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 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.
Harder
D R
DR
Department of Physiology, Medical College of Wisconsin, Milwaukee 53226.
Gebremedhin
D
D
Narayanan
J
J
Jefcoat
C
C
Falck
J R
JR
Campbell
W B
WB
Roman
R
R
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D002415:Cats; D002560:Cerebrovascular Circulation / Q000187:drug effects; D042926:Cytochrome P-450 CYP4A; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005260:Female; D005290:Ferric Compounds; D006893:Hydroxyeicosatetraenoic Acids / Q000032:analysis / Q000378:metabolism* / Q000494:pharmacology*; D008297:Male; D008564:Membrane Potentials / Q000502:physiology; D008833:Microcirculation / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D015221:Potassium Channels / Q000502:physiology*
D005290:Ferric Compounds; D006893:Hydroxyeicosatetraenoic Acids; D015221:Potassium Channels; C000499:ferric oxide; D016718:Arachidonic Acid; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D042926:Cytochrome P-450 CYP4A