Characterization of an anandamide degradation system in prostate epithelial PC-3 cells: synthesis of new transporter inhibitors as tools for this study

Authors

  • Lidia Ruiz-Llorente,

    1. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain
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    • 6

      Contributed equally to this study

  • Silvia Ortega-Gutiérrez,

    1. Departamento de Química Orgánica, Facultad de Químicas, Universidad Complutense, 28040 Madrid, Spain
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    • 6

      Contributed equally to this study

  • Alma Viso,

    1. Departamento de Química Orgánica, Facultad de Químicas, Universidad Complutense, 28040 Madrid, Spain
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  • María G Sánchez,

    1. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain
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  • Ana M Sánchez,

    1. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain
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  • Carlos Fernández,

    1. Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, 28034 Madrid, Spain
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  • José A Ramos,

    1. Departamento de Bioquímica y Biología Molecular III, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain
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  • Cecilia Hillard,

    1. Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank Rd, Milwaukee, WI 53226-0509, U.S.A.
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  • Miguel A Lasunción,

    1. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain
    2. Servicio de Bioquímica-Investigación, Hospital Ramón y Cajal, 28034 Madrid, Spain
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  • María L López-Rodríguez,

    1. Departamento de Química Orgánica, Facultad de Químicas, Universidad Complutense, 28040 Madrid, Spain
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  • Inés Díaz-Laviada

    Corresponding author
    1. Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain
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Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain. E-mail: ines.diazlaviada@uah.es

Abstract

  • 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 μM and Vmax=3.3±0.3 pmol min−1 (106 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 18 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 μM, 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

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