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The cellular processing of the endocannabinoid anandamide and its pharmacological manipulation
Umeå University, Faculty of Medicine, Pharmacology and Clinical Neuroscience, Pharmacology. (Christopher J Fowler)
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 [en]
Endocannabinoid, anandamide, cellular processing, pain, flavonoids, fatty acid amide hydrolase
National Category
Pharmacology and Toxicology
Research subject
Medical Pharmacology
Identifiers
URN: urn:nbn:se:umu:diva-22221ISBN: 978-91-7264-732-9 (print)OAI: oai:DiVA.org:umu-22221DiVA: diva2:213724
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
List of papers
1. Characterization of anandamide retention in synthetic liposomes
Open this publication in new window or tab >>Characterization of anandamide retention in synthetic liposomes
(English)Manuscript (Other academic)
Abstract [en]

Anandamide (AEA) is an endogenous ligand for cannabinoid receptors. Prior to termination of signalling activity AEA has to move across the plasma membrane to the reach its intracellular hydrolytic enzyme. The mechanism underlying this transport is under considerable debate. In the present study, we have examined the properties of AEA retention using synthetic large unilamellar liposomes (LUVs). Retention of AEA in LUVs was saturable, time- and temperature-dependent. Preincubation of LUVs with inhibitors of AEA uptake and metabolism did not decrease the tritium retention. These results mimic the “initial” cellular uptake of AEA and indeed argue against the need of a specific membrane transporter protein.

Keyword
Liposome, endocannabinoid, anandamide, membrane retention
National Category
Pharmacology and Toxicology
Research subject
biokemisk farmakologi
Identifiers
urn:nbn:se:umu:diva-22170 (URN)
Available from: 2009-04-28 Created: 2009-04-27 Last updated: 2010-01-14Bibliographically approved
2. The 'specific' tyrosine kinase inhibitor genistein inhibits the enzymic hydrolysis of anandamide: implications for anandamide uptake
Open this publication in new window or tab >>The 'specific' tyrosine kinase inhibitor genistein inhibits the enzymic hydrolysis of anandamide: implications for anandamide uptake
2007 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 150, no 7, 951-960 p.Article in journal (Refereed) Published
Abstract [en]

Background and Purpose: The cellular uptake of anandamide is reduced by inhibitors of fatty acid amide hydrolase (FAAH) and by agents disrupting endocytotic mechanisms. However, it is not clear if these events occur over the same time frame and if they occur to the same extent in different cells. We have therefore investigated the effects of such compounds in three cell lines of different origins using different assay incubation times and temperatures.

Experimental approach: FAAH activity and cellular uptake of anandamide was measured using anandamide, radio-labelled either in the ethanolamine or arachidonoyl part of the molecule.

Key results: The FAAH inhibitor URB597 inhibited the uptake of anandamide into C6 glioma, RBL2H3 basophilic leukaemia cells and P19 embryonic carcinoma cells at incubation time 4 min. However, a time-dependent and temperature-sensitive residual uptake remained after URB597 treatment. The combination of progesterone and nystatin reduced the uptake, but also decreased the amount of anandamide retained by the wells. Genistein inhibited anandamide uptake in a manner that was not additive to that of URB597. However, genistein was a potent competitive inhibitor of FAAH (Ki value 8 mM).

Conclusions and implications: The reduction of anandamide uptake by genistein can be explained by its ability to inhibit FAAH with a potency which overlaps that for inhibition of tyrosine kinase. The FAAH- resistant but time-dependent uptake of anandamide is seen in all three cell lines studied and is thus presumably a generally occurring process.

Keyword
Amidohydrolases/*antagonists & inhibitors/metabolism, Animals, Arachidonic Acids/*metabolism, Benzamides/pharmacology, Brain/enzymology, Carbamates/pharmacology, Cell Line; Tumor, Genistein/*pharmacology, Hydrolysis, Mice, Nystatin/pharmacology, Polyunsaturated Alkamides/*metabolism, Progesterone/pharmacology, Protein Kinase Inhibitors/pharmacology, Protein-Tyrosine Kinases/antagonists & inhibitors, Rats
Identifiers
urn:nbn:se:umu:diva-5987 (URN)10.1038/sj.bjp.0707172 (DOI)17325653 (PubMedID)
Available from: 2007-12-04 Created: 2007-12-04 Last updated: 2017-12-14Bibliographically approved
3. 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
Open this publication in new window or tab >>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
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.

Keyword
endocannabinoid; anandamide; cellular uptake; fatty acid amide hydrolase; tyrosine kinase; genistein; daidzein
Identifiers
urn:nbn:se:umu:diva-5983 (URN)10.1038/sj.bjp.0707401 (DOI)17676056 (PubMedID)
Available from: 2007-12-04 Created: 2007-12-04 Last updated: 2017-12-14Bibliographically approved
4. Inhibition of fatty acid amide hydrolase by kaempferol and related naturally occurring flavonoids
Open this publication in new window or tab >>Inhibition of fatty acid amide hydrolase by kaempferol and related naturally occurring flavonoids
2008 (English)In: British Journal of Pharmacology, ISSN 0007-1188, E-ISSN 1476-5381, Vol. 155, no 2, 244-252 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND AND PURPOSE: Recent studies have demonstrated that the naturally occurring isoflavone compounds genistein and daidzein inhibit the hydrolysis of anandamide by fatty acid amide hydrolase (FAAH) in the low micromolar concentration range. The purpose of the present study was to determine whether this property is shared by flavonoids.

EXPERIMENTAL APPROACH:The hydrolysis of anandamide in homogenates and intact cells was measured using the substrate labelled in the ethanolamine part of the molecule.

KEY RESULTS: Twenty compounds were tested. Among the commonly occurring flavonoids, kaempferol was the most potent, inhibiting FAAH in a competitive manner with a K(i) value of 5 microM. Among flavonoids with a more restricted distribution in nature, the two most active toward FAAH were 7-hydroxyflavone (IC(50) value of 0.5-1 microM depending on the solvent used) and 3,7-dihydroxyflavone (IC(50) value 2.2 microM). All three compounds reduced the FAAH-dependent uptake of anandamide and its metabolism by intact RBL2H3 basophilic leukaemia cells.

CONCLUSIONS AND IMPLICATIONS: Inhibition of FAAH is an additional in vitro biochemical property of flavonoids. Kaempferol, 7-hydroxyflavone and 3,7-dihydroxyflavone may be useful as templates for the synthesis of novel compounds, which target several systems that are involved in the control of inflammation and cancer.

Keyword
anandamide; cannabinoid; fatty acid amide hydrolase; flavonoids; kaempferol; apigenin
Identifiers
urn:nbn:se:umu:diva-10516 (URN)10.1038/bjp.2008.237 (DOI)18552875 (PubMedID)
Available from: 2008-09-23 Created: 2008-09-23 Last updated: 2017-12-14Bibliographically approved
5. Biochanin A acts as a peripheral inhibitor of fatty acid amide hydrolase
Open this publication in new window or tab >>Biochanin A acts as a peripheral inhibitor of fatty acid amide hydrolase
Show others...
(English)Manuscript (Other academic)
Abstract [en]

Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme primarily responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide, are effective in animal models of inflammatory, cancer, visceral and neuropathic pain. No peripherallyrestricted FAAH inhibitors have yet been reported. The isoflavones are a class of naturallyoccurring compounds that show little brain penetration, and two of these, genistein and daidzein, inhibit FAAH at low micromolar concentrations. Here, we report that the related isoflavone biochanin A inhibits the hydrolysis of 0.5 μM anandamide by mouse, rat and human FAAH with IC50 values of 1.8, 1.4, and 2.4 μM, respectively. The inhibition of rat FAAH by biochanin A was mixed type in nature, with Ki slope and Ki intercept values of 1.1 and 8.2 μM, respectively. Anandamide hydrolysis was also inhibited in intact basophilic leukaemia cells at sub- to low- micromolar concentrations. The compound did not interact to any major extent with CB1 or CB2 receptors. In vivo, biochanin A (10 mg/kg i.v.) did not increase brain anandamide concentrations, but produced a modest potentiation of the effects of 10 mg/kg i.v. anandamide in the tetrad test. In anaesthetized mice, URB597 (30 μg/paw) and biochanin A (100 μg/paw) both inhibited the spinal phosphorylation of extracellular receptor kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB1 receptor antagonist/inverse agonist AM251 (30 μg/paw). It is concluded that biochanin A may be useful as a template for the design of novel, peripherally-acting FAAH inhibitors.

Keyword
fatty acid amide hydrolase; inflammatory pain; cannabinoid; isoflavones, biochanin A; anandamide
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:umu:diva-22172 (URN)
Available from: 2009-04-28 Created: 2009-04-27 Last updated: 2010-01-14Bibliographically approved
6. Effect of nitric oxide donors on membrane tritium accumulation of endocannabinoids and related endogenous lipids
Open this publication in new window or tab >>Effect of nitric oxide donors on membrane tritium accumulation of endocannabinoids and related endogenous lipids
(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
Endocannabinoids; anandamide; nitric oxide; membrane accumulation; fatty acid amide hydrolase
National Category
Pharmacology and Toxicology
Research subject
biokemisk farmakologi
Identifiers
urn:nbn:se:umu:diva-22171 (URN)
Available from: 2009-04-28 Created: 2009-04-27 Last updated: 2010-01-14Bibliographically approved

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