title: Exploiting nanotechnologies and TRPV1 channels to investigate the putative anandamide membrane transporter.
abstract: 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.
authors.affiliation: Institute of Biomolecular Chemistry, Consiglio Nazionale delle Ricerche, Pozzuoli, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Ligresti
authors.forename: Luciano
authors.initials: L
authors.lastname: De Petrocellis
authors.forename: Dolores
authors.initials: D
authors.lastname: Hernán Pérez de la Ossa
authors.forename: Rosario
authors.initials: R
authors.lastname: Aberturas
authors.forename: Luigia
authors.initials: L
authors.lastname: Cristino
authors.forename: Aniello Schiano
authors.initials: AS
authors.lastname: Moriello
authors.forename: Andrea
authors.initials: A
authors.lastname: Finizio
authors.forename: M Esther
authors.initials: ME
authors.lastname: Gil
authors.forename: Ana-Isabel
authors.initials: AI
authors.lastname: Torres
authors.forename: Jesús
authors.initials: J
authors.lastname: Molpeceres
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Di Marzo
mesh_terms: 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
chemical_list: 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