EDHF function in the ductus arteriosus: evidence against involvement of epoxyeicosatrienoic acids and 12S-hydroxyeicosatetraenoic acid.
We have previously shown (Ref. 2) that endothelium-derived hyperpolarizing factor (EDHF) becomes functional in the fetal ductus arteriosus on removal of nitric oxide and carbon monoxide. From this, it was proposed that EDHF originates from a cytochrome P-450 (CYP450)-catalyzed reaction being inhibited by the two agents. Here, we have examined in the mouse ductus whether EDHF can be identified as an arachidonic acid product of a CYP450 epoxygenase and allied pathways. We did not detect transcripts of the mouse CYP2C subfamily in vessel, while CYP2J subfamily transcripts were expressed with CYP2J6 and CYP2J9. These CYP2J hemoproteins were also detected in the ductus by immunofluorescence microscopy, being colocalized with the endoplasmic reticulum in both endothelial and muscle cells. Distinct CYP450 transcripts were also detected and were responsible for omega-hydroxylation (CYP4A31) and 12R-hydroxylation (CYP4B1). Mass spectrometric analysis showed formation of epoxyeicosatrienoic acids (EETs) in the intact ductus, with 11,12- and 14,15-EETs being more prominent than 5,6- and 8,9-EETs. However, their yield did not increase with nitric oxide/carbon monoxide suppression, nor did it abate with endothelium removal. No evidence was obtained for formation of 12R-hydroxyeicosatrienoic acid and omega-hydroxylation products. 2S-hydroxyeicosatetraenoic acid was instead detected, and, contrary to data implicating this compound as an alternative EDHF, its suppression with baicalein did not modify the EDHF-mediated relaxation to bradykinin. We conclude that none of the more common CYP450-linked arachidonic acid metabolites appears to qualify as EDHF in mouse ductus. We speculate that some novel eicosanoid or a totally unrelated compound requiring CYP450 for its synthesis accounts for EDHF in this vessel.
Baragatti
Barbara
B
Scuola Superiore Sant'Anna, Piazza Martiri della Libertà 33, 56127 Pisa, Italy.
Schwartzman
Michal Laniado
ML
Angeloni
Debora
D
Scebba
Francesca
F
Ciofini
Enrica
E
Sodini
Daria
D
Ottaviano
Virginia
V
Nencioni
Simona
S
Paolicchi
Aldo
A
Graves
Joan P
JP
Zeldin
Darryl C
DC
Gotlinger
Katherine
K
Luin
Stefano
S
Coceani
Flavio
F
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid / Q000378:metabolism*; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001685:Biological Factors / Q000378:metabolism*; D001920:Bradykinin / Q000378:metabolism; D000090762:Cytochrome P-450 CYP2J2; D042926:Cytochrome P-450 CYP4A / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004373:Ductus Arteriosus / Q000201:enzymology*; D042783:Endothelial Cells / Q000201:enzymology; D019317:Evidence-Based Medicine; D015971:Gene Expression Regulation, Enzymologic; D006900:Hydroxylation; D051379:Mice; D008810:Mice, Inbred C57BL; D006899:Mixed Function Oxygenases / Q000378:metabolism; D009131:Muscle, Smooth, Vascular / Q000201:enzymology; D014664:Vasodilation
D001685:Biological Factors; C000717353:Cyp2j6 protein, mouse; C400322:cytochrome P-450 CYP2C subfamily; C067410:endothelium-dependent hyperpolarization factor; D016718:Arachidonic Acid; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D003577:Cytochrome P-450 Enzyme System; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; D000090762:Cytochrome P-450 CYP2J2; C095029:cytochrome P-450 CYP4B1; D042926:Cytochrome P-450 CYP4A; C431943:cytochrome P-450 CYP2J9 (murine); D001920:Bradykinin