Exploiting Nanotechnologies and TRPV1 Channels to Investigate the Putative Anandamide Membrane Transporter

Abstract

BackgroundConsiderable 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 FindingsWe 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-ε-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.ConclusionsWe 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.

From Phytocannabinoids to Cannabinoid Receptors and Endocannabinoids: Pleiotropic Physiological and Pathological Roles Through Complex Pharmacology

“…In one case, nanotechnologies and the AEA cytosolic binding site on TRPV1 channels were exploited to detect AEA entry into cells. This study demonstrated how uptake inhibitors lose their efficacy when AEA is prevented from interacting directly with plasma membrane proteins because masked into nanoparticles, thus arguing in favor of the existence of membrane carrier (476). Different models of AEA cellular uptake and transport have been proposed and argued over.…”

Pitfalls and solutions in assaying anandamide transport in cells

“…buffer (a procedure very useful to achieve really short incubation times), it is possible to save even more money by reducing the amount of radioactivity used for each assay. During the preparation of this manuscript, a novel procedure for investigating the uptake of AEA in intact cells was reported ( 51 ). In this study, Ligresti et al used TRPV1 channel as a biosensor to detect AEA and developed nanoparticles to deliver AEA into the cytosol, by-passing any interaction with membrane proteins.…”

“…In this study, Ligresti et al used TRPV1 channel as a biosensor to detect AEA and developed nanoparticles to deliver AEA into the cytosol, by-passing any interaction with membrane proteins. Using this approach, they found that several agents previously reported to inhibit AEA uptake lose their effi cacy when AEA is prevented from interacting with membrane proteins, thus providing novel and more direct evidence for the existence of an AEA transporter (51). Interestingly, because they kept cells in quartz cuvettes or Petri dishes for fl uorescence and digital holographic quantitative phase microscopy, respectively, it is tempting to suggest that glass coverslips or chambers could represent a better support also for these novel procedures.…”

Evidence for Bidirectional Endocannabinoid Transport across Cell Membranes

“…Despite an increasing knowledge about the receptors and signaling pathways involved in the ECS, the movement of AEA and 2-AG across the cell membrane remains to be elucidated and is subject to ongoing controversy (12). Previous studies have addressed the issue of intracellular AEA uptake, providing strong evidence in favor of a carrier-mediated transport that is linked to but distinctly independent of the enzymatic hydrolysis of AEA by FAAH (13)(14)(15)(16)(17)(18). Importantly, sev-eral apparently selective cellular AEA uptake inhibitors have been reported, which at nanomolar or low micromolar concentrations do not inhibit FAAH but still inhibit cellular AEA uptake (19 -21).…”