Introduction

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Monoamine oxidase (MAO; monoamine oxido-reductase, EC 1.4.3.4) is an FAD-dependent enzyme responsible for the oxidative deamination of biogenic amines that have a neurotransmitter function in the central nervous system, and of other xenobiotic amines.

The existence of two isoforms, MAO-A and MAO-B, according to pharmacological criteria (Johnston, 1968) was subsequently confirmed by sequencing and cloning studies (Bach et al., 1988). Both isoforms present a 70% degree of homology in their primary structure and are coded by two different genes located on the X chromosome. In terms of substrate specificity, it is well established that MAO-A preferentially deaminates serotonin and noradrenaline (NA), and is sensitive to inhibition by nanomolar concentration of clorgyline, whereas MAO-B deaminates phenylethylamine (PEA) and benzylamine, and is inhibited by a nanomolar concentration of l-deprenyl. Dopamine (DA) and tyramine are thought to be oxidized by both MAO isoforms.

Low levels of serotoninergic and noradrenergic transmission, correlated with depression, make selective MAO-A inhibitors potential therapeutic agents to be used in the treatment of this affective disorder (Tipton, 1989). It is worth pointing out that the excess of H₂O₂ generated by a MAO-B increase under certain circumstances, such as aging, Alzheimer dementia or increased DA turnover in Parkinsonism could contribute to oxidative stress and the subsequent neuronal death. In this context, MAO-B inhibitors could be used to delay the progressive degeneration of specific neural circuits affected in these neurological diseases.

With the aim of identifying the most potent and selective novel MAO inhibitors to be used in the therapy of neurological and affective disorders, a series of indolealkylamine derivatives has been designed and evaluated biologically and labeled as MAO-A and MAO-B inhibitors (Balsa et al., 1990, 1994; Avila et al., 1993; Pérez et al., 1996; Morón et al., 1998). The corresponding structure-activity relationship studies allow us to conclude that the introduction of an OH group at position 5 of the indole ring is determinant for the increasing selectivity toward MAO-A inhibition, whereas the introduction of a bulky group at the same position, such as the benzyloxy group, is determinant for changing selectivity toward MAO-B inhibition (Perez et al., 1999). Among these novel compounds, the 5-hydroxy-indolealkylamine derivative has been selected (Fig. 1), and its potency and selectivity as an MAO-A inhibitor have been evaluated in vitro and ex vivo.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Molecular structure of the 5-hydroxy-indolealkylamine derivative FA70.

The ex vivo effect of FA70 after acute and chronic administration on the amine metabolism in mouse cerebral cortex also has been studied to elucidate the role of both MAO isoforms on aminergic metabolism. To understand the DA content after these treatments, the involvement of tyrosine hydroxylase has been determined by protein expression studies. That no alteration in the tyrosine hydroxylase expression was observed after chronic and acute treatment allows us to postulate a possible modulatory effect on the tyrosine hydroxylase enzyme induced by this highly selective MAO-A inhibitor.

	Materials and Methods

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Animals. Male Sprague-Dawley rats weighing between 200 and 250 g were used for in vitro MAO-activity determination. Male OF1 mice weighing between 20 and 30 g were used for the ex vivo study of MAO and tyrosine hydroxylase activities, and for the quantification of biogenic amines and metabolites. All animals were housed in a controlled environment (light/dark cycles of 12 h and temperature of 21°C) with food and water ad libitum.

Chemicals and Drugs. FA70 was synthesized by Cruces et al. (1988) at the Instituto de Química Orgánica, Consejo Superior de Investigaciones Cientificas, Madrid, Spain. [¹⁴C]5-hydroxytryptamine (5-HT) creatinine sulfate (55 mCi/mmol; 50 µCi/ml) was purchased from Amersham (Arlington Heights, IL) and [¹⁴C]PEA (50 mCi/mmol; 0.1 mCi/ml) was purchased from New England Nuclear (Boston, MA). Kynuramine dihydrobromide, benzylamine HCl, DA, NA, 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyramine (3-MT), 5-hydroxyindole acetic acid (5-HIAA), 5-HT, 3,4-dyhydroxybenzylamine (DHBA), N-methyl-5-HT, L-tyrosine, alumina (Al₂O₃, activity grade 1), and clorgyline hydrochloride were obtained from Sigma Chemical Co. (St. Louis, MO) l-Deprenyl was obtained from Research Biochemicals (Natick, MA). Mouse monoclonal antityrosine hydroxylase antibody was purchased from Incstar Co. (Stillwater, MN), whereas horseradish peroxidase-conjugated goat anti-mouse IgG was obtained from Sigma. High-molecular-mass standard proteins for SDS gels were obtained from Bio-Rad (Richmond, CA).

Drug Treatments. FA70 and clorgyline were dissolved in 9% (w/v) saline solution and administered by the i.p. route. Doses are always expressed in milligrams per kilogram body weight. For the study of the effect of chronic versus acute treatment with FA70 and clorgyline, mice were divided into three groups: chronic, acute, and control. The chronic group received daily 5 mg/kg FA70 or clorgyline during 21 days; the acute group received 20 days' treatment with an equivalent volume of saline on a daily basis and 5 mg/kg FA70/clorgyline on day 21. The control group received saline solution during 21 days.

Determination of Kinetic Parameters of MAO Inhibition In Vitro. These studies were performed in rat-liver mitochondrial fractions. Rat-liver homogenates were prepared in 10 volumes of ice-cold 0.05 mM potassium phosphate buffer (pH 7.2). The mitochondrial fraction was prepared by a standard differential centrifugation method (Gómez et al., 1986). The pellets were resuspended in the same buffer and frozen as small aliquots at 20°C until required. Kinetic parameters of MAO inhibition were determined spectrophotometrically. Determination of MAO-B activity was performed at 37°C with 333 µM benzylamine as substrate by measuring the appearance of benzaldehyde as the final product at 250 nm by a modification (Avila et al., 1993) of the Tabor method (Tabor et al., 1954). This modification allowed the sequential determination of six samples with the corresponding blank. MAO-A activity was determined with 40 µM kynuramine as a substrate (Weissbach et al., 1960) by measuring the appearance of the product at 324 nm (Avila et al., 1993). Because kynuramine is a common substrate of both MAO forms, to make sure that only MAO A activity was present, it was necessary to inhibit MAO-B activity beforehand with 3.10⁷ l-deprenyl.

Determination of MAO Activity Ex Vivo. Mice were treated i.p. with FA70 or clorgyline and were decapitated at specified times after treatment. Brain cortex and liver were dissected out and frozen rapidly. The samples were kept at 80°C until MAO-A and MAO-B assays. The tissues were homogenized in 10 volumes of ice-cold 0.05 M potassium phosphate buffer (pH 7.2). MAO activities were then determined radiochemically in triplicate. MAO-A and MAO-B activities were assayed as described previously by Fowler and Tipton (1981). Briefly, aliquots (0.05 ml) of the homogenate were incubated with [¹⁴C]5-HT (final concentration, 100 µM) for 15 min and with [¹⁴C]PEA (final concentration, 20 µM) for 4 min in a total volume of 225 µl at 37°C. The product formation was lineal during both periods of time. The reaction was stopped with 100 µl of 2 M citric acid and deaminated metabolites were extracted by vigorous shaking with 4 ml of toluene-ethyl acetate (v/v) containing 0.6% (w/v) 2,5-diphenyloxazol. After extraction, the aqueous phase was frozen and the organic layer was poured into a scintillation vial. Radioactivity was measured in a scintillation counter (LKB; Wallac, Gaithersburg, MD). Blank values were obtained by adding citric acid before adding the substrate. ED₅₀ values were determined from the log dose-inhibition plots. MAO activity was expressed as a percentage of control ± S.E.M.

Determination of Levels of Biogenic Amines and Their Major Metabolites in Mouse Cortex. Mice were treated i.p. with FA70 or clorgyline and decapitated 24 h after last administration. Cortex samples were rapidly dissected out and frozen in liquid N₂ to determine the levels of 5-HT, 5-HIAA, DA, DOPAC, HVA, 3-MT, and NA with an HPLC system with electrochemical detection following a procedure mainly based on the method described by Pastó and Sabrià (1990). Briefly, samples were homogenized in 10 volumes of 0.25 M HClO₄ containing 0.25 mM disodium EDTA, 0.1 mM sodium metabisulphite, 1000 ng/ml of 3,4-dihydroxybenzylamine, and 100 ng/ml of N-methyl-5-HT as the internal standards for catecholamine and indolealkylamine, respectively. The homogenate was stored at 80°C overnight. At the time of analysis, samples were spun in an Eppendorf microcentrifuge for 10 min, and 20 µl of the supernatants was injected directly into the HPLC system. Analyses were performed at room temperature (20-25°C). The mobile phase consisted of 0.1 M citric acid, 0.05 mM disodium EDTA_, 1.2 mM sodium octyl sulfate, and triethylamine to adjust pH to 2.65. Acetonitrile was added to reach 1.0 to 1.5% (v/v). Elution was performed at a flow rate of 1 ml/min.

Determination of Tyrosine Hydroxylase Activity. Mice were treated i.p. with FA70 or clorgyline and decapitated 24 h after the last administration. Their brain cortices were removed and rapidly homogenized in 1 M sodium acetate buffer (pH 6.0). Tyrosine hydroxylase activity was assayed as described by Cho et al. (1996) with some modifications. Briefly, aliquots of the homogenate were added to the incubation mixture containing 1 M sodium acetate buffer (pH 6.0), 1 mM 6-methyl-5,6,7,8-tetrahydropteridine, 0.1 mg/ml catalase, and 0.4 mM L-tyrosine. After 15 min of incubation at 37°C, the reaction was terminated by adding 0.4 M HClO₄. The mixture was centrifuged and the supernatant was added to alumina columns to isolate the L-dopa accumulated. After two washings with 0.5 M Tris-HCl buffer (pH 8.6), the L-dopa was eluted with 0.25 M of HClO₄. The eluate was centrifuged and the supernatant was injected into the HPLC-electrochemical detection system described above.

Quantification of Tyrosine Hydroxylase Protein. Mice were treated i.p. with FA70 or clorgyline and were decapitated 24 h after the final administration. Brain cortices were dissected out and homogenized in 10 volumes of ice-cold 0.05 M potassium phosphate buffer (pH 7.2). Homogenates were diluted 1:10 (v/v) in Laemli sample buffer and boiled for 5 min. Proteins were separated by electrophoresis in 10% SDS polyacrylamide gel, in parallel with molecular-mass standard proteins. Gels were transferred to 0.45-µm nitrocellulose membranes, and blots were incubated at 4°C overnight in Tris-buffered saline-Tween (20 mM Tris, 0.15 M NaCl, 0.1% Tween-20) containing 5% (w/v) dry milk as a blocking agent. Blots were sequentially incubated for 90 min at room temperature with mouse monoclonal, anti-tyrosine hydroxylase and horseradish peroxidase-conjugated anti-mouse IgG antibodies, with extensive washing in Tris-buffered saline-Tween after incubation with each antibody. The bound antibody was detected by incubation with a solution containing H₂O₂ and diaminobenzidine. After color development, the membrane was washed out with distilled water, air dried, and photographed. The intensity of the bands was determined by using a densitometer, and optical density was calculated per milligram of protein. The statistical significance of the data was evaluated by one-way ANOVA.

	Results

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Effect of FA70 on MAO Activity in Rat-Liver Mitochondrial Fraction In Vitro. In rat mitochondrial fraction, FA70 behaved as an irreversible, time-dependent and mechanism-based inhibitor toward both MAO forms (data not shown). Table 1 shows the kinetic inhibition parameters of FA70 and clorgyline for comparative purposes. FA70 showed 1544 times more affinity toward MAO-A than toward MAO-B. Compared with clorgyline, as an example of a potent and selective MAO-A irreversible inhibitor, FA70 showed five times more affinity toward MAO-A. However, the selectivity of FA70 toward MAO-A was slightly lower than that observed toward clorgyline. The velocity constants (k_inact) were similar for both MAO inhibitors and no differences were observed between either MAO isoform.

Ex Vivo Effect of FA70 on MAO Activity in Mouse Cortex and Liver. As shown in Fig. 2, FA70, when given at the 10-mg/kg i.p. dose, induced a maximal inhibition of brain cortex MAO-A (95%) as early as 30 min after its administration without affecting MAO-B activity. This inhibition remained up to 24 h after administration, exhibiting a typical profile of an irreversible MAO inhibitor. Under similar experimental conditions, the time course of MAO-A inhibition by clorgyline (10 mg/kg i.p.) in mouse cortex was comparable to that of FA70. For MAO-B activity, clorgyline, at the dose of 10 mg/kg i.p., caused a partial inhibition (15%) of this enzymatic form. In contrast to the cortex, MAO-A activity was only partially inhibited (30%) in liver after treatment with FA70 at a dose of 10 mg/kg i.p. At this dose, clorgyline produced 85 and 10% inhibition of MAO-A and MAO-B, respectively, in this tissue. No recovery of MAO activity was observed in liver after 24 h of treatment with either FA70 or clorgyline.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Time course of the effects of FA70/clorgyline administration (10 mg/kg i.p.) on the ex vivo deamination of 5-HT (MAO-A) and PEA (MAO-B) in mouse cortex and liver. The results are expressed as a percentage of respective control values. Each point represents the mean value ± S.E.; n = 6-10 animals. Control activities (picomoles per minute · milligram protein) were cortex (5-HT) 794 ± 10 and (PEA) 480 ± 8; liver (5-HT) 205 ± 5 and (PEA) 1140 ± 25. *P < .001 compared with control. FA70 (5-HT), ; FA70 (PEA), ; clorgyline (5-HT), ; clorgyline (PEA), .

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effects of the administration of graded doses of FA70/clorgyline (0.125-10 mg/kg i.p.) on the ex vivo deamination of 5-HT (MAO-A) and PEA (MAO-B) in mouse cortex and liver. Animals were sacrificed 2 h after the administration of the drugs. The results are expressed as a percentage of respective control values. Each point represents the mean value ± S.E.; n = 6-10 animals. Control activities (picomoles per minute · milligram protein) were cortex (5-HT) 794 ± 10 and (PEA) 480 ± 8; liver (5-HT) 205 ± 5 and (PEA) 1140 ± 25. *P < .001 compared with control. FA70 (5-HT), ; FA70 (PEA), ; clorgyline (5-HT), ; clorgyline (PEA), .

Ex Vivo Effect of FA70 on Level of Monoamines and Their Metabolites in Mouse Cortex. As shown in Fig. 4, acute treatment with FA70 (5 mg/kg i.p.), 24 h before sacrifice, induced significant increases in cortical concentrations of 5-HT (51 ± 2%) and NA (33 ± 3%), with concomitant decreases in 5-HIAA (47 ± 6%), DOPAC (76 ± 2%), and HVA (45 ± 3%) levels. 3-MT levels increased significantly (129 ± 5%), whereas DA levels remained unaltered compared with controls.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of chronic and acute administration of FA70 (5 mg/kg i.p.) on mouse cortical concentrations of NA, DOPAC, DA, 5-HIAA, HVA, 3-MT, and 5-HT. Animals were sacrificed 24 h after the last administration. The results are expressed as a percentage of variation versus respective controls. Each point represents the mean value ± S.E.; n = 6-10 animals. Control levels (picomoles per milligram tissue): NA, 298 ± 12; DOPAC, 113 ± 9; DA, 913 ± 60; 5-HIAA, 211 ± 15; HVA, 159 ± 6; 3-MT, 41 ± 5; and 5-HT, 340 ± 12. *P < .001 compared with control, +P < .01 compared with acute treatment. , control; , acute; , chronic.

Effect of FA70 on Tyrosine Hydroxylase Activity in Mouse Cortex. The results in Fig. 5 show that acute treatment (5 mg/kg i.p.) with FA70, 24 h before sacrifice, produced a significant increase (60 ± 3%) in tyrosine hydroxylase activity in the cortex. This effect was significantly more pronounced (114 ± 4%) after repeated i.p. administration of FA70 (5 mg/kg i.p.) for 21 days than that obtained after acute treatment. Intraperitoneal administration of clorgyline induced similar effects on tyrosine hydroxylase activity in the cortex when given acutely (5 mg/kg i.p.), 24 h before sacrifice, or chronically (5 mg/kg i.p.) for 21 days (Fig. 5). The increase in tyrosine hydroxylase observed after both treatments with clorgyline differed significantly, in the same way as that of FA70.

View larger version (27K): [in this window] [in a new window]   Fig. 5.   Effects of chronic and acute administration of FA70/clorgyline (5 mg/kg i.p.) on tyrosine hydroxylase activity in mouse cortex. Animals were sacrificed 24 h after the last administration. The results are expressed as percentage of variation versus respective controls. Each point represents the mean value ± S.E.; n = 6-10 animals. Control activity (picograms of L-dopa per minute · milligram protein): 0.47 ± 0.04. *P < .01 compared with control, +P < .01 compared with acute treatment. , control; , acute; , chronic.

Effect of FA70 on Tyrosine Hydroxylase Protein Expression in Mouse Cortex. Acute i.p. administration of FA70 (5 mg/kg i.p.) did not have any effect on the quantity of tyrosine hydroxylase protein in the cortex as can be seen from the Western blot obtained after incubation with monoclonal tyrosine hydroxylase antibody (Table 4). Similarly, chronic i.p. administration of FA70 (5 mg/kg i.p.) for 21 days did not show significant differences in tyrosine hydroxylase protein compared with control. As shown in Table 4, no significant differences were observed after acute and chronic i.p. treatment with clorgyline (5 mg/kg i.p.) in tyrosine hydroxylase protein in the cortex.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

This study shows that FA70, a 5-hydroxy-indolealkylamine derivative (Fig. 1) is a new, irreversible, and highly selective MAO-A inhibitor. It behaves in vitro as a mechanism-based inhibitor of both MAO isoforms, and it has proved to be a more potent MAO-A inhibitor than clorgyline. These results allow us to conclude that the introduction of an OH group at position 5 of the indole ring is determinant to significantly increase selectivity toward MAO-A when making comparisons with other similar indolealkylamine derivatives that present an H- (FA26) or a CH₃O group (FA 42) at this position (Balsa et al., 1990, 1994; Pérez et al., 1999).

The ex vivo effect of FA70 in mouse cerebral cortex after i.p. administration shows high selectivity toward MAO-A, due to the total inhibition observed, whereas MAO-B activity remained unaltered. However, clorgyline partially inhibited MAO-B under the same experimental conditions. These data are in agreement with those observed in vitro and allow us to conclude that FA70 is a novel, irreversible MAO-A inhibitor that acts ex vivo in mouse cerebral cortex in a more potent and selective way than clorgyline.

The ex vivo effect of FA70 on the liver showed a different inhibitory behavior compared with brain. This difference must be explained under the pharmacokinetic perspective. FA70 administered i.p. would reach the liver first and after the interaction with MAO-A, the corresponding metabolite would result in a more active molecule than the parent compound. This should be reflected in the amount of the active material reaching and entering the brain. Thus, MAO-A present in brain would be inhibited to a greater extent than MAO-A present in liver. However, the ex vivo inhibitory effect of clorgyline was similar in both tissues, indicating that this MAO-A inhibitor is not metabolized into a more active product.

These results lead us to assert that FA70 behaves as a more active MAO-A inhibitor in mouse cerebral cortex than in the liver. In this context it would be assumed that FA 70 could act as a prodrug that renders the active inhibitor of MAO-A in the brain after its hepatic metabolism.

The acute and chronic effect of FA70 on MAO activities were studied in mouse cerebral cortex, and no differences were found between either treatment. MAO-A was totally inhibited, whereas MAO-B was not altered; however, with clorgyline this activity was affected and, furthermore, displaying less selectivity than FA70.

It has been suggested that the repetitive administration of irreversible inhibitors increases enzyme inhibition (Felner and Waldmeier, 1979; Finberg et al., 1995). Nevertheless, the chronic treatment by FA70 did not increase MAO-A inhibition and could be explained because the inhibitor concentration administered rendered maximum enzyme inhibition.

The chronic and acute effects of FA70 on amine metabolism in mouse cortex also was studied. It has been described that MAO-A is the main MAO isoform responsible for amine metabolism in the rat striatum (Butcher et al., 1990; Lamensdorf et al., 1996). However, Wolf et al. (1985) and Curet et al. (1996) have suggested that MAO-B could play an important role in brain amine metabolism when MAO-A is inhibited.

When FA70 was administered in acute form, a decrease in the amine metabolites such as DOPAC, 5HIAA, and HVA confirmed MAO-A as the main responsible enzyme of DA, NA, and 5-HT metabolism. The observed increase of 3-MT would confirm that once MAO-A was inhibited, the alternative metabolic pathway of DA by catechol-O-methyl-transferase activity is working. Nevertheless, that a constant DA level remained during acute treatment is not completely justified and requires further clarification.

To assess that MAO-A was the main responsible enzyme of amine metabolism in mouse cortex, a repeated acute administration of l-deprenyl, as the selective MAO-B inhibitor, was performed and the amines and metabolites determined. No variation in the amine and metabolites content was observed compared with control. These results are in agreement with those reported by Butcher et al. (1990), Lamensdorf et al. (1996), and Molinengo and Ghi (1997). The present data assessed definitively that MAO-B is not involved in the amine metabolism in mouse cortex. Nevertheless, to discard whether acute FA70 effect could be potentiated by simultaneously inhibiting MAO-B, as has been suggested to happen in rat striatum by Schoepp and Azzaro (1983) and Wolf et al. (1985), the effect of acute administration of both inhibitors simultaneously was studied. This experiment rendered the same results as those observed when FA70 was administered separately. These results definitively confirm that MAO-A was the main responsible enzyme of amine metabolism in mouse cerebral cortex. The deamination of a given substrate by both MAO forms depends on the relative concentration of each MAO isoform present in the brain regions. Thus, the differences observed between both studies could be explained in terms of the different animal species and the brain regions used.

When the chronic effect of FA70 on amine metabolism was studied, no differences were found between acute and chronic treatments for NA, 5-HT, and 5-HIAA content, whereas DA metabolites DOPAC, HVA, and 3-MT showed significant differences among themselves. The constant level of DA and the great increase of 3-MT could be attributed to an increase in the DA synthesis, via stimulation of tyrosine hydroxylase by FA70. This activity was determined after chronic and acute FA70 administration and showed a significant increase in both cases. The same results were observed when clorgyline was assayed. Tyrosine hydroxylase is the limiting step of catecholamine synthesis, and in this context the same effect would be expected for NA, but it was not the case. However, it has been described that chronic treatment with clorgyline decreases DA -hydroxylase activity (Lerner et al., 1979, 1980), and this might be the reason that an increase in NA content was not observed.

To elucidate whether the increasing effect of FA70 on tyrosine hydroxylase has a direct effect on the activity or whether this stimulation is the result of an inductory effect at the transcriptional level, the expression of the enzyme was determined. No differences were observed when using specific tyrosine hydroxylase antibodies and Western blot techniques in the intensity of the 60-kDA band corresponding to the subunit of the tetrameric tyrosine hydroxylase enzyme (Kumer and Vrana, 1996). These results confirmed that the increase of the activity could be a direct effect of FA70 on the enzyme and not provoked by an inductory effect on protein synthesis.

Some DA receptors show a regulatory effect on tyrosine hydroxylase present in dopaminergic neurons (Roth et al., 1987), especially those belonging to the D₂ family (O'Hara et al., 1996; Cho et al., 1997). These articles have reported that the activation of such receptors in the presence of specific agonists diminished tyrosine hydroxylase activity. This effect would be due through the activation of G protein, which inhibits adenylate cyclase (Tang et al., 1994; O'Hara et al., 1996), diminishing cAMP and cAMP-protein kinase dependence, and producing inactivation of tyrosine hydroxylase by lack of phosphorylation.

The regulatory effect of tyrosine hydroxylase via D₂ receptors has been widely described in rat striatum, but few reports have been published on the cerebral cortex. These results suggest that the activation of tyrosine hydroxylase observed in mouse cerebral cortex after acute and chronic treatment with FA70 and clorgyline could be related to the presence of D₂ receptors in this tissue demonstrated by in situ hybridization studies (Tarazi et al., 1997).

Previous work has indicated that the chronic administration of MAO inhibitors diminished the D₂ receptor concentration (Paetsch and Greenshaw, 1993; Martin et al., 1995). In this context, it has been reported that clorgyline inhibited the binding of the quinpirole, an agonist of D₂ receptors (Levant and Bancroft, 1998).

The activation effect of tyrosine hydroxylase by clorgyline and FA70 reported in this study could be explained by a decreasing D₂ receptor-concentration effect or by an induced change in its functionality. It has been described by Ebmeier and Ebert (1996) that an MAO inhibitor treatment induces a change in the D₂ receptor conformation from the high-affinity to the low-affinity state and, consequently, affecting tyrosine hydroxylase activity. Because the high increase of tyrosine hydroxylase activity did not correlate with an increase of the corresponding protein expression, these results would be in agreement with the existence of a pool of inactive enzymes that becomes activated under situations in which a catecholamine increase would be needed (Okuno and Fujisawa, 1991; Kumer and Vrana, 1996).

These results show that acute and chronic treatment with the novel, irreversible, and highly selective MAO-A inhibitor FA70 produces an alteration of endogenous amine levels, especially those related to DA, in mouse cerebral cortex. This effect involves a stimulation of tyrosine hydroxylase activity probably via D₂ receptors. More work must be done to definitively elucidate this mechanism.

These results widely described in rat striatum and to a lesser extent, in the cerebral cortex, could be of interest with regard to the therapy of affective disorders, because some depressive dysfunctions associated with a decrease of tyrosine hydroxylase activity in the cerebral cortex (Charlton, 1997) have been observed.