Cytochrome P450 2D Catalyze Steroid 21-Hydroxylation in the Brain

Abstract

mRNA of cytochrome P450 21-hydroxylase (P450c21) is expressed in the brain, but little is known about the enzymatic properties of P450c21 in the brain. In the present study, we showed, by using various recombinant cytochrome P450 (CYP)2D enzymes and anti-CYP2D4- or P450c21-specific antibodies, that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D isoforms. Rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain, possess 21-hydroxylation activity for both progesterone and 17alpha-hydroxyprogesterone. In rat brain microsomes, these activities were not inhibited by anti-P450c21 antibodies, but they were effectively inhibited by the CYP2D-specific chemical inhibitor quinidine and by anti-CYP2D4 antibodies. mRNA and protein of CYP2D4 were expressed throughout the brain, especially in cerebellum, striatum, pons, and medulla oblongata, whereas the mRNA and protein levels of P450c21 were extremely low or undetectable. These results support the idea that CYP2D4, not P450c21, works as steroid 21-hydroxylase in the brain. Allopregnanolone, a representative gamma-aminobutyric acid receptor modulator, was also hydroxylated at the C-21 position by recombinant CYP2D4 and CYP2D6. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited by fluoxetine with an IC(50) value of 2 microm, suggesting the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation is modified by central nervous system-active drugs such as fluoxetine.

Effect of Psychotropic Drugs on the 21-Hydroxylation of Neurosteroids, Progesterone and Allopregnanolone, Catalyzed by Rat CYP2D4 and Human CYP2D6 in the Brain

“…15,18) Enzyme Assay and Quantification of 21-Hydroxylated Metabolites of PROG and ALLO Progesterone 21-hydroxylation activity was measured by the method described previously 29) with a minor modification. The incubation mixture consisted of microsomes from cells containing recombinant CYP2D4 (50 pmol/ml) or CYP2D6 (30 pmol/ml), 2, 5, 10, or 20 mM (for CYP2D4) or 4, 10, 25, or 100 mM (for CYP2D6) PROG, 0, 2, 5, 20 or 50 mM of the tested psychotropic drug, 1 mM NADPH, and 100 mM potassium phosphate buffer (pH 7.4) in a final volume of 0.5 ml.…”

“…In addition, CYP2D4 mRNA is expressed in the rat brain, 14,15) and the reverse transcriptase-polymerase chain reaction (RT-PCR) product from CYP2D4, the predominant CYP2D isoform in the rat brain, is more abundant in the cerebellum, striatum, pons, and medulla oblongata.…”

“…5,8,11,12) Interestingly, CYP2D6 is expressed in the human brain, especially the midbrain, 13) as well as in the liver. In addition, CYP2D4 mRNA is expressed in the rat brain, 14,15) and the reverse transcriptase-polymerase chain reaction (RT-PCR) product from CYP2D4, the predominant CYP2D isoform in the rat brain, is more abundant in the cerebellum, striatum, pons, and medulla oblongata. 16) However, the physiological and pharmacological functions of CYP2D isoforms in the brain are still unknown.…”

“…5,15) Many of the psychotropic drugs, including fluoxetine (a selective serotonin reuptake inhibitor, SSRI), imipramine (a tricyclic antidepressant and a serotonin and noradrenaline reuptake inhibitor), and desipramine (a noradrenaline reuptake inhibitor), are metabolized by CYP2D and/or inhibit the CYP2D-mediated metabolic activities. 8,15,[19][20][21][22][23] One of the dopamine uptake sites in the brain displays high affinity for dopamine uptake inhibitors such as mazindol, a noradrenaline reuptake inhibitor. 24,25) The other site displays rather high affinity for piperizine derivatives and has been termed the piperazine acceptor site.…”

Temporal control of glucocorticoid neurodynamics and its relevance for brain homeostasis, neuropathology and glucocorticoid-based therapeutics

“…Despite the fact that the enzymatic activity of Cytochrome P450 21-hydroxylase (P450c21), the main cytochrome 450 enzyme responsible for converting 17-OH-progesterone and progesterone to GC precursors (11-deoxycortisol and 11-deoxycorticosterone respectively) in AGs, is almost absent from CNS tissues (Mellon and Miller, 1989), various brain areas are capable of locally altering the GCs levels via (i) cytochrome P450 2D (CYP2D) isoforms (like the CYP2D6 isoform in human brain, found in most corticolimbic areas, basal forebrain and cerebellum) (Miksys and Tyndale, 2004) which perform the steroid 21-hydroxylation (replacing P450c21 lack of activity within the brain) (Kishimoto et al, 2004), (ii) 11β-hydroxysteroid dehydrogenase (11βHSD), that either increase the turnover between active and inactive GCs (isoform 1, found in corticolimbic regions, hypothalamic areas, brainstem and cerebellum) or solely degrade active GCs to inactive molecules (isoform 2, found in GC-insensitive brain regions like the circumventricular organs) (de Kloet et al, 2009), (iii) cytochrome P450 11-betahydroxylase (P450c11β) enzyme (found in neocortex) responsible for converting GC precursors to active hormones (cortisol and corticosterone respectively), and (iv) 5a-reductase (found in hypothalamic areas, corticolimbic regions and circumventricular organs), which directs corticosterone precursors to other metabolic pathways (Mensah-Nyagan et al, 1999) (Table 2).…”