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Effect of nitric oxide donors on membrane tritium accumulation of endocannabinoids and related endogenous lipids
Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Pharmacology. (Christopher Fowler)
Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Pharmacology.
(English)Manuscript (Other academic)
Abstract [en]

Endocannabinoids refers to a group of lipids with the ability of binding the two known cannabinoid receptors and synthesized by hydrolysis of lipid precursors. But still is the processing of the endocannabinoid metabolites following degradation poorly understood. Using tritium labelled anandamide (AEA) we discovered that nitric oxide (NO) donors significantly increased the amount of tritium accumulated in the cell membranes of RBL2H3 rat basophilic cells, 3T3-L1 mouse fibroblast cells and b.End5 mouse brain endothelioma cells following incubation. In addition, utilizing AEA labelled in either the arachidonate or ethanolamine part of the molecule revealed that the effect of NO donors mainly involved the arachidonate part of the molecule. In enzyme studies, pretreatment of intact cells with NO donors did not increase the activity of the enzyme mainly responsible for metabolism of AEA, fatty acid amide hydrolase (FAAH). Furthermore, inhibition of FAAH completely diminished the effect produced by NO donors in cells with a large FAAH component, indicating that the effects were downstream of the enzyme. Similar results of the NO donors were also obtained when radiolabelled 2-arachidonoylglycerol (2-AG) was used. These data showed that the processing of endocannabinoid metabolites can be regulated by NO donors and may therefore be of importance in recycling lipid compounds to elevate the tissue levels.

Keyword [en]
Endocannabinoids; anandamide; nitric oxide; membrane accumulation; fatty acid amide hydrolase
National Category
Pharmacology and Toxicology
Research subject
biokemisk farmakologi
Identifiers
URN: urn:nbn:se:umu:diva-22171OAI: oai:DiVA.org:umu-22171DiVA: diva2:213030
Available from: 2009-04-28 Created: 2009-04-27 Last updated: 2010-01-14Bibliographically 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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