FORMATION AND ACTION OF A P-450 4A METABOLITE OF ARACHIDONIC-ACID IN CAT CEREBRAL MICROVESSELS

The purpose of this study was to determine whether arachidonic acid ca n be converted to 20-hydroxyeicosatetraenoic acid (HETE) by P-450 enzy mes in cat cerebral microvasculature, to identify the P-450 isoforms r esponsible for the formation of this metabolite, and to characterize t he vasoactive effects of 80-HETE on these vessels. Cerebral microvesse ls were isolated by filling them with a suspension of magnetized iron oxide (particle size = 10 mu m) and separated from minced cerebral cor tical tissue using a magnet. Cat cerebral microvessels were homogenize d and incubated with [C-14]arachidonic acid (AA), and cytochrome P-450 -dependent metabolites of AA were separated by reverse-phase high-pres sure liquid chromatography. A major metabolite that coeluted with synt hetic 20-HETE was identified. The formation of this metabolite was dep endent on NADPH and was inhibited by 17-octadecynoic acid (ODYA), a sp ecific suicide-substrate inhibitor of the omega-hydroxylation of AA by P-450 enzymes. Western blot analysis confirmed the presence of a P-45 0 enzyme of the 4A gene family in cat cerebral microvessels. Gas chrom atography/mass spectrometry analysis revealed that this metabolite has an identical mass-to-charge ratio (391 m/z) as that of standard 20-HE TE. Exogenous 20-HETE constricted pressurized cat pial arteries in a c oncentration-dependent manner with a threshold concentration of < 1.0 nM. 20-HETE (1 nM) inhibited the activity of a 217-pS K+ channel recor ded in cell-attached patches of isolated cat cerebral microvascular mu scle cells. Blockade of endogenous P-450 activity with 17-ODYA markedl y increased the activity of the 217 pS K+ channel in these cells, an a ction 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 dem onstrate the presence of P-450 4A enzyme activity in the cerebral micr ovasculature of the cat that converts AA to 20-HETE. The potent vasoco nstrictor effects of 20-HETE on cerebral vessels suggests that metabol ites of P-450 enzymes of the 4A gene family could play an important ro le in regulating cerebral microvascular tone.