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Involvement of Fatty Acid Amide Hydrolase and Fatty Acid Binding Protein 5 in the Uptake of Anandamide by Cell Lines with Different Levels of Fatty Acid Amide Hydrolase Expression: A Pharmacological Study
Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
Umeå universitet, Medicinska fakulteten, Institutionen för farmakologi och klinisk neurovetenskap, Farmakologi.
Umeå universitet, Medicinska fakulteten, Institutionen för strålningsvetenskaper, Onkologi.
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2014 (Engelska)Ingår i: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, nr 7, s. e103479-Artikel i tidskrift (Refereegranskat) Published
Abstract [en]

Background:

The endocannabinoid ligand anandamide (AEA) is removed from the extracellular space by a process ofcellular uptake followed by metabolism. In many cells, such as the RBL-2H3 cell line, inhibition of FAAH activity reduces theobserved uptake, indicating that the enzyme regulates uptake by controlling the intra- : extracellular AEA concentrationgradient. However, in other FAAH-expressing cells, no such effect is seen. It is not clear, however, whether these differencesare methodological in nature or due to properties of the cells themselves. In consequence, we have reinvestigated the roleof FAAH in gating the uptake of AEA.Methodology/Principal Findings: The effects of FAAH inhibition upon AEA uptake were investigated in four cell lines: AT1rat prostate cancer, RBL-2H3 rat basophilic leukaemia, rat C6 glioma and mouse P19 embryonic carcinoma cells. SemiquantitativePCR for the cells and for a rat brain lysate confirmed the expression of FAAH. No obvious expression of atranscript with the expected molecular weight of FLAT was seen. FAAH expression differed between cells, but all four couldaccumulate AEA in a manner inhibitable by the selective FAAH inhibitor URB597. However, there was a difference in thesensitivities seen in the reduction of uptake for a given degree of FAAH inhibition produced by a reversible FAAH inhibitor,with C6 cells being more sensitive than RBL-2H3 cells, despite rather similar expression levels and activities of FAAH. Thefour cell lines all expressed FABP5, and AEA uptake was reduced in the presence of the FABP5 inhibitor SB-FI-26, suggestingthat the different sensitivities to FAAH inhibition for C6 and RBL2H3 cells is not due to differences at the level of FABP-5.Conclusions/Significance: When assayed using the same methodology, different FAAH-expressing cells display differentsensitivities of uptake to FAAH inhibition.

Ort, förlag, år, upplaga, sidor
PLoS ONE , 2014. Vol. 9, nr 7, s. e103479-
Nationell ämneskategori
Farmakologi och toxikologi
Identifikatorer
URN: urn:nbn:se:umu:diva-91572DOI: 10.1371/journal.pone.0103479ISI: 000339954800048PubMedID: 25078278OAI: oai:DiVA.org:umu-91572DiVA, id: diva2:737031
Tillgänglig från: 2014-08-11 Skapad: 2014-08-11 Senast uppdaterad: 2018-06-07Bibliografiskt granskad
Ingår i avhandling
1. The endocannabinoid system: a translational study from Achilles tendinosis to cyclooxygenase
Öppna denna publikation i ny flik eller fönster >>The endocannabinoid system: a translational study from Achilles tendinosis to cyclooxygenase
2014 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
Abstract [en]

The endogenous cannabinoids anandamide (arachidonoyl ethanolamide, AEA) and 2-arachidonoyl glycerol (2-AG) exert their effect by activating cannabinoid receptors (CB). These receptors mediate a broad range of physiological functions such as beneficial effects in pain and inflammation, although little is known about the expression of CB receptors in human pain conditions. AEA and 2-AG are short- lived molecules due to their rapid cellular accumulation and metabolism. The enzymes primarily responsible for their degradation are fatty acid amide hydrolase (FAAH) for AEA and monoacylglycerol lipase (MGL) for 2-AG. Inhibition of endocannabinoid metabolism is a potential approach for drug development, and there is a need for the identification of novel compounds with inhibitory effects upon FAAH and MGL.

In Paper I of this thesis, the expression of CB1 receptors in human Achilles tendon was examined. We found expression of CB1 receptors in tenocytes, blood vessel wall as well as in the perineurium of the nerve. A semi-quantitative analysis showed an increase of CB1 receptors in painful human Achilles tendinosis.

In papers II and III, termination of AEA signalling was investigated via inhibition of FAAH. In Paper II, Flu-AM1, an analogue of flurbiprofen, was investigated. The compound inhibited both FAAH and the oxygenation of 2-AG by cyclooxygenase-2. In Paper III the antifungal compound ketoconazole was shown to inhibit the cellular uptake of AEA in HepG2, CaCo-2 and C6 cell lines in a manner consistent with inhibition of FAAH.

The role of FAAH in gating the cellular accumulation of AEA was investigated in Paper IV. FAAH has been shown to control the concentration gradient of AEA across the plasmamembrane in RBL2H3 cells, whereas no such effect is seen in other FAAH-expressing cell lines. To determine whether this effect is assay dependent or due to intrinsic differences between the cell lines, we assayed four cell lines with different levels of FAAH expression using the same methodology. We found that the sensitivity of FAAH uptake inhibition was not dependent on the expression level of FAAH, suggesting that factors other than FAAH are important for uptake.

Paper V is focused on the inhibition of MGL. Prior to this study no selective inhibitors of the enzyme had been described. Thus, we screened a number of compounds for their inhibitory effect on MGL. Troglitazone was found to be an inhibitor of MGL, although its potency was dependent upon the enzyme assay used. 

Ort, förlag, år, upplaga, sidor
Umeå: Umeå universitet, 2014. s. 72
Serie
Umeå University medical dissertations, ISSN 0346-6612 ; 1663
Nationell ämneskategori
Farmakologi och toxikologi
Identifikatorer
urn:nbn:se:umu:diva-91573 (URN)978-91-7601-089-1 (ISBN)
Disputation
2014-09-05, Hörsal E04 Unod R1, Byggnad 6E, Norrlands universitetssjukhus, Umeå, 09:00 (Svenska)
Opponent
Handledare
Tillgänglig från: 2014-08-15 Skapad: 2014-08-11 Senast uppdaterad: 2018-06-07Bibliografiskt granskad

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Involvement of Fatty Acid Amide Hydrolase and Fatty Acid Binding Protein 5 in the Uptake of Anandamide by Cell Lines with Different Levels of Fatty Acid Amide Hydrolase Expression: A Pharmacological Study(1633 kB)211 nedladdningar
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