Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study.
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 KM=4.7+/-0.2 microm and Vmax=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 (9Z,12Z)-N-(fur-3-ylmethyl)octadeca-9,12-dienamide (4, UCM119) being the most efficacious, with maximal inhibition and IC50 values of 49% and 11.3+/-0.5 microM, 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
Ruiz-Llorente
Lidia
L
Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain.
Ortega-Gutiérrez
Silvia
S
Viso
Alma
A
Sánchez
María G
MG
Sánchez
Ana M
AM
Fernández
Carlos
C
Ramos
José A
JA
Hillard
Cecilia
C
Lasunción
Miguel A
MA
López-Rodríguez
María L
ML
Díaz-Laviada
Inés
I
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000378:metabolism; D001095:Arachidonic Acids / Q000378:metabolism* / Q000494:pharmacology; D001711:Biotransformation; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002186:Cannabinoids / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D005663:Furans / Q000494:pharmacology; D006801:Humans; D008297:Male; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D053284:Polyunsaturated Alkamides; D011467:Prostate / Q000187:drug effects / Q000378:metabolism*; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D002121:Calcium Channel Blockers; D002186:Cannabinoids; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005663:Furans; D049990:Membrane Transport Modulators; D026901:Membrane Transport Proteins; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C443680:N-(3-furylmethyl)eicosa-5,8,11,14-tetraenamide; C078814:anandamide