Promoting Effect and Potential Mechanism of Lactobacillus pentosus LPQ1-Produced Active Compounds on the Secretion of 5-Hydroxytryptophan.

5-hydroxytryptophan (5-HTP) is an important substance thought to improve depression. It has been shown that Lactobacillus can promote the secretion of 5-HTP in the body and thus ameliorate depression-like behavior in mice. However, the mechanism by which Lactobacillus promotes the secretion of 5-HTP is unclear. In this study, we investigated the promoting effect and mechanism of Lactobacillus, isolated from Chinese fermented foods, on the secretion of 5-HTP. The results showed that Lactobacillus (L.) pentosus LPQ1 exhibited the strongest 5-HTP secretion-promoting effect ((9.44 ± 0.69)-fold), which was dependent on the mixture of compounds secreted by L. pentosus LPQ1 (termed SLPQ1). In addition, the results of the RNA sequencing (RNA-seq) and quantitative real-time polymerase chain reaction (qRT-PCR) analyses indicated that SLPQ1 alters the TNF and oxidative phosphorylation signaling pathways. Moreover, the SLPQ1 ultrafiltration fraction (>10 kDa) showed a similar 5-HTP promoting effect as SLPQ1. Furthermore, reverse-phase liquid chromatography-tandem mass spectrometry (RPLC-MS/MS) identified 29 compounds of >10 kDa in SLPQ1, including DUF488 domain-containing protein, BspA family leucine-rich repeat surface protein, and 30S ribosomal protein S5, which together accounted for up to 62.51%. This study reports new findings on the mechanism by which L. pentosus LPQ1 promotes 5-HTP production in some cell lines in vitro.

A simple, efficient and rapid HPLC-UV method for the detection of 5-HT in RIN-14B cell extract and cell culture medium.

5-Hydroxytryptamine (also known as 5-HT, serotonin) is one of the monoamine neurotransmitters which is distributed widely in plasma and brain of mammals and plays important roles in physiological manipulations. In the present method, we describe the development of a simple, efficient and rapid high performance liquid chromatographic method coupled with ultraviolet (HPLC-UV) detector for the qualitative and quantitative analysis of 5-HT in both cell extract and cell culture medium (RIN-14B). The experiments use repeated freeze-thaw cycles followed by centrifugation and direct injection of the supernatant into the chromatography. An analytical C18 column (Agilent Zorbax Extend, 4.6 × 250 mm, 5 µm.) was taken for chromatographic separation; the mobile phase was 0.05 mol/L potassium dihydrogen phosphate (KH2PO4)/acetonitrile (90:10 v/v). Isocratic elution is established at the flow rate of 1.0 mL/min. The time required for this chromatographic run is 8 min. Over the concentration range of 0.1-10 µg/mL, the calibration curve is linear in this method. Other unique characteristics and advantages include high accuracy (92.02-103.28%) and high precision (intra- and inter-day coefficients of variation ≤ 4.69%). This method is applicable for the investigation of drug/condition-response relationships in the function of synthesis and secretion of 5-HT in cultured RIN-14B cells in various in vitro studies.

Bidirectional effects of hydrogen sulfide via ATP-sensitive K(+) channels and transient receptor potential A1 channels in RIN14B cells.

Hydrogen sulfide (H2S) reportedly acts as a gasotransmitter because it mediates various cellular responses through several ion channels including ATP-sensitive K(+) (KATP) channels and transient receptor potential (TRP) A1 channels. H2S can activate both KATP and TRPA1 channels at a similar concentration range. In a single cell expressing both channels, however, it remains unknown what happens when both channels are simultaneously activated by H2S. In this study, we examined the effects of H2S on RIN14B cells that express both KATP and TRPA1 channels. RIN14B cells showed several intracellular Ca(2+) concentration ([Ca(2+)]i) responses to NaHS (300 µM), an H2S donor, i.e., inhibition of spontaneous Ca(2+) oscillations (37%), inhibition followed by [Ca(2+)]i increase (24%), and a rapid increase in [Ca(2+)]i (25%). KATP channel blockers, glibenclamide or tolbutamide, abolished any inhibitory effects of NaHS and enhanced NaHS-mediated [Ca(2+)]i increases, which were inhibited by extracellular Ca(2+) removal, HC030031 (a TRPA1 antagonist), and disulfide bond-reducing agents. NaHS induced 5-hydroxytryptamine (5-HT) release from RIN14B cells, which was also inhibited by TRPA1 antagonists. These results indicate that H2S has both inhibitory and excitatory effects by opening KATP and TRPA1 channels, respectively, in RIN14B cells, suggesting potential bidirectional modulation of secretory functions.