Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes.
OBJECTIVE
Brain parenchymal tissue metabolizes arachidonic acid (AA) via the cytochrome P450 (P450) epoxygenase to epoxyeicosatrienoic acids (EETs). EETs dilate cerebral arterioles and enhance K+ current in vascular smooth muscle cells from large cerebral arteries. Because of the close association between astrocytes and the cerebral microcirculation, we hypothesized that brain epoxygenase activity originates from astrocytes. This study was designed to identify and localize an AA epoxygenase in rat brain astrocytes. We also tested the effect of EETs on whole-cell K+ current in rat cerebral microvascular smooth muscle cells.


METHODS
A functional assay was used to demonstrate endogenous epoxygenase activity of intact astrocytes in culture. Oligonucleotide primers derived from the sequence of a known hepatic epoxygenase, P450 2C11, were used in reverse transcription/polymerase chain reaction of RNA isolated from cultured rat astrocytes. The appropriate size reverse transcription/polymerase chain reaction product was cloned into a plasmid vector and sequenced. A polyclonal peptide antibody was raised against P450 2C11 and used in Western blotting and immunocytochemical staining of cultured astrocytes. A voltage-clamp technique was used to test the effect of EETs on whole-cell K+ current recorded from rat cerebral microvascular muscle cells.


RESULTS
Based on elution time of known standards and inhibition by miconazole, an inhibitor of P450 AA epoxygenase, cultured astrocytes produce 11,12- and 14,15-EETs when incubated with AA. The sequence of a cDNA derived from RNA isolated from cultured rat astrocytes was 100% identical to P450 2C11. Immunoreactivity to glial fibrillary acidic protein, a marker for astrocytes, colocalized with 2C11 immunoreactivity in double immunochemical staining of cultured astrocytes. EETs enhanced outward K+ current in muscle cells from rat brain microvessels.


CONCLUSIONS
Our results demonstrate that a P450 2C11 mRNA is expressed in astrocytes and may be responsible for astrocyte epoxygenase activity. Given the vasodilatory effect of EETs, our findings suggest a role for astrocytes in the control of cerebral microcirculation mediated by P450 2C11-catalyzed conversion of AA to EETs. The mechanism of EET-induced dilation of rat cerebral microvessels may involve activation of K+ channels.
Alkayed
N J
NJ
Department of Physiology, Medical College of Wisconsin, Milwaukee 53226, USA.
Narayanan
J
J
Gebremedhin
D
D
Medhora
M
M
Roman
R J
RJ
Harder
D R
DR
D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D000906:Antibodies; D001189:Aryl Hydrocarbon Hydroxylases; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000201:enzymology*; D002560:Cerebrovascular Circulation / Q000187:drug effects / Q000502:physiology*; D003001:Cloning, Molecular; D000090762:Cytochrome P-450 CYP2J2; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D017931:DNA Primers; D018076:DNA, Complementary; D007150:Immunohistochemistry; D008099:Liver / Q000201:enzymology; D008564:Membrane Potentials / Q000187:drug effects; D008833:Microcirculation / Q000187:drug effects / Q000502:physiology; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000502:physiology; D010105:Oxygenases / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D018408:Patch-Clamp Techniques; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000276:immunology; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000187:drug effects / Q000502:physiology*; D051381:Rats; D017207:Rats, Sprague-Dawley; D039181:Steroid 16-alpha-Hydroxylase; D013250:Steroid Hydroxylases / Q000235:genetics
D000906:Antibodies; D017931:DNA Primers; D018076:DNA, Complementary; D010446:Peptide Fragments; D015221:Potassium Channels; C046782:14,15-epoxy-5,8,11-eicosatrienoic acid; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D000090762:Cytochrome P-450 CYP2J2; D039181:Steroid 16-alpha-Hydroxylase; D015126:8,11,14-Eicosatrienoic Acid