ß-Catenin and SOX2 Interaction Regulate Visual Experience-Dependent Cell Homeostasis in the Developing Xenopus Thalamus.

In the vertebrate brain, sensory experience plays a crucial role in shaping thalamocortical connections for visual processing. However, it is still not clear how visual experience influences tissue homeostasis and neurogenesis in the developing thalamus. Here, we reported that the majority of SOX2-positive cells in the thalamus are differentiated neurons that receive visual inputs as early as stage 47 Xenopus. Visual deprivation (VD) for 2 days shifts the neurogenic balance toward proliferation at the expense of differentiation, which is accompanied by a reduction in nuclear-accumulated ß-catenin in SOX2-positive neurons. The knockdown of ß-catenin decreases the expression of SOX2 and increases the number of progenitor cells. Coimmunoprecipitation studies reveal the evolutionary conservation of strong interactions between ß-catenin and SOX2. These findings indicate that ß-catenin interacts with SOX2 to maintain homeostatic neurogenesis during thalamus development.

Spirulina (Arthrospira maxima) mitigates the toxicity induced by a mixture of metal and NSAID in Xenopus laevis.

Cadmium (Cd) is often detected in the environment due to its wide use in industry; also, NSAIDs are one of the most consumed pharmaceuticals, particularly diclofenac (DCF). Several studies have reported the presence of both contaminants in water bodies at concentrations ranging from ng L-1 to µg L-1; in addition, they have shown that they can induce oxidative stress in aquatic species and disturb signal transduction, cell proliferation, and intercellular communication, which could lead to teratogenesis. Spirulina has been consumed as a dietary supplement; its antioxidant, anti-inflammatory, neuroprotective, and nutritional properties are well documented. This work aimed to evaluate if Spirulina reduces the damage induced by Cd and DCF mixture in Xenopus laevis at early life stages. FETAX assay was carried out: 20 fertilized oocytes were exposed to seven different treatments on triplicate, control, Cd (24.5 µg L-1), DCF (149 µg L-1), Cd + DCF, Cd+DCF+Spirulina (2 mg L-1), Cd+DCF+Spirulina (4 mg L-1), Cd+DCF+Spirulina (10 mg L-1), malformations, mortality, and growth were evaluated after 96 h, also lipid peroxidation, superoxide dismutase and catalase activity were determined after 192 h. Cd increased DCF mortality, Cd and DCF mixture increased the incidence of malformations as well as oxidative damage; on the other hand, the results obtained show that Spirulina can be used to reduce the damage caused by the mixture of Cd and DCF since it promotes growth, reduce mortality, malformations, and oxidative stress in X. laevis.

Mortality and malformation effects of acute vanadium (V) exposure on the African clawed frog (Xenopus laevis) embryos.

Vanadium (V) is a transition metal that is found in low concentrations in aquatic ecosystems. These levels increase due to anthropogenic activities. The mortality and teratogenicity effects of V remain unexplored in amphibian species. To address this gap in the knowledge base, a standard Frog Embryo Teratogenic Index - Xenopus (FETAX) assessment was conducted. Vanadium pentoxide (V2O5) was chosen for its known toxicity in other aquatic biota and its solubility in water. A range-finding test was conducted in two different mediums, V2O5 in distilled water (VDH2O) and V2O5 in FETAX medium (VMED), to determine concentration ranges where effects occurred. Thereafter, definitive tests were conducted using two separate breeding pairs, with two replicate dishes per concentration containing 15 embryos each. Multiple endpoints were assessed including mortality, malformations, minimum concentration to inhibit growth (MCIG), and the teratogenic index (TI). Mortality and malformation effects occurred at different ranges, and therefore, the exposures were conducted in low dose and high dose ranges. The high dose range for mortality effects was conducted at 0, 10, 20, 40, 80, and 160 mg/L of V. The low dose exposures to assess malformation effects were conducted at 0.0001, 0.00025, 0.0005, 0.00075, and 0.001 mg/L. Binary logistic regression was used to determine the LC50 and EC50 for the two sets of definitive tests. The LC50s were determined to be 46.10 mg/L and 26.91 mg/L for VDH2O and 34.50 and 25.25 for VMED for the two breeding pairs respectively. The EC50 was calculated as 0.00053 mg/L and 0.00037 mg/L for VDH2O and 0.00036 mg/L and 0.00017 mg/L for VMED for the two definitive tests respectively. The TI was calculated as 86,981 and 72,729 for VDH2O and 95,833 and 148,526 for VMED. Ultimately, there were severe malformation effects in embryos exposed to low doses of V and V was determined to be a very strong teratogen.

Importance of diet in amphibian metamorphosis-based studies designed to assess the risk of thyroid active substances.

The present study evaluated the hypothesis that dietary quality used in historical studies may impact the effects of chemical stressors on premetamorphic development and metamorphosis due to suboptimal nutritional quality. A modified Amphibian Metamorphosis Assay (AMA) was performed in which Nieuwkoop and Faber (NF) Stage 47 tadpoles of Xenopus laevis were exposed for 32 days to iodide (I- )-deficient FETAX solution supplemented with <0.025, 0.17, 0.52, 1.58, and 4.80 µg I- /L (measured concentrations 0.061, 0.220, 0.614, 1.65, and 4.73 µg I- /L) and fed a pureed Frog Brittle (FB) diet. An AMA guideline benchmark group (four replicates) exposed to dechlorinated tap water and fed standard Sera Micron Nature® (SMN) diet was evaluated concurrently. Developmental delay, observed as changes in stage distribution or median developmental stage, occurred in FB treatments with 0.061, 0.220, and 0.614 µg/L I- , respectively. Developmental rates and hind limb length of the 1.65 and 4.73 µg/L I- groups were similar to each other, but both treatments fell short of the developmental rate achieved by the SMN benchmark. Iodide supplementation also had no impact on nonthyroidal growth endpoints, which were markedly reduced in FB-fed frogs compared with their SMN-fed counterparts. All larvae that received the FB diet had mildly to severely hypoplastic/atrophic thyroids, a condition for which iodine supplementation had little if any ameliorative effect. Collectively, these results suggested that nutritional deficiencies in the FB diet negatively affected both growth and metamorphic development, the latter of which was only compensated to a limited extent by iodine supplementation.

Xenopus GLP-1-based glycopeptides as dual glucagon-like peptide 1 receptor/glucagon receptor agonists with improved in vivo stability for treating diabetes and obesity.

Peptide dual agonists toward both glucagon-like peptide 1 receptor (GLP-1R) and glucagon receptor (GCGR) are emerging as novel therapeutics for the treatment of type 2 diabetes mellitus (T2DM) patients with obesity. Our previous work identified a Xenopus GLP-1-based dual GLP-1R/GCGR agonist termed xGLP/GCG-13, which showed decent hypoglycemic and body weight lowering activity. However, the clinical utility of xGLP/GCG-13 is limited due to its short in vivo half-life. Inspired by the fact that O-GlcNAcylation of intracellular proteins leads to increased stability of secreted proteins, we rationally designed a panel of O-GlcNAcylated xGLP/GCG-13 analogs as potential long-acting GLP-1R/ GCGR dual agonists. One of the synthesized glycopeptides 1f was found to be equipotent to xGLP/GCG-13 in cell-based receptor activation assays. As expected, O-GlcNAcylation effectively improved the stability of xGLP/GCG-13 in vivo. Importantly, chronic administration of 1f potently induced body weight loss and hypoglycemic effects, improved glucose tolerance, and normalized lipid metabolism and adiposity in both db/db and diet induced obesity (DIO) mice models. These results supported the hypothesis that glycosylation is a useful strategy for improving the in vivo stability of GLP-1-based peptides and promoted the development of dual GLP-1R/GCGR agonists as antidiabetic/antiobesity drugs.

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery.

Epilepsy is an international public health concern that greatly affects patients' health and lifestyle. About 30% of patients do not respond to available therapies, making new research models important for further drug discovery. Aquatic vertebrates present a promising avenue for improved seizure drug screening and discovery. Zebrafish (Danio rerio) and African clawed frogs (Xenopus laevis and tropicalis) are increasing in popularity for seizure research due to their cost-effective housing and rearing, similar genome to humans, ease of genetic manipulation, and simplicity of drug dosing. These organisms have demonstrated utility in a variety of seizure-induction models including chemical and genetic methods. Past studies with these methods have produced promising data and generated questions for further applications of these models to promote discovery of drug-resistant seizure pathology and lead to effective treatments for these patients.

Established Protocols for cRNA Expression and Voltage-Clamp Characterization of the P2X7 Receptor in Xenopus laevis Oocytes.

P2X7 receptors (P2X7Rs) are fast ATP4--gated ion channels that, like other members of the P2X receptor family, function as homotrimers. A high-resolution cryo-EM structure of the full-length rat P2X7R is available. Using voltage-clamp experiments in Xenopus laevis oocytes, even the earliest steps of P2X7R activation can be quantitatively recorded in the millisecond range. Site-directed mutagenesis combined with voltage-clamp recordings can reveal residues and domains of the P2X7R involved in ATP4- binding, gating (i.e., opening and closing of the channel pore) and ion selectivity. We present here proven voltage-clamp protocols that take into account requirements that are important at the levels of cDNA and vector sequences, cRNA synthesis, and Xenopus laevis oocyte isolation for reliable results.

Anesthetic Pharmacology of the Mint Extracts L-Carvone and Methyl Salicylate.

INTRODUCTION: Hydrocarbons with sufficient water solubility allosterically modulate anesthetic-sensitive ion channels. Mint extracts L-carvone and methyl salicylate water solubility exceeds modulation cutoff values for γ-amino butyric acid type A (GABAA) receptors, N-methyl-D-aspartate (NMDA) receptors, and type-2 voltage-gated sodium (Nav1.2) channels. We hypothesized that mint extracts modulate these channels at concentrations that anesthetize rats. METHODS: Channels were expressed separately in frog oocytes and studied using 2-electrode voltage clamp techniques at drug concentrations up to 10 mM. Normalized current effects were fit to Hill equations. Mint compounds were formulated in a lipid emulsion and administered IV to rats. When unresponsive to the tail clamp, rats were exsanguinated, and plasma drug concentrations were measured. RESULTS: Both mint compounds caused concentration-dependent inhibition of all channels except for methyl salicylate which inhibited GABAA receptors at low concentrations and potentiated at high concentrations. Plasma drug concentrations in anesthetized rats were 7.9 mM for L-carvone and 2.7 mM for methyl salicylate. This corresponded to ≥53% NMDA receptor inhibition and ≥78% Nav1.2 channel inhibition by both compounds and 30% potentiation of GABAA receptors by methyl salicylate. CONCLUSION: L-Carvone and methyl salicylate allosterically modulate cell receptor targets important to molecular actions of conventional anesthetics at concentrations that also induce general anesthesia in rats.

Peimine, an Anti-Inflammatory Compound from Chinese Herbal Extracts, Modulates Muscle-Type Nicotinic Receptors.

Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (IAChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked IACh, with an IC50 in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of IAch, (ii) enhancement of nAChR desensitization, revealed by both an accelerated IACh decay and a decelerated IACh deactivation, and (iii) resting-nAChR blockade, deduced from the IACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.

Cucumis sativus extract elicits chloride secretion by stimulation of the intestinal TMEM16A ion channel.

CONTEXT: Cucumber (Cucumis sativus Linn. [Cucurbitaceae]) is widely known for its purgative, antidiabetic, antioxidant, and anticancer therapeutic potential. However, its effect on gastrointestinal (GI) disease is unrecognised. OBJECTIVE: This study investigated the effect of C. sativus fruit extract (CCE) on intestinal chloride secretion, motility, and motor function, and the role of TMEM16A chloride channels. MATERIALS AND METHODS: CCE extracts were obtained from commercially available cucumber. Active fractions were then purified by HPLC and analysed by high resolution mass spectrometry. The effect of CCE on intestinal chloride secretion was investigated in human colonic T84 cells, ex vivo mouse intestinal tissue using an Ussing chamber, and the two-electrode voltage-clamp technique to record calcium sensitive TMEM16A chloride currents in Xenopus laevis oocytes. In vivo, intestinal motility was investigated using the loperamide-induced C57BL/6 constipation mouse model. Ex vivo contractility of mouse colonic smooth muscles was assessed by isometric force measurements. RESULTS: CCE increased the short-circuit current (ΔIsc 34.47 ± µA/cm2) and apical membrane chloride conductance (ΔICl 95 ± 8.1 µA/cm2) in intestinal epithelial cells. The effect was dose-dependent, with an EC50 value of 0.06 µg/mL. CCE stimulated the endogenous TMEM16A-induced Cl- current in Xenopus laevis oocytes. Moreover, CCE increased the contractility of smooth muscle in mouse colonic tissue and enhanced small bowel transit in CCE treated mice compared to loperamide controls. Mass spectrometry suggested a cucurbitacin-like analogue with a mass of 512.07 g/mol underlying the bioactivity of CCE. CONCLUSION: A cucurbitacin-like analog present in CCE activates TMEM16A channels, which may have therapeutic potential in cystic fibrosis and intestinal hypodynamic disorders.

Stigmasterol can be new steroidal drug for neurological disorders: Evidence of the GABAergic mechanism via receptor modulation.

BACKGROUND: Gamma-aminobutyric acid A (GABAA) receptors have been implicated in anxiety and epileptic disorders. HYPOTHESIS/PURPOSE: This study aimed to investigate the effects of stigmasterol, a plant sterol (phytosterol) isolated from Artemisia indica Linn on neurological disorders. METHODS: Stigmasterol was evaluated on various recombinant GABAA receptor subtypes expressed in Xenopus laevis oocytes and its anxiolytic and anticonvulsant potential was assessed using the elevated plus maze (EPM), light-dark box (LDB) test, and pentylenetetrazole- (PTZ-) induced seizure paradigms. Furthermore, computational modeling of α2ß2γ2L, α4ß3δ, and α4ß3 subtypes was performed to gain insights into the GABAergic mechanism of stigmasterol. For the first time, a model of GABAδ subtype was generated. Stigmasterol was targeted to all the binding sites (neurotransmitters, positive and negative modulator binding sites) of GABAA α2ß2γ2L, α4ß3, and α4ß3δ complexes by in silico docking. RESULTS: Stigmasterol enhanced GABA-induced currents at ternary α2ß2γ2L, α4ß3δ, and binary α4ß3 GABAAR subtypes. The potentiation of GABA-induced currents at extrasynaptic α4ß3δ was significantly higher compared to the binary α4ß3 subtype, indicating that the δ subunit is important for efficacy. Stigmasterol was found to be a potent positive modulator of the extrasynaptic α4ß3δ subtype, which was also confirmed by computational analysis. The computational analysis reveals that stigmasterol preferentially binds at the transmembrane region shared by positive modulators or a binding site constituted by the M2-M3 region of α4 and M1-M2 of ß3 at α4ß3δ complex. In in vivo studies, Stigmasterol (0.5-3.0 mg/kg, i.p.) exerted significant anxiolytic and anticonvulsant effects in an identical manner of allopregnanolone, indicating the involvement of a GABAergic mechanism. CONCLUSION: To our knowledge, this is the first study reporting the positive modulation of GABAA receptors, anxiolytic and anticonvulsant potential of stigmasterol. Thus, stigmasterol is considered to be a candidate steroidal drug for the treatment of neurological disorders due to its positive modulation of GABA receptors.

Use of an electrophysiological technique for stepwise detection of trace agonist constituents of Hochuekkito in Xenopus oocytes injected with serotonin 2C receptor mRNA.

An electrophysiological bioassay was used to isolate the active compound from Hochuekkito (HET), which the current authors previously described as having potent agonist action against serotonin 2C receptors (5-HT2CR). Synthetic 5-HT2CR mRNA was injected into Xenopus oocytes to specifically express these receptors. Crude extracts and purified products were subjected to an electrophysiological bioassay using the voltage clamp method. HET stimulated a 5-HT2CR-induced current response, whereas Juzentaohoto (JTT), which has anti-depressive action similar to that of HET, did not. Current responses were not observed with an extract mixed with five types of herbal medicines common to HET and JTT but were detected with an extract with the five types of herbal medicines found in HET alone (Hoc5). When the responses to each of the five types of Hoc5 were examined, current responses were noted with Cimicifugae rhizoma (CR) and Citrus unshiu Markovich extracts. Since efficacy and the EC50 value were higher for CR, its constituents were separated using three-dimensional high-performance liquid chromatography and the current response at each of the isolated peaks was examined. One constituent displayed a strong response and was identified as a single substance with a molecular weight of 283.1393 based on liquid chromatography/mass spectrometry. These results will contribute to the isolation of 5-HT2CR-stimulating constituents in HET and the identification of trace constituents with agonist action.

Rice amino acid transporter-like 6 (OsATL6) is involved in amino acid homeostasis by modulating the vacuolar storage of glutamine in roots.

Glutamine is a product of ammonium (NH4+ ) assimilation catalyzed by glutamine synthetase (GS) and glutamate synthase (GOGAT). The growth of NH4+ -preferring paddy rice (Oryza sativa L.) depends on root NH4+ assimilation and the subsequent root-to-shoot allocation of glutamine; however, little is known about the mechanism of glutamine storage in roots. Here, using transcriptome and reverse genetics analyses, we show that the rice amino acid transporter-like 6 (OsATL6) protein exports glutamine to the root vacuoles under NH4+ -replete conditions. OsATL6 was expressed, along with OsGS1;2 and OsNADH-GOGAT1, in wild-type (WT) roots fed with sufficient NH4 Cl, and was induced by glutamine treatment. We generated two independent Tos17 retrotransposon insertion mutants showing reduced OsATL6 expression to determine the function of OsATL6. Compared with segregants lacking the Tos17 insertion, the OsATL6 knock-down mutant seedlings exhibited lower root glutamine content but higher glutamine concentration in the xylem sap and greater shoot growth under NH4+ -replete conditions. The transient expression of monomeric red fluorescent protein-fused OsATL6 in onion epidermal cells confirmed the tonoplast localization of OsATL6. When OsATL6 was expressed in Xenopus laevis oocytes, glutamine efflux from the cell into the acidic bath solution increased. Under sufficient NH4+ supply, OsATL6 transiently accumulated in sclerenchyma and pericycle cells, which are located adjacent to the Casparian strip, thus obstructing the apoplastic solute path, and in vascular parenchyma cells of WT roots before the peak accumulation of GS1;2 and NADH-GOGAT1 occurred. These findings suggest that OsATL6 temporarily stores excess glutamine, produced by NH4+ assimilation, in root vacuoles before it can be translocated to the shoot.

Allosteric Inhibition of a Vesicular Glutamate Transporter by an Isoform-Specific Antibody.

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.

Identification and molecular study on the interaction of Schisandrin C with human 5-HT3A receptor.

Schisandrin C (Sch C) is one of the main components of Schisandra chinensis (Schisandra). Since the olden times, Schisandra has been used as a traditional herbal medicine in Asia. Recent studies have shown that Schisandra is effective against irritable bowel syndrome (IBS) in an animal model and affects IBS through the 5-HT3A pathway in the IBS rat model. However, there lacks fundamental research on the interaction of specific components of Schisandra with the 5-HT3A receptor for the treatment of IBS. We hypothesized that a component of Schisandra binds to the 5-HT3A receptor and identified Sch C via a screening work using two electrode-voltage clamps (TEVC). Thus, we aimed to elucidate the neuropharmacological actions between Sch C and the 5-HT3A receptor at molecular and cellular levels. Co-treatment of Sch C with 5-HT inhibited I5-HT in a reversible, concentrate-dependent, like-competition, and voltage-independent manner, and IC50 values of Sch C. Besides, the main binding positions of Sch C were identified through 3D modeling and point mutation were V225A and V288Y on 5-HT3A receptor. Thus, we suggest the potential of Sch C in treating IBS in a manner that suppresses excessive neuronal serotonin signaling in the synapse of sensory neurons and enterochromaffin (EC) cells. In conclusion, the results demonstrate the mechanism of interaction between Sch C and 5-HT3A receptor and reveal Sch C as a novel antagonist.

N-acetylcysteine, xCT and suppression of Maxi-chloride channel activity in human placenta.

INTRODUCTION: Placental oxidative stress features in pregnancy pathologies but in clinical trials antioxidant supplementation has not improved outcomes. N-acetylcysteine (NAC) stimulates glutathione production and is proposed as a therapeutic agent in pregnancy. However, key elements of N-acetylcysteine biology, including its cellular uptake mechanism, remains unclear. This study explores how the cystine/glutamate transporter xCT may mediate N-acetylcysteine uptake and how N-acetylcysteine alters placental redox status. METHODS: The involvement of xCT in NAC uptake by the human placenta was studied in perfused placenta and Xenopus oocytes. The effect of short-term N-acetylcysteine exposure on the placental villous proteome was determined using LC-MS. The effect of N-acetylcysteine on Maxi-chloride channel activity was investigated in perfused placenta, villous fragments and cell culture. RESULTS: Maternoplacental N-acetylcysteine administration stimulated intracellular glutamate efflux suggesting a role of the exchange transporter xCT, which was localised to the microvillous membrane of the placental syncytiotrophoblast. Placental exposure to a bolus of N-acetylcysteine inhibited subsequent activation of the redox sensitive Maxi-chloride channel independently of glutathione synthesis. Stable isotope quantitative proteomics of placental villi treated with N-acetylcysteine demonstrated changes in pathways associated with oxidative stress, apoptosis and the acute phase response. DISCUSSION: This study suggests that xCT mediates N-acetylcysteine uptake into the placenta and that N-acetylcysteine treatment of placental tissue alters the placental proteome while regulating the redox sensitive Maxi-chloride channel. Interestingly N-acetylcysteine had antioxidant effects independent of the glutathione pathway. Effective placental antioxidant therapy in pregnancy may require maintaining the balance between normalising redox status without inhibiting physiological redox signalling.

Glycine agonism in ionotropic glutamate receptors.

Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in the vertebrate CNS. Classified as AMPA, kainate, delta and NMDA receptors, iGluRs are central drivers of synaptic plasticity widely considered as a major cellular substrate of learning and memory. Surprisingly however, five out of the eighteen vertebrate iGluR subunits do not bind glutamate but glycine, a neurotransmitter known to mediate inhibitory neurotransmission through its action on pentameric glycine receptors (GlyRs). This is the case of GluN1, GluN3A, GluN3B, GluD1 and GluD2 subunits, all also binding the D amino acid d-serine endogenously present in many brain regions. Glycine and d-serine action and affinities broadly differ between glycinergic iGluR subtypes. On 'conventional' GluN1/GluN2 NMDA receptors, glycine (or d-serine) acts in concert with glutamate as a mandatory co-agonist to set the level of receptor activity. It also regulates the receptor's trafficking and expression independently of glutamate. On 'unconventional' GluN1/GluN3 NMDARs, glycine acts as the sole agonist directly triggering opening of excitatory glycinergic channels recently shown to be physiologically relevant. On GluD receptors, d-serine on its own mediates non-ionotropic signaling involved in excitatory and inhibitory synaptogenesis, further reinforcing the concept of glutamate-insensitive iGluRs. Here we present an overview of our current knowledge on glycine and d-serine agonism in iGluRs emphasizing aspects related to molecular mechanisms, cellular function and pharmacological profile. The growing appreciation of the critical influence of glycine and d-serine on iGluR biology reshapes our understanding of iGluR signaling diversity and complexity, with important implications in neuropharmacology.

Identification of Triterpene Acids in Poria cocos Extract as Bile Acid Uptake Transporter Inhibitors.

Literature reports that Poria cocos reduces blood lipid levels; however, the underlying mechanism remains unclear. Blood lipid levels are closely related to the enterohepatic circulation of bile acids, where uptake transporters playing a significant role. P. cocos extract is commonly used in traditional prescriptions and food supplements in China. We investigated the effects of P. cocos and its five triterpene acids on bile acid uptake transporters, including intestinal apical sodium-dependent bile acid transporter (ASBT) and hepatic sodium/taurocholate cotransporting polypeptide (NTCP). Triterpene acids were fingerprinted by high-performance liquid chromatography-TripleTOF and quantified by ultraperformance liquid chromatography/tandem mass spectrometry. The inhibitory effect of P. cocos and its five major representative triterpene acids on ASBT and NTCP was investigated by in vitro assays using Xenopus oocytes expressing ASBT and NTCP. P. cocos extract exhibited significant inhibitory effects with half-maximum inhibition constants of 5.89 µg/ml and 14.6 µg/ml for NTCP and ASBT, respectively. Among five triterpene acids, poricoic acid A, poricoic acid B, and polyporenic acid C significantly inhibited NTCP function. Poricoic acid A, poricoic acid B, and dehydrotumulosic acid significantly inhibited ASBT function. The representative triterpene acid, poricoic acid A, was identified as a competitive inhibitor of NTCP with an inhibitory constant of 63.4 ± 18.7 µM. In conclusion, our results indicate that both P. cocos extract and its major triterpenes are competitive inhibitors of ASBT and NTCP. Accordingly, it was suggested that competitive inhibition of these bile acid transporters is one of the underlying mechanisms for the hypolipidemic effect of P. cocos. SIGNIFICANCE STATEMENT: Poria cocos, a commonly used Chinese herbal medicine and food supplement, demonstrates significantly inhibitory effects on the function of apical sodium-dependent bile acid transporter and sodium/taurocholate cotransporting polypeptide. P. cocos has potential to reduce the blood lipid through inhibition of these uptake transporters in enterohepatic circulation of bile acid.

KCNQ5 Potassium Channel Activation Underlies Vasodilation by Tea.

BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation. METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone. RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries. CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.

Geoffroea decorticans fruit extracts inhibit the wnt/ß-catenin pathway, a therapeutic target in cancer.

Geoffroea decorticans (chañar) is commonly used for culinary and medicinal purposes in rural communities. The aim of this work was to chemically characterize three Geoffroea decorticans extracts and determine their capacity to modulate the wnt/ß-catenin pathway. This signaling pathway plays a key role in embryonic development but its overactivation leads to cancer cell growth. Phytochemical analysis of extracts showed presence of major classes of phytochemicals. Gas chromatography-mass spectrometry results revealed the presence of acids, esters and furanic compounds. Using Xenopus embryos as in vivo model organisms, we found that the extracts modulated dorso-ventral axis formation and rescued hyperdorsalized phenotypes produced by LiCl treatment. In agreement with these findings, Geoffroea decorticans extracts decreased ß-catenin levels and suppressed the expression of wnt target genes such as xnr3 and chordin, thus demonstrating an inhibitory regulation of the wnt/ß-catenin signaling pathway. All these results support a new role for Geoffroea decorticans fruit derivatives with possible anti-carcinogenic actions.