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Chem. Res. Toxicol. 1998, 11, 608-613
Inhibition of Nitric Oxide Formation by Neuronal Nitric Oxide Synthase by Quinones: Nitric Oxide Synthase as a Quinone Reductase† Yoshito Kumagai,*,‡ Hiromi Nakajima,§,| Kazumi Midorikawa,§ Shino Homma-Takeda,‡ and Nobuhiro Shimojo‡ Department of Environmental Medicine, Institute of Community Medicine, and Master’s Program in Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305, Japan Received July 10, 1997
Inhibitory action of a variety of quinoid compounds on neuronal nitric oxide synthase (nNOS) activity was examined with a 20000g rat cerebellar supernatant preparation and purified nNOS. The inhibition of citrulline formation from L-arginine by quinones, which exhibit one-electron reduction potentials (E17) ranging between -240 and -100 mV, increased at a more positive one-electron reduction potential, suggesting that quinone appears to act as an electron acceptor for nNOS. Among the quinones tested, 9,10-phenanthraquinone (PQ), corresponding to an E17 value of -124 mV, exhibited the most potent inhibiton of citrulline formation (IC50 value ) 10 µM). A kinetic study revealed that PQ is a competitive inhibitor with respect to NADPH, with a Ki value of 0.38 ( 0.12 µM, and a noncompetitive inhibitor with respect to L-arginine, with a Ki value of 9.63 ( 0.20 µM. Partial purification of the enzymes which are responsible for reducing PQ in 20000g supernatant of rat cerebellum by anion-exchange column chromatography indicated that one catalyst for PQ reduction was nNOS. Reductase activity of PQ by purified nNOS required CaCl2/calmodulin and was markedly suppressed by the flavoprotein inhibitor diphenyleneiodonium but not by L-nitroarginine which is a specific inhibitor for NO formation. nNOS effectively reduced the quinones as well as PQ causing a marked decrease in the production of NO from L-arginine, while 1,4-benzoquinone, 9,10-anthraquinone, mitomycin C, and lapachol, which show negligible inhibitory action on nNOS activity, were poor substrates for the enzyme on reduction. These results indicate that PQ and other quinones used in the present study interact with the NADPH-cytochrome P450 reductase domain on nNOS and thus probably inhibit NO formation by shunting electrons away from the normal catalytic pathway. Therefore, our study suggests that quinones could possibly affect NOdependent physiological and/or pathophysiological actions in vivo.
Introduction Quinones represent an important class of naturally occurring compounds that are found in plants, fungi, and bacteria, primarily as important components of the electron-transport chains involved in cellular respiration and photosynthesis. Synthetic or quinoid compounds extracted from plants, such as 1,4-benzoquinone (1,4BQ)1 and 1,4-naphthoquinone (1,4-NQ), have been utilized as dyes. 9,10-Phenanthraquinone (PQ), 9,10anthraquinone (AQ), and various unidentified quinones † Presented in part at the 4th International Symposium on Metal Ions in Biology and Medicine, Barcelona, Spain, May 19-22, 1996, and also at the 5th International Meeting on Biology of Nitric Oxide, Kyoto, Japan, Sept 15-19, 1997. * To whom correspondence should be addressed: Yoshito Kumagai, Ph.D. Tel: 0298-53-3297 (Japan). Fax: 0298-53-3039 (Japan). Email:
[email protected]. ‡ Department of Environmental Medicine. § Master’s Program in Environmental Sciences. | Present address: Central Research Institute of Electric Power Industry, Chiba 270-11, Japan. 1Abbreviations: NO, nitric oxide; nNOS, neuronal nitric oxide synthase; BQ, benzoquinone; NQ, naphthoquinone; PQ, 9,10-phenanthraquinone; AQ, 9,10-anthraquinone; AZQ, 2,5-diaziridinyl-3,6-bis(carboxyamino)-1,4-benzoquinone; E17, one-electron reduction potential; CaM, calmodulin; P450 reductase, NADPH-cytochrome P450 reductase; DPI, diphenyleneiodonium; DTT, dithiothreitol; PMSF, phenylmethanesulfonyl fluoride; BH4, (6R)-5,6,7,8-tetrahydro-L-biopterin.
are ubiquitous contaminants in urban air along with other polycyclic aromatic hydrocarbons (1-4); PQ can be formed from phenanthrene via biotransformation in mammals (5). AQ or BQ derivatives including mitomycin C represent the largest class of clinically useful antitumor agents (6). For these reasons, biological effects of quinones have been studied extensively. In general, the toxicity of quinones can result from different mechanisms (e.g., direct alkylation, oxidation-reduction cycling, and resulting generation of active oxygen species) (7, 8). Cytosolic enzymes, carbonyl reductase, DT-diaphorase, and 15-hydroxyprostaglandin dehydrogenase have been suggested as being involved in the reduction of PQ (914). Most of the quinones can also undergo one-electron reduction by the membrane-bound flavoprotein NADPHcytochrome P450 reductase (P450 reductase) to form a semiquinone radical which can reduce oxygen to superoxide (7, 8, 11, 15). Nitric oxide (NO) is biosynthesized from L-arginine by the enzyme nitric oxide synthase (NOS, EC 1.14.13.39). Citrulline is the coproduct of NO biosynthesis and is generated stoichiometrically with NO. It has been established that NO is involved in a variety of physiological processes including cardiovascular function, neurotransmission, and immune response (16-18). Neu-
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ronal NOS (nNOS) has a calmodulin (CaM)-dependent FAD- and FMN-containing reductase domain which is highly homologous with P450 reductase (19); this domain is capable of transferring electrons from NADPH to artificial acceptor molecules such as cytochrome c (20), 2,6-dichlorophenol indophenol (20), methylene blue (21), and nitroblue tetrazolium (22). It has been found that antineoplastic anthracyclines, doxorubicin and aclarubicin, which possess a quinone moiety in the strucutre, affected NOS activity in rat cerebellum (23). Thus, these findings led us to propose that quinoid compounds could interact with the P450 reductase domain on nNOS, resulting in a decrease in NO formation from L-arginine because these chemicals are able to accept electrons from NADPH during enzymatic reaction with P450 reductase. The present study indicates that a variety of quinones with different substituent groups or structures are effectively reduced by nNOS, resulting in suppression of citrulline formation from L-arginine in the presence of these compounds.
Experimental Procedures Materials. Chemicals were obtained as follows: PQ, 1,4BQ, and 2-methyl-1,4-NQ (menadione) from Nacalai Tesque, Inc. (Kyoto, Japan); AQ from Wako Pure Chemical Industries, Ltd. (Osaka, Japan); 1,2-NQ, 1,4-NQ, and 2-chloro-AQ from Tokyo Kasei Industries, Ltd. (Tokyo, Japan); 2-chloro-1,4-BQ, 2,3,5,6tetramethyl-1,4-BQ, 2-methyl-1,4-BQ, mitomycin C, 5,8-dihydroxy-1,4-NQ, 5-hydroxy-2-methyl-1,4-NQ (plumbagin), 2,3dichloro-1,4-NQ, 2-chloro-3-pyrrolidino-1,4-NQ, 2-hydroxy-3-(3methyl-2-butenyl)-1,4-NQ (lapachol), and 5,12-naphthacenequinone from Aldorich Chemical Co. Inc. (Milwaukee, WI); 1,4NQ-2-sulfonate from Estman Kodak Co. (Rochester, NY); 5-hydroxy-1,4-BQ, 2,5-diaziridinyl-3,6-bis(carboxyamino)-1,4-benzoquinone (AZQ), pyrroloquinoline quinone (methoxatin), L-arginine, and diphenyleneiodonium (DPI) from Sigma Chemical Co. (St. Louis, MO); L-[2,3-3H]arginine from DuPont/NEN Research Products (Boston, MA). Phenanthraquinol was synthesized by addition of sodium borohydride to PQ in ethanol, and the reaction product was purified by silica gel column chromatography with chloroform/methanol (9:1, v/v) as eluate [λmax ) 271, 239 nm; MS (FAB) m/z 212 (M+). Anal. Calcd for C14H12O2: C, 79.20; H, 5.66; O, 15.1. Found: C, 78.49; H, 5.62; O, 15.83]. DEAE-Sephacel, 2′,5′-ADP Sepharose 4B, and CaM Sepharose 4B were obtained from Pharmacia LKB Biotechnology, Inc. (Uppsala, Sweden). CaM was purified from bovine brain by the method of Gopalakrishna and Anderson (24). All other chemicals used were of the highest grade available. Apparent octanol/ H2O partition coefficient of each quinone was determined by the method of Casy and Wright (25). Enzyme Preparation. Wistar male rats (5 weeks) were used. The cerebellum was homogenized with 2 vol of 50 mM Tris-HCl (pH 7.4)-0.1 mM EDTA-0.1 mM EGTA-0.5 mM dithiothreitol (DTT)-protease mix consisting of 1 µM pepstatin-2 µM leupeptin-1 mM phenylmethanesulfonyl fluoride (PMSF). The homogenate was centrifuged at 20000g for 60 min. Supernatants obtained were frozen under liquid nitrogen and kept at -70 °C before use. Purification of nNOS was performed according to the methods of Bredt and Snyder (26) and Schmidt et al. (27). P450 reductase was purified from liver microsomes of phenobarbital-treated rats as reported previously (28). Protein concentration was measured by the method of Bradford with bovine serum albumin as a standard (29). NOS Assay. Incubation mixtures (0.1 mL) contained enzyme preparation, various concentrations of quinoid compounds, complete medium [20 nM [2,3-3H]arginine, 50 µM L-arginine, 100 µM NADPH, 10 µM (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4), 2 mM CaCl2, 1 µg of CaM], and 20 mM Hepes (pH 7.4). Reactions were initiated by addition of the complete medium
Chem. Res. Toxicol., Vol. 11, No. 6, 1998 609 Table 1. Inhibition Potencies of a Variety of Quinones on the Citrulline Formation by 20000g Supernatant of Rat Cerebellum and Their One-Electron Reduction Potentials (E17) and Apparent Octanol/H2O Partition Coefficientsa E17 (mV)b
quinone benzoquinone 1,4-BQ 2-methyl-1,4-BQ 2-chloro-1,4-BQ 2,3,5,6-tetramethyl-1,4-BQ AZQ methoxatin naphthoquinone 1,2-NQ 1,4-NQ menadione 5-hydroxy-1,4-NQ 5,8-dihydroxy-1,4-NQ plumbagin 1,4-NQ-2-sulfonate 2,3-dichloro-1,4-NQ 2-anilino-1,4-NQ lapachol 2-chloro-3-pyrrolidino-1,4-NQ polycyclicquinone PQ AQ 2-chloro-AQ 5,12-naphthacenequinone mitomycin C
IC50 (µM)c
+99 >100 +23 63.8 61.3 -240 93.1 -168 83.9 -122 15.1
partition coefficientd 1.5 13.9 nd 144.3 1.3 0.2
11.5 -140 25.9 -203 49.5 -93 11.1 -110 10.9 -156 27.4 -60 44.4 -36 27.6 - >100 - >100 - >100
nd 38.7 75.9 16.5 1.3 28.4 0.08 26.8 nd nd nd
-124 -348 -271
18.6 82.3 nd nd nd
10.0 >100 >100 >100 >100
a NOS activity in the absence of quinones was 0.49 nmol/min/ mg of protein (n ) 5). b These single-electron reduction potentials are literature values as shown in the text. c Each value was determined from an equation obtained from, at least, four data points (duplicate determinations) exhibiting inhibition degree to NO formation caused by quinones. d Apparent octanol/H2O partition coefficients were determined under the conditions described in Experimental Procedures. -, no data available; nd, not determined.
and carried out at 37 °C for 10 min. Quinones were dissolved in DMSO, and the maximal volume of DMSO was maintained at 20 µL/mL of assay mixture: Under these conditions, the NOS activity was slightly affected by DMSO (10.7 ( 3.6% inhibition of control, n ) 5). After reaction, production of [3H]citrulline from [3H]arginine was performed by the method of Bredt and Snyder (26). Quinone Reduction Assay. Reductions of PQ and other quinoid compounds were determind by NADPH oxidation at 340 nm using an extinction coefficient of 6.22 mM-1 cm-1, employing a Shimadzu UV-1600 double-beam spectrophotometer (Kyoto, Japan), in a sample compartment thermostated at 25 °C. Briefly, incubation mixtures (1.2 mL) contained purified nNOS (but this is added to the sample cuvette only), 10-100 µM PQ, complete medium described above, and 20 mM Hepes (pH 7.4). Reactions were initiated by addition of the complete medium to the reference and sample cuvettes. The quinone-dependent NADPH oxidation was estimated by deducting the enzymatic NADPH consumption in the presence of DMSO only.
Results Table 1 shows the IC50 values for various quinoid compounds on the NO formation determined by production of citrulline from L-arginine by 20000g supernatant preparation of rat cerebellum. Among 22 quinones tested, 14 quinones suppressed nNOS activity in a concentration-dependent manner. Methoxatin, 1,2-NQ, 5-hydroxy-1,4-NQ, 5,8-dihydroxy-1,4-NQ, and PQ were potent inhibitors of citrulline formation with IC50 values of around 10 µM. IC50 values for 1,4-NQ, plumbagin, and
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Figure 1. Relationship between inhibition potencies of quinones on the NOS activity and their octanol/H2O partition coefficients (left side) or their one-electron reduction potentials (right side): 1, AQ; 2, mitomycin C; 3, 2,3,5,6-tetramethyl-1,4BQ; 4, menadione; 5, AZQ; 6, plumbagin 7, 1,4-NQ; 8, PQ; 9, methoxatin; 10, 5,8-dihydroxy-1,4-NQ; 11, 5-hydroxy-1,4-NQ; 12, 1,4-NQ-2-sulfonate; 13, 2,3-dichloro-1,4-NQ; 14, 2-methyl-1,4BQ; 15, 1,4-BQ. These values of IC50, single-electron reduction potential (E17), and octanol/H2O partition coefficient for quinones examined are listed in Table 1. IC50 values of quinones which give negligible inhibitory action on NOS activity were plotted as 100.
2,3-dichloro-1,4-NQ were about 3-fold greater. 2-Methyl1,4-BQ, 2-chloro-1,4-BQ, 2,3,5,6-tetramethyl-1,4-BQ, AZQ, menadione, and 1,4-NQ-2-sulfonate were even less potent, showing IC50 values ranging from approximately 50 to 90 µM. By contrast, 1,4-BQ, 2-anilino-1,4-BQ, lapachol, 2-chloro-3-pyrrolidino-1,4-NQ, AQ, 2-chloro-AQ, 5,12-naphthacenequinone, and mitomycin C at concentrations up to even 100 µM were without inhibitory action on nNOS activity. A plot of IC50 values of quinones versus their octanol/H2O partition coefficients is presented in Figure 1 (left side) and shows that no correlation exists between two parameters. In contrast, a bellsharp dependency of the inhibition potencies of quinones on their one-electron reduction potentials (E17) obtained by pulse radiolysis studies (30-32) was found (Figure 1, right side). The IC50 values of quinones on NOS activity were correlated (γ ) 0.737) with one-electron reduction potentials of these quinones for molecules having E17 values ranging between -240 and -100 mV. A maximal inhibition was observed with PQ, corresponding to an E17 value of -124 mV. However, compounds with potentials more than positive than -36 mV were shown to deviate from the correlation. At E17 either lower than -270 mV, as was the case for mitomycin C and AQ, or greater than approximately +100 mV, as was the case for 1,4-BQ, negligible inhibition of citrulline formation by quinones was seen at even 100 µM. The inhibition of nNOS by PQ was found to be reversible since full enzyme activity was restored following dialysis (data not shown). Thus, the observed inhibition of nNOS was not due to irreversible enzyme modification resulting in active oxygen species (i.e., superoxide, hydrogen peroxide, hydroxyl radical, etc.) generated from PQ redox cycling. PQ was found to be a noncompetitive inhibitor with respect to L-arginine (Figure 2); the Ki value estimated by Dixon plot analysis was 9.63 ( 0.20 µM. Inhibition of citrulline formation by PQ, however, exhibited competitive behavior (Ki ) 0.38 ( 0.12 µM) versus NADPH (Figure 2). Figure 3 shows an elution pattern of enzymes which are capable of reducing PQ from a DEAE-Sephacel column. There were at least three peaks showing PQ reductase activity. The reductase activity contained in
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Figure 2. Lineweaver-Burk plots of production of citrulline from L-arginine by the 20000g supernatant of rat cerebellum in the presence of PQ. V, NO formation as estimated by citrulline formed from L-arginine. Incubations were performed under the conditions described in Experimental Procedures. Each point is the average of duplicate determinations.
Figure 3. Separation of enzymes responsible for PQ reduction from rat cerebellum enzyme preparation by DEAE-Sephacel column chromatography. Open circle, closed circle, and solid line indicate NOS activity, PQ reductase activity, and absorbance at 280 nm, respectively. The 20000g supernatant fraction (197 mg) of rat cerebellum was applied on a DEAE-Sephacel column (4- × 2.5-cm i.d.), which had been equilibrated with 50 mM TrisHCl (pH 7.4)-10% glycerol-0.1 mM EDTA-0.1 mM EGTA-1 mM DTT-protease mix, as described in Experimental Procedures, at a flow rate of 1.5 mL/min. After the column was washed with equilibration buffer until absorbance in eluates showed less than 0.10, then a linear gradient of NaCl ranging from 0 to 0.4 M was started from fraction 37.
the first fractions (11-15) required either NADPH or NADH as cofactor and was inhibited by the DT-diaphorase inhibitor dicumarol at 0.01 mM (33). Also, an inhibitor of carbonyl reductase, quercetin (34), inhibited reductase by 24% in these fractions. The PQ reductase activity which eluted later (fractions 50-67) coincided with NOS activity. The enzymes did not bind to a BlueSepharose column, to which DT-diaphorase is selectively bound (35), but exhibited high affinity for 2′,5′-ADP Sepharose 4B and CaM Sepharose 4B gels (data not shown). Thus, isolation steps were performed according to the established method for purification of nNOS (26, 27). Final enzyme preparation gave a single band with a molecular weight of 164 kDa on SDS-PAGE (Figure 4A) and was positive against specific anti-nNOS (Figure 4B). As expected, the purified enzyme catalyzed reduction of PQ with a specific activity of 6802 nmol/mg/min. Results of PQ reduction by nNOS and inhibition of citrulline formation by quinones are shown in Table 2. PQ reduction by nNOS required CaCl2/CaM. No reductase activity was measurable when NADPH was replaced by the same concentration of NADH. The reverse reaction, i.e., the dehydrogenation of phenanthraquinol to PQ, was tested in the presence of NADP, but no NADPH was
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Chem. Res. Toxicol., Vol. 11, No. 6, 1998 611 Table 3. Reductase Activities of Quinones by nNOS and P450 Reductasea reductase activity (µmol/mg/min) compound
Figure 4. SDS-polyacrylamide gel (15%) electrophoreses of enzyme preparations responsible for PQ reduction: A, silver stain; B, Western blot analysis. Lanes 1, 20000g supernatant of rat cerebellum (2.7 µg); 2, DEAE-Sepacel fraction (1.3 µg); 3, 2′,5′-ADP Sepharose fraction (216 ng); 4, CaM Sepharose fraction (117 ng). DEAE-bound fraction (no. 55-67, 33 mg) was applied on a 2′,5′-ADP Sepharose 4B column (2.8- × 1-cm i.d.), which had been equilibrated with 10 mM Tris-HCl (pH 7.4)10% glycerol-4 µM FAD-4 µM BH4-1 mM DTT-protease mix (buffer A). The column was successively washed with buffer A, followed by buffer A-0.6 M NaCl (25 mL), buffer A-1.1 mM NADP+ (25 mL). Then nNOS was eluted by 16 mL of buffer A-10 mM NADPH at a flow rate of 0.7 mL/min. Fractions (25 µg) containing not only NOS activity but also PQ reduction activity were mixed with 1/9 vol of buffer A-1 M NaCl-20 mM CaCl2. The mixture was further applied on a CaM Sepharose 4B column (3- × 1-cm i.d.), which had been equilibrated with 50 mM Tris-HCl (pH 7.4)-10% glycerol-0.1 M NaCl-4 µM FAD-4 µM BH4-1 mM DTT-protease mix (buffer B) containing 2 mM CaCl2. After the column was extensively washed with the equilibration buffer, nNOS was eluted with buffer B containing 5 mM EGTA at 0.7 mL/min. Finally, fractions (6.5 µg) which contained only single protein showing a molecular weight of 164 kDa on the electrophoresis were collected. The specific activities of each purification step during purification were as follows. NOS: 20000g supernatant fraction, 0.45; DEAE-Sephacel fraction, 1.07; 2′,5′-ADP Sepharose fraction, 355; CaM Sepharose fraction, 447 nmol/mg/min. PQ reductase: 20000g supernatant fraction, 89; CaM Sepharose fraction, 6802 nmol/mg/min. Table 2. Effects of a Variety of Compounds on NO Formation and PQ Reduction by Purified nNOSa enzyme activity (% of control) conditions
NO formation
PQ reduction
complete (control) + NADH (0.1 mM)b - (Ca+/CaM) + PQ (0.01 mM) + PQ (0.1 mM) + phenanthraquinol (0.1 mM) + 1,4-NQ (0.1 mM) + menadione (0.1 mM) + lapachol (0.1 mM) + mitomycin C (0.1 mM) + L-arginine (0.05 mM) + L-nitroarginine (0.05 mM) + diphenyleneiodonium (0.01 mM)
3 4 54 5 105 3 10 126 117 100 4 3
0 0 100 112c 0c 107 132 32
a Incubations were performed under the conditions described in Experimental Procedures. NO formation was estimated as citrulline formed from L-arginine. Control enzyme activities of NO formation (with 0.05 mM L-arginine) and quinone reduction (with 0.01 mM PQ) were 447 and 6802 nmol/min/mg of protein, respectively. b Used instead of NADPH. c Incubated in the presence of 0.1 mM NADP. Each value is the mean of two to three determinations.
produced, suggesting that nNOS can only catalyze the reduction of PQ and not its oxidation of phenanthraquinol. Citrulline formation by nNOS was markedly inhibited by PQ, 1,4-NQ, and menadione (0.1 mM). In
benzoquinone 1,4-BQ 2-methyl-1,4-BQ 2,3,5,6-tetramethyl-1,4-BQ methoxatin AZQ naphthoquinone 1,4-NQ menadione 5-hydroxy-1,4-NQ 5,8-dihydroxy-1,4-NQ plumbagin 1,4-NQ-2-sulfonate 2,3-dichloro-1,4-NQ polycyclic quinone PQ AQ mitomycin C lapachol
nNOS
P450 reductase
0 0 4.83 5.82 7.80
9.70 4.35 11.85 4.35 13.48
8.00 11.72 15.60 7.87 8.18 23.42 13.28
11.63 11.53 13.66 8.06 13.08 8.83 9.70
5.55 0 0 0
14.04 0 0.83 0.47
a Incubations were performed under the conditions described in Experimental Procedures. Quinone reductase activity was estimated as quinone-dependent NADPH consumption. Quinoneindependent NADPH oxidations in the absence and presence of CaCl2/CaM by nNOS were 0.07 and 0.98 µmol/mg/min, respectively. Final concentration of each quinone was 0.1 mM. Specific activities of nNOS and P450 reductase were 364 nmol of Lcitrulline formed/mg/min and 34.53 U/mg, respectively. Each value is the mean of two determinations.
contrast, mitomycin C and lapachol gave negligible inhibition to citrulline formation. L-Nitroarginine (0.05 mM), a competitive inhibitor of L-arginine binding on nNOS (36), was without effect on the reduction of PQ (0.01 mM) by nNOS, although citrulline formation by the enzyme was suppressed by 96% by the enzyme inhibitor. However, the addition of 0.01 mM DPI, which competes with a nucleotide cofactor for binding to NOS (37), to the reaction mixture resulted in a significant decrease in PQ reduction. To confirm that not only PQ but also quinones which show inhibitory action on NOS activity would serve as electron acceptors for nNOS, reductase activities of various quinones by nNOS were investigated (Table 3). As expected, nNOS catalyzed reduction of quinoid compounds having inhibition of citrulline formation from L-arginine, whereas quinones except for 2-methyl-1,4-BQ, which did not affect NOS activity, were as poor substrates for nNOS as quinone reductase. The reductase activities of nNOS were relatively comparable to those of P450 reductase, an enzyme catalyzing one-electron reduction of quinone, although 1,4-BQ, 2-methyl-1,4-BQ, mitomycin C, and lapachol were reduced by P450 reductase but not by nNOS (Table 3).
Discussion The inhibition of the NOS isozymes can be accomplished in a number of ways by numerous classes of compounds (38-40). The results presented here indicate that quinones are potent inhibitors of citrulline formation from L-arginine by nNOS and, therefore, represent another class of NOS inhibitor. It was evident from the data in Figure 1 that inhibition potencies of quinones which exhibit E17 values ranging between -240 and -100 mV on NOS activity increase with the increase in their
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one-electron reduction potentials, reaching maximal values at E17 of approximately -100 mV. Such a relationship has been observed between reductase activities of various quinones by P450 reductase and their oneelectron reduction potentials (41-43). Iyanagi et al. (44) and Vermilion et al. (45) have found the redox couple FMNH•/FMNH2 (-270 mV) of P450 reductase can function as a one-electron carrier in its catalytic cycle. NOS flavins, FAD and FMN, are also thought to shuttle electrons obtained from NADPH onto the enzyme’s heme group, enabling heme-dependent oxygen activation and NO formation from L-arginine to take place (46-48). Because electron acceptance favors high potentials and the same acceptance makes the compound more easily reduced, it was suggested that the one-electron reduction potential (between approximately -100 and -350 mV) of each quinone reflects the observed magnitude of inhibition potency: In fact, quinones which are reduced by nNOS exhibited inhibitory action on citrulline formation, whereas mitomycin C or AQ that served as poor substrates for the enzyme did not. As shown in Tables 1 and 3, however, there was not a good correlation between the reduction rate and the IC50 of quinones. Although it has been reported that an approximate linear relationship is observed between Vmax/Km or kcat/Km and E17 of oxidants (43, 49), we did not estimate kinetic parameters for nNOS-catalyzed quinone reduction in the present study. The covalent modification of nNOS protein via thiol groups and the generation of potentially destructive active oxygen species during reduction of quinone by nNOS should be noted as possible inhibitory actions of quinone on enzyme activity (7, 8), but it seems unlikely because (1) there was an excess amount of DTT in the incubation mixture and (2) scavenging agents for superoxide, hydrogen peroxide, and hydroxyl radical did not prevent the decrease in NO synthesis caused by quinone. For these reasons, the mechanistic details of quinone-mediated decrease in nNOS activity still remain obscure in this study. It has been shown that nNOS is capable of catalyzing electron transfer to artifical electron acceptors such as cytochrome c (20), 2,6-dichlorophenol indophenol (20), methylene blue (21), nitroblue tetrazolium (22), and now quinone; the reductase activities of 2,6-dichlorophenol indophenol, methylene blue, and nitroblue tetrazolium by nNOS were CaCl2/CaM-independent, whereas these cofactors were essential for the reduction of cytochrome c or PQ. This may indicate a differential role of CaCl2/ CaM in the nNOS-catalyzed electron transfer to each acceptor. Among the quinones employed in this study, PQ exhibited the most potent inhibiton of citrulline formation with a IC50 value of 10 µM. The inhibition of nNOS by PQ was noncompetitive with respect to L-arginine and competitive with respect to NADPH indicating that it does not bind to the substrate binding site of nNOS but rather binds to or near the NADPH binding site. This hypothesis was supported by experiments with L-nitroarginine and DPI. Taken together, it is suggested that PQ binds to the P450 reductase domain close to the C-terminal of nNOS, thereby shunting the electron flow from the cofactor resulting in the inhibition of NO formation. In our preliminary examination, electron-spin resonance studies showed that addition of PQ to an incubation mixture of nNOS, CaCl2/CaM, and NADPH resulted in a stimulation of superoxide production (Y.
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Kumagai et al., unpublished observation). This observation indicates that the resulting semiquinone radical formed, during reduction of PQ by nNOS, transfers in turn its unpaired electron to molecular oxygen, thus forming superoxide. Niwa et al. (50) have recently found that Triptoquinone A, a naturally occurring diterpene, alters the expression of inducible NOS by decreasing the mRNA levels. Further study is, therefore, required to reveal whether the inhibition of NO production caused by quinones with different structures involves suppression of gene expression and/or the reduction of the electron transfer by binding to NOS isozymes. In conclusion, present data suggest that quinones can act as artificial electron acceptors and thus inhibit NO synthesis. We have recently found that PQ (1 µg/mL) suppressed relaxation of rat aortic rings with intact endothelium by acetylcholine significantly, whereas rings denuded of endothelium by nitroglycerin were unaffected by this quinone (51), suggesting that PQ inhibits not only nNOS but also endothelial NOS by affecting the CaCl2/ CaM-dependent electron transfer. Also, our preliminary studies found that a subcutaneous administration of PQ (75 mg/kg) to rats caused a significant elevation of blood pressure (Y. Kumagai et al., unpublished observation). Thus these observations may indicate a potential for quinones to elicit physiological and/or pathophysiological affects via an alteration on NO-mediated events.
Acknowledgment. We wish to thank Dr. Masaru Shinyashiki for his excellent contribution to this work, Dr. Jon M. Fukuto, Department of Molecular and Medical Pharmacology, UCLA School of Medicine, for helpful comments, and Dr. Takashi Miyauchi and Dr. Satoshi Sakai, Cardiovascular Division, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, for encouragement. This research was supported in part by a Grant-in-Aid (08670385) for scientific research from the Ministry of Education, Science and Culture of Japan and by a fund (University Research Project) from the University of Tsukuba. Note Added in Proof. During the review of this manuscript, Va´squez-Vivar et al. [Biochemistry 36, 1129311297 (1997)] showed that the endothelial NOS reduces adriamycin, a quinone derivative, to the semiquinone radical.
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