Characterization of biotin-anandamide, a novel tool for the visualization of anandamide accumulation

Abstract

Anandamide (N-arachidonoylethanolamide; AEA) acts as an endogenous agonist of both cannabinoid and vanilloid receptors. During the last two decades, its metabolic pathways and biological activity have been investigated extensively and relatively well characterized. In contrast, at present, the effective nature and mechanism of AEA transport remain controversial and still unsolved issues. Here, we report the characterization of a biotinylated analog of AEA (b-AEA) that has the same lipophilicity of the parent compound. In addition, by means of biochemical assays and fluorescence microscopy, we show that b-AEA is accumulated inside the cells in a way superimposable on that of AEA. Conversely, b-AEA does not interact or interfere with the other components of the endocannabinoid system, such as type-1 and type-2 cannabinoid receptors, vanilloid receptor, AEA synthetase (N-acylphosphatidylethanolamine-hydrolyzing phospholipase D), or AEA hydrolase (fatty acid amide hydrolase). Together, our data suggest that b-AEA could be a very useful probe for visualizing the accumulation and intracellular distribution of this endocannabinoid.-Fezza,

Fatty Acid-binding Proteins Transport N-Acylethanolamines to Nuclear Receptors and Are Targets of Endocannabinoid Transport Inhibitors

“…Our study focused upon FABP5 as it is widely expressed across multiple cell types and mammalian tissues (6,22,44) and its inhibition likely underlies the efficacy of transport inhibitors in many cells. For example, AEA uptake is reduced by transport inhibitors in neurons and HaCaT keratinocytes, which express FABP5 (15,22,(45)(46)(47). Although our study focused upon FABP5, it is noteworthy that FABP3 and FABP7 bind to NAEs with relatively similar affinities (Table 1), and a similar trend may exist for transport inhibitors.…”

Pitfalls and solutions in assaying anandamide transport in cells

“…Finally, we took advantage of the glass support to develop a fl uorescence-based method for studying import and export of AEA by confocal microscopy, using a biotinylated analog of AEA (b-AEA). This substance provides a nonradioactive tool for visualizing the movement of this lipid in intact cells ( 28,39 ). The time-dependent accumulation assay showed that the uptake of b-AEA in both cell types occurred within minutes ( Table 2 ).…”

“…This new approach was successfully applied also to the measurement of AEA export from cells. Additionally, we demonstrated that a biotinylated derivative of AEA (b-AEA) ( 28,39 ) can be used to visualize the in-and-out traffi c of this endocannabinoid by immunofl uorescence techniques, providing a nonradioactive, rapid, and easy-to-use alternative for the study of anandamide metabolism.…”

Identification of intracellular carriers for the endocannabinoid anandamide

“…The results presented thus far suggest that the transmembrane transport and subsequent intracellular delivery of AEA to FAAH is rapid. The hydrophobicity of AEA (estimated LogP value of 5.1 (41)) suggests that it is unlikely to diffuse efficiently from the plasma membrane to intracellular membranes containing FAAH unaided. Because AEA is taken up and hydrolyzed by all cells that natively express and those transfected with FAAH (23)(24)(25), it is likely that AEA may share an intracellular delivery system used by other endogenous lipophilic molecules.…”