Faculty Collaboration Database

Formation and action of a P-450 4A metabolite of arachidonic acid in cat cerebral microvessels. Am J Physiol 1994 May;266(5 Pt 2):H2098-107

Date

05/01/1994

Pubmed ID

8203608

DOI

10.1152/ajpheart.1994.266.5.H2098

Scopus ID

2-s2.0-0028155755 (requires institutional sign-in at Scopus site)   236 Citations

Abstract

The purpose of this study was to determine whether arachidonic acid can be converted to 20-hydroxyeicosatetraenoic acid (HETE) by P-450 enzymes in cat cerebral microvasculature, to identify the P-450 isoforms responsible for the formation of this metabolite, and to characterize the vasoactive effects of 20-HETE on these vessels. Cerebral microvessels were isolated by filling them with a suspension of magnetized iron oxide (particle size = 10 microns) and separated from minced cerebral cortical tissue using a magnet. Cat cerebral microvessels were homogenized and incubated with [14C]arachidonic acid (AA), and cytochrome P-450-dependent metabolites of AA were separated by reverse-phase high-pressure liquid chromatography. A major metabolite that coeluted with synthetic 20-HETE was identified. The formation of this metabolite was dependent on NADPH and was inhibited by 17-octadecynoic acid (ODYA), a specific suicide-substrate inhibitor of the omega-hydroxylation of AA by P-450 enzymes. Western blot analysis confirmed the presence of a P-450 enzyme of the 4A gene family in cat cerebral microvessels. Gas chromatography/mass spectrometry analysis revealed that this metabolite has an identical mass-to-charge ratio (391 m/z) as that of standard 20-HETE. Exogenous 20-HETE constricted pressurized cat pial arteries in a concentration-dependent manner with a threshold concentration of < 1.0 nM. 20-HETE (1 nM) inhibited the activity of a 217-pS K+ channel recorded in cell-attached patches of isolated cat cerebral microvascular muscle cells. Blockade of endogenous P-450 activity with 17-ODYA markedly increased the activity of the 217 pS K+ channel in these cells, an action that was completely reversed by a nanomolar concentration of 20-HETE, suggesting that 20-HETE might be an endogenous modulator of the 217 pS K+ channel in cerebral arterial muscle cells. These results demonstrate the presence of P-450 4A enzyme activity in the cerebral microvasculature of the cat that converts AA to 20-HETE. The potent vasoconstrictor effects of 20-HETE on cerebral vessels suggests that metabolites of P-450 enzymes of the 4A gene family could play an important role in regulating cerebral microvascular tone.

Author List

Harder DR, Gebremedhin D, Narayanan J, Jefcoat C, Falck JR, Campbell WB, Roman R

Author

William B. Campbell PhD Professor in the Pharmacology and Toxicology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Arachidonic Acid
Cats
Cerebrovascular Circulation
Cytochrome P-450 CYP4A
Cytochrome P-450 Enzyme System
Female
Ferric Compounds
Hydroxyeicosatetraenoic Acids
Male
Membrane Potentials
Microcirculation
Mixed Function Oxygenases
Muscle, Smooth, Vascular
Potassium Channels