Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. Stroke 1996 May;27(5):971-9
Date
05/01/1996Pubmed ID
8623121DOI
10.1161/01.str.27.5.971Scopus ID
2-s2.0-0029930387 (requires institutional sign-in at Scopus site) 182 CitationsAbstract
BACKGROUND AND PURPOSE: 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.
Author List
Alkayed NJ, Narayanan J, Gebremedhin D, Medhora M, Roman RJ, Harder DRAuthors
David Harder PhD, MS Emeritus Professor in the Physiology department at Medical College of WisconsinMeetha Medhora Professor in the Radiation Oncology department at Medical College of Wisconsin
MESH terms used to index this publication - Major topics in bold
8,11,14-Eicosatrienoic AcidAmino Acid Sequence
Animals
Animals, Newborn
Antibodies
Aryl Hydrocarbon Hydroxylases
Astrocytes
Base Sequence
Blotting, Southern
Blotting, Western
Cells, Cultured
Cerebral Cortex
Cerebrovascular Circulation
Cloning, Molecular
Cytochrome P-450 Enzyme System
DNA Primers
DNA, Complementary
Immunohistochemistry
Liver
Membrane Potentials
Microcirculation
Molecular Sequence Data
Muscle, Smooth, Vascular
Oxygenases
Patch-Clamp Techniques
Peptide Fragments
Polymerase Chain Reaction
Potassium Channels
Rats
Rats, Sprague-Dawley
Steroid 16-alpha-Hydroxylase
Steroid Hydroxylases
