help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2003-1109
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/2/699    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kishimoto, W.
Right arrow Articles by Funae, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kishimoto, W.
Right arrow Articles by Funae, Y.
Endocrinology Vol. 145, No. 2 699-705
Copyright © 2004 by The Endocrine Society

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

Wataru Kishimoto, Toyoko Hiroi, Masakazu Shiraishi, Mayuko Osada, Susumu Imaoka, Shiro Kominami, Takashi Igarashi and Yoshihiko Funae

Department of Chemical Biology (W.K., T.H., M.S., M.O., S.I., Y.F.), Osaka City University Medical School, Osaka 545-8585, Japan; Department of Drug Metabolism and Pharmacokinetics (W.K., T.I.), Kawanishi Pharma Research Institute, Nippon Boehringer Ingelheim Co., Ltd., Hyogo 666-0193, Japan; and Faculty of Integrated Arts and Sciences (S.K.), Hiroshima University, Higashi-Hiroshima 739-8521, Japan

Address all correspondence and requests for reprints to: Yoshihiko Funae, Ph.D., Department of Chemical Biology, Osaka City University Medical School, 1-4-3 Asahimachi, Abeno-ku, Osaka 545-8585, Japan. E-mail: funae{at}med.osaka-cu.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 17{alpha}-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 IC50 value of 2 µM, 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
STEROID 21-HYDROXYLATION IS an obligatory conversion for the biosynthesis of the main adrenal steroids, aldosterone, cortisol, and corticosterone. In the adrenal gland, cytochrome P450 21-hydroxylase (P450c21) converts progesterone and 17{alpha}-hydroxyprogesterone to 11-deoxycorticosterone and 11-deoxycortisol, respectively. However, there is no published evidence concerning de novo synthesis of adrenal steroids by P450c21 in the brain, and although the brain possesses 21-hydroxylase activity (1), it expresses only a very small amount of P450c21 mRNA (2, 3). These findings, and the residual 21-hydroxylase activity found in human patients with congenital adrenal hyperplasia due to a defective P450c21 gene (4, 5), suggest the existence in the brain of another 21-hydroxylase different from the adrenal P450c21.

Cytochrome P450 (CYP)2D4 and CYP2D6 are CYP2D isoforms predominantly expressed in the brains of rats and humans, respectively (6, 7, 8, 9, 10). Recent studies have revealed that CYP2D isoforms contribute not only to the metabolism of a large number of clinically relevant drugs in the liver, but also to the biosynthesis of endogenous substances such as dopamine (11). In addition, we showed previously that CYP2D4 and CYP2D6 generate 11-deoxycorticosterone through 21-hydroxylation of progesterone, and that this activity in rat brain microsomes could be effectively inhibited by anti-CYP2D4 antibodies (12). Thus, we hypothesized that the CYP2Ds, not P450c21, are responsible for steroid 21-hydroxylation in the brain. The physiological functions of the brain CYP2D isoforms have not so far been fully elucidated.

In the present study, we investigated whether CYP2D isoforms catalyze steroid 21-hydroxylation in the brain by using various recombinant CYP2D enzymes. In addition to progesterone and 17{alpha}-hydroxyprogesterone, allopregnanolone was recognized to be a substrate for brain CYP2D, based on the effects of specific antibodies against CYP2D4 or P450c21. We also examined regional variations of CYP2D4 and P450c21 in brain at the mRNA and protein levels by using RT-PCR and Western blot analysis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Chemicals and animals
Progesterone, 11-deoxycorticosterone, 17{alpha}-hydroxyprogesterone, 11-deoxycortisol, allopregnanolone, 3{alpha},5{alpha}-tetrahydrodeoxycorticosterone, 5{alpha}-dihydroprogesterone, 5{alpha}-dihydrodeoxycorticosterone, quinidine, and fluoxetine were purchased from Sigma-Aldrich (St. Louis, MO). Bufuralol was purchased from Daiichi Pure Chemicals Co., Ltd. (Tokyo, Japan). Nicotinamide adenine dinucleotide phosphate (reduced) (NADPH) was obtained from the Oriental Yeast Co., Ltd. (Tokyo, Japan). Other reagents and organic solvents were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Male Sprague Dawley rats, obtained from Nippon Clea (Kyoto, Japan), were maintained in a 12-h light, 12-h dark cycle (lights on at 0600 h) with free access to water and pelleted food.

Recombinant CYP2D-expressing microsomes and antibody
Rat CYP2D1 and CYP2D3 cDNAs were isolated from a rat liver cDNA library. Rat CYP2D2 cDNA was amplified from a rat liver cDNA library by PCR. Rat CYP2D4 cDNA was amplified from rat brain total RNA by RT-PCR. Human CYP2D6 cDNA was amplified from a human liver cDNA library by PCR. CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D6 enzymes were expressed in Saccharomyces cerevisiae, as reported previously (13, 14), and the microsomal fraction was prepared from the yeast cells and used as recombinant enzymes. The contents of recombinant CYP2D1, CYP2D2, CYP2D3, CYP2D4, and CYP2D6 were 95.5, 49.0, 22.8, 90.7, and 56.4 nmol/mg, respectively.

Anti-CYP2D4 antibodies were raised in a female Japanese White rabbit using recombinant CYP2D4 as the immunogen. Anti-CYP2D4 antibodies recognized rat CYP2D4 and inhibited CYP2D4 catalytic activity. Anti-P450c21 antibodies, elicited in a male white rabbit using purified bovine adrenocortical microsomes as the immunogen (15), recognized and cross-reacted to rat P450c21.

Preparation of rat brain microsomes
Adult male rats were killed by decapitation, and their brains, livers, and adrenal glands were immediately removed by dissection. Brains were divided into nine regions: cerebrum, cerebellum, midbrain, medulla oblongata, hippocampus, thalamus, hypothalamus, pons, and striatum. Rat tissue microsomes from each brain region were prepared as described previously, with some minor modifications (16). All animal treatments were performed according to standard methods of humane animal care. The protocol for this study was approved by the Committee on Animal Care and Use of Osaka City University Medical School.

Microsomal steroid 21-hydroxylation
Progesterone, 17{alpha}-hydroxyprogesterone, 5{alpha}-dihydroprogesterone, or allopregnanolone (12.5 nmol) was incubated at 37 C with rat brain microsomes (0.5 mg protein) or recombinant CYP2D isoforms (90 pmol) in a final volume of 0.5 ml 0.1 M potassium phosphate buffer (pH 7.4). To study chemical inhibition, various amounts of quinidine or fluoxetine were added at a final concentration of organic solvent of less than 0.5%. For the immunoinhibition study, anti-CYP2D4 and anti-P450c21 antibodies were used. Rat brain microsomes were preincubated with various amounts of anti-CYP2D4 or anti-P450c21 antibodies at room temperature for 15 min. After the preincubation, substrate and 0.1 M potassium phosphate buffer (pH 7.4) were added to the mixture on ice. The reaction was initiated by adding NADPH (0.5 µmol) and terminated with the addition of 2 ml ethyl acetate. After extraction with ethyl acetate, the organic phase was collected and evaporated under vacuum on a rotary evaporator. The resulting residue was dissolved in methanol and injected onto a HPLC apparatus with an ODS-80Ts column (2.0 x 150 mm; Tosoh Corporation, Tokyo, Japan). Bufuralol 1'2'-ethenylation activity (a probe specific to rat CYP2D4) was measured as described previously (17).

Quantification of metabolites were performed by liquid chromatography/tandem mass spectrometry (LC-MS/MS) methods as described previously, with minor modifications (12). To quantify metabolites, selected-reaction monitoring mode was applied for 11-deoxycorticosterone (m/z 331.2–295.2), 11-deoxycortisol (m/z 347.2–311.2), 5{alpha}-dihydrodeoxycorticosterone (m/z 315.2–278.2), and 3{alpha},5{alpha}-tetradeoxycorticosterone (m/z 317.2–281.2). The LCQ instrument was interfaced to a computer workstation running Finnigan Mat LCQ Navigator software. Microsomal activity was quantified from the peak area, using linear calibration plots obtained by LC-MS/MS.

Expression of mRNA and protein in the brain
Total RNA was extracted from frozen tissue homogenates using total RNA isolation reagent (Nippon Gene, Toyama, Japan). After deoxyribonuclease treatment, total RNA was reverse-transcribed using an RNA PCR kit (Takara Shuzo Co., Ltd., Kyoto, Japan) according to the manufacturer’s instructions. For conversion of total RNA to cDNA, a reaction mixture (20 µl) was prepared containing 5 U reverse transcriptase (RT) avian myeloblastosis virus (Takara Shuzo), 1x RT-PCR buffer [10 mM Tris-HCl (pH 8.3), 50 mM KCl], 1.5 mM MgCl2, 1 mM deoxynucleotide triphosphates, 2.5 µM random 9-mer primers, 20 U ribonuclease inhibitor, and 2 µg total RNA. The reaction was carried out at 55 C for 60 min. RT was terminated by heating the reaction mixture to 99 C for 5 min, followed by rapid chilling on ice. RT reaction mixtures, including cDNA products, were stored at -20 C until used. A single cDNA produced from total RNA was amplified by PCR with primers for CYP2D4, and P450c21. Specific oligonucleotide primer pairs for these isoforms were synthesized. Sense and antisense primer sequences for CYP2D4 were 5'-GACCAGTCGGGCTTTGGACCAC-3' and 5'-CGAAGGCCTTCTTTCCAGAG-3' (nucleotide positions 3094–3115 and 4179–4198 in the rat CYP2D4 gene sequence), respectively (18). Those for P450c21 were 5'-AGGAGCTGAAGAGGCACAAG-3' and 5'-GAGGTAGCTGCATTCGGTTC-3' (nucleotide positions 719–738 and 1005–1024 in the rat CYP21 gene sequence), respectively (19). The expected product sizes from CYP2D4 and P450c21 were 420 and 306 bp, respectively. For PCR amplification of cDNA, the PCR was allowed to proceed for 35 cycles in 50-µl aliquots of reaction mixture containing 1x polymerase reaction buffer (10 mM Tris-HCl (pH 8.3), 50 mM KCl), 1.5 mM MgCl2, 0.2 mM deoxynucleotide triphosphates, 1 U Gold Taq DNA polymerase (Applied Biosystems, Foster City, CA), 0.1 µg cDNA and 200 nM concentration of the specific primers. Aliquots (10 µl) of amplified cDNA products were separated by electrophoresis using 2.0% agarose gels. Gels were stained with ethidium bromide and visualized under UV light. Images of the gels were recorded with a Color Image Freezer (Atto Corp., Tokyo, Japan).

The microsomal fractions were denatured for 5 min at 90 C and subjected to electrophoresis using 10% polyacrylamide gels. After electrophoresis, proteins were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA) for 90 min. Blotted membranes were washed three times with PBS containing 0.05% Tween 20 and blocked for 15 min. Blots were then probed with antibodies against CYP2D4 (1:500) or P450c21 (1:500) for 60 min at room temperature. After primary antibody treatment, the blots were washed and treated with a Vectastain avidin/biotin complex kit (Vector, Burlingame, CA) according to the manufacturer’s protocol. The protein bands were detected with 4-chloro-1-naphthol (Bio-Rad). For quantitative analysis, images of the bands were captured with a scanner, and densitometric analysis was performed using NIH Image 1.61 software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Steroid 21-hydroxylation by recombinant CYP2D isoforms
One isoform (CYP2D6) in humans and four major isoforms (CYP2D1, CYP2D2, CYP2D3, and CYP2D4) in rats have been identified (18, 20). We investigated whether these rat and human CYP2D isoforms catalyze steroid 21-hydroxylation. The microsomal fraction containing CYP2D1, CYP2D2, CYP2D3, CYP2D4, or CYP2D6 prepared from yeast cells, was used as recombinant enzyme (13, 14). Each recombinant enzyme was incubated with progesterone or 17{alpha}-hydroxyprogesterone, and the 21-hydroxylated product, 11-deoxycorticosterone or 11-deoxycortisol, respectively, was identified by LC-MS/MS and quantified by comparison with an authentic sample. As shown in Fig. 1Go, rat CYP2D1, CYP2D2, and CYP2D3 had no 21-hydroxylation activity toward progesterone or 17{alpha}-hydroxyprogesterone. In contrast, rat CYP2D4 possessed 21-hydroxylation activity toward both progesterone and 17{alpha}-hydroxyprogesterone, amounting to 1.06 ± 0.17 and 0.085 ± 0.005 pmol/min·pmol P450, respectively. In humans, the progesterone and 17{alpha}-hydroxyprogesterone 21-hydroxylation activities of CYP2D6 were 0.18 ± 0.09 and 0.062 ± 0.005 pmol/min·pmol P450, respectively. These results indicate that rat CYP2D4 and human CYP2D6, the predominant CYP2D isoforms in the brain, can catalyze 21-hydroxylation of steroids.



View larger version (22K):
[in this window]
[in a new window]
  		FIG. 1. Steroid 21-hydroxylation by recombinant CYP2D-expressing microsomes. Recombinant microsomes expressing various CYP2D isoforms (CYP2D1, -2D2, -2D3, -2D4, and -2D6) were incubated with 12.5 nmol progesterone (A) or 17{alpha}-hydroxyprogesterone (B) in a final volume of 0.5 ml 0.1 M potassium phosphate buffer (pH 7.4). Data are expressed as the mean ± SD (n = 3).

 
The kinetic parameters of recombinant CYP2D4-mediated 21-hydroxylation of progesterone and 17{alpha}-hydroxyprogesterone were calculated (Table 1Go). The Michaelis-Menten constant (Km) values for progesterone and 17{alpha}-hydroxyprogesterone were 15.4 ± 2.9 and 17.6 ± 5.6 µM, respectively, which were higher than those obtained from P450c21 recombinant enzyme expressed in the yeast (i.e. 0.33 and 0.23 µM, respectively) (21). The maximum velocity (Vmax) values were 1.58 ± 0.10 and 0.14 ± 0.02 pmol/min·pmol P450, respectively. The levels of steroid hormones in the blood circulation are very low (22); however, the levels in the steroidogenic tissues are thought to be relatively high. In fact, the Km value of the steroidogenic CYP isoform CYP7 for steroid hormone was reported to be about 13 µM (23). The Km values of CYP2D4 for these 21-hydroxylations are probably sufficient for the in vivo reaction to be significant.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Kinetic parameters of steroid 21-hydroxylation by CYP2D4

 
Steroid 21-hydroxylation by rat brain microsomes
CYP2D4 is known to be expressed in rat brain (6, 8, 9). We therefore examined the contribution of CYP2D4 to rat brain microsomal steroid 21-hydroxylation. Rat brain microsomes catalyzed 21-hydroxylation of progesterone and 17{alpha}-hydroxyprogesterone with activities of 0.051 ± 0.035 and 0.024 ± 0.010 pmol/min·mg protein, respectively, at the substrate concentration of 25 µM. To investigate which enzyme (i.e. CYP2D4 or P450c21) is the main mediator of rat brain microsomal 21-hydroxylation, the CYP2D-specific inhibitor quinidine, or anti-CYP2D4 antibodies, or anti-P450c21 antibodies were added to the reaction mixture of rat brain microsomes. Quinidine and anti-CYP2D antibodies did not inhibit rat adrenal progesterone or 17{alpha}-hydroxyprogesterone 21-hydroxylation, which is catalyzed by adrenal P450c21 (data not shown). Furthermore, anti-P450c21 antibodies did not inhibit rat brain microsomal bufuralol 1'2'-ethynylation, which is a specific reaction for CYP2D4 (17), but inhibited rat adrenal microsomal P450c21 catalytic activities (data not shown). As shown in Fig. 2Go, A and B, the rat brain microsomal 21-hydroxylation of both progesterone and 17{alpha}-hydroxyprogesterone was almost completely inhibited by quinidine in a dose-dependent manner. These activities were not inhibited by anti-P450c21 antibodies, but were effectively inhibited by anti-CYP2D antibodies (Fig. 2Go, C and D). These results support the view that rat brain microsomal steroid 21-hydroxylation is catalyzed not by P450c21, but by CYP2D4.



View larger version (32K):
[in this window]
[in a new window]
  		FIG. 2. Chemical inhibition and immunoinhibition of rat brain microsomal steroid 21-hydroxylation. Rat brain microsomes (0.5 mg) were incubated with 12.5 nmol progesterone (A) or 17{alpha}-hydroxyprogesterone (B) in the presence of various concentrations of the CYP2D-specific inhibitor quinidine in a final volume of 0.5 ml 0.1 M potassium phosphate buffer (pH 7.4). Rat brain microsomes (0.5 mg) were preincubated with various amounts of anti-CYP2D antibodies (filled circle) or anti-P450c21 antibodies (open circle) at room temperature for 15 min. After preincubation, 12.5 nmol progesterone (C) or 17{alpha}-hydroxyprogesterone (D), and 0.1 M potassium phosphate buffer (pH 7.4) were added to the mixture on ice. The reaction was initiated by adding NADPH (0.5 µmol). Progesterone (A and C) and 17{alpha}-hydroxyprogesterone (B and D) 21-hydroxylation activities were expressed as percentage of the control.

 
Regional distribution of CYP2D4 and P450c21 in rat brain
To examine the expression of CYP2D4 and P450c21 mRNAs in rat brain, total RNA extracted from each brain region was subjected to RT-PCR. RNA extracts of the liver and adrenal gland served as positive controls of CYP2D4 and P450c21, respectively. The initial RNA amount provided for RT-PCR was adjusted on the basis of spectrophotometric measurement, and RT-PCR for glyceraldehyde-3-phosphate dehydrogenase was performed as an internal standard. In gel electrophoresis of the RT-PCR product for the CYP2D4 gene (Fig. 3Go, A and B), a single band corresponding in size to CYP2D4 mRNA, but not the genomic size, was detected in all regions assayed here, i.e. cerebrum, cerebellum, midbrain, medulla oblongata, hippocampus, thalamus, hypothalamus, pons, and striatum. The density of the band in each brain region was lower than that in the liver, but all regions except for cerebellum gave bands of similar density. The expression of P450c21 mRNA was also examined using the same RNA samples (Fig. 3Go, A and C). P450c21 mRNA expression was not detected in the cerebrum, medulla oblongata, or hypothalamus. In other brain regions assayed here, P450c21 mRNA was very weakly expressed.



View larger version (44K):
[in this window]
[in a new window]
  		FIG. 3. Expression of CYP2D4 and P450c21 mRNAs in rat brain regions. Expression of mRNAs of CYP2D4 and P450c21 in rat brain, liver, and adrenal was estimated by RT-PCR (A) and expressed as the ratio of CYP2D4 (B) or P450c21 (C) to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Brain regions: lane 1, cerebrum; 2, cerebellum; 3, midbrain; 4, medulla oblongata; 5, thalamus; 6, hypothalamus; 7, hippocampus; 8, pons; 9, striatum. L, Liver; A, adrenal gland.

 
We further verified the presence of CYP2D4 and P450c21 proteins in rat brain using CYP2D- and P450c21-specific antibodies. A Western blot analysis revealed high levels of CYP2D4 protein in cerebellum, striatum, pons, and medulla oblongata compared with other regions (Fig. 4AGo). The CYP2D4 content was calculated as 1.27, 0.97, 0.93, and 0.85 pmol/mg protein for cerebellum, striatum, pons, and medulla oblongata, respectively (Fig. 4BGo). Contents of CYP2D4 were well correlated to activity for bufuralol 1'2'-ethenylation, which is a CYP2D4-specific reaction (17), among the brain regions, and the correlation coefficient was calculated as r = 0.861 (Fig. 4CGo). In contrast, no band of P450c21 protein was detected in any rat brain region under conditions such that P450c21 protein from minute amounts of adrenal microsomes could be detected (Fig. 4AGo). These results demonstrated biochemically that CYP2D4 is widely distributed to all brain regions, especially cerebellum, striatum, and pons, and is more highly expressed than P450c21.



View larger version (42K):
[in this window]
[in a new window]
  		FIG. 4. Regional distribution of CYP2D4 protein in rat brain and its correlation to CYP2D4-specific metabolic activities. A, Levels of CYP2D4 (upper) and P450c21 (lower) protein in each rat brain region were determined by immunoblot analysis. Microsomes (50 µg) from various rat brain regions (lane 1, cerebrum; 2, cerebellum; 3, midbrain; 4, medulla oblongata; 5, thalamus; 6, hypothalamus; 7, hippocampus; 8, pons; 9, striatum) were applied to a polyacrylamide gel, and immunoblotting was performed using anti-CYP2D4 or anti-P450c21 antibodies. Microsomes from recombinant CYP2D4 (S1, 0.5 pmol; S2, 1 pmol; S3, 2 pmol) or adrenal gland (S1, 0.1 µg; S2, 1 µg; S3, 10 µg) were also applied. B, CYP2D4 protein was quantitated based on the band density, using recombinant CYP2D4 protein as an authentic standard. C, Bufuralol (10 nmol) was incubated with microsomes from each rat brain region (0.5 mg) in a final volume of 0.5 ml 0.1 M potassium phosphate buffer (pH 7.4). The reaction was initiated by adding NADPH (0.5 µmol).

 
Allopregnanolone 21-hydroxylation by brain CYP2D
Neurosteroids such as allopregnanolone promptly decrease brain excitability, acting as potent positive allosteric modulators of {gamma}-aminobutyric acid (GABA) action at GABAA receptors (24, 25, 26). Consequently, they have potential value in the treatment of various neuropsychiatric disorders (27, 28). Because allopregnanolone is a C-21 steroid, CYP2D4 might show 21-hydroxylation activity toward these GABA modulators. Among the CYP2D isoforms used here, only CYP2D4 and CYP2D6 showed 21-hydroxylation activity toward allopregnanolone (Fig. 5AGo), and also toward 5{alpha}-dihydroprogesterone (data not shown). The Km value of CYP2D4 is sufficient for in vivo reaction, i.e. 6.05 ± 0.83 µM (Fig. 5BGo). Rat brain microsomal allopregnanolone 21-hydroxylation was not inhibited by anti-P450c21 antibodies, but was inhibited by quinidine and by anti-CYP2D4 antibodies (Fig. 5Go, C and D). Furthermore, fluoxetine, a representative selective serotonin reuptake inhibitor that is known to inhibit CYP2D activity (29), inhibited rat brain microsomal allopregnanolone 21-hydroxylation activity in a dose-dependent manner, and its IC50 value was estimated to be around 2 µM (Fig. 5DGo). These results raise the possibility that the brain CYP2D isoforms regulate levels of neurosteroids such as allopregnanolone, and that this regulation would be modified by central nervous system-active drugs such as fluoxetine.



View larger version (28K):
[in this window]
[in a new window]
  		FIG. 5. Allopregnanolone 21-hydroxylation catalyzed by brain CYP2D isoforms. A, Recombinant microsomes from various CYP2D isoforms were incubated with 12.5 nmol allopregnanolone in a final volume of 0.5 ml 0.1 M potassium phosphate buffer (pH 7.4). Data are expressed as the mean ± SD (n = 3). B, Km values and Vmax values were calculated by means of nonlinear regression: 6.05 ± 0.83 µM and 0.33 ± 0.016 pmol/min·pmol P450, respectively (mean ± SD; n = 3). C, Rat brain microsomes (0.5 mg) were preincubated with various amounts of anti-CYP2D antibodies (filled circle) or anti-P450c21 antibodies (open circle) at room temperature for 15 min. After preincubation, 12.5 nmol allopregnanolone and 0.1 M potassium phosphate buffer (pH 7.4) were added to the mixture on ice. The reaction was initiated by adding NADPH (0.5 µmol). Allopregnanolone 21-hydroxylation activities were expressed as percentage of the control. D, Rat brain microsomes (0.5 mg) were incubated with 12.5 nmol allopregnanolone in the presence of various concentrations of the CYP2D-specific inhibitor quinidine (filled circle) or fluoxetine (open circle) in a final volume of 0.5 ml 0.1 M potassium phosphate buffer (pH 7.4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
P450c21 mRNA was detected in the brain, although at approximately 100- to 10,000-fold concentration lower than that in the adrenal gland, by using quantitative RT-PCR methods (2, 3). Microsome-enriched fractions from human fetal tissues showed 21-hydroxylase activity, converting progesterone to 11-deoxycorticosterone (30). Several areas of the rat brain produced very small amounts of corticosterone and aldosterone from endogenous precursors through a synthetic cascade that must include 21-hydroxylase activity (31). However, it has not been established that P450c21 enzyme is involved in steroid 21-hydroxylation in the brain.

In our studies, rat CYP2D4 and human CYP2D6, which are the predominant CYP2D isoforms in the brain (6, 7, 8, 9, 10), were shown to hydroxylate progesterone and 17{alpha}-hydroxyprogesterone at the C-21 position. In rat brain microsomes, these progesterone and 17{alpha}-hydroxyprogesterone 21-hydroxylation activities were not inhibited by anti-P450c21 antibodies, but were effectively inhibited by anti-CYP2D4 antibodies, indicating that these activities are due to CYP2D isoforms in the brain. P450c21 mRNA was detected in the brain using RT-PCR (2, 3), but the amount was very small. The immunohistochemical staining for P450c21 protein was observed in the thalamus and pons of rat (32). Indeed, our results confirmed that the expression of P450c21 mRNA and protein in the brain was extremely low compared with that of CYP2D4. These facts support our hypothesis that steroid 21-hydroxylation in the brain is mediated by CYP2Ds, not P450c21. However, the physiological relevance of the potential for adrenal steroid production in the brain is as yet unknown.

In addition to the function of CYP2D4 as a steroid 21-hydroxylase, CYP2D4 was revealed to metabolize neurosteroids such as allopregnanolone. Allopregnanolone is synthesized from progesterone via two sequential A-ring reductions. A 5{alpha}-reductase first converts progesterone to 5{alpha}-dihydroprogesterone, which is then further reduced by 3{alpha}-hydroxysteroid oxidoreductase to form allopregnanolone. Enzymes involved in allopregnanolone formation were reported to be expressed in the brain (33, 34), and their distribution pattern is in good agreement with that of CYP2D4 (Figs. 3Go and 4Go). Moreover, GABAA receptor (35) and glutamic acid decarboxylase (36), involved in GABA synthesis, are also highly expressed in CYP2D4-rich brain regions. These results strongly supported the view that CYP2D4 plays an important role in regulating brain levels of GABA modulators in cooperation with 5{alpha}-reductase and 3{alpha}-hydroxysteroid oxidoreductase.

Several recent studies have pointed to commonly used selective serotonin reuptake inhibitors as potential modulators of neurosteroid synthesis in the brain. Rat brain microsomal allopregnanolone 21-hydroxylation was inhibited not only by quinidine and anti-CYP2D4 antibodies, but also by fluoxetine, which increased the level of allopregnanolone in the brain of rats (37) and humans (38). However, the mechanism of this effect of fluoxetine is poorly understood: one report claimed that fluoxetine allosterically activates biosynthesis of allopregnanolone catalyzed by 3{alpha}-hydroxysteroid oxidoreductase (39), whereas another study found no activation of 3{alpha}-hydroxysteroid oxidoreductase (40). The concentration of fluoxetine that completely inhibits rat brain microsomal allopregnanolone 21-hydroxylation is approximately equivalent to the brain concentration of this drug at the dose used in the study that observed the fluoxetine-elicited increase of allopregnanolone (41). These results are consistent with the idea that the inhibition of CYP2D-mediated allopregnanolone degradation in the brain by fluoxetine caused the increase of allopregnanolone in the brain. Several other compounds, such as paroxetine and clozapine, also increase the brain allopregnanolone level in vivo (37, 42). It will be intriguing to investigate the effects of other central nervous system-active drugs on brain neurosteroid levels, in relation to the affinity of the drugs for CYP2D.

In conclusion, our findings suggest that CYP2D isoforms, not P450c21, function as steroid 21-hydroxylase in the brain, and may positively regulate the levels of neurosteroids such as GABA modulators (Fig. 6Go).



View larger version (23K):
[in this window]
[in a new window]
  		FIG. 6. Proposed scheme for involvement of CYP2D isoforms in the steroid synthesis in the brain.

 


   Acknowledgments
 
We thank Ms. Atsuko Tominaga for excellent technical assistance.


   Footnotes
 
This work was supported in part by a grant-in-aid from the Ministry of Education, Science, and Culture of Japan.

Abbreviations: CYP, Cytochrome P450; GABA, {gamma}-aminobutyric acid; LC-MS/MS, liquid chromatography/tandem mass spectrometry; NADPH, nicotinamide adenine dinucleotide phosphate (reduced); P450c21, cytochrome P450 21-hydroxylase; RT, reverse transcriptase; Vmax, maximum velocity.

Received August 26, 2003.

Accepted for publication October 7, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Mellon SH, Miller WL 1989 Extraadrenal steroid 21-hydroxylation is not mediated by P450c21. J Clin Invest 84:1497–1502
  2. Beyenburg S, Watzka M, Clusmann H, Blumcke I, Bidlingmaier F, Elger CE, Stoffel-Wagner B 2001 Messenger RNA of steroid 21-hydroxylase (CYP21) is expressed in the human hippocampus. Neurosci Lett 308:111–114[CrossRef][Medline]
  3. Yu L, Romero DG, Gomez-Sanchez CE, Gomez-Sanchez EP 2002 Steroidogenic enzyme gene expression in the human brain. Mol Cell Endocrinol 190:9–17[CrossRef][Medline]
  4. Rogoff D, Gomez-Sanchez CE, Foecking MF, Wortsman J, Slominski A 2001 Steroidogenesis in the human skin: 21-hydroxylation in cultured keratinocytes. J Steroid Biochem Mol Biol 78:77–81[CrossRef][Medline]
  5. Speiser PW, Agdere L, Ueshiba H, White PC, New MI 1991 Aldosterone synthesis in salt-wasting congenital adrenal hyperplasia with complete absence of adrenal 21-hydroxylase. N Engl J Med 324:145–149[Abstract]
  6. Hiroi T, Imaoka S, Chow T, Funae Y 1998 Tissue distributions of CYP2D1, 2D2, 2D3 and 2D4 mRNA in rats detected by RT-PCR. Biochim Biophys Acta 1380:305–312[Medline]
  7. Miksys S, Rao Y, Sellers EM, Kwan M, Mendis D, Tyndale RF 2000 Regional and cellular distribution of CYP2D subfamily members in rat brain. Xenobiotica 30:547–564[CrossRef][Medline]
  8. Komori M 1993 A novel P450 expressed at the high level in rat brain. Biochem Biophys Res Commun 196:721–728[CrossRef][Medline]
  9. Hedlund E, Wyss A, Kainu T, Backlund M, Kohler C, Pelto-Huikko M, Gustafsson JA, Warner M 1996 Cytochrome P4502D4 in the brain: specific neuronal regulation by clozapine and toluene. Mol Pharmacol 50:342–350[Abstract]
  10. McFayden MC, Melvin WT, Murray GI 1998 Regional distribution of individual forms of cytochrome P450 mRNA in normal adult human brain. Biochem Pharmacol 55:825–830[CrossRef][Medline]
  11. Hiroi T, Imaoka S, Funae Y 1998 Dopamine formation from tyramine by CYP2D6. Biochem Biophys Res Commun 249:838–843[CrossRef][Medline]
  12. Hiroi T, Kishimoto W, Chow T, Imaoka S, Igarashi T, Funae Y 2001 Progesterone oxidation by cytochrome P450 2D isoforms in the brain. Endocrinology 142:3901–3908[Abstract/Free Full Text]
  13. Wan J, Imaoka S, Chow T, Hiroi T, Yabusaki Y, Funae Y 1997 Expression of four rat CYP2D isoforms in Saccharomyces cerevisiae and their catalytic specificity. Arch Biochem Biophys 348:383–390[CrossRef][Medline]
  14. Imaoka S, Yamada T, Hiroi T, Hayashi K, Sakaki T, Yabusaki Y, Funae Y 1996 Multiple forms of human P450 expressed in Saccharomyces cerevisiae. Systematic characterization and comparison with those of the rat. Biochem Pharmacol 51:1041–1050[CrossRef][Medline]
  15. Kominami S, Shinzawa K, Takemori S 1983 Immunochemical studies on cytochrome P-450 in adrenal microsomes. Biochim Biophys Acta 755:163–169[Medline]
  16. Funae Y, Imaoka S 1985 Simultaneous purification of multiple forms of rat liver microsomal cytochrome P-450 by high-performance liquid chromatography. Biochim Biophys Acta 842:119–132[Medline]
  17. Hiroi T, Chow T, Imaoka S, Funae Y 2002 Catalytic specificity of CYP2D isoforms in rat and human. Drug Metab Dispos 30:970–976[Abstract/Free Full Text]
  18. Matsunaga E, Umeno M, Gonzalez FJ 1990 The rat P450 IID subfamily: complete sequences of four closely linked genes and evidence that gene conversions maintained sequence homogeneity at the heme-binding region of the cytochrome P450 active site. J Mol Evol 30:155–169[CrossRef][Medline]
  19. Zhou MY, del Carmen Vila M, Gomez-Sanchez EP, Gomez-Sanchez CE 1997 Cloning of two alternatively spliced 21-hydroxylase cDNAs from rat adrenal. J Steroid Biochem Mol Biol 62:277–286[CrossRef][Medline]
  20. Gonzalez FJ, Matsunaga T, Nagata K, Meyer UA, Nebert DW, Pastewka J, Kozak CA, Gillette J, Gelboin HV, Hardwick JP 1987 Debrisoquine 4-hydroxylase: characterization of a new P450 gene subfamily, regulation, chromosomal mapping, and molecular analysis of the DA rat polymorphism. DNA 6:149–161[Medline]
  21. Wu DA, Hu MC, Chung BC 1991 Expression and functional study of wild-type and mutant human cytochrome P450c21 in Saccharomyces cerevisiae. DNA Cell Biol 10:201–209[Medline]
  22. Baulieu EE 1998 Neurosteroids: a novel function of the brain. Psychoneuroendocrinology 23:963–987[CrossRef][Medline]
  23. Rose KA, Stapleton G, Dott K, Kieny MP, Best R, Schwarz M, Russell DW, Bjorkhem I, Seckl J, Lathe R 1997 Cyp7b, a novel brain cytochrome P450, catalyzes the synthesis of neurosteroids 7{alpha}-hydroxy dehydroepiandrosterone and 7{alpha}-hydroxy pregnenolone. Proc Natl Acad Sci USA 94:4925–4930[Abstract/Free Full Text]
  24. Paul SM, Purdy RH 1992 Neuroactive steroids. FASEB J 6:2311–2322[Abstract]
  25. Majewska MD 1992 Neurosteroids: endogenous bimodal modulators of the GABAA receptor. Mechanism of action and physiological significance. Prog Neurobiol 38:379–395[CrossRef][Medline]
  26. Rupprecht R, Holsboer F 1999 Neuroactive steroids: mechanisms of action and neuropsychopharmacological perspectives. Trends Neurosci 22:410–416[CrossRef][Medline]
  27. Costa E, Cheney DL, Grayson DR, Korneyev A, Longone P, Pani L, Romeo E, Zivkovich E, Guidotti A 1994 Pharmacology of neurosteroid biosynthesis. Role of the mitochondrial DBI receptor (MDR) complex. Ann NY Acad Sci 746:223–242[Medline]
  28. Gee KW, McCauley LD, Lan NC 1995 A putative receptor for neurosteroids on the GABAA receptor complex: the pharmacological properties and therapeutic potential of epalons. Crit Rev Neurobiol 9:207–227[Medline]
  29. Otton SV, Wu D, Joffe RT, Cheung SW, Sellers EM 1993 Inhibition by fluoxetine of cytochrome P450 2D6 activity. Clin Pharmacol Ther 53:401–409[Medline]
  30. Casey ML, Winkel CA, MacDonald PC 1983 Conversion of progesterone to deoxycorticosterone in the human fetus: steroid 21-hydroxylase activity in fetal tissues. J Steroid Biochem 18:449–452[CrossRef][Medline]
  31. Gomez-Sanchez CE, Zhou MY, Cozza EN, Morita H, Foecking MF, Gomez-Sanchez EP 1997 Aldosterone biosynthesis in the rat brain. Endocrinology 138:3369–3373[Abstract/Free Full Text]
  32. Iwahashi K, Kawai Y, Suwaki H, Hosokawa K, Ichikawa Y 1993 A localization study of the cytochrome P-450(21)-linked monooxygenase system in adult rat brain. J Steroid Biochem Mol Biol 44:163–169[CrossRef][Medline]
  33. Pelletier G, Luu-The V, Labrie F 1994 Immunocytochemical localization of 5{alpha}-reductase in rat brain. Mol Cell Neurosci 5:394–399[CrossRef][Medline]
  34. Khanna M, Qin KN, Cheng KC 1995 Distribution of 3{alpha}-hydroxysteroid dehydrogenase in rat brain and molecular cloning of multiple cDNAs encoding structurally related proteins in humans. J Steroid Biochem Mol Biol 53:41–46[CrossRef][Medline]
  35. Enna SJ, Snyder SH 1975 Properties of {gamma}-aminobutyric acid (GABA) receptor binding in rat brain synaptic membrane fractions. Brain Res 100:81–97[CrossRef][Medline]
  36. Wood TL, Frantz GD, Menkes JH, Tobin AJ 1986 Regional distribution of messenger RNAs in postmortem human brain. J Neurosci Res 16:311–324[CrossRef][Medline]
  37. Uzunov DP, Cooper TB, Costa E, Guidotti A 1996 Fluoxetine-elicited changes in brain neurosteroid content measured by negative ion mass fragmentography. Proc Natl Acad Sci USA 93:12599–12604[Abstract/Free Full Text]
  38. Uzunova V, Sheline Y, Davis JM, Rasmusson A, Uzunov DP, Costa E, Guidotti A 1998 Increase in the cerebrospinal fluid content of neurosteroids in patients with unipolar major depression who are receiving fluoxetine or fluvoxamine. Proc Natl Acad Sci USA 95:3239–3244[Abstract/Free Full Text]
  39. Griffin LD, Mellon SH 1999 Selective serotonin reuptake inhibitors directly alter activity of neurosteroidogenic enzymes. Proc Natl Acad Sci USA 96:13512–13517[Abstract/Free Full Text]
  40. Trauger JW, Jiang A, Stearns BA, LoGrasso PV 2002 Kinetics of allopregnanolone formation catalyzed by human 3{alpha}-hydroxysteroid dehydrogenase type III (AKR1C2). Biochemistry 41:13451–13459[CrossRef][Medline]
  41. Caccia S, Bizzi A, Coltro G, Fracasso C, Frittoli E, Mennini T, Garattini S 1992 Anorectic activity of fluoxetine and norfluoxetine in rats: relationship between brain concentrations and in-vitro potencies on monoaminergic mechanisms. J Pharm Pharmacol 44:250–254[Medline]
  42. Barbaccia ML, Affricano D, Purdy RH, Maciocco E, Spiga F, Biggio G 2001 Clozapine, but not haloperidol, increases brain concentrations of neuroactive steroids in the rat. Neuropsychopharmacology 25:489–497[CrossRef][Medline]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Regul. Integr. Comp. Physiol.Home page
E. P. Gomez-Sanchez, J. Samuel, G. Vergara, and N. Ahmad
Effect of 3{beta}-hydroxysteroid dehydrogenase inhibition by trilostane on blood pressure in the Dahl salt-sensitive rat
Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2005; 288(2): R389 - R393.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
145/2/699    most recent
Author Manuscript (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kishimoto, W.
Right arrow Articles by Funae, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Kishimoto, W.
Right arrow Articles by Funae, Y.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals