Campbell William B, Spitzbarth Nancy, Gauthier Kathryn M, Pfister Sandra L
Department of Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226,USA.
Am J Physiol Heart Circ Physiol. 2003 Dec;285(6):H2648-56. doi: 10.1152/ajpheart.00412.2003. Epub 2003 Aug 7.
Rabbit aortic endothelium metabolizes arachidonic acid (AA) by the 15-lipoxygenase pathway to vasodilatory eicosanoids, hydroxyepoxyeicosatrienoic acids (HEETAs), and trihydroxyeicosatrienoic acids (THETAs). The present study determined the chemical identity of the vasoactive THETA and investigated its role in ACh-induced relaxation in the rabbit aorta. AA caused endothelium-dependent, concentration-related relaxations of the rabbit aorta. Increasing the extracellular KCl concentration from 4.8 to 20 mM inhibited the relaxations to AA by approximately 60%, thereby implicating K+-channel activation in the relaxations. In addition, AA caused an endothelium-dependent hyperpolarization of aortic smooth muscle from -39.6 +/- 2.7 to -56.1 +/- 3.4 mV. In rabbit aortic rings, [14C]AA was metabolized to prostaglandins, HEETAs, THETAs, and 15-hydroxyeicosatetraenoic acid. Additional purification of the THETAs by HPLC resolved the mixture into its 14C-labeled products. Gas chromatography/mass spectrometry identified the metabolites as isomers of 11,12,15-THETA and 11,14,15-THETA. The 11,12,15-THETA relaxed and hyperpolarized the rabbit aorta, whereas 11,14,15-THETA had no vasoactive effect. The relaxations to 11,12,15-THETA were blocked by 20 mM KCl. In aortic rings pretreated with inhibitors of nitric oxide and prostaglandin synthesis, ACh caused a concentration-related relaxation that was completely blocked by 20 mM KCl. Pretreatment with the phospholipase A2 inhibitors mepacrine and 7,7-dimethyl-5,8-eicosadienoic acid, the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate, nordihydroguaiaretic acid, and ebselen, or the hydroperoxide isomerase inhibitors miconazole and clotrimazole also blocked ACh-induced relaxations. ACh caused a threefold increase in THETA release. These studies indicate that AA is metabolized by endothelial cells to 11,12,15-THETA, which activates K+ channels to hyperpolarize the aortic smooth muscle membrane and induce relaxation. Additionally, this lipoxygenase pathway mediates the nonnitric oxide, nonprostaglandin relaxations to ACh in the rabbit aorta by acting as a source of an endothelium-derived hyperpolarizing factor.
兔主动脉内皮细胞通过15-脂氧合酶途径将花生四烯酸（AA）代谢为血管舒张性类二十烷酸、羟基环氧二十碳三烯酸（HEETAs）和三羟基二十碳三烯酸（THETAs）。本研究确定了具有血管活性的THETA的化学特性，并研究了其在兔主动脉中乙酰胆碱（ACh）诱导的舒张反应中的作用。AA引起兔主动脉的内皮依赖性、浓度相关的舒张反应。将细胞外氯化钾浓度从4.8 mM增加到20 mM可使对AA的舒张反应抑制约60%，从而表明钾通道激活参与了舒张反应。此外，AA引起主动脉平滑肌的内皮依赖性超极化，从-39.6±2.7 mV变为-56.1±3.4 mV。在兔主动脉环中，[14C]AA被代谢为前列腺素、HEETAs、THETAs和15-羟基二十碳四烯酸。通过高效液相色谱（HPLC）对THETAs进行进一步纯化，将混合物分离为其14C标记的产物。气相色谱/质谱分析确定这些代谢产物为11,12,15-THETA和11,14,15-THETA的异构体。11,12,15-THETA使兔主动脉舒张并使其超极化，而11,14,15-THETA没有血管活性作用。对11,12,15-THETA的舒张反应被20 mM氯化钾阻断。在用一氧化氮和前列腺素合成抑制剂预处理的主动脉环中，ACh引起浓度相关的舒张反应，该反应被20 mM氯化钾完全阻断。用磷脂酶A2抑制剂米帕林和7,7-二甲基-5,8-二十碳二烯酸、脂氧合酶抑制剂肉桂基-3,4-二羟基-α-氰基肉桂酸、去甲二氢愈创木酸和依布硒啉，或氢过氧化物异构酶抑制剂咪康唑和克霉唑预处理也可阻断ACh诱导的舒张反应。ACh使THETA释放增加了三倍。这些研究表明，AA被内皮细胞代谢为11,12,15-THETA，后者激活钾通道使主动脉平滑肌膜超极化并诱导舒张。此外，这条脂氧合酶途径通过作为内皮源性超极化因子的来源，介导兔主动脉中对ACh的非一氧化氮、非前列腺素介导的舒张反应。
