INTRODUCTION

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EPIDEMIOLOGIC STUDIES have suggested that exposure of humans to ambient particulate matter is associated with cardiopulmonary-related diseases and mortality (1, 7, 25). Ikeda et al. (14) reported that incubation of aortic rings of rats with suspensions of diesel exhaust particles caused suppression of endothelium-dependent vasorelaxation caused by acetylcholine. However, mechanistic details of this phenomenon still remain obscure.

Nitric oxide (NO), which is synthesized by NO synthase (NOS), plays an important role in neurotransmission, vasorelaxation, and immune response. This gas produced in endothelial cells is involved in the regulation of blood pressure, inhibition of platelet aggregation, inhibition of smooth muscle migration, and ischemic protection (22, 23, 28). Reduction of NO formation by NOS inhibitors or disruption of the gene encoding endothelial NOS (eNOS) results in vasoconstriction and increase in blood pressure (13, 26, 27). It has been shown that impairment of NO production in the endothelium is implicated in the pathophysiological actions of vascular diseases (16, 32).

We demonstrated that a variety of quinones interact with the reductase domain of neuronal NOS (nNOS), which is highly homologous with NADPH-cytochrome P-450 reductase (3) and thus inhibited NO formation by shunting the electron flow from NADPH (18). We (17) also reported that NADPH oxidation was stimulated during interaction of NADPH-cytochrome P-450 reductase with organic components extracted from diesel exhaust particles by methanol, suggesting that quinones, which are good substrates for the enzyme, are contained in diesel exhaust particles. Since it was reported that the sequence of the P-450 reductase domain of nNOS is similar to that of eNOS (3, 20), we hypothesized that quinones would inhibit enzyme activity of not only nNOS but also eNOS, thereby resulting in alteration in NO formation, which could lead to suppression of eNOS-dependent vasorelaxation and could increase blood pressure. Thus the present study was designed to evaluate the effect of phenanthraquinone on eNOS activity and NO-dependent vascular relaxation, because phenanthraquinone 1) has been found to be a relatively abundant quinone contained in diesel exhaust particles (29, 30) and 2) was the most potent inhibitor of nNOS activity among 22 quinones tested (18).

	MATERIALS AND METHODS

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Materials. Chemicals were obtained as follows: phenanthraquinone, 1,4-benzoquinone, and 2-methyl-1,4-naphthoquinone from Nacalai Tesque (Kyoto, Japan); anthraquinone from Wako Pure Chemical Industries (Osaka, Japan); 1,4-naphthoquinone from Tokyo Kasei Industries (Tokyo, Japan); 2-methyl-1,4-benzoquinone and mitomycin C from Aldrich Chemical (Milwaukee, WI); 1,4-naphthoquinone- 2-sulfonate from Eastman Kodak (Rochester, NY); 5-hydroxy-1,4-benzoquinone, AZQ, ACh, nitrate reductase, and L-arginine from Sigma Chemical (St. Louis, MO); nitroglycerin (NG) from Nihon Kayaku (Tokyo, Japan); L-2,3-[³H]arginine from DuPont-NEN Research Products (Boston, MA). AG50W-X8 resin was obtained from Bio-Rad Laboratories (Richmond, CA). Calmodulin was purified from bovine brain by the method of Gopalakrishna and Anderson (8). All other chemicals used were of the highest grade available.

Preparation of enzyme. Bovine aortic endothelial cells (BAEC) were obtained from Dainippon Pharmaceutical Industrial (Tokyo, Japan). BAEC were maintained in Dulbecco's modified Eagle's medium-nutrient mixture F-12 (1:1, vol/vol)-15% heat-inactivated fetal bovine serum-penicillin (100 U/ml)-streptomycin (100 µg/ml), fibroblast growth factor-acidic (5 ng/ml)-heparin (10 U/ml). Cells were incubated in a humidified atmosphere of 95% air-5% CO₂. The medium was changed every 2-3 days, and cells were routinely passaged by trypsin-EDTA with a split ratio of 1:4. BAEC between passages 3 and 6 were scraped from culture plates and homogenized in 50 mM Tris · HCl (pH 7.4)-0.1 mM EDTA-0.1 mM EGTA-1 mM phenylmethylsulfonyl fluoride-leupeptin (1 µg/ml). The homogenate was centrifuged at 100,000 g for 60 min. The total membrane fractions obtained were suspended in the homogenate buffer containing 2.5 mM CaCl₂ according to the method of Patel and Block (24). Suspensions obtained were frozen under liquid nitrogen and kept at 70°C until use.

NOS activity. Incubation mixtures (0.1 ml) consisted of suspension of the membrane fraction of BAEC (0.11-0.13 mg of protein), various concentrations of phenanthraquinone, complete medium (20 nM 2,3-[³H]arginine, 50 µM L-arginine, 100 µM NADPH, 10 µM tetrahydrobiopterin, 2 mM CaCl₂, 1 µg of calmodulin), and 20 mM HEPES (pH 7.4). After the enzyme solution was preincubated with phenanthraquinone at 37°C for 5 min, reactions were initiated by the addition of the complete medium and carried out at 37°C for 30 min. Under these conditions, NO production determined by formation of citrulline was linear with time and protein concentration. Phenanthraquinone was dissolved in DMSO, and the maximal volume of DMSO was maintained at 20 µl/ml of assay mixture, because DMSO slightly affected NOS activity. Production of [³H]citrulline from L-[³H]arginine was performed by the method of Bredt and Snyder (4). Briefly, each incubation was terminated by addition of 2 ml of cold stop buffer [20 mM sodium acetate buffer (pH 5.5)-1 mM citrulline-2 mM EDTA-0.2 mM EGTA]. A portion (2 ml) of the mixture was applied to a column packed with AG50W-X8 resin (1 ml), which had been extensively equilibrated with the stop buffer, and then the column was washed with 2 ml of water. The sample (1 ml) of eluates collected was mixed with 5 ml of scintillation cocktail, and radioactivity was determined using a Beckman LS-600 scintillation counter. Protein concentration was measured by the method of Bradford (2), with bovine serum albumin as the standard. To calculate IC₅₀ value for each quinone on eNOS activity and to determine kinetic parameters, values obtained from eNOS activity in the presence of different concentrations of quinones were analyzed by a nonlinear regression program using PRISM version 3.0 (Graph Pad Software, San Diego, CA). Data are expressed as the means ± SE, and a t-test and one-way ANOVA were performed. When statistically significant F values were obtained with the ANOVA, Bonferroni's correction was used.

Measurement of vascular relaxation. A total of eight male Wistar rats (8-10 wk old), weighing 200-250 g, were obtained from Kitayama Rabbis (Ina, Nagano, Japan). The rats were killed by exsanguination after being anesthetized with pentobarbital sodium (50 mg/kg ip). The thoracic aortas were removed carefully to protect the endothelial lining, cleared of adhering fat and connective tissue, and cut into 2-mm-wide transverse rings (12). The optimal passive load was determined as the contractile response to 122 mM KCl as described previously (11). Before experiments, the rings were stretched to their predetermined optimal tension, mounted on stainless-steel hooks in 20 ml-capacity muscle chambers, and bathed in oxygenated (95% O₂-5% CO₂) Krebs-Henseleit solution (in mM: 118 NaCl, 4.7 KCl, 1.5 CaCl₂, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 11 glucose, and 2 µM EDTA, pH 7.4) at 37°C for 1 h. Tension was measured isometrically using a force-displacement transducer (model DSA-603, Minebea, Tokyo, Japan). Experiments were conducted to determine the responsiveness of endothelium-intact aortic rings to an endothelium-dependent vasodilator, ACh. Then endothelium-dependent relaxation by ACh was done after preincubation with phenanthraquinone (5 µM) for 30 min. After washing the organ bath with Krebs-Henseleit solution three times, the endothelium-dependent relaxation by ACh was carried out with or without preincubation of L-arginine (1 mM). The responsiveness of endothelium-denuded aortic rings to the endothelium-independent vasodilator NG was also measured. In these experiments, phenylephrine (0.1 µM) initially induced submaximal tension (10). In some cases, indomethacin (5 µM) was added to muscle chambers for 60 min to rule out the contribution of prostanoids. Relaxation was measured as the percentage of decrease in tension below the tension evoked by phenylephrine in arterial rings. Values are expressed as means ± SE for vascular responses, and they represent unpaired measurements. Means were compared by ANOVA with repeated measurements. If a significant F value was found, Scheffé's test for multiple comparisons was used to identify differences among groups.

Measurement of blood pressure. Rats were anesthetized with urethane (750 mg/kg) and -chloralose (80 mg/kg). A polyethylene catheter (SP-31, Natsume, Tokyo, Japan) was inserted into the right carotid artery to measure arterial blood pressure. Hemodynamic measurements were recorded by means of a polygraph system (AP-601G amplifier and WT-687G thermal pen recorder, Nihon Koden, Tokyo, Japan). Phenanthraquinone (0.36 mmol/kg), dissolved in corn oil (3 ml/kg), was administered to rats by intraperitoneal injection; subsequently, blood pressure was measured for 30 min. In control rats, vehicle (corn oil) was injected. After hemodynamic measurements, a blood sample was collected from the right carotid artery.

Determination of NO metabolites. The plasma level of nitrite (NO)/nitrate (NO) was measured by the method of Conrad et al. (6). After blood samples from rats after treatment with and without phenanthraquinone were centrifuged at 700 g for 5 min, plasma (80 µl each) obtained was incubated with 2.8 mM FAD, 0.1 mM NADPH, nitrate reductase (0.035 U), and 50 mM potassium phosphate buffer (pH 7.5). Reaction (total volume, 0.35 ml) was performed at 25°C for 60 min and then terminated by addition of 0.8 ml of Griess reagent consisting of 5% phosphoric acid, 2% sulfanilamide-0.2% N-(1-naphthyl)-ethylenediamine dihydrochloride (1:1, vol/vol), and 0.45 ml of water. The mixture was kept at room temperature, and the concentration of NO/NO was measured at 542 nm. Sodium nitrate was used as the standard. For measurement of blood pressure and determination of NO/NO concentration, values are expressed as means ± SE (n = 6), and they represent paired measurements. Means were compared by ANOVA with repeated measurements. If a significant F value was found, Scheffé's test for multiple comparisons was used to identify differences among groups.

	RESULTS

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Inhibition of eNOS activity. NO production determined by citrulline formation from L-arginine by membrane fraction of BAEC was suppressed by phenanthraquinone in a concentration-dependent manner. Phenanthraquinone (1 µM) inhibited the eNOS activity by ~80%. When the effects of other quinones on eNOS activity were examined, it was found that 2-methyl-1,4-benzoquinone, AZQ, 5-hydroxy-1,4-naphthoquinone, 1,4-naphthoquinone-2-sulfonate, and mitomycin C were also potent inhibitors of eNOS activity, with IC₅₀ values ranging from 0.8 to 68.8 µM, whereas 1,4-benzoquinone and anthraquinone at concentrations up to even 100 µM did not affect NO formation by membrane fraction of BAEC. Inhibition potencies of AZQ, phenanthraquinone, 5-hydroxy-1,4-naphthoquinone, and 1,4-naphthoquinone-2-sulfonate on eNOS activity were significantly greater than those on nNOS. Because our previous findings indicated that inhibition potency of NO formation by quinones by rat cerebellar enzyme preparation for nNOS was associated with their one-electron potentials (18), IC₅₀ values of quinones tested vs. their one-electron potential values obtained by pulse radiolysis studies (15, 31, 34) were plotted as shown in Fig. 1. A bell-shaped dependency of the inhibition potencies of quinones on enzyme activity of eNOS, similar to nNOS, was found. Inhibition potencies of quinones, which have one-electron potential values ranging between 348 and 124 mV on eNOS activity increased with the increase in their one-electron reduction potentials (r = 0.959, P < 0.05). A maximal inhibition potency by quinones was seen with phenanthraquinone (IC₅₀ value = 0.6 µM), corresponding to a one-electron reduction potential of 124 mV in the case of both nNOS and eNOS. In contrast, quinones with one-electron reduction potentials more positive than 60 mV were shown to deviate from the correlation in either case.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Relationship between the inhibition of quinoid compounds on the enzyme activities for neuronal and endothelial nitric oxide synthase (nNOS and eNOS, respectively) and their one-electron reduction potentials. E, one-electron reduction potential; 1, anthraquinone; 2, mitomycin C; 3, AZQ; 4, phenanthraquinone (PQ); 5, 5-hydroxy-1,4-naphthoquinone; 6, 1,4-naphthoquinone-2-sulfonate; 7, 2-methyl-1,4-benzoquinone; 8, 1,4-benzoquinone. IC50 values for quinones that show a negligible inhibitory action on NOS activity are plotted as 100. The IC50 values for quinones on nNOS activity are cited from our previous study (22). Each point is the mean ± SE (n = 4). **Significantly different from eNOS (P < 0.01).

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Lineweaver-Burk plots of NO formed from L-arginine by membrane fraction of bovine aortic endothelial cells (BAEC) in the absence and presence of PQ. V, NO formation estimated by citrulline formed from L-arginine. Incubations were performed under conditions described in MATERIALS AND METHODS. Each point is the mean ± SE (n = 4).

Alterations in vasorelaxation. In all experimental groups, ACh produced a concentration-dependent relaxation of precontracted aortic rings with phenylephrine under intact endothelium (Fig. 3A). The magnitude of the relaxation of aortic rings preincubated with phenanthraquinone (5 µM), however, was significantly diminished (Fig. 3A). Phenanthraquinone impaired the maximum response of relaxation by ACh (control, 78.8 ± 2.9%; + phenanthraquinone, 51.8 ± 4.3%, P < 0.01) without affecting the EC₅₀ values significantly (control, 0.21 ± 0.16 µM; + phenanthraquinone, 2.72 ± 1.58 µM). No significant differences in endothelium-dependent relaxation were observed among the aortic rings obtained from the control groups or from the recovery arteries (EC₅₀ value = 0.23 ± 0.22 µM, maximum response = 69.3 ± 4.4%), indicating a reversible decrease in the endothelium-dependent relaxation by phenanthraquinone. Pretreatment with L-arginine (1 mM) did not significantly restore the suppressed endothelium-dependent relaxation by phenanthraquinone (EC₅₀ value = 0.70 ± 0.17 µM, maximum response = 68.3 ± 3.7%). In contrast, phenanthraquinone did not affect the endothelium-independent relaxation caused by NG under the same conditions (Fig. 3B).

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effects of PQ on the endothelium-dependent relaxation by ACh and on endothelium-independent relaxation by nitroglycerin. A, with endothelium-intact aortic rings; B, with endothelium-denuded aortic rings; , control; , + PQ. Relaxations caused by ACh and nitroglycerin in the absence and presence of PQ (5 µM) are expressed as percent decreases in tension from the concentration evoked by phenylephrine. Each value is the mean ± SE of 6 or 7 rats. **P < 0.01 vs.  PQ.

View larger version (15K): [in this window] [in a new window]   Fig. 4.   Change in plasma levels of NO metabolites after injection of PQ. Open bar, without PQ; closed bar, with PQ. PQ (0.36 mmol/kg) was intraperitoneally administered to rats. Plasma NO/NO was determined under conditions described in MATERIALS AND METHODS. **P < 0.01 compared with controls ( PQ). Each bar is the mean ± SE of 6 animals.

	DISCUSSION

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It was suggested that exposure of humans to ambient particulate matter was associated with cardiopulmonary-related diseases and mortality (1, 7, 25). Diesel exhaust particles have been shown to suppress endothelium-dependent relaxation of rat aorta (14). Cheng and Kang (5) reported recently that chemical components extracted from motorcycle exhaust particles inhibit endothelium-dependent relaxation of rat aorta by ACh. From these findings it is suggested that component(s) in these urban air particles play a critical role in the impairment of vasorelaxation. The findings presented here indicate that phenanthraquinone, a component of diesel exhaust particles (29, 30), inhibits constitutive NOS isozymes with different potency. Phenanthraquinone was a more potent inhibitor of eNOS (IC₅₀ = 0.6 µM) than nNOS (IC₅₀ = 10 µM). We showed previously that IC₅₀ values of quinones on nNOS activity were well correlated with one-electron reduction potentials of these quinones for molecules having these values ranging from 240 and 100 mV (18). In the present study, the inhibition potencies of anthraquinone, mitomycin C, AZQ, and phenanthraquinone on eNOS activity increased with the increase in their one-electron reduction potentials. The IC₅₀ values for AZQ, phenanthraquinone, 5-hydroxy-1,4-naphthoquinone, and 1,4-naphthoquinone-2-sulfonate on eNOS activity were significantly smaller than those on nNOS. These observations suggest that quinones are more potent inhibitors of eNOS than of nNOS and, thus, attention must be given to cardiovascular alterations by quinones in vivo.

It was reported that an anthraquinone derivative aclarubicin (5.9 µM) suppressed endothelium-dependent relaxation by ACh but this quinone had no effect on NO donor-mediated endothelium-independent relaxation under those conditions (33). Lee et al. (21) recently reported that 2-methyl-1,4-naphthoquinone (menadione) was capable of suppressing endothelium-dependent, but not endothelium-independent, relaxation of the aortic ring in rats by either ACh or histamine. Consistent with these observations, phenanthraquinone (5 µM) showed the same pharmacological action on the vasorelaxation of the rat aorta (Fig. 3). The alteration in the concentration-dependent curve for ACh-mediated vasodilation by phenanthraquinone exhibited the suppression of the maximum response without changing the EC₅₀ value, suggesting that the pharmacological action of phenanthraquinone does not involve competition at the acetylcholine receptor of the rat aorta. In our previous study, we demonstrated that menadione (100 µM) inhibited NO formation of >90% by purified nNOS (18) and that this quinone was a noncompetitive inhibitor with regard to L-arginine, with a K_i value of 27.3 µM (19). The present kinetic study revealed that the inhibition of eNOS by phenanthraquinone was noncompetitive with respect to L-arginine and competitive with respect to NADPH, as observed with nNOS enzyme preparation (18). Taken together, it is suggested that phenanthraquinone binds to the P-450 reductase domain of eNOS as well as nNOS, thereby inhibiting NO production by shunting electrons away from the normal catalytic pathway: as a consequence, quinoid compounds such as aclarubicin, menadione, and now phenanthraquinone appear to act as modulators in the vasorelaxation conducted by NO formed from constitutive NOS isozymes. Furthermore, in vivo experiments have indicated that intraperitoneal administration of phenanthraquinone (0.36 mmol/kg) to rats elevated the mean blood pressure by 1.4 times the control level; under identical conditions, the plasma level of NO/NO, which is an index for NO production in vivo (9, 35), was reduced to 68% of the control. This observation further supports a possibility that the significant increase in blood pressure of rats after phenanthraquinone exposure appears to be, in part, attributable to decreased production of NO, which regulates basal vascular tone (13, 26, 27), by inhibiting eNOS activity.

In conclusion, the present findings indicate that phenanthraquinone may be a candidate for the diesel exhaust particles-mediated dysfunction of vasodilation. Since it was reported that a variety of quinones were contained in diesel exhaust particles (29, 30), identification of the quinones, which are potent inhibitors of eNOS activity and impair cardiovascular functions, is in progress in our laboratory.

Perspectives

To our knowledge, no study has reported an elevated blood pressure by exposure to diesel exhaust particle or determination of plasma levels of phenanthraquinone in humans exposed to diesel exhaust particles. In the present study, it was shown that an intraperitoneal administration of phenanthraquinone (0.36 mmol/kg) to rats resulted in the significant elevation of blood pressure (Table 1). In our preliminary study, we found that the plasma level of phenanthraquinone 1 h after the injection of phenanthraquinone (0.36 mmol/kg) to rats was ~0.3 µM (Kumagai et al., unpublished observation); under the concentration of phenanthraquinone, the enzyme activity of eNOS in the total membrane fraction of BAEC is inhibited by 30% of control level. However, it is uncertain whether phenanthraquinone is ever elevated to the level used in the present study during exposure of animals or humans to diesel fumes. Further study is required to address this issue.

Injection of phenanthraquinone to rats decreased plasma levels of NO/NO, an indicator of NO production in vivo. This suggests that it is of great importance to investigate whether phenanthraquinone affects the plasma level of NO/NO in humans because impairment of NO production in endothelium is thought to be implicated in the pathophysiological actions of vascular diseases (16, 32). Such a study in humans and the quantitative determination of phenanthraquinone in diesel exhaust particles would be required to elucidate contribution of phenanthraquinone to vascular dysfunction caused by exposure to urban air particles.