Effects of flurbiprofen on the functional regulation of serotonin transporter and its misfolded mutant.

Flurbiprofen, a nonsteroidal anti-inflammatory drug, reportedly exhibits chemical chaperone activity. Herein, we investigated the role of flurbiprofen in regulating serotonin transporter (SERT) function via membrane trafficking. We used COS-7 cells transiently expressing wild-type (WT) SERT or a C-terminus-deleted mutant of SERT (SERTΔCT), a misfolded protein. Flurbiprofen treatment reduced the expression of immaturely glycosylated SERT and enhanced the expression of maturely glycosylated SERT. In addition, we observed increased serotonin uptake in SERT-expressing cells. These results suggest that flurbiprofen modulates SERT function by promoting membrane trafficking. In SERTΔCT-expressing cells, flurbiprofen reduced the protein expression and uptake activity of SERTΔCT. Furthermore, flurbiprofen inhibited the formation of SERTΔCT aggregates. Studies using flurbiprofen enantiomers suggested that these effects of flurbiprofen on SERT were not mediated via cyclooxygenase inhibition. The levels of GRP78/BiP, an endoplasmic reticulum (ER) stress marker, were assessed to elucidate whether flurbiprofen can ameliorate SERTΔCT-induced ER stress. Interestingly, flurbiprofen induced GRP78/BiP expression only under ER stress conditions and not under steady-state conditions. In HRD1 E3 ubiquitin ligase knockdown cells, flurbiprofen affected the ER-associated degradation system. Collectively, the findings suggest that flurbiprofen may function as an inducer of molecular chaperones, in addition to functioning as a chemical chaperone.

Syntaxin 3 interacts with serotonin transporter and regulates its function.

Herein, we investigated the functional association of the serotonin transporter (SERT) with syntaxin-3 (STX3). We first overexpressed SERT and STX3 in various cells and examined their interaction, localization, and functional association. Immunoprecipitation studies revealed that STX3 interacted with SERT when expressed in COS-7 cells. Immunocytochemical studies revealed that SERT and STX3 were colocalized in the endoplasmic reticulum (ER) and Golgi apparatus. STX3 overexpression significantly reduced the uptake activity of SERT by attenuating its plasma membrane expression, suggesting that overexpressed STX3 anchors SERT in the ER and Golgi apparatus. STX3 knockdown did not affect the uptake activity of SERT but altered its glycosylation state. To elucidate the association of STX3 with SERT under physiological conditions, rather than overexpressing cells, we investigated this interaction in polarized Caco-2 cells, which endogenously express both proteins. Immunocytochemical studies revealed that SERT and STX3 were localized in microvilli-like structures at the apical plasma membrane. STX3 knockdown marginally but significantly decreased the serotonin uptake activity of Caco-2 cells, suggesting that STX3 positively regulates SERT function in Caco-2 cells, as opposed to SERT regulation by STX3 in overexpressing cells. Collectively, STX3 may colocalize with SERT during SERT membrane trafficking and regulate SERT function in an STX3-expressing lesion-dependent manner.

SKF-10047, a prototype Sigma-1 receptor agonist, augmented the membrane trafficking and uptake activity of the serotonin transporter and its C-terminus-deleted mutant via a Sigma-1 receptor-independent mechanism.

The serotonin transporter (SERT) is functionally regulated via membrane trafficking. Our previous studies have demonstrated that the SERT C-terminal deletion mutant (SERTΔCT) showed a robust decrease in its membrane trafficking and was retained in the endoplasmic reticulum (ER), suggesting that SERTΔCT is an unfolded protein that may cause ER stress. The Sigma-1 receptor (SigR1) has been reported to attenuate ER stress via its chaperone activity. In this study, we investigated the effects of SKF-10047, a prototype SigR1 agonist, on the membrane trafficking and uptake activity of SERT and SERTΔCT expressed in COS-7 cells. Twenty-four hours of SKF-10047 treatment (>200 µM) accelerated SERT membrane trafficking and robustly upregulated SERTΔCT activity. Interestingly, these effects of SKF-10047 on SERT functions were also found in cells in which SigR1 expression was knocked down by shRNA, suggesting that SKF-10047 exerted these effects on SERT via a mechanism independent of SigR1. A cDNA array study identified several candidate genes involved in the mechanism of action of SKF-10047. Among them, Syntaxin3, a member of the SNARE complex, was significantly upregulated by 48 h of SKF-10047 treatment. These results suggest that SKF-10047 is a candidate for ER stress relief.

The Development of Screening Methods to Identify Drugs to Limit ER Stress Using Wild-type and Mutant Serotonin Transporter.

The function of the serotonin transporter (SERT) is regulated by its membrane trafficking. Previously, we showed that the C-terminus-deleted mutant of SERT (SERTΔCT) exhibited an aberrant membrane trafficking and subsequent retention at the endoplasmic reticulum (ER). In addition, we found that proteasome inhibitor-induced ER stress resulted in the impairment of SERT membrane trafficking and retention of SERT at the ER, an impairment very similar to that of SERTΔCT. Based on the result that the chemical chaperone 4-phnylbutulic acid (4-PBA), which relieves ER stress, accelerated the membrane trafficking and upregulated SERT activity, we hypothesized that drugs that facilitate the membrane trafficking of SERT would have potential therapeutic effects on an ER stress-related disease. In this study, we aimed to develop simple screening methods for such drugs using SERT. We first validated the serotonin uptake assay using fluorescent substrates. This simple and reliable assay method was useful for screening for drugs that affected the wild-type SERT but not SERTΔCT. In addition, we verified an assay focusing on the formation of SERTΔCT aggregates. The drugs 4-PBA and SKF-10047 facilitated the trafficking of SERT to the membrane and reduced SERTΔCT aggregates, indicating that the drugs with such characters could be potential candidates for ER stress relief. For both assays, we clarified the usefulness of a high-content screening microscope. These results could pave the way for high-throughput screening for such drugs.

Impact of intensive high-fat ingestion in the early stage of recovery from exercise training on substrate metabolism during exercise in humans.

Not only increasing body carbohydrate (CHO) stores before exercise but also suppressing CHO oxidation during exercise is important for improving endurance performance. We tested the hypothesis that intensive high-fat ingestion in the early stage of recovery from exercise training (ET) for 2 d would suppress CHO oxidation during exercise by increasing whole body lipolysis and/or fat oxidation. In a randomized crossover design, on days 1 and 2, six male subjects performed cycle ET at 50% peak oxygen consumption (VO(2 peak)) for 60-90 min, and consumed a control diet (CON: 1,224 kcal, 55% carbohydrate, 30% fat) or the same diet supplemented with high fat (HF: 1,974 kcal, 34% carbohydrate, 56% fat) 1 h after ET, with the diet other than post-ET similar in both trials. On day 3, subjects performed cycle exercise at 65% VO(2 peak) until exhaustion. Exercise time to exhaustion was longer in the HF trial than in the CON trial (CON: 48.9 ± 6.7 vs. HF: 55.8 ± 7.7 min, p<0.05). In the HF trial, total fat oxidation until exhaustion was higher, accompanied by higher post-exercise plasma glycerol concentration, than in the CON trial (CON: 213 ± 54 vs. HF: 286 ± 63 kcal, p<0.05), whereas total carbohydrate oxidation until exhaustion was not different between trials. These results suggest that intensive high-fat ingestion in the early stage of recovery from ET for a few days until the day before exercise was an effective means of eliciting a CHO-sparing effect during exercise by enhancing fat metabolism.