Molecular and Cellular Pharmacology
The ability of cytochrome P450 2D isoforms to synthesize dopamine in the brain: An in vitro study

https://doi.org/10.1016/j.ejphar.2009.09.062Get rights and content

Abstract

The present study was aimed at determining which rat cytochrome P450 (CYP) isoforms are involved in the hydroxylation of tyramine to dopamine and at determining whether the reaction can take place in the brain. Moreover, we examined the relative distribution of the CYP2D subfamily's activity in the rat brain, focusing our attention on dopaminergic structures. The study was conducted with cDNA-expressed CYP isoforms (rat CYP1A1, 2A2, 2B1, 2C6/11/13, 2D1/2/4/18, 2E1, 3A2 and human CYP2D6) and with rat brain microsomes. Of the rat CYP isoforms tested, only CYP2D2, 2D4 and 2D18 (but not CYP2D1) were capable of forming dopamine from tyramine. The rat CYP2D isoforms were less efficient than the human CYP2D6 and the efficiency of both human and rat enzymes was higher for m-tyramine (Km = 256, 143 and 87 μM; Vmax = 0.47, 0.23 and 9.55 pmol/pmol CYP/min for CYP2D4, 2D18 and 2D6, respectively) than for p-tyramine (Km = 433 and 688 μM, Vmax = 0.12 and 0.19 pmol/pmol CYP/min for CYP2D4 and 2D18, respectively). Brain microsomes were able to metabolize tyramine to dopamine. The reaction was inhibited by the CYP2D inhibitor quinine and by anti-CYP2D4 antibodies, which suggests that CYP2D4 is the isoform governing tyramine hydroxylation to dopamine in the rat brain. A relatively high level of CYP2D activity (bufuralol 1′-hydroxylation) was found in the substantia nigra, the cerebellum, the nucleus accumbens and the truncus cerebri. The results are discussed in the context of the likelihood of CYP2D-mediated dopamine synthesis in vivo, the implications for Parkinson's disease and the addiction process.

Introduction

The CYP2D subfamily of cytochrome P450 (CYP) enzymes consists of six isoforms in the rat (CYP2D1–5 and CYP2D18), but it has only one representative isoform, CYP2D6, in man (Funae et al., 2003). The mentioned CYP2D isoforms share 71–99% overall amino acid sequence identity. They often show an overlap in substrate specificity, e.g., the 1′-hydroxylation of bufuralol or the O-demethylation of dextromethorphan, but they differ in their catalytic competency for specific reactions such as debrisoquine 4-hydroxylation (catalyzed by 2D2 and 2D6), bufuralol 1′2′-ethenylation, steroid 21-hydroxylation (catalyzed by 2D4 and 2D6) and lidocaine 3-hydroxylation (catalyzed by 2D1–2D4, but not by 2D6) (Hiroi et al., 2002, Kishimoto et al., 2004, Kobayashi et al., 2002, Schulz-Utermoehl et al., 1999, Wan et al., 1997).

The CYP2D subfamily's isoforms are present in hepatic and extrahepatic tissues (Hiroi et al., 1998a, Miksys et al., 2000, Norris et al., 1996, Riedl et al., 1999). CYP2D4 is regarded as the main CYP2D isoform in the rat brain, while CYP2D1 and CYP2D2 are the most abundant CYP2D isoforms in the liver (Funae et al., 2003, Hiroi et al., 1998a, Komori, 1993, Wyss et al., 1995). The presence of CYP2D protein in the brain was observed predominantly in the cerebellum, the cortex, the amygdaloid complex, the olfactory bulbs, as well as in the hippocampus, the substantia nigra and the striatum (Miksys et al., 2000, Siegle et al., 2001). Brain CYP2D isoforms, like their hepatic homologues, exhibit an enzymatic activity toward endo- and xenobiotics (Miksys and Tyndale, 2002, Voirol et al., 2000; Strobel et al., 2001).

It has been suggested that CYP2D6 polymorphism may influence personality traits, which suggests the importance of CYP2D in the metabolism of endogenous neuroactive substrates in the brain (Bertilsson et al., 1989, Dorado et al., 2007, Gonzáles et al., 2008, Llerena et al., 1993). The brain CYP2D isoforms seem to play an important role in the local metabolism of neurosteroids (Hiroi et al., 2001, Kishimoto et al., 2004), serotonin (Yu et al., 2003) and dopamine (Hiroi et al., 1998b, Thompson et al., 2000). Hiroi et al., 1998b, Niwa et al., 2004 showed that human cDNA-expressed CYP2D6 and liver microsomes were capable of catalyzing the aromatic hydroxylation of tyramine to dopamine.

Since both tyramine and CYP2D are present in the brain, it is possible that the hydroxylation of tyramine to dopamine takes place in this organ, hence this pathway may constitute an alternative to tyrosine hydroxylase-mediated dopamine synthesis. The present study was aimed at determining which rat CYP isoforms are involved in the hydroxylation of tyramine to dopamine and at determining whether the reaction can take place in brain microsomes. It was of particular interest to us to find out whether CYP2D4, the main brain isoform of the six rat CYP2D isoforms, was capable of catalyzing that reaction. Rat CYP2D isoforms were compared to a human CYP2D6. Moreover, we examined the relative distribution of the CYP2D subfamily's activity in the brain, focusing our attention on dopaminergic structures. The results constitute a base for further in vivo study.

Section snippets

Chemicals

Dopamine hydrochloride, quinine, p-tyramine, m-tyramine and NADPH were purchased from Sigma (St. Louis, USA). Both tyramines were free from dopamine contamination, which was tested using a high performance liquid chromatography (HPLC) method described below. The polyclonal antibody, anti-rat CYP2D4 rabbit antiserum (Kishimoto et al., 2004), bufuralol and 1′-hydroxybufuralol (Hiroi et al., 2002) were donated by Dr. Y. Funae of the Osaka City University Medical School, Japan. All the organic

Hydroxylation of tyramine to dopamine by cDNA-expressed CYP isoforms

The main rat CYP isoforms, expressed in insect (Supersomes) or bacteria cells (Bactosomes), were tested to ascertain which were capable of hydroxylating tyramine to dopamine. m-Tyramine and p-tyramine were used as substrates. In the case of both m-tyramine and p-tyramine, only CYP2D2, CYP2D4 and CYP2D18 displayed some activity for dopamine formation. The other nine CYP isoforms (1A1, 2A2, 2B1, 2C6, 2C11, 2C13, 2D1, 2E1, 3A2) tested had no such activity. Control Supersomes or Bactosomes (not

Discussion

This is the first report to demonstrate that the hydroxylation of tyramine to dopamine may take place in the rat brain microsomes, and to indicate that of the rat CYP isoforms tested, only CYP2D2, CYP2D4 — the main brain isoform and CYP2D18 (but not CYP2D1) are capable of forming dopamine from tyramine. Moreover, this study shows that the highest level of CYP2D activity in the brain is reached in the substantia nigra — the beginning of the nigrostriatal pathway involved in Parkinson's disease.

Acknowledgements

The authors are very grateful to Dr. Y. Funae of the Osaka City University Medical School, Japan for the generous donation of a polyclonal antibody (anti-rat CYP2D4), as well as bufuralol and 1′-hydroxybufuralol.

This study was supported by Grant no. 2 P05F 002 29 from the Ministry of Science and Higher Education (Warszawa, Poland) and by statutory funds from the Institute of Pharmacology, Polish Academy of Sciences (Kraków, Poland). The sponsors had no further role in the study design; in the

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