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Aldose reductase: An aldehyde scavenging enzyme in the intraneuronal metabolism of norepinephrine in human sympathetic ganglia
Autonomic Neuroscience: Basic and Clinical 96 (2002) 131 – 139 www.elsevier.com/locate/autneu
Aldose reductase: An aldehyde scavenging enzyme in the intraneuronal metabolism of norepinephrine in human sympathetic ganglia Minoru Kawamura a,b, Graeme Eisenhofer a, Irwin J. Kopin a, Peter F. Kador c, Yong S. Lee d, Shigeki Fujisawa c, Sanai Sato c,* a
Clinical Neurocardiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda MD 20892, USA b Institute of Bio-Active Science, Nippon Zoki Pharmaceutical Co., Ltd., Hyogo 673-1461, Japan c Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, 10/10B09, 10 Center Drive, MSC 1850, Bethesda MD 20892-1850, USA d Center for Molecular Modeling, Center for Information Technology, National Institutes of Health, Bethesda MD 20892, USA Received 27 June 2001; received in revised form 4 October 2001; accepted 31 October 2001
Abstract The neurotransmitter norepinephrine is metabolized by monoamine oxidase into an aldehyde intermediate that is further metabolized to the stable glycol derivative, 3,4-dihydroxyphenylglycol (DHPG). In this study, the possible role of aldose reductase in reducing this aldehyde intermediate in human sympathetic neurons has been examined. DHPG is formed when norepinephrine is incubated with aldose reductase in the presence of monoamine oxidase. DHPG metabolism is inhibited by the monoamine oxidase inhibitor, pargyline which prevents the deamination of norepinephrine, and by the aldose reductase inhibitor AL 1576, which inhibits DHPG formation without affecting the deamination of norepinephrine. Although similar formation of DHPG was observed with human liver aldehyde reductase, the production of DHPG was more effective with aldose reductase than aldehyde reductase. Two peaks of reductase activity corresponding to aldose reductase and aldehyde reductase were observed when sympathetic ganglia were chromatofocused. Molecular modeling studies indicate that the energy-minimized structure of 3,4-dihydroxymandelaldehyde bound to aldose reductase is similar to that of glyceraldehyde where the 2Vhydroxyl group forms hydrogen bonds with Trp111 and NADPH. These results suggest that aldose reductase may be important in metabolizing the potentially toxic aldehyde intermediate formed from norepinephrine in human sympathetic ganglia. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Aldose reductase; Aldehyde reductase; Norepinephrine; 3,4-Dihydroxyphenylglycol; Sympathetic nervous system
1. Introduction Monoamine neurotransmitters produced in aminergic nerves are primarily metabolized within the cytoplasm of nerve terminals (Dong et al., 1993, 1995; Graefe and Henseling, 1983). Because monoamine oxidase (EC 1.4.3.4, MAO) is the only catecholamine-metabolizing enzyme in this intraneuronal location, the first step of the intraneuronal metabolism of these neurotransmitters is oxidative deamination by MAO. This deamination step generates aldehyde intermediates that are further metabolized to stable acid and/or glycol derivatives by either dehydrogenase(s) or reductase(s). Bec-
*
Corresponding author. Tel.: +1-301-402-9849; fax: +1-301-402-2399. E-mail address: [email protected] (S. Sato).
ause of their highly reactive nature, the rapid and complete removal of these aldehyde intermediates is crucial for eliminating possible cytotoxicity that can initiate the process of neurodegeneration (Eisenhofer et al., 2000). Aldose reductase (alditol:NAD+ 1-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) are members of the aldo-keto reductase (AKR) superfamily (Jez et al., 1997). The role of these enzymes in biogenic amine metabolism was extensively studied in 1970 –1980s using mammalian brain tissues. In these early studies, two NADPH-dependent reductases (high Km aldehyde reductase and low Km aldehyde reductase) utilizing bhydroxylated aldehydes as substrate were detected (Duncan et al., 1975; Turner and Tipton, 1972a,b). These two enzymes were later identified as aldehyde reductase (AR3, ALR1) and aldose reductase (AR2, ALR2) (Hoffman et al., 1980;
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 1 ) 0 0 3 8 5 - X
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Cromlish and Flynn, 1985). Other reductases such as succinic semialdehyde reductase are also present in the brain tissue, but have negligible activity in utilizing b-hydroxylated aldehydes as substrate (Hoffman et al., 1980). The above described studies consistently indicated that, kinetically, the favored pathway of biogenic amine reduction involves aldose reductase rather than aldehyde reductase (Turner and Tipton, 1972a,b; Hoffman et al., 1980; Wermuth et al., 1982; Wermuth, 1985). It was also reported that the formation of 3-methoxy-4-hydroxyphenylglycol (MHPG) from normetanephrine by rat brain homogenates is inhibited by aldose reductase inhibitors such as quercetin and quercitrin, but not by the aldehyde reductase specific inhibitor sodium valproate (Whittle and Turner, 1981). Although the kinetic evidence favors the importance of aldose reductase over aldehyde reductase for reduction of bhydroxylated aldehydes, whether either or both enzymes are present at the sites of biogenic amine metabolism is not clear. Reductase activity is widely detected in the whole brain (Erwin et al., 1972; Wirth and Wermuth, 1985); however, aldose reductase activity is not associated with either the nerve terminals of the striatum, the major site of catecholamine production (Duncan et al., 1975) or with the synaptosomal fractions of brain homogenates (Turner and Tipton, 1972a,b). In the retina, aldose reductase activity is greater in glial cells than ganglion cells (Akagi et al., 1984). Similarly, the principal site for aldose reductase in peripheral nerves is the Schwann cells rather than nerve fibers (Powell et al., 1991). The above findings suggest a role of aldose reductase in the extraneuronal metabolism of monoamine neurotransmitters. It remains unclear whether aldose reductase is involved in the intraneuronal metabolism of monoamine neurotransmitters. In peripheral sympathetic neurons, the neurotransmitter norepinephrine is predominantly metabolized to the glycol metabolite, 3,4-dihydroxyphenylglycol (DHPG). In humans, plasma DHPG is primarily derived from peripheral sympathetic neurons, with contributions from brain tissue being minor (Goldstein et al., 1988). Previously, we reported that aldose reductase is the primary enzyme in rat sympathetic neurons (Kawamura et al., 1999) and that aldose reductase inhibitors reduce levels of DHPG and increase those of 3,4-dihydroxymandelic acid (DHMA) (Kawamura et al., 1997). To assess the importance of aldose reductase in human tissues, species differences with respect to the kinetic properties of aldose reductase must be determined, along with its specific localization in human sympathetic neurons. Here, we report that, just like rat aldose reductase, human aldose reductase generates DHPG from norepinephrine through an aldehyde intermediate generated by MAO. In addition, aldose reductase is localized in human sympathetic neurons. To obtain insight into the structural basis of the reduction of catechol aldehydes by human aldose reductase, molecular modeling studies were also conducted.
2. Materials and methods 2.1. Chemicals and materials Unless otherwise stated, all reagents utilized in this study were analytical grade. Norepinephrine bitartrate (NE), epinephrine bitartrate (EPI), DL-metanephrine hydrochloride, 3,4-dihydroxyphenylglycol (DHPG), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxybenzylamine (DHBA), pargyline and NADPH were all obtained from Sigma (St. Louis, MO). Polybuffer 74 was product of Pharmacia-LKB Biotechnology, (Piscataway, NJ). The aldose reductase inhibitor, AL 1576 (2,7-difluorospirofluorene-9,5V-imidazolidine2V,4V-dione: Imirestat), was a gift from Alcon Laboratories (Fort Worth, TX). Recombinant human muscle aldose reductase was purchased from Wako (Osaka, Japan). Human liver aldehyde reductase was prepared as described previously (Sato and Kador, 1993). Human liver tissues utilized for enzyme purification were obtained through the National Diabetes Research Interchange (Philadelphia, PA). Mitochondrial membranes, utilized as a source of MAO, were isolated from rat liver as described previously (Kobayashi and Fujisawa, 1994). Samples of pheochromocytoma tumor tissue were obtained within 1 h of surgical removal under a research protocol approved by the Intramural Review Board (IRB) of the National Cancer Institute, National Institutes of Health (NIH). All patients from whom tumor tissue was obtained provided written informed consent for their participation in the study. Dimensions of tumors were recorded, and small samples (50 –400 mg) were dissected away from surrounding tissue, placed on dry ice, and then stored at 80 C. Samples of human sympathetic ganglia were obtained during routine autopsies of two patients at the NIH. Samples were obtained from a 74-year-old female within 8 h of death due to cardiorespiratory failure and a 49-year-old female within 20 h of death due to complications of malignant melanoma. Collection of these samples was approved under an exemption from NIH IRB review based on a written ‘‘Request for Patient Related Materials’’ and under the condition that collections of specimens would not require identification of patients. 2.2. Enzyme assay Reductase activities were spectrophotometrically assayed on a Shimadzu UV-2101PC spectrophotometer (Shimadzu, Kyoto, Japan) by following the decrease of NADPH. The reaction mixture (1 ml) consisted of 0.3 mM NADPH, 10 mM aldehyde substrate, 0.1 M phosphate buffer, pH 6.2, and adequate amounts of enzyme (approximately 2– 3 mU as DLglyceraldehyde as substrate). The enzyme reaction was started by adding substrate and incubation was continued at 25 C for 4 min. One enzyme unit (U) was defined as the activity consuming 1 mmol of NADPH per min under the assay condition.
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2.3. In vitro production of 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) Unless otherwise stated, the reaction mixture (100 ml) containing 2 mM ascorbic acid, 1 mM catecholamine, 0.1 mM NADPH, 10 ml of MAO and aldose reductase (10 mU as DLglyceraldehyde as substrate) in 0.1 M phosphate buffer, pH 7.4, was incubated at 37 C for 1 h. The reaction was terminated by the addition of 50 ml of 0.4 M perchloric acid containing 0.5 mM EDTA. After centrifugation at 10,000 g for 15 min, the supernatant was stored at 80 C for catechol analysis by HPLC. DHPG and MHPG were analyzed by HPLC with electrochemical detection as previously reported (Eisenhofer et al., 1986, 1994; Kawamura et al., 1997).
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assumed to be negatively charged while arginine and lysine were positively charged. Since (R)-(+)-glyceraldehyde and (R)-( )-norepinephrine are physiologically active, D-forms of glyceraldehyde, 3,4-dihydroxymandelaldehyde (DHMAL), 3-methoxy-4-hydroxymandelaldehyde (MHMAL) and 3,4-dihydroxyphenylacetaldehyde were modeled at the active site. Charge assignment on NADPH, substrates and the hydration of the complex of aldose reductase-NADPH with a substrate as well as the subsequent energy minimization by CHARMM with an all-atom parameter set (Molecular Simulations, 1992) were carried out according to procedures published elsewhere (Lee et al., 1998a,b).
3. Results
2.4. Sephacryl S-100 chromatography and chromatofocusing on Mono P
3.1. In the presence of MAO, human aldose reductase generates DHPG from norepinephrine in vitro
Approximately 1 g of either human sympathetic ganglia or pheochromocytoma tissue was homogenized with 3 volumes of 20 mM imidazole-HCl buffer, pH 7.5 containing 10 mM 2mercaptoethanol. After centrifugation at 30,000 g for 10 min, the supernatant was applied to a Sephacryl S-100 column (HiPrepTM 16/60, Pharmacia-LKB Biotechnology) and eluted with the same imidazole buffer at the flow rate of 1.0 ml/min. The eluant was collected into 35-drop aliquots (approximately 1.7 ml) and the fractions containing reductase activity were applied to a Mono P column (HR 5/20, Pharmacia-LKB Biotechnology) equilibrated with 20 mM imidazole-HCl buffer, pH 7.5, containing 10 mM 2-mercaptoethanol. The column was developed at a flow rate of 1.0 ml/ min with Polybuffer 74 diluted 10 times with 10 mM 2mercaptoethanol and the eluant was collected into 20-drop aliquots (approximately 1.0 ml). The reductase activity of each fraction was assayed with DL-glyceraldehyde and Dglucuronate as substrates.
To confirm the formation of DHPG by human aldose reductase, norepinephrine was incubated with human muscle aldose reductase in the presence of MAO (Fig. 1). No DHPG was formed in the absence of MAO and aldose reductase and the HPLC chromatogram of the reaction mixture only displayed peaks corresponding to non-metabolized norepinephrine and the internal standard, dihydroxybenzylamine (DHBA). When the reaction mixture contained both MAO and aldose reductase, a new peak corresponding to DHPG was clearly detected and the peak corresponding to norepinephrine was decreased. Formation of DHPG along with the decrease in norepinephrine was completely prevented by the MAO inhibitor, pargyline. The aldose reductase inhibitor, AL 1576, also significantly reduced the formation of DHPG; however, this inhibitor failed to prevent the loss of norepinephrine. This confirms that norepinephrine is first metabolized to an aldehyde intermediate by MAO and that this aldehyde intermediate is metabolized to DHPG by aldose reductase.
2.5. Structural modeling
3.2. Human aldose reductase also generates DHPG from epinephrine
The coordinates of human aldose reductase complexed with cofactor and crystal waters (1ADS) (Wilson et al., 1992) were obtained from the Brookhaven Protein Data Bank. Hydrogens were added to the amino acid residues of human aldose reductase utilizing the HBUILD routine of CHARMM (Brooks et al., 1983). With the exception of His110, all histidine residues were treated as neutral with hydrogen assigned to the Nd1 of neutral histidine. His110 was treated as positively charged based on the crystallographic evidence that the carboxylate of zopolrestat is salt-linked to the Ne2 atom of His110 (Wilson et al., 1993, 1995). Further support has also come form the evidence by the computer simulation reduction mechanism of aldose reductase (Lee et al., 1998a,b) and mutagenic and kinetic studies on 3a-hydroxysteroid dehydrogenase, a member of aldo-keto reductase, (Schlegel et al., 1998a,b). All aspartate and glutamate residues were
MAO also utilizes epinephrine as substrate. The deaminated product formed from epinephrine is the same aldehyde intermediate, DHMAL, as formed from norepinephrine. Since aldose reductase utilizes this aldehyde intermediate as substrate to generate DHPG, the final metabolite formed from epinephrine by MAO and aldose reductase should also be DHPG. This was confirmed by incubating epinephrine with MAO and aldose reductase (Fig. 2). Similar to formation from norepinephrine, DHPG formation from epinephrine was only detected in the presence of both MAO and aldose reductase. Because epinephrine is not as good as norepinephrine as a substrate for MAO, the decrease in epinephrine was slower than that of norepinephrine. Pargyline prevented both DHPG formation and the decrease of epinephrine. AL 1576 reduced the formation of DHPG but
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3.3. Aldose reductase is more active than aldehyde reductase in producing DHPG Since the reduction of DHMAL to DHPG is also catalyzed by aldehyde reductase, the activities of aldose reductase and aldehyde reductase in producing DHPG were compared in the same in vitro incubation system (Fig. 3). The levels of DHPG produced by the incubation of norepinephrine with 0.01, 0.1 and 1.0 mU of human muscle aldose reductase were 87 F 18, 261 F 53 and 1507 F 61 pmol/ml (n = 4), respectively. In contrast, the amounts of DHPG generated by aldehyde reductase were far less than
Fig. 1. HPLC chromatograms obtained from a mixture containing 1 mM norepinephrine (NE), monoamine oxidase (MAO) and 10 mU human muscle aldose reductase (AR) incubated at 37 C for 1 h as described in Materials and methods. (A) represents the reaction mixture containing only NE while (B) is the complete reaction mixture containing NE, MAO and AR. In addition to the complete reaction mixture, (C) contains 1 mM of the MAO inhibitor pargyline while (D) contains 100 mM of the aldose reductase inhibitor AL 1576. DHPG; 3,4-dihydroxyphenylglycol. DHBA; 3,4-dihydroxybenzylamine.
did not affect the decrease of epinephrine. This again confirms that aldose reductase generates DHPG by reducing the aldehyde intermediate, DHMAL formed by MAO.
Fig. 2. HPLC chromatograms obtained from a mixture containing 1 mM epinephrine (EPI), monoamine oxidase (MAO) and 10 mU human muscle aldose reductase (AR) incubated at 37 C for 1 h. (A) represents the reaction mixture that contains only EPI while (B) contains the complete reaction mixture NE, MAO and AR. In addition to the complete mixture, (C) contains 1 mM of the MAO inhibitor pargyline and (D) contains 100 mM aldose reductase inhibitor AL 1576. DHPG; 3,4-dihydroxyphenylglycol. DHBA; 3,4-dihydroxybenzylamine.
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reductase displays higher activity with D-glucuronate compared to DL-glyceraldehyde. This indicates that both aldose reductase and aldehyde reductase are present in human sympathetic ganglions. However, there may be some individual variations in the relative amounts of aldose reductase versus aldehyde reductase. 3.5. Aldose reductase also utilizes 3-methoxy-aldehyde substrate derived from metanephrine
Fig. 3. 3,4-Dihydroxyphenylglycol (DHPG) formed by the in vitro incubation at 37 C for 1 h of 10 mM norepinephrine with 1.0, 0.1 and 0.01 mU of human muscle aldose reductase (light gray) and 1.0 and 0.1 mU of human liver aldehyde reductase (dark gray). Enzyme units (U) are expressed as mmol/min with 10 mM DL-glyceraldehyde as substrate. Mean F S.D. (n = 4).
those generated by aldose reductase. Human liver aldehyde reductase, in 0.1 and 1.0 mU amount produced only 64 F 6 and 218 F 10 pmol/ml of DHPG (n = 4), respectively. The dependence of our in vitro reaction system on amine precursors (e.g. norepinephrine and epinephrine) to generate aldehyde intermediates did not make it possible to provide saturating concentration of substrate for aldose reductase or aldehyde reductase. This explains the ‘apparent’ non-linear kinetics of reductase activity, as assessed by DHPG production. Thus, although the correlation between the amounts of enzyme and the levels of DHPG was not linear, the levels of DHPG generated by aldose reductase were consistently higher than those by aldehyde reductase. This result was consistent with our previous reports using rat enzymes (Kawamura et al., 1999).
Normetanephrine and metanephrine are O-methylated metabolites that are formed by catechol-O-methyltransferase (COMT) from norepinephrine and epinephrine, respectively. These two O-methylated amines are also deaminated by MAO to another aldehyde intermediate, 3-methoxy-4hydroxymandelaldehyde (MHMAL) (Tipton, 1986; Eisenhofer and Finberg, 1994). To examine whether this 3methoxyaldehyde intermediate is further metabolized by aldose reductase to its glycol metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), human muscle aldose reductase was also incubated with metanephrine in the presence of MAO (Fig. 5).
3.4. Both aldose and aldehyde reductases are present in human sympathetic neurons Because of overlapping substrate specificity, aldose reductase activity cannot be distinguished from aldehyde reductase activity by the traditional photometric enzyme assay. These two enzymes are separated only by procedure(s) utilizing their charge differences (Sato and Kador, 1993). In this study, to confirm the presence of aldose reductase and to estimate the relative amounts of aldose reductase versus aldehyde reductase, chromatofocusing of human sympathetic neurons was conducted (Fig. 4). The chromatogram obtained from chromatofocusing resulted in the appearance of two activity peaks with DLglyceraldehyde as substrate that corresponded to aldose reductase (the first peak, pI 6.0) and aldehyde reductase (the second peak, pI 5.5), respectively. The identity of these two activity peaks was confirmed by Western blot and by substrate specificity characteristics in which the activity with DLglyceraldehyde is approximately two-fold higher than that with D-glucuronate for aldose reductase while aldehyde
Fig. 4. Chromatofocusing of human sympathetic ganglia homogenates illustrating the presence of both aldose reductase and aldehyde reductase. Closed circles indicate the activity with DL-glyceraldehyde as substrate and broken line indicates pH of eluents. The first activity peak corresponds to aldose reductase (AR) while the second peak corresponds to aldehyde reductase (ALR).
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When metanephrine was incubated with both aldose reductase and MAO, MHPG was produced. Although there was no exact linearity, the MHPG production was increased in dose-dependent manner when the enzyme amounts were increased. MHPG levels produced by 1.0, 0.1 and 0.01 mU of human muscle aldose reductase were 130 F 0.5, 59 F 3 and 39 F 0.7 pmol/ml (n = 4), respectively. MHPC was also generated by human liver aldehyde reductase; however, the levels produced were lower than those produced with aldose reductase. MHPG levels produced by 1.0 and 0.1 mU of human liver aldehyde reductase were only 41 F 0.6 and 35 F 1 pmol/ml (n = 4), respectively. This confirms that both human aldose reductase and aldehyde reductase can utilize Fig. 5. 3-Methoxy-4-hydroxypbenylglycol (MHPG) formation by the in vitro incubation at 37 C for 1 h of 1 mM metanephrine with 1.0, 0.1 and 0.01 mU of human muscle aldose reductase (light gray) and 1.0 and 0.1 mU of human liver aldehyde reductase (dark gray). Enzyme units (U) are expressed as mmol/min with 10 mM DL-glyceraldehyde as substrate. Mean F S.D. (n = 4).
Fig. 6. Chromatofocusing of homogenized pheochromocytoma cells illustrating the presence of both aldose reductase and aldehyde reductase. Closed circles indicate activity with DL-glyceraldehyde as substrate and broken line indicates pH of eluents. The first activity peak corresponds to aldose reductase (AR) while the second peak corresponds to aldehyde reductase (ALR).
Fig. 7. CHARMM energy minimized structure of human aldose reductase complexed with D-glyceraldehyde (A) and 3,4-dihydroxymandelaldehyde (DHMAL, B). Atoms are represented by colors: white, carbon; green, hydrogen; blue, nitrogen; red, oxygen; yellow, sulfur. Yellow dot line indicates hydrogen-bonding interaction.
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the aldehyde intermediate from metanephrine, but that its reduction is more favorable with aldose reductase compared to aldehyde reductase.
3.7. Hydrogen-bonding interactions between substrates and aldose reductase indicate that the 2V-hydroxyl group is the key motif recognized by aldose reductase
3.6. Aldose reductase is also detectable in pheochromocytoma cells
In molecular modeling studies of DHMAL in position at the active site of aldose reductase, the carbonyl of the aldehyde intermediate hydrogen-bond with His110 while the 2V-hydroxyl group hydrogen-bond with both Trp 111 and the amide oxygen of NADPH (Fig. 7). The CHARMM energy minimized structure further indicates that the side chains of Trp20, Trp111, and Phe122 are positioned for aromatic –aromatic and van der Waals interactions with the aromatic portion of DHMAL (Lee et al., 1998a,b). This hydrogen bonding pattern is essentially identical to that observed with D-glyceraldehyde, a commonly used substrate for aldose reductase. Similar hydrogen bonding interactions with aldose reductase are also formed by MHMAL, which has a methoxy group on the three carbon position on the aromatic ring (Fig. 8). However, unlike DHMAL or MHMAL, the carbonyl of 3,4-dihydroxyphenylacetaldehyde only forms a hydrogen bond with His110 (Fig. 8). These optimized structures all suggest that the substrate carbonyl is properly oriented to accept the pro-R hydride transfer from NADPH and a proton from His110.
Another major source for catecholamines is the adrenal medulla. Chromatofocusing of the crude extract of pheochromocytoma also displayed two peaks of reductase activity corresponding to aldose reductase and aldehyde reductase (Fig. 6).
4. Discussion
Fig. 8. CHARMM energy minimized structure of human aldose reductase complexed with 3-methoxy-4-hydroxymandelaldehyde (MHMAL, A) and 3,4-dihydroxyphenylacetaldehyde (B). Atoms are represented by colors: white, carbon; green, hydrogen; blue, nitrogen; red, oxygen; yellow, sulfur. Yellow dot line indicates hydrogen-bonding interaction.
Both aldose reductase and aldehyde reductase are members of aldo-keto reductase (AKR) superfamily (Jez et al., 1997). Although the amino acid sequence homology between aldose reductase and aldehyde reductase is only about 65% (Bohren et al., 1989), the tertiary structures of these two enzymes are highly conserved with arrangements of the key amino acids, His110, Tyr48 and Typ111, in their active sites being virtually identical (el-Kabbani et al., 1991; Rondeau et al., 1992; Wilson et al., 1992). As a consequence, both enzymes share a wide range of substrates. The present study confirms that both human aldose reductase and aldehyde reductase can utilize the aldehyde intermediate formed from the neurotransmitter, norepinephrine, as substrate to form the stable glycol metabolite, DHPG. However, the in vitro data also indicate that DHPG formation occurs more favorably with aldose reductase rather than with aldehyde reductase. Molecular modeling studies at the atomic level provide the structural basis for the reduction of DHMAL by aldose reductase. The hydrogen bonding interactions between DHMAL and aldose reductase are similar to those with D-glyceraldehyde, suggesting that DHMAL can be a good substrate for aldose reductase. This also suggests that aldose reductase reduces DHMAL in a similar manner to the reduction of glyceraldehyde to glycerol. Importantly, the energy-minimized structure demonstrates hydrogen bonding between the 2V-hydroxyl group of DHMAL and Trp111 and the amide oxygen of NADPH. The 2V-hydroxyl group appears to be crucial in correctly orienting the substrate for catalysis. In
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general, catecholamines lacking the 2V-hydroxyl group are preferentially metabolized to acids while those containing a 2V-hydroxyl group are reduced to glycols (Rutledge and Jonason, 1967; Turner et al., 1974). For example, dopamine is almost exclusively oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In contrast, both norepinephrine and epinephrine are primarily metabolized to DHPG and MHPG (Kopin, 1985). Failure in forming hydrogen bonds without the 2V-hydroxyl group may explain this experimental observation. Molecular modeling also indicates that the 3V-methoxy group of MHMAL has no effect on the hydrogen-bonding interactions between the substrate and enzyme (Fig. 8). This suggests that the aldehyde intermediate formed from either normetanephrine or metanephrine can be a good substrate for aldose reductase. This is again consistent with the observation that human aldose reductase generates MHPG from metanephrine when incubated in the presence of MAO. Aldose reductase is known as the first enzyme in the polyol pathway where glucose is metabolized to fructose through the intermediate sugar alcohol, sorbitol. Aldose reductase also plays a key role in the pathogenesis of various diabetic complications by initiating the abnormal intracellular accumulation of sorbitol (Kador, 1988; Yabe-Nishimura, 1998). In sympathetic neurons, however, aldose reductase appears to be important as an aldehyde-scavenging enzyme. Like many biological aldehydes, the aldehyde intermediates formed in neurotransmitter metabolism are potentially toxic (Lamensdorf et al., 2000a,b). The rapid and complete removal of these aldehyde metabolites is important (Eisenhofer et al., 2000). Under physiological conditions, the aldehyde intermediate, DHMAL, formed from norepinephrine is primarily metabolized to the glycol metabolite, DHPG. However, when the reduction pathway to DHPG is blocked, the pathway quickly shifts to produce 3,4-dihydroxymandelic acid, DHMA, through oxidation by aldehyde dehydrogenase (Kawamura et al., 1999; Sato et al., 1999). This metabolic redundancy helps ensure the appropriate removal of toxic aldehyde metabolites. The present study confirms that both aldose reductase and aldehyde reductase are present in human sympathetic nerves. Since both enzymes utilize DHMAL as substrate, the presence of both aldose reductase and aldehyde reductase, in addition to aldehyde dehydrogenase, adds another level of metabolic redundancy to ensure appropriate removal of toxic aldehyde metabolites. Thus, aldose reductase and aldehyde reductase are both potentially important in the intraneuronal metabolism of the neurotransmitter norepinephrine in human sympathetic neurons. However, under normal conditions, most DHPG in sympathetic neurons is likely formed by aldose reductase. Aldose reductase appears to be more active than aldehyde reductase in reducing aldehyde intermediates formed from catecholamines. There may also be, however, some individual variation in the levels of aldose reductase and aldehyde reductase (Fig. 4). The large-scale clinical trials on aldose reductase inhibitors completed over the last few decades failed to show
abnormal catecholamine metabolism or adrenergic dysfunction. Presumably, this indicates the importance of the redundancy in catecholamine metabolism that secures efficient removal of toxic aldehyde intermediates under various circumstances. The role of aldose reductase in catecholamine metabolism may not be limited to the intraneuronal metabolism of norepinephrine. Aldose reductase may be also important in the adrenal glands. A substantial portion of free metanephrine and normetanephrine are formed within the adrenals. Similar to intraneuronal metabolism, the major metabolite of these 3methoxy compounds in adrenal glands is the glycol derivative, MHPG (Eisenhofer et al., 1995a,b). In the present study, human aldose reductase was observed to generate MHPG from metanephrine in the presence of MAO. Molecular modeling studies support the premise that the aldehyde intermediate formed from either normetanephrine or metanephrine is also a good substrate for aldose reductase. It is well known that adrenal glands contain high levels of aldose reductase (Grimshaw and Mathur, 1989; Matsuura et al., 1995), and pheochromocytoma cells originating from the adrenal medulla also contain the significant amounts of aldose reductase (Fig. 6). In the adrenal cortex, aldose reductase appears to be important in steroid metabolism (Matsuura et al., 1996; Petrash et al., 1997). In the medulla, however, aldose reductase (together with aldehyde reductase) may contribute to catecholamine metabolism by reducing an aldehyde intermediate, MHMAL, to the glycol derivative, MHPG. References Akagi, Y., Yajima, Y., Kador, P.F., Kuwabara, T., Kinoshita, J.H., 1984. Localization of aldose reductase in the human eye. Diabetes 33, 562 – 566. Bohren, K.M., Bullock, B., Wermuth, B., Gabbay, K.H., 1989. The aldoketo reductase superfamily. cDNAs and deduced amino acid sequences of human aldehyde and aldose reductases. J. Biol. Chem. 264, 9547 – 9551. Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., Karplus, M., 1983. A program for maculomolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187. Cromlish, J.A., Flynn, T.G., 1985. Identification of pig brain aldehyde reductases with the high-Km aldehyde reductase, the low-Km aldehyde reductase and aldose reductase, carbonyl reductase, and succinic semialdehyde reductase. J. Neurochem. 44, 1485 – 1493. Dong, W.X., Schneider, J., Lacolley, P., Brisac, A.M., Safar, M., Cuche, J.L., 1993. Neuronal metabolism of catecholamines: plasma DHPG, DOMA and DOPAC. J. Auton. Nerv. Syst. 44, 109 – 117. Dong, W.X., Schneider, J., Dabire, H., Safar, M., Cuche, J.L., 1995. Neuronal metabolism of catecholamines in pithed and electrically stimulated rats. J. Auton. Nerv. Syst. 54, 41 – 48. Duncan, R.J., Sourkes, T.L., Dubrovsky, B.O., Quik, M., 1975. Activity of aldehyde dehydrogenase, aldehyde reductase, and acetylcholine esterase in striatum of rats bearing electrolytic lesions of the medial forebrain bundle. J. Neurochem. 24, 143 – 147. Eisenhofer, G., Finberg, J.P., 1994. Different metabolism of norepinephrine and epinephrine by catechol-O-methyltransferase and monoamine oxidase in rats. J. Pharmacol. Exp. Ther. 268, 1242 – 1251. Eisenhofer, G., Goldstein, D.S., Stull, R., Keiser, H.R., Sunderland, T.,
M. Kawamura et al. / Autonomic Neuroscience: Basic and Clinical 96 (2002) 131–139 Yabe-Nishimura, C., Kopin, I.J., 1986. Simultaneous liquid-chromatographic determination of 3,4-dihydroxyphenylglycol, catecholamines, and 3,4-dihydroxyphenylalanine in plasma, and their responses to inhibition of monoamine oxidase. Clin. Chem. 32, 2030 – 2033. Eisenhofer, G., Pecorella, W., Pacak, K., Hooper, D., Kopin, I.J., Goldstein, D.S., 1994. The neuronal and extraneuronal origins of plasma 3-methoxy-4-hydroxyphenylglycol in rats. J. Auton. Nerv. Syst. 50, 93 – 107. Eisenhofer, G., Friberg, P., Pacak, K., Goldstein, D.S., Murphy, D.L., Tsigos, C., Quyyumi, A.A., Brunner, H.G., Lenders, J.W., 1995a. Plasma metadrenalines: do they provide useful information about sympathoadrenal function and catecholamine metabolism? Clin. Sci. 88, 533 – 542. Eisenhofer, G., Rundquist, B., Aneman, A., Friberg, P., Dakak, N., Kopin, I.J., Jacobs, M.C., Lenders, J.W., 1995b. Regional release and removal of catecholamines and extraneuronal metabolism to metanephrines. J. Clin. Endocrinol. Metab. 80, 3009 – 3017. Eisenhofer, G.E., Lamensdorf, I., Kierk, K.L., Kawamura, M., Sato, S., 2000. Oxidative deamination of monoamines and biogenic aldehydes in neurodegeneration. Role Catechol Quinone Species Cell. Toxic. 103 – 145. el-Kabbani, O., Narayana, S.V., Babu, Y.S., Moore, K.M., Flynn, T.G., Petrash, J.M., Westbrook, E.M., DeLucas, L.J., Bugg, C.E., 1991. Purification, crystallization and preliminary crystallographic analysis of porcine aldose reductase. J. Mol. Biol. 218, 695 – 698. Erwin, V.G., Heston, W.D., Tabakoff, B., 1972. Purification and characterization of an NADH-linked aldehyde reductase from bovine brain. J. Neurochem. 19, 2269 – 2278. Goldstein, D.S., Eisenhofer, G., Stull, R., Folio, C.J., Keiser, H.R., Kopin, I.J., 1988. Plasma dihydroxyphenylglycol and the intraneuronal disposition of norepinephrine in humans. J. Clin. Invest. 81, 213 – 220. Graefe, K.H., Henseling, M., 1983. Neuronal and extraneuronal uptake and metabolism of catecholamines. Gen. Pharmacol. 14, 27 – 33. Grimshaw, C.E., Mathur, E.J., 1989. Immunoquantitation of aldose reductase in human tissues. Anal. Biochem. 176, 66 – 71. Hoffman, P.l., Wermuth, B., von Wartburg, J.P., 1980. Human brain aldehyde reductases: relationship to succinic semialdehyde reductase and aldose reductase. J. Neurochem. 35, 354 – 366. Jez, J.M., Flynn, T.G., Penning, T.M., 1997. A new nomenclature for the aldo-keto reductase superfamily. Biochem. Pharmacol. 54, 639 – 647. Kador, P.F., 1988. The role of aldose reductase in the development of diabetic complications. Med. Res. Rev. 8, 325 – 352. Kawamura, M., Kopin, I.J., Kador, P.F., Sato, S., Tjurmina, O., Eisenhofer, G., 1997. Effects of aldehyde/aldose reductase inhibition on neuronal metabolism of norepinephrine. J. Auton. Nerv. Syst. 66, 145 – 148. Kawamura, M., Eisenhofer, G., Kopin, I.J., Kador, P.F., Lee, Y.S., Tsai, J.Y., Fujisawa, S., Lizak, M.J., Sinz, A., Sato, S., 1999. Aldose reductase, a key enzyme in the oxidative deamination of norepinephrine in rats. Biochem. Pharmacol. 58, 517 – 524. Kobayashi, A., Fujisawa, S., 1994. Effect of L-carnitine on mitochondrial acyl CoA esters in the ischemic dog heart. J. Mol. Cell. Cardiol. 26, 499 – 508. Kopin, I.J., 1985. Catecholamine metabolism: basic aspects and clinical significance. Pharmacol. Rev. 37, 333 – 364. Lamensdorf, I., Eisenhofer, G., Harvey-White, J., Hayakawa, Y., Kirk, K., Kopin, I.J., 2000a. Metabolic stress in PC12 cells induces the formation of the endogenous dopaminergic neurotoxin, 3,4-dihydroxyphenylacetaldehyde. J. Neurosci. Res. 60, 552 – 558. Lamensdorf, I., Eisenhofer, G., Harvey-White, J., Nechustan, A., Kirk, K., Kopin, I.J., 2000b. 3,4-Dihydroxyphenylacetaldehyde potentiates the toxic effects of metabolic stress in PC12 cells. Brain Res. 868, 191 – 201. Lee, Y.S., Chen, Z., Kador, P.F., 1998a. Molecular modeling studies of the binding modes of aldose reductase inhibitors at the active site of human aldose reductase. Bioorg. Med. Chem. 6, 1811 – 1819. Lee, Y.S., Hodoscek, M., Brooks, B.R., Kador, P.F., 1998b. Catalytic mechanism of aldose reductase studied by the combined potentials of quantum mechanics and molecular mechanics. Biophys. Chem. 70, 203 – 216.
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Matsuura, K., Sato, K., Deyashiki, Y., Nakanishi, M., Hara, A., 1995. 3Deoxyglucosone reductase in dog adrenal glands. Identification as aldose reductase. Biol. Pharm. Bull. 18, 1765 – 1767. Matsuura, K., Deyashiki, Y., Bunai, Y., Ohya, I., Hara, A., 1996. Aldose reductase is a major reductase for isocaproaldehyde, a product of sidechain cleavage of cholesterol, in human and animal adrenal glands. Arch. Biochem. Biophys. 328, 265 – 271. Petrash, J.M., Harter, T.M., Murdock, G.L., 1997. A potential role for aldose reductase in steroid metabolism. Adv. Exp. Med. Biol. 414, 465 – 473. Powell, H.C., Garrett, R.S., Kador, P.F., Mizisin, A.P., 1991. Fine-structural localization of aldose reductase and ouabain-sensitive, K(+)-dependent p-nitro-phenylphosphatase in rat peripheral nerve. Acta Neuropathol. 81, 529 – 539. Rondeau, J.M., Tete-Favier, F., Podjarny, A., Reymann, J.M., Barth, P., Biellmann, J.F., Moras, D., 1992. Novel NADPH-binding domain revealed by the crystal structure of aldose reductase. Nature 355, 469 – 472. Rutledge, C.O., Jonason, J., 1967. Metabolic pathways of dopamine and norepinephrine in rabbit brain in vitro. J. Pharmacol. Exp. Ther. 157, 493 – 502. Sato, S., Kador, P.F., 1993. Human kidney aldose and aldehyde reductases. J. Diabetes Complications 7, 179 – 187. Sato, S., Kawamura, M., Eisenhofer, G., Kopin, I.J., Fujisawa, S., Kador, P.F., 1999. Aldo-keto reductases in norepinephrine metabolism. Adv. Exp. Med. Biol. 463, 459 – 463. Schlegel, B.P., Jez, J.M., Penning, T.M., 1998a. Mutagenesis of 3 alphahydroxysteroid dehydrogenase reveals a ‘‘push – pull’’ mechanism for proton transfer in aldo-keto reductases. Biochemistry 37, 3538 – 3548. Schlegel, B.P., Ratnam, K., Penning, T.M., 1998b. Retention of NADPHlinked quinone reductase activity in an aldo-keto reductase following mutation of the catalytic tyrosine. Biochemistry 37, 11003 – 11011. Tipton, K.F., 1986. Enzymology of monoamine oxidase. Cell Biochem. Funct. 4, 79 – 87. Turner, A.J., Tipton, K.F., 1972a. The characterization of two reduced nicotinamide-adenine dinucleotide phosphate-linked aldehyde reductases from pig brain. Biochem. J. 130, 765 – 772. Turner, A.J., Tipton, K.F., 1972b. The purification and properties of an NADPH-linked aldehyde reductase from pig brain. Eur. J. Biochem. 30, 361 – 368. Turner, A.J., Illingworth, J.A., Tipton, K.F., 1974. Simulation of biogenic amine metabolism in the brain. Biochem. J. 144, 353 – 360. Wermuth, B., 1985. Aldo-keto reductases. Prog. Clin. Biol. Res. 174, 209 – 230. Wermuth, B., Burgisser, H., Bohren, K., von Wartburg, J.P., 1982. Purification and characterization of human-brain aldose reductase. Eur. J. Biochem. 127, 279 – 284. Whittle, S.R., Turner, A.J., 1981. Biogenic aldehyde metabolism in rat brain. Differential sensitivity of aldehyde reductase isoenzymes to sodium valproate. Biochim. Biophys. Acta 657, 94 – 105. Wilson, D.K., Bohren, K.M., Gabbay, K.H., Quiocho, F.A., 1992. An unlikely sugar substrate site in the 1.65 A structure of the human aldose reductase holoenzyme implicated in diabetic complications. Science 257, 81 – 84. Wilson, D.K., Tarle, I., Petrash, J.M., Quiocho, F.A., 1993. Refined 1.8 A structure of human aldose reductase complexed with the potent inhibitor zopolrestat. Proc. Natl. Acad. Sci. U. S. A. 90, 9847 – 9851. Wilson, D.K., Nakano, T., Petrash, J.M., Quiocho, F.A., 1995. 1.7 A structure of FR-1, a fibroblast growth factor-induced member of the aldoketo reductase family, complexed with coenzyme and inhibitor. Biochemistry 34, 14323 – 14330. Wirth, H.P., Wermuth, B., 1985. Immunohistochemical localisation of aldehyde and aldose reductase in human tissues. Prog. Clin. Biol. Res. 174, 231 – 239. Yabe-Nishimura, C., 1998. Aldose reductase in glucose toxicity: a potential target for the prevention of diabetic complications. Pharmacol. Rev. 50, 21 – 33.
Aldose reductase: An aldehyde scavenging enzyme in the intraneuronal metabolism of norepinephrine in human sympathetic ganglia Minoru Kawamura a,b, Graeme Eisenhofer a, Irwin J. Kopin a, Peter F. Kador c, Yong S. Lee d, Shigeki Fujisawa c, Sanai Sato c,* a
Clinical Neurocardiology Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda MD 20892, USA b Institute of Bio-Active Science, Nippon Zoki Pharmaceutical Co., Ltd., Hyogo 673-1461, Japan c Laboratory of Ocular Therapeutics, National Eye Institute, National Institutes of Health, 10/10B09, 10 Center Drive, MSC 1850, Bethesda MD 20892-1850, USA d Center for Molecular Modeling, Center for Information Technology, National Institutes of Health, Bethesda MD 20892, USA Received 27 June 2001; received in revised form 4 October 2001; accepted 31 October 2001
Abstract The neurotransmitter norepinephrine is metabolized by monoamine oxidase into an aldehyde intermediate that is further metabolized to the stable glycol derivative, 3,4-dihydroxyphenylglycol (DHPG). In this study, the possible role of aldose reductase in reducing this aldehyde intermediate in human sympathetic neurons has been examined. DHPG is formed when norepinephrine is incubated with aldose reductase in the presence of monoamine oxidase. DHPG metabolism is inhibited by the monoamine oxidase inhibitor, pargyline which prevents the deamination of norepinephrine, and by the aldose reductase inhibitor AL 1576, which inhibits DHPG formation without affecting the deamination of norepinephrine. Although similar formation of DHPG was observed with human liver aldehyde reductase, the production of DHPG was more effective with aldose reductase than aldehyde reductase. Two peaks of reductase activity corresponding to aldose reductase and aldehyde reductase were observed when sympathetic ganglia were chromatofocused. Molecular modeling studies indicate that the energy-minimized structure of 3,4-dihydroxymandelaldehyde bound to aldose reductase is similar to that of glyceraldehyde where the 2Vhydroxyl group forms hydrogen bonds with Trp111 and NADPH. These results suggest that aldose reductase may be important in metabolizing the potentially toxic aldehyde intermediate formed from norepinephrine in human sympathetic ganglia. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Aldose reductase; Aldehyde reductase; Norepinephrine; 3,4-Dihydroxyphenylglycol; Sympathetic nervous system
1. Introduction Monoamine neurotransmitters produced in aminergic nerves are primarily metabolized within the cytoplasm of nerve terminals (Dong et al., 1993, 1995; Graefe and Henseling, 1983). Because monoamine oxidase (EC 1.4.3.4, MAO) is the only catecholamine-metabolizing enzyme in this intraneuronal location, the first step of the intraneuronal metabolism of these neurotransmitters is oxidative deamination by MAO. This deamination step generates aldehyde intermediates that are further metabolized to stable acid and/or glycol derivatives by either dehydrogenase(s) or reductase(s). Bec-
*
Corresponding author. Tel.: +1-301-402-9849; fax: +1-301-402-2399. E-mail address: [email protected] (S. Sato).
ause of their highly reactive nature, the rapid and complete removal of these aldehyde intermediates is crucial for eliminating possible cytotoxicity that can initiate the process of neurodegeneration (Eisenhofer et al., 2000). Aldose reductase (alditol:NAD+ 1-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) are members of the aldo-keto reductase (AKR) superfamily (Jez et al., 1997). The role of these enzymes in biogenic amine metabolism was extensively studied in 1970 –1980s using mammalian brain tissues. In these early studies, two NADPH-dependent reductases (high Km aldehyde reductase and low Km aldehyde reductase) utilizing bhydroxylated aldehydes as substrate were detected (Duncan et al., 1975; Turner and Tipton, 1972a,b). These two enzymes were later identified as aldehyde reductase (AR3, ALR1) and aldose reductase (AR2, ALR2) (Hoffman et al., 1980;
1566-0702/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 1 5 6 6 - 0 7 0 2 ( 0 1 ) 0 0 3 8 5 - X
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Cromlish and Flynn, 1985). Other reductases such as succinic semialdehyde reductase are also present in the brain tissue, but have negligible activity in utilizing b-hydroxylated aldehydes as substrate (Hoffman et al., 1980). The above described studies consistently indicated that, kinetically, the favored pathway of biogenic amine reduction involves aldose reductase rather than aldehyde reductase (Turner and Tipton, 1972a,b; Hoffman et al., 1980; Wermuth et al., 1982; Wermuth, 1985). It was also reported that the formation of 3-methoxy-4-hydroxyphenylglycol (MHPG) from normetanephrine by rat brain homogenates is inhibited by aldose reductase inhibitors such as quercetin and quercitrin, but not by the aldehyde reductase specific inhibitor sodium valproate (Whittle and Turner, 1981). Although the kinetic evidence favors the importance of aldose reductase over aldehyde reductase for reduction of bhydroxylated aldehydes, whether either or both enzymes are present at the sites of biogenic amine metabolism is not clear. Reductase activity is widely detected in the whole brain (Erwin et al., 1972; Wirth and Wermuth, 1985); however, aldose reductase activity is not associated with either the nerve terminals of the striatum, the major site of catecholamine production (Duncan et al., 1975) or with the synaptosomal fractions of brain homogenates (Turner and Tipton, 1972a,b). In the retina, aldose reductase activity is greater in glial cells than ganglion cells (Akagi et al., 1984). Similarly, the principal site for aldose reductase in peripheral nerves is the Schwann cells rather than nerve fibers (Powell et al., 1991). The above findings suggest a role of aldose reductase in the extraneuronal metabolism of monoamine neurotransmitters. It remains unclear whether aldose reductase is involved in the intraneuronal metabolism of monoamine neurotransmitters. In peripheral sympathetic neurons, the neurotransmitter norepinephrine is predominantly metabolized to the glycol metabolite, 3,4-dihydroxyphenylglycol (DHPG). In humans, plasma DHPG is primarily derived from peripheral sympathetic neurons, with contributions from brain tissue being minor (Goldstein et al., 1988). Previously, we reported that aldose reductase is the primary enzyme in rat sympathetic neurons (Kawamura et al., 1999) and that aldose reductase inhibitors reduce levels of DHPG and increase those of 3,4-dihydroxymandelic acid (DHMA) (Kawamura et al., 1997). To assess the importance of aldose reductase in human tissues, species differences with respect to the kinetic properties of aldose reductase must be determined, along with its specific localization in human sympathetic neurons. Here, we report that, just like rat aldose reductase, human aldose reductase generates DHPG from norepinephrine through an aldehyde intermediate generated by MAO. In addition, aldose reductase is localized in human sympathetic neurons. To obtain insight into the structural basis of the reduction of catechol aldehydes by human aldose reductase, molecular modeling studies were also conducted.
2. Materials and methods 2.1. Chemicals and materials Unless otherwise stated, all reagents utilized in this study were analytical grade. Norepinephrine bitartrate (NE), epinephrine bitartrate (EPI), DL-metanephrine hydrochloride, 3,4-dihydroxyphenylglycol (DHPG), 3-methoxy-4-hydroxyphenylglycol (MHPG), 3,4-dihydroxybenzylamine (DHBA), pargyline and NADPH were all obtained from Sigma (St. Louis, MO). Polybuffer 74 was product of Pharmacia-LKB Biotechnology, (Piscataway, NJ). The aldose reductase inhibitor, AL 1576 (2,7-difluorospirofluorene-9,5V-imidazolidine2V,4V-dione: Imirestat), was a gift from Alcon Laboratories (Fort Worth, TX). Recombinant human muscle aldose reductase was purchased from Wako (Osaka, Japan). Human liver aldehyde reductase was prepared as described previously (Sato and Kador, 1993). Human liver tissues utilized for enzyme purification were obtained through the National Diabetes Research Interchange (Philadelphia, PA). Mitochondrial membranes, utilized as a source of MAO, were isolated from rat liver as described previously (Kobayashi and Fujisawa, 1994). Samples of pheochromocytoma tumor tissue were obtained within 1 h of surgical removal under a research protocol approved by the Intramural Review Board (IRB) of the National Cancer Institute, National Institutes of Health (NIH). All patients from whom tumor tissue was obtained provided written informed consent for their participation in the study. Dimensions of tumors were recorded, and small samples (50 –400 mg) were dissected away from surrounding tissue, placed on dry ice, and then stored at 80 C. Samples of human sympathetic ganglia were obtained during routine autopsies of two patients at the NIH. Samples were obtained from a 74-year-old female within 8 h of death due to cardiorespiratory failure and a 49-year-old female within 20 h of death due to complications of malignant melanoma. Collection of these samples was approved under an exemption from NIH IRB review based on a written ‘‘Request for Patient Related Materials’’ and under the condition that collections of specimens would not require identification of patients. 2.2. Enzyme assay Reductase activities were spectrophotometrically assayed on a Shimadzu UV-2101PC spectrophotometer (Shimadzu, Kyoto, Japan) by following the decrease of NADPH. The reaction mixture (1 ml) consisted of 0.3 mM NADPH, 10 mM aldehyde substrate, 0.1 M phosphate buffer, pH 6.2, and adequate amounts of enzyme (approximately 2– 3 mU as DLglyceraldehyde as substrate). The enzyme reaction was started by adding substrate and incubation was continued at 25 C for 4 min. One enzyme unit (U) was defined as the activity consuming 1 mmol of NADPH per min under the assay condition.
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2.3. In vitro production of 3,4-dihydroxyphenylglycol (DHPG) and 3-methoxy-4-hydroxyphenylglycol (MHPG) Unless otherwise stated, the reaction mixture (100 ml) containing 2 mM ascorbic acid, 1 mM catecholamine, 0.1 mM NADPH, 10 ml of MAO and aldose reductase (10 mU as DLglyceraldehyde as substrate) in 0.1 M phosphate buffer, pH 7.4, was incubated at 37 C for 1 h. The reaction was terminated by the addition of 50 ml of 0.4 M perchloric acid containing 0.5 mM EDTA. After centrifugation at 10,000 g for 15 min, the supernatant was stored at 80 C for catechol analysis by HPLC. DHPG and MHPG were analyzed by HPLC with electrochemical detection as previously reported (Eisenhofer et al., 1986, 1994; Kawamura et al., 1997).
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assumed to be negatively charged while arginine and lysine were positively charged. Since (R)-(+)-glyceraldehyde and (R)-( )-norepinephrine are physiologically active, D-forms of glyceraldehyde, 3,4-dihydroxymandelaldehyde (DHMAL), 3-methoxy-4-hydroxymandelaldehyde (MHMAL) and 3,4-dihydroxyphenylacetaldehyde were modeled at the active site. Charge assignment on NADPH, substrates and the hydration of the complex of aldose reductase-NADPH with a substrate as well as the subsequent energy minimization by CHARMM with an all-atom parameter set (Molecular Simulations, 1992) were carried out according to procedures published elsewhere (Lee et al., 1998a,b).
3. Results
2.4. Sephacryl S-100 chromatography and chromatofocusing on Mono P
3.1. In the presence of MAO, human aldose reductase generates DHPG from norepinephrine in vitro
Approximately 1 g of either human sympathetic ganglia or pheochromocytoma tissue was homogenized with 3 volumes of 20 mM imidazole-HCl buffer, pH 7.5 containing 10 mM 2mercaptoethanol. After centrifugation at 30,000 g for 10 min, the supernatant was applied to a Sephacryl S-100 column (HiPrepTM 16/60, Pharmacia-LKB Biotechnology) and eluted with the same imidazole buffer at the flow rate of 1.0 ml/min. The eluant was collected into 35-drop aliquots (approximately 1.7 ml) and the fractions containing reductase activity were applied to a Mono P column (HR 5/20, Pharmacia-LKB Biotechnology) equilibrated with 20 mM imidazole-HCl buffer, pH 7.5, containing 10 mM 2-mercaptoethanol. The column was developed at a flow rate of 1.0 ml/ min with Polybuffer 74 diluted 10 times with 10 mM 2mercaptoethanol and the eluant was collected into 20-drop aliquots (approximately 1.0 ml). The reductase activity of each fraction was assayed with DL-glyceraldehyde and Dglucuronate as substrates.
To confirm the formation of DHPG by human aldose reductase, norepinephrine was incubated with human muscle aldose reductase in the presence of MAO (Fig. 1). No DHPG was formed in the absence of MAO and aldose reductase and the HPLC chromatogram of the reaction mixture only displayed peaks corresponding to non-metabolized norepinephrine and the internal standard, dihydroxybenzylamine (DHBA). When the reaction mixture contained both MAO and aldose reductase, a new peak corresponding to DHPG was clearly detected and the peak corresponding to norepinephrine was decreased. Formation of DHPG along with the decrease in norepinephrine was completely prevented by the MAO inhibitor, pargyline. The aldose reductase inhibitor, AL 1576, also significantly reduced the formation of DHPG; however, this inhibitor failed to prevent the loss of norepinephrine. This confirms that norepinephrine is first metabolized to an aldehyde intermediate by MAO and that this aldehyde intermediate is metabolized to DHPG by aldose reductase.
2.5. Structural modeling
3.2. Human aldose reductase also generates DHPG from epinephrine
The coordinates of human aldose reductase complexed with cofactor and crystal waters (1ADS) (Wilson et al., 1992) were obtained from the Brookhaven Protein Data Bank. Hydrogens were added to the amino acid residues of human aldose reductase utilizing the HBUILD routine of CHARMM (Brooks et al., 1983). With the exception of His110, all histidine residues were treated as neutral with hydrogen assigned to the Nd1 of neutral histidine. His110 was treated as positively charged based on the crystallographic evidence that the carboxylate of zopolrestat is salt-linked to the Ne2 atom of His110 (Wilson et al., 1993, 1995). Further support has also come form the evidence by the computer simulation reduction mechanism of aldose reductase (Lee et al., 1998a,b) and mutagenic and kinetic studies on 3a-hydroxysteroid dehydrogenase, a member of aldo-keto reductase, (Schlegel et al., 1998a,b). All aspartate and glutamate residues were
MAO also utilizes epinephrine as substrate. The deaminated product formed from epinephrine is the same aldehyde intermediate, DHMAL, as formed from norepinephrine. Since aldose reductase utilizes this aldehyde intermediate as substrate to generate DHPG, the final metabolite formed from epinephrine by MAO and aldose reductase should also be DHPG. This was confirmed by incubating epinephrine with MAO and aldose reductase (Fig. 2). Similar to formation from norepinephrine, DHPG formation from epinephrine was only detected in the presence of both MAO and aldose reductase. Because epinephrine is not as good as norepinephrine as a substrate for MAO, the decrease in epinephrine was slower than that of norepinephrine. Pargyline prevented both DHPG formation and the decrease of epinephrine. AL 1576 reduced the formation of DHPG but
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3.3. Aldose reductase is more active than aldehyde reductase in producing DHPG Since the reduction of DHMAL to DHPG is also catalyzed by aldehyde reductase, the activities of aldose reductase and aldehyde reductase in producing DHPG were compared in the same in vitro incubation system (Fig. 3). The levels of DHPG produced by the incubation of norepinephrine with 0.01, 0.1 and 1.0 mU of human muscle aldose reductase were 87 F 18, 261 F 53 and 1507 F 61 pmol/ml (n = 4), respectively. In contrast, the amounts of DHPG generated by aldehyde reductase were far less than
Fig. 1. HPLC chromatograms obtained from a mixture containing 1 mM norepinephrine (NE), monoamine oxidase (MAO) and 10 mU human muscle aldose reductase (AR) incubated at 37 C for 1 h as described in Materials and methods. (A) represents the reaction mixture containing only NE while (B) is the complete reaction mixture containing NE, MAO and AR. In addition to the complete reaction mixture, (C) contains 1 mM of the MAO inhibitor pargyline while (D) contains 100 mM of the aldose reductase inhibitor AL 1576. DHPG; 3,4-dihydroxyphenylglycol. DHBA; 3,4-dihydroxybenzylamine.
did not affect the decrease of epinephrine. This again confirms that aldose reductase generates DHPG by reducing the aldehyde intermediate, DHMAL formed by MAO.
Fig. 2. HPLC chromatograms obtained from a mixture containing 1 mM epinephrine (EPI), monoamine oxidase (MAO) and 10 mU human muscle aldose reductase (AR) incubated at 37 C for 1 h. (A) represents the reaction mixture that contains only EPI while (B) contains the complete reaction mixture NE, MAO and AR. In addition to the complete mixture, (C) contains 1 mM of the MAO inhibitor pargyline and (D) contains 100 mM aldose reductase inhibitor AL 1576. DHPG; 3,4-dihydroxyphenylglycol. DHBA; 3,4-dihydroxybenzylamine.
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reductase displays higher activity with D-glucuronate compared to DL-glyceraldehyde. This indicates that both aldose reductase and aldehyde reductase are present in human sympathetic ganglions. However, there may be some individual variations in the relative amounts of aldose reductase versus aldehyde reductase. 3.5. Aldose reductase also utilizes 3-methoxy-aldehyde substrate derived from metanephrine
Fig. 3. 3,4-Dihydroxyphenylglycol (DHPG) formed by the in vitro incubation at 37 C for 1 h of 10 mM norepinephrine with 1.0, 0.1 and 0.01 mU of human muscle aldose reductase (light gray) and 1.0 and 0.1 mU of human liver aldehyde reductase (dark gray). Enzyme units (U) are expressed as mmol/min with 10 mM DL-glyceraldehyde as substrate. Mean F S.D. (n = 4).
those generated by aldose reductase. Human liver aldehyde reductase, in 0.1 and 1.0 mU amount produced only 64 F 6 and 218 F 10 pmol/ml of DHPG (n = 4), respectively. The dependence of our in vitro reaction system on amine precursors (e.g. norepinephrine and epinephrine) to generate aldehyde intermediates did not make it possible to provide saturating concentration of substrate for aldose reductase or aldehyde reductase. This explains the ‘apparent’ non-linear kinetics of reductase activity, as assessed by DHPG production. Thus, although the correlation between the amounts of enzyme and the levels of DHPG was not linear, the levels of DHPG generated by aldose reductase were consistently higher than those by aldehyde reductase. This result was consistent with our previous reports using rat enzymes (Kawamura et al., 1999).
Normetanephrine and metanephrine are O-methylated metabolites that are formed by catechol-O-methyltransferase (COMT) from norepinephrine and epinephrine, respectively. These two O-methylated amines are also deaminated by MAO to another aldehyde intermediate, 3-methoxy-4hydroxymandelaldehyde (MHMAL) (Tipton, 1986; Eisenhofer and Finberg, 1994). To examine whether this 3methoxyaldehyde intermediate is further metabolized by aldose reductase to its glycol metabolite, 3-methoxy-4-hydroxyphenylglycol (MHPG), human muscle aldose reductase was also incubated with metanephrine in the presence of MAO (Fig. 5).
3.4. Both aldose and aldehyde reductases are present in human sympathetic neurons Because of overlapping substrate specificity, aldose reductase activity cannot be distinguished from aldehyde reductase activity by the traditional photometric enzyme assay. These two enzymes are separated only by procedure(s) utilizing their charge differences (Sato and Kador, 1993). In this study, to confirm the presence of aldose reductase and to estimate the relative amounts of aldose reductase versus aldehyde reductase, chromatofocusing of human sympathetic neurons was conducted (Fig. 4). The chromatogram obtained from chromatofocusing resulted in the appearance of two activity peaks with DLglyceraldehyde as substrate that corresponded to aldose reductase (the first peak, pI 6.0) and aldehyde reductase (the second peak, pI 5.5), respectively. The identity of these two activity peaks was confirmed by Western blot and by substrate specificity characteristics in which the activity with DLglyceraldehyde is approximately two-fold higher than that with D-glucuronate for aldose reductase while aldehyde
Fig. 4. Chromatofocusing of human sympathetic ganglia homogenates illustrating the presence of both aldose reductase and aldehyde reductase. Closed circles indicate the activity with DL-glyceraldehyde as substrate and broken line indicates pH of eluents. The first activity peak corresponds to aldose reductase (AR) while the second peak corresponds to aldehyde reductase (ALR).
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When metanephrine was incubated with both aldose reductase and MAO, MHPG was produced. Although there was no exact linearity, the MHPG production was increased in dose-dependent manner when the enzyme amounts were increased. MHPG levels produced by 1.0, 0.1 and 0.01 mU of human muscle aldose reductase were 130 F 0.5, 59 F 3 and 39 F 0.7 pmol/ml (n = 4), respectively. MHPC was also generated by human liver aldehyde reductase; however, the levels produced were lower than those produced with aldose reductase. MHPG levels produced by 1.0 and 0.1 mU of human liver aldehyde reductase were only 41 F 0.6 and 35 F 1 pmol/ml (n = 4), respectively. This confirms that both human aldose reductase and aldehyde reductase can utilize Fig. 5. 3-Methoxy-4-hydroxypbenylglycol (MHPG) formation by the in vitro incubation at 37 C for 1 h of 1 mM metanephrine with 1.0, 0.1 and 0.01 mU of human muscle aldose reductase (light gray) and 1.0 and 0.1 mU of human liver aldehyde reductase (dark gray). Enzyme units (U) are expressed as mmol/min with 10 mM DL-glyceraldehyde as substrate. Mean F S.D. (n = 4).
Fig. 6. Chromatofocusing of homogenized pheochromocytoma cells illustrating the presence of both aldose reductase and aldehyde reductase. Closed circles indicate activity with DL-glyceraldehyde as substrate and broken line indicates pH of eluents. The first activity peak corresponds to aldose reductase (AR) while the second peak corresponds to aldehyde reductase (ALR).
Fig. 7. CHARMM energy minimized structure of human aldose reductase complexed with D-glyceraldehyde (A) and 3,4-dihydroxymandelaldehyde (DHMAL, B). Atoms are represented by colors: white, carbon; green, hydrogen; blue, nitrogen; red, oxygen; yellow, sulfur. Yellow dot line indicates hydrogen-bonding interaction.
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the aldehyde intermediate from metanephrine, but that its reduction is more favorable with aldose reductase compared to aldehyde reductase.
3.7. Hydrogen-bonding interactions between substrates and aldose reductase indicate that the 2V-hydroxyl group is the key motif recognized by aldose reductase
3.6. Aldose reductase is also detectable in pheochromocytoma cells
In molecular modeling studies of DHMAL in position at the active site of aldose reductase, the carbonyl of the aldehyde intermediate hydrogen-bond with His110 while the 2V-hydroxyl group hydrogen-bond with both Trp 111 and the amide oxygen of NADPH (Fig. 7). The CHARMM energy minimized structure further indicates that the side chains of Trp20, Trp111, and Phe122 are positioned for aromatic –aromatic and van der Waals interactions with the aromatic portion of DHMAL (Lee et al., 1998a,b). This hydrogen bonding pattern is essentially identical to that observed with D-glyceraldehyde, a commonly used substrate for aldose reductase. Similar hydrogen bonding interactions with aldose reductase are also formed by MHMAL, which has a methoxy group on the three carbon position on the aromatic ring (Fig. 8). However, unlike DHMAL or MHMAL, the carbonyl of 3,4-dihydroxyphenylacetaldehyde only forms a hydrogen bond with His110 (Fig. 8). These optimized structures all suggest that the substrate carbonyl is properly oriented to accept the pro-R hydride transfer from NADPH and a proton from His110.
Another major source for catecholamines is the adrenal medulla. Chromatofocusing of the crude extract of pheochromocytoma also displayed two peaks of reductase activity corresponding to aldose reductase and aldehyde reductase (Fig. 6).
4. Discussion
Fig. 8. CHARMM energy minimized structure of human aldose reductase complexed with 3-methoxy-4-hydroxymandelaldehyde (MHMAL, A) and 3,4-dihydroxyphenylacetaldehyde (B). Atoms are represented by colors: white, carbon; green, hydrogen; blue, nitrogen; red, oxygen; yellow, sulfur. Yellow dot line indicates hydrogen-bonding interaction.
Both aldose reductase and aldehyde reductase are members of aldo-keto reductase (AKR) superfamily (Jez et al., 1997). Although the amino acid sequence homology between aldose reductase and aldehyde reductase is only about 65% (Bohren et al., 1989), the tertiary structures of these two enzymes are highly conserved with arrangements of the key amino acids, His110, Tyr48 and Typ111, in their active sites being virtually identical (el-Kabbani et al., 1991; Rondeau et al., 1992; Wilson et al., 1992). As a consequence, both enzymes share a wide range of substrates. The present study confirms that both human aldose reductase and aldehyde reductase can utilize the aldehyde intermediate formed from the neurotransmitter, norepinephrine, as substrate to form the stable glycol metabolite, DHPG. However, the in vitro data also indicate that DHPG formation occurs more favorably with aldose reductase rather than with aldehyde reductase. Molecular modeling studies at the atomic level provide the structural basis for the reduction of DHMAL by aldose reductase. The hydrogen bonding interactions between DHMAL and aldose reductase are similar to those with D-glyceraldehyde, suggesting that DHMAL can be a good substrate for aldose reductase. This also suggests that aldose reductase reduces DHMAL in a similar manner to the reduction of glyceraldehyde to glycerol. Importantly, the energy-minimized structure demonstrates hydrogen bonding between the 2V-hydroxyl group of DHMAL and Trp111 and the amide oxygen of NADPH. The 2V-hydroxyl group appears to be crucial in correctly orienting the substrate for catalysis. In
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general, catecholamines lacking the 2V-hydroxyl group are preferentially metabolized to acids while those containing a 2V-hydroxyl group are reduced to glycols (Rutledge and Jonason, 1967; Turner et al., 1974). For example, dopamine is almost exclusively oxidized to 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). In contrast, both norepinephrine and epinephrine are primarily metabolized to DHPG and MHPG (Kopin, 1985). Failure in forming hydrogen bonds without the 2V-hydroxyl group may explain this experimental observation. Molecular modeling also indicates that the 3V-methoxy group of MHMAL has no effect on the hydrogen-bonding interactions between the substrate and enzyme (Fig. 8). This suggests that the aldehyde intermediate formed from either normetanephrine or metanephrine can be a good substrate for aldose reductase. This is again consistent with the observation that human aldose reductase generates MHPG from metanephrine when incubated in the presence of MAO. Aldose reductase is known as the first enzyme in the polyol pathway where glucose is metabolized to fructose through the intermediate sugar alcohol, sorbitol. Aldose reductase also plays a key role in the pathogenesis of various diabetic complications by initiating the abnormal intracellular accumulation of sorbitol (Kador, 1988; Yabe-Nishimura, 1998). In sympathetic neurons, however, aldose reductase appears to be important as an aldehyde-scavenging enzyme. Like many biological aldehydes, the aldehyde intermediates formed in neurotransmitter metabolism are potentially toxic (Lamensdorf et al., 2000a,b). The rapid and complete removal of these aldehyde metabolites is important (Eisenhofer et al., 2000). Under physiological conditions, the aldehyde intermediate, DHMAL, formed from norepinephrine is primarily metabolized to the glycol metabolite, DHPG. However, when the reduction pathway to DHPG is blocked, the pathway quickly shifts to produce 3,4-dihydroxymandelic acid, DHMA, through oxidation by aldehyde dehydrogenase (Kawamura et al., 1999; Sato et al., 1999). This metabolic redundancy helps ensure the appropriate removal of toxic aldehyde metabolites. The present study confirms that both aldose reductase and aldehyde reductase are present in human sympathetic nerves. Since both enzymes utilize DHMAL as substrate, the presence of both aldose reductase and aldehyde reductase, in addition to aldehyde dehydrogenase, adds another level of metabolic redundancy to ensure appropriate removal of toxic aldehyde metabolites. Thus, aldose reductase and aldehyde reductase are both potentially important in the intraneuronal metabolism of the neurotransmitter norepinephrine in human sympathetic neurons. However, under normal conditions, most DHPG in sympathetic neurons is likely formed by aldose reductase. Aldose reductase appears to be more active than aldehyde reductase in reducing aldehyde intermediates formed from catecholamines. There may also be, however, some individual variation in the levels of aldose reductase and aldehyde reductase (Fig. 4). The large-scale clinical trials on aldose reductase inhibitors completed over the last few decades failed to show
abnormal catecholamine metabolism or adrenergic dysfunction. Presumably, this indicates the importance of the redundancy in catecholamine metabolism that secures efficient removal of toxic aldehyde intermediates under various circumstances. The role of aldose reductase in catecholamine metabolism may not be limited to the intraneuronal metabolism of norepinephrine. Aldose reductase may be also important in the adrenal glands. A substantial portion of free metanephrine and normetanephrine are formed within the adrenals. Similar to intraneuronal metabolism, the major metabolite of these 3methoxy compounds in adrenal glands is the glycol derivative, MHPG (Eisenhofer et al., 1995a,b). In the present study, human aldose reductase was observed to generate MHPG from metanephrine in the presence of MAO. Molecular modeling studies support the premise that the aldehyde intermediate formed from either normetanephrine or metanephrine is also a good substrate for aldose reductase. It is well known that adrenal glands contain high levels of aldose reductase (Grimshaw and Mathur, 1989; Matsuura et al., 1995), and pheochromocytoma cells originating from the adrenal medulla also contain the significant amounts of aldose reductase (Fig. 6). In the adrenal cortex, aldose reductase appears to be important in steroid metabolism (Matsuura et al., 1996; Petrash et al., 1997). In the medulla, however, aldose reductase (together with aldehyde reductase) may contribute to catecholamine metabolism by reducing an aldehyde intermediate, MHMAL, to the glycol derivative, MHPG. References Akagi, Y., Yajima, Y., Kador, P.F., Kuwabara, T., Kinoshita, J.H., 1984. Localization of aldose reductase in the human eye. Diabetes 33, 562 – 566. Bohren, K.M., Bullock, B., Wermuth, B., Gabbay, K.H., 1989. The aldoketo reductase superfamily. cDNAs and deduced amino acid sequences of human aldehyde and aldose reductases. J. Biol. Chem. 264, 9547 – 9551. Brooks, B.R., Bruccoleri, R.E., Olafson, B.D., States, D.J., Swaminathan, S., Karplus, M., 1983. A program for maculomolecular energy, minimization, and dynamics calculations. J. Comput. Chem. 4, 187. Cromlish, J.A., Flynn, T.G., 1985. Identification of pig brain aldehyde reductases with the high-Km aldehyde reductase, the low-Km aldehyde reductase and aldose reductase, carbonyl reductase, and succinic semialdehyde reductase. J. Neurochem. 44, 1485 – 1493. Dong, W.X., Schneider, J., Lacolley, P., Brisac, A.M., Safar, M., Cuche, J.L., 1993. Neuronal metabolism of catecholamines: plasma DHPG, DOMA and DOPAC. J. Auton. Nerv. Syst. 44, 109 – 117. Dong, W.X., Schneider, J., Dabire, H., Safar, M., Cuche, J.L., 1995. Neuronal metabolism of catecholamines in pithed and electrically stimulated rats. J. Auton. Nerv. Syst. 54, 41 – 48. Duncan, R.J., Sourkes, T.L., Dubrovsky, B.O., Quik, M., 1975. Activity of aldehyde dehydrogenase, aldehyde reductase, and acetylcholine esterase in striatum of rats bearing electrolytic lesions of the medial forebrain bundle. J. Neurochem. 24, 143 – 147. Eisenhofer, G., Finberg, J.P., 1994. Different metabolism of norepinephrine and epinephrine by catechol-O-methyltransferase and monoamine oxidase in rats. J. Pharmacol. Exp. Ther. 268, 1242 – 1251. Eisenhofer, G., Goldstein, D.S., Stull, R., Keiser, H.R., Sunderland, T.,
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