Amruthesh S C, Boerschel M F, McKinney J S, Willoughby K A, Ellis E F
Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0613.
J Neurochem. 1993 Jul;61(1):150-9. doi: 10.1111/j.1471-4159.1993.tb03550.x.
We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxygenase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [3H]arachidonic acid, formed products that eluted in four major groups designated as R17-30, R42-50, R51-82, and R83-90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impact-mass spectrometry analysis, the more polar fractions (R17-30) contained prostaglandin D2 as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F1 alpha, prostaglandin E2, prostaglandin F2 alpha, and thromboxane B2. Fractions R42-50, R51-82, and R83-90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R83-90 was 5,6-epoxyeicosatrienoic acid (EET). Fractions R51-82 contained 14,15- and 8,9-EETs, 12- and 5-hydroxyeicosatetraenoic acids, and 8,9- and 5,6-dihydroxyeicosatrienoic acids (DHETs). In fractions R42-50, 14,15-DHET was the major product. When radiolabeled [3H]14,15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [3H]14,15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxy alcohols from 14,15-DHET was not observed. In conclusion, astrocytes readily metabolize arachidonic acid to 14,15-EET, 5,6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.
我们最近发现脑片能够通过环氧合酶途径代谢花生四烯酸。本研究的目的是开始确定单个脑细胞类型形成环氧合酶代谢产物的能力。我们研究了星形胶质细胞的环氧合酶途径，还证实了其通过环氧化酶和脂氧化酶系统进行的代谢。培养的大鼠海马星形胶质细胞匀浆与放射性标记的[3H]花生四烯酸一起孵育时，会形成产物，根据其在反相高效液相色谱中的保留时间，这些产物可分为四个主要组，分别命名为R17 - 30、R42 - 50、R51 - 82和R83 - 90。通过正相高效液相色谱和第二个反相高效液相色谱系统，这些馏分进一步分离出多达13个峰。每个峰中的主要成分通过气相色谱/电子轰击质谱进行结构表征。基于高效液相色谱保留时间和气相色谱/电子轰击质谱分析，极性较强的馏分（R17 - 30）中主要的环氧化酶产物是前列腺素D2。次要产物包括6 - 酮前列腺素F1α、前列腺素E2、前列腺素F2α和血栓素B2。馏分R42 - 50、R51 - 82和R83 - 90含有环氧合酶和脂氧化酶样产物。馏分R83 - 90中的主要代谢产物是5,6 - 环氧二十碳三烯酸（EET）。馏分R51 - 82含有14,15 - 和8,9 - EETs、12 - 和5 - 羟基二十碳四烯酸以及8,9 - 和5,6 - 二羟基二十碳三烯酸（DHETs）。在馏分R42 - 50中，14,15 - DHET是主要产物。当放射性标记的[3H]14, fifteen - EET与星形胶质细胞匀浆一起孵育时，它会迅速代谢为[3H]14,15 - DHET。这种代谢受到亚微摩尔浓度的4 - 苯基查尔酮氧化物（一种环氧水解酶活性的强效抑制剂）的抑制。未观察到由14,15 - DHET形成其他极性代谢产物，如三醇或环氧醇。总之，星形胶质细胞很容易将花生四烯酸代谢为14,15 - EET、5,6 - EET及其邻二醇。先前的研究表明这些产物可能会影响神经元功能和脑血流量。
