The Effects of Phenelzine and Other Monoamine Oxidase Inhibitor

Home , Iproniazid, Isocarboxazid, Moclobemide, Monoamine oxidase A

British Journal of Phammcology (1995) 114. 837-845 B 1995 Stockton Press All rights reserved 0007-1188/95 $9.00 The effects of phenelzine and other monoamine oxidase inhibitor antidepressants on brain and liver 12 imidazoline-preferring receptors Regina Alemany, Gabriel Olmos & 'Jesu's A. Garcia-Sevilla

Laboratory of Neuropharmacology, Department of Fundamental Biology and Health Sciences, University of the Balearic Islands, E-07071 Palma de Mallorca, Spain 1 The binding of [3H]-idazoxan in the presence of 106 M (-)-adrenaline was used to quantitate 12 imidazoline-preferring receptors in the rat brain and liver after chronic treatment with various irreversible and reversible monoamine oxidase (MAO) inhibitors. 2 Chronic treatment (7-14 days) with the irreversible MAO inhibitors, phenelzine (1-20 mg kg-', i.p.), isocarboxazid (10 mg kg-', i.p.), clorgyline (3 mg kg-', i.p.) and tranylcypromine (10mg kg-', i.p.) markedly decreased (21-71%) the density of 12 imidazoline-preferring receptors in the rat brain and liver. In contrast, chronic treatment (7 days) with the reversible MAO-A inhibitors, moclobemide (1 and 10 mg kg-', i.p.) or chlordimeform (10 mg kg-', i.p.) or with the reversible MAO-B inhibitor Ro 16-6491 (1 and 10 mg kg-', i.p.) did not alter the density of 12 imidazoline-preferring receptors in the rat brain and liver; except for the higher dose of Ro 16-6491 which only decreased the density of these putative receptors in the liver (38%). 3 In vitro, phenelzine, clorgyline, 3-phenylpropargylamine, tranylcypromine and chlordimeform displaced the binding of [3H]-idazoxan to brain and liver I2 imidazoline-preferring receptors from two distinct binding sites. Phenelzine, 3-phenylpropargylamine and tranylcypromine displayed moderate affinity (KiH = 0.3-6 JLM) for brain and liver I2 imidazoline-preferring receptors; whereas chlordimeform displayed high affinity (KiH = 6 nM) for these receptors in the two tissues studied, Clorgyline displayed very high affinity for rat brain (KiH = 40 pM) but not for rat liver I2 imidazoline-preferring receptors (KiH = 169 nM). 4 Preincubation of cortical or liver membranes with phenelzine (10-4 M for 30 min) did not alter the total density of I2 imidazoline-preferring receptors, indicating that this irreversible MAO inhibitor does not irreversibly bind to I2 imidazoline-preferring receptors. In contrast, preincubation with 10-6 M clorgyline reduced by 40% the Bmax of [3H]-idazoxan to brain and liver I2 imidazoline-preferring receptors. 5 Chronic treatment (7 days) with the inducers of cytochrome P-450 enzymes phenobarbitone (40 or 80 mg kg-', i.p.), 3-methylcholanthrene (20 mg kg-', i.p.) or 2-methylimidazole (40 mg kg-', i.p.) did not alter the binding parameters of [3H]-idazoxan to brain and liver 12 imidazoline-preferring receptors. The compound SKF 525A, a potent inhibitor of cytochrome P-450 enzymes which forms a tight but reversible complex with the haemoprotein, completely displaced with moderate affinity (KiH = 2-10 ;LM) the specific binding of [3H]-idazoxan to brain and liver 12 imidazoline-preferring receptors. Preincubation of total liver homogenates with 3 x 10-4 M phenelzine in the presence of 10-3 M NADH, a treatment that irreversibly inactivates the haeme group of cytochrome P-450, did not reduce the density of liver I2 imidazoline-preferring receptors. These results discounted a possible interaction of [3H]-idazoxan with the haeme group of cytochrome P-450 enzymes. 6 Together the results indicate that the down-regulation of I2 imidazoline-preferring receptors is associated with an irreversible inactivation of MAO (at least in the brain) that is not related either to the affinity of the MAO inhibitors for I2 imidazoline-preferring receptors or to an irreversible binding to these putative receptors. These findings indicate a novel effect of irreversible MAO inhibitors in the brain and suggest a new target for these compounds that could be of relevance in the treatment of depression, a disease in which an increased density of brain I2 imidazoline-preferring receptors has been reported. Keywords: Imidazoline-preferring receptors; phenelzine; clorgyline; tranylcypromine; moclobemide; monoamine oxidase (MAO); [3H]-idazoxan

Introduction Several tissues and cell lines express specific and functional mined and identified as agmatine (Li et al., 1994); this sub- binding sites for imidazoli(di)ne/guanidine compounds which stance is bioactive, promoting release of catecholamines from have been termed imidazoline-preferring receptors (Michel & adrenal chromaffin cells and its biosynthetic enzyme, arginine Ernsberger, 1992). The structure of a possible endogenous decarboxylase is expressed in the rat brain (Li et al., 1994). ligand for these receptors (a component of the so-called The imidazoline-preferring receptors appear to comprise at clonidine-displacing substance; CDS) has been recently deter- least two subtypes, recently designated as I, and I2 (Erns-

berger,- I 1992), which differ in their pharmacological profiles 'Author for correspondence at: Lab Neurofarmacolgia, Dept. and also in their tissue and subcellular distributions (for a Biologia Fonamental i Ciencies de la Salut, Universitat de les Illes review see Michel & Insel, 1989; Atlas, 1991; Hieble & Balears, Cra, Valldemossa Km 7.5, E-07071 Palma de Mallorca, Ruffolo, 1992; Kilpatrick et al., 1992). Spain. The I, imidazoline-preferring receptors have been found to 838 R. Alemany et al Pheneizine down-regulates 12 imidazoline receptors be increased in platelet membranes from depressed patients with the cytochrome P-450 inducing agents phenobarbitone and down-regulated after desipramine treatment (Piletz et al., (40 or 80 mg kg-', i.p., every 24 h for 7 days), 3- 1990; 1991). The 12 imidazoline-preferring receptors might methylcholanthrene (20 mg kg-', i.p., every 12 h for 7 days) also be increased in the frontal cortex of depressed suicide or 2-methylimidazole (40 mg kg-', i.p. every 24 h for 7 days). victims (Meana et al., 1993) and recently, down-regulation of Both in the acute and chronic treatments the rats were killed these putative receptors after chronic treatment with the 24 h after the last injection. irreversible monoamine oxidase (MAO) inhibitors clorgyline and pargyline has been reported in the rat brain (Olmos et Rat cortical and liver membrane preparation al., 1993). The I2 imidazoline-preferring receptors and the enzyme MAO show some similarities: (1) they are located on The animals were decapitated and the parieto-occipital cortex the mitochondrial outer membrane (Tesson & Parini, 1991; and portions of liver were rapidly removed into ice-cold Tesson et al., 1991); (2) in the rat and human brain the Tris-sucrose buffer (5 mM Tris-HC1; 250 mM sucrose; 1 mM regional distribution of 12 imidazoline-preferring receptors MgCl2; pH 7.4) and frozen at - 80TC until required. Cortical correlates well with that of the B but not A isoform of the and liver membranes (P2 fractions) were prepared by estab- MAO (Sastre & Garcia-Sevilla, 1993); and (3) the density of lished methods with modifications (Giralt & Garcia-Sevilla, 12 imidazoline-preferring receptors and MAO-B increases 1989). Briefly, the tissue samples were homogenized in 5 ml with age in the human brain (Sastre & Garcia-Sevilla, 1993). of ice-cold Tris-sucrose buffer containing 2 mM of the pro- These findings suggest some relationship between these two tease inhibitor, phenylmethylsulphonyl fluoride (PMSF). The proteins; however, a direct interaction of imidazoline ligands homogenates were centrifuged at 1,100 g for 10min, and the with the active centre of MAO-A and B isoenzymes has been supernatants were then recentrifuged at 40,000 g for 10min. previously discounted (Olmos et al., 1993; Sastre & Garcia- The resulting pellet was washed twice with 2 ml of fresh Sevilla, 1993). incubation buffer (50 mM Tris-HCl, 0.1% ascorbic acid, The MAO inhibitors consist of a structurally diverse group pH 7.5). The final pellet was resuspended in an appropriate of compounds whose antidepressant properties have been volume of this buffer to a final protein content of attributed to their common ability to increase synaptic 800-1OOO gml-' for the cortical membranes and 900- availability of certain monoamine neurotransmitters. How- 1600 g ml1' for the liver membranes. Protein was deter- ever, maximal inhibition of MAO activity usually precedes mined by the method of Lowry et al. (1951), with bovine antidepressant effects, suggesting that the therapeutic effects serum albumin as the standard. of these compounds may be mediated by more than one target (for a review see Baker et al., 1992). Thus, interactions of MAO inhibitors with cytochrome P-450 enzymes (Muak- [3H]-idazoxan binding assay kassah & Yang, 1981; Belanger & Atitse-Gbeasson, 1982; Dupont et al., 1987) have been described. The synthesis of Total [3H]-idazoxan binding was measured in 1.1 ml-aliquots different cytochrome P-450 forms can be induced by a variety (50 mM Tris-HCl, 0.1% ascorbic acid, pH 7.5) of the cortical of compounds in the mitochondria and endoplasmic reticu- or liver membranes which were incubated with shaking for lum of the liver and other tissues (Murrey & Reidy, 1990). 30 min at 25C. Binding of [3H]-idazoxan to brain I2 Therefore, if the 12 imidazoline-preferring receptors were a imidazoline-preferring receptors was always done in the form of cytochrome P-450 enzymes, their density in the liver presence of 10-6 M (-)-adrenaline to prevent the binding of and brain might be modulated by chronic treatment with one the radioligand to M2-adrenoceptors (Olmos et al., 1992; of these inducing agents. Miralles et al., 1993a). In the liver it was found that (-)- The present study was thus designed (1) to assess further adrenaline (10-'° to 10-3 M) did not compete against the and extend the effects of chronic treatments with irreversible binding of [3H]-idazoxan (10-8 M) (only 10% of inhibition at and reversible MAO inhibitors on rat brain and liver I2 1i-0M) suggesting that the radioligand does not label a2- imidazoline-preferring receptors; (2) to investigate in vitro the adrenoceptors in this tissue, as previously described (Zon- pharmacological properties of the interaction of these drugs nenschein et al., 1990). Because of this, total binding was with the imidazoline-preferring receptors and (3) to seek for a defined in the liver membranes in the absence of (-)-adrena- possible link between 12 imidazoline-preferring receptors and line. Nonspecific binding was determined in the presence of cytochrome P-450 enzymes. 10- M naphazoline, as previously described (Olmos et al., 1992). In the saturation studies, cortical and liver membranes were incubated with eight concentrations of [3H]-idazoxan Methods (6 X 10-10 M to 4 X 10- M) as above. The specific binding was defined as the difference between total binding (liver Animals and treatments membranes) or total non-adrenergic binding (cortical membranes) and nonspecific binding and was plotted as a function Male Sprague-Dawley rats (250-300 g) were used. The of increasing concentrations of the radioligand. Specific bin- animals received a standard diet with water freely available ding represented 70% to 40% of total binding in cortical and were housed at 20 ± 2°C with a 12 h light/dark cycle. In membranes and 90% to 80% in liver membranes. In the drug one series of experiments and for the chronic treatments the competition studies, membranes were incubated as above animals received i.p., every 12 h either 0.9% saline vehicle, with [3H]-idazoxan (10-8 M) and in the absence or presence the irreversible MAO inhibitors, phenelzine (0.3-20 mg kg-' of various concentrations of the competing drugs (10-14M or for 7 days), isocarboxazid (10 mg kg-' for 7 days), clorgyline 10-12 M to 10-3 M; 15-23 concentrations). Total binding was (3 mg kg-' for 14 days) or tranylcypromine (10 mg kg-' for 7 determined as above and plotted as a function of the drug days). The time-course for the effect of phenelzine concentration. (3 mg kg-') was studied on days 1 (acute), 7, 14 and 21 after In some experiments, the effect of in vitro preincubation treatment. Chronic treatment with these MAO inhibitors has with phenelzine, clorgyline or chlordimeform on [3H]-ida- been shown to result in a profound and sustained inhibition zoxan binding to brain and liver I2 imidazoline-preferring of MAO-A/B (80-90%) in the rat brain and liver (Mousseau receptors was assessed as described previously (Olmos et al., et al., 1993). 1993). The pellet resulting from the last centrifugation (see In another series of experiments rats were treated i.p., preparation of membranes) was resuspended in 10 ml of fresh every 12 h for 7 days with the reversible MAO-A inhibitors, incubation buffer and incubated for 30 min at 25°C in the moclobemide (1 and 10 mg kg-') or chlordimeform presence of phenelzine (10-4 M), clorgyline (10-6 M) or chlor- (10 mg kg-') or with the reversible MAO-B inhibitor, Ro 16- dimeform (10-6 M). Then, the membranes were washed twice 6491 (1 and 1Omg kg'). Rats were also treated chronically with 10 ml of fresh incubation buffer and the final pellet was R. Alemany et al Pheneizine down-regulates 12 imidazoline receptors 839 was used for the statistical evaluations. The level of resuspended as above and used in saturation or competition was used for the statistical evaluations. The level of binding experiments. significance was P = 0.05. Incubations were terminated by diluting the samples with 5 ml of ice-cold Tris incubation buffer (4C). Membrane- Drugs bound [3HJ-idazoxan was measured by vacuum filtration through Whatman GF/C glass fibre filters which had been [3H]-idazoxan (specific activity, 41-46 Ci mmol-'; batches 42 presoaked with 0.5% polyethylenimine (Bruns et al., 1983), to 47) was purchased from Amersham International plc using a Brandel 48R cell harvester (Biomedical Research & (U.K.). [3H]-Ro 41-1049 [N-(2-aminoethyl)-5-(m-fluoro- Development Laboratories, U.S.A.). Then the filters were phenyl)-4-thiazole carboxamide HCl] (specific activity, rinsed twice with 5 ml of incubation buffer, air-dried, trans- 30.8 Ci mmol-') and [3H]-Ro 19-6327 [N-(2-aminoethyl)-5- ferred to minivials containing 5 ml of OptiPhase 'HiSafe' II chloro-2-pyridine carboxamide HCl] (specific activity, cocktail (LKB, England) and counted for radioactivity by 20.2 Ci mmol ') were generous gifts from Dr J. Richards and liquid scintillation spectrometry at 57% efficiency (Packard Dr J. Saura (F. Hoffmann-La Roche Ltd., Switzerland). For model 1900 TR). the binding assays, appropriate amounts of the stock solu- tions were diluted with distilled and purified water (Milli-Q) 41-1049 and [3H]-Ro 19-6327 binding assays containing 2.5 mM HC1 and 6% ethanol. Other drugs (and [3H1-Ro their sources) included: (-)-adrenaline bitartrate (Sigma In some experiments, the effect of in vitro preincubation with Chemical Co., U.S.A.); chlordimeform HC1 (Ciba-Geigy, phenelzine (10-4 M) as above was assessed on the binding of Switzerland); clorgyline HC1 (Sigma); iproniazid (RBI, [3H]-Ro 41-1049 to liver MAO-A and that of [3H]-Ro 19- U.S.A.); isocarboxazid HCl (F. Hoffmann-La Roche Ltd.); 6327 to liver MAO-B. Binding assays of these radioligands 3-methylcholanthrene (Sigma); 2-methylimidazole (Sigma); were performed as described previously (Sastre & Garcia- moclobemide base (F. Hoffmann-La Roche Ltd.); naphazo- Sevilla, 1993). line HCI (Sigma); phenelzine sulphate (Sigma); phenobarbitone (Sigma); 3-phenylpropargylamine HC1 (RBI); Ro 16- binding data and statistics 6491 [N-(2-aminoethyl-p-chlorobenzamide] HCl (F. Hoffmann- Analyses of La Roche Ltd.); semicarbazide HCl (RBI); SKF 525A [2- Analyses of saturation isotherms (Kd, dissociation constant; diethylaminoethyl diphenylpropylacetate] HCI (proadifen) Bmax, maximum density of binding sites) and competition (RBI); tranylcypromine sulphate (Smith-Kline, France). experiments (Ki, inhibition constant) as well as the fitting of Other reagents were obtained from Sigma Chemical Co. data to the appropriate binding models were performed by (U.S.A.). computer-assisted nonlinear regression using the EBDA- LIGAND (Munson & Rodbard, 1980; McPherson, 1985) programmes. All experiments were initially analysed assum- Results ing a one-site model of radioligand binding and then assum- ing a two-site binding model. The selection between the In vivo effects of irreversible and reversible MAO different binding models was made statistically using the inhibitors on brain and liver I, imidazoline-preferring extra sum of squares principle (F test) as outlined by Munson receptors & Rodbard (1980). The more complex model was accepted if the P value resulting from the F test was less than 0.05. Chronic treatment (7 days) with the irreversible non-selective Results are expressed as mean ± s.e.mean. One-way MAO inhibitor, phenelzine (1-20mg kg-') dose-dependently analysis of variance (ANOVA), followed by Scheffe's test, decreased the density of I2 imidazoline-preferring receptors in

Table 1 Effects of chronic treatments with irreversible monoamine oxidase (MAO) inhibitors on I2 imidazoline-preferring receptors in the rat cerebral cortex and liver Cerebral cortex Liver [3HJidazoxan [3H]-idazoxan Dose Kd B,= Kd Bx Treatment (mg kg-') (nM) (fmol mg-' protein) n (nM) (fmol mg-' protein) n ± Saline - 14.4 ± 0.7 68 ± 3 24 9.0 ± 1.0 335 21 24 Hydrazines ± 5 237 ± 17 3 Phenelzine 0.3 13.7 0.7 75±4 4.9±0.3 3 1 12.1 ± 2.2 49± 3 4 8.4±0.1 264± 10 3 12.8 ± 2.9 42± 5* 4 8.8±0.8 147 ± 10* 4 10 9.2 ± 1.6 36 ± 2** 3 7.7±0.4 133±9*** 6 ± 30 ± 2*** 6 8.2 ± 0.5 172 ± 16* 5 20 7.5 0.5 ± Isocarboxazid 10 9.5 ± 1.5 20 ± 3*** 3 16.1 ±0.7 124 9* 3 Propargylamines ± 4 Clorgyline 3 13.3 ± 1.9 34 ± 3*** 6 7.1±0.7 156 14* Cyclopropylamines Tranylcypromine 10 13.7±4.9 31 ± 6*** 4 12.0± 0.9 134± 13* 3 killed Each drug was administered i.p., every 12 h for 7 days, except for the clorgyline treatment which was for 14 days. The rats were 24 h after the last injection. Cortical and liver membranes were incubated at 25'C for 30 min with eight concentrations of was defined in the [3H]-idazoxan (6 x 10-1' M to 4 x 10-8 M). Total binding of [3H]-idazoxan to 12 imidazoline-preferring receptors was defined in of 10-6 M adrenaline for the cortical membranes or buffer only for the liver membranes; non-specific binding presence analysis the presence of 10-4 M naphazoline. Binding parameters (Kd, Bmu) were determined directly by computer-assisted nonlinear from untransformed data using the EBDA-LIGAND programmes. Each value represents the mean ± s.e.mean of n experiments per with an animal per experiment. Data for clorgyline treatment in the brain were taken from Olmos et al. (1993). One-way group irreversible ANOVA followed by a multiple comparison test detected a significant decrease in B.. after chronic treatment with the MAO inhibitors (F[8,50] = 20.86, P = 0.0001 and F18,46] = 10.33; P = 0.0001 for the cortical and liver membranes, respectively). *P <0.05, **P <0.01 and ***P <0.001 as compared with saline-treated group (ANOVA followed by Scheffe's test). 840 0R. Alemany et al Phenezine down-regulates 12 imidazoline receptors the rat cerebral cortex (Bma, values reduced by 28%-56%) effect was also induced by chronic treatment (7 days) with and liver (Bmax values reduced by 21%-60%) (Table 1). This the cyclopropylamine, tranylcypromine (10 mg kg-l) which effect of phenelzine was similar in both tissues, in spite of the reduced by 55%-60% the density of brain and liver I2 marked different densities of 12 imidazoline-preferring recep- imidazoline-preferring receptors (Table 1). No significant tors in brain and liver (five times greater density in the liver; alterations of the Kd values for the binding of [3H]-idazoxan Table 1). The effect of phenelzine (3 mg kg-') was rapid (1 to cortical or liver membranes were observed after the day) in the liver but not in the brain (7 days) and the different chronic treatments with the irreversible MAO down-regulation of I2 imidazoline-preferring receptors was inhibitors (Table 1). However, chronic treatment with phenel- maintained in both tissues after 14 or 21 days of repeated zine also tended to increase the affinity of the radioligand for treatment (Bmax values reduced by 35% in brain and 60% in the brain 12 imidazoline-preferring receptors (Table 1) liver) (Figure 1). Similar chronic treatment with isocarbox- (ANOVA for Kd values: F15,40] = 4.07, P = 0.004). azid (10 mg kg-1), a hydrazine structurally related to phenel- In order to determine further (Olmos et al., 1993) if the zine, also significantly decreased the density of brain and down-regulation of I2 imidazoline-preferring receptors found liver 12 imidazoline-preferring receptors (Bmin values reduced after chronic treatment with these MAO inhibitors was by 71% and 63%, respectively) (Table 1). related to their ability to inactivate irreversibly the enzyme, In addition to hydrazines the study was extended to other the effects of chronic treatments with three reversible MAO irreversible MAO inhibitors including propargylamines and inhibitors were studied. Chronic treatment (7 days) with the cyclopropylamines. Chronic treatment (14 days) with the pro- selective MAO-A inhibitors, moclobemide (1 and 10mg kg-') pargylamine, clorgyline (3 mg kg-') decreased by 50% the or chlordimeform (10 mg kg-') or with the selective MAO-B density of 12 imidazoline-preferring receptors in the rat brain inhibitor, Ro 16-6491 (1 and 10mg kg-') did not induce any (data taken from Olmos et al., 1993) and liver (Table 1). This significant change in the binding parameters (Kd, B~n) of [3H]-idazoxan to rat brain membranes (Table 2). In the liver only the higher dose of Ro 16-6491 reduced significantly the density of I2 imidazoline-preferring receptors (38%) (Table 2). 120

100 I In vitro effects of irreversible and reversible MAO inhibitors on brain and liver I2 imidazoline-preferring o 80 receptors M(UCo0 In vitro competition curves for various irreversible and rever- _X 60 sible MAO inhibitors against [3H]-idazoxan binding to rat cerebral cortex and liver I2 imidazoline-preferring receptors 20 were biphasic (Table 3, Figures 2 and 3). Phenelzine, 3- phenylpropargylamine, tranylcypromine and the reversible MAO-B inhibitor, Ro 16-6491 displayed moderate affinity 20- (low micromolar range) for I2 imidazoline-preferring receptors with similar inhibition constants (Ki) in rat brain and 1 7 14 21 liver (Table 3 and Figure 2). The hydrazine non-selective Days MAO inhibitors isocarboxazid, iproniazid and semicarbazide and the reversible MAO-A inhibitor moclobemide did not Figure 1 Time-course for the effect of phenelzine (3 mg kg-', i.p., compete against [3H]-idazoxan binding to 12 imidazoline- every 12 h) on the density (Bmax) of 12 imidazoline-preferring recep- preferring receptors in the two tissues studied (Table 3). tors in the rat cerebral cortex (solid columns) and rat liver (hatched Clorgyline competition curves against [3H]-idazoxan binding columns). Saturation curves of [3H]-idazoxan binding (6 x 10- " M to to rat brain I2 imidazoline-preferring receptors revealed the 4 x 10-8 M in the presence of 10-6 M adrenaline) to 12 imidazoline- of a = 40 site for this preferring receptors were determined in P2 fractions of cortical or existence very high affinity (KiH pM) liver membranes as described in Table 1. Bmax values are expressed as drug that represented about a 40% of total binding (Olmos percentage of that from saline-treated rats and represent et al., 1993); in contrast, clorgyline only displayed moderate mean ± s.e.mean of 3 to 4 experiments with an animal per experi- affinity (KiH = 169 nM) for the same proportion of sites in the ment. *P<0.05 and **P<0.001 as compared with saline-treated liver (Table 3 and Figure 3a). These differences between brain group (ANOVA followed by Scheff6's test). and liver I2 imidazoline-preferring receptors were further

Table 2 Effects of chronic treatments with reversible monoamine oxidase (MAO) inhibitors on 12 imidazoline-preferring receptors in the rat cerebral cortex and liver Cerebral cortex Liver [3H]-idazoxan [3H]-idazoxan Dose Kd B,,, Kd Bmax Treatment (mg kg-') (nM) (fmol mg-' protein) n (nM) (fmol mg-' protein) n

Saline - 15.8 ± 1.8 75± 6 7 7.9±0.5 272± 16 9 Moclobemide 1 16.3±2.2 77 ± 7 3 8.3±0.5 203 ± 7 4 ± 10 12.7 ± 1.6 69± 12 3 10.0± 1.1 197 16 4 Chlordimeform 10 15.4± 1.6 76 ± 7 3 9.8±0.6 307 ± 32 4

Ro 16-6491 1 16.5 ± 3.8 75 ± 9 3 6.4 ± 0.4 288 ± 31 4 10 12.8±3.0 70± 11 4 9.9±0.2 170± 19* 4

Each drug was administered i.p., every 12 h for 7 days. The rats were killed 24 h after the last injection. Binding of [3H]-idazoxan to brain and liver 12 imidazoline-preferring receptors and estimation of binding parameters (Kd, Bm,) were done as described in Table 1. Each value represents the mean ± s.e.mean of n experiments per group with an animal per experiment. One-way ANOVA followed by a multiple comparison test detected a significant decrease in B.k only after chronic treatment with Ro 16-6491 (10 mg kg-') in the liver (F[5,23] = 6.31; P = 0.0008). *P <0.05 as compared with saline-treated group (ANOVA followed by Scheffe's test). R. Alemany et al Pheneizine down-regulates 12 imidazoline receptors 841 Table 3 Binding parameters of various monoamine oxidase (MAO) inhibitors on I2 imidazoline-preferring receptors in the rat cerebral cortex and liver Cerebral cortex Liver [3H1-idazoxan [3H1-idazoxan KiH (rim) KiL A#m) %RH n KH (nM) KiL (lM) %RH n MAO inhibitors Irreversibles Hydrazines Phenelzine 5,900 38.4 78 3 3,000 179 84 4 Isocarboxazid > 1000 2 > 1000 2 Iproniazid >1000 2 > 1000 2 Semicarbazide > 1000 2 > 1000 - 2 Propargylamines Clorgyline 0.04 10.6 38 10 169 15.0 45 4 3-Phenylpropargylamine 356 116 63 4 793 128 92 2 Cyclopropylanines Tranylcypromine 357 771 85 2 592 685 83 3 Reversibles Moclobemide >1000 _ 2 > 1000 _ 2 Chlordimeform 6.8 45.9 60 4 6.1 18.8 90 3 Ro 16-6491 1,900 100 2 Cortical or liver membranes were incubated at 25°C for 30 min with [3H]-idazoxan (10-8 M in the presence of 10-6 M adrenaline) and in the absence or the presence of the competing drugs (10-'4 M or 10-12 M to 10-3 M). Binding parameters (KiH, KiL and %RH, defined as the percentage of high affinity sites for a given drug) were determined directly by simultaneous analysis of n independent experiments for each drug using the EBDA-LIGAND programmes. For the drugs tested (except Ro 16-6491) that completely inhibited radioligand binding computer-assisted curve fitting demonstrated that a two-site fit was significantly better than a one-site binding model (P< 0.001, F test). The binding parameters for clorgyline and Ro 16-6491 competition curves in the brain are taken from Olmos et al. (1993). a a 120 r 120 r

Cu 100 ou P 40 100 Ad) 0- 1-00 801- 80 - 0 .0 601- .0 60w- C x x 0 0 N 401- N 401- co

C,I 201- m 201-

0 1-10 10-8 10-4 10-4 10-2 lo-14 1o-12 10 10 lo-- 10-6 104 10-2 Pheneizine (M) Clorgyline (M) b 120 r 0 mCu 4-0 0 0 0-0 0 801 80 - 0 3 .0c 10 601 .0x 60 - Cu x x 0 0 CN 401 co 401- . I V 201 201-

10-8 106 1C 10-14 1O-12 10-10 10-8 10-6 10-4 10-2 Tranylcypromine (M) Chlordimeform (M) Figure 2 Inhibition of binding of [3H]-idazoxan (in the presence of Figure 3 Inhibition of binding of [3H]-idazoxan (in the presence of 10-6 M adrenaline) to 12 imidazoline-preferring receptors by (a) 10-6 M adrenaline) to I2 imidazoline-preferring receptors by (a) phenelzine or (b) tranylcypromine in the rat cerebral cortex mem- clorgyline or (b) chlordimeform in the rat cerebral cortex membranes branes (0) and in the rat liver membranes (0). Computer-assisted (0) and in the rat liver membranes (0). Computer-assisted curve curve fitting (EBDA-LIGAND programmes) demonstrated that in fitting (EBDA-LIGAND programmes) demonstrated that in all cases all cases a two site fit was significantly better than a one site fit a two site fit was significantly better than a one site fit (P <0.001; F (P <0.001; F test). Data shown are mean ± s.d. or s.e.mean of 2 to test). Data shown are mean ± s.e.mean of 3 to 10 experiments. See 4 experiments. See Table 3 for Ki values and other details. Table 3 for Ki values and other details. 842 R. Alemany et al Pheneizine down-regulates 12 imidazoline receptors revealed by competition curves for chlordimeform against imidazoline-preferring receptors (Figure 4). These results dis- [3H]-idazoxan binding. The pesticide and MAO inhibitor, counted an irreversible binding of phenelzine to 12 imidazo- chlordimeform (Hollingworth et al., 1979) displayed high line sites. Under the same experimental conditions (30 min at affinity (KiH-6nM) for the brain and liver I2 imidazoline- 25°C), however, preincubation with 10-4 M phenelzine de- preferring receptors. However, the proportion of high affinity creased by 95% and 30% the binding of [3H]-Ro 41-1049 and sites for the drug represented 60% in the rat bain but 90% in [3H]-Ro 19-6327 to liver MAO-A and B, respectively. In the rat liver (Table 3 and Figure 3b). contrast, preincubation with 10-6 M clorgyline reduced by 40% (P <0.01) the B, of [3H]-idazoxan binding to brain and liver membranes (Figure 4). In vitro preincubation with phenelzine and Similar to the irreversible MAO-A inhibitor clorgyline, chlordimeform on brain and liver I2 chlordimeform, a reversible MAO-A inhibitor (Hollingworth et al., 1979), also displayed high affinity (low nanomolar imidazoline-preferring receptors range) for 12 imidazoline-preferring receptors (Table 3). In To assess further the mechanisms of action involved in the in order to determine whether this high affinity of the drug for vivo down-regulation of brain and liver 12 imidazoline- I2 sites could also result in irreversible binding, cortical mem- preferring receptors induced by phenelzine, incubation branes were preincubated with 10-6 M chlordimeform and experiments were performed to determine if, consistent with reassessed for [3H]-idazoxan binding. Preincubation with its actions on MAO, this compound could also bind irrever- chlordimeform altered neither the density of sites nor the sibly to 12 imidazoline-preferring receptors as described for competition parameters of cirazoline ([3H]-idazoxan binding, clorgyline and pargyline (Olmos et al., 1993). However, control membranes: KiH = 4 nM; KiL = 3.3 pM; %RH = 63; preincubation (30 min at 250C) of brain or liver membranes n = 3; preincubated membranes: KiH = 8 nM; KiL = 2.3 jtM; with 10-' M phenelzine did not alter the total density of 12 %RH= 69; n = 3).

120 r

/ 100 -o 0 0 4-0

X 80 - 80 - E* C 0 - 60 60 x 40 ._0 0 aN 40- V 20 'a 20 - 0 0 L / 010 10-8 10-6 0- 10-2 Brain Liver SKF 525A (M) Figure 4 Effects of preincubation (30 min at 25'C) of cortical or liver membranes in buffer containing 10-4M phenelzine (hatched Figure 5 Inhibition of binding of [3H]-idazoxan (in the presence of columns) or 10-6 M clorgyline (solid columns) on the total density 10-6 M adrenaline) to I2 imidazoline-preferring receptors by SKF (Bmax) of 12 imidazoline-preferring receptors. Saturation curves of 525A in the rat cerebral cortex membranes (0) and in the rat liver [3H]-idazoxan binding (6 x 10-' M to 4 X IO8 M in the presence of membranes (0). Computer-assisted curve fitting (EBDA-LIGAND 10-6 M adrenaline) to 12 imidazoline-preferring receptors were deter- programmes) demonstrated that for the competition curves in the rat mined in rat cerebral cortex or liver membranes after preincubation liver a two site fit was significantly better than a one site binding and washing as described in methods. Bma, values are expressed as model (P <0.001; F test). The binding parameters obtained were the percentage of that preincubated in buffer only (control) and Ki = 10 lm in the rat cerebral cortex; and KiH = 1.6 LM, KiL = 36 pM; represent mean ± s.e.mean of 3 to 6 experiments. *P <0.05 as %RH = 43 in the rat liver. Data shown are mean ± s.d. of 2 experi- compared with control group (ANOVA followed by Scheffe's test). ments.

Table 4 Effects of chronic treatments with various inducers of cytochrome P-450 enzymes on 12 imidazoline-preferring receptors in the rat cerebral cortex and liver Cerebral cortex Liver [3H]-idazoxan [3HJ-idazoxan Dose Kd B, Kd Ba, Treatment (mg kg-') (nM) (fmol mg-' protein) n (nM) (fmol mg-' protein) n Saline - 13.2±0.8 62 ± 3 17 12.8±2.1 440 ± 24 9 Phenobarbitone 40 10.8 ±0.7 62 ± 1 3 15.1 ± 3.2 370 ± 41 3 80 11.3 ± 1.6 62 ± 6 4 3-Methylcholanthrene 20 16.9± 1.8 52 ± 8 3 15.2± 1.1 474 ± 34 3 2-Methylimidazole 40 10.8±0.2 60± 3 2 - Each drug was administered i.p., every 24 h (phenobarbitone and 2-methylimidazole) or every 12 h (3-methylcholanthrene) for 7 days. One-way ANOVA did not detect any significant change in the binding parameters of [3H]-idazoxan to brain or liver 12 imidazoline-preferring receptors after the various treatments. Other details as for Table 1. R. Alemany et al Pheneizine down-regulates 12 imidazoline receptors 843

Effects of manipulation of cytochrome P-450 enzymes on affinities for I2 imidazoline-preferring receptors (Table 3) but brain and liver I2 imidazoline-preferring receptors chronic treatment resulted in similar down-regulation of 12 imidazoline-preferring receptors (Table 1); isocarboxazid did Chronic treatments (7 days) with the non-selective inducers not compete for [3H]-idazoxan binding to these receptors of cytochrome P-450 forms phenobarbitone (40 or (Table 3) but it was more effective than phenelzine and 80 mg kg-', i.p., every 24 h) and 3-methylcholanthrene tranylcypromine to down-regulate in vivo 12 imidazoline- (20 mg kg-', i.p., every 12 h) or with 2-methylimidazole preferring receptors (Table 1). Thirdly, the down-regulation (40 mg kg-', i.p., every 24 h), a selective inducer of the IIE observed after chronic treatment with MAO inhibitors is not form (Koop et al., 1985), with doses enough to increase related to an irreversible binding of these compounds to 12 hepatic cytochrome P-450 activity by several fold (Cresteil et imidazoline-preferring receptors (see also Olmos et al., 1993). al., 1986) did not significantly alter the binding parameters of Thus, phenelzine, in contrast to clorgyline, does not irrever- [3H]-idazoxan binding to rat liver and brain 12 imidazoline- sibly bind in vitro to brain or liver 12 imidazoline-preferring preferring receptors (Table 4). In vitro, however, the com- receptors (Figure 4) but was able to down-regulate brain and pound SKF 525A, a potent inhibitor of cytochrome P-450 liver 12 imidazoline-preferring receptors after chronic treat- enzymes which forms a tight but reversible complex with the ment. Fourthly, the binding of [3H]-idazoxan to brain and haemoprotein, completely displaced with moderate affinity liver 12 imidazoline-preferring receptors is heterogeneous in (low micromolar range) the specific binding of [3H]-idazoxan nature and competition curves with most MAO inhibitors to I2 imidazoline-preferring receptors in brain and liver were biphasic as previously described for other drugs (Figure 5). (Miralles et al., 1993a); the differences reported here for the Phenelzine and probably other hydrazine MAO inhibitors in vitro interaction of clorgyline and chlordimeform with (isocarboxazid and iproniazid) have been shown to directly brain and liver 12 imidazoline-preferring receptors indeed sug- bind to the ferrous haeme of cytochrome P-450 and to induce gest that they may represent different molecular-pharmaco- its irreversible inactivation in the presence of NADPH or logical entities (see also Escribi et al., 1994). NADH (Muakkassah & Yang, 1981; Jonen et al., 1982). In The possibility that [3H]-idazoxan binds to the active site order to assess if this mechanism could also underline the of MAO-A and/or B isoenzymes has been previously dis- interaction of phenelzine with I2 imidazoline-preferring recep- counted (Olmos et al., 1993; Sastre & Garcia-Sevilla, 1993); tors, total homogenates of rat liver were preincubated for however, it has been suggested that this radioligand could 30 min at 37°C in the presence of 3 x IO-'M phenelzine bind to another site on the MAO protein (Renouard et al., alone or with 10-3 M NADH; therafter P2 fractions were 1993). This possibility is very unlikely, mainly because obtained and assessed for [3H]-idazoxan binding. Preincuba- idazoxan, contrary to other imidazoline derivatives (e.g. tion with phenelzine and NADH did not reduce the density cirazoline), did not inhibit up to 10-3 M rat liver basal MAO of liver I2 imidazoline-preferring receptors (phenelzine: activity (Carpene et al., 1994). Moreover, the proposed Kd= 18.7 ± 0.9 nM; Bm_ =444+ 18fmol mg-' protein; molecular mass for the MAO-B (59 kDa) (Bach et al., 1988; n = 3; phenelzine plus NADH; Kd= 19.0 ± 1.5 nM; Bx = Chen et al., 1993) does not correspond with those proposed 501 ± 34 fmol mg-' protein; n = 3), suggesting that [3H]- for the main 12 imidazoline-preferring receptors (29/30 and idazoxan does not bind to the haeme group of cytochrome 70kDa proteins) (Wang et al., 1992; Escriba et al., 1994). P-450. Recently, polyclonal antibodies have been raised against the 70 kDa imidazoline-receptor protein (Wang et al., 1993) which in the present laboratory failed to recognize a purified Discussion fraction (up to 0.7 pg) of MAO from bovine plasma (Sigma Chemical Co.). Moreover, 12 imidazoline-preferring receptors The present study demonstrates that chronic treatments with and the two isoforms of MAO display different isoelectric the MAO inhibitor antidepressant, phenelzine and other points (Limon et al., 1992). On the other hand, the 12 clinically effective irreversible MAO inhibitors including imidazoline-preferring receptors and the MAO-B are not sub- isocarboxazid, clorgyline and tranylcypromine are associated jected to the same regulation. Thus, chronic treatments with with down-regulation of brain and liver 12 imidazoline- cirazoline and idazoxan that up-regulate the density of brain preferring receptors. However, the clinically effective I2 imidazoline-preferring receptors did not modify that of antidepressant and reversible MAO inhibitor, moclobemide brain MAO-B (Olmos et al., 1992; 1994). Moreover, the did not interact with brain and liver I2 sites. These observa- quinoline derivative EEDQ alkylates in vivo a significant tions indicate a novel effect of irreversible MAO inhibitors in proportion (50%) of 12 imidazoline-preferring receptors with- the brain and suggest a new target for these compounds that out altering the density of MAO-B sites (Miralles et al., could be of relevance in the pathophysiology and treatment 1993b). Together these findings indicate that although 12 of depression, a disease in which an increased density of imidazoline-preferring receptors and MAO-B are co-ex- brain 12 imidazoline-preferring receptors has been reported pressed in the same brain areas (Sastre & Garcia-Sevilla, (Meana et al., 1993). 1993) they represent different proteins. Several conclusions can be deduced from the present data. Irreversible MAO inhibitors including clorgyline, phenel- Firstly, the reported down-regulation of brain I2 imidazoline- zine and tranylcypromine have been shown also to interact preferring receptors is related to an irreversible inactivation with hepatic cytochrome P-450 enzymes (Muakkassah & of the enzyme MAO. Thus, chronic treatment with irreversi- Yang, 1981; Belanger & Atitse-Gbeasson, 1982; Dupont et ble (phenelzine, isocarboxazid and tranylcypromine, present al., 1987). The rat brain also expresses these enzymes study; or clorgyline and pargyline, Olmos et al., 1993) but (Warner et al., 1988; Bergh et al., 1992) and a significant not with reversible MAO-A (moclobemide, chlordimeform, proportion are located on mitochondria (Walther et al., present study; or Ro 41-1049, Olmos et al., 1993) and MAO- 1986). Thus, the possibility that 12 imidazoline-preferring B inhibitors (Ro 16-6491, present study) resulted in down- receptors could be a form of these enzymes was investigated. regulation of 12 imidazoline-preferring receptors. This Chronic treatment with the enzyme inducers phenobarbitone, conclusion also applies to these putative receptors in the 3-methylcholanthrene or 2-methylimidazole did not modulate liver, except for the chronic effects of a high dose of the the density of 12 sites in brain and liver. On the other hand, compound Ro 16-6491 (Table 2). Secondly, among the irre- phenelzine, SKF 525A and imidazole compounds have been versible MAO inhibitors there is no relationship between the shown to interact with the haeme group of cytochrome P-450 affinity displayed in vitro against [3H]-idazoxan binding to (Muakkassah & Yang, 1981; Yoshida & Aoyama, 1987). For brain and liver I2 imidazoline-preferring receptors and the instance, SKF 525A inhibited with moderate affinity ability to down-regulate these receptors in vivo. Thus, clorgy- (Ki = 1-38 gM) the binding of [3H]-GBR-12935 to cyto- line, tranylcypromine and phenelzine markedly differ in their chrome P-450 in human brain (Allard et al., 1994). Although 844 R. Alemany et al Pheneizine down-regulates 12 imidazoline receptors SKF 525A completely displaced with similar moderate dazoline-preferring receptors. The endogenous amine 2- affinity the specific binding of [3H]-idazoxan to 12 phenylethylamine is also a metabolite of phenelzine and has imidazoline-preferring receptors in brain and liver (Figure 5), been proposed as one of the mediators of its antidepressant preincubation with phenelzine in the presence of NADH did effects (McManus et al., 1991; Paetsch et al., 1993). However, not reduce the density of liver 12 imidazoline-preferring recep- 2-phenylethylamine displayed low affinity for the I2 tors, indicating that [3H]-idazoxan does not bind to the imidazoline-preferring receptors in the rat brain (Ki = 2 PM). haeme group of cytochrome P-450 enzymes. Moreover, there Further work is needed to unravel the mechanisms by which is no correlation between the densities of cytochrome P-450 irreversible MAO inhibitors down-regulate 12 imidazoline- (Allard et al., 1994) and those of II/2 imidazoline-preferring preferring receptors in the brain. receptors (De Vos et al., 1994) in the various regions of the human brain (r = 0.20 and 0.03 for the II- and I2 imidazoline-preferring receptors; n = 10). Together these results are compatible with the view that an This study was supported by DGICYT Grant PM 91-0069, FIS indirect mechanism is responsible for the in vivo down- Grant 93-0641 and by CDTI-S.A. LASA Laboratorios Grant 92- regulation of brain I2 imidazoline-preferring receptors found 0198, Barcelona, Spain. R.A. was supported by a fellowship from after chronic treatment with irreversible MAO inhibitors. In Fondo de Investigaciones Sanitarias, Spain. We are grateful to Dr this sense, there is the possibility that some metabolite(s) of D.J. Reis (Cornell University Medical College) for generously sup- the irreversible MAO inhibitors could be active on I2 imi- plying the antibody against imidazoline receptor proteins.

References ALLARD, P., MARCUSON, J.O. & ROSS, S.B. (1994). [3H]GBR-12935 HIEBLE, J.P. & RUFFOLO, R.R. Jr. (1992). Imidazoline receptors: binding to cytochrome P-450 in the human brain. J. Neurochem., historical perspective. Fundam. Clin. Pharmacol., 6 (Suppl 1), 62, 342-348. 7s- 13s. ATLAS, D. (1991). Clonidine-displacing substance (CDS) and its HOLLINGWORTH, R.M., LEISTER, J. & GHALI, G. (1979). Mode of putative imidazoline receptor. New leads for further divergence of action of formamidine pesticides: an evaluation of monoamine a2-adrenergic receptor activity. Biochem. Pharmacol., 41, oxidase as the target. Chem. Biol. Interactions, 24, 35-49. 1541-1549. JONEN, H.G., WERRINGLOER, J., PROUGH, R.A. & ESTABROOK, BACH, A.W., LAN, N.C., JOHNSON, D.L., ABELL, C.W., BEMBENEK, R.W. (1982). The reaction of phenyihydrazine with microsomal M.E., KWAN, S.W., SEEBURG, P.H. & SHIH, J.C. (1988). cDNA cytochrome P-450. J. Biol. Chem., 257, 4404-4411. cloning of human liver monoamine oxidase A and B. Molecular KILPATRICK, A.T., BROWN, C.C. & MACKINNON, A.C. (1992). Non- basis of differences in enzymatic properties. Proc. Natl. Acad. Sci. M2-adrenoceptor idazoxan binding sites: a new target for drug U.S.A., 85, 4934-4938. development. Biochem. Soc. Trans., 20, 113-118. BAKER, G.B., COUrrS, R.T., McKENNA, K.F. & SHERRY-MCKENNA, KOOP, D.R., CRUMP, B.L., NORDBLOM, G.D. & COON, M.J. (1985). R.L. (1992). Insights into the mechanisms of action of the MAO Immunochemical evidence for induction of the alcohol-oxidizing inhibitors phenelzine and tranylcypromine: a review. J. Psychiatr. cytochrome P450 of rabbit liver microsomes by diverse agents: Neurosci., 17, 206-214. ethanol, imidazole, trichloroethylene, acetone, pyrazole, and BtLANGER, P.M. & ATITS&-GBEASSON, A. (1982). Inhibitory effect isoniazid. Proc. Nati. Acad. Sci. U.S.A., 82, 4065-4069. of tranylcypromine on hepatic drug metabolism in the rat. LI, G., REGUNATHAN, S., BARROW, C.J., ESHRAGHI, J., COOPER, R. Biochem. Pharmacol., 31, 2679-2683. & REIS, D.J. (1994). Agmatine - an endogenous clonidine- BERGH, A.F. & STROBEL, H.W. (1992). Reconstitution of the brain displacing substance in the brain. Science, 263, 966-969. mixed function oxidase system: purification of NADPH- LIMON, I., COUPRY, I., LANIER, S.M. & PARINI, A. (1992). cytochrome P450 reductase and partial purification of cyto- Purification and characterization of mitochondrial imidazoline- chrome P450 from whole brain. J. Neurochem., 59, 575-581. guanidinium receptive site from rabbit kidney. J. Biol. Chem., BRUNS, R.F., LAWSON-WENDLING, K. & PUGSLEY, T.A. (1983). A 267, 21645-21649. rapid filtration assay for soluble receptors using poly- LOWRY, O.H., ROSEBROUGH, N.F., FARR, A.G. & RANDALL, R.J. ethylenimine-treated filtres. Anal. Biochem., 132, 74-81. (1951). Protein measurement with the Folin phenol reagent. J. CARPENE, C., COLLON, P., REMAURY, A., CORDI, A., HUDSON, A., Biol. Chem., 193, 265-275. NUTT, D. & LAFONTAN, M. (1994). Inhibition of amine oxidase MCMANUS, D.J., MOUSSEAU, D.D., PAETSCH, P.R., WISHART, T.B. & activity by derivatives recognizing 12-imidazoline sites. J. Phar- GREENSHAW, A.J. (1991). P-adrenoceptors and antidepressants: macol. Exp. Ther. (in press). possible 2-phenylethylamine mediation of chronic phenelzine CHEN, K., WU, H.-F. & SHIH, J. (1993). The deduced amino acid effects. Biol. Psychiatry, 30, 1122-1130. sequences of human platelet and frontal cortex monoamine MCPHERSON, G.A. (1985). Analysis of radioligand binding oxidase B are identical. J. Neurochem., 61, 187-190. experiments: a collection of computer programs for IBM PC. J. CRESTEIL, T., BEAUNE, P., CELIER, C., LEROUX, J.P. & Pharmacol. Methods, 14, 213-228. GUENGERICH, F.P. (1986). Cytochrome P-450 isoenzyme content MEANA, J.J., BARTUREN, F., MARTIN, I. & GARCIA-SEVILLA, J.A. and monooxygenase activities in rat liver: effect of ontogenesis (1993). Evidence of increased non-adrenoceptor [3H]idazoxan and pretreatment by phenobarbital and 3-methylcholanthrene. J. binding sites in the frontal cortex of depressed suicide victims. Pharmacol. Exp. Ther., 236, 269-276. Biol. Psychiatry, 34, 498-501. DE VOS H., BRICCA, G., DE KEYSER, J., DE BACKER, J.P., BOUS- MICHEL, M.C. & ERNSBERGER, P. (1992). Keeping an eye on the I QUET, P. & VAUQUELIN, G. (1994). Imidazoline receptors, non- site: imidazoline-preferring receptors. Trends Pharmacol. Sci., 13, adrenergic idazoxan binding sites and a2-adrenoceptors in the 369-370. human central nervous system. Neuroscience, 59, 589-598. MICHEL, M.C. & INSEL, P.A. (1989). Are there multiple imidazoline DUPONT, H., DAVIES, D.S. & STROLIN-BENEDETTI, M. (1987). binding sites? Trends Pharmcol. Sci., 10, 342-344. Inhibition of cytochrome P-450-dependent oxidation reactions by MIRALLES, A., OLMOS, G., SASTRE, M., BARTUREN, F., MARTIN, I. MAO inhibitors in rat liver microsomes. Biochem. Pharmacol., & GARCIA-SEVILLA, J.A. (1993a). Discrimination and pharmaco- 36, 1651-1657. logical characterization of I2-imidazoline sites with [3H]idazoxan ERNSBERGER, P. (1992). Heterogeneity of imidazoline binding sites: and alpha-2 adrenoceptors with [3H]RX821002 (2-methoxy proposed I1 and 12 subtypes. Fundam. Clin. Pharmacol., 6 idazoxan) in the human and rat brains. J. Pharmacol. Exp. Ther., (Suppl 1), 55s, P18. 264, 1187-1197. ESCRIBA, P.V., SASTRE, M., WANG, H., REGUNATHAN, S., REIS, D.J. MIRALLES, A., RIBAS, C., OLMOS, G. & GARCIA-SEVILLA, J.A. & GARCIA-SEVILLA, J.A. (1994). Immunodetection of putative (1993b). Differential effects of the alkylating agent N-ethoxy- imidazoline receptors proteins in the human and rat brain and carbonyl-2-ethoxy-1,2-dihydroquinoline on brain M2-adreno- other tissues. Neurosci. Lett., 178, 81-84. ceptors and I2-imidazoline sites in vitro and in vivo. J. GIRALT, M.T. & GARCIA-SEVILLA, J.A. (1989). Acute and long-term Neurochem., 61, 1602-1610. regulation of brain z2-adrenoceptors after manipulation of noradrenergic transmission in the rat. Eur. J. Pharmacol., 164, 455-466. R. Alemany et al Pheneizine down-regulates 12 imidazoline receptors 845

MOUSSEAU, D.D, MCMANUS, D.J., BAKER, G.B., JUORIO, A.V., RENOUARD, A., WIDDOWSON, P.S. & CORDI, A. (1993). [3H]- DEWHURST, W.G. & GREENSHAW, A.J. (1993). Effects of age and idazoxan binding to rabbit cerebral cortex recognises multiple of chronic antidepressant treatment on [3H]tryptamine and imidazoline 12-type receptors: pharmacological characterization [3H]dihydroalprenolol binding to rat cortical membranes. Cell. and relationship to monoamine oxidase. Br. J. Pharmacol., 109, Mol. Neurobiol., 13, 3-13. 625-631. MUAKKASSAH, S.F. & YANG, W.C.T. (1981). Mechanism of the SASTRE, M. & GARCiA-SEVILLA, J.A. (1993). Opposite age- inhibitory action of phenelzine on microsomal drug metabolism. dependent changes of M2A-adrenoceptors and non-adrenoceptor J. Pharmacol. Exp. Ther., 219, 147-155. [3Hlidazoxan binding sites (12-imidazoline sites) in the human MUNSON, P.J. & RODBARD, D. (1980). Ligand: a versatile com- brain: strong correlation of 12 with MAO-B sites. J. Neurochem., puterized approach for the characterization of ligand binding 61, 881-889. system. Anal. Biochem., 107, 220-239. TESSON, F. & PARINI, A. (1991). Identification of an imidazoline- MURRAY, M. & REIDY, G.F. (1990). Selectivity in the inhibition of guanidinium receptive site in mitochondria from rabbit cerebral mammalian cytochromes P-450 by chemical agents. Pharmacol. cortex. Eur. J. Pharmacol., 208, 81-83. Rev., 42, 85-101. TESSON, F., PRIP-BUUS, C., LEMOINE, A., PEGORIER J.-P. & PARINI, OLMOS, G., ALEMANY, R., ESCRIBA, P.V. & GARCIA-SEVILLA, J.A. A. (1991). Subcellular distribution of imidazoline-guanidinium- (1994). The effects of chronic imidazoline drug treatment on glial receptive sites in human and rabbit liver. J. Biol. Chem., 266, fibrillary acidic protein concentrations in rat brain. Br. J. Phar- 155-160. macol., 111, 997-1002. WALTHER, B., GHERSI-EGEA, J.F., MINN, A. & SIEST, G. (1986). OLMOS, G., GABILONDO, A.M., MIRALLES, A., ESCRIBA, P.V. & Subcellular distribution of cytochrome P-450 in the brain. Brain GARCIA-SEVILLA, J.A. (1993). Chronic treatment with the Res., 375, 338-344. monoamine oxidase inhibitors clorgyline and pargyline down- WANG, H., REGUNATHAN, S., MEELEY, M.P. & REIS, D.J. (1992). regulates non-adrenoceptor [3H]-idazoxan binding sites in the rat Isolation and characterization of imidazoline receptor protein brain. Br. J. Pharmacol., 108, 597-603. from bovine adrenal chromaffin cells. Mol. Pharmacol., 42, OLMOS, G., MIRALLES, A., BARTUREN, F. & GARCIA-SEVILLA, J.A. 792-801. (1992). Characterization of brain imidazoline receptors in nor- WANG, H., REGUNATHAN, S., RUGGIERO, D.A. & REIS, D.J. (1993). motensive and hypertensive rats: differential regulation by Production and characterization of antibodies specific for the chronic imidazoline drug treatment. J. Pharmacol. Exp. Ther., imidazoline receptor protein. Mol. Pharmacol., 43, 509-515. 260, 1000-1007. WARNER, M., KOHLER, C., HANSSON, T. & GUSTAFSSON, J.-A. PAETSCH, P.R., BAKER, G.B. & GREENSHAW, A.J. (1993). Induction (1988). Regional distribution of cytochrome P-450 in the rat of functional down-regulation of P-adrenoceptors in rats by 2- brain: spectral quantitation and contribution of P-450b,e and phenylethylamine. J. Pharmacol. Sci., 82, 22-24. P-450c,d. J. Neurochem., 50, 1057-1065. PILETZ, J.E., HALARIS, A., SARAN, A. & MARLER, M.R. (1990). YOSHIDA, Y. & AOYAMA, Y. (1987). Interaction of azole antifungal Elevated 3H-para-aminoclonidine binding to platelet purified agents with cytochrome P-45014DM purified from Saccahromyces plasma membranes from depressed patients. Neuropsychopharma- cerevisiae microsomes. Biochem. Pharmacol., 36, 229-235. cology, 3, 201-210. ZONNENSCHEIN, R., DIAMANT, S. & ATLAS, D. (1990). Imidazoline PILETZ, JE., HALARIS, A., SARAN, A. & MARLER, M.R. (1991). receptors in rat liver cells: a novel receptor or a subtype of Desipramine lowers tritiated para-aminoclonidine binding in M2-adrenoceptor? Eur. J. Pharmacol., 190, 203-215. platelets of depressed patients. Arch. Gen. Psychiatry, 48, 813-820. (Received September 19, 1994 Revised October 10, 1994 Accepted October 17, 1994)