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

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

Expression of FAAH and sensitivity of AEA uptake to the FAAH inhibitor URB597 in AT1, C6, RBL-2H3 and P19 cells.

<p>In Panels A and B semi-quantitative PCR analysis of the mRNA expression of FAAH are shown for the cells, with rat brain lysates (two different lysates, lanes 2–5, in Panel B) as positive controls. Molecular size markers are also shown. Total RNA was isolated and reverse transcribed into cDNA. Samples were pooled (n = 4) and the PCR analysis was performed using primers designed to recognize rat or mouse FAAH, as appropriate. The PCR products were analyzed by agarose gel electrophoresis and fragment size estimated using a 100 bp marker. The arrows show the expected sizes for FAAH (both Panels) and FLAT (Panel B) with the primer pair used. In Panel B, the main gel shows an overexposure of the gels, with the small panel above showing the band corresponding to FAAH at normal levels of exposure. The photographs have been inverted to show the minor bands. In Panel C, the effect of 1 µM URB597 upon the uptake of [³H]AEA is shown for AT1, C6, RBL2H3 and P19 cells. Cells (or wells alone) were preincubated with URB597 for 10 min 37°C followed by addition of 100 nM [³H]AEA and incubation for further 4 min at 37°C. Shown are means and s.e.m., n = 5. The statistical treatment of the data is presented in Results.</p

Time-dependent uptake of [³H]AEA in AT1, C6, RBL-2H3 and P19 cells treated with either buffer alone, vehicle, or 1 µM URB597.

<p>The cells were preincubated for 10[³H]AEA. Values are mean and s.e.m. (when not enclosed by the symbols), n = 4 (AT1, C6), 3 (RBL-2H3), or 5 (P19). The concentration of DMSO for the vehicle was 0.2%. For the cell data with vehicle and 1 µM URB597 as variables, two-way repeated-measure ANOVAs matching both time and treatment indicated significant effects of time (P<0.01 for all cells), treatment (P<0.005 for C6, RBL-2H3 and P19; P<0.05 for AT1) and the interaction term time×treatment (P<0.01 for all cells). *P<0.05, **P<0.01, <i>post-hoc</i> comparisons using Šídák’s multiple comparison for URB597-treated <i>vs.</i> corresponding vehicle-treated cells.</p

Effect of compound 33 upon AEA uptake using either [³H-ethanolamine]- or [³H-arachidonoyl]- labelled ligand and hydrolysis of [³H-ethanolamine]-AEA by cell lysates.

<p>The same conditions as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103479#pone-0103479-g002" target="_blank">figure 2</a> were used. Shown are means ± s.e.m., n = 3–4. The data for [³H-arachidonoyl]- labelled AEA uptake is the same as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103479#pone-0103479-g002" target="_blank">Fig. 2</a>, but in this case the x-axes are for cell lysates rather than intact cells. The values (± s.e.m.) of the slopes determined from the regression lines of the pooled data for [³H-ethanolamine]- or [³H-arachidonoyl]- labelled AEA, respectively, were: C6 cells,, 1.03±0.10 and 1.11±0.31; RBL-2H3 cells, 0.49±0.14 and 0.46±0.14. The concentration of EtOH for the vehicle was 0.2% (cells) and 1% (lysates).</p

Effect of compound 33 upon AEA uptake and hydrolysis by intact cells.

<p>Cells (or wells alone) were pre-incubated with Compound 33, a paracetamol ester with a 2-(4-(2-(trifluoromethyl)pyridin-4-ylamino)phenyl)acetic acid substituent <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103479#pone.0103479-Onnis1" target="_blank">[20]</a>, for 10 minutes followed by further incubation with [³H]AEA for an additional 4 (uptake; [³H-arachidonoyl]-AEA) or 10 (hydrolysis, [³H-ethanolamine]-AEA) minutes. The longer time used for the hydrolysis measurements was to allow a sufficient assay: blank ratio. The lack of time-dependency of the inhibition of FAAH by compound 33 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103479#pone.0103479-Onnis1" target="_blank">[20]</a> permits this difference in incubation times. Shown are means ± s.e.m., n = 3–4. The concentration of EtOH for the vehicle was 0.2% (cells). The values (± s.e.m.) of the slopes determined from the regression lines of the pooled data for each cell were: AT1, 0.58±0.21; C6, 0.85±0.18; RBL-2H3, 0.30±0.12.</p