Vascular hepoxilin and trioxilins mediate vasorelaxation through TP receptor inhibition in mouse arteries.

AIM: 12/15-lipoxygenase (12/15-LO) metabolizes arachidonic acid (AA) into several vasoactive eicosanoids. In mouse arteries, we previously characterized the enzyme's 15-LO metabolites 12(S)-hydroxyeicosatetraenoic acid (HETE), 15-HETE, hydroxyepoxyeicosatrienoic acids (HEETAs) and 11,12,15-trihydroxyeicosatrienoic acids (11,12,15-THETAs) as endothelium-derived relaxing factors. However, the observed 12-LO metabolites remained uncharacterized. The purpose of this study was to determine the structure and biological functions of eicosanoids generated by the enzyme's 12-LO activity. METHODS: Metabolites extracted from aortas of C57BL/6 male mice were separated using a series of reverse and normal phase chromatographic steps and identified as hepoxilin A3 , trioxilin A3 and trioxilin C3 by mass spectrometry. Activities of these natural compounds were tested on isometric tension and intracellular calcium release. The role of thromboxane (TP) receptor was determined in HEK293 cells overexpressing TPα receptor (TPα -HEK). RESULTS: All identified vascular 12-LO metabolites were biologically active. In mouse mesenteric arteries, trioxilin A3 , C3 and hepoxilin A3 (3 µm) relaxed arteries constricted with the thromboxane mimetic, U46619-constricted arteries (maximum relaxations of 78.9 ± 3.2, 29.7 ± 4.6, 82.2 ± 5.0 and 88.0 ± 2.4% respectively), but not phenylephrine-constricted arteries. In TPα-HEK cells, trioxilin A3 , C3 and hepoxilin A3 (10 µm) inhibited U46619 (10 nM)-induced increases in intracellular calcium by 53.0 ± 7.2%, 32.8 ± 5.0% and 37.9 ± 13.5% respectively. In contrast, trioxilin B3 and hepoxilin B3 were not synthesized in arteries and exhibited little biological activity. CONCLUSION: Trioxilin A3 and C3 and hepoxilin A3 are endogenous vascular relaxing factors. They are not endothelium-derived hyperpolarizing factors but mediate vascular relaxation by inhibiting TP agonist-induced increases in intracellular calcium. Thus, they regulate vascular homeostasis by acting as endogenous TP antagonists.

Inhibition of 2-arachidonoylglycerol catabolism modulates vasoconstriction of rat middle cerebral artery by the thromboxane mimetic, U-46619.

BACKGROUND AND PURPOSE: Cerebrovascular smooth muscle cells express the CB1 cannabinoid receptor and CB1 agonists produce vasodilatation of the middle cerebral artery (MCA). The thromboxane A2 mimetic, U-46619, increased the content of the endocannabinoid, 2-arachidonoylglycerol (2-AG) in the MCA and 2-AG moderated the vasoconstriction produced by U46619 in this tissue. The purposes of this study were to examine the extent to which 2-AG is catabolized by cerebral arteries and to determine whether blockade of 2-AG inactivation potentiates its feedback inhibition of U-44619-mediated vasoconstriction. EXPERIMENTAL APPROACH: The diameters of isolated, perfused MCA from male rats were measured using videomicroscopy. KEY RESULTS: Exogenous 2-AG produces a CB1 receptor-dependent and concentration-related increase in the diameter of MCA constricted with 5-HT. The E (max) for 2-AG dilation is increased 4-fold in the presence of the metabolic inhibitors 3-(decylthio)-1,1,1-trifluropropan-2-one (DETFP), URB754 and URB597. To examine the role of catabolism in the effects of endogenous 2-AG, vasoconstriction induced by U-46619 was studied. DETFP and URB754, but not the fatty acid amide hydrolase inhibitor, URB597, significantly increased the EC(50) for U-46619. These data support a physiological role for endocannabinoid feedback inhibition in the effects of U-46619 and indicate that endogenously produced 2-AG is also efficiently catabolized within the MCA. CONCLUSIONS AND IMPLICATIONS: MCA express mechanisms for the efficient inactivation of 2-AG, providing further support for an endocannabinoid feedback mechanism that opposes thromboxane-mediated vasoconstriction. These data suggest that potentiation of endogenously produced 2-AG could be a novel therapeutic approach to the treatment of thrombotic stroke.