Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries

Abstract

Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined. ACh caused concentration-dependent relaxations of the mouse thoracic and abdominal aorta and carotid, femoral, and mesentery arteries (maximal relaxation: 57 ± 4%, 72 ± 4%, 82 ± 3%, 80 ± 3%, and 85 ± 3%, respectively). The NO synthase inhibitor nitro-L-arginine (L-NA; 30 μM) blocked relaxations in the thoracic aorta, and L-NA plus the COX inhibitor indomethacin (10 μM) inhibited relaxations in the abdominal aorta and carotid, femoral, and mesenteric arteries (maximal relaxation: 31 ± 10%, 33 ± 5%, 41 ± 8%, and 73 ± 3%, respectively). In mesenteric arteries, NO- and COX-independent relaxations to ACh were inhibited by the lipoxygenase (LO) inhibitors nordihydroguaiaretic acid (NDGA; 10 μM) and BW-755C (200 μM), the K(+) channel inhibitor apamin (1 μM), and 60 mM KCl and eliminated by endothelium removal. They were not altered by the cytochrome P-450 inhibitor N-methylsulfonyl-6-(2-propargyloxyphenyl)hexanamide (20 μM) or the epoxyeicosatrienoic acid antagonist 14,15-epoxyeicosa-5(Z)-enoic acid (10 μM). AA relaxations were attenuated by NDGA or apamin and eliminated by 60 mM KCl. Reverse-phase HPLC analysis revealed arterial [(14)C]AA metabolites that comigrated with prostaglandins, trihydroxyeicosatrienoic acids (THETAs), hydroxyepoxyeicosatrienoic acids (HEETAs), and hydroxyeicosatetraenoic acids (HETEs). Epoxyeicosatrienoic acids were not observed. Mass spectrometry confirmed the identity of 6-keto-PGF(1α), PGE(2), 12-HETE, 15-HETE, HEETAs, 11,12,15-THETA, and 11,14,15-THETA. AA metabolism was blocked by NDGA and endothelium removal. 11(R),12(S),15(S)-THETA relaxations (maximal relaxation: 73 ± 3%) were endothelium independent and blocked by 60 mM KCl. Western immunoblot analysis and RT-PCR of the aorta and mesenteric arteries demonstrated protein and mRNA expression of leukocyte-type 12/15-LO. Thus, in mouse resistance arteries, 12/15-LO AA metabolites mediate endothelium-dependent relaxations to ACh and AA.

A vasoconstrictor role for cyclooxygenase-1-mediated prostacyclin synthesis in mouse renal arteries

“…Part of the reason for this could be the altered level of COX-1, which is found to be lower in renal than in carotid arteries. Moreover, we find that the relaxation evoked by ACh in L-NAME-treated COX-1 Ϫ/Ϫ vessels is abolished by 60 mM K ϩ , concurring with the dilation mediated by EDHF previously shown in mouse vascular beds (1,10,24). On the other hand, our results with AA in COX-1 Ϫ/Ϫ vessels may not only imply little function for COX-2 but also suggest that non-COX AA metabolites, which are proposed to function as EDHF in mouse vascular beds and vessels of some other species (10,15,41), have little role in mouse renal arteries under the in vitro experimental condition, consistent with findings we previously reported in several other mouse vessels (42).…”

“…Moreover, we find that the relaxation evoked by ACh in L-NAME-treated COX-1 Ϫ/Ϫ vessels is abolished by 60 mM K ϩ , concurring with the dilation mediated by EDHF previously shown in mouse vascular beds (1,10,24). On the other hand, our results with AA in COX-1 Ϫ/Ϫ vessels may not only imply little function for COX-2 but also suggest that non-COX AA metabolites, which are proposed to function as EDHF in mouse vascular beds and vessels of some other species (10,15,41), have little role in mouse renal arteries under the in vitro experimental condition, consistent with findings we previously reported in several other mouse vessels (42). It should be noted that although non-COX AA metabolites have been considered to act as EDHF in renal vasculature (15), a direct vasodilator effect of AA on isolated renal arteries following COX blockade has not yet been reported, possibly resulting from conditions that are different from those of in vivo experiments.…”

“…Response evoked by AA in C57BL/6 and COX-1 Ϫ/Ϫ renal arteries. To further confirm the functional predominance of COX-1 and determine whether non-COX AA metabolites, which have been suggested to be endothelium-derived hyperpolarizing factors (EDHF), cause relaxation in mouse renal arteries (10,15,41), the responses evoked by AA in vessels from C57BL/6 and COX-1 Ϫ/Ϫ mice were analyzed. Vessels were also treated with L-NAME (1 mM) and contracted with PE (2 M).…”

Reduction of Mitochondria–Endoplasmic Reticulum Interactions by Acetylcholine Protects Human Umbilical Vein Endothelial Cells From Hypoxia/Reoxygenation Injury

“…35 Acetylcholine stimulates the release of soluble mediators in vascular endothelium; the main relaxing factors include NO, arachidonic acid metabolite, and endothelium-derived hyperpolarizing factors. 36 In the present study, we explored the potential role of NO in mediating the protective effects of acetylcholine on endothelial cells. However, it remains possible that other factors, such as arachidonic acid metabolites and endothelium-derived hyperpolarizing factors, contribute to acetylcholine-mediated endothelial protection.…”

Endothelial 12(S)-HETE vasorelaxation is mediated by thromboxane receptor inhibition in mouse mesenteric arteries

“…The preparation of protein lysates was carried out as previously described (10). Briefly, cleaned mouse mesenteric arteries were homogenized and lysed in lysis buffer [in mM: 50 HEPES, 150 NaCl, 1.5 MgCl2, and 1 EGTA and 10% glycerol, 1% Triton X-100, and protease inhibitor cocktail (Roche Molecular Biochemicals, Germany)].…”