Exploiting nanotechnologies and TRPV1 channels to investigate the putative anandamide membrane transporter.
BACKGROUND
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.


RESULTS
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.


CONCLUSIONS
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.
Ligresti
Alessia
A
Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.
De Petrocellis
Luciano
L
Hernán Pérez de la Ossa
Dolores
D
Aberturas
Rosario
R
Cristino
Luigia
L
Moriello
Aniello Schiano
AS
Finizio
Andrea
A
Gil
M Esther
ME
Torres
Ana-Isabel
AI
Molpeceres
Jesús
J
Di Marzo
Vincenzo
V
D001095:Arachidonic Acids / Q000008:administration & dosage / Q000378:metabolism*; D001665:Binding Sites; D002121:Calcium Channel Blockers; D063385:Cannabinoid Receptor Modulators; D002460:Cell Line; D004337:Drug Carriers / Q000737:chemistry / Q000494:pharmacology*; D063388:Endocannabinoids; D006801:Humans; D026901:Membrane Transport Proteins / Q000378:metabolism*; D011091:Polyesters; D053284:Polyunsaturated Alkamides / Q000008:administration & dosage / Q000378:metabolism*; D050916:TRPV Cation Channels / Q000378:metabolism* / Q000493:pharmacokinetics
D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D063385:Cannabinoid Receptor Modulators; D004337:Drug Carriers; D063388:Endocannabinoids; D026901:Membrane Transport Proteins; D011091:Polyesters; D053284:Polyunsaturated Alkamides; D050916:TRPV Cation Channels; C016240:polycaprolactone; C078814:anandamide