McFarland Matthew J, Bardell Tamera K, Yates Marla L, Placzek Ekaterina A, Barker Eric L
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, 575 Stadium Mall Dr., West Lafayette, IN 47907-2091, USA.
Mol Pharmacol. 2008 Jul;74(1):101-8. doi: 10.1124/mol.108.044834. Epub 2008 Apr 24.
The precise mechanism by which the cellular uptake of the endocannabinoid anandamide (AEA) occurs has been the source of much debate. In the current study, we show that neuronal differentiated CAD (dCAD) cells accumulate anandamide by a process that is inhibited in a dose-dependent manner by N-(4-hydroxyphenyl)arachidonylamide (AM404). We also show that dCAD cells express functional fatty acid amide hydrolase, the enzyme primarily responsible for anandamide metabolism. Previous data from our laboratory indicated that anandamide uptake occurs by a caveolae-related endocytic mechanism in RBL-2H3 cells. In the current study, we show that anandamide uptake by dCAD cells may also occur by an endocytic process that is associated with detergent-resistant membrane microdomains or lipid rafts. Nystatin and progesterone pretreatment of dCAD cells significantly inhibited anandamide accumulation. Furthermore, RNA interference (RNAi)-mediated knockdown of dynamin 2, a protein involved in endocytosis, blocked the internalization of the fluorescently labeled anandamide analog SKM 4-45-1 ([3',6'-bis(acetyloxy)-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen-5-yl]-2-[[1-oxo-5Z,8Z,11Z,14Z-eicosatetraenyl]amino]ethyl ester carbamic acid). RNAi-mediated knockdown of the beta2 subunit of the clathrin-associated activator protein 2 complex had no effect on SKM 4-45-1 internalization. We were surprised to find that dynamin 2 knockdown in dCAD cells did not affect [3H]AEA uptake. However, dynamin 2 knockdown caused a significant increase in the overall levels of intact [3H]AEA associated with the cells, suggesting that trafficking of [3H]AEA to FAAH had been disrupted. This finding may be the result of an accumulation of the anandamide carrier protein in detergent-resistant membranes after dynamin 2 knockdown. Our studies provide evidence that the cellular uptake of anandamide may occur by a dynamin 2-dependent, caveolae-related endocytic process in dCAD cells.
内源性大麻素花生四烯乙醇胺（AEA）的细胞摄取的确切机制一直是诸多争论的源头。在当前研究中，我们发现神经元分化的CAD（dCAD）细胞通过一个过程积累花生四烯乙醇胺，该过程会被N-(4-羟基苯基)花生四烯酸酰胺（AM404）以剂量依赖性方式抑制。我们还表明dCAD细胞表达功能性脂肪酸酰胺水解酶，该酶是主要负责花生四烯乙醇胺代谢的酶。我们实验室之前的数据表明，在RBL-2H3细胞中，花生四烯乙醇胺摄取是通过一种与小窝相关的内吞机制发生的。在当前研究中，我们表明dCAD细胞对花生四烯乙醇胺的摄取也可能通过一种与耐去污剂膜微区或脂筏相关的内吞过程发生。用制霉菌素和孕酮预处理dCAD细胞可显著抑制花生四烯乙醇胺的积累。此外，RNA干扰（RNAi）介导的发动蛋白2（一种参与内吞作用的蛋白质）敲低，阻断了荧光标记的花生四烯乙醇胺类似物SKM 4-45-1（[3',6'-双(乙酰氧基)-3-氧代螺[异苯并呋喃-1(3H),9'-[9H]呫吨-5-基]-2-[[1-氧代-5Z,8Z,11Z,14Z-二十碳四烯基]氨基]乙酯氨基甲酸）的内化。RNAi介导的网格蛋白相关激活蛋白2复合物的β2亚基敲低对SKM 4-45-1的内化没有影响。我们惊讶地发现，dCAD细胞中发动蛋白2敲低并不影响[3H]AEA的摄取。然而，发动蛋白2敲低导致与细胞相关的完整[3H]AEA的总体水平显著增加，这表明[3H]AEA向脂肪酸酰胺水解酶的转运被破坏。这一发现可能是发动蛋白2敲低后花生四烯乙醇胺载体蛋白在耐去污剂膜中积累的结果。我们的研究提供了证据，表明在dCAD细胞中，花生四烯乙醇胺的细胞摄取可能通过一种依赖发动蛋白2的、与小窝相关的内吞过程发生。
