Cytochrome P450 CYP2J9, a new mouse arachidonic acid omega-1 hydroxylase predominantly expressed in brain.
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 Ca(2+) 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 Ca(2+) channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.
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Division of Intramural Research, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Bradbury
J A
JA
Tsao
C C
CC
Maronpot
R
R
Harry
G J
GJ
Parker
C E
CE
Davis
L S
LS
Breyer
M D
MD
Waalkes
M P
MP
Falck
J R
JR
Chen
J
J
Rosenberg
R L
RL
Zeldin
D C
DC
D000595:Amino Acid Sequence; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016367:Baculoviridae; D001483:Base Sequence; D001921:Brain / Q000201:enzymology*; D015220:Calcium Channels / Q000378:metabolism; D002460:Cell Line; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D017403:In Situ Hybridization; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011689:Purkinje Cells / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017422:Sequence Analysis, DNA; D018411:Spodoptera; D014162:Transfection
D015220:Calcium Channels; D018076:DNA, Complementary; D006893:Hydroxyeicosatetraenoic Acids; D012333:RNA, Messenger; D016718:Arachidonic Acid; C055986:19-hydroxy-5,8,11,14-eicosatetraenoic acid; D006899:Mixed Function Oxygenases; C431943:cytochrome P-450 CYP2J9 (murine)