EDHF function in the ductus arteriosus: evidence against involvement of epoxyeicosatrienoic acids and 12S-hydroxyeicosatetraenoic acid

Abstract

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.

Figures

Fig. 1.

Quantitative RT-PCR for CYP2J6 and CYP2J9 from the ductus arteriosus of the untreated preterm (PT) and term (T) mouse, and the preterm mouse treated with retinoic acid (PT+RA) (for details, see materials and methods). With every condition, relative levels of transcript refer to five experiments (each in triplicate) and are expressed in arbitrary units after normalization by the reference β-actin.

Fig. 2.

Immunofluorescence micrographs of ductus arteriosus from term mouse. A and B: epifluorescence images of sections incubated, respectively, with CYP2J9 and CYP2J6 antibody. C: negative control for CYP2J9 image. D–F: confocal images of sections presenting in sequence the staining with CYP2J9 antibody, the endoplasmic reticulum marker BODIPY 558/568 brefeldin A, and the superimposition of the two. Note the colocalization of CYP2J9 with the endoplasmic reticulum; no colocalization was instead observed with the plasma membrane (data not shown). et, Endothelium; smc, smooth muscle. Original magnification: ×20 (A–C) and ×40 (D–F).

Fig. 3.

Release of epoxyeicosatrienoic acids (EETs) from the intact (•) and endothelium-denuded (○) term mouse ductus arteriosus under basal conditions and during treatment with either, or both, compound 1555 (epoxide hydrolase inhibitor) and a cocktail of inhibitors (Cockt; indomethacin, NG-nitro-l-arginine methyl ester, zinc protoporphyrin) (for details, see materials and methods). A: 5,6-EET. B: 8,9-EET. C: 11,12-EET. D: 14,15-EET. For each compound, differences among experimental conditions are not significant. Conversely, when comparing control values for different compounds, a significant difference (*P < 0.05) was found between 5,6-EET and 11,12-EET. Note that the analysis of a fivefold larger pool of specimens, treated with cocktail and compound 1555, yielded comparable results. With endothelium/without endothelium (values in pg/μg protein): 5,6-EET, 0.2/0.1; 8,9-EET, 0/0; 11,12-EET, 0.3/0.4; 14,15-EET, 0.4/0.5.

Fig. 4.

Release of 12-hydroxytetraenoic acids (12-HETE) and its metabolite, 12-hydroxyeicosatrienoic (12-HETrE), from the intact (solid bars) and endothelium-denuded (open bars) term mouse ductus arteriosus under basal conditions and during treatment with a cocktail of inhibitors (Cockt), alone or with baicalein (for details, see materials and methods). *P < 0.01 and P < 0.001 vs. control and cocktail groups, respectively; †P < 0.05 and P < 0.001 vs. control and cocktail/baicalein groups, respectively. Note that differences between +endothelium/cocktail and −endothelium/cocktail for both compounds are not significant.

Fig. 5.

Isolated ductus arteriosus from term fetal mouse. Concentration-response curves to bradykinin (BK) in preparations precontracted with the combination indomethacin (2.8 μM)/NG-nitro-l-arginine methyl ester (100 μM)/zinc protoporphyrin (10 μM), in the absence and presence of baicalein (10 μM) (n = 4 for both groups) are shown. Wall tension before BK was as follows: without baicalein, 0.59 ± 0.07 mN/mm; with baicalein, 0.93 ± 0.14 mN/mm. Difference between the two curves is not significant.