Alkayed N J, Narayanan J, Gebremedhin D, Medhora M, Roman R J, Harder D R
Department of Physiology, Medical College of Wisconsin, Milwaukee 53226, USA.
Stroke. 1996 May;27(5):971-9. doi: 10.1161/01.str.27.5.971.
Brain parenchymal tissue metabolizes arachidonic acid (AA) via the cytochrome P450 (P450) 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.
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, P450 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 P450 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.
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.
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.
脑实质组织通过细胞色素P450（P450）环氧合酶将花生四烯酸（AA）代谢为环氧二十碳三烯酸（EETs）。EETs可使脑动脉扩张，并增强大脑大动脉血管平滑肌细胞中的钾离子电流。由于星形胶质细胞与脑微循环联系紧密，我们推测脑环氧合酶活性源自星形胶质细胞。本研究旨在鉴定并定位大鼠脑星形胶质细胞中的AA环氧合酶。我们还测试了EETs对大鼠脑微血管平滑肌细胞全细胞钾离子电流的影响。
采用功能测定法证明培养的完整星形胶质细胞的内源性环氧合酶活性。从已知肝环氧合酶P450 2C11的序列中获得寡核苷酸引物，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。将合适大小的逆转录/聚合酶链反应产物克隆到质粒载体中并测序。制备针对P450 2C11的多克隆肽抗体，并用于培养的星形胶质细胞的蛋白质免疫印迹和免疫细胞化学染色。采用电压钳技术测试EETs对从大鼠脑微血管肌细胞记录的全细胞钾离子电流的影响。
根据已知标准品的洗脱时间以及P450 AA环氧合酶抑制剂咪康唑的抑制作用，培养的星形胶质细胞与AA孵育时可产生11,12-和14,15-EETs。从培养的大鼠星形胶质细胞中分离的RNA衍生的cDNA序列与P450 2C11完全相同。在培养的星形胶质细胞的双重免疫化学染色中，对星形胶质细胞标志物胶质纤维酸性蛋白的免疫反应性与2C11免疫反应性共定位。EETs增强了大鼠脑微血管肌细胞的外向钾离子电流。
我们的结果表明，P450 2C11 mRNA在星形胶质细胞中表达，可能是星形胶质细胞环氧合酶活性的原因。鉴于EETs的血管舒张作用，我们的研究结果表明星形胶质细胞在由P45� 2C11催化的AA转化为EETs介导的脑微循环控制中发挥作用。EETs诱导大鼠脑微血管扩张的机制可能涉及钾离子通道的激活。
