MOLECULAR CHARACTERIZATION OF AN ARACHIDONIC-ACID EPOXYGENASE IN RAT-BRAIN ASTROCYTES

Background and Purpose Brain parenchymal tissue metabolizes arachidoni c acid (AA) via the cytochrome P350 (P350) epoxygenase to epoxyeicosat rienoic acids (EETs). EETs dilate cerebral arterioles and enhance K+ c urrent in vascular smooth muscle cells from large cerebral arteries. B ecause of the close association between astrocytes and the cerebral mi crocirculation, we hypothesized that brain epoxygenase activity origin ates 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 endog enous epoxygenase activity of intact astrocytes in culture. Oligonucle otide primers derived from the sequence of a known hepatic epoxygenase , P350 2C11, were used in reverse transcription/polymerase chain react ion of RNA isolated from cultured rat astrocytes. The appropriate size reverse transcription/polymerase chain reaction product was cloned in to a plasmid vector and sequenced. A polyclonal peptide antibody was r aised against P350 2C11 and used in Western blotting and immunocytoche mical staining of cultured astrocytes. A voltage-clamp technique was u sed 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 whe n incubated with AA. The sequence of a cDNA derived from RNA isolated from cultured rat astrocytes was 100% identical to P450 2C11. Immunore activity to glial fibrillary acidic protein, a marker for astrocytes, colocalized with 2C11 immunoreactivity in double immunochemical staini ng 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 responsib le for astrocyte epoxygenase activity. Given the vasodilatory effect o f EETs, our findings suggest a role for astrocytes in the control of c erebral microcirculation mediated by P450 2C11-catalyzed conversion of AA to EETs. The mechanism of EET-induced dilation of rat cerebral mic rovessels may involve activation of K+ channels.