Copyright © 2004 by Institute of Pharmacology Polish Journal of Pharmacology Polish Academy of Sciences Pol. J. Pharmacol., 2004, 56, 727734 ISSN 1230-6002 INHIBITION OF RODENT BRAIN MONOAMINE OXIDASE AND TYROSINE HYDROXYLASE BY ENDOGENOUS COMPOUNDS – 1,2,3,4-TETRAHYDROISOQUINOLINE ALKALOIDS Antoni Patsenka, Lucyna Antkiewicz-Michaluk Department of Biochemistry, Institute of Pharmacology, Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland Inhibition of rodent brain monoamine oxidase and tyrosine hydroxylase by endogenous compounds – 1,2,3,4-tetrahydroisoquinoline alkaloids. A. PATSENKA, L. ANTKIEWICZ-MICHALUK. Pol. J. Pharmacol., 2004, 56, 727–734. Four different noncatecholic and one catecholic tetrahydroisoquinolines (TIQs), cyclic condensation derivatives of b-phenylethylamine and dopa- mine with aldehydes or keto acids, were examined for the inhibition of rat and mouse brain monoamine oxidase (MAO) and rat striatum tyrosine hy- droxylase (TH) activity. Simple noncatecholic TIQs were found to act as moderate (TIQ, N-methyl-TIQ, 1-methyl-TIQ) or weak (1-benzyl-TIQ), MAO B and MAO A inhibitors. 1-Methyl-TIQ inhibited more potently MAO-A than MAO-B; the similar but more modest effect was exerted by salsolinol. Only salsolinol markedly inhibited TH activity, being competitive with the enzyme biopterin cofactor. The inhibition of MAO and TH by TIQs is discussed in relation to their ability to regulate monoamine metabolism. Key words: monoamine oxidase, tyrosine hydroxylase, inhibition of en- zyme activity, tetrahydroisoquinoline derivatives correspondence; e-mail: [email protected] A. Patsenka, L. Antkiewicz-Michaluk Abbreviations: BBB – blood-brain barrier,BH" been found to be toxic to dopaminergic neurons – 5,6,7,8-tetrahydrobiopterin, CSF – cerebrospinal and their concentration has been shown to increase fluid, 5-HT – 5-hydroxytryptamine, MAO – mono- in parkinsonian brains or cerebrospinal fluid (CSF) amine oxidase, MPTP – 1-methyl-4-phenyl-1,2,3,6- [12]. Others, such as 1MeTIQ, show a neuroprotec- tetrahydropyridine, b-PEA – 2-phenylethylamine, tive effect against the rotenone-induced neurode- TH – tyrosine hydroxylase, THP – 1,2,3,4-tetrahydro- generation and mortality in rats [1, 5]. papaveroline, TIQ 1,2,3,4- tetrahydroisoquinoline Tyrosine hydroxylase (TH) and MAO are cru- cial enzymes for catecholaminergic neurons. The first step of catecholamines synthesis is catalyzed INTRODUCTION by TH, a rate-limiting regulatory enzyme. TH is an iron-containing 3-monooxygenase (tyrosine-tetra- In physiological conditions catecholamines hydrobiopteridine (BH4): oxygen oxidoreductase, condense with a variety of simple and biogenic al- EC 1.14.16.2, TH) expressed in catecholamine- dehydes to form tetrahydroisoquinoline (TIQ) alka- neurons in mammalian brain, adrenal medulla and loids [7, 9]. The presence of TIQs in mammals in- peripheral sympathetic nerve terminals. The main cluding humans has been widely reported [26, 28]. catabolic enzyme for brain monoamines is mono- Using high-performance liquid chromatography amine oxidase (amine: oxygen oxidoreductase, (HPLC) with electrochemical, fluorescence or gas flavin-containing, EC 1.4.3.4, MAO). It occurs chromatography with mass spectrometry detection usually as the dimer and is classified into isoen- many authors have shown the presence of TIQ de- zymes A and B with a different substrates and in- rivatives such as TIQ, 1-methyl-TIQ (1MeTIQ), hibitors, which are encoded by two different genes. N-methyl-TIQ (NMeTIQ), 1-benzyl-TIQ (1BnTIQ), These enzymes can also produce hydrogen perox- 1-methyl-6,7,-dihydroxy-TIQ (salsolinol) and tet- ide and other reactive oxygen species as additional rahydropapaveroline (THP) in rodent and human products of their reactions; oxidative deamination brain (see a review by Nagatsu [24]). On the other also results in the production of very reactive bio- hand, TIQs are also widely distributed in the envi- genic aldehydes [8, 13, 35]. ronment, being present in many plants and food- Salsolinol derivatives have been reported to in- stuffs, such as port wine, bananas, beer and milk hibit the activity of tryptophan hydroxylase and TH [17, 27]. The exogenously administered TIQs eas- [30, 34, 40], some hydroxylated TIQs have shown ily cross the blood-brain barrier (BBB) and migrate to affect MAO activity [6, 22, 23]. into the brain, producing behavioral and biochemi- However, till now it has not been clarified cal effects in monoamine systems [2, 3, 11, 21]. whether TIQs without a catechol structure can dis- Some of them, e.g. salsolinol, a product of the con- turb the monoamine synthesizing and metabolizing densation of dopamine (DA) with acetaldehyde enzymes. The aim of the present study was to de- and/or pyruvate, have aroused considerable interest termine the effect of four noncatecholic TIQs (TIQ, as it may be involved in the pathogenesis of alco- 1MeTIQ, NMeTIQ and 1BnTIQ) on the activity of holism and neurodegeneration [24]. Other-than- MAO and TH and compare them with salsolinol. aromatic amines (phenylethylamines) may also un- dergo condensation with aldehydes yielding such TIQs as TIQ and 1MeTIQ. These alkaloids are pre- MATERIALS and METHODS sumably activated via N-methylation and are oxi- Chemicals and animals dized by monoamine oxidase (MAO) to form a neurotoxic N-methylisoquinolinium ion, the same L-[3,5-H] tyrosine (51 Ci/mmol), 5-hydroxy-3-in- as MPP+ (1-methyl-4-phenyl-1,2,3,6-tetrahydropy- dolyl [14C] ethyl-2-amine creatinine sulfate ([14C]- ridinium ion) from 1-methyl-4-phenyl-1,2,3,6-tetra- 5-HT, 56.7 mCi/mmol) and 2-phenyl[14C]ethylami- hydropyridine (MPTP) neurotoxin, which produces ne hydrochloride ([14C-b-PEA, 57 mCi/mmol) was a clinical syndrome similar to Parkinson’s disease. purchased from Amersham (UK). L-tyrosine, 5-HT, Indeed, it has been shown that salsolinol is a weak b-PEA, a-methyl-p-tyrosine, salsolinol. HCl, TIQ, substrate for N-methyltransferase in the rat sub- THP, BH4, dithiotreitol, bovine serum albumin stantia nigra, but its endogenous concentration is (BSA) and catalase were obtained from Sigma- minute [26]. Some TIQs, such as 1BnTIQ, have Aldrich Chemical Co. (St. Louis, MO, USA). Clor- 728 Pol. J. Pharmacol., 2004, 56, 727734 INHIBITION OF MAO AND TH BY TETRAHYDROISOQUINOLINES giline and L-deprenyl came from RBI (MA, USA). ethyl acetate/toluene (1:1, v/v) (POCH, Poland) 1MeTIQ, NMeTIQ and 1BnTIQ were synthesized and 1 ml of the organic layer was withdrawn and in the Department of Medicinal Chemistry of the mixed with 4 ml of the scintillation fluid. Radioac- Institute of Pharmacology, Polish Academy of Sci- tivity was determined by liquid scintillation spec- ences in Kraków. All other reagents were of ana- trometry (Beckman LS 6500 counter). Data were lytical grade. expressed in nmol/min/mg of protein of 5-TH- and Male mice (18–20 g) and Wistar rats (240–280 g) b-PEA-oxidized products for MAO-A and B, were used for tissue preparation. All the procedures respectively. were carried out according to the National Institutes of Health Guide for Care and Use of Laboratory Animals, and were approved by the Institute’s In- Determination of tyrosine hydroxylase activity ternal Bioethics Commission. TH activity was determined by a radiochemical assay according to the method of Rejnhard et al. [33] with further modifications. Determination of monoamine oxidase activity The tissue was prepared in the same way as for MAO-A and MAO-B activities were deter- the MAO assay. After the last centrifugation, the mined by a radiochemical assay according to the supernatant was used in TH assay and the protein method of Tipton and Yodim [38], with further concentration was about 3.0 mg/ml. TH reactions modifications. were carried out at a total volume of 100 mlin 5-Hydroxy-3-indolyl [14C] ethyl-2-amine cre- Brand 1.5 ml polyethylene tubes. Reaction mixture atinine sulfate and 2-phenyl[14C]ethylamine hydro- contained the following components: 75–80 mgof chloride were used as the substrates for MAO-A protein; MES buffer, 50 mM, pH 6.0; dithiothreitol, and MAO-B, respectively. Animals (rats or mice) 5 mM; catalase (Sigma C-100, 47 000 U/mg), 90 mg; 3 were sacrificed by decapitation, brain tissue was re- L-tyrosine/L-[ring-3,5- H]tyrosine, 50 mM/1 mCi moved, dissected and frozen till further assayed. per sample; (6R)-L-erythro-5,6,7,8,-tetrahydrobio- Pieces of the frontal cortex, striatum and brainstem pterin(BH4), 125–500 mM. Blank values, obtained of rats or whole brains, except for the cerebellum of by omitting BH, equaled the blank values when tis- mice, were homogenized in 50 mM phosphate sue was discarded. Thus, the last blank was rou- buffer (pH 7.4, 10% w/v) using a Teflon ho- tinely used. The reaction was initiated by adding mogenizer. All manipulations were carried out at BH4, incubated at 37°C for 20 min, and was termi- 4°C. Homogenates were centrifuged at 1000 × g nated with 0.7 ml of a stirred suspension of 7.5% for 5 min; the sediment was discarded and the su- (w/v) charcoal (POCh, Poland) in 0.1 M HCl. The pernatant was recentrifuged at 11800 × g for mixtures were then rapidly stirred for 20 s and cen- 30 min, at +4°C. The pellet (mitochondrial fraction) trifuged at 10 000g for 3 min. Aliquots 0.4 ml of was resuspended in the same buffer to final protein the clear supernatant were transferred to 5 ml scin- concentration of 1.0–1.5 mg/ml, determined by tillation vials, and 4.0 ml of Aquascint (BIOCARE, Lowry’s et al. method [15]. Poland) were added. TH utilizes O2 to produce 3 3 The preincubation medium (50 mM phosphate DOPA and H-H2O from H-tyrosine. Unreacted ty- buffer, pH 7.4) contained 100 ml of displacing solu- rosine and catecholamines were absorbed with an tion with TIQ-alkaloids, ranging from 1.0 mMto aqueous slurry of activated charcoal, and the re- 3 1.0 mM, or the MAO inhibitors clorgiline or leased H-H2O was analyzed by liquid scintillation L-deprenyl (0.1 nM–0.5 mM) and 100 ml of resus- counting.