(Received for publication, September 6, 1995; and in revised form, November 30, 1995)
From the
A cDNA encoding a human cytochrome P450 arachidonic acid epoxygenase was isolated from a human liver cDNA library. Sequence analysis revealed that this 1,876-base pair cDNA contained an open reading frame and encoded a new 502-amino acid protein designated CYP2J2. Blot hybridization analysis of RNA prepared from human tissues revealed that CYP2J2 was highly expressed in the heart. Recombinant CYP2J2 protein was prepared using the baculovirus expression system and purified to near electrophoretic homogeneity. The enzyme metabolized arachidonic acid predominantly via olefin epoxidation to all four regioisomeric cis-epoxyeicosatrienoic acids (catalytic turnover 65 pmol of product formed/nmol of cytochrome P450/min at 30 °C). Epoxidation of arachidonic acid by CYP2J2 at the 14,15-olefin was highly enantioselective for (14R,15S)-epoxyeicosatrienoic acid (76% optical purity). Immunoblotting of microsomal fractions prepared from human tissues using a polyclonal antibody raised against the recombinant hemoprotein confirmed primary expression of CYP2J2 protein in human heart. The in vivo significance of CYP2J2 was suggested by documenting the presence of epoxyeicosatrienoic acids in the human heart using gas chromatography/mass spectroscopy. Importantly, the chirality of CYP2J2 products matched that of the epoxyeicosatrienoic acid enantiomers present, in vivo, in human heart. We propose that CYP2J2 is one of the enzymes responsible for epoxidation of endogenous arachidonic acid pools in human heart and that epoxyeicosatrienoic acids may, therefore, play important functional roles in cardiac physiology.
The role of P450 ()in the NADPH-dependent epoxidation
of arachidonic acid is well
documented(1, 2, 3) . The primary products
formed are four regioisomeric cis-epoxyeicosatrienoic acids
(5,6-, 8,9-, 11,12-, and
14,15-EET)(1, 2, 3) . Thus far, arachidonic
acid epoxygenase activity has been demonstrated in microsomal fractions
prepared from several organs including liver, kidney, lung, and
pituitary(4, 5, 6, 7, 8) .
Studies utilizing both purified and recombinant P450 enzymes have shown
that (a) the epoxygenase reaction is enantioselective, (b) the reaction asymmetry is P450 enzyme specific, and (c) the predominant epoxygenase isoforms belong to the CYP2
gene
family(4, 6, 9, 10, 11, 12, 13) . (
)The chiral nature of endogenous EET pools in liver,
kidney, lung, and plasma confirms the biosynthetic origin of these
eicosanoids and documents an endogenous role for microsomal P450 in the
bioactivation of arachidonic
acid(6, 11, 14, 15) .
The potential physiological significance of the epoxygenase reaction is highlighted by the fact that the EETs possess numerous biological activities including modulation of membrane ion fluxes, stimulation of peptide hormone release, and effects on airway smooth muscle (Refs. 6 and 16-19 and references therein). Recent studies demonstrating that (a) the rat renal epoxygenase is under regulatory control by dietary salt, (b) alterations of the rat renal epoxygenase induce hypertension in rats fed a high salt diet, and (c) urinary excretion of epoxygenase metabolites is increased during pregnancy-induced hypertension in humans have supported the hypothesis that P450-derived arachidonic acid metabolites may be involved in the pathophysiology of hypertension(5, 19, 20, 21) . The EETs have also been shown to have other cardiovascular effects. For example, EETs cause renal artery vasoconstriction(14) , cerebral, intestinal, and coronary artery vasodilation(22, 23, 24, 25) , inhibition of platelet aggregation(26) , and cardiac myocyte shortening(27) . Importantly, the EETs have been shown to exacerbate the response of the heart to ischemia and reperfusion(27) .
P450 has been identified using spectral, immunologic, and/or monooxygenase activity assays in heart microsomal fractions from several vertebrate species including scup, rat, rabbit, and pig (28, 29, 30, 31) . In addition, aromatic hydrocarbons have been shown to induce P450 activity in scup, chick embryo, and rabbit heart(30, 32, 33) . Despite these studies, the identity of the P450 isoforms present in heart tissues has not been reported. Furthermore, the function of this ubiquitous enzyme system in the heart remains unknown. In this report, we describe the cloning and cDNA-directed expression of a new human P450 arachidonic acid epoxygenase that is highly expressed in human heart. We also show that human heart contains substantial quantities of endogenous EETs and that the chirality of these EETs matches that of those produced by the recombinant enzyme.
For purification of recombinant CYP2J2,
the P450 and sodium cholate concentrations of the crude cell lysate
were adjusted to 1 µM and 0.4%, respectively. The
resulting suspension was loaded by gravity at room temperature onto a 3
5-cm
-aminooctyl-agarose (Sigma) column equilibrated with
0.1 M potassium phosphate (pH 7.4) containing 20% (v/v)
glycerol, 0.1 µM EDTA, 0.1 µM dithiothreitol,
and 0.4% (w/v) sodium cholate (buffer A). The column was washed with 4
column volumes of buffer A, and the bound CYP2J2 was eluted with buffer
A containing 0.4% (v/v) Emulgen 911 (Kao Chemical Co., Tokyo). After
dialysis versus 200 volumes of 10 mM Tris-Cl buffer
(pH 7.4) containing 20% (v/v) glycerol, 0.1% (w/v) sodium cholate, 0.1
µM EDTA, and 0.1 µM dithiothreitol (buffer
B), the CYP2J2 sample was loaded onto a 1
5-cm hydroxylapatite
(Bio-Rad) column equilibrated with buffer B containing 0.4% (v/v)
Emulgen 911. The column was washed with 6 column volumes of
equilibration buffer, 4 column volumes of 0.04 M sodium
phosphate (pH 7.4) containing 20% (v/v) glycerol, 0.1 µM EDTA, 0.1 µM dithiothreitol, 0.1% (w/v) sodium
cholate, and 0.4% (v/v) Emulgen 911, and the bound CYP2J2 was eluted
with 0.1 M sodium phosphate (pH 7.4) containing 20% (v/v)
glycerol, 0.1 µM EDTA, 0.1 µM dithiothreitol,
0.1% (w/v) sodium cholate, and 0.4% (v/v) Emulgen 911 and dialyzed versus 200 volumes of buffer B. Alternatively, the recombinant
P450 was eluted using a stepwise gradient from 0.02 to 0.1 M sodium phosphate containing buffer. To remove free Emulgen 911,
dialyzed CYP2J2 was loaded onto a second hydroxylapatite column
equilibrated with buffer B and washed with 20-30 column volumes
of buffer B (until the eluent absorbance at 280 nm reached a constant
minimum). The CYP2J2 was eluted with 0.2 M sodium phosphate
(pH 7.4) containing 20% (v/v) glycerol, 0.1 µM EDTA, 0.1
µM dithiothreitol, and 0.1% (w/v) sodium cholate, dialyzed versus 400 column volumes of cholate-free buffer B, and
concentrated using a Centricon-30 microconcentrator (Amicon).
Recombinant CYP2J2 was subjected to N-terminal amino acid analysis and
molecular mass determination by mass spectrometry. For N-terminal amino
acid analysis(38) , partially purified CYP2J2 was
electrophoresed on SDS, 10% (w/v) polyacrylamide slab gels (200
200
1 mm), electroblotted onto Immobilon-P polyvinylidene
difluoride membranes (Millipore), stained with Coomassie Brilliant Blue
R-250 (Bio-Rad), and sequenced directly in the membrane after removal
of the protein stain using an Applied Biosystems 475A protein sequencer
(Perkin-Elmer Corp.). Cycle yields were calculated by comparison with
internal standards. For molecular mass determination, recombinant
CYP2J2 was analyzed on a PerSeptive Biosystems Voyager RP
matrix-assisted laser desorption/ionization time-of-flight mass
spectrometer (PerSeptive Biosystems, Houston, TX) using bovine serum
albumin (M+H and M+2H) as an external mass calibrant and
-cyano-4-hydroxycinnamic acid as a matrix.
Studies on the importance and functional role of the human P450 arachidonic acid epoxygenases in human physiology and pathophysiology required a detailed knowledge of the molecular and catalytic properties of the enzymes involved and access to biospecific probes to study the regulation of the relevant enzymes at the gene and/or protein level. With these goals in mind, we screened a human liver library with a 0.9-kb human cDNA fragment that shared 83% identity with the previously cloned rabbit P450, CYP2J1(36) . Five of the clones contained nucleotide sequences that were identical. One of these clones (clone SW2-14, 1.9 kb) was selected for further study.
Complete nucleic acid sequence analysis of clone SW2-14 revealed that the cDNA was 1876 nucleotides long, contained an open reading frame between nucleotides 6 and 1511 flanked by initiation (ATG) and termination (TAA) codons, and contained a short 5`-end untranslated region and a 364-nucleotide 3`-end untranslated region with a polyadenylation tail (Fig. 1). The cDNA encoded a 502-amino acid protein that had a derived molecular mass of 57,653 Da. The deduced amino acid sequence for SW2-14 contained a putative heme binding peptide (FSIGKRACLGEOLA), with the underlined conserved residues and the invariant cysteine at position 448 (Fig. 1). A comparison of the SW2-14 nucleotide sequence with those of other human P450s indicated that the extent of similarity was limited. Thus, human CYP1A2, CYP2A6, CYP2B6, and CYP2C10 exhibited 43, 48, 48, and 50% nucleic acid sequence identity with SW2-14, respectively. The differences in nucleotide sequence between SW2-14 and other human P450s were randomly distributed along the entire length of the cDNA. In contrast, SW2-14 was 83% identical to rabbit CYP2J1 with the highest variability occurring in the 3`-end untranslated region. Comparison of the deduced amino acid sequence encoded by SW2-14 with those of other P450s demonstrated the following: (a) 19-30% sequence identity with hemoproteins belonging to the CYP1, CYP3, CYP4, CYP5, and CYP6 families; (b) 40-46% sequence identity to hemoproteins belonging to the CYP2 family; and (c) 80% sequence identity to rabbit CYP2J1. Furthermore, amino acid alignment of the protein encoded by SW2-14 with that of rabbit CYP2J1 demonstrated that most of the differences represented conservative changes, i.e. replacement with residues with overall similar chemical properties. Based on the amino acid sequence homology with rabbit CYP2J1, the human hemoprotein has been designated CYP2J2(56) .
Figure 1: Nucleotide and deduced amino acid sequence for clone SW2-14. The putative heme-binding peptide is underlined. The termination codon is marked with three asterisks.
Figure 2:
Nucleic acid blot hybridization analysis
of total RNA prepared from various human tissues. Total RNA (20 µg)
was isolated from various human tissues as described under
``Experimental Procedures,'' denatured, and electrophoresed
in a 1.2% agarose gel containing 0.2 M formaldehyde. After
capillary pressure transfer to nylon membranes, the blot was hybridized
with the cloned 1.9-kb SW2-14 cDNA insert labeled with
[-
P]dATP by nick translation. Top
panel, autoradiograph of blot after 72 h exposure time. Bottom
panel, ethidium bromide-stained gel prior to transfer. Lane
1, brain; lane 2, heart; lane 3, lung; lane
4, kidney; lane 5, liver; lane 6, ileum; lane 7, jejunum; lane 8, colon; lane 9,
ovary; lane 10, testes.
Kikuta and co-workers (36) reported that CYP2J1 was selectively expressed in rabbit small intestine with low or undetectable expression in other rabbit tissues including colon and liver. Expression of CYP2J1 in rabbit heart was not reported. To confirm that rabbit CYP2J1 and human CYP2J2 had different tissue-specific distributions, we performed nucleic acid blot hybridization analysis on rabbit mRNA. Using both a sequence-specific oligonucleotide probe to rabbit CYP2J1 and the human CYP2J2 cDNA probe, we detected mRNA transcript levels, albeit at low levels, in rabbit liver, lung, and kidney. The expression of CYP2J1 in rabbit heart was detectable but was clearly lower than in other rabbit tissues (data not shown). Based on these data, we conclude that rabbit CYP2J1 and human CYP2J2 have different tissue-specific distributions and that only the human CYP2J2 cDNA is predominately expressed in the heart.
For purification of
recombinant CYP2J2, infected SF9 cells were lysed in the
presence of 1% sodium cholate, and the crude protein lysate (specific
content, 0.33 nmol of P450/mg of protein) was loaded onto an
-aminooctyl-agarose column as described under ``Experimental
Procedures.'' The large majority of the recombinant protein
remained bound to the
-aminooctyl-agarose, while most insect
proteins eluted during sample loading and washing with the column
equilibration buffer. The CYP2J2 was eluted as a single brown band
after addition of 0.4% Emulgen 911 to the washing buffer. As shown in Fig. 3, this simple chromatographic step produced a
substantially purified protein in 85% yield. Following dialysis, the
-aminooctyl-agarose purified protein was loaded onto a
hydroxylapatite column, washed with a buffer containing 0.04 M sodium phosphate, and the recombinant P450 eluted as a narrow band
with a 0.10 M sodium phosphate-containing buffer affording a
slightly more purified protein in 35-40% overall yield (Fig. 3). Alternatively, the recombinant P450 was eluted using a
stepwise gradient from 0.02 to 0.1 M sodium
phosphate-containing buffer affording a protein that was nearly
electrophoretically pure in 30% overall yield (Fig. 3). The
purified protein was dialyzed, passed over a second hydroxylapatite
column to remove free Emulgen 911, dialyzed against detergent-free
buffer, and concentrated. The resulting protein had a specific content
of 7.14 nmol of P450/mg of protein and was obtained in 10% overall
yield. Purified CYP2J2 migrated as a discrete band on
SDS-polyacrylamide gels with an estimated molecular mass of 57,000 Da (Fig. 3). Based on the P450 specific content, recombinant CYP2J2
was estimated to be approximately 40% pure. The molecular mass of the
purified, recombinant protein was determined by matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry to confirm that
the protein expressed was, indeed, recombinant CYP2J2. The
experimentally obtained molecular mass (57,666 Da) was within 0.02% of
the derived molecular mass (57,653 Da). We further confirmed that the
recombinant protein was identical with CYP2J2 by performing N-terminal
amino acid sequence analysis on the purified protein. The
experimentally obtained amino acid sequence (MLAAMGSLAAALWAVVHPRT) was
identical to the amino acid sequence predicted from the nucleic acid
sequence of clone SW2-14 (Fig. 1).
Figure 3: SDS-polyacrylamide gel electrophoresis of fractions obtained during the purification of recombinant CYP2J2 from insect cell lysates. Fractions obtained during the purification of recombinant CYP2J2 from insect cell lysates (6-8 pmol of P450/lane) were electrophoresed on SDS, 10% polyacrylamide gels as described under ``Experimental Procedures.'' Gels were stained for 2 h in a 10% solution of Coomassie Brilliant Blue R250 dye and destained in 0.7% acetic acid containing 10% methanol. STD, molecular weight standards in Da; lane 1, CYP2J2-infected SF9 cell lysate; lane 2, aminooctyl-agarose purified CYP2J2; lane 3, hydroxylapatite-purified CYP2J2; lane 4, sodium phosphate stepwise gradient (0.02-0.1 M) hydroxylapatite-purified CYP2J2.
Figure 4:
Reversed-phase HPLC chromatogram of the
organic soluble metabolites generated during incubation of purified,
recombinant CYP2J2 with [1-C]arachidonic acid.
Purified, recombinant CYP2J2 and NADPH-P450 reductase (1 µM final concentration, each) were reconstituted in the presence of L-
-dilauroyl-sn-glycero-3-phosphocholine (50
µg/ml) and incubated at 30 °C with
[1-
C]arachidonic acid (100 µM,
final concentration) with/without NADPH (1 mM, final
concentration). After 1 h, the reaction products were extracted and
resolved by reversed-phase HPLC as described under ``Experimental
Procedures.'' Peak identifications were made by comparisons of the
HPLC properties of individual peaks with those of authentic standards
and by gas chromatography/mass spectroscopy. Ordinate,
radioactivity in cpm; abscissa, time in min. Left
panel, incubation with NADPH; right panel, incubation
without NADPH.
The regio- and stereochemical composition of EETs produced by
recombinant CYP2J2 is shown in Table 1. Metabolism of arachidonic
acid by the hemoprotein was only moderately regioselective with
epoxidation occurring preferentially at the 14,15-olefin (37% of total
EET products) and less often at the 11,12-, 8,9-, and 5,6-olefins (18,
24, and 21% of total EET products, respectively). Thus, CYP2J2 is less
regioselective than rabbit CYP2C2 and human CYP2C8, which generate only
14,15- and 11,12-EETs(10, 13, 55) . The
regioselectivity of CYP2J2 also is different from (a) rat
CYP2C23, which generates primarily 11,12-EET(9, 12) ; (b) rabbit CYP2B4, which has a unique preference for the
5,6-olefin(6) ; and (c) human CYP2C9 and CYP2C10,
which do not generate significant quantities of
5,6-EET(10, 55) . Epoxidation by CYP2J2 at the
14,15-olefin was highly enantioselective for
(14R,15S)-EET (ratio of antipodes 3:1) (Table 1). In contrast, epoxidation at the 11,12- and 8,9-olefins
was non-enantioselective and thus generated racemic EETs (Table 1). The stereoselectivity of epoxidation at the 5,6-olefin
could not be evaluated because 5,6-EET underwent rapid, chemical
hydration to 5,6-DHET and the -lactone of 5,6-DHET. The
stereoselectivity of CYP2J2 is different than that previously reported
for several purified and recombinant rodent P450 epoxygenases including
CYP1A1, CYP2B1, CYP2C11, and CYP2C23(4, 9) . The
stereoselectivity of CYP2J2 is also different from that reported for
the human epoxygenases CYP2C8, CYP2C9, and
CYP2C10(10, 55) .
Kikuta and co-workers (36) have previously reported that rabbit CYP2J1 rapidly
catalyzed the N-demethylation of benzphetamine to
formaldehyde. The same group also found that CYP2J1 did not catalyze
lauric acid or arachidonic acid -oxidation. (
)To
confirm that rabbit CYP2J1 and human CYP2J2 had different substrate
specificities, we incubated CYP2J2 with benzphetamine in the presence
of NADPH-P450 reductase and NADPH. Despite multiple attempts and
prolonged incubation times, we were unable to demonstrate significant
enzymatic metabolism of benzphetamine by recombinant CYP2J2. Based on
these data, we conclude that rabbit CYP2J1 and human CYP2J2 have
different enzymological properties.
Figure 5: Tissue-specific expression of CYP2J2 by protein immunoblotting. Purified recombinant CYP2J2 (0.25 pmol/lane) or microsomal fractions prepared from human lung, heart, aorta, vena cava, kidney, jejunum, liver, and skeletal muscle (30 µg of microsomal protein/lane) were electrophoresed on SDS, 10% polyacrylamide gels, and the resolved proteins were transferred to nitrocellulose membranes as described under ``Experimental Procedures.'' Membranes were immunoblotted using affinity-purified rabbit anti-human CYP2J2 IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase. The immunoreactive proteins were visualized using the ECL detection system and autoradiography. Lane 1, purified, recombinant CYP2J2; lane 2, lung; lane 3, heart; lane 4, aorta; lane 5, vena cava; lane 6, kidney; lane 7, jejunum; lane 8, liver; lane 9, skeletal muscle.
With the exception of human kidney, CYP2J2 protein levels correlated well with CYP2J2 mRNA levels ( Fig. 2and Fig. 5). In human kidney, CYP2J2 protein was expressed at moderate levels despite low mRNA expression. These results are particularly interesting given the potential relevance of the epoxygenase enzyme system to kidney salt and/or water metabolism and to the pathophysiology of human hypertension(5, 8, 9, 10, 14, 19, 20, 21) . Other investigators have noted the lack of correlation between protein and mRNA levels for some human P450s and have proposed that translation rate and/or protein turnover may be important in determining human P450 hemoprotein levels(64) . Fig. 5also demonstrates that the purified, recombinant CYP2J2 protein produces an immunoreactive band that migrates with a slightly lower mobility (higher molecular mass) than the bands produced by endogenous CYP2J2 present in human tissues. We have determined that the molecular mass of the purified, recombinant CYP2J2 protein is nearly identical to that calculated from the amino acid sequence derived from the CYP2J2 cDNA. The differences in electrophoretic mobility between recombinant CYP2J2 and endogenous CYP2J2, although minor, suggest that (a) the endogenous hemoprotein is produced in a truncated form, (b) the endogenous protein is post-translationally modified, or (c) the clone that we isolated (clone SW2-14) encodes a P450 that shares antigenic determinants with a related, slightly lower molecular weight protein that is more abundant in human tissues and is predominately expressed in heart. Further investigation will be necessary to determine if these explanations, or others, can account for the minor differences in electrophoretic mobility between the endogenous and the recombinant proteins.
To evaluate interindividual differences in expression of CYP2J2 protein, we performed immunoblotting on microsomal fractions prepared from an additional seven human liver and three human heart specimens. Protein immunoblotting of the human liver microsomal fractions revealed remarkably low interindividual variation in the expression of CYP2J2 protein (middle band) in human liver tissue (Fig. 6A). In contrast, the expression of the cross-reactive liver proteins (upper and lower bands) were more variable (Fig. 6A). Thus, while some livers contained roughly equal amounts of CYP2J2 and the two cross-reactive proteins (e.g. L3-L7), others primarily expressed CYP2J2 and the lower mobility protein (upper band) (L1-L2). Protein immunoblotting of the human heart microsomal fractions revealed that, while CYP2J2 was expressed at high levels in each of the heart tissues, there was greater interindividual variation in CYP2J2 expression in human heart than in human liver (Fig. 6B). In a given individual, however, CYP2J2 expression was always significantly higher in the heart than in extracardiac tissues. Many factors are known to alter the levels of expression of human P450 genes including genetic polymorphism, enzyme induction, and/or inhibition and developmental factors(65, 66, 67) . A number of investigators have reported large (3-115-fold) interindividual variation in expression of human P450s of the CYP1, CYP2, CYP3, and CYP4 gene families(64, 68, 69, 70) .
Figure 6: Interindividual variation in expression of CYP2J2 protein in different human livers and hearts by protein immunoblotting. Panel A, purified, recombinant CYP2J2 or human liver microsomal fractions (50 µg of microsomal protein/lane) prepared from seven different human livers (L1-L7) were electrophoresed on SDS, 10% polyacrylamide gels, and the resolved proteins were transferred to nitrocellulose membranes, immunoblotted with affinity-purified rabbit anti-human CYP2J2 IgG and goat anti-rabbit IgG conjugated to horseradish peroxidase, and detected using the ECL detection system as described under ``Experimental Procedures.'' Panel B, purified, recombinant CYP2J2 or human heart microsomal fractions (30 µg of microsomal protein/lane) prepared from three different human hearts (H1-H3) were electrophoresed, transferred to nitrocellulose, and immunoblotted with rabbit anti-human CYP2J2 IgG as described under ``Experimental Procedures.''
Compared with human kidney cortex, human heart contains approximately 5-fold less total EETs and has a distinctly different regio- and stereochemical profile(76) . Thus, whereas both tissues favor epoxidation at the re, si face of the 14,15-olefin and produce racemic 8,9-EET, only human heart produces racemic 11,12-EET. The chirality of endogenous EETs recovered from human heart also differs from those isolated from rat liver and rabbit lung in which (14R,15S)-, (11S,12R)-, and (8S,9R)-EET were the predominant antipodes(6, 11) . In fact, our data suggest that the regio- and stereochemical properties of the arachidonic acid epoxygenases are organism and tissue specific. These findings have important implications given that many of the biological actions of the EETs appear to be both regio- and stereoselective(6, 14, 26) . Stereoselective formation of eicosanoids is a sufficient criterion to establish their enzymatic origin(11) . Therefore, based on the data presented in Table 2, we conclude that 14,15-EET was produced in vivo by the human heart epoxygenase. Although the racemic nature of endogenous human heart 11,12- and 8,9-EETs precludes a definitive statement regarding their biosynthetic origin, the fact that CYP2J2, an epoxygenase highly expressed in human heart, also produces racemic 11,12- and 8,9-EETs supports the contention that these eicosanoids are formed enzymatically.
The P450 monooxygenases have long been thought to function primarily in the metabolism of exogenous compounds including drugs and carcinogens (66, 67) . Over the past 10-15 years, there has been an increased awareness that this ubiquitous enzyme system may also be involved in the bioactivation of endogenous substrates such as steroids and fatty acids(16, 17, 18) . The documentation of P450 monooxygenases in vertebrate heart tissue(28, 29, 30, 31, 32, 33) , together with the known cardiovascular effects of P450 arachidonic acid epoxygenase metabolites(14, 22, 23, 24, 25, 27, 74, 75) , suggests that this enzyme system may play important functional roles in cardiac physiology and pathophysiology. We report here the cDNA cloning and cDNA-directed expression of CYP2J2, a new human P450 that is highly and constitutively expressed in heart. We demonstrate that the recombinant hemoprotein catalyzes the regio- and stereoselective epoxidation of arachidonic acid and show that the chirality of CYP2J2 products matches that of the enantiomers present in vivo in human heart. We conclude, therefore, that CYP2J2 is one of the predominant enzymes responsible for epoxidation of endogenous arachidonic acid pools in human heart and suggest that, in addition to the cyclooxygenase and lipoxygenase pathways, the P450 monooxygenase pathway is an important member of the cardiac arachidonic acid metabolic cascade. We speculate that epoxygenase metabolites may be important in maintaining cardiac homeostasis and that altered local concentration of these eicosanoids may lead to cardiac dysfunction. As pathologically normal human heart tissue becomes available, it will be important to (a) evaluate the metabolism of arachidonic acid by cardiac microsomal fractions, (b) localize expression of CYP2J2 within the heart by immunohistochemistry and in situ hybridization, and (c) further examine the role that the EETs may play in cardiac physiology.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U37143[GenBank].