Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes
Molecular characterization of an arachidonic acid epoxygenase in rat brain astrocytes
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DOI：
10.1161/01.str.27.5.971
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发表时间：
1996-05-01
期刊：
影响因子：
8.3
通讯作者：
Harder, DR
中科院分区：
文献类型：
--
作者：
Alkayed, NJ;Narayanan, J;Harder, DR
背景和目的的脑实质组织通过细胞色素P350（P350）环氧酶代谢蛛网膜酸（AA）为环氧酸磷脂酸（EETS）。 Eets扩张大脑动脉，并增强来自大脑动脉的血管平滑肌细胞中的K+电流。由于星形胶质细胞与脑微循环之间的密切关联，我们假设脑环氧酶活性起源于星形胶质细胞。这项研究旨在识别和定位在大鼠脑星形胶质细胞中的AA环氧酶。我们还测试了EET对大鼠脑微血管平滑肌细胞中全细胞K+电流的影响。方法用于证明培养中完整星形胶质细胞的内源性环氧酶活性。寡核苷酸引物源自已知的肝环氧酶p350 2c11的序列，用于从培养的大鼠星形胶质细胞中分离的RNA的逆转录/聚合酶链反应中。将适当的大小逆转录/聚合酶链反应产物克隆到质粒载体中并进行测序。对P350 2C11提出多克隆肽抗体，并用于培养的星形胶质细胞的蛋白质印迹和免疫细胞化学染色。使用一种电压钳技术来测试EET对从大鼠脑微血管肌肉细胞记录的全细胞K+电流的影响。基于已知标准的洗脱时间和miconazole的抑制作用，P450 AA Epoxygegenase，培养的星形镜会产生P450 AA的抑制剂。与AA一起孵育时，11,12-和14,15欧元。从培养的大鼠星形胶质细胞分离的RNA的cDNA的序列与P450 2C11相同100％。对神经纤维纤维酸性蛋白的免疫反应性是星形胶质细胞的标志物，与培养的星形胶质细胞的双重免疫化学染色中与2C11免疫反应性共定位。 Eets从大鼠脑微血管中增强了肌肉细胞中的外向K+电流。结论我们的结果表明，P450 2C11 mRNA在星形胶质细胞中表达，可能导致星形胶质细胞环氧酶活性。鉴于EET的血管舒张作用，我们的发现表明，星形胶质细胞在控制P450 2C11催化AA转换为EET的脑微循环中的作用。 EET诱导的大鼠脑微血管扩张的机制可能涉及K+通道的激活。
Background and Purpose Brain parenchymal tissue metabolizes arachidonic acid (AA) via the cytochrome P350 (P350) epoxygenase to epoxyeicosatrienoic acids (EETs). EETs dilate cerebral arterioles and enhance K+ current in vascular smooth muscle cells from large cerebral arteries. Because of the close association between astrocytes and the cerebral microcirculation, we hypothesized that brain epoxygenase activity originates from astrocytes. This study was designed to identify and localize an AA epoxygenase in rat brain astrocytes. We also tested the effect of EETs on whole-cell K+ current in rat cerebral microvascular smooth muscle cells.Methods A functional assay was used to demonstrate endogenous epoxygenase activity of intact astrocytes in culture. Oligonucleotide primers derived from the sequence of a known hepatic epoxygenase, P350 2C11, were used in reverse transcription/polymerase chain reaction of RNA isolated from cultured rat astrocytes. The appropriate size reverse transcription/polymerase chain reaction product was cloned into a plasmid vector and sequenced. A polyclonal peptide antibody was raised against P350 2C11 and used in Western blotting and immunocytochemical staining of cultured astrocytes. A voltage-clamp technique was used to test the effect of EETs on whole-cell K+ current recorded from rat cerebral microvascular muscle cells.Results Based on elution time of known standards and inhibition by miconazole, an inhibitor of P450 AA epoxygenase, cultured astrocytes produce 11,12- and 14,15-EETs when incubated with AA. The sequence of a cDNA derived from RNA isolated from cultured rat astrocytes was 100% identical to P450 2C11. Immunoreactivity to glial fibrillary acidic protein, a marker for astrocytes, colocalized with 2C11 immunoreactivity in double immunochemical staining of cultured astrocytes. EETs enhanced outward K+ current in muscle cells from rat brain microvessels.Conclusions Our results demonstrate that a P450 2C11 mRNA is expressed in astrocytes and may be responsible for astrocyte epoxygenase activity. Given the vasodilatory effect of EETs, our findings suggest a role for astrocytes in the control of cerebral microcirculation mediated by P450 2C11-catalyzed conversion of AA to EETs. The mechanism of EET-induced dilation of rat cerebral microvessels may involve activation of K+ channels.