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

Abstract

1 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. 2 Saturable uptake of PEA and AEA into both cell lines were demonstrated with apparent K M values of 28 mM (PEA) and 10 mM (AEA) in Neuro-2a cells, and 30 mM (PEA) and 9.3 mM (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¯uoride. 3 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 mM, did not a ect AEA uptake in either cell line. AEA, 2-AG, arachidonic acid, R1-methanandamide, D 9 -THC, and cannabidiol inhibited PEA transport in both cell lines. The non-steroidal anti-in¯ammatory drug indomethacin inhibited the AEA uptake but had very weak e ects on the uptake of PEA. 4 From these data, it can be concluded that PEA is transported in to cells both by passive di usion and by a facilitated transport that is pharmacologically distinguishable from AEA uptake.

Identification of a high-affinity binding site involved in the transport of endocannabinoids

“…The Michaelis constant (K m ϭ 4.69 Ϯ 0.460 M) (Fig. 2a) of anandamide uptake saturation is lower than any reported value for RBL-2H3 cells, which range from 9.3 (47) to 33 (48) M. Similar to other studies, anandamide uptake is saturable, as well as time-and temperature-dependent (34,43,45,48,49). When [ 14 C]anandamide uptake is measured in the presence of LY2318912 (vehicle, 5 and 10 nM), K m is shifted to the right (4.69 Ϯ 0.460, 13.4 Ϯ 2.23, and 26.6 Ϯ 5.24 M, respectively), with little change in V max (2.15 ϫ 10 Ϫ17 , 1.91 ϫ 10 Ϫ17 , and 1.83 ϫ 10 Ϫ17 mol͞min per cell, respectively) ( Fig.…”

Molecular targets for cannabidiol and its synthetic analogues: effect on vanilloid VR1 receptors and on the cellular uptake and enzymatic hydrolysis of anandamide

“…The (7)-5'DMH-CBD is obtained by a facile, high yield synthesis (Baek et al, 1985) and may ®nd application as therapeutic agent for those disorders where AEA exerts an endogenous tone with bene®cial e ects. The novel AMT inhibitors developed here should be tested also on the cellular uptake of palmitoylethanolamide, a natural anti-in¯ammatory AEA congener (Lambert & Di Marzo, 1999), since a recent study showed that CBD inhibits the facilitated transport of this compound into RBL ± 2H3 cells (Jacobsson & Fowler, 2001).…”

Involvement of N-acylethanolamine-hydrolyzing acid amidase in the degradation of anandamide and other N-acylethanolamines in macrophages

“…Further studies will be required to clarify the reason why substrate specificity of the crude enzymes from RAW264.7 cells\ is different from those of pure enzymes previously reported. Moreover, the mechanism and rate of cellular uptake appeared to differ among various N-acylethanolamines [51,52]. Thus, it seems that the degradation rate of each N-acylethanolamine in intact cells cannot be explained simply by substrate specificity of pure enzymes and is determined by multiple factors.…”