Evidence for Bidirectional Endocannabinoid Transport across Cell Membranes
Evidence for Bidirectional Endocannabinoid Transport across Cell Membranes
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DOI：
10.1074/jbc.m112.373241
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发表时间：
2012-10-05
影响因子：
4.8
通讯作者：
Gertsch, Juerg
中科院分区：
文献类型：
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作者：
Chicca, Andrea;Marazzi, Janine;Gertsch, Juerg
尽管对大麻素 (AEA) 跨细胞膜的运输进行了广泛的研究，但对其他内源性大麻素（例如 2-花生四烯酰甘油 (2-AG)）的膜运输知之甚少。先前的研究使用不同的方法提供了支持和反对细胞膜载体介导的内源性大麻素转运的数据。由于 AEA 和 2-AG 经历快速且几乎完全的细胞内水解，因此我们采用了放射性配体测定和细胞和细胞外内源性大麻素水平的绝对定量相结合的方法。在人 U937 白血病细胞中，100 nM AEA 和 1 muM 2-AG 通过快速且可饱和的过程被吸收，5 分钟后达到稳定水平。通过对内源性大麻素的摄取、分解和与细胞内结合蛋白的相互作用进行不同的药理学阻断，我们发现含有花生四烯酰链的类二十烷酸内源性大麻素会竞争调节其转运的共同膜靶点，而其他N-酰基乙醇胺不会干扰AEA和2-AG的摄取。通过将脂肪酸酰胺水解酶或单酰基甘油脂肪酶抑制剂与水解酶失活浓度的 AEA 转运抑制剂 UCM707 (1 μM) 和 OMDM-2 (5 μM) 组合，观察到细胞 AEA 和 2-AG 摄取的功能协同作用。有趣的是，结构上不相关的 AEA 摄取抑制剂也阻止了 AEA 和 2-AG 的细胞释放。我们首次证明 UCM707 和 OMDM-2 抑制 AEA 和 2-AG 跨细胞膜的双向运动。我们的研究结果表明，假定的内源性大麻素细胞膜转运蛋白控制细胞 AEA 和 2-AG 的运输和代谢。
Despite extensive research on the trafficking of anandamide (AEA) across cell membranes, little is known about the membrane transport of other endocannabinoids, such as 2-arachidonoylglycerol (2-AG). Previous studies have provided data both in favor and against a cell membrane carrier-mediated transport of endocannabinoids, using different methodological approaches. Because AEA and 2-AG undergo rapid and almost complete intracellular hydrolysis, we employed a combination of radioligand assays and absolute quantification of cellular and extracellular endocannabinoid levels. In human U937 leukemia cells, 100 nM AEA and 1 mu M 2-AG were taken up through a fast and saturable process, reaching a plateau after 5 min. Employing differential pharmacological blockage of endocannabinoid uptake, breakdown, and interaction with intracellular binding proteins, we show that eicosanoid endocannabinoids harboring an arachidonoyl chain compete for a common membrane target that regulates their transport, whereas other N-acylethanolamines did not interfere with AEA and 2-AG uptake. By combining fatty acid amide hydrolase or monoacyl glycerol lipase inhibitors with hydrolase-inactive concentrations of the AEA transport inhibitors UCM707 (1 mu M) and OMDM-2 (5 mu M), a functional synergism on cellular AEA and 2-AG uptake was observed. Intriguingly, structurally unrelated AEA uptake inhibitors also blocked the cellular release of AEA and 2-AG. We show, for the first time, that UCM707 and OMDM-2 inhibit the bidirectional movement of AEA and 2-AG across cell membranes. Our findings suggest that a putative endocannabinoid cell membrane transporter controls the cellular AEA and 2-AG trafficking and metabolism.