Introduction

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The flavoproteins monoamine oxidase A and B (MAO-A and -B) are the principal enzymes responsible for the metabolism of the neurotransmitters dopamine, norepinephrine, epinephrine, and serotonin (Waldmeier, 1987; Kopin, 1994). These enzymes are found throughout the body and are localized in the outer mitochondrial membrane. For reasons that remain unclear, often only one form of the enzyme is present in a specific organ and/or within a specific cell type (Trendelenburg et al., 1987; Yu et al., 1992). Extensive studies have been performed over the years to characterize the properties of MAO-A and -B (Kalgutkar and Castagnoli, 1995; Silverman, 1995; Castagnoli et al., 1997; Wouters, 1998).

In addition to their roles in the regulation of neurotransmitter levels, these enzymes also catalyze the oxidation of xenobiotic amines (Strolin Benedetti and Tipton, 1998) including dietary tyramine (Hauptmann et al., 1996). Furthermore, it is well known that the parkinsonian inducing neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine [MPTP (1)] is an excellent MAO-B substrate (Trevor et al., 1987). The neurotoxicity of MPTP is dependent on its MAO-B-catalyzed oxidation to the dihydropyridinium intermediate 1-methyl-4-phenyl-2,3-dihydropyridinium species (MPDP⁺; 2), which undergoes further oxidation to the 1-methyl-4-phenylpyridinium species (MPP⁺; 3), the ultimate toxin.

In view of the diverse nature of MAO substrates and inhibitors, a better appreciation of the factors that may influence the interactions of MAO-A and MAO-B with xenobiotics, as has been detailed for the cytochrome P-450 family of oxidases (Guengerich, 1997), could prove useful in the design and development of new therapeutic agents. As an initial effort toward this goal, we have undertaken a series of systematic studies aimed at characterizing species- and organ-dependent differences in MAO activity. Some evidence for such differences has been reported. Garrick and Murphy (1980) have observed species differences in the inhibition curves of clorgyline, (R)-deprenyl, and pargyline, all potent mechanism-based inactivators of MAO-A and/or MAO-B, on the deamination of dopamine, serotonin, tyramine, and 2-phenylethylamine. Dramatic species differences in the inhibition of MAO-B by various oxadiazolones and oxadiazolethiones also have been described (Krueger et al., 1995). Furthermore, the substrate and inhibition properties of a series of -aminoamides were shown to be tissue dependent (O'Brien et al., 1995).

In the present study, the rates of -carbon oxidation of a panel of tetrahydropyridinyl substrates have been compared. These substrates were selected in part because of our interests in MAO-catalyzed metabolic bioactivation processes (Castagnoli et al., 1997). All of the corresponding dihydropyridinium metabolites absorb light at wavelengths considerably longer than those of the substrate molecules, making it possible to estimate the rates of oxidation with the spectrophotometric assay described below.

	Materials and Methods

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Chemicals. The following tetrahydropyridinyl derivatives (see Fig. 1 for structures), used as substrates in these studies, were synthesized in our laboratory: 1,1-methyl-4-(3-methoxyphenyl)-1,2,3,6-tetrahydropyridine (4; Youngster et al., 1987), 1-methyl-4-(1-methyl-2-pyrrolyl)-1,2,3,6-tetrahydropyridine (5; Nimkar et al., 1996), 1-methyl-4-(3-ethyl-2-furanyl)-1,2,3,6-tetrahydropyridine (6; Yu and Castagnoli, 1999), and, as described below, 1-methyl-4-(2-isopropylphenyl)-1,2,3,6-tetrahydropyridine (7). Spectroscopic data and elemental analyses for the oxalate salt of 4 has not been reported and is as follows: 4 · C₂H₂O₄ is a white solid (MeOH/Et₂O): m.p.: 147°C; ¹H NMR [dimethyl sulfoxide (DMSO)-d₆]  7.28 (1 H, t, J = 8.0 Hz), 7.08 (1 H, d, J = 7.7 Hz), 6.99 (1 H, t, J = 2.2 Hz), 6.88 (1 H, dd, J = 2.4 Hz, J = 8.0 Hz), 6.04 (1 H, M), 3.79 (2 H, M), 3.77 (3 H, s), 3.34 (2 H, M), 2.81 (3 H, s), 2.73 (2 H, bs); ¹³C NMR (DMSO-d₆)  165.1, 159.5, 140.0, 133.6, 129.6, 117.2, 117.1, 113.4, 110.5, 55.1, 51.2, 49.5, 41.6, 23.8; gas chromatography (GC)-EIMS, m/z (rel intensities) 203 (100%), 202 (63), 159 (20), 145 (24), 115 (20), 96 (59), 82 (15), 51 (11); UV (nm, MeOH) 213, 246, 288. Analysis calculated (C₁₅H₁₉NO₅): C, 61.42; H, 6.53; N, 4.78. Found: C, 61.28; H, 6.49; N, 4.73. Compound 7 was prepared by the condensation of the lithium salt derived from 2-isopropylbromobenzene with 1-methyl-4-piperidone followed by acid catalyzed dehydration of the resulting carbinol. The product was purified as its oxalate salt: 7 · C₂H₂O₄ (MeOH/Et₂O): m.p.: 204-205°C; ¹H NMR (DMSO-d₆)  7.30 (1 H, dt, J = 7.8 Hz, J = 1.5 Hz), 7.24 (1 H, dd, J = 7.8 Hz, J = 1.5 Hz), 7.13 (1 H, dt, J = 7.6 Hz, J = 1.5 Hz), 7.02 (1 H, ddd, J = 7.6 Hz, J = 1.5 Hz, J = 0.4 Hz), 5.49 (1 H, M), 3.70 (2 H, M), 3.29 (2 H, t, J = 6.0 Hz), 3.09 (1 H, sept, J = 6.9 Hz), 2.80 (3 H, s), 2.53 (2 H, M), 1.14 (6 H, d, J = 6.9 Hz); ¹³C NMR (DMSO-d₆)  163.8, 145.5, 136.3, 127.6, 127.3, 125.1, 118.7, 51.1, 49.6, 41.8, 28.6, 28.2, 24.0; GC-EIMS, m/z (rel intensities) 215 (95), 214 (85), 200 (86), 172 (45), 157 (100), 142 (52), 128 (48), 115 (42), 96 (53); UV (nm, MeOH) 212, 242, 265. Analysis calculated (C₁₇H₂₃NO₄): C, 66.86; H, 7.59; N, 4.59. Found: C, 67.02; H, 7.42; N, 4.43. 

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Chemical structures of study compounds.

Preparation of Mitochondria and Semipurified MAO-A and -B. Protocols for obtaining tissues were approved by the appropriate committee at the institution of origin. In addition, these were reviewed and approved by the Virginia Tech Animal Care Committee, as were the protocols for all animal procedures performed at Virginia Tech. Receipt of the human tissue was approved by the Virginia Tech Committee on Human Experimentation. Experiments were carried out with mitochondria prepared from brains and livers of male C57BL/6 and ICR mice, Sprague-Dawley rats, New Zealand White rabbits, Beagle dogs, cynomolgus monkeys, and baboons (Papio papio ursinus). For human and bovine preparations, only liver samples were available. The preparation of the mitochondrial fractions followed literature procedures (Salach and Weyler, 1987) except that minor volume changes were made with the brain preparations. Tissue homogenates from individual animals were prepared as follows (dog, n = 2; baboon, n = 3; monkey, n = 2). Pooled tissue homogenates were prepared using the following numbers of animals per pooled preparation (number of pooled preparations): rabbit, n = 2 (2); rat, n = 2 to 10 (3); ICR mice, n = 5 (2); and C57BL/6 mice, n = 3 to 12 (3). Individual human liver homogenates were prepared from tissues obtained from five donors. Data derived from individual animals or humans are indicated in the tables. These preparations were stored in aliquots of 100 to 200 µl at 70°C. The aliquoted samples were thawed as needed and mixed with glycerol-containing buffer [50 mM sodium phosphate buffer, pH 7.4, containing 50% (w/v) glycerol] for the initial protein concentration. All protein concentrations were determined according to the Coomassie Brilliant Blue dye binding method of Bradford (1976) with BSA used as standard. For the assays, these were further diluted using 50 mM sodium phosphate buffer, pH 7.4, to a concentration appropriate for the experiment. Solutions of all inhibitors and substrates also were prepared in the same phosphate buffer. Each assay was run in duplicate, and the results differed by less than 5%.

Optimization of Preincubation Conditions to Inhibit MAO-A or -B Activity by Selective Inhibitors. These studies used the 1-methyl-2-pyrrolyl analog 5 because it is a good substrate for both forms of the enzyme. The stability of the dihydropyridinium metabolite 9, its relatively large molar extinction coefficient ( = 25,000 M¹), and its maximal absorbance at 420 nm, which is far removed from mitochondrial background, made 5 an excellent substrate for these experiments (Flaherty et al., 1996).

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Studies on 2-Isopropylphenyl Analog 7. Compound 7 was examined as a potential MAO-A-selective substrate. Evidence of the instability of the resulting dihydropyridinium metabolite 11, however, led us to attempt to identify the corresponding pyridinium oxidation product 14. A 1-h postincubation mixture of 7 (100 µM) and MAO-A (0.13 µM) in glycerol-containing buffer was treated with NaBD₄ in 500 µl of methanol. The mixture was left at room temperature for an additional hour and then extracted with 1.5 ml of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, filtered, and evaporated under a gentle stream of nitrogen. The residue was reconstituted with 100 µl of methanol, and 1 µl was analyzed by GC (model HP 5890)-mass spectrometry (model HP 5970; GC-MS) using an HP-1 capillary column (12.5 M × 0.2 mm × 0.33 µM). The injection port temperature was 225°C. The GC oven temperature was 60°C for 1 min and then was increased at 25°C/min to 275°C.

Determination of MAO-A/MAO-B (12) in Mitochondrial Preparations. These studies were performed using the 1-methyl-2-pyrrolyl analog 5 as substrate (see above) at 2 mM, a concentration that is well above the K_m value for this compound in all the species examined (see Table 3). The activities of total MAO, MAO-A, or MAO-B were measured after 15-min preincubations of mitochondria with no inhibitor or with 3 × 10⁷ M (R)-deprenyl (12) or 3 × 10⁸ M clorgyline (13), respectively. The activity remaining when both inhibitors were present was defined as the residual activity. When significant, the residual activity was subtracted from the estimated MAO-A and -B activities. The percentages of MAO-A, MAO-B, and residual activities were calculated with the sum of these activities as 100%. In related experiments, mitochondrial preparations were preincubated with the iron chelator deferoxamine (2 × 10⁴ M, final concentration) in the presence or absence of both inhibitors to examine the influence of adventitious iron on the oxidation of the tetrahydropyridinyl substrates (Di Monte et al., 1995).

Determination of Mitochondrial MAO Activity of Selected Tetrahydropyridinyl Substrates. Mitochondrial preparations (500 µl final volume containing 0.15 mg protein/ml for liver samples and 0.3 mg protein/ml for brain samples) were incubated with various concentrations of the tetrahydropyridinyl substrates. In some instances, higher protein concentrations were used to give rates of oxidation that were sufficiently fast to measure with confidence. The incubations were run for 15 to 60 min, depending on the rate of product formation, with gentle agitation in a 37°C water bath. When assaying for ratios of MAO-A/MAO-B activity with the mixed substrates, the 1-methyl-2-pyrrolyl (5) and the 3-ethyl-2-furanyl (6) analogs, the mitochondrial preparations were preincubated in 237.5 µl of the buffer containing either no inhibitor, 3 × 10⁸ M clorgyline, 3 × 10⁷ M (R)-deprenyl, or both inhibitors for 15 min, followed by the addition of 237.5 µl of the substrate solution. In all experiments, the reactions were quenched by the addition of 20 µl of 70% perchloric acid, and the denatured protein was removed by centrifugation at 16,000g for 5 min. The incubation times for each substrate were 45 min for the methoxyphenyl analog 4 and MPTP, 5 to 15 min for the pyrrolyl analog, and 6 to 60 min for the furanyl analog. The _max values and the molar extinction coefficients () of the dihydropyridinium metabolites generated in these MAO-catalyzed reactions were 343 nm and 16,000 M¹ for 2 (Kalgutkar et al., 1994) and 8, 420 nm and 25,000 M¹ for 9 (Bai, 1991), and 400 nm and 23,000 M¹ for 10 (Yu and Castagnoli, 1998). A synthetic standard of 8 was not available, so the  value of 2 was used. Values for V_max and K_m were obtained from Lineweaver-Burk double reciprocal plots of velocities versus substrate concentrations. Duplicate analyses gave V_max/K_m values that differed by 10% or less.

	Results

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Stability of MAO Activity in Mitochondria. MAO-B activities in dog brain and liver were assayed at 0, 1, 2, 4, 6, 9, and 12 months in triplicate. Under the conditions of the present study, MAO-B activities in dog brain and liver were stable over a period of 12 months when stored at 70°C. The ranges for V_max/K_m (mean ± S.E.) given as nmol MPDP⁺ formed/min/mg protein of the activities observed in these studies were 21.0 to 25.7 (23.6 ± 0.69) for brain and 62.2 to 83.0 (69.0 ± 2.77) for liver. Our general observations suggest that the other mitochondrial preparations also were stable under these storage conditions.

Time- and Concentration-Dependent Inhibition of MAO by Clorgyline and (R)-Deprenyl. Estimations of the MAO-A/MAO-B activity ratios required that kinetic measurements be made when one form of the enzyme was selectively and quantitatively inhibited in the presence of the other form. These studies used the pyrrolyl analog 5 (2 mM), a mixed MAO-A/MAO-B substrate.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Time course for the inhibition of the MAO-A-catalyzed oxidation of the pyrrolyl substrate 5 (2 mM) by 3 × 108 M clorgyline in rat liver mitochondrial preparations (0.15 mg protein/ml) preincubated for 15 min with 3 × 107 M (R)-deprenyl (A) and that of the corresponding MAO-B-catalyzed reaction by 3 × 107 M (R)-deprenyl in mitochondria preincubated with 3 × 108 M clorgyline (B). In both plots, the 5% residual activity observed when both inhibitors were present was subtracted.

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View larger version (18K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent inhibition by clorgyline (A) and by (R)-deprenyl (B) of the MAO-catalyzed oxidation of the pyrrolyl substrate 5 (1 mM) in rat liver mitochondrial preparations (0.15 mg protein/ml). Mitochondria were preincubated with the inhibitors for 15 min at 37°C before assay for remaining activity.

Linearity of Production of Metabolites with Time. The linearity of the rates of dihydropyridinium metabolite formation in these mitochondrial preparations was examined to determine whether the kinetic measurements were being made under steady-state conditions. Figure 4 illustrates the time-dependent increase in the concentration of MPDP⁺ (2) from MPTP (1) by human liver mitochondria. The increase in absorption at _max 343 nm with time was reasonably linear (r² = 0.995) for at least 50 min. Because no changes in the shape of absorption curves were observed during this period of time, we concluded that MPDP⁺ is stable under these conditions. Similar results were observed with the other substrates (4-6) used in this study. This was not the case, however, with the 2-isopropylphenyl analog 7, a reported MAO-A-selective substrate (Singer and Ramsay, 1991), as discussed below.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Time-dependent increase in the dihydropyridinium metabolite MPDP+ (2) formed from 200 µM MPTP (1) by human liver mitochondria (0.15 mg protein/ml). After subtraction of the background absorption of the mixture, the spectrum were recorded every 5 min for 50 min at 37°C.

Attempts to Develop an MAO-A-Selective Substrate. MPTP and its 3-methoxy analog 4 are both highly selective MAO-B substrates. In the C57BL/6 mouse brain preparation, which contains about 15% MAO-A activity by our estimates, the V_max/K_m values for MPTP in the presence of 3.3 × 10⁸ M clorgyline (38.0 nmol product formed/min-mg protein) and in the absence of clorgyline (37.8 nmol product formed/min-mg protein) were essentially the same. The corresponding values for the 3-methoxy analog 4 using human liver mitochondria (18% mean MAO-A activity) were 99.7 nmol/min-mg protein in the presence of 3.3 × 10⁷ M clorgyline and 97.3 nmol/min-mg protein in the absence of clorgyline. Consequently, it was possible to assay MAO-B activity without inhibiting MAO-A.

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Time-dependent oxidation of 1-methyl-4-(2-isopropylphenyl)-1,2,3,6-tetrahydropyridine (7) by MAO-A. Semipurified MAO-A (20 nM) was mixed with the substrate (1 mM) in a quartz cell. A UV-VIS spectrum of the mixture was immediately recorded as blank. Then, after subtraction of the blank, the production of the metabolite was measured every 3 min for 30 min at 37°C. Note the shift in max with time corresponding to the conversion of the initially formed dihydropyridinium metabolite 11 to the corresponding pyridinium compound 14.

View larger version (7K): [in this window] [in a new window]   Fig. 6.   The metabolic fate of 1-methyl-4-(2-isopropylphenyl)-1,2,3,6-tetrahydropyridine (7) and subsequent oxidation of the resulting dihydropyridinium metabolite 11 to form the pyridinium species 14.

Species Differences in MAO-A and -B Catalytic Activity. Studies to estimate the MAO-A and -B activities in the tissues of interest used the pyrrolyl substrate 5, a mixed A/B substrate, and the selective MAO-B inhibitor (R)-deprenyl (12) to estimate MAO-A activity and the selective MAO-A inhibitor clorgyline (13) to estimate MAO-B activity. Figure 7 summarizes the results obtained with dog brain mitochondria and are typical of the quality of the data generated in these studies. The results obtained with all of the tissues examined are summarized in Table 1 numerically and in Fig. 8 graphically. These results were obtained with a saturating concentration of the substrate. Estimates of V_max and K_m also were obtained for 5 and the second mixed substrate, the furanyl analog 6 (Table 2). In general, all of the catalytic activity could be accounted for in terms of the A and B forms of the enzyme. The brain and liver mitochondrial preparations isolated from the rabbit were exceptional in that a high percentage of "residual" oxidase activity was present when both MAO-A and -B were inhibited as illustrated in Fig. 9 for the liver preparation.

View larger version (27K): [in this window] [in a new window]   Fig. 7.   UV-VIS scans of incubation mixtures containing the 1-methyl-2-pyrrolyl analog 5 (2 mM) in the presence of mitochondrial preparations from dog brain with and without inhibitors. Mitochondrial preparation (0.3 mg protein/ml at final concentration) were preincubated with either no inhibitor, 3 × 108 M clorgyline (Clo), 3 × 107 (R)-deprenyl (Dep), or clorgyline plus (R)-deprenyl for 15 min at 37°C, following which 2 mM concentration of the 1-methyl-2-pyrrolyl analog in phosphate buffer was added. The incubation was continued for an additional 15 min, and the reaction was quenched by the addition of perchloric acid.

View this table: [in this window] [in a new window]   TABLE 1 MAO-A and MAO-B activities observed with various mitochondrial preparations as estimated by the rates of oxidation of the MAO-A/MAO-B mixed substrate 5 after selective inhibition of each form of the enzyme Because we do not know the actual concentration of MAO-A and MAO-B in the mitochondrial preparations, the reported rates of substrate oxidations are expressed as nmol product formed/min/mg protein. Therefore, these comparisons do not necessarily reflect the relative abundances of these enzymes. Total activities were measured after preincubation of mitochondria with no inhibitor. MAO-A and MAO-B activities were measured after preincubation with 3 × 107 M (R)-deprenyl and 3 × 108 M clorgyline, respectively. The percentages of MAO-A, MAO-B, and residual activities were calculated using the sum of these activities as 100%.

View larger version (78K): [in this window] [in a new window]   Fig. 8.   Percent brain and liver MAO-A, MAO-B, and residual activity observed in the mitochondrial preparations of various species. Technical details were as described in Table 1.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   UV-VIS scans of incubation mixtures containing the 1-methyl-2-pyrrolyl analog 5 (2 mM at final concentration) in the presence of mitochondrial preparations from rabbit liver (0.15 mg protein/ml at final concentration) with and without inhibitors. Technical details were as described in Fig. 7.

Species-Dependent MAO Kinetic Profiles Observed with Various Tetrahydropyridinyl Substrates. A second major thrust of these studies has been to focus on the characterization of the species-dependent differences in liver and brain mitochondrial MAO activity as defined by the kinetic behavior of a panel of tetrahydropyridinyl substrates. In these experiments, we measured total enzyme activity for all four substrates. The results reported in Table 3 were obtained by estimates of reaction rates using substrate concentrations that bracketed their apparent K_m values. The V_max and K_m values (both of which are listed) were determined from Lineweaver-Burke plots; included is the range of the ratios of V_max/K_m.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Species Differences in MAO-A and -B Catalytic Activity. One of the principal goals of this study was to characterize the MAO-A and -B activities present in the mitochondrial preparations obtained from the species under study. Several measurements of the levels of MAO-A and -B activity using radiographic-based enzyme activities (Saura et al., 1992) have appeared in the literature. Results from a specific inhibitor binding assay indicate that up to 80% of the total MAO activity in human liver is due to MAO-B (Saura et al., 1996). Histochemical studies have shown that MAO activity in the marmoset brain is essentially all due to MAO-B (Willoughby et al., 1988). Similar results have been reported for other subhuman primates by Riachi and Harik (1992), who used a [³H]pargyline binding assay to measure MAO activity. In contrast to the above results, Murphy et al. (1979), who estimated activity by the extent to which clorgyline and (R)-deprenyl inhibited tyramine deamination, reported up to 30% MAO-A activity in various regions of the vervet monkey brain.

Species-Dependent MAO Kinetic Profiles Observed with Various Tetrahydropyridinyl Substrates. An inspection of Table 3 leads to several observations. Liver activity was higher than brain activity in all animals examined except for the C57BL/6 mouse, the only animal with a liver/brain ratio of V_max/K_m of less than 1 (range, 0.3-0.7). The particularly low value of V_max/K_m for the oxidation of MPTP by liver mitochondria is principally due to the high K_m value (520 µM) for this substrate. It may be reasonable to speculate that the increased susceptibility of the C57BL/6 mouse to the neurotoxic effects of MPTP compared with other rodents may be due in part to the limited systemic detoxification of this compound by liver MAO-B.

Summary. Perhaps the most significant finding of these comparative studies is the greater similarities in the MAO activity profiles between humans and rodents (particularly the rat) than that between humans and subhuman primates. The livers obtained from baboons and monkeys were essentially devoid of MAO-A activity, whereas the human and rat preparations were relatively rich in MAO-A activity, although, as with all tissues examined, MAO-B also was the dominant enzyme present in these species. A comparison of the substrate profiles among the various species documented many similarities. The ratios of V_max/K_m were higher in the liver (on a per-mg protein basis) than in the brain in all species except the C57BL/6 strain of mouse. This difference was not due to lower K_m values for the liver enzymes. The factors contributing to these tissue-dependent differences in activity remain to be identified. Similar studies with other MAO-containing tissues (gut, heart, lung, platelet) would help to identify potentially important tissue- and species-dependent differences in the interactions of xenobiotics with these flavoenzymes. The relatively weak MAO-B activity observed in the liver mitochondria isolated from the C57BL/6 mouse, which is due primarily to the very high K_m value for the liver enzyme, opens the possibility of limited systemic detoxification of MAO-B substrates in this strain of mouse. Other significant differences in MAO activity identified by these studies include the unusually low MAO-B activity displayed by brain and liver mitochondrial preparations isolated from the New Zealand White rabbit. These low activities were coupled with a resistance to inactivation by (R)-deprenyl. At the other end of the spectrum was the exceptionally robust activity of the bovine liver preparations, which reflects primarily the high V_max values for the substrates examined. Overall, the results of this effort should form the basis for future studies that will help to further identify the tissue- and species-dependent interactions of xenobiotics with MAO-A and -B.