Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes

Nabil Alkayed, Jayashree Narayanan, Debebe Gebremedhin, Meetha Medhora, Richard J. Roman, David R. Harder

Research output: Contribution to journalArticle

160 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)971-979
Number of pages9
JournalStroke
Volume27
Issue number5
StatePublished - May 1996
Externally publishedYes

Fingerprint

Astrocytes
Brain
Arachidonic Acid
Microcirculation
Microvessels
arachidonate epoxygenase
Muscle Cells
Reverse Transcription
Smooth Muscle Myocytes
RNA
Staining and Labeling
Miconazole
Polymerase Chain Reaction
DNA Primers
Cerebral Arteries
Glial Fibrillary Acidic Protein
Arterioles
Patch-Clamp Techniques
Vascular Smooth Muscle
Cytochrome P-450 Enzyme System

Keywords

  • astrocytes
  • microcirculation
  • potassium channels
  • rats

ASJC Scopus subject areas

  • Cardiology and Cardiovascular Medicine
  • Neuroscience(all)

Cite this

Alkayed, N., Narayanan, J., Gebremedhin, D., Medhora, M., Roman, R. J., & Harder, D. R. (1996). Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. Stroke, 27(5), 971-979.

Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. / Alkayed, Nabil; Narayanan, Jayashree; Gebremedhin, Debebe; Medhora, Meetha; Roman, Richard J.; Harder, David R.

In: Stroke, Vol. 27, No. 5, 05.1996, p. 971-979.

Research output: Contribution to journalArticle

Alkayed, N, Narayanan, J, Gebremedhin, D, Medhora, M, Roman, RJ & Harder, DR 1996, 'Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes', Stroke, vol. 27, no. 5, pp. 971-979.
Alkayed N, Narayanan J, Gebremedhin D, Medhora M, Roman RJ, Harder DR. Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. Stroke. 1996 May;27(5):971-979.
Alkayed, Nabil ; Narayanan, Jayashree ; Gebremedhin, Debebe ; Medhora, Meetha ; Roman, Richard J. ; Harder, David R. / Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes. In: Stroke. 1996 ; Vol. 27, No. 5. pp. 971-979.
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