Reduction of Nω-hydroxy-l -arginine to l -arginine by pig liver microsomes, mitochondria, and human liver microsomes

BBRC Biochemical and Biophysical Research Communications 349 (2006) 869–873 www.elsevier.com/locate/ybbrc

Reduction of Nx-hydroxy-L-arginine to L-arginine by pig liver microsomes, mitochondria, and human liver microsomes q Bernd Clement *, Thomas Kunze, Sabine Heberling Pharmazeutisches Institut, Christian-Albrechts-Universita¨t zu Kiel, Gutenbergstraße 76, D-24118 Kiel, Germany Received 7 August 2006 Available online 31 August 2006

Abstract Nx-Hydroxy-L-arginine, the intermediate in nitric oxide formation from L-arginine catalyzed by NO synthase, can be released into the extracellular space. It has been suggested that it can circulate and exert paracrine effects. Since it cannot only be used as substrate by NO synthases, but can also be oxidized by cytochrome P450 and other hemoproteins in a superoxide-dependent manner, it has been proposed that it can serve as NO donor. In the present study, the in vitro reduction of Nx-hydroxy-L-arginine was examined. Pig and human liver microsomes as well as pig liver mitochondria were capable of reducing Nx-hydroxy-L-arginine to L-arginine in an oxygen-insensitive enzymatic reaction. These results demonstrate that this metabolic pathway has to be considered when suggesting Nx-hydroxy-L-arginine as NO-precursor. The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce L-arginine from Nx-hydroxy-L-arginine.  2006 Elsevier Inc. All rights reserved. Keywords: Nx-Hydroxy-L-arginine; Reduction; Nitric oxide; NO synthase

Nx-Hydroxy-L-arginine (NOHA) is an intermediate compound formed during the two-step enzymatic reaction leading to the production of nitric oxide (NO) and L-citrulline from L-arginine (L-Arg) by NO synthase [1]. This NADPH-dependent NOS-catalyzed L-Arg/NO pathway is resumed in Fig. 1. Nitric oxide synthase has a cytochrome P450-type active site and catalyzes the monooxygenation of L-Arg to NOHA in a normal P450-type reaction in the first step of NO synthesis. This first step is NOS-dependent, since there was no other enzyme found so far leading to the formation of Nx-hydroxy-L-arginine from L-Arg. The fact that NOHA can act as a good substrate of NOS which represents the second step of NO-formation has been shown by Moali et al. [2] using the purified recombinant neuronal q Abbreviations: L-Arg, L-arginine; NO, nitric oxide; NOHA, Nx-hydroxy-L-arginine; NOS, nitric oxide synthase. * Corresponding author. Fax: +49 431 880 1352. E-mail address: [email protected] (B. Clement).

0006-291X/$ - see front matter  2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.08.123

and macrophage isoforms. Rodriguez-Crespo et al. [3] completed these results demonstrating that also the recombinant endothelial isoform is capable of forming nitric oxide from Nx-hydroxy-L-arginine. In contrast to NOHA-formation from L-Arg, alternative pathways for the oxidation of Nx-hydroxy-L-arginine to nitric oxide have been proposed which include the involvement of cytochrome P450 or other hemoproteins and of superoxide ions [4–6]. Additionally, it has been demonstrated that NOHA can be liberated from the active site of NOS and is released into the extracellular medium [7,8]. These reports suggest that NOHA might exert paracrine or endocrine effects and can act as NO donor also in cells not containing NOS, provided that it can enter other cells once released in the extracellular fluid. It has also been discussed that Nx-hydroxy-L-arginine can be regarded as a distinct biological effector molecule that functions to inhibit arginase [9,10]. Previous reports have shown that inflammatory stimuli increase serum NOHA significantly, suggesting that circulating NOHA might represent a specific marker of

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B. Clement et al. / Biochemical and Biophysical Research Communications 349 (2006) 869–873

H2N

NH

H2N

NH

+

H2N

NH

COO-

L-arginine

O NH +

0.5 equivalent NADPH + O2

NADPH + O2 H3N

N~ OH

H3N

+

COO-

N -hydroxy-L-arginine

H3N

+

.NO

COO-

citrulline

nitric oxide

Fig. 1. L-Arginine/NO pathway catalyzed by NO synthase.

NOS activity in vivo [8]. In humans, serum Nx-hydroxy-Larginine concentration, an index of an increased NO synthase activity or expression, or both, has been shown to be important in pathophysiology. Indeed, in diseases of high inflammatory activity, such as rheumatoid arthritis and systemic lupus erythematosus, serum NOHA was significantly increased [11]. Nitric serves as intra- and intercellular mediator [12,13]. It displays potent activities in mammalian cardiovascular and nervous systems and is an important cytotoxic agent produced by activated macrophages [12]. It participates in important physiological processes, such as vasodilatation, neuronal signalling, and inhibition of tumor cells. Previous studies have shown that N-hydroxylated derivatives of strongly basic functional groups can be reduced both in vivo and in vitro by microsomal enzyme systems present in all mammalian species (rats, rabbits, pigs, and humans) tested to date [14–18]. This oxygen-insensitive liver microsomal enzyme system that requires NADH-cytochrome b5-reductase, cytochrome b5, and a third protein component, named the benzamidoxime reductase, was shown to be one of the catalysts responsible for the efficient reduction of primary N-hydroxylated structures such as amidoximes, hydroxylamines, and N-hydroxyguanidines [15]. The third protein component, isolated and purified from pig liver microsomes, has been identified as an isoenzyme of the cytochrome P4502D subfamily [19]. The question arose if the endogenous N-hydroxyguanidine, Nx-hydroxy-L-arginine, can also be reduced like the xenobiotic compounds studied before. Thus, the object of the study presented here was to determine if Nx-hydroxy-L-arginine is being reduced back to L-arginine by liver microsomal homogenates. Since it has been demonstrated that NOHA can be liberated from the active site of NOS and is released into the extracellular medium [7,8], this possible reduction is of interest when suggesting that NOHA can act as NO donor also in cells not containing NOS, provided that it can enter other cells once released in the extracellular fluid. Nx-Hydroxy-L-arginine containing a N-hydroxylated guanidinium function was assumed to be a substrate for N-reductive microsomal enzymes such as the benzamidoxime reductase. In the present study, we were able to show that pig and human liver microsomes as well as pig liver mitochondria were capable of reducing

Nx-hydroxy-L-arginine to L-Arg in an oxygen-insensitive enzymatic reaction. Materials and methods Chemicals. Nx-Hydroxy-L-arginine, octanesulfonic acid, and DLPC were purchased from Sigma–Aldrich (Steinheim, Germany) and NADPH from Merck (Darmstadt, Germany). NADH and L-arginine were obtained from Fluka (Buchs, Switzerland). All other chemicals and solvents were of analytical grade, methanol was of HPLC grade. Preparation of pig liver microsomes and mitochondria. Pig liver microsomes were obtained by ultracentrifugation as published before [20]. Mitochondria were prepared according to the procedure of Hovius et al. [21]. Human liver microsomes. Human liver was obtained from the Medical Department of the University of Kiel. Prior consent of the Local Medical Ethics Committee and the donors before removal of the liver pieces was granted. Liver pieces from partial hepatectomies (Chirurgische Klinik der CAU, Kiel, Germany) were worked up to receive human microsomes as described before [20]. Preparation of cytochrome b5, NADH-cytochrome b5-reductase, and CYP2D enzyme. Cytochrome b5, NADH-cytochrome b5-reductase, and CYP2D enzyme were fractionated by the procedure of Clement et al. [19]. Thesit (Fluka, Buchs, Switzerland) was used to solubilize the microsomal proteins in the elution buffers. All purification steps were performed at 4 C. Cytochrome b5. Cytochrome b5 was purified on a DEAE–cellulose column as described previously [19]. The final cytochrome b5 charge stocks contained 26.38, 36.42, and 139.22 nmol of cytochrome b5/mg of protein, respectively. CYP2D enzyme. Benzamidoxime reductase was chromatographed of an anion exchange column by preparative HPLC with a conventional HPLC system (L-6210 Intelligent Pump, 655 A-22 UV detector, D-2500 integrator; Merck/Hitachi, Darmstadt, Germany) as described before [19] using a semi-preparative Fractogel EMD TMAE 650 (S) column (160 · 50 mm; particle size, 25–40 lM; Merck, Darmstadt, Germany). The first fraction contained the CYP2D enzyme, the second fraction contained the NADH-cytochrome b5-reductase. NADH-cytochrome b5-reductase. NADH-cytochrome b5-reductase was purified to homogeneity by affinity chromatography on 5 0 -AMPSepharose 4B (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to the procedure of Yasukochi and Masters [22] with slight modifications [19]. The fractions containing the highest NADH-ferricyanide-reductase activity were combined and concentrated, followed by gel filtration (NAP 25, Amersham Pharmacia Biotech AB, Uppsala, Sweden). The specific activity of the purified reductase was 99 U/mg of protein. Detergents were removed from the purified enzymes by shaking the concentrated fractions with Calbiosorb (Calbiochem, La Jolla, CA, USA) at 4 C. The microsomal and the enzyme fractions were stored at 80 C in aliquots.

B. Clement et al. / Biochemical and Biophysical Research Communications 349 (2006) 869–873 Analytical procedures. (1) Protein concentration. Protein concentrations were measured using the method described by Smith et al. [23] with bicinchoninic acid (BCA reagent kit, Pierce Chemical Co., Rockford, IL, USA). All photometric measurements were performed with an Uvicon 930 (Kontron Instruments, Neufahrn, Germany) spectrophotometer. (2) Cytochrome P450 concentrations. The P450 concentration was analyzed using the method of Omura and Sato [24]. (3) Cytochrome b5 concentrations. The cytochrome b5 concentration was determined by recording the reduced minus the oxidized spectrum (absorbance at 185 nM1 cm1) as described by Estabrook and Werringloer [25]. (4) NADH-cytochrome b5-reductase. NADH-cytochrome b5-reductase was assessed by its NADH-ferricyanide-reductase activity (1 U = 1 lmol of reduced ferricyanide/min) as described by Mihara and Sato [26]. (5) CYP2D enzyme. CYP2D enzyme activity was measured by reduction of benzamidoxime to benzamidine and monitored by HPLC [19]. Incubation procedures. A typical incubation procedure with pig liver microsomes or pig liver mitochondria is as follows: incubation mixtures consisted of 220 lg active protein and substrate 1 mM Nx-hydroxy-L-arginine in 100 mM phosphate buffer, pH 6.3 or 7.4. After a preincubation time of 2 min at 37 C, NADH or NADPH (final concentration: 1 mM) was added to a total volume of 150 lL and incubations were performed for 30 min at 37 C. Incubations were terminated by the addition of an equal volume of cold methanol and centrifuged for 5 min at 9000 rpm. Ten microliter aliquots of the resulting supernatant were analyzed by reversed phase HPLC. A typical incubation procedure using the reconstituted enzyme system (benzamidoxime reductase) is as follows: to a buffered suspension (phosphate buffer, 100 mM, pH 6.3) containing 75, 112.5, 150 or 300 pmol cytochrome b5, NADH-cytochrome b5-reductase (0.55, 1.1 or 1.6 U) and 5 or 10 lg of the CYP2D enzyme, DLPC (usual concentration: 40 lM), were added. After a preincubation time of 3 min at 37 C, the substrate (final concentration: 1 mM) was added and the mixture was preincubated again for another 2 min. The reaction was started adding NADH (1 mM), and incubations were performed for 30 min at 37 C. Reactions were terminated by the addition of an equal volume of cold methanol. The samples were centrifuged for 5 min at 9000 rpm, and the supernatants were used for HPLC analysis.

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Aerobic incubations were exposed to laboratory air while anaerobic incubations were performed in argon-degassed buffers, were gassed with argon, and the reaction tubes were closed during incubation. Determination of L-arginine. The assay is based on the principle that amino acids derivatized with o-phthaldialdehyde and 2-mercaptoethanol form highly fluorescent 1-alkylthio-2-alkyl-substituted isoindoles, and the amino acids are separated and quantified by reversed phase high-performance liquid chromatography (HPLC). o-Phthaldialdehyde/2-mercaptoethanol derivatives were made with a solution of 1.5 mM o-phthaldialdehyde (o-PA) and 7 mM of 2-mercaptoethanol in 0.2 M sodium borate buffer (pH 9.25, flow rate 1 mL/min) and 10 lL of the sample. L-Arginine standards were used to quantify the samples. The HPLC system used consisted of a 600 HPLC pump and a controller 600 E from Waters (Milford, CT, USA), equipped with an autosampler (Waters 717 plus) and a fluorescence detector (Waters 470). The fluorometer was set at an excitation wavelength of 340 nm and an emission wavelength of 455 nm. The areas under the peak were integrated with the EZChrom Chromatography Data System (EZChrom Elite SS42ox Interface, Scientific Software Inc., Pleasanton, CA, USA). Nx-Hydroxy-L-arginine and L-arginine (retention times of 18.6 and 22.2 min, respectively) were separated using a 250 · 4 mm ODS Hypersil column (5 lm) (Merck, Darmstadt, Germany) with a RP-select B precolumn (4 · 4 mm). Separation was carried out at room temperature using a 69/31 (v/v) mixture of water containing 100 mM octanesulfonic acid (pH 3.0) and methanol as eluent. The column flow rate was 1 mL/min.

Results Reduction of N-hydroxy-L-arginine Aerobic and anaerobic incubations of 1 mM Nx-hydroxy-L-arginine containing NADH in the presence of liver microsomes or mitochondria (220 lg) led to the formation of L-arginine. The relative rates of in vitro reduction of NOHA to L-Arg by microsomal fractions from pig and human livers as well as the relative rates received from incubations with pig liver mitochondria are listed in Table 1. A representative HPLC chromatogram recorded after the anaerobic incubation of Nx-hydroxy-L-arginine with pig liver microsomes (Fig. 2) showed that the retention

Table 1 In vitro reduction of Nx-hydroxy-L-arginine to L-arginine by pig liver microsomes and mitochondria and by human liver microsomesa (nmol min1 [mg of protein]1)

Preparation

Composition

Nb

L-Arginine

Pig liver microsomes

Complete mixture NADH/+NADPH pH 7.4 Without NADH Without substrate Without oxygen

12 4 4 8 4 4

3.86 ± 0.76 2.67 ± 0.57 2.48 ± 0.48 NDc NDc 3.55 ± 0.82

Human

Liver complete mixture NADH/+NADPH pH 7.4 Without substrate

8 8 4 4

0.63 ± 0.13 0.20 ± 0.16 0.16 ± 0.04 NDc

Pig liver mitochondria

Complete mixture NADH/+NADPH

4 4

9.59 ± 0.18 10.33 ± 0.20

a

A complete incubation mixture consisted of 1 mM Nx-hydroxy-L-arginine, 220 lg of microsomal and mitochondrial protein, respectively, and 1 mM NADH in 150 lL of 100 mM potassium phosphate buffer (pH 6.3), as described in Materials and methods. Data are means ± standard deviation from N different determinations. b Number of determinations. c Not detected.

B. Clement et al. / Biochemical and Biophysical Research Communications 349 (2006) 869–873 Table 2 Apparent Vmax and Km values of the in vitro reduction of Nx-hydroxy-Larginine to L-arginine by pig liver microsomes and mitochondriaa

L-arginine 22.2 min

N -hydroxy-L-arginine 18.6 min

872

0

Preparation

Vmax (nmol min1 [mg of protein]1)

Km (lM)

Pig liver microsomes Pig liver mitochondria

4.51 ± 1.84 12.1 ± 5.84

707 ± 211 244 ± 34.3

a Incubation mixture consisted of 0.25–4 mM Nx-hydroxy-L-arginine, 220 lg of microsomal and mitochondrial protein, respectively, and 1 mM NADH in 150 lL of 100 mM potassium phosphate buffer (pH 6.3), as described in Materials and methods. Data are means ± standard deviation from three different determinations.

45

time [min] Fig. 2. Representative HPLC chromatogram of the incubation of Nx-hydroxy-L-arginine with pig liver microsomes under anaerobic conditions. The incubation mixture was as described in Materials and methods.

N -hydroxy-L-arginine 18.6 min

time for the metabolite (22.2 min) agreed with that of the synthetic material. Addition of the reference substance to the incubation mixture led to an increase in the area of the metabolite peak. Control incubations without protein (Fig. 3) or cofactor (data not shown) exhibited a smaller peak for L-arginine which resulted from impurities of the substrate Nx-hydroxy-L-arginine. It is assumed that the signal with a retention time of 5 min (Fig. 2) belongs to citrul-

line which can be formed in the presence of microsomal preparations containing P450 enzymes and superoxide [4]. Formation of L-arginine was an oxygen-insensitive enzymatic reaction. The presence of a reducing agent, NADH or NADPH, was also required. In incubations with microsomes as active protein component the use of NADPH instead of NADH gave much lower activity as shown in Table 1. The highest reduction activity was obtained when incubating NOHA at pH 6.3, but also at the physiological pH value 7.4 measurable amounts of L-Arg could be detected (Table 1). The enzymatic formation of L-arginine followed the Michaelis-kinetics. The apparent Vmax and Km values were determined using pig liver microsomes and mitochondria (Table 2). The reconstituted liver microsomal system of a pig liver CYP2D enzyme, the benzamidoxime reductase, was unable to replace microsomes to produce L-Arg from NOHA. Different stocks of the isolated and purified components of the enzyme system in various concentrations, as described in Materials and methods, were used, but under stated conditions, no reduction of NOHA could be observed.

L-arginine 22.2 min

Discussion

0

45

time [min] Fig. 3. Representative HPLC chromatogram of the incubation of Nx-hydroxy-L-arginine without protein.

In the literature, several NOS-independent enzymatic pathways of Nx-hydroxy-L-arginine have been reported [4–6]. It has been hypothesized that the oxidation of NOHA to NO by hemoproteins might be important for the formation of NO in cells or compartments not containing NOS, suggesting that NOHA would thus act as a transportable precursor of NO. The objective of this study was to investigate a possible reduction of Nx-hydroxy-L-arginine, the intermediate in NO formation from L-arginine by NO synthases, to its parent guanidine L-Arg. Human and pig liver microsomes were capable of catalyzing this reaction, suggesting that high concentrations of Nx-hydroxy-L-arginine in cells without NOS activity but containing these reductive enzymes are unlikely. As the reduction is not inhibited by oxygen the in vivo relevance is without doubt. Since we were able to demonstrate the reduction of NOHA to L-Arg, the question arose whether the enzyme system of the benzamidoxime reductase is responsible for this reaction. This microsomal enzyme system is present in liver and other organs of all species studied so far [15]. It consists of

B. Clement et al. / Biochemical and Biophysical Research Communications 349 (2006) 869–873

cytochrome b5 and cytochrome b5-reductase and a third protein needed for optimal activity [15]. This third component has been isolated from pig liver and identified as a cytochrome P450 belonging to the subfamily 2D [19]. The protein has not been obtained in a recombinant form so far. Thus the purified enzyme was employed in this study. The human equivalent is still not identified, it is not CYP2D6 [19]. It could also be demonstrated that cytochrome b5 and cytochrome b5-reductase alone can catalyze the reduction of certain N-hydroxylated derivatives [15]. However, proteins alone or in combination with the purified third component were not a catalyst of the reduction of Nx-hydroxy-L-arginine. Despite the facts that optimized conditions for this reduction are close to those for the benzamidoxime reductase (i.e. preference of the same cofactor and the optimized pH), and although NOHA shows structural similarities to benzamidoxime, the reconstituted liver microsomal system of the pig liver CYP2D enzyme seems incapable of catalyzing this conversion of NOHA to L-Arg. Nx-Hydroxy-L-arginine is charged under physiological conditions which might hinder this amino acid in contrast to uncharged non-physiological N-hydroxyamidines (amidoximes) and N-hydroxyguanidines to enter the active site of the P450 enzyme. Further studies will be directed towards the identification of the microsomal and mitochondrial enzyme involved in the reduction of Nx-hydroxy-L-arginine.

[8]

[9]

[10]

[11]

[12]

[13] [14] [15]

[16]

[17]

Acknowledgments This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie. We are indebted to Sven Wichmann for his excellent technical assistance.

[18]

[19]

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