition of DTT or 2ME (to a lesser extent) but not by
`glutathione.
`Human mtALDH has two cysteines immediately adjacent to
`the active site thiol. The formation of an intramolecular disulfide
`bond has been demonstrated by MS (30, 31) and accounts for the
`‘‘inactive form’’ of ALDH that is produced by ALDH inhibitors.
`Notably, active site participation in a disulfide bond has been
`previously implicated in the mechanism-based inhibition of
`mtALDH by NO (32), SNO (33), and GTN (21, 34), accounting
`for disulfram-like reactions in GTN-treated patients ingesting
`alcohol. But whereas the generation of disulfide has been viewed
`as indicative of enzyme inactivation (i.e., loss of dehydrogenase
`and esterase activities), the new data suggest that it can represent
`a reaction intermediate in a novel reductase reaction requiring
`thiol (and perhaps other reductant) cofactor (Scheme 1). That
`is, GTN is an inhibitor of mtALDH activity because it serves as
`a substrate for its reductase activity. Collectively, our data
`indicate that mtALDH is in fact a GTN reductase.
`The specific conversion of GTN to 1,2-GDN and dependence
`on thiol cofactor (as reductant) make mtALDH a compelling
`candidate for the elusive enzymatic system that is responsible for
`catalyzing the bioactivation of GTN in the vasculature. We tested
`this hypothesis by using an aortic ring bioassay. Preincubation of
`blood vessels with mtALDH inhibitors chloral hydrate and
`cyanamide or with the mtALDH substrate acetaldehyde (a
`competitive inhibitor of GTN metabolizing activity) produced a
`rightward shift in the GTN relaxation curves (Fig. 2 a1–c1) that
`depended on inhibitor concentration (Fig. 2a1 and data not
`shown). The mtALDH inhibitor disulfiram also blocked GTN
`relaxations, but interpretation of these data was confounded by
`a direct affect on vessel tone. By contrast, preincubation with the
`mtALDH inhibitors did not attenuate relaxation induced by the
`nitrosovasodilator SNP (Fig. 2 a2–c2), by NO solutions or
`
`(a) Purification of mtALDH from mouse macrophage RAW264.7 cells.
`Fig. 1.
`The gradual enrichment of the 53-kDa band is shown on a 7% SDS polyacryl-
`amide gel. Lane A, protein molecular mass marker (kDa); lane B, 100,000-g
`supernatant; lane C, after DEAE-cellulose; lane D, after Q-Sepharose; lane E,
`after butyl-Sepharose; lane F, after hydroxyapatite. (b) Kinetic analysis of
`purified GTN reductase in the absence of NAD⫹ (KM ⫽ 12 ␮M; Vmax ⫽ 3
`nmol兾mg per min).Vmax in the presence of NAD⫹ ⫽ ⬇30 nmol兾mg per min.
`
`⫺ ⫹ Eox.
`
`GTN ⫹ Ered
`
`1,2-GDN兾NOx (nitrite plus nitrate) (0.73 ⫾ 0.05, n ⫽ 3). Thus,
`the overall reaction can be expressed as:
`3 1,2-GDN ⫹ NO2
`To show that the GTN metabolizing activity and enantiomeric
`selectivity of mtALDH is not a function of cell type or species,
`we purified the enzyme from bovine liver (22). Bovine mtALDH
`also specifically catalyzes 1,2-GDN formation (1,2-GDN兾1,3-
`GDN ⫽ ⬇8:1) from GTN (0.1 to 10 ␮M), with classical
`Michaelis–Menten kinetics (Km ⫽ 11.98 ⫾ 3.04 ␮M and Vmax ⫽
`3.03 ⫾ 0.20 nmol兾min per mg) (Fig. 1b) [and with the same pH
`dependence as the dehydrogenase activity (optimum pH of 9.0)].
`Addition of NAD⫹ (20–1,000 ␮M) to the reaction mixture
`increases the rate of 1,2-GDN formation by about 10-fold (i.e.,
`Vmax ⬇ 30 nmol兾min per mg), easily adequate for NO bioactivity
`such as vasodilation. By comparison, the purified bovine enzyme
`has a specific ALDH activity of 0.54 ␮mol兾min per mg. It is
`important to appreciate that whereas the GTN reductase activity
`is ⬇15- to 20-fold lower than the classical dehydrogenase activity
`of mtALDH, bioactive NO concentrations are as much as
`10,000-fold lower than circulating or tissue acetaldehyde con-
`centrations (23), and the mitochondrial GTN reductase activity
`is large from the standpoint of NO biology, being significantly
`greater than that of mitochondrial NO synthase activity (24, 25).
`We reasoned that acetaldehyde (the natural enzyme sub-
`strate) should competitively inhibit GTN turnover by mtALDH,
`and we also tested the effects of the classical ALDH inhibitors
`on GTN-metabolizing activity of the purified bovine enzyme.
`Both chloral hydrate and acetaldehyde inhibited 1,2-GDN for-
`mation, with IC50 ⫽ 99.5 and 815.5 ␮M, respectively. The
`relatively high IC50s in the reductase assay [as compared with the
`
`8308 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.122225199
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`Chen et al.
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`Page 3 of 6
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`
`Effects of inhibitor treatment and GTN tolerance on GTN-induced (Left) and SNP-induced (Right) relaxation in rabbit aorta. The dose–response curves
`Fig. 2.
`of control rings are shown as F, and inhibitor-treated rings (1 mM) are shown as E. (a) Chloral hydrate dose–response (E, 1 mM; Œ, 5 mM). (b) Cyanamide. (c)
`Acetaldehyde. (d) GTN tolerant. All inhibitor curves (a–c) are significantly different from GTN control (P ⬍ 0.01), but none are different from SNP control (P ⫽
`not significant). The GTN tolerance curve (d) is significantly different from control to P ⬍ 0.05. Data are expressed as the means ⫾ SD of 4 – 6 aortic rings.
`
`MEDICALSCIENCES
`
`verapamil, which works through a NO-independent mechanism
`(not shown). These results suggest a specific inhibition of the
`organic nitrate ester reductase activity in the arterial vascula-
`ture. Indeed, we confirmed that 1,2-GDN generation in the
`aortic rings was diminished by chloral hydrate, cyanamide, and
`acetaldehyde (1 mM of each) to 27.6%, 56.9%, and 52.5% of the
`control activity, respectively (Fig. 3a). The GTN reductase
`inhibitors and competitive substrate also significantly decreased
`cGMP accumulation in aortic rings (Fig. 4). For example, chloral
`hydrate and acetaldehyde completely blocked the increase in
`cGMP produced by GTN (1 ␮M for 1 min) and cyanamide
`abrogated the cGMP increase by 46%. Thus, we conclude that
`the reductase activity of mtALDH is largely responsible for
`the increase in cGMP that at least partly mediates vasorelaxation
`by GTN.
`It has been reported that vascular rings also generate some
`1,3-GDN from GTN, and we verified that a small amount
`of 1,3-GDN was produced under our assay conditions (1,2-
`GDN兾1,3-GDN ⫽ 4.98 ⫾ 0.64, n ⫽ 6). However, 1,3-GDN
`
`formation was not mechanism based, because heat-inactivated
`rings generated the same amounts.
`Chronic therapy with organic nitrates produces ‘‘tolerance,’’ a
`desensitization of blood vessels that compromises antianginal
`efficacy. This poorly understood phenomenon is at least partly
`attributable to impaired bioactivation of GTN in both small
`animals and humans and is readily recapitulated in vitro by
`lengthy high-dose exposures. We induced tolerance with a
`regimen of 0.3 mM GTN for 30 min, followed by washout.
`Whereas these conditions had no effect on SNP-induced rela-
`tions (Fig. 2d2), the EC50 value for GTN increased significantly
`(Fig. 2d1). Furthermore, in aorta made tolerant to GTN, both
`GTN reductase activity (Fig. 3b), as measured by 1,2-GDN-
`formation, and classical mtALDH activity (Fig. 3c) decreased
`significantly (and comparably), and cGMP accumulation was
`abolished (Fig. 4). Reports that ALDH (dehydrogenase activity)
`is also potently inhibited in patients undergoing chronic treat-
`ment with GTN and other nitrate esters (34) provide strong
`support for the clinical relevance of these findings.
`
`Chen et al.
`
`PNAS 兩
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`June 11, 2002 兩 vol. 99 兩 no. 12 兩 8309
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`Page 4 of 6
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`

`
`Inhibition of cGMP accumulation in rabbit aorta by ALDH inhibitors
`Fig. 4.
`(1 mM ⫻ 20 min, followed by washout) and tolerance (Tol)-inducing amounts
`of GTN (0.3 mM ⫻ 30 min, followed by washout). Rings were (⫹) or were not
`(⫺) exposed to 1 ␮M GTN for 1 min. All bars are significantly less than control
`(Con) ⫹ GTN to P ⬍ 0.01, except cyanamide (Cya) ⫹ GTN, where P ⬍ 0.05. Data
`are presented as the means ⫾ SD of three aortic rings. Chl, chloral hydrate;
`Ace, acetaldehyde.
`
`(iv) offered alternative interpretations for the effects of thiols on
`GTN bioactivity, and (v) suggested new mechanisms of tolerance
`(29). There is even recent evidence to suggest that GTN acti-
`vation of guanylate cyclase may be independent of NO (38).
`None of these explanations, however, can reconcile the entire set
`of data. In contrast,
`the identification of
`the sulfhydryl-
`dependent mtALDH with GTN metabolic activity provides a
`viewpoint from which the models of Needleman et al. and
`Ignarro et al. are revived and shown to be in line with the original
`ideas of Murad et al. (1) as well as the newer data on enzymatic
`
`Inhibition of GTN (1 ␮M) biotransformation by ALDH inhibitors and
`Fig. 3.
`tolerance induction in rabbit aorta. (a) Concentration-dependent inhibition
`of 1,2-GDN formation by chloral hydrate (F), cyanamide (I), and acetalde-
`hyde (Œ). Activities are presented as the percentage of control rings (without
`inhibitor treatment). Data are the average of two experiments. (b) 1,2-GDN
`formation is reduced in tolerant vessels relative to control (P ⬍ 0.01). Data are
`the means ⫾ SD of three experiments. (c) mtALDH activity is inhibited in
`tolerant vessels (P ⬍ 0.01). Data are the means ⫾ SD of three experiments.
`
`As a further test of the physiological role of mtALDH in
`bioactivation of GTN, we assessed the effects of the mtALDH
`inhibitors cyanamide and chloral hydrate on the systemic effects
`of GTN infusions in anesthetized rabbits. Both inhibitors sig-
`nificantly attenuated the hypotensive effects of GTN (Fig. 5a),
`whereas they had little or no effect on SNP-mediated (i.e., NO
`dependent) or adenosine-mediated (i.e., NO independent) (not
`shown) hypotension (Fig. 5b). Similar results were obtained in
`rats, suggesting a generalized role for mtALDH in mammalian
`GTN metabolism (data not shown).
`The classical studies by Needleman et al. (27) and Ignarro et
`al. (2, 35), suggesting a sulfhydryl requirement for relaxation by
`GTN, have been interpreted in terms of a ‘‘thiol receptor model’’
`for organic nitrates in vascular smooth muscle, which provided
`for the possibility of an enzyme that generated inorganic nitrite
`as an intermediate and rationalized nitrate tolerance in terms of
`a depletion of intracellular thiols (27). Almost every feature of
`this model has been challenged in recent years by studies that
`have: (i) implicated alternative metalloenzymes in the mecha-
`nism of GTN bioactivation (11, 12); (ii) failed to detect a
`depletion of intracellular thiol
`in tolerant vessels (28); (iii)
`rejected the role of inorganic nitrite as an intermediate (36, 37);
`
`Fig. 5. mtALDH inhibitors block the hypotensive effects of GTN in vivo. (a)
`i.v. infusions of GTN in rabbits produce dose-dependent decrements in blood
`pressure (before treatment, F) that are attenuated significantly (P ⬍ 0.05) by
`both cyanamide (17 mg兾kg, E) and chloral hydrate (66 mg兾kg, ‚) (n ⫽ 5–7). (b)
`Cyanamide and chloral hydrate have no effect on SNP-mediated decreases in
`blood pressure (n ⫽ 7) (data for cyanamide treatment are shown). F, before
`treatment; E, after treatment.
`
`8310 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.122225199
`
`Chen et al.
`
`Page 5 of 6
`
`

`
`MEDICALSCIENCES
`
`biotransformation of GTN and impairment of this mechanism in
`tolerant vessels. In particular, our findings are strongly sup-
`ported by recent studies in GTN-treated patients, which have
`shown a profound decrease in mitochondrial-type (low Km)
`ALDH activity in the blood (34) and lower amounts of 1,2-GDN
`in tolerant vessels (8).
`Several lines of evidence have been offered against the original
`proposal (2, 39) that GTN acts via liberation of inorganic nitrite,
`including studies showing that GTN is readily transformed into
`NO bioactivity within cellular homogenates and subcellular
`fractions whereas nitrite is not (36, 37). However, these previous
`studies did not consider the possibility of biotransformation
`within the mitochondria, which is highly competent for nitrite
`bioactivation to NO. It has been shown that members of mito-
`chondria electron transport chain, cytochrome bc1 complex
`(complex III) and cytochrome c oxidase (complex IV) can
`reduce nitrite to NO (40, 41), and nitrite might also be trans-
`ported to the intermembrane space where the higher proton
`concentration may facilitate its conversion to NO or SNO.
`Mitochondria can also reduce NO to HNO, which has been
`shown to produce both reversible [e.g., intramolecular disulfide
`(32)] and irreversible [e.g., sulfinamide (33)] modifications of the
`active site cysteine in mtALDH and thus may explain why
`complete reversal of nitrate tolerance requires new protein
`synthesis (42). Interestingly, the organic nitrate nicorandil [N-(2-
`hydroxyethyl)nicotinamide nitrate ester] was recently shown to
`generate NO in rat myocardial mitochondria (43). It is therefore
`conceivable that other nitrates and endogenous nitrogen oxides
`may be ALDH substrates. More generally, our studies provide
`new insights into subcellular processing of NO metabolites and
`suggest new approaches for generating NO bioactivity and
`overcoming nitrate tolerance.
`
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`Residual, albeit attenuated, relaxations by GTN were ob-
`served in the presence of biotransformation inhibitors that
`completely blocked the GTN-mediated increase in cGMP. Like-
`wise, the relaxations in tolerant blood vessels appeared to be
`partly cGMP independent. These data are consistent with a
`number of studies showing that the mechanism of smooth muscle
`relaxation by NO and GTN is partly mediated by a direct effect
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`Our discovery that GTN is metabolized within mitochondria
`by the actions of ALDH has implications for patient care. It has
`long been a mystery why nitrates do not provide survival
`benefits, unlike agents that increase NO synthase activity, and
`the possibility that they may impair mitochondrial metabolism
`would provide an explanation. Indeed, this notion is supported
`by the findings of mitochondrial damage, impaired respiration,
`and increased production of proatherogenic superoxide in GTN-
`exposed tissues (29, 47). In addition, our studies suggest that
`certain classes of drugs such as sulfonylurea hypoglycemics,
`chloral hydrate, and acetaminophen (48), which inhibit
`mtALDH activity, may be contraindicated in patients taking
`organic nitrates esters for the treatment of acute ischemic
`syndromes and congestive heart failure. Finally, a polymorphism
`in the mtALDH gene (ALDH2) has been linked to impairments
`in ethanol metabolism and risk of developing cancer and de-
`mentia (48), and it follows that the ALDH genotype may predict
`GTN responsiveness and even therapeutic efficacy. A variety of
`new approaches to providing NO bioactivity and overcoming
`tolerance are suggested by our results.
`
`We thank Drs. D. Hess, T. McMahon, A. Hausladen, J. Eu, and L. Liu
`for their support and many valuable discussions and M. Dewhirst for
`assistance.
`
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