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Characterization of an anandamide degradation system in prostate epithelial PC‐3 cells: synthesis of new transporter inhibitors as tools for this study
Author(s) -
RuizLlorente Lidia,
OrtegaGutiérrez Silvia,
Viso Alma,
Sánchez María G,
Sánchez Ana M,
Fernández Carlos,
Ramos José A,
Hillard Cecilia,
Lasunción Miguel A,
LópezRodríguez María L,
DíazLaviada Inés
Publication year - 2004
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1038/sj.bjp.0705628
Subject(s) - anandamide , endocannabinoid system , chemistry , fatty acid amide hydrolase , cannabinoid , cannabinoid receptor , biochemistry , transporter , pharmacology , biology , agonist , receptor , gene
The response of anandamide is terminated by a carrier‐mediated transport followed by degradation catalyzed by the cloned enzyme fatty acid amidohydrolase (FAAH). In this study, we provide biochemical data showing an anandamide uptake process and the expression of FAAH in human prostate. Anandamide was accumulated in PC‐3 cells by a saturable and temperature‐dependent process. Kinetic studies of anandamide uptake, determined in the presence of cannabinoid and vanilloid antagonists, revealed apparent parameters of K M =4.7±0.2 μ M and V max =3.3±0.3 pmol min −1 (10 6 cells) −1 . The accumulation of anandamide was moderately inhibited by previously characterized anandamide transporter inhibitors (AM404, UCM707 and VDM11) but was unaffected by inhibitors of other lipid transport systems (phloretin or verapamil) and moderately affected by the FAAH inhibitor methyl arachidonyl fluorophosphonate. The presence of FAAH in human prostate epithelial PC‐3 cells was confirmed by analyzing its expression by Western blot and measuring FAAH activity. To further study the structural requirements of the putative carrier, we synthesized a series of structurally different compounds 1 – 8 and evaluated their capacity as uptake inhibitors. They showed different inhibitory capacity in PC‐3 cells, with (9 Z ,12 Z )‐ N ‐(fur‐3‐ylmethyl)octadeca‐9,12‐dienamide ( 4 , UCM119 ) being the most efficacious, with maximal inhibition and IC 50 values of 49% and 11.3±0.5 μ M , respectively. In conclusion, PC‐3 cells possess a complete inactivation system for anandamide formed by an uptake process and the enzyme FAAH. These results suggest a possible physiological function of anandamide in the prostate, reinforcing the role of endocannabinoid system as a neuroendocrine modulator.British Journal of Pharmacology (2004) 141 , 457–467. doi: 10.1038/sj.bjp.0705628