Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.
PLoS One. 2010 Apr 22;5(4):e10239. doi: 10.1371/journal.pone.0010239.
Considerable efforts have been made to characterize the pathways regulating the extracellular levels of the endocannabinoid anandamide. However, none of such pathways has been so argued as the existence of a carrier-mediated transport of anandamide across the membrane. Apart from the lack of molecular evidence for such a carrier, the main reasons of this controversy lie in the methodologies currently used to study anandamide cellular uptake. Furthermore, the main evidence in favor of the existence of an "anandamide transporter" relies on synthetic inhibitors of this process, the selectivity of which has been questioned.
METHODOLOGY/PRINCIPAL FINDINGS: We used the cytosolic binding site for anandamide on TRPV1 channels as a biosensor to detect anandamide entry into cells, and exploited nanotechnologies to study anandamide membrane transport into intact TRPV1-overexpressing HEK-293 cells. Both fluorescence and digital holographic (DH) quantitative phase microscopy were used to study TRPV1 activation. Poly-epsilon-caprolactone nanoparticles (PCL-NPs) were used to incorporate anandamide, which could thus enter the cell and activate TRPV1 channels bypassing any possible specific protein(s) involved in the uptake process. We reasoned that in the absence of such protein(s), pharmacological tools previously shown to inhibit the "anandamide transporter" would affect in the same way the uptake of anandamide and PCL-NP-anandamide, and hence the activation of TRPV1. However, when masked into PCL-NPs, anandamide cellular uptake became much less sensitive to these agents, although it maintained the same pharmacokinetics and pharmacodynamics as that of "free" anandamide.
We found here that several agents previously reported to inhibit anandamide cellular uptake lose their efficacy when anandamide is prevented from interacting directly with plasma membrane proteins, thus arguing in favor of the specificity of such agents for the putative "anandamide transporter", and of the existence of such mechanism.
人们已经做出了相当大的努力来描述调节内源性大麻素大麻酰胺细胞外水平的途径。然而，还没有一种途径被认为是大麻酰胺穿过细胞膜的载体介导转运。除了缺乏这种载体的分子证据外，这种争议的主要原因在于目前用于研究大麻酰胺细胞摄取的方法学。此外，支持“大麻酰胺转运体”存在的主要证据依赖于该过程的合成抑制剂，其选择性受到质疑。
方法/主要发现：我们使用 TRPV1 通道上大麻酰胺的胞质结合位点作为生物传感器来检测大麻酰胺进入细胞，利用纳米技术来研究完整的 TRPV1 过表达 HEK-293 细胞中大麻酰胺的膜转运。荧光和数字全息（DH）定量相位显微镜都用于研究 TRPV1 的激活。聚己内酯纳米颗粒（PCL-NPs）用于包裹大麻酰胺，从而可以进入细胞并激活 TRPV1 通道，而无需任何可能参与摄取过程的特定蛋白。我们推断，在没有这种蛋白的情况下，先前显示抑制“大麻酰胺转运体”的药理学工具将以相同的方式影响大麻酰胺和 PCL-NP-大麻酰胺的摄取，从而影响 TRPV1 的激活。然而，当被包裹在 PCL-NPs 中时，大麻酰胺的细胞摄取对这些药物的敏感性降低了很多，尽管它保持了与“游离”大麻酰胺相同的药代动力学和药效学。
我们在这里发现，当大麻酰胺被阻止与质膜蛋白直接相互作用时，先前报道的几种抑制大麻酰胺细胞摄取的药物失去了其效力，从而支持了这些药物对所谓的“大麻酰胺转运体”的特异性，以及这种机制的存在。
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