1. PreparationsIsoforms m-tyramine hydroxylation K m [µM] V max [pmol/pmol CYP/min] Cl V max / K m Supersomes (Gentest) ( 0,36 mg protein/ml) Rat CYP2D2 – high reductase 129.404.960.0383 Hum. CYP2D6 – high reductase 121.0015.820.1307 Bactosomes (Cypex) (0,10 mg protein/ml) Rat CYP2D4 – low reductase 256.000.470.0018 Rat CYP2D18 – low reductase 142.800.230.0016 Hum. CYP2D6 – low reductase 86.669.550.1102 Hum. CYP2D6 – high reductase 115.4042.200.3657 Rat brain microsomes (6 mg protein/ml) 954.220.940.0010 PreparationsIsoforms p-tyramine hydroxylation K m [µM] V max [pmol/pmol CYP/min] Cl V max / K m Supersomes (Gentest) ( 0,36 mg protein/ml) Rat CYP2D2 – high reductase 563.306.270.0111 Hum. CYP2D6 – high reductase 564.1018.980.0336 Bactosomes (Cypex) (0,10 mg protein/ml) Rat CYP2D4 – low reductase 433.000.120.0003 Rat CYP2D18 – low reductase 687.900.190.0003 Rat brain microsomes (6 mg protein/ml) 1303.00.540.0004 Hydroxylation of tyramine to dopamine Izoenzyme: 1A12A22B12C62C112C132D12D22D42D182E13A2 m-tyramine –––––––+++–– p-tyramine –––––––+++–– Drug CYP2D2 Ki [µM] CYP2D4 Ki [µM] Brain microsomes Ki [µM] fluoxetine1,2510255 imipramine2,7525340 nefazodoneNo effect5350 mirtazapineNo effect thioridazine0,52,8485 HVA pg/mg tissue ** DOPAC pg/mg tissue ** p<0,01 vs control # p<0,01 vs res ControlRes αMT Res quinine Res αMT parg Res αMT quinine parg** ** # ** # **$ **+ ** ControlRes αMT Res quinine Res αMT parg Res αMT quinine parg ** p<0,01 vs control # p<0,01 vs res $ p<0,01 vs res + α MT + p<0,05 vs res+ α MT + parg DA pg/mg tissue ** ControlRes αMT Res quinine Res αMT parg Res αMT quinine parg ** ** ** # $ ** + ** p<0,01 vs control # p<0,01 vs res $ p<0,01 vs res + α MT + p<0,05 vs res+ α MT + parg Formation of dopamine from tyramine in rat brain microsomes m-tyramine [  M] hum. CYP2D6 Bactosome – low (LR) and high (HR) reductase 0100200300400500 0 5 10 15 20 25 30 V 2D6HR 2D6LR dopamine [pmol/pmolCYP/min] S tyramine [  M] 02004006008001000 m-tyramine p-tyramine S 0.00 0.05 0.10 0.15 0.20 0.25 0.30 dopamine [pmol/pmolCYP/min] V rat CYP2D18 (Bactosome – low reductase) rat CYP2D4 (Bactosome – low reductase) S m-tyramine 0200 4006008001000 0.0 0.1 0.2 0.3 0.4 0.5 p-tyramine dopamine [pmol/pmolCYP/min] tyramine [  M] V m-tyramine p-tyramine 0200400 6008001000 0.0 0.1 0.2 0.3 0.4 0.5 S dopamine [pmol/mg protein/min] tyramine [  M] quinine [  M] K i = 250 µM I -500-250 0 2505007501000 5 10 15 20 25 30 1/V m-tyr [250  M] m-tyr [500  M] m-tyr [1000  M] dopamine [pmol / mg protein / min] mg IgG / mg mikrosomal protein Anty-CYP2D4 IgG Control IgG 0.000.020.040.06 0 20 40 60 80 100 Activity (% control) Bromek E, Haduch A, Daniel WA Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland Smetna 12, 31-343 Krakow, Poland CYP2D-SYNTHESIZED DOPAMINE IN THE BRAIN. THE EFFECT OF PSYCHOTROPICS REFERENCES 1.Miksys S, Rao Y, Sellers E M, Kwan M, Mendis D, Tyndale R F: Regional and cellular distribution of CYP2D subfamily members in rat brain. Xenobiotica, 2000, 30(6), 547-564. 2.Hiroi T, Imaoka S, Funae Y: Dopamine formation from tyramine by CYP2D6. Biochem. Biophys.Research Communications, 1998, 249, 838-843. 3.Hiroi T, Kishimoto W, Chow T, Imaoka S, Igarashi T, Funae Y: Progesterone oxidation by cytochrome P450 2D isoforms in the brain. Endocrinology, 2001, 142, 3901-3908. MATERIALS AND METHODS: 1. Hydroxylation of tyramine to dopamine by cDNA-expressed CYPs: Supersomes (Gentest): - rat 1A1, 2A2, 2B1, 2C6, 2C11, 2C13, 2D1, 2D2, 2E1, 3A2 expressed from rat CYP cDNA using Baculovirus infected insect cells (high expression of NADPH P450 reductase), - human 2D6 expressed from human CYP cDNA using Baculovirus infected insect cells (high expression of NADPH P450 reductase), Bactosomes (Cypex): - rat 2D4, 2D18 - expressed from rat cDNA in Escherichia coli (low expression of NADPH P450 reductase) - human 2D6 - expressed from rat cDNA in Escherichia coli (low or high expression of NADPH P450 reductase) 2. Hydroxylation of tyramine to dopamine by rat brain microsomes: microsome preparation from the whole rat brain [3] inhibitor – quinine (50-1000  M) anti-CYP2D4 antibodies (100-400 µg IgG/ml) The amount of dopamine formed in vitro was measured using the HPLC method with electrochemical detection. 3. Hydroxylation of tyramine to dopamine in the brain in vivo: Experimental groups: Control ip Reserpine (10 mg/kg ip) Reserpine + quinine (50 mg/kg ip) Reserpine + α-mehtyl-p-tyrosine (300 mg/kg ip) Reserpine + α-methyl-p-tyrosine + pargyline (150 mg/kg ip) Reserpine + α-methyl-p-tyrosine+ quinine + pargyline The brains were isolated and divided into the structures (nucleus accumbens, substancia nigra, striatum, prefrontal cortex, brain steam, cerebellum, olfactory bulbs and rest of brain) Dopamine and its metabolits were measured using HPLC method. 4. Influence of the selected antidepressant drugs on CYP2D activity: microsome preparation from the whole rat brain cDNA-expressed CYPs (Supersomes CYP2D2 from Gentest, Bactosomes CYP2D4 from Cypex) in the absence and presence of drugs (imipramine, fluoxetine, mirtazapine or nefazodone) added in vitro. Bufuralol and its metabolite 1`-hydroxybufuralol were measured using HPLC with fluorometric detection. CONCLUSIONS: Among the rat CYP isoforms tested only CYP2D2, CYP2D4 and 2D18 were able to form dopamine from tyramine. The rat CYP2D isoforms were less efficient than human CYP2D6. The efficiency of CYP2D2/2D4/2D18 and CYP2D6 to hydroxylate tyramine to dopamine was higher for m-tyramine than p-tyramine. Brain microsomes were able to metabolise tyramine to dopamine, m-tyramine being a more favourite substrate than p-tyramine. The reaction was inhibited by a CYP2D inhibitor – quinine and anti-CYP2D4 antibodies indicating a role of CYP2D in dopamine formation Quinine, a specific CYP2D inhibitor, given to reserpinized rats after inhibition of the classic pathway of dopamine synthesis, decreased dopamine level in the striatum, which indicates dopamine formation from tyramine in the brain in vivo. INTRODUCTION: The CYP2D subfamily of cytochrome P450 in the rat consists of six isoforms (CYP2D1-5 and CYP2D18), whereas in man it has only one representative: isoform CYP2D6. CYP2D4 is regarded as a main CYP2D isoform in rat brain [1]. The CYP2D isoforms which are capable of metabolizing a number of drugs such as psychotropics, the drugs of abuse codeine and amphetamine, and neurotoxins. Furthermore, recent studies suggest that human recombinant CYP2D6 is able to catalyze the formation of dopamine from the endogenous neurochemical substrate tyramine [2]. Since both cytochrome P450 and tyramine are present in the brain, this alternative pathway of dopamine synthesis may contribute to the overall level of this neurotransmitter in the brain. The present study was aimed at determining which rat CYP isoforms (CYPs) were involved in the formation of dopamine from tyramine and wheather the reaction could take place in the brain; moreover, we investigated the influence of the antidepressant drugs imipramine, fluoxetine, mirtazapine and nefazodone on the activity of CYP2D in the brain. Dopamine plays an important role in the pharmacological and therapeutic action of psychotropic drugs. The aim of the present study was to identify and characterize rat CYP2D isoforms engaged in the hydroxylation of tyramine to dopamine, as well as to investigate the effect of selected psychotropics on the activity of CYP2D, assessed by measuring the rate of bufuralol 1’- hydroxylation (HPLC with electrochemical or fluorescent detection, respectively). The study was conducted on rat brain microsomes and two biotechnologically different preparations of c-DNA- expressed CYPs: a) rat CYP2D1 and CYP2D2 and human CYP2D6, (Supersomes, Gentest; a high expression of NADPH P450 reductase); b) rat CYP2D4 and CYP2D18, (Bactosomes, Cypex; a low expression of NADPH P450 reductase). Of the CYP2Ds isoforms tested, only CYP2D1 displayed no activity towards dopamine formation. The efficacy of all the CYP2D isoforms engaged in dopamine formation was higher for m-tyramine than for p-tyramine. The affinity of tyramine for the CYP2D isoforms tested (K m ) was as follows: CYP2D6 > CYP2D2 > CYP2D18 > CYP2D4 for m- tyramine, and CYP2D18 > CYP2D4 > CYP2D2 > CYP2D6 for p-tyramine. The V max values were compared between isoforms with a similar expression of NADPH P450 reductase, i.e. between Supersomes (CYP2D6 > CYP2D2) and Bactosomes (CYP2D4 > CYP2D18), which referred to both m- and p-tyramine. Brain microsomes catalyzed the hydroxylation of tyramine to dopamine, m- tyramine being more efficiently metabolized than p-tyramine. The reaction was inhibited by the CYP2D inhibitor quinine and CYP2D4-directed antibodies, which testifies to the contribution of CYP2D to dopamine formation in the brain. Antidepressants - fluoxetine and imipramine, as well as neuroleptics - thioridazine and perazine decreased the activity of CYP2D in brain microsomes. Using recombinant CYPs, we showed that the investigated psychotropics decreased the activity of CYP2D2 (K i = 1.25, 2.75, 0.5, 17.4  M, respectively) and CYP2D4 (K i = 10, 25, 2.8, 7.1  M, respectively) with different potency. Nefazodone inhibited only CYP2D4 (K i = 5  M), while mirtazapine was not active in this respect. Our study provides direct evidence that dopamine can be formed from tyramine by CYP2D isoforms in rat brain. That alternative pathway may be inhibited by some psychotropics which may affect their pharmacological profile. ABSTRACT: Tyrosine (phenylala.) hydroxylase Classic pathway Alternative pathway Aromatic L-amino acid decarboxylase Tyrosine hydroxylase Aromatic L-amino acid decarboxylase Aromatic L-amino acid decarboxylase rat CYP2D ? brain? dihydroxyphenylalanine phenylalanine tyrosine dopamine phenylethylamine tyramine