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.

Figure 1

Saturation kinetics of AEA (a,c) and PEA (b,c) uptake in Neuro-2a cells (a,b) and RBL-2H3 cells (c,d). The cells were incubated with increasing concentrations of [³H]-AEA or [³H]-PEA for 15 min at 37°C or 4°C. The effect of 100 μM AM404 upon AEA uptake was determined by pre-incubating the cells for 15 min at 37°C with AM404 followed by the addition of [³H]-AEA. Each value represents the mean±s.e.mean (n=3 – 6). Insets, Hanes-Woolf plots of the respective saturation curves obtained at 37°C.

Figure 2

Competition of [³H]-WIN 55,212-2 binding to rat brain (a), Neuro-2a cell (b), and RBL-2H3 cell (c) membranes by the cannabinoid receptor antagonists SR 141716A and SR 144528. The control values were obtained in the absence of inhibitors and CP 55,940 values were obtained in the presence of 1 μM of this compound. Data represent the mean±s.e.mean (n=3). The radiolabelled ligand concentration used was in the range of 0.6 – 0.8 nM.

Figure 3

Uptake of 10 μM [³H]-AEA in various concentrations of ethanol. Stock solutions of AEA in ethanol was added to the RBL-2H3 cells and the cultures were incubated 15 min at 37°C. Data represent means±s.e.mean (n=4). Statistically significant differences (one-way ANOVA with Dunnett's multiple comparison test) are indicated as ^**P<0.01 when compared with corresponding cellular uptake of AEA in 0.07% ethanol (26±1.3 pmol min⁻¹ well⁻¹).

Figure 4

Concentration-dependent effects of putative endocannabinoids and arachidonic acid on [³H]-AEA (a,c) and [³H]-PEA (b,d) uptake in Neuro-2a cells (a,b) and RBL-2H3 cells (c,d). The cells were incubated 15 min with the indicated concentration of the test compound, followed by the addition of 10 μM [³H]-AEA or 20 μM [³H]-PEA. After 15 min at 37°C, the cellular uptake of AEA and PEA was determined. Data are plotted as percentage of total [³H]-AEA or [³H]-PEA uptake and represent means±s.e.mean (n=3 – 5).

Figure 5

Concentration-dependent effects of AEA derivatives and the synthetic vanilloid agonist olvanil on [³H]-AEA (a,c) and [³H]-PEA (b,d) uptake in Neuro-2a cells (a,b) and RBL-2H3 cells (c,d). The experiments were undertaken as described in the legend to Figure 4. Data are plotted as percentage of total [³H]-AEA or [³H]-PEA uptake and represent means±s.e.mean (n=3 – 5).

Figure 6

Concentration-dependent effects of the nonsteroidal anti-inflammatory drugs ibuprofen and indomethacin on [³H]-AEA (a,d) and [³H]-PEA (b,e) uptake and LDH release (c,f) in Neuro-2a cells (a,b,c) and RBL-2H3 cells (d,e,f). The experiments were undertaken as described in the legend to Figure 4. Data are plotted as percentage of total [³H]-AEA or [³H]-PEA uptake and represent means±s.e.mean (n=3 – 5).

Figure 7

Concentration-dependent effects of cannabinoids on [³H]-PEA uptake in Neuro-2a cells (a) and RBL-2H3 cells (b). The experiments were undertaken as described in the legend to Figure 4. Data are plotted as percentage of total [³H]-PEA uptake and represent means±s.e.mean (n=3 – 5).

Figure 8

The effects of treatment with Pronase, PMSF and 4°C upon cell viability, AEA and PEA transport in Neuro-2a cells. The cells were incubated with the indicated concentration of Pronase or PMSF (b), followed by the addition of 10 μM [³H]-AEA or 20 μM [³H]-PEA. After 15 min at 37°C or 4°C, the cellular uptake of AEA and PEA and the cellular release of LDH or cellular MTT reduction were determined. Data are plotted as percentage of untreated control cultures and represent means±s.e.mean (n=3 – 8). Statistically significant differences (one-way ANOVA with Bonferroni multiple comparison test) are indicated: ^***P<0.001 when cellular AEA or PEA uptake is compared with corresponding cell viability values, and ^•••P<0.001 between AEA and PEA uptake at respective treatment.