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Inhibition of the cellular uptake of anandamide by genistein and its analogue daidzein in cells with different levels of fatty acid amide hydrolase-driven uptake
Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Pharmacology. Farmakologi.
Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Pharmacology. Farmakologi.
2007 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 152, no 5, 744-750 p.Article in journal (Refereed) Published
Abstract [en]

Background and purpose: Genistein, a tyrosine kinase inhibitor used to block caveolae dependent endocytosis, reduces the cellular uptake of anandamide in RBL2H3 basophilic leukaemia cells. However, genistein is also a competitive inhibitor of fatty acid amide hydrolase, the enzyme responsible for anandamide hydrolysis. Here we have investigated whether inhibition of fatty acid amide hydrolase rather than inhibition of endocytosis is the primary determinant of genistein actions upon anandamide uptake.

Experimental approach: Cellular uptake of anandamide, labelled in the arachidonoyl part of the molecule was assessed in four different cell lines using a standard method. Fatty acid amide hydrolase activity in homogenates and intact cells was measured using anandamide labelled in the ethanolamine part of the molecule.

Key results: The fatty acid amide hydrolase inhibitor URB597 inhibited anandamide uptake into RBL2H3 cells and R3327 AT-1 prostate cancer cells, but not into 3T3-L1 preadipocytes or PC-3 prostate cancer cells. An identical pattern was seen with genistein. The related compound daidzein inhibited anandamide hydrolysis in homogenates and intact cells, and reduced its uptake into RBL2H3 and R3327 AT-1, but not PC-3 cells. Anandamide hydrolysis by cell homogenates was in the order RBL2H3 4 R3327 AT-1 4 PC-3 E3T3-L1.

Conclusions and implications: The ability of genistein to inhibit anandamide uptake is mimicked by daidzein (which does not affect tyrosine kinase), and is only seen in cells that show sensitivity to URB597. This indicates that blockade of fatty acid amide hydrolase is the primary determinant of the effects of genistein on cellular anandamide uptake.

Place, publisher, year, edition, pages
2007. Vol. 152, no 5, 744-750 p.
Keyword [en]
endocannabinoid; anandamide; cellular uptake; fatty acid amide hydrolase; tyrosine kinase; genistein; daidzein
Identifiers
URN: urn:nbn:se:umu:diva-5983DOI: 10.1038/sj.bjp.0707401PubMedID: 17676056OAI: oai:DiVA.org:umu-5983DiVA: diva2:145651
Available from: 2007-12-04 Created: 2007-12-04 Last updated: 2017-12-14Bibliographically approved
In thesis
1. The cellular processing of the endocannabinoid anandamide and its pharmacological manipulation
Open this publication in new window or tab >>The cellular processing of the endocannabinoid anandamide and its pharmacological manipulation
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert most of their actions by binding to cannabinoid receptors. The effects of the endocannabinoids are short-lived due to rapid cellular accumulation and metabolism, for AEA, primarily by the enzymes fatty acid amide hydrolase (FAAH). This has led to the hypothesis that by inhibition of the cellular processing of AEA, beneficial effects in conditions such as pain and inflammation can be enhanced. The overall aim of the present thesis has been to examine the mechanisms involved in the cellular processing of AEA and how they can be influenced pharmacologically by both synthetic natural compounds.

Liposomes, artificial membranes, were used in paper I to study the membrane retention of AEA. The AEA retention mimicked the early properties of AEA accumulation, such as temperature-dependency and saturability.

In paper II, FAAH was blocked by a selective inhibitor, URB597, and reduced the accumulation of AEA into RBL2H3 basophilic leukaemia cells by approximately half. Treating intact cells with the tyrosine kinase inhibitor genistein, an isoflavone found in soy plants and known to disrupt caveolae-related endocytosis, reduced the AEA accumulation by half, but in combination with URB597 no further decrease was seen. Further on, the effects of genistein upon uptake were secondary to inhibition of FAAH. The ability to inhibit the accumulation and metabolism of AEA was shared by several flavonoids (shown in paper III). In paper IV, the isoflavone biochanin A and URB597 had effects in vivo, in a model of persistent pain, effects decreased by the cannabinoid receptor 1 antagonist AM251.

In paper VI, the cellular processing of the endocannabinoid metabolites following degradation was examined, a mechanism poorly understood. It was found that nitric oxide (NO) donors significantly increased the retention of tritium in cell membranes following incubation with either tritiated AEA or 2-AG. Further experiments revealed that the effect of NO donors mainly involves the arachidonate part of the molecules. Inhibition of FAAH completely reduced the effect of NO donors in cells with a large FAAH component, indicating that the effects were downstream of the enzyme.

These results suggest that the cellular processing of endocannabinoids can be affected in a manner of different ways by pharmacological manipulation in vitro and that naturally occurring flavonoid compounds can interact with the endocannabinoid system.

Place, publisher, year, edition, pages
Umeå: Farmakologi och klinisk neurovetenskap, 2009. 80 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1247
Keyword
Endocannabinoid, anandamide, cellular processing, pain, flavonoids, fatty acid amide hydrolase
National Category
Pharmacology and Toxicology
Research subject
Medical Pharmacology
Identifiers
urn:nbn:se:umu:diva-22221 (URN)978-91-7264-732-9 (ISBN)
Distributor:
Farmakologi, 901 87, Umeå
Public defence
2009-05-22, Sal E04, byggnad 6E, Umeå universitetssjukhus, 901 89 Umeå, Umeå, 09:00 (English)
Opponent
Supervisors
Available from: 2009-04-30 Created: 2009-04-28 Last updated: 2010-01-18Bibliographically approved

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