Di Pasquale Eric, Chahinian Henri, Sanchez Patrick, Fantini Jacques
Université de la Méditerranée (Aix-Marseille 2), Centre de Recherche en Neurobiologie et Neurophysiologie de Marseille, CNRS UMR 6231, INRA USC 2027, Interactions Moléculaires et Systèmes Membranaires, Faculté des Sciences Saint-Jérôme, Marseille, France.
PLoS One. 2009;4(3):e4989. doi: 10.1371/journal.pone.0004989. Epub 2009 Mar 30.
Anandamide is a lipid neurotransmitter which belongs to a class of molecules termed the endocannabinoids involved in multiple physiological functions. Anandamide is readily taken up into cells, but there is considerable controversy as to the nature of this transport process (passive diffusion through the lipid bilayer vs. involvement of putative proteic transporters). This issue is of major importance since anandamide transport through the plasma membrane is crucial for its biological activity and intracellular degradation. The aim of the present study was to evaluate the involvement of cholesterol in membrane uptake and transport of anandamide.
METHODOLOGY/PRINCIPAL FINDINGS: Molecular modeling simulations suggested that anandamide can adopt a shape that is remarkably complementary to cholesterol. Physicochemical studies showed that in the nanomolar concentration range, anandamide strongly interacted with cholesterol monolayers at the air-water interface. The specificity of this interaction was assessed by: i) the lack of activity of structurally related unsaturated fatty acids (oleic acid and arachidonic acid at 50 nM) on cholesterol monolayers, and ii) the weak insertion of anandamide into phosphatidylcholine or sphingomyelin monolayers. In agreement with these data, the presence of cholesterol in reconstituted planar lipid bilayers triggered the stable insertion of anandamide detected as an increase in bilayer capacitance. Kinetics transport studies showed that pure phosphatidylcholine bilayers were weakly permeable to anandamide. The incorporation of cholesterol in phosphatidylcholine bilayers dose-dependently stimulated the translocation of anandamide.
CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that cholesterol stimulates both the insertion of anandamide into synthetic lipid monolayers and bilayers, and its transport across bilayer membranes. In this respect, we suggest that besides putative anandamide protein-transporters, cholesterol could be an important component of the anandamide transport machinery. Finally, this study provides a mechanistic explanation for the key regulatory activity played by membrane cholesterol in the responsiveness of cells to anandamide.
花生四烯乙醇胺是一种脂质神经递质，属于一类被称为内源性大麻素的分子，参与多种生理功能。花生四烯乙醇胺很容易被细胞摄取，但关于这种转运过程的性质（通过脂质双层的被动扩散与假定的蛋白质转运体的参与）存在相当大的争议。这个问题至关重要，因为花生四烯乙醇胺通过质膜的转运对其生物活性和细胞内降解至关重要。本研究的目的是评估胆固醇在花生四烯乙醇胺的膜摄取和转运中的作用。
方法/主要发现：分子模拟表明，花生四烯乙醇胺可以呈现出与胆固醇显著互补的形状。物理化学研究表明，在纳摩尔浓度范围内，花生四烯乙醇胺在气-水界面与胆固醇单层强烈相互作用。这种相互作用的特异性通过以下方式评估：i）结构相关的不饱和脂肪酸（50 nM的油酸和花生四烯酸）对胆固醇单层无活性，ii）花生四烯乙醇胺在磷脂酰胆碱或鞘磷脂单层中的弱插入。与这些数据一致，在重构的平面脂质双层中存在胆固醇会引发花生四烯乙醇胺的稳定插入，表现为双层电容增加。动力学转运研究表明，纯磷脂酰胆碱双层对花生四烯乙醇胺的通透性较弱。在磷脂酰胆碱双层中加入胆固醇剂量依赖性地刺激了花生四烯乙醇胺的转运。
结论/意义：我们的结果表明，胆固醇既刺激花生四烯乙醇胺插入合成脂质单层和双层，也刺激其跨双层膜的转运。在这方面，我们认为除了假定的花生四烯乙醇胺蛋白质转运体之外，胆固醇可能是花生四烯乙醇胺转运机制的重要组成部分。最后，本研究为膜胆固醇在细胞对花生四烯乙醇胺反应性中的关键调节活性提供了一个机制解释。
