Expression of serotonin transporter and monoamine oxidase A in P19 cells and P19 neurons.

<p>(A) Reverse transcription PCR analysis of the mRNA expressions of SERT in P19 cells and P19 neurons (at day 8 in the serum-free media) with a mouse brain lysate (MB) as a positive control. Total RNA was isolated and reverse transcribed into cDNA. The PCR products were analysed by agarose gel electrophoresis and fragment size estimated using a 100 bp marker. The arrow shows the expected amplicon size for SERT (127 bp) with the primer pair used. (B) qPCR analysis of mRNA expression levels of SERT in P19 cells and neuronally differentiated P19 cells (at days 8 and 10 in the serum-free media) with RPL19 as housekeeping gene. Results are expressed as a percentage of P19 cells. Values are means ± SEM of n = 5 independent experiments. (C) Expression of SERT in P19 cells and P19 neurons (at day 8 in the serum-free media) as measured with ELISA. Data are means ± SEM of n = 4 independent cell preparations (P19 neurons) and 5 (P19 cells). Statistical analysis (unpaired t test) showed a significant difference between P19 cells and neurons (***p< 0.001). (D) Effects of the selective SERT inhibitor citalopram and MDMA on 5-HT uptake in P19 neurons (at day 8 in the serum-free media). The cells (or wells without cells) were preincubated for 10 min with 1 μM citalopram, 1 mM MDMA or 0.002% DMSO as vehicle control followed by 30 min incubation with 100 nM [³H]-5-HT at 37°C. Data are means ± SEM of n = 8 independent experiments. Statistical analysis was performed using one-way repeated measures ANOVA with post hoc Bonferroni’s multiple comparison test (***p<0.001 for citalopram- and MDMA- vs. vehicle-treated control cells, no statistically significant difference was observed between citalopram-treated and MDMA-treated cells). (E) Western blot analysis with rabbit anti-monoamine oxidase A monoclonal antibody (ab126751) (Abcam). Comparison of immunoreactivity between P19 cells, P19 neurons (at day 10 in the serum-free media) and the positive control human liver hepatocellular carcinoma cell line (HepG2). Cell lysates: 10 μg per lane. The arrow shows the expected size of MAO-A (60 kDA).</p

Effects of clorgyline, deprenyl, ketanserin, α-tocopherol (VitE) and N-acetylcysteine (NAC) on MDMA-induced toxicity in P19 neurons.

<p>P19 neurons were cultured for eight days in the serum-free media and incubated for 48 h with the test compounds in absence or presence of 1 mM MDMA. Cell viability was measured with (A) MTT reduction assay and (B) LDH activity assay. Data are means ± SEM of n = 3–4 independent experiments. For MTT reduction, data are expressed as percentage of non-treated control cells. For LDH release, the results are presented as percentage of total cell death (cells treated with 2% Triton X-100). Statistical analysis was performed using repeated measures one-way ANOVA with post hoc Bonferroni's multiple comparisons test (#p< 0.0001 for comparison between untreated control cells and the treatments, and *p< 0.05 when treatments in presence of MDMA are compared to 1 mM MDMA).</p

Effects of MDMA, clorgyline, deprenyl, fluoxetine and the combinations on MTT reduction in P19 neurons.

<p>Effects of MDMA, clorgyline, deprenyl, fluoxetine and the combinations on MTT reduction in P19 neurons.</p

Time-dependent effects of MDMA on mitochondrial membrane potential.

<p>P19 neurons, cultured for seven to nine days in serum-free medium, were exposed to 1 mM MDMA for 10 min up to 48 h. Mitochondrial membrane potential was measured by using the TMRE assay. The mitochondrial oxidative phosphorylation uncoupler FCCP (5 μM) applied for 10 min was used as a positive control. Data are means ± SEM of n = 4–6 independent experiments. Statistical analysis was performed using one-way ANOVA with post hoc Dunnett’s multiple comparisons test (****p< 0.0001) compared to the untreated control.</p

Effects of MDMA treatment prior to high temperature upon the MTT reduction produced by P19 neurons.

<p>P19 neurons were treated with MDMA on day 7 in the serum-free media at 37°C, 5% CO₂ for 48 h without (white bars) or with (grey bars) an additional step whereby medium was changed to remove MDMA and the cells were incubated for a further 24 h at 42°C, in a humified atmosphere with 5% CO₂. Cell viability was assessed with MTT reduction assay. Data (means ± SEM of n = 4 independent experiments) are presented as percentage of non-treated control cells. Statistical analysis was performed using two-way repeated-measures ANOVA matching both treatment and temperature. The interaction term treatment x temperature was not significant. The significant difference within temperature groups were followed up with post hoc Dunnett’s multiple comparison test compared to non-treated control cells (*p< 0.05, ****p< 0.0001).</p

Concentration-, time- and temperature- dependent effects of MDMA on the viability of P19 neurons.

<p>MTT reduction and LDH release were measured in P19-derived neurons exposed to MDMA at day 7 in the serum-free media for 24 h, 48 h and 72 h at the temperatures 37°C, 40°C and 42°C, in a humified atmosphere with 5% CO₂. Data are means ± SEM of n = 4–5 independent experiments. For MTT reduction, data are expressed as percentage of non-treated control cells. For LDH release, the results are presented as percentage of total cell death (cells treated with 2% Triton X-100). Statistical analysis was performed using one-way ANOVA with post hoc Dunnett’s multiple comparisons test (*p< 0.05, †p< 0.01, ‡p< 0.001) compared to corresponding controls.</p

Inhibition of A. [³H]PEA and B. [³H]2-AG uptake by ketoconazole.

<p>The panels show the data for the uptake into HepG2 cells (filled symbols) or adsorbtion by wells (open symbols). The cells (or wells) were preincubated with ketoconazole for 10 min prior to addition of [³H]PEA or [³H]2-AG as appropriate (assay concentration 100 nM) and incubation for a further 4 min. For comparative purposes, the effects of the selective FAAH inhibitor URB597 (“U”, 1 µM) and (for 2-AG) the selective MGL inhibitor JZL184 (“J”, 100 µM) are indicated. Shown are means ± s.e.m. (when not enclosed by the symbols), n = 3.</p

Effects of ketoconazole, nefazodone, AM404 and URB597 (“U”, 1 µM) upon A, the uptake of [³H]AEA; B, the hydrolysis of [³H]AEA and C, the hydrolysis of [³H]2-AG by C6 glioma cells.

<p>The compounds were preincubated with the cells for 10-AG (100 nM final concentration) and incubation for a further 10 min at 37°C. For the uptake experiments, the tritium label was on the arachidonoyl side chain, whilst for the hydrolysis experiments, the label was on the ethanolamine group (AEA) or the glycerol group (2-AG). Shown are means ± s.e.m. (when not enclosed by the symbols), n = 3, except for ketoconazole in Panel B, where n = 4.</p

The effects of ketoconazole and nefazodone upon FAAH activity.

<p>Panel A. Hydrolysis of [³H]AEA by lysates of HepG2 and CaCo2 cells. Shown are means ± s.e.m., n = 3. Concurrent data for human SH-SY5Y neuroblastoma and rat C6 glioma cells are shown for comparison. Panel B. Inhibition of the hydrolysis of [³H]AEA in lysates of HepG2 cells by ketoconazole and nefazodone. Shown are means ± s.e.m. (unless enclosed by the symbols), n = 3.</p

Time course of the effects of ketoconazole and URB597 upon the accumulation of [³H]AEA by HepG2 cells.

<p> In Panels A and C, the individual values at each time point are shown (means ± s.e.m., unless enclosed by the symbols, n = 6). For both Panels, two-way ANOVA with repeated time measures for the data with the cells gave a significant (P<0.05) interaction time x treatment. Regression lines for each experiment were determined and the slopes, i.e. the rates of uptake are shown in Panels B and D as means ± s.e.m., n = 6. The vehicles used were A, ethanol; B, DMSO. *P<0.05, **P<0.01 <i>vs</i>. vehicle, either using Dunnett's multiple comparisons test following significant one-way repeated ANOVA not assuming sphericity for the three conditions with cells (Panel C) or for a two-tailed paired t-test for the two conditions with cells (Panel D).</p