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Characterization of palmitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells: comparison with anandamide.

ABSTRACT: The endogenous cannabinoid receptor agonist anandamide (AEA) and the related compound palmitoylethanolamide (PEA) are inactivated by transport into cells followed by metabolism by fatty acid amide hydrolase (FAAH). The cellular uptake of AEA has been characterized in detail, whereas less is known about the properties of the PEA uptake, in particular in neuronal cells. In the present study, the pharmacological and functional properties of PEA and AEA uptake have been investigated in mouse Neuro-2a neuroblastoma and, for comparison, in rat RBL-2H3 basophilic leukaemia cells. Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K(M) values of 28 microM (PEA) and 10 microM (AEA) in Neuro-2a cells, and 30 microM (PEA) and 9.3 microM (AEA) in RBL-2H3 cells. Both PEA and AEA uptake showed temperature-dependence but only the AEA uptake was sensitive to treatment with Pronase and phenylmethylsulfonyl fluoride. The AEA uptake was inhibited by AM404, 2-arachidonoylglycerol (2-AG), R1- and S1-methanandamide, arachidonic acid and olvanil with similar potencies for the two cell types. PEA, up to a concentration of 100 microM, did not affect AEA uptake in either cell line. AEA, 2-AG, arachidonic acid, R1-methanandamide, (9)-THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-inflammatory drug indomethacin inhibited the AEA uptake but had very weak effects on the uptake of PEA. From these data, it can be concluded that PEA is transported in to cells both by passive diffusion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

SUBMITTER: Jacobsson SO

PROVIDER: S-EPMC1572744 | biostudies-other | 2001 Apr

REPOSITORIES: biostudies-other

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Project description:Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

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Characterization of palmitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells: comparison with anandamide.

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