Cerebrovascular ResearchDepartments of Cell Biology Molecular Medicine Neurological Surgery Epilepsy Center, Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Epilepsia. 2011 Mar;52(3):562-71. doi: 10.1111/j.1528-1167.2010.02956.x. Epub 2011 Feb 5.
Compelling evidence supports the presence of P450 enzymes (CYPs) in the central nervous system (CNS). However, little information is available on the localization and function of CYPs in the drug-resistant epileptic brain. We have evaluated the pattern of expression of the specific enzyme CYP3A4 and studied its co-localization with MDR1. We also determined whether an association exists between CYP3A4 expression and cell survival.
Brain specimens were obtained from eight patients undergoing resection to relieve drug-resistant seizures or to remove a cavernous angioma. Each specimen was partitioned for either immunostaining or primary culture of human endothelial cells and astrocytes. Immunostaining was performed using anti-CYP3A4, MDR1, GFAP, or NeuN antibodies. High performance liquid chromatography-ultraviolet (HPLC-UV) analysis was used to quantify carbamazepine (CBZ) metabolism by these cells. CYP3A4 expression was correlated to DAPI) condensation, a marker of cell viability. Human embryonic kidney (HEK) cells were transfected with 4',6-diamidino-2-phenylindole (CYP3A4 to further evaluate the link between CYP3A4 levels, CBZ metabolism, and cell viability.
CYP3A4 was expressed by blood-brain barrier (BBB) endothelial cells and by the majority of neurons (75 ± 10%). Fluorescent immunostaining showed coexpression of CYP3A4 and MDR1 in endothelial cells and neurons. CYP3A4 expression inversely correlated with DAPI nuclear condensation. CYP3A4 overexpression in HEK cells conferred resistance to cytotoxic levels of carbamazepine. CYP3A4 levels positively correlated with the amount of CBZ metabolized.
CYP3A4 brain expression is not only associated with drug metabolism but may also represent a cytoprotective mechanism. Coexpression of CYP3A4 and MDR1 may be involved in cell survival in the diseased brain.
有强有力的证据表明细胞色素 P450 酶（CYPs）存在于中枢神经系统（CNS）中。然而，关于耐药性癫痫脑中 CYPs 的定位和功能的信息很少。我们评估了特定酶 CYP3A4 的表达模式，并研究了其与 MDR1 的共定位。我们还确定了 CYP3A4 表达与细胞存活之间是否存在关联。
从 8 名接受手术以缓解耐药性癫痫发作或切除海绵状血管畸形的患者中获得脑标本。每个标本都分为用于免疫染色或人内皮细胞和星形胶质细胞原代培养的部分。使用抗 CYP3A4、MDR1、GFAP 或 NeuN 抗体进行免疫染色。高效液相色谱-紫外（HPLC-UV）分析用于定量这些细胞的卡马西平（CBZ）代谢。CYP3A4 表达与 DAPI 凝聚相关，DAPI 凝聚是细胞活力的标志物。用 4'，6-二脒基-2-苯基吲哚（CYP3A4）转染人胚肾（HEK）细胞，以进一步评估 CYP3A4 水平、CBZ 代谢和细胞活力之间的联系。
CYP3A4 由血脑屏障（BBB）内皮细胞和大多数神经元（75±10%）表达。荧光免疫染色显示 CYP3A4 和 MDR1 在内皮细胞和神经元中共同表达。CYP3A4 表达与 DAPI 核凝聚呈负相关。HEK 细胞中 CYP3A4 的过表达赋予对细胞毒性水平的卡马西平的抗性。CYP3A4 水平与 CBZ 代谢量呈正相关。
CYP3A4 在大脑中的表达不仅与药物代谢有关，而且可能代表一种细胞保护机制。CYP3A4 和 MDR1 的共表达可能参与了患病大脑中的细胞存活。
