Identification of the Enzymatic Mechanism of Nitroglycerin Bioactivation
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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 synthesized according to ref. 17 and purified by TLC. 1,2-GDN Alfred Nobel, has been used to treat angina and heart failure for and 1,3-GDN was prepared by hydrolysis of GTN and purified over 130 years. However, the molecular mechanism of GTN bio- by TLC (18). [2-14C] GTN (55 mCi͞mmol) was obtained from transformation has remained a mystery and it is not well under- American Radiolabeled Chemicals (St. Louis). Disulfiram, chlo- stood why ‘‘tolerance’’ (i.e., loss of clinical efficacy) manifests over ral hydrate, cyanamide, acetaldehyde, phenylephrine, sodium time. Here we purify a nitrate reductase that specifically catalyzes nitroprusside (SNP), Q-Sepharose, DEAE-cellulose, and butyl- the formation of 1,2-glyceryl dinitrate and nitrite from GTN, lead- Sepharose were obtained from Sigma. Hydroxyapatite and ing to production of cGMP and relaxation of vascular smooth protease inhibitor (mixture set III) were from CalBiochem. muscle both in vitro and in vivo, and we identify it as mitochondrial The cGMP assay (125I) kit was purchased from Amersham aldehyde dehydrogenase (mtALDH). We also show that mtALDH is Pharmacia. inhibited in blood vessels made tolerant by GTN. These results demonstrate that the biotransformation of GTN occurs predomi- Enzyme Purification. Mouse RAW264.7 cells (50-g pellet; Ϸ1010 nantly in mitochondria through a novel reductase action of cells) were disrupted by sonication in 30 mM phosphate buffer mtALDH and suggest that nitrite is an obligate intermediate in (KPi), pH 7.5 containing 1 mM DTT, 0.5 mM EDTA, and generation of NO bioactivity. The data also indicate that attenu- protease inhibitors (1:200 dilution). The 100,000-g supernatant ated biotransformation of GTN by mtALDH underlies the induction was loaded onto a DEAE-cellulose column, and the flow- of nitrate tolerance. More generally, our studies provide new through fractions, which contained the enzyme activity, were insights into subcellular processing of NO metabolites and suggest new approaches to generating NO bioactivity and overcoming pooled and concentrated by ultrafiltration (Amicon, 10-kDa nitrate tolerance. 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, t is generally assumed that glyceryl trinitrate (GTN) is con- pH 7.5); the active fractions were pooled and solid potassium Iverted in vascular smooth muscle cells to NO or an NO chloride (KCl) was added to 1.25 M. The sample was then loaded congener (S-nitrosothiol, SNO), which activates guanylate cy- onto a butyl-Sepharose column, and the enzyme activity was clase and thus relaxes vascular smooth muscle (1, 2). However, eluted by decreasing the salt concentration. The concentrated the molecular mechanism involved is not known, and there is no active fractions were then diluted and loaded onto a hydroxy- unifying explanation for the pharmacological profile of the drug apatite column. After washing the column with 10 mM KPi͞0.4 (e.g., the preferential effects on capacitance vessels and sulfhy- M KCl, the enzyme was eluted by a phosphate gradient from 10 dryl requirement for relaxation) or the phenomenon of nitrate tolerance (i.e., the loss of clinical efficacy over time), which mM KPi to 150 mM KPi, pH 7.5. All steps were performed at leaves patients vulnerable to ischemia and may contribute to 4°C, and the elution buffers contained 1 mM DTT and 0.5 mM other deleterious consequences of long-term nitrate use (3, 4). EDTA unless otherwise specified. GTN biotransformation is tissue and cell specific and dose dependent, and it yields 1,2-glyceryl dinitrate (1,2-GDN), 1,3- GTN Biotransformation. 1,2-GDN and 1,3-GDN formation were GDN, inorganic nitrite, and NO (or SNO) in differing amounts measured by the TLC and liquid scintillation spectrometry and ratios. In vascular smooth muscle cells, 1,2-GDN is the method as described by Brien et al. (19) with a few modifications. predominant dinitrate metabolite (2, 5, 6) and the vasorelax- During protein purification, the assay mixture (1 ml) contained: ation-related formation of 1,2-GDN is diminished in GTN- 100 mM KPi (pH 7.5), 0.5 mM EDTA, 1 mM NADH, 1 mM tolerant blood vessels (7, 8). These data suggest that an enzyme NADPH, 0.1 or 1 M GTN, and protein (with DTT) from with product specificity for 1,2-GDN (over 1,3-GDN) generates column fractions. After incubation at 37°C for 10–30 min, the NO bioactivity from GTN and that loss of this activity at least reaction was stopped (on dry ice or 4°C) and GTN and its partly accounts for tolerance. Several enzymatic mechanisms of metabolites were extracted with 3 ϫ 4 ml ether and pooled, and vascular smooth muscle relaxation by GTN have been proposed the solvent was evaporated by a stream of nitrogen. The final (9–15), and candidate enzymes include glutathione S- volume was kept to less than 100 l in ethanol for subsequent transferases, cytochrome P450 reductase, cytochrome P450, TLC separation and scintillation counting. For the activation of xanthine oxidoreductase, and an unidentified microsomal pro- dialyzed enzyme samples and assay of the activity of purified tein. But none of these enzymes catalyzes the selective formation enzyme, the assay mixture contained: 100 mM KPi (pH 7.5), 0.5 of 1,2-GDN (12, 15, 16) or is inhibited in tolerant vessels, and the mM EDTA, 0–1 mM DTT or other reductants (0.5 mM DTT for roles of these enzymes in generating NO bioactivity remain the bovine enzyme), and 1 M GTN. For assays in tissues such controversial. Here we report on the purification of a GTN as rabbit aorta, rings were blotted and weighed after sitting for reductase that specifically generates 1,2-GDN, identify it as 1 h in Krebs solution (control) (composition described below). mitochondrial aldehyde dehydrogenase (mtALDH), and estab- When used, inhibitors were added for 20 min before the addition lish its central role in GTN bioactivation, vasorelaxation, and tolerance. Abbreviations: GTN, glyceryl trinitrate; GDN, glyceryl dinitrate; mtALDH, mitochondrial Materials Methods aldehyde dehydrogenase; SNO, S-nitrosothiol; SNP, sodium nitroprusside. Chemicals and Drugs. GTN, 1,2-GDN, and 1,3-GDN standards See commentary on page 7816. were bought from Radian International (Austin, TX). GTN was ‡To whom reprint requests should be addressed. E-mail: [email protected]. 8306–8311 ͉ PNAS ͉ June 11, 2002 ͉ vol. 99 ͉ no. 12 www.pnas.org͞cgi͞doi͞10.1073͞pnas.122225199 Downloaded by guest on September 25, 2021 Table 1. Purification of GTN reductase from mouse macrophage RAW264.7 cells Total units, Total protein, Specific activity, Yield, Fold Procedures nmol͞h mg nmol͞h per mg % purification 100,000 g supernatant 837.4 2,265 0.37 100 1.0 DEAE-cellulose 756.0 345 2.19 90.3 5.9 Q-Sepharose 528.5 123 4.29 63.1 11.6 Butyl-Sepharose 205.4 10.9 18.81 24.5 50.8 Hydroxyapatite 120.9 3.6 33.36 14.4 90.2 Starting material: 50-g cell pellet. of 1 M GTN; the mixture (1 ml) was then kept at 37°C for 5 min. Statistical Analysis. Results were statistically analyzed by the The extraction and TLC-liquid scintillation spectrometry anal- Student’s t test, and two sets of data were considered statistically ysis were as described above. Buffer control (Krebs buffer plus different when P Ͻ 0.05. GTN) and nonspecific biotransformation (heat-inactivated rings plus GTN) activities were also measured, and the results were Results and Discussion corrected accordingly. We reasoned that the difficulty in isolating the enzyme respon- sible for GTN biotransformation from blood vessels may reflect Aortic Ring Bioassays. New Zealand White rabbits (2.5–3 kg) were a lack of starting material and͞or exposure to excessive concen- killed by carbon dioxide inhalation. Thoracic aorta were re- trations of GTN (under which conditions GTN metabolism may moved, cleaned of fat and connecting tissue, and cut into 3-mm not represent biotransformation), and we thus screened alter- rings. The rings were mounted under2gofresting tension in native animal tissues and cell lines by monitoring 1,2-GDN tissue baths (25 ml) filled with Krebs solution (37°C) containing: formation from physiological amounts of GTN (0.1 M). We 118 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, found that mouse macrophage RAW264.7 cells, which can be 2.5 mM CaCl2, 25 mM NaHCO3, and 11 mM glucose, pH 7.4. grown in large numbers, resemble vascular smooth muscle cells The solution was bubbled with 20% O2,5%CO2, and balance N2. in generating mainly 1,2-GDN, and that after cellular disruption Changes in isometric tension were recorded with Statham (Hato by sonication, the enzyme activity was present predominantly in Rey, PR) transducers and a Grass Instruments (Quincy, MA) the 100,000-g supernatant. We then developed a four-step polygraph, and contractions were initiated with phenylephrine. purification procedure to isolate the enzyme from RAW264.7 Tolerance to GTN was induced by incubating rings with 0.3 mM cells (Table 1). The enzyme activity passed freely through a GTN for 30 min. Rings were then washed several times with DEAE-cellulose column and was preserved as a single peak after Krebs solution and left for 1 h before being further tested.