Journal: PLoS ONE

Article Title: Serotonin and Dopamine Protect from Hypothermia/Rewarming Damage through the CBS/ H 2 S Pathway

doi: 10.1371/journal.pone.0022568

Figure Lengend Snippet: Cells subjected to hypothermia (black bars) were incubated at 3°C for 24 h, followed by rewarming to 37°C for 3 h, and compared to non-cooled control cells (37°C, gray bars). Cell survival was assessed by adding MTS to the cells upon rewarming and spectrophotometrical formazon measurement. ( A ) DDT-1 cells show natural resistance to hypothermia, which is abrogated by the serotonin transporter (SERT) inhibitor fluoxetine (Fluox, 1 µM, 15 min) and pretreatment with the tryptophan hydroxylase inhibitor parachlorophenylalanine (PCPA, 24 h). ( B to C ) Concentration-dependent inhibition of hypothermic cells death by serotonin (B) and dopamine (C) in SMAC. ( D ) The protective effect of serotonin (30 µM, 15 min) and dopamine (20 µM, 15 min) pretreatment on hypothermic cell death is precluded by inhibition of their respective transporters with fluoxetine (1 µM, 15 min) and vanoxerine (1 µM, 15 min), but unaffected by non-specific receptor antagonists ketanserin (1 µM, 15 min) and spiperone (1 µM, 15 min). ( E ) Serotonin (30 µM, 15 min) and dopamine (20 µM, 15 min) pretreatment prevent caspase3/7 activation induced by hypothermia in SMAC cells, which is precluded by inhibition of their uptake by fluoxetine (1 µM, 15 min) and vanoxerine (1 µM, 15 min). ANOVA tests, different from non-cooled cells (37°C) P<0.05 (*); different from untreated hypothermic cells (Con) P<0.05 (#). Experiments consist of n≥4. Means ± SEM.

Article Snippet: Inhibition of the serotonin transporter SERT (fluoxetine, 1 μM) and dopamine transporter DAT (vanoxerine, 1 μM), however, completely abrogated serotonin and dopamine induced resistance to hypothermic damage in SMAC ( ).

Techniques: Incubation, Control, Concentration Assay, Inhibition, Activation Assay

Journal: PLoS ONE

Article Title: Serotonin and Dopamine Protect from Hypothermia/Rewarming Damage through the CBS/ H 2 S Pathway

doi: 10.1371/journal.pone.0022568

Figure Lengend Snippet: H 2 S content was measured in cell medium of non-cooled cells (gray bars, 37°C) and hypothermic cells (black bars, 3°C) after incubation for 24 h. ( A ) H 2 S content in DDT cells was unaffected by cooling, but reduced by the serotonin transporter (SERT) inhibitor fluoxetine (1 µM, 15 min pretreatment prior to cooling). ( B to C ) Serotonin (30 µM) and dopamine (20 µM) induce H 2 S production in SMAC both at 37°C and 3°C compared o untreated cells (control), which is blocked by inhibition of their respective transporters fluoxetine (1 µM, 15 min) and vanoxerine (1 µM, 15 min). ANOVA tests, different from non-cooled cells (37°C or control) P<0.05 (*); different from untreated hypothermic cells (Con) P<0.05 (#). Experiments consist of n≥4. Means ± SEM.

Article Snippet: Inhibition of the serotonin transporter SERT (fluoxetine, 1 μM) and dopamine transporter DAT (vanoxerine, 1 μM), however, completely abrogated serotonin and dopamine induced resistance to hypothermic damage in SMAC ( ).

Techniques: Incubation, Control, Inhibition

Journal: PLoS ONE

Article Title: Non-Serotonergic Neurotoxicity by MDMA (Ecstasy) in Neurons Derived from Mouse P19 Embryonal Carcinoma Cells

doi: 10.1371/journal.pone.0166750

Figure Lengend Snippet: (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 [ 3 H]-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).

Article Snippet: ELISA kit for serotonin transporter (SERT) (mus musculus) was purchased from USCN Life Science Inc. (Hubei, P.R.

Techniques: Reverse Transcription, Positive Control, Isolation, Agarose Gel Electrophoresis, Marker, Amplification, Expressing, Enzyme-linked Immunosorbent Assay, Control, Incubation, Comparison, Western Blot