From the Division of Intramural Research, NIEHS,
National Institutes of Health, Research Triangle Park,
North Carolina 27709, the § Department of Medicine,
Vanderbilt University, Nashville, Tennessee 37232, the
¶ Inorganic Carcinogenesis Section, NCI at NIEHS, National
Institutes of Health, Research Triangle Park, North Carolina, 27709, the
Department of Biochemistry, University of Texas Southwestern
Medical Center, Dallas, Texas 75235, and the ** Department of
Pharmacology, University of North Carolina,
Chapel Hill, North Carolina 27599
Received for publication, January 19, 2001, and in revised form, February 24, 2001
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ABSTRACT |
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A cDNA encoding a new cytochrome
P450 was isolated from a mouse brain library. Sequence analysis reveals
that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid
polypeptide that is 70-91% identical to CYP2J subfamily P450s and is
designated CYP2J9. Recombinant CYP2J9 was co-expressed with
NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9
cells using a baculovirus system. Microsomes of
CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to
19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically
distinct from other P450s. Northern analysis reveals that CYP2J9
transcripts are present at high levels in mouse brain. Mouse brain
microsomes biosynthesize 19-HETE. RNA polymerase chain reaction
analysis demonstrates that CYP2J9 mRNAs are widely distributed in
brain and most abundant in the cerebellum. Immunoblotting using an
antibody raised against human CYP2J2 that cross-reacts with CYP2J9
detects a 56-kDa protein band that is expressed in cerebellum and other
brain segments and is regulated during postnatal development. In
situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2
IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje
cells. Importantly, 19-HETE inhibits the activity of recombinant
P/Q-type Ca2+ channels that are known to be expressed
preferentially in cerebellar Purkinje cells and are involved in
triggering neurotransmitter release. Based on these data, we conclude
that CYP2J9 is a developmentally regulated P450 that is abundant in
brain, localized to cerebellar Purkinje cells, and active in the
biosynthesis of 19-HETE, an eicosanoid that inhibits activity of
P/Q-type Ca2+ channels. We postulate that CYP2J9
arachidonic acid products play important functional roles in the brain.
Cytochromes P450 catalyze the NADPH-dependent
oxidation of arachidonic acid to cis-epoxyeicosatrienoic
acids (5,6-, 8,9-, 11,12-, and
14,15-EET),1 mid-chain
cis-trans-conjugated dienols (5-, 8-, 9-, 11-, 12-, and
15-HETE), and/or Remarkably little is known about cytochrome P450-dependent
arachidonic acid metabolism in the central nervous system. Multiple different P450 isoforms capable of arachidonic acid metabolism are
known to be expressed constitutively in brain tissue including members
of the CYP1A, CYP2B, CYP2C, CYP2D, CYP2E, CYP4A, and CYP4F subfamilies
(18-25). In addition, CYP1A, CYP2B, CYP2C, CYP2E, and CYP4A subfamily
members have been shown to be inducible by xenochemicals in the brain
(18, 26, 27). EETs are biosynthesized in the pituitary and hypothalamus
where they have been shown to stimulate the release of various
neuropeptides including somatostatin, arginine vasopressin, oxytocin,
and luteinizing hormone-releasing hormone (28-30). EETs are also
produced by a cytochrome P450 (most likely a CYP2C isoform) in brain
astrocytes, cells that are anatomically and functionally associated
with cerebral microvessels (31-33). Harder and colleagues (32, 33)
have proposed that astrocyte-derived EETs participate in functional
hyperemia and the local regulation of cerebral blood flow by dilating
adjacent cerebral arterioles through a mechanism that involves
activation of vascular smooth muscle K+ channels. One of
the EETs has also been proposed as calcium influx factor, the elusive
link between release of Ca2+ from intracellular stores and
capacitative Ca2+ influx, in astrocytes (34). In contrast,
20-HETE, the primary arachidonic acid metabolite produced in cerebral
arteries by a CYP4A isoform, activates vascular smooth muscle L-type
Ca2+ channels and promotes cerebral vasoconstriction
(35).
Herein, we report the cDNA cloning and heterologous expression of
CYP2J9, a new mouse P450 that is primarily expressed in the brain,
regulated during postnatal brain development, and active in the Materials--
[ Cloning of the CYP2J9 cDNA--
An oligo(dT)-primed Heterologous Expression of Recombinant CYP2J9--
Co-expression
of the protein encoded by the cloned 1.958-kb WQ2J9-7 cDNA insert
(CYP2J9) with CYPOR in Sf9 insect cells was accomplished with the pAcUW51-CYPOR shuttle vector (kindly provided by
Dr. Cosette Serabjit-Singh, GlaxoSmithKline, Research Triangle Park, NC) and the BaculoGold Baculovirus Expression System (PharMingen, San Diego, CA) using methods similar to those described previously (37-39). The CYP2J9 cDNA (nucleotides 130-1649) including the
entire coding region was amplified by PCR and ligated into a slightly modified pAcUW51-CYPOR vector at the NheI and
KpnI sites. The orientation and identity of the resulting
expression vector (pAcUW51-CYPOR-CYP2J9) was confirmed by sequence
analysis. In this construct, the expression of CYPOR is controlled by
the p10 promoter, whereas the expression of CYP2J9 is independently
controlled by the polyhedrin promoter. Cultured Sf9
cells were co-transfected with the pAcUw51-CYPOR-CYP2J9 vector and
linear wild-type BaculoGold viral DNA, and recombinant viruses were
purified as described (37, 38). Cultured Sf9 cells
grown in spinner flasks at a density of 1.5-2 × 106
cells/ml were then infected with high titer CYP2J9-CYPOR recombinant baculovirus stock in the presence of Incubations of Recombinant CYP2J9 with Arachidonic Acid, Product
Characterization--
Reaction mixtures containing 0.05 M
Tris-Cl buffer (pH 7.5), 0.15 M KCl, 0.01 M
MgCl2, 8 mM sodium isocitrate, 0.5 IU/ml
isocitrate dehydrogenase, CYP2J9-CYPOR-transfected
Sf9 cell microsomes (an amount corresponding to
0.1-0.2 nmol P450/ml, final concentration), and
[1-14C]arachidonic acid (55-56 µCi/µmole; 100 µM, final concentration) were constantly stirred at
37 °C. After temperature equilibration, NADPH (1 mM,
final concentration) was added to initiate the reaction. At different
time points (5-20 min, selected to ensure that the quantitative
assessment of the rates of product formation reflect initial rates),
aliquots were withdrawn, and the reaction products were extracted and
analyzed by reverse-phase HPLC as described (37, 38). All products were
identified by comparing their reverse- and normal-phase HPLC properties
with those of authentic EET and HETE standards. In addition, the
CYP2J9-derived 19-HETE was analyzed by negative ion chemical ionization
GC/MS using methane as the reagent gas as described (41). Briefly, the
19-HETE was derivatized to the corresponding pentafluorobenzyl (PFB)
ester, trimethylsilyl ether, and injected on a ThermoQuest Trace MS
equipped with a 30-m Supelco SPB-5 capillary column (0.32 mm inner
diameter, 0.25-µm film thickness). The gas chromatograph was
programmed from 150 to 300 °C at a rate of 10 °C/min with an
initial hold time of 1 min. Both full scan and selected ion monitoring
data were collected. For chiral analysis, the EETs and 19-HETE were collected from the HPLC eluent, derivatized to the corresponding PFB or
methyl esters, and purified by normal-phase HPLC. The 11,12-EET-PFB, 8,9-EET-PFB, and 14,15-EET-methyl esters were resolved into
corresponding antipodes by chiral-phase HPLC and quantified
by liquid scintillation as described previously (42, 43). Chiral
analysis of 19-HETE-methyl ester was performed on naphthylated
derivatives using a Pirkle covalent D-phenyl glycine column
(5 µm, 4.6 × 250 mm, Regis Chemical Co., Morton Grove, IL)
equilibrated with 99.75% hexane, 0.25% isopropyl alcohol at 1 ml/min as described (44). Under these conditions, the retention times
for authentic (19R)- and (19S)-HETEs are 76 and
80 min, respectively, with a resolution factor of 0.93. Control studies
were performed by incubating uninfected Sf9 cell microsomes and baculovirus-infected Sf9 cell
microsomes expressing recombinant CYPOR but containing no spectrally
evident P450 with arachidonic acid under identical conditions. In some
experiments, [1-14C]linoleic acid (55 µCi/µmol; 100 µM, final concentration) was substituted for
[1-14C]arachidonic acid, and the products were identified
by co-elution with authentic hydroxyoctadecenoic acid and
epoxyoctadecenoic acid standards on reverse-phase HPLC.
Incubations of Mouse Brain Microsomes with Arachidonic
Acid--
Microsomal fractions were prepared from freshly isolated
mouse brains by differential centrifugation and resuspended in 50 mM Tris-Cl buffer (pH 7.4), 1 mM
dithiothreitol, 1 mM EDTA, and 20% glycerol. Reaction
mixtures containing 0.05 M Tris-Cl buffer (pH 7.5), 0.15 M KCl, 0.01 M MgCl2, 8 mM sodium isocitrate, 0.5 IU/ml isocitrate dehydrogenase, 1 mM NADPH, 5 mg of brain microsomal protein/ml, and 100 µM [1-14C]arachidonic acid were incubated
at 37 °C for 30-90 min. Reaction products were extracted and
analyzed by HPLC as described (37, 38). All products were identified by
comparing their reverse- and normal-phase HPLC properties with those of
authentic EET and HETE standards.
Northern Blot Hybridization and RNA PCR Analysis--
Normal
mouse tissues (lung, brain, liver, kidney, spleen, heart, stomach,
small intestine, colon, and testes) were obtained from adult male and
female C57BL/6J mice fed NIH 31 rodent chow (Agway, St. Mary, OH)
ad libitum and sacrificed by lethal CO2 inhalation. For some experiments, the brain was subdivided into the
following anatomic sections using conventional morphologic criteria as
follows: cerebellum, cerebral cortex, hippocampus, striatum,
hypothalamus, midbrain, brainstem, and pituitary. RNA was prepared
using TRIreagent (Molecular Research Center, Cincinnati, OH) according
to the manufacturer's instructions. For Northern analysis, total RNA
(20 µg) was denatured and electrophoresed in 1.2% agarose gels
containing 2.2 M formaldehyde as described (45). After
capillary-pressure transfer to Hybond-N+ membranes
(Amersham Pharmacia Biotech), the blots were hybridized with the cloned
1.958-kb CYP2J9 cDNA insert. Hybridizations were performed at
42 °C in 50% formamide containing 0.7 M NaCl, 70 mM sodium citrate, 5× Denhardt's solution, 0.5% SDS, and
0.1 mg of heat-denatured salmon sperm DNA/ml. The cDNA probe was
labeled with [ Protein Immunoblotting--
Lysates were prepared from frozen
mouse (3-day to 7-month postnatal) cerebellum, cerebral cortex,
hippocampus, striatum, hypothalamus, midbrain, and brainstem by
homogenization in a buffer containing 50 mM Tris-HCl (pH
7.4), 1% Triton X-100, 150 mM NaCl, 1 mM EGTA, 0.25% sodium deoxycholate, 1 mM NaF, 0.25 M
phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml aprotinin, 1 mg/ml pepstatin, and 100 mM Na3VO4.
Polyclonal antibodies against the partially purified, recombinant human
CYP2J2 protein (anti-CYP2J2 IgG) were raised in New Zealand White
rabbits and affinity purified as described previously (46). This
antibody has been shown previously to immunoreact with known CYP2J
subfamily P450s (including CYP2J2, CYP2J3, CYP2J5, CYP2J6, and CYP2J8)
but does not cross-react with members of the CYP1A, CYP2A, CYP2B,
CYP2C, CYP2D, CYP2E, and CYP4A subfamilies (12, 37, 38, 46). A
polypeptide corresponding to amino acids 346-357 of the deduced CYP2J9
sequence was designed based upon sequence alignments with known CYP2
family P450s. This peptide was synthesized, HPLC-purified, and coupled
to keyhole limpet hemocyanin to enhance antigenicity by Research
Genetics (Huntsville, AL). Polyclonal antibodies against this
CYP2J9-specific peptide (anti-CYP2J9pep2 IgG) were raised in New
Zealand White rabbits using methods described previously (38).
Microsomes prepared from Sf9 insect cells infected
with recombinant CYP2J2, CYP2J3, CYP2J5, CYP2J6 and CYP2J8
baculoviruses were prepared as previously described (12, 37, 38, 46).
For immunoblotting, tissue lysates or recombinant P450s were
electrophoresed in 8-16% Tris glycine gels (80 × 80 × 1 mm) purchased from NOVEX (San Diego, CA), and the resolved proteins
were transferred electrophoretically onto nitrocellulose membranes.
Membranes were immunoblotted using either rabbit anti-CYP2J2 IgG or
rabbit anti-CYP2J9pep2 IgG, goat anti-rabbit IgG conjugated to
horseradish peroxidase (Bio-Rad), and the ECL Western blotting
Detection System (Amersham Pharmacia Biotech) as described (38, 46).
Protein determinations were performed according to the method of
Bradford (47). Preimmune serum, collected from the rabbits prior to
immunization, did not cross-react with CYP2J9 or with microsomal
fractions prepared from mouse tissues.
In Situ Hybridization and Immunohistochemistry--
Radiolabeled
sense and antisense RNA probes to mouse CYP2J9 were transcribed from
the linearized CYP2J9 (clone WQ24-1)/pBluescript construct using T7
and T3 RNA polymerases and [
For immunohistochemistry, adult mouse brain was fixed in 10% neutral
buffered formalin, processed routinely, and embedded in paraffin.
Localization of CYP2J protein expression was investigated on serial
sections (5-6 µm) with the anti-CYP2J2 IgG (1:100 dilution) using
methods described previously (12, 37, 38). Preimmune rabbit IgG was
used as the negative control in place of the primary antibody.
Exposure of Mice to Mercury Vapor--
Female mice (7 weeks old)
were randomly divided into mercury-exposed and control groups
(n = 5 each). Mice were exposed to mercury vapor (4.0 mg/m3) by nose only for 2 h/day for 3 consecutive days.
Control mice where placed in holding tubes in a nose only exposure
system and exposed to conditioned air at the same flow rates as the
animals receiving mercury. After the final exposure, all animals were immediately euthanized by CO2 asphyxiation. Brains were
quickly removed, frozen in liquid nitrogen, and used to prepare RNA as described above. All animal studies were conducted in accordance with
principles and procedures outlined in the NIH Guide for the Care and
Use of Laboratory Animals and approved by the NIEHS Animal Care and Use Committee.
Expression of Recombinant Ca2+ Channel Subunits in
Xenopus Oocytes--
cDNAs for the Two-electrode Voltage Clamp--
Two-microelectrode voltage
clamp was performed with a GeneClamp 500 amplifier driven by pCLAMP6
software (Axon Instruments, Foster City, CA). Microelectrodes were
filled with 3 M KCl and had resistances of 0.5-2.1
megohms. To minimize the contribution of endogenous
Ca2+-activated chloride channels to the electrical
recordings, oocytes were injected with 46 nl of 50 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (final intracellular concentration ~5 mM) ~15 min
before recording (49). After two-electrode voltage clamp was
established, oocytes were superfused with a chloride-free
Ba2+ recording solution (5 mM
Ba(OH)2, 102.5 mM NaOH, 1 mM KOH,
10 mM Na-HEPES, methanesulfonic acid (pH 7.2)). The holding
potentials were Synthetic Procedures--
The [1-14C]EET internal
standards were synthesized from [1-14C]arachidonic acid
(56-57 µCi/µmol) by nonselective epoxidation as described
previously (50). EET, mid-chain HETE, and 20-HETE standards were
prepared by total chemical synthesis according to published procedures
(51-53). 19-HETE enantiomers were synthesized as described (17). All
synthetic compounds were purified by reverse-phase HPLC prior to use
(37, 38, 54).
Statistical Analysis--
All values are expressed as means ± S.E. Data were analyzed by analysis of variance using SYSTAT
software. When F values indicated that a significant
difference was present, Tukey's HSD test for multiple comparisons was
used. Values were considered significantly different if
p < 0.05.
Cloning of Mouse CYP2J9 cDNA--
Screening of a mouse
brain cDNA library with the CYP2J8 cDNA probe yielded a novel
clone (clone WQ24-1) that shared some sequence homology with several
different human and rodent P450s; however, this clone did not contain
an open reading frame and, based on alignment with other P450s, was
missing the 5'-end including the start codon. The full-length cDNA
(clone WQ2J9-7) was obtained by 5'-rapid amplification of cDNA
ends. Complete nucleic acid sequence analysis of clone WQ2J9-7 revealed
that the cDNA was 1958 nucleotides long, contained an open
reading frame between nucleotides 141 and 1649 flanked by initiation
(ATG) and termination (TGA) codons, had a 140 nucleotide
5'-untranslated region, and had a 308 nucleotide 3'-untranslated region
with a polyadenylation tail (Fig. 1). The
cDNA encoded a 502-amino acid polypeptide that had a derived
molecular mass of 57,935 Da. The deduced amino acid sequence for
WQ2J9-7 contained a putative heme-binding peptide (FSMGKRACLGEQLA)
with the underlined conserved residues and the invariant cysteine at
position 448. The polypeptide encoded by WQ2J9-7 also contained other
structural features associated with CYP2 family P450s including
an N-terminal hydrophobic peptide and a proline cluster between
residues 40 and 51 (Fig. 1). A comparison of the deduced WQ2J9-7 amino
acid sequence with that of other P450s indicated that it was
(a) <31% identical to members of the CYP1, CYP3, CYP4,
CYP5, CYP6, CYP7, and CYP11 families; (b) 40-43% identical
with members of the CYP2A, CYP2B, CYP2C, CYP2D, CYP2E, CYP2F, and CYP2G
subfamilies; and (c) 72-91% identical to human, rabbit,
rat, and mouse CYP2J subfamily P450s (37, 38, 46, 55-57). Based on the
amino acid sequence homology with other CYP2Js, the new mouse
hemoprotein encoded by clone WQ2J9-7 was designated CYP2J9 by the
Committee on Standardized Cytochrome P450 Nomenclature.
Heterologous Expression and Enzymatic Characterization of
CYP2J9--
The protein encoded by clone WQ2J9-7 (CYP2J9) was
co-expressed with CYPOR in Sf9 insect cells using the
baculovirus expression system. The level of expression of recombinant
CYP2J9 was ~5 nmol of P450/liter of infected cells. Coomassie
Brilliant Blue-stained Tris glycine gels of lysates prepared from
CYP2J9 baculovirus-infected Sf9 cells revealed a
prominent band with an estimated molecular mass of 57-58 kDa that was
not present in uninfected insect cell lysates (data not shown).
To reconstitute CYP2J9 activity and to ascertain the catalytic
properties of the recombinant heme-thiolate protein, we incubated microsomal fractions prepared from CYP2J9/CYPOR-transfected
Sf9 cells with radiolabeled arachidonic acid in the
presence of NADPH and an NADPH-regenerating system. CYP2J9 metabolized
arachidonic acid in a time-dependent manner with linear
reaction kinetics up to 20-min incubation times at P450 concentrations
up to 200 pmol/ml. The catalytic turnover was 0.37 nmol of product
formed/nmol P450/min at 37 °C and 100 µM arachidonic
acid. The principal product formed was 19-HETE (retention time = 16-17 min) which was 66% of the total (Fig.
2A). This compound was
identified based upon the following: (a) co-elution with
authentic 19-HETE on reverse-phase HPLC (Fig. 2A);
(b) co-elution with authentic 19-HETE on normal-phase HPLC
(Fig. 2B); and (c) analysis of its PFB ester,
trimethylsilyl ether derivative, by negative ion chemical ionization
GC/MS in both full scan (data not shown) and single ion monitoring
modes (Fig. 2C). EETs and other HETEs were formed in lower
amounts (22 and 12% of the total, respectively). None of the
metabolites were formed when NADPH was omitted from the reaction, when
uninfected Sf9 cell microsomes were used, or when
baculovirus-infected Sf9 cell microsomes expressing
only CYPOR were used (data not shown).
Stereochemical analysis of the CYP2J9-derived 19-HETE revealed a
preference for the (19R)-HETE enantiomer (optical purity 78%). Regiochemical analysis of the CYP2J9-derived EETs revealed a
slight preference for epoxidation at the 14,15-olefin (42% of total
EETs), whereas epoxidation at the 11,12- and 8,9-olefins occurred less
often (28 and 30% of total EETs, respectively). Epoxidation at the
5,6-olefin occurred only rarely (<1% of the total EETs).
Stereochemical analysis of the EETs revealed a slight preference for
(14S,15R)-, (11R,12S)-, and
(8R,9S)-EETs (optical purities, 60, 60, and 54%,
respectively). Based on the above data, we conclude the following:
(a) CYP2J9 is predominantly an arachidonic acid
To determine if arachidonic acid was a preferred endogenous substrate
for CYP2J9, we looked at other possible substrates. Recombinant CYP2J9
metabolized [1-14C]linoleic acid to mixtures of
hydroxyoctadecenoic acids and epoxyoctadecenoic acids. Interestingly,
the rate of linoleic acid metabolism by CYP2J9 was nearly 5-fold lower
(0.08 nmol product/nmol P450/min) than the rate of arachidonic acid
metabolism by CYP2J9. We also examined several xenobiotic substrates,
but without exception, the rates of metabolism were low-undetectable.
Based on these data, we conclude that arachidonic acid is a preferred
substrate for CYP2J9.
Metabolism of Arachidonic Acid by Mouse Brain Microsomes--
To
examine if 19-HETE is formed in mouse brain, we incubated mouse brain
microsomes with radiolabeled arachidonic acid in the presence of NADPH.
Reverse-phase HPLC analysis of the resulting products revealed that the
major brain metabolite (retention time = 16-17 min) co-elutes
with authentic 19- and 20-HETE (Fig. 2D) and the major
19-HETE peak produced by recombinant CYP2J9 (Fig. 2A).
Indeed, the similarities between mouse brain and recombinant CYP2J9
chromatograms are striking. Since the HETEs are not well resolved under
the reverse-phase HPLC conditions used, we collected the radioactive
material generated by mouse brain microsomes which eluted between 15 and 20 min and re-injected it on a normal-phase HPLC system that
resolves individual HETE regioisomers. The results show that this
fraction contains mainly 20-HETE (45%) and 19-HETE (19%), with other
mid-chain HETEs formed in lower amounts (Fig. 2E). Thus, the
major CYP2J9 product 19-HETE is produced by mouse brain microsomes.
We also examined whether the anti-CYP2J9pep2 could inhibit brain
microsomal arachidonic acid metabolism. Unfortunately, the anti-CYP2J9pep2 did not inhibit arachidonic acid metabolism by recombinant CYP2J9, even at concentrations as high as 5 mg of IgG/nmol
of P450. Thus, although the anti-CYP2J9pep2 is specific for CYP2J9 on
Western blots, it is not inhibitory. Moreover, the anti-CYP2J2 IgG is
not inhibitory. Therefore, with currently available reagents, we are
unable to determine the exact contribution of CYP2J9 to 19-HETE
biosynthesis in the brain. Other P450s may also contribute to brain
19-HETE production.
Tissue Distribution of CYP2J9 mRNA--
To determine the
relative organ abundance of CYP2J9 transcripts, total RNA extracted
from adult male mouse tissues was blot-hybridized under high stringency
conditions with the full-length CYP2J9 cDNA probe. As shown in Fig.
3A, the CYP2J9 cDNA
hybridized strongly with brain RNA to produce two transcripts as
follows: (a) an abundant 2.0-kb transcript consistent with
the size of the CYP2J9 mRNA; and (b) a larger 3.5-kb
transcript of lower intensity. The identity of the 3.5-kb transcript is
unknown but may represent another mouse P450 that shares nucleic acid
sequence homology with CYP2J9 or an alternate splice variant of CYP2J9.
The 2.0- and 3.5-kb CYP2J9 transcripts were also present, albeit at
much lower levels, in mouse kidney RNA but were absent from RNA
prepared from other mouse tissues including lung, liver, spleen, heart,
stomach, small intestine, colon, and testis (Fig. 3A). The
differences in mRNA abundance were not due to differences in the
amount of RNA applied to each lane as assessed by ethidium bromide
staining of the gel prior to transfer (Fig. 3A) and the
membrane following transfer (data not shown). To determine if there
were interanimal or gender differences in the abundance of CYP2J9
transcripts in brain or kidney, we performed Northern analysis on
tissues collected from several adult male and female mice. As shown in
Fig. 3B, whereas there was some interanimal variability in
expression of the 2.0-kb CYP2J9 transcript in mouse brain, there were
no consistent differences between the two sexes. In contrast, the
2.0-kb CYP2J9 transcript was more abundant in male versus
female kidney RNA (Fig. 3C).
To confirm independently the tissue distribution of CYP2J9 mRNAs,
we used a sensitive RNA PCR method that was highly specific for CYP2J9.
We were able to amplify a 451-bp DNA fragment (predicted size) from
reverse-transcribed mouse brain RNA and to a lesser extent from mouse
kidney and colon RNA (Fig.
4A). We were unable to amplify
a fragment from lung, liver, spleen, heart, stomach, small intestine,
or testis RNA indicating that CYP2J5 mRNA expression was below the
limit of detection of the RNA PCR assay in these tissues. RNA-PCR
analysis using
To examine the relative distribution of CYP2J9 transcripts within the
central nervous system, we applied the above RNA PCR method to
different portions of the mouse brain. As shown in Fig. 4B,
we amplified a 451-bp DNA fragment from all brain sections including
the cerebellum, cerebral cortex, hippocampus, striatum, hypothalamus,
midbrain, brainstem, and pituitary. Densitometric analysis based on
data from 12 mice and normalized to the corresponding Expression of CYP2J9 Protein in Mouse Brain--
Previous work
(12, 37, 38, 46) has shown that the anti-CYP2J2 IgG is immunospecific
for the CYP2J subfamily (i.e. it reacts with known CYP2J
subfamily P450s in human, rat, and mouse but does not cross-react with
non-CYP2Js). Immunoblotting of microsomal fractions prepared from
Sf9 insect cells infected with recombinant CYP2J9
baculovirus using this anti-CYP2J2 IgG produced a band at ~57-58 kDa
indicating that this antibody also cross-reacted with mouse CYP2J9
(Fig. 5A). In order to examine the distribution of CYP2J protein within the mouse brain, we performed immunoblotting of lysates prepared from different brain sections. The
anti-CYP2J2 IgG detected a single protein at ~56 kDa in hippocampus, hypothalamus, frontal lobe, cerebral cortex, brainstem, cerebellum, and
striatum (Fig. 5B). This suggests that CYP2J
immunoreactivity is widely distributed in the mouse brain and is
consistent with the RNA PCR data (Fig. 4, B and
C) showing the presence of CYP2J9 mRNAs in all brain
sections. Immunoblotting of lysates prepared from the hippocampus,
hypothalamus, frontal lobe, cerebral cortex, brainstem, and cerebellum
of mice ages 3 days to 7 months showed that brain CYP2J
immunoreactivity was present shortly after birth, gradually increased
during the first 3 weeks of life, and achieved maximal levels during
adulthood (Fig. 5C). These data indicate that brain CYP2J
expression is regulated during postnatal development and that the
developmental expression patterns are similar in different portions of
the brain.
The electrophoretic mobility of the recombinant CYP2J9 protein was
slightly lower (i.e. slightly higher molecular mass) than that of the CYP2J immunoreactive protein band detected in mouse brain
tissues by the anti-CYP2J2 IgG (Fig. 5A). These differences, although minor, suggest that (a) the endogenous brain CYP2J9
protein is produced in a truncated form or post-translationally
modified, or (b) the endogenous protein detected in mouse
brain by the anti-CYP2J2 IgG is a related, slightly lower molecular
weight protein that shares antigenic determinants with CYP2J2. Indeed,
the mouse CYP2J subfamily is known to be complex and contains at least
5 different members including CYP2J9 (57). To clarify this further, we
developed a polyclonal antibody to a CYP2J9-specific peptide
(anti-CYP2J9pep2 IgG) (Fig. 1). As shown in Fig. 5D, this
antibody strongly immunoreacts with recombinant CYP2J9 and rat CYP2J3
but does not cross-react with human CYP2J2 or other known murine CYP2J
isoforms including CYP2J5, CYP2J6, and CYP2J8. Immunoblotting of
lysates prepared from mouse brain sections with the anti-CYP2J9pep2 IgG
produced a prominent 56-kDa band that co-migrated with the endogenous
protein detected in mouse brain tissues by the anti-CYP2J2 IgG (Fig. 5, A and E). These data provide further evidence
that the 56-kDa protein detected in brain tissue by both anti-CYP2J2
IgG and anti-CYP2J9pep2 IgG is CYP2J9 or a closely related mouse CYP2J
isoform (but not CYP2J5, CYP2J6, or CYP2J8). Interestingly, the
anti-CYP2J9pep2 IgG also detected a 59-kDa protein in lysates prepared
from mouse brain sections (Fig. 5E). Although the identity
of this protein band is unknown, it is unlikely to be a mouse CYP2J
isoform because it is not detected by the anti-CYP2J2 IgG that is known
to immunoreact with other CYP2J subfamily members.
Localization of CYP2J9 mRNA and CYP2J Protein Expression in the
Brain--
To ascertain the distribution of CYP2J9 mRNAs within
the brain, we performed in situ hybridization on
paraformaldehyde-fixed, paraffin-embedded mouse brain sections with the
35S-labeled antisense and sense CYP2J9 RNA probes. Uniform
silver grain deposition was noted throughout the entire brain in
sections hybridized with the antisense CYP2J9 RNA probe (data not
shown). In contrast, the sense CYP2J9 RNA probe produced very low
background silver staining. These data are consistent with the results
in Fig. 4 showing wide distribution of CYP2J9 mRNAs in the brain. We showed earlier that CYP2J9 transcripts were most abundant in the
mouse cerebellum by RNA PCR. Upon closer examination under dark-field
illumination, we noted intense silver grain deposition at the interface
of the granular and molecular layer in the cerebellum with the
antisense CYP2J9 RNA probe (Fig.
6A). In this region, the sense
probe produced only low level background deposition (Fig.
6B). At higher magnification, intense labeling was observed primarily in cerebellar Purkinje cells (Fig. 6C).
To determine the distribution of CYP2J proteins within the brain, we
stained formalin-fixed paraffin-embedded mouse brain sections with the
anti-CYP2J2 IgG. Consistent with the results of Fig. 5 showing
widespread distribution of CYP2J immunoreactivity by Western blot,
immunohistochemical staining showed that CYP2J reactivity was diffusely
present throughout the mouse brain (data not shown). By comparison,
pre-immune IgG produced only low level background staining. In
agreement with the in situ hybridization results, CYP2J
immunostaining in the cerebellum was highly localized to the Purkinje
cells (Fig. 6D).
To our knowledge, this report is the first to localize expression of a
P450 involved in arachidonic acid metabolism to cerebellar Purkinje
cells. The localization of CYP2J9 mRNAs and CYP2J protein to these
critical cells that integrate information from other neurons within and
outside the cerebellum, and are the major projection neurons to motor
nuclei, may have important functional implications.
Regulation of Brain CYP2J9 Expression by Mercury--
To
investigate whether mercury, a potent neurotoxin and important
environmental pollutant, regulates brain CYP2J9 expression, we
performed Northern blot analysis of total brain RNA isolated from
mercury-exposed and control mice. As shown in Fig.
7A, mercury treatment resulted
in a significant increase in CYP2J9 mRNA levels. Densitometric
normalization to the Inhibition of Recombinant Ca2+ Channels by
19-HETE--
We have shown previously that 11,12- and 14,15-EET
inhibit cardiac L-type Ca2+ channels, probably via a direct
mechanism (58). In order to determine if 19-HETE, the major metabolite
of CYP2J9, could also inhibit voltage-gated Ca2+ channels,
we tested its effects on various Ca2+ channel types
expressed in Xenopus oocytes. Fig.
8 shows Ba2+ currents through
P/Q-type Ca2+ channels (expressed as a combination of
To determine if the effect of 19-HETE was stereoselective, we added
synthetic (19R)- and (19S)-HETE to
Xenopus oocytes expressing recombinant P/Q-type
Ca2+ channels. Interestingly, we found that whereas both
(19R)- and (19S)-HETE significantly inhibited
these channels, the effect of (19R)-HETE was much more
pronounced (Fig. 9). Thus, the maximal inhibition observed with 100 nM (19R)-HETE
(11.0 ± 1.2%, n = 4) was comparable to that
observed with racemic 19-HETE, whereas the maximal inhibition observed
with 100 nM (19S)-HETE was only 5.0 ± 0.7% (n = 3, p < 0.05 versus, (19R)-HETE). Based on these data, we
conclude that the major CYP2J9 enantiomer, (19R)-HETE, was
also the most active in inhibiting P/Q-type Ca2+
channels.
Cytochromes P450 have been the subject of intense investigation by
toxicologists and pharmacologists in the past because they catalyze the
metabolism of a wide variety of drugs, industrial chemicals,
environmental pollutants, and carcinogens (36, 61). The highest
concentrations of P450 enzymes are found in the liver, and as a result,
most studies have focused on the molecular and biochemical
characterization of hepatic heme-thiolate proteins involved in
xenobiotic metabolism and on their regulatory properties. Some P450s
can also catalyze the NADPH-dependent oxidation of arachidonic acid to biologically active eicosanoids (EETs and HETEs)
that have been shown to play critical roles in renal, pulmonary, intestinal, and cardiovascular function (1-17, 37, 57, 58). Recently,
there has been growing interest in studying P450 metabolism of
arachidonic acid in the brain in light of well documented effects of
P450-derived eicosanoids in the release of neuropeptides and in the
control of cerebral blood flow (28-35). In this report, we describe
the cDNA cloning of CYP2J9, a new mouse P450 that is primarily
expressed in the brain, regulated during postnatal brain development
and active in the biosynthesis of 19-HETE. We further demonstrate that
CYP2J9 is particularly abundant in cerebellar Purkinje cells and that
19-HETE inhibits P/Q-type Ca2+ channels, voltage-gated
channels that are known to be expressed preferentially in Purkinje
cells and are involved in triggering the release of neurotransmitters
(59, 60).
We found that CYP2J9 transcripts are most abundant in the brain and
present at lower levels in extracranial tissues. Although a number of
P450 isoforms have been reported to be expressed in the central nervous
system (18-27, 62-64), most of these enzymes are present at much
higher levels in the liver and/or kidney. There are, however, a few
notable exceptions. Lathe and co-workers (62, 63) have reported that
CYP7B is primarily expressed in the brain, where it catalyzes the
biosynthesis of the neurosteroids 7 We found that CYP2J9 expression is particularly abundant in the
cerebellum and highly localized to Purkinje cells, neurons that
integrate information from within and outside the cerebellum, and
communicate with motor nuclei. Tirumalai and co-workers (22) have shown
that brain P450 content varies depending on the region, with the
brainstem and cerebellum showing the highest levels. P450RAI-2 appears
to be highly localized to the cerebellum, although its cellular
localization within the cerebellum is unknown (65). Several other P450s
have been reported to be present in cerebellar Purkinje cells including
CYP2E1, CYP2B1, and CYP11A1 (P450scc) (66-68). CYPOR, which is
required for mammalian P450 enzyme activity, is also abundantly
expressed in Purkinje cells (68).
The recombinant CYP2J9 protein catalyzes
the NADPH-dependent metabolism of arachidonic acid to
19-HETE as the principal reaction product; other HETEs and EETs are
formed in much lower amounts. Hence, CYP2J9 is mainly an arachidonic
acid Little is known about the functional role of P450-derived eicosanoids
in the brain. In the pituitary and hypothalamus, EETs are known to
stimulate the release of neuropeptides, most likely through mechanisms
that involve release of cAMP and increased intracellular calcium
(28-30, 74). EETs are also potent vasodilators in the brain where they
are believed to regulate local cerebral blood flow by enhancing
K+ outward current, hyperpolarizing the resting membrane
potential, and inhibiting voltage-gated Ca2+ channels in
vascular smooth muscle cells (31-34). Indeed, astrocyte-derived EETs
have been proposed to be the elusive link between neuronal metabolic
activation and increased nutritive blood flow (i.e. mediators of functional hyperemia) (75). In contrast, 20-HETE is a
known cerebral vasoconstrictor and is thought to act by enhancing L-type Ca2+ channel currents (35). We are unaware of
previous studies evaluating the physiological role of 19-HETE in the brain.
The localization of CYP2J9 expression to Purkinje cells in the
cerebellum suggested that CYP2J9 products may be essential for Purkinje
cell function. An important finding of this study is that the major
CYP2J9 product (19-HETE) significantly inhibits voltage-gated
Ca2+ channels. It is now well established that
Ca2+ influx through voltage-gated channels is important in
triggering release of neurotransmitters, stimulating contraction of
smooth and cardiac muscle, initiating the transcription of numerous
genes, and controlling other critical cellular processes (60). In the central nervous system, Ca2+ influx through L-type
Ca2+ channels may initiate specific gene expression,
whereas Ca2+ influx through P/Q- and N-type
Ca2+ channels is more important for triggering
neurotransmitter release (60). The effect of 19-HETE was to inhibit all
three types of voltage-gated Ca2+ channels. The P/Q-, N-,
and L-type Ca2+ channels were each significantly inhibited
by 19-HETE. The largest effect was observed in P/Q-type channels that
are preferentially expressed in cerebellar Purkinje cells. Importantly,
these effects occurred at nanomolar concentrations, levels of 19-HETE
that are likely to be achievable in vivo (16). Although the
level of inhibition that we observed (10-20%) is not very large,
there is a steep power relationship between the rise in intracellular Ca2+ mediated by the influx through the voltage-gated
channels and the rate of neurotransmitter release (76). Thus, even
modest inhibition of Ca2+ channel activity by 19-HETE is
likely to cause a profound inhibition of downstream events like
neurotransmitter release and gene expression. Previous work has
documented effects of 19-HETE on renal vascular tone and
Na+/K+-ATPase activity (16, 17).
Mercury compounds are common pollutants and are considered to be some
of the most toxic substances in the environment (77). Following mercury
vapor exposure, the central nervous system is affected, and significant
neurologic abnormalities can result (77). In fact, the cerebellum is a
primary target for mercury-induced lesions, and Purkinje cells show
pronounced mercury accumulation after mercury vapor exposure (77). The
present study demonstrates that CYP2J9 expression is particularly
abundant in the cerebellum and highly localized in Purkinje cells.
Interestingly, mercury vapor exposure in vivo caused
enhanced expression of CYP2J9 mRNA in mouse brain. Thus, an
important environmental toxicant can regulate CYP2J9 expression at a
pretranslational level. Further work will be necessary to define the
molecular mechanisms underlying mercury-induced alteration in CYP2J9
expression and its potential toxicological and functional consequences,
but the possibility exists that CYP2J9 up-regulation could be a
sensitive biochemical marker of exposure to this toxic transition metal.
In summary, we report the cDNA cloning and heterologous expression
of CYP2J9, a new mouse P450 that is primarily expressed in the brain,
regulated during postnatal brain development, and active in the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-terminal alcohols of arachidonic acid (16-, 17-, 18-, 19-, and 20-HETE) (1-3). These eicosanoids are biosynthesized in
numerous tissues where they possess a myriad of potent biological activities. For example, the EETs have been shown to control peptide hormone secretion in the pancreas (4), regulate vascular tone in the
intestine, kidney, heart, and lung (5-9), affect ion transport in the
kidney (6, 10, 11), and have anti-inflammatory properties (12). 20-HETE
constricts renal and aortic vessels by inhibiting smooth muscle cell
large conductance Ca2+-activated K+ channels
(13, 14) and affects renal tubular ion transport (15). 19-HETE has also
been reported to have effects on vascular tone and ion transport in the
kidney (16, 17).
-1
hydroxylation of arachidonic acid to 19-HETE. We further demonstrate,
using in situ hybridization and immunohistochemical techniques, that CYP2J9 mRNA and protein are localized to Purkinje cells in the cerebellum. Importantly, we show that 19-HETE
significantly inhibits the activity of recombinant P/Q-type
Ca2+ channels, voltage-gated channels which are known to be
preferentially expressed in Purkinje cells and are involved in
triggering the release of neurotransmitters. Based on these data, we
postulate that CYP2J9 products play important functional roles in the brain.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-32P]dATP,
[1-14C]arachidonic acid, and
[1-14C]linoleic acid were purchased from PerkinElmer Life
Sciences. Restriction enzymes were purchased from New England Biolabs
(Beverly, MA). PCR reagents including AmpliTaq® DNA polymerase were
purchased from PerkinElmer Life Sciences. Oligonucleotides were
purchased from Life Technologies, Inc. All other chemicals and reagents
were purchased from Sigma unless otherwise specified.
gt10
C57B6 mouse brain cDNA library (a gift from Dr. David Burt,
University of Maryland) was screened with a radiolabeled 1.6-kb CYP2J8
cDNA fragment (GenBankTM accession number AF218857).
Nucleic acid hybridizations were done at 57 °C in 0.9 M
NaCl containing 0.05 M
NaH2PO4/Na2HPO4 (pH
7.0), 0.5% SDS, 0.01 M EDTA, 5× Denhardt's solution, and
0.1 mg of heat-denatured salmon sperm DNA/ml. Fourteen duplicate
positive clones were identified, of which 10 clones, selected at
random, were plaque-purified and rescued into pBluescript SK(+)
(Stratagene). Plasmid DNAs were isolated using a Qiagen Plasmid
Purification Kit (Qiagen Inc., Chatsworth, CA), and insert sizes were
determined by agarose gel electrophoresis after EcoRI
digestion. The pBluescript cDNA inserts were partially sequenced on
an ABI model 377 automated DNA sequencer using the ABI PRISM Dye
Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq® DNA
polymerase (PerkinElmer Life Sciences) and T3/T7 universal
oligonucleotide primers. Nucleotide sequences were analyzed by
searching GenBankTM and EMBL data bases utilizing GCG
software (Genetics Computer Group, Inc., Madison, WI). One of the
duplicate positive clones (clone WQ24-1, ~1.8-kb) contained a novel
sequence that shared homology with several human and rodent CYP2 family
P450s (36). Based on alignment with other CYP2J subfamily members, it
was determined that this clone was missing ~120 nucleotides at the 5'-end of the cDNA including the start codon. The full-length cDNA was obtained by 5'-rapid amplification of cDNA ends using Mouse Brain Marathon-ReadyTM cDNA
(CLONTECH Laboratories, Palo Alto, CA) and the
following gene-specific primer,
5'-GTCTCATTGCACGCACTCTGTGTCACC-3'. The resulting ~2.0-kb
cDNA was gel-purified, cloned into the pCRTM2.1 vector
using the Original TA Cloning kit (Invitrogen Corp., Carlsbad, CA), and
replicated in DH5
-competent Escherichia coli. Ten of the
resulting clones contained identical sequences, one of which
(clone WQ2J9-7) was completely sequenced utilizing a total of 14 oligonucleotide primers (20-25 nucleotides, each) that spanned the
entire length of the sense and antisense cDNA strands.
-aminolevulinic acid and iron
citrate (100 µM each). Cells co-expressing recombinant
CYP2J9 and CYPOR were harvested 72 h after infection, washed twice
with phosphate-buffered saline, and used to prepare microsomal
fractions by differential centrifugation at 4 °C. P450 content was
determined spectrally according to the method of Omura and Sato (40)
using a Shimadzu UV-3000 dual-wavelength/double-beam spectrophotometer (Shimadzu Scientific Instruments, Columbia, MD). Sf9
cells expressing only recombinant CYPOR (no P450) were prepared as
described (39).
-32P]dATP using the
MegaprimeTM DNA labeling system (Amersham Pharmacia
Biotech) and purified using G-50 Sephadex columns (Stratagene).
Northern blot results were confirmed by PCR amplification of
reverse-transcribed mouse RNAs using the GeneAmp® RNA PCR kit
(PerkinElmer Life Sciences). The following CYP2J9 sequence-specific
oligonucleotides were used: forward primer,
5'-CAACCTAACTGCACTGCACAG-3', corresponding to nucleotides 1403-1423 of
the CYP2J9 cDNA; reverse primer, 5'-GGCTTTCTACTTACTGAACCC-3', complementary to nucleotides 1834-1854 of the CYP2J9 cDNA. Reverse transcription was performed with 1 µg of total RNA in a buffer containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl,
5 mM MgCl2, 2.5 µM random
hexamers, 1 mM each of dGTP, dATP, dTTP, and dCTP, and 50 units of Moloney murine leukemia virus reverse transcriptase incubated
at 42 °C for 15 min. The PCR amplifications were performed in the
presence of 2 mM MgCl2, 0.15 µM
forward and reverse primers, and 2.5 units of AmpliTaq® DNA
polymerase. Following an initial incubation for 120 s at 95 °C,
samples were subjected to 30 cycles of 25 s at 95 °C, 45 s
at 64 °C, and 45 s at 72 °C, followed by a final 7-min
incubation at 72 °C. The PCR products were electrophoresed on 1.2%
agarose gels containing ethidium bromide. As a positive control, clone
WQ2J9-7 (CYP2J9) was substituted for the first strand products.
Negative controls included omission of RNA, omission of primers, and
use of CYP2J5, CYP2J6, CYP2J7, and CYP2J8 cDNAs as the template.
The following
-actin sequence-specific primers were used to control
for the quality and amount of RNA: forward primer,
5'-GAGCTATGAGCTGCCTGACG-3', corresponding to nucleotides 776-795 of
the mouse
-actin cDNA; reverse primer,
5'-AGCACTTGCGGTGCACGATG-3', complementary to nucleotides 1166-1185 of
the mouse
-actin cDNA. Following electrophoresis, gels were
scanned using a ChemiImager 4000 Imaging System (Alpha Innotech Corp.,
San Leandro, CA), and relative P450 mRNA levels were determined by
normalization to the
-actin signal.
-35S]UTP. The
35S-labeled probes were then subjected to alkaline
hydrolysis. In situ hybridization was performed on 4%
paraformaldehyde-fixed, paraffin-embedded adult mouse brain sections as
described previously (48). Briefly, 6-7-µm sections were
deparaffinized, refixed in paraformaldehyde, treated with proteinase K
(20 µg/ml), washed with phosphate-buffered saline, refixed in
paraformaldehyde, treated with triethanolamine plus acetic anhydride
(0.25% v/v), and then dehydrated with 100% ethanol. Sense or
antisense RNA probes were hybridized to the sections at 55 °C for
~18 h. After hybridization, the sections were washed once in 5× SSC
plus 10 mM
-mercaptoethanol for 30 min and once more in
50% formamide, 2× SSC, and 100 mM
-mercaptoethanol for
60 min at 65 °C. After two additional washes in 10 mM
Tris-Cl, 5 mM EDTA, 500 mM NaCl (TEN) at
37 °C, sections were treated with RNase A (10 µg/ml) at 37 °C
for 30 min, followed by another wash in TEN at 37 °C. Sections were
then washed twice in 2× SSC and two more times in 0.1× SSC at
65 °C. Slides were dehydrated with graded ethanol containing 300 mM ammonium acetate, dipped in photoemulsion (Ilford K5,
Knutsford, Cheshire, UK) diluted 1:1 with 2% glycerol, and exposed for
4-5 days at 4 °C. After developing in Kodak D-19, slides were
counterstained with hematoxylin.
1A,
1B,
1C, and
1a
subunits of voltage-gated Ca2+ channels, subcloned into
pBluescript SK(+) (
1A and
1a), pBSTA (
1B), and pGEM-3 (
1C),
were generous gifts from Drs. William Horne (North Carolina State
University;
1A and
1a), Diane Lipscombe (Brown University;
1B), and Lucie Parent (University of Montreal;
1C). Linearized
cDNA templates were obtained by digestion with XbaI
(
1A and
1a), VspI (
1B), or HindIII
(
1C). Capped cRNA was transcribed in vitro with T7 RNA
polymerase using the mMessage mMachine kit (Ambion, Austin, TX)
according to the manufacturer's instructions and resuspended in water.
Oocytes were surgically removed from female African clawed frogs,
Xenopus laevis (Nasco, Atkinson, WI), and treated with
collagenase (Sigma) to remove the follicular cell layer. Oocytes were
injected with 10-15 ng of
1A,
1B, or
1C and 2-3 ng of
1a
cRNA (molar ratio ~1:1) and incubated at 19 °C for 2-5 days in
ND96 media (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2,
and 5 mM Na-HEPES (pH 7.5)) supplemented with 50 µg/ml
gentamicin and 0.55 mg/ml sodium pyruvate.
80 mV (
1A or
1B) or
60 mV (
1C). Test
pulses of 650-ms duration were applied every 30 s. Test potentials
were
10 mV (
1A or
1B) or +10 mV (
1C); these test potentials
were at the peak of the current-voltage curve for the respective
channel type. Currents were filtered at 1 kHz with an 8-pole Bessel
low-pass filter and digitized at 2 kHz with a Digidata 1200 A/D
converter (Axon Instruments). Ba2+ currents were recorded
for 6-8 min to obtain a stable base line and then the solution was
switched to Ba2+ recording solution containing either 100 nM racemic 19-HETE, (19R)-HETE, or
(19S)-HETE. Because addition of 0.01% ethanol (vehicle for
19-HETE) caused a small, transient inhibition of channels in some
oocytes, we recorded currents in a solution containing 0.01% ethanol
prior to switching to the solution containing 19-HETE. In this way, the
concentration of ethanol was the same in control and 19-HETE-containing solutions.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Nucleotide sequences for CYP2J9. The
putative heme-binding peptide is underlined, and the
conserved residues are in bold. An asterisk
indicates the termination codon. The location of the peptide used to
prepare the anti-CYP2J9pep2 IgG is enclosed within a
box.
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Fig. 2.
Metabolism of arachidonic acid by recombinant
CYP2J9 and mouse brain microsomes. A,
reverse-phase HPLC chromatogram of the metabolites generated during an
incubation of Sf9 insect cell microsomes containing
recombinant CYP2J9 and CYPOR with [1-14C]arachidonic acid
(AA). Under the HPLC conditions used, authentic 19-HETE
elutes at 16-17 min. Chromatogram is representative of 14 incubations
with four independent preparations of enzyme. B,
normal-phase HPLC chromatogram of the 16-17-min peak produced during
incubation of CYP2J9 with [1-14C]arachidonic acid. The
retention times of authentic 19- and 20-HETE are 16 and 20 min,
respectively, under the normal-phase conditions employed. Chromatogram
is representative of three independent experiments. C,
negative ion chemical ionization GC/MS analysis of the PFB ester,
trimethylsilyl ether derivative of the 16-17-min peak produced during
incubation of CYP2J9 with [1-14C]arachidonic acid. The
top chromatogram shows the retention times of derivatized
19-HETE and 20-HETE standards (selected ion monitoring at
m/z 391), and the bottom chromatogram shows
co-elution of the derivatized 14C-labeled 16-17-min peak
with 19-HETE (selected ion monitoring at m/z 393).
D, reverse-phase HPLC chromatogram of the metabolites
generated during an incubation of mouse brain microsomes with
[1-14C]arachidonic acid. Chromatogram is representative
of nine incubations with three different preparations of microsomes,
each prepared from brains of six mice. E, normal-phase HPLC
chromatogram of the the 15-20-min fraction produced during incubation
of mouse brain microsomes with [1-14C]arachidonic acid.
The retention times of authentic 12-, 15-, 11-, 19- and 20-HETE
standards are 7, 8, 12, 16, and 20 min, respectively. Chromatogram is
representative of three independent experiments.
-1
hydroxylase, and (b) CYP2J9 is enzymologically distinct from
other known P450s.
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Fig. 3.
Tissue distribution of CYP2J9 mRNAs by
Northern analysis. A, total RNA (20 µg)
isolated from adult male mouse tissues was denatured, electrophoresed,
transferred to nylon membranes, and hybridized with the radiolabeled
CYP2J9 cDNA probe under high stringency conditions. Top,
autoradiograph of blot after 24 h of exposure time.
Bottom, ethidium bromide-stained membrane after transfer.
Results are representative of four independent experiments.
B, total RNA (20 µg) isolated from adult male and female
mouse brains was blot-hybridized with the CYP2J9 cDNA as described.
Top, autoradiograph of blot after 12 h of exposure
time. Bottom, ethidium bromide-stained membrane after
transfer. C, total RNA (20 µg) isolated from adult male
and female mouse kidneys was blot-hybridized with the CYP2J9 cDNA
as described. Top, autoradiograph of blot after 72 h of
exposure time. Bottom, ethidium bromide-stained membrane
after transfer.
-actin sequence-specific primers confirmed that the
observations were not due to differences in the quality or amount of
RNA used (Fig. 4A).
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Fig. 4.
Tissue distribution of CYP2J9 transcripts by
RNA PCR. Total RNA (1 µg) was reverse-transcribed, and PCR
amplifications were then performed using either CYP2J9 or mouse
-actin sequence-specific oligonucleotide primers as described under
"Experimental Procedures." The resulting PCR products were
electrophoresed on 1.2% agarose gels containing ethidium bromide.
A, ethidium bromide-stained gels of DNAs amplified from
adult male mouse tissues. Top, CYP2J9-specific primers.
Bottom,
-actin-specific primers. Results are
representative of three independent experiments. bp, base
pairs. B, ethidium bromide-stained gels of DNAs amplified
from different portions of adult mouse brain. Top,
CYP2J9-specific primers. Bottom,
-actin-specific primers.
C, autoradiographs were scanned, and relative CYP2J9
mRNA levels were determined by normalization to the
-actin
signal. Values shown are the means ± S.E. of three independent
experiments performed with pooled RNA prepared from brain sections of
12 mice. The * indicates p < 0.0001 by analysis of
variance.
-actin signals
demonstrated that CYP2J9 transcripts were most abundant in the
cerebellum (p < 0.0001) (Fig. 4C). Thus,
based upon the Northern analysis and RNA PCR, we conclude that CYP2J9 mRNA is primarily present in mouse brain, expressed at lower levels in mouse kidney and colon, and absent from other mouse tissues. Moreover, within the brain, CYP2J9 transcripts are widely distributed but particularly abundant in the cerebellum.
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Fig. 5.
Expression of CYP2J9 protein in mouse brain
by immunoblotting. A, microsomal fractions
prepared from Sf9 cells infected with recombinant
CYP2J2 or CYP2J9 baculovirus (0.5 pmol of P450/lane) or lysates
prepared from mouse cerebral cortex (30 µg/lane) were electrophoresed
on Tris glycine gels, and the resolved proteins were transferred to
nitrocellulose membranes. Membranes were cut and immunoblotted with
either anti-CYP2J2 IgG (left side) or anti-CYP2J9pep2 IgG
(right side). B, lysates prepared from mouse
hippocampus, hypothalamus, frontal lobe, cerebral cortex, brainstem,
cerebellum, and striatum (30 µg/lane) were electrophoresed,
transferred to nitrocellulose, and immunoblotted with the anti-CYP2J2
IgG. Results are representative of five independent experiments.
C, lysates prepared from 3-, 9-, 12-, and 21-day-old and
7-month-old mouse brain sections (30 µg/lane) were electrophoresed,
transferred to nitrocellulose, and immunoblotted with the anti-CYP2J2
IgG. Results are representative of three independent experiments.
D, microsomal fractions prepared from Sf9
cells infected with recombinant CYP2J2, CYP2J3, CYP2J5, CYP2J6, CYP2J8,
and CYP2J9 baculovirus (0.5 pmol P450/lane) were electrophoresed,
transferred to nitrocellulose, and immunoblotted with the
anti-CYP2J9pep2 IgG. E, lysates prepared from mouse cerebral
cortex, striatum, cerebellum, brainstem, frontal lobe, hypothalamus,
and hippocampus (30 µg/lane) or microsomes prepared from
Sf9 cells infected with recombinant CYP2J9 (0.5 pmol
P450/lane) were electrophoresed, transferred to nitrocellulose, and
immunoblotted with the anti-CYP2J9pep2 IgG. Results are representative
of four independent experiments.
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Fig. 6.
Localization of CYP2J9 mRNA and
CYP2J protein in mouse brain. A, dark-field
photomicrograph of a mouse cerebellar section stained with an
35S-labeled antisense CYP2J9 RNA probe. There is intense
silver grain deposition at the interface of the granular and molecular
layer of the cerebellum. B, dark-field photomicrograph of an
adjacent mouse cerebellar section stained with an
35S-labeled sense CYP2J9 RNA probe showing only low level
background silver grain deposition (negative control). C,
light-field illumination of the section in A at higher
magnification showing localization of silver grain deposition to
Purkinje cells. D, adjacent mouse cerebellar section stained
with anti-CYP2J2 IgG showing strong immunostaining in Purkinje cells.
Magnifications, × 92 (A and B) and 185 (C and D).
-actin signal for quantitation revealed that
CYP2J9 mRNA was increased by 52% in mercury-treated mice compared
with control animals (Fig. 7B). These data demonstrate that
mercury exposure can up-regulate the expression of CYP2J9 at a
pretranslational level.
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Fig. 7.
Effect of mercury exposure on
CYP2J9 mRNA expression. A, total RNA (20 µg) isolated from control and mercury-exposed mouse brains was
blot-hybridized with the CYP2J9 cDNA probe as described in Fig. 3.
After autoradiography, the radiolabeled CYP2J9 probe were removed by
boiling, and the blot was rehybridized with a mouse -actin probe.
Results are representative of three independent experiments.
B, autoradiographs were scanned, and relative CYP2J9
mRNA levels were determined by normalization to the
-actin
signal. Values are the mean ± S.E. (n = 5). The *
indicates p < 0.05 for comparison between control and
mercury-treated groups by Student t test.
1A/
1a subunits), N-type Ca2+ channels (
1B/
1a),
and L-type Ca2+ channels (
1C/
1a). P/Q-type
Ca2+ channels, which are preferentially expressed in
cerebellar Purkinje neurons (59), were significantly inhibited by 100 nM racemic 19-HETE (maximal inhibition: 14.8 ± 2.2%,
n = 5, p < 0.05) (Fig. 8A).
Similarly, N-type Ca2+ channels that are expressed in a
variety of central neurons and are present in large numbers in
sympathetic neurons (59), were also significantly inhibited by 100 nM racemic 19-HETE, albeit to a lesser extent than P/Q-type
Ca2+ channels (maximal inhibition, 10.0 ± 2.7%,
n = 3, p < 0.05) (Fig. 8B).
Finally, L-type Ca2+ channels that are expressed in heart,
muscle, and neurons (60) were inhibited by 100 nM racemic
19-HETE (maximal inhibition, 11.4 ± 2.2%, n = 3, p < 0.05) (Fig. 8C). The kinetics of
voltage-dependent activation and inactivation of each
channel type were not altered by 19-HETE. For each channel type,
inhibition by racemic 19-HETE was apparent within 30 s of
application and reached maximal levels in 1-2 min (Fig. 8D,
shown only for the P/Q-type Ca2+ channel). During sustained
application of racemic 19-HETE, there was a small, variable recovery of
the current magnitude. After removal of the 19-HETE, current amplitudes
recovered partially to values that were approximately halfway between
the pretreatment level and the level of maximal inhibition (Fig.
8D). The extent of inhibition by 100 nM racemic
19-HETE was approximately the same as that of 100 nM
11,12-EET on these channels (data not shown). Together, these data
demonstrate that racemic 19-HETE, like the EETs, inhibits voltage-gated
Ca2+ channels, including P/Q-types that are preferentially
expressed in cerebellar Purkinje neurons. To our knowledge, this is the first demonstration of a physiologically relevant effect of a P450
-1 hydroxylase metabolite in the brain.
View larger version (17K):
[in a new window]
Fig. 8.
Inhibition by 19-HETE of voltage-activated
Ca2+ channels expressed in Xenopus
oocytes. Inward Ba2+ currents (shown as
downward deflections) of P/Q-type Ca2+ channels
(A), N-type Ca2+ channels (B), and
L-type Ca2+ channels (C) were evoked by
depolarization from a holding potential (HP) of 80 mV
(A and B) or
60 mV (C) to a test
potential of
10 mV (A and B) or +10 mV
(C) during the interval shown by the bar at the
top. In each panel, the larger inward current amplitude
(lower trace) was obtained before application of the racemic
19-HETE, and the smaller current amplitude (upper trace) was
obtained 1-2 min after changing the bathing solution to one containing
100 nM racemic 19-HETE. The time/amplitude scale is the
same for A-C. Results shown are representative of 3-5
independent experiments. D shows a typical time course of
racemic 19-HETE inhibition of P/Q-type Ca2+ channels
(
1A/
1a). Normalized peak inward current amplitude during the
depolarization is plotted versus time, with the duration of
application of 100 nM 19-HETE shown by the bar.
For this experiment, the maximal current amplitude was
2.25
µA.
View larger version (20K):
[in a new window]
Fig. 9.
Inhibition of P/Q-type Ca2+
channels by (19R)- and
(19S)-HETE. A, inward Ba2+
currents of P/Q-type Ca2+ channels, evoked by
depolarizations from 80 to
10 mV, were recorded before (lower
trace) and 3 min after (upper trace) addition of 100 nM (19R)-HETE. The inhibition was ~13% in
this experiment. B, P/Q-type Ca2+ channels in a
different oocyte before and 3 min after exposure to 100 nM
(19S)-HETE. The inhibition was ~5% in this
experiment.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-hydroxydehydroepiandrosterone
and 7
-hydroxypregnenolone which are known to influence cognition and
behavior. CYP2D18, which is expressed in rat brain but not rat liver,
has been reported to metabolize a variety of central nervous
system-acting compounds including imipramine, chlorpromazine,
chlorzoxazone, and dopamine (24, 64). Recently, White et al.
(65) have identified a novel P450 (P450RAI-2) that is
predominantly expressed in the brain and is responsible for the
metabolism of all-trans-retinoic acid to more polar,
inactive metabolites. Among these enzymes, only CYP2D18 has been
reported to metabolize arachidonic acid (64).
-1 hydroxylase. The catalytic turnover of CYP2J9 is comparable
to that of other P450s that are known to metabolize arachidonic acid
including members of the CYP1A, CYP2B, CYP2C, CYP2E, CYP2G, and CYP4A
subfamilies, other CYP2J isoforms, and CYP2D18 (37, 46, 64, 69-71).
Importantly, the CYP2J9 product profile is distinct from that
previously reported for other CYP2J enzymes. Thus, CYP2J1, CYP2J6, and
CYP2J8 do not significantly metabolize arachidonic acid
(55)2; CYP2J2 is
predominantly an arachidonic acid epoxygenase (46); CYP2J3 and CYP2J4
are arachidonic acid epoxygenases and
-1 hydroxylases (37, 56); and
CYP2J5 is an arachidonic acid epoxygenase and mid-chain hydroxylase
(38). Moreover, the CYP2J9 product profile is different from non-CYP2J
P450s that metabolize arachidonic acid (64, 69-73). Among these,
CYP2D18 biosynthesizes mainly EETs and forms only low amounts of
19-HETE (64); CYP4A1, CYP4A2, and CYP4A3 make primarily 20-HETE (71);
and CYP2E1 forms mainly (19S)-HETE and (18R)-HETE
(73). Therefore, we conclude that CYP2J9 possesses unique enzymological properties.
-1
hydroxylation of arachidonic acid to 19-HETE. We further show, using
in situ hybridization and immunohistochemical techniques,
that CYP2J9 mRNA and protein are localized to Purkinje cells in the
cerebellum. Importantly, we demonstrate that 19-HETE significantly
inhibits the activity of recombinant P/Q-type Ca2+
channels, voltage-gated channels that are known to be expressed preferentially in Purkinje cells and are involved in triggering the
release of neurotransmitters. Based on these data, we conclude that
CYP2J9 is an enzymologically distinct heme-thiolate protein that
contributes to the
-1 hydroxylation of arachidonic acid in
cerebellar Purkinje cells. In light of the effects of 19-HETE on
voltage-gated Ca2+ channel activity, and the documented
role of intracellular Ca2+ mediated by the influx through
these channels on rates of neurotransmitter release, we postulate that
CYP2J9 products play important functional roles in the brain.
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ACKNOWLEDGEMENTS |
---|
Mercury vapor exposures were performed at the
NIEHS inhalation facility under contract to ManTech Environmental
Technology, Inc., Research Triangle Park, NC. We thank Dr. David Burt
for providing the mouse brain cDNA library; Dr. William Horne for providing the cDNAs to 1A and
1a; Dr. Diane Lipscombe for
providing the cDNA to
1B; and Dr. Lucie Parent for providing the
cDNA to
1C. We are also grateful to Drs. Thomas Eling and Robert
Langenbach for their helpful comments during preparation of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants P01-DK38226 (to M. D. B.), GM-31278 (to J. R. F.), and HL49449 (to R. L. R.) and the NIEHS Division of Intramural Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF336850.
The sequence reported in this paper was submitted to the Committee on Standardized Cytochrome P450 Nomenclature and has been designated CYP2J9.
To whom correspondence should be addressed: Laboratory of
Pulmonary Pathobiology, NIEHS, National Institutes of Health, Bldg. 101, Rm. D236, 111 T.W. Alexander Dr., Research Triangle Park, NC
27709. Tel.: 919-541-1169; Fax: 919-541-4133; E-mail:
zeldin@niehs.nih.gov.
Published, JBC Papers in Press, April 27, 2001, DOI 10.1074/jbc.M100545200
2 D. C. Zeldin, unpublished observations.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: EET, cis-epoxyeicosatrienoic acid; HETE, hydroxyeicosatetraenoic acid; HPLC, high performance liquid chromatography; P450, cytochrome P450; CYPOR, NADPH-cytochrome P450 oxidoreductase; PFB, pentafluorobenzyl; kb, kilobase; PCR, polymerase chain reaction; GC/MS, gas chromatography/mass spectroscopy.
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