Omega-oxidation of 20-hydroxyeicosatetraenoic acid (20-HETE) in cerebral microvascular smooth muscle and endothelium by alcohol dehydrogenase 4.
20-Carboxyeicosatetraenoic acid (20-COOH-AA) is a bioactive metabolite of 20-hydroxyeicosatetraenoic acid (20-HETE), an eicosanoid that produces vasoconstriction in the cerebral circulation. We found that smooth muscle (MSMC) and endothelial (MEC) cultures obtained from mouse brain microvessels convert [3H]20-HETE to 20-COOH-AA, indicating that the cerebral vasculature can produce this metabolite. The [3H]20-COOH-AA accumulated primarily in the culture medium, together with additional radiolabeled metabolites identified as the chain-shortened dicarboxylic acids 18-COOH-18:4, 18-COOH-18:3, and 16-COOH-16:3. N-Heptylformamide, a potent inhibitor of alcohol dehydrogenase (ADH), decreased the conversion of [3H]20-HETE to 20-COOH-AA by the MSMC and MEC and also by isolated mouse brain microvessels. Purified mouse and human ADH4, human ADH3, and horse liver ADH1 efficiently oxidized 20-HETE, and ADH4 and ADH3 were detected in MSMC and MEC by Western blotting. N-Heptylformamide inhibited the oxidation of 20-HETE by mouse and human ADH4 but not by ADH3. These results demonstrated that cerebral microvessels convert 20-HETE to 20-COOH-AA and that ADH catalyzes the reaction. Although ADH4 and ADH3 are expressed in MSMC and MEC, the inhibition produced by N-heptylformamide suggests that ADH4 is primarily responsible for 20-COOH-AA formation in the cerebral microvasculature.
Collins
Xixuan H
XH
Department of Biochemistry, Carver College of Medicine, The University of Iowa, Iowa City, Iowa 52242, USA.
Harmon
Shawn D
SD
Kaduce
Terry L
TL
Berst
Kristine B
KB
Fang
Xiang
X
Moore
Steven A
SA
Raju
T Verugopal
TV
Falck
John R
JR
Weintraub
Neal L
NL
Duester
Gregg
G
Plapp
Bryce V
BV
Spector
Arthur A
AA
D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002384:Catalysis; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003470:Culture Media / Q000032:analysis; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005559:Formamides / Q000494:pharmacology; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism*; D007700:Kinetics; D008055:Lipids / Q000032:analysis; D051379:Mice; D008833:Microcirculation / Q000187:drug effects / Q000201:enzymology*; D009131:Muscle, Smooth, Vascular / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013687:Telencephalon / Q000098:blood supply*; D014316:Tritium / Q000378:metabolism
D003470:Culture Media; D004791:Enzyme Inhibitors; D005559:Formamides; D006893:Hydroxyeicosatetraenoic Acids; D008055:Lipids; D011994:Recombinant Proteins; D014316:Tritium; C055987:20-hydroxy-5,8,11,14-eicosatetraenoic acid; D000426:Alcohol Dehydrogenase