Targeting Vasohibins to Promote Axon Regeneration.

Treatments accelerating axon regeneration in the nervous system are still clinically unavailable. However, parthenolide promotes adult sensory neurons' axon growth in culture by inhibiting microtubule detyrosination. Here, we show that overexpression of vasohibins increases microtubule detyrosination in growth cones and compromises growth in culture and in vivo. Moreover, overexpression of these proteins increases the required parthenolide concentrations to promote axon regeneration. At the same time, the partial knockdown of endogenous vasohibins or their enhancer SVBP in neurons facilitates axon growth, verifying them as pharmacological targets for promoting axon growth. In vivo, repeated intravenous application of parthenolide or its prodrug di-methyl-amino-parthenolide (DMAPT) markedly facilitates the regeneration of sensory, motor, and sympathetic axons in injured murine and rat nerves, leading to acceleration of functional recovery. Moreover, orally applied DMAPT was similarly effective in promoting nerve regeneration. Thus, pharmacological inhibition of vasohibins facilitates axon regeneration in different species and nerves, making parthenolide and DMAPT the first promising drugs for curing nerve injury.

Functional expression and ligand identification of homo- and heteromeric Drosophila melanogaster CO2 receptors in the Xenopus laevis oocyte system.

Carbon dioxide (CO2) is an important olfactory cue in Drosophila melanogaster and can elicit both attractive and aversive behaviors. It is detected by gustatory receptors, Gr21a and Gr63a, found in the ab1C neuron in basiconic sensilla on the third antennal segment. Volatile substances that modulate the receptors' function are of interest for pest control. While several substances block ab1C neurons or mimic the activating effect of carbon dioxide, it is not known if these substances are indeed ligands of the CO2 receptor or might act on other proteins in the receptor neuron. In this study, we used the recombinant Xenopus laevis expression system and two-electrode voltage-clamp technology to investigate the receptor function. We found that application of sodium bicarbonate evokes large inward currents in oocytes co-expressing Gr21a and Gr63a. The receptors most likely form hetromultimeric complexes. Homomultimeric receptors of Gr21a or Gr63a are sufficient for receptor functionality, although oocytes gave significantly lower current responses compared to the probable heteromultimeric receptor. We screened for putative blockers of the sodium bicarbonate response and confirmed that some of the substances identified by spike recordings of olfactory receptor neurons, such as 1-hexanol, are also blockers in the Xenopus oocyte system. We also identified a new blocking substance, citronellol, which is related to insect repellents. Many substances that activate receptor neurons were inactive in the Xenopus oocyte system, indicating that they may not be ligands for the receptor, but may act on other proteins. However, methyl pyruvate and n-hexylamine were found to be activators of the recombinant Gr21a/Gr63a receptor.

Transneuronal delivery of hyper-interleukin-6 enables functional recovery after severe spinal cord injury in mice.

Spinal cord injury (SCI) often causes severe and permanent disabilities due to the regenerative failure of severed axons. Here we report significant locomotor recovery of both hindlimbs after a complete spinal cord crush. This is achieved by the unilateral transduction of cortical motoneurons with an AAV expressing hyper-IL-6 (hIL-6), a potent designer cytokine stimulating JAK/STAT3 signaling and axon regeneration. We find collaterals of these AAV-transduced motoneurons projecting to serotonergic neurons in both sides of the raphe nuclei. Hence, the transduction of cortical neurons facilitates the axonal transport and release of hIL-6 at innervated neurons in the brain stem. Therefore, this transneuronal delivery of hIL-6 promotes the regeneration of corticospinal and raphespinal fibers after injury, with the latter being essential for hIL-6-induced functional recovery. Thus, transneuronal delivery enables regenerative stimulation of neurons in the deep brain stem that are otherwise challenging to access, yet highly relevant for functional recovery after SCI.

Effect of 158 herbal remedies on human TRPV1 and the two-pore domain potassium channels KCNK2, 3 and 9.

BACKGROUND AND AIM: Herbal medicines are used to treat a broad number of maladies. However, the pharmacological profile of most remedies is poorly understood. We investigated the effect of herbal remedies from kampo, traditional Chinese medicine (TCM) and other phytotherapies on human two-pore domain potassium channels (KCNK channels; TREK-1, TASK-1 and TASK-3) as well as the human TRPV1 channel. KCNK channels are responsible for the background potassium current of excitable cells, thus essential for the maintenance of the resting membrane potential. Hence, modulators of KCNK channels are of medical significance, e.g. for the treatment of sleep disorders and pain. The transient receptor potential channel TRPV1 is a pain detector for noxious heat. Agonists of this receptor are still used for the treatment of pain in ectopic applications. EXPERIMENTAL PROCEDURE: We evaluated the effect of 158 herbal remedies on these channels in a heterologous expression system (Xenopus laevis oocytes) using the two-electrode voltage-clamp technique with the aim of increasing the comprehension of their pharmacological profile. RESULTS AND CONCLUSION: Some remedies with modulating effects were identified such as Angelica pubescens (radix), which inhibit TASK-1 and TASK-3 channels. Furthermore, the modulatory effects of the most effective remedies on the two TASK family members TASK-1 and TASK-3 correlate positively, reflecting their close relation. For the TRPV1 channel Terminalia chebula and Alchemilla xanthochlora were identified as potentiators. This study identifies a variety of herbal remedies as modulators of human K2P and TRPV1 channels and gives new insights into the pharmacological profile of these herbal remedies.

Effects of Quinine, Quinidine and Chloroquine on Human Muscle Nicotinic Acetylcholine Receptors.

The genus Cinchona is known for a range of alkaloids, such as quinine, quinidine, cinchonine, and cinchonidine. Cinchona bark has been used as an antimalarial agent for more than 400 years. Quinine was first isolated in 1820 and is still acknowledged in the therapy of chloroquine-resistant falciparum malaria; in lower dosage quinine has been used as treatment for leg cramps since the 1940s. Here we report the effects of the quinoline derivatives quinine, quinidine, and chloroquine on human adult and fetal muscle nicotinic acetylcholine receptors (nAChRs). It could be demonstrated that the compounds blocked acetylcholine (ACh)-evoked responses in Xenopus laevis oocytes expressing the adult nAChR composed of αß𝜀δ subunits in a concentration-dependent manner, with a ranked potency of quinine (IC50 = 1.70 µM), chloroquine (IC50 = 2.22 µM) and quinidine (IC50 = 3.96 µM). At the fetal nAChR composed of αßγδ subunits, the IC50 for quinine was found to be 2.30 µM. The efficacy of the block by quinine was independent of the ACh concentration. Therefore, quinine is proposed to inhibit ACh-evoked currents in a non-competitive manner. The present results add to the pharmacological characterization of muscle nAChRs and indicate that quinine is effective at the muscular nAChRs close to therapeutic blood concentrations required for the therapy and prophylaxis of nocturnal leg cramps, suggesting that the clinically proven efficacy of quinine could be based on targeting nAChRs.

Characterization of the Olfactory Receptor OR10H1 in Human Urinary Bladder Cancer.

Olfactory receptors (ORs) are a large group of G-protein coupled receptors predominantly found in the olfactory epithelium. Many ORs are, however, ectopically expressed in other tissues and involved in several diseases including cancer. In this study, we describe that one OR, OR10H1, is predominantly expressed in the human urinary bladder with a notably higher expression at mRNA and protein level in bladder cancer tissues. Interestingly, also significantly higher amounts of OR10H1 transcripts were detectable in the urine of bladder cancer patients than in the urine of control persons. We identified the sandalwood-related compound Sandranol as a specific agonist of OR10H1. This deorphanization allowed the functional characterization of OR10H1 in BFTC905 bladder cancer cells. The effect of receptor activation was morphologically apparent in cell rounding, accompanied by changes in the cytoskeleton detected by ß-actin, T-cadherin and ß-Catenin staining. In addition, Sandranol treatment significantly diminished cell viability, cell proliferation and migration and induced a limited degree of apoptosis. Cell cycle analysis revealed an increased G1 fraction. In a concentration-dependent manner, Sandranol application elevated cAMP levels, which was reduced by inhibition of adenylyl cyclase, and elicited intracellular Ca2+ concentration increase. Furthermore, activation of OR10H1 enhanced secretion of ATP and serotonin. Our results suggest OR10H1 as a potential biomarker and therapeutic target for bladder cancer.

Olfactory Receptors as Biomarkers in Human Breast Carcinoma Tissues.

Olfactory receptors (ORs) are known to be expressed in a variety of human tissues and act on different physiological processes, such as cell migration, proliferation, or secretion and have been found to function as biomarkers for carcinoma tissues of prostate, lung, and small intestine. In this study, we analyzed the OR expression profiles of several different carcinoma tissues, with a focus on breast cancer. The expression of OR2B6 was detectable in breast carcinoma tissues; here, transcripts of OR2B6 were detected in 73% of all breast carcinoma cell lines and in over 80% of all of the breast carcinoma tissues analyzed. Interestingly, there was no expression of OR2B6 observed in healthy tissues. Immunohistochemical staining of OR2B6 in breast carcinoma tissues revealed a distinct staining pattern of carcinoma cells. Furthermore, we detected a fusion transcript containing part of the coding exon of OR2B6 as a part of a splice variant of the histone HIST1H2BO transcript. In addition, in cancer tissues and cell lines derived from lung, pancreas, and brain, OR expression patterns were compared to that of corresponding healthy tissues. The number of ORs detected in lung carcinoma tissues was significantly reduced in comparison to the surrounding healthy tissues. In pancreatic carcinoma tissues, OR4C6 was considerably more highly expressed in comparison to the respective healthy tissues. We detected OR2B6 as a potential biomarker for breast carcinoma tissues.

Odorant Receptor 51E2 Agonist ß-ionone Regulates RPE Cell Migration and Proliferation.

The odorant receptor 51E2 (OR51E2), which is well-characterized in prostate cancer cells and epidermal pigment cells, was identified for the first time as the most highly expressed OR in human fetal and adult retinal pigment epithelial (RPE) cells. Immunofluorescence staining and Western blot analysis revealed OR51E2 localization throughout the cytosol and in the plasma membrane. Additionally, immunohistochemical staining of diverse layers of the eye showed that the expression of OR51E2 is restricted to the pigment cells of the RPE and choroid. The results of Ca2+-imaging experiments demonstrate that activation of OR51E2 triggers a Ca2+ dependent signal pathway in RPE cells. Downstream signaling of OR51E2 involves the activation of adenylyl cyclase, ERK1/2 and AKT. The activity of these protein kinases likely accounts for the demonstrated increase in the migration and proliferation of RPE cells upon stimulation with the OR51E2 ligand ß-ionone. These findings suggest that OR51E2 is involved in the regulation of RPE cell growth. Thus, OR51E2 represents a potential target for the treatment of proliferative disorders.

The Effect of Pungent and Tingling Compounds from Piper nigrum L. on Background K+ Currents.

Black peppercorns (Piper nigrum L.) elicit a pungent and tingling oral impression. Their pungency is partially explained by the agonist activity of some of their active principles, especially piperine, on TRP channels. However, we recently showed that piperine, as well as other pungent compounds, also possess a marked effect on two-pore domain (KCNK, K2P) K+ channels. Members of this family play a key role in maintaining the resting membrane potential of excitable cells. Interestingly, tingling compounds have been shown to induce neuronal excitation by inhibiting KCNK channels. We addressed the question of whether it was plausible that KCNK channels could constitute a physiologically relevant target for the sensory active compounds present in black peppercorns. Because previous studies have demonstrated that mouse trigeminal neurons respond to several pungent compounds, to which humans are also sensitive, we used a primary culture of mouse trigeminal neurons to investigate whether the effect of piperine on these cell types could also be mediated by KCNK channels. We observed that even in the presence of classical TRP-antagonists, piperine was still able to activate a fraction of trigeminal neurons. Furthermore, our results showed that piperine is capable of inducing neuronal depolarization by a mechanism that does not require extracellular Na+ or Ca2+. This depolarization was mediated by the inhibition of a background K+ conductance, most likely corresponding to the KCNK channels of the TASK subfamily. We then performed a screening with 12 other pungent and/or tingling chemosensates isolated from black peppercorns. These compounds were evaluated on Xenopus laevis oocytes expressing the human orthologues of KCNK3, KNCK9 and KCNK18, which we previously showed to be inhibited by piperine. Remarkably, almost all of the isolated chemosensates inhibited the basal activity of hKCNK3, with 1-(octadeca-2E,4E,13/12Z-trienoyl)pyrrolidine acting as one of the most potent natural blockers for hKCNK3 found to date. Our results suggest that KCNK channels, especially KCNK3, are likely to play a complementary role to TRP channels in the complex orosensory impression elicited by black peppercorns, while they also help to expand the pharmacological knowledge of KCNK channels.

Activation of odorant receptor in colorectal cancer cells leads to inhibition of cell proliferation and apoptosis.

The analysis and functional characterization of ectopically expressed human olfactory receptors (ORs) is becoming increasingly important, as many ORs have been identified in several healthy and cancerous tissues. OR activation has been demonstrated to have influence on cancer cell growth and progression. Here, ORs were identified using RNA-Seq analyses and RT-PCR. We demonstrated the OR protein localization in HCT116 cells using immunocytochemistry (IHC). In order to analyze the physiological role of OR51B4, we deorphanized the receptor by the use of CRE-Luciferase assays, conducted calcium imaging experiments as well as scratch- and proliferation assays. Furthermore, western blot analyses revealed the involvement of different protein kinases in the ligand-dependent signaling pathway. Receptor knockdown via shRNA was used to analyze the involvement of OR51B4. We identified OR51B4, which is highly expressed in the colon cancer cell line HCT116 and in native human colon cancer tissues. We deorphanized the receptor and identified Troenan as an effective ligand. Troenan stimulation of HCT116 cells has anti-proliferative, anti-migratory and pro-apoptotic effects, mediated by changes in the intracellular calcium level upon PLC activation. These effects cause changes in the phosphorylation levels of p38, mTor and Akt kinases. Knockdown of the receptor via shRNA confirmed the involvement of OR51B4. This study emphasizes the importance of ectopically expressed ORs in the therapy for several diseases. The findings provide the basis for alternative treatments of colorectal cancer.

Deep Sequencing of the Human Retinae Reveals the Expression of Odorant Receptors.

Several studies have demonstrated that the expression of odorant receptors (ORs) occurs in various tissues. These findings have served as a basis for functional studies that demonstrate the potential of ORs as drug targets for a clinical application. To the best of our knowledge, this report describes the first evaluation of the mRNA expression of ORs and the localization of OR proteins in the human retina that set a stage for subsequent functional analyses. RNA-Sequencing datasets of three individual neural retinae were generated using Next-generation sequencing and were compared to previously published but reanalyzed datasets of the peripheral and the macular human retina and to reference tissues. The protein localization of several ORs was investigated by immunohistochemistry. The transcriptome analyses detected an average of 14 OR transcripts in the neural retina, of which OR6B3 is one of the most highly expressed ORs. Immunohistochemical stainings of retina sections localized OR2W3 to the photosensitive outer segment membranes of cones, whereas OR6B3 was found in various cell types. OR5P3 and OR10AD1 were detected at the base of the photoreceptor connecting cilium, and OR10AD1 was also localized to the nuclear envelope of all of the nuclei of the retina. The cell type-specific expression of the ORs in the retina suggests that there are unique biological functions for those receptors.

Two olfactory receptors-OR2A4/7 and OR51B5-differentially affect epidermal proliferation and differentiation.

Olfactory receptors (ORs), which belong to the G-protein coupled receptor family, are expressed in various human tissues, including skin. Cells in non-olfactory tissues tend to express more than one individual OR gene, but function and interaction of two or more ORs in the same cell type has only been marginally analysed. Here, we revealed OR2A4/7 and OR51B5 as two new ORs in human skin cells and identified cyclohexyl salicylate and isononyl alcohol as agonists of these receptors. In cultured human keratinocytes, both odorants induce strong Ca2+ signals that are mediated by OR2A4/7 and OR51B5, as demonstrated by the receptor knockdown experiments. Activation of corresponding receptors induces a cAMP-dependent pathway. Localization studies and functional characterization of both receptors revealed several differences. OR2A4/7 is expressed in suprabasal keratinocytes and basal melanocytes of the epidermis and influences cytokinesis, cell proliferation, phosphorylation of AKT and Chk-2 and secretion of IL-1. In contrast, OR51B5 is exclusively expressed in suprabasal keratinocytes, supports cell migration and regeneration of keratinocyte monolayers, influences Hsp27, AMPK1 and p38MAPK phosphorylation and interestingly, IL-6 secretion. These findings underline that different ORs perform diverse functions in cutaneous cells, and thus offering an approach for the modulated treatment of skin diseases and wound repair.

Expression and functionality of TRPV1 in breast cancer cells.

Transient receptor potential (TRP) channels contribute to the regulation of intracellular calcium, which can promote cancer hallmarks in cases of dysregulation of gene transcription and calcium-dependent pro-proliferative or anti-apoptotic mechanisms. Several studies have begun to elucidate the roles of TRPV1, TRPV6, TRPM8, and TRPC1 in cancer progression; however, no study has examined the expression profiles of human TRP channels in breast cancer on a large scale. This study focused on the expression and functionality of TRPV1, a nonselective cation channel that was found to be expressed in different carcinoma tissues. Next-generation sequencing analyses revealed the expression of TRPV1 in several native breast cancer tissues, which was subsequently validated via reverse transcriptase-polymerase chain reaction. Activation of TRPV1 by its ligand capsaicin was associated with the growth inhibition of some cancer cell types; however, the signaling components involved are complex. In this study, stimulation by the TRPV1 agonist, capsaicin, of SUM149PT cells, a model system for the most aggressive breast cancer subtype, triple-negative breast cancer, led to intracellular calcium signals that were diminished by the specific TRPV1 antagonist, capsazepin. Activation of TRPV1 by capsaicin caused significant inhibition of cancer cell growth and induced apoptosis and necrosis. In conclusion, the current study revealed the expression profiles of human TRP channels in 60 different breast cancer tissues and cell lines and furthermore validated the antitumor activity of TRPV1 against SUM149PT breast cancer cells, indicating that activation of TRPV1 could be used as a therapeutic target, even in the most aggressive breast cancer types.

Kampo Medicine: Evaluation of the Pharmacological Activity of 121 Herbal Drugs on GABAA and 5-HT3A Receptors.

Kampo medicine is a form of Japanese phytotherapy originating from traditional Chinese medicine (TCM). During the last several decades, much attention has been paid to the pharmacological effects of these medical plants and their constituents. However, in many cases, a systematic screening of Kampo remedies to determine pharmacologically relevant targets is still lacking. In this study, a broad screening of Kampo remedies was performed to look for pharmacologically relevant 5-HT3A and GABAA receptor ligands. Several of the Kampo remedies are currently used for symptoms such as nausea, emesis, gastrointestinal motility disorders, anxiety, restlessness, or insomnia. Therefore, the pharmacological effects of 121 herbal drugs from Kampo medicine were analyzed as ethanol tinctures on heterologously expressed 5-HT3A and GABAA receptors, due to the involvement of these receptors in such pathophysiological processes. The tinctures of Lindera aggregata (radix) and Leonurus japonicus (herba) were the most effective inhibitory compounds on the 5-HT3A receptor. Further investigation of known ingredients in these compounds led to the identification of leonurine from Leonurus as a new natural 5-HT3A receptor antagonist. Several potentiating herbs (e.g., Magnolia officinalis (cortex), Syzygium aromaticum (flos), and Panax ginseng (radix)) were also identified for the GABAA receptor, which are all traditionally used for their sedative or anxiolytic effects. A variety of tinctures with antagonistic effects Salvia miltiorrhiza (radix) were also detected. Therefore, this study reveals new insights into the pharmacological action of a broad spectrum of herbal drugs from Kampo, allowing for a better understanding of their physiological effects and clinical applications.

Characterization of non-olfactory GPCRs in human sperm with a focus on GPR18.

G protein-coupled receptors (GPCRs) transduce external chemical cues into intracellular signals and are involved in a plethora of physiological processes, but knowledge regarding the function of these receptors in spermatozoa is limited. In the present study, we performed RNA-Seq and analyzed the expression of the all GPCRs except olfactory receptors in human spermatozoa. We revealed the expression of up to 223 different GPCR transcripts in human spermatozoa (FPKM > 0.1) and identified GPR18, a newly described cannabinoid receptor, together with GPR137 and GPR135, as one of the three most highly expressed GPCRs. To date, the expression of GPR18 was completely unknown in human spermatozoa. We confirmed GPR18 expression using RT-PCR and immuncytochemistry experiments and localized the GPR18 protein in the midpiece of human spermatozoa. Stimulation of human spermatozoa with the GPR18 ligand N-arachidonoylglycine induced the phosphorylation of 12 protein kinases, some of them are for example known to be involved in the acrosome reaction. In line with this, N-arachidonoylglycine affected the cytoskeleton by changing levels of F-actin and inducing the acrosome reaction in human spermatozoa in a concentration-dependent manner. Our results indicate that GPR18 might be involved in physiological processes of human spermatozoa, suggesting GPR18 to be a potential player in sperm physiology.

Modulatory Effects of Sex Steroids Progesterone and Estradiol on Odorant Evoked Responses in Olfactory Receptor Neurons.

The influence of the sex steroid hormones progesterone and estradiol on physiology and behavior during menstrual cycles and pregnancy is well known. Several studies indicate that olfactory performance changes with cyclically fluctuating steroid hormone levels in females. Knowledge of the exact mechanisms behind how female sex steroids modulate olfactory signaling is limited. A number of different known genomic and non-genomic actions that are mediated by progesterone and estradiol via interactions with different receptors may be responsible for this modulation. Next generation sequencing-based RNA-Seq transcriptome data from the murine olfactory epithelium (OE) and olfactory receptor neurons (ORNs) revealed the expression of several membrane progestin receptors and the estradiol receptor Gpr30. These receptors are known to mediate rapid non-genomic effects through interactions with G proteins. RT-PCR and immunohistochemical staining results provide evidence for progestin and estradiol receptors in the ORNs. These data support the hypothesis that steroid hormones are capable of modulating the odorant-evoked activity of ORNs. Here, we validated this hypothesis through the investigation of steroid hormone effects by submerged electro-olfactogram and whole cell patch-clamp recordings of ORNs. For the first time, we demonstrate that the sex steroid hormones progesterone and estradiol decrease odorant-evoked signals in the OE and ORNs of mice at low nanomolar concentrations. Thus, both of these sex steroids can rapidly modulate the odor responsiveness of ORNs through membrane progestin receptors and the estradiol receptor Gpr30.

The activation of OR51E1 causes growth suppression of human prostate cancer cells.

The development of prostate cancer (PCa) is regulated by the androgen-dependent activity of the androgen receptor (AR). Androgen-deprivation therapy (ADT) is therefore the gold standard treatment to suppress malignant progression of PCa. Nevertheless, due to the development of castration resistance, recurrence of disease after initial response to ADT is a major obstacle to successful treatment. As G-protein coupled receptors play a fundamental role in PCa physiology, they might represent promising alternative or combinatorial targets for advanced diseases. Here, we verified gene expression of the olfactory receptors (ORs) OR51E1 [prostate-specific G-protein coupled receptor 2 (PSGR2)] and OR51E2 (PSGR) in human PCa tissue by RNA-Seq analysis and RT-PCR and elucidated the subcellular localization of both receptor proteins in human prostate tissue. The OR51E1 agonist nonanoic acid (NA) leads to the phosphorylation of various protein kinases and growth suppression of the PCa cell line LNCaP. Furthermore, treatment with NA causes reduction of androgen-mediated AR target gene expression. Interestingly, NA induces cellular senescence, which coincides with reduced E2F1 mRNA levels. In contrast, treatment with the structurally related compound 1-nonanol or the OR2AG1 agonist amyl butyrate, neither of which activates OR51E1, did not lead to reduced cell growth or an induction of cellular senescence. However, decanoic acid, another OR51E1 agonist, also induces cellular senescence. Thus, our results suggest the involvement of OR51E1 in growth processes of PCa cells and its impact on AR-mediated signaling. These findings provide novel evidences to support the functional importance of ORs in PCa pathogenesis.

Potentiating effect of glabridin from Glycyrrhiza glabra on GABAA receptors.

Extracts from Glycyrrhiza are traditionally used for the treatment of insomnia and anxiety. Glabridin is one of the main flavonoid compounds from Glycyrrhiza glabra and displays a broad range of biological properties. In the present work, we investigated the effect of glabridin on GABAA receptors. For this purpose, we employed the two-electrode voltage-clamp technique on Xenopus laevis oocytes expressing recombinant GABAA receptors. Through this approach, we observed that glabridin presents a strong potentiating effect on GABAA α1ß(1-3)γ2 receptors. The potentiation was slightly dependent on the ß subunit and was most pronounced at the α1ß2γ2 subunit combination, which forms the most abundant GABAA receptor in the CNS. Glabridin potentiated with an EC50 of 6.3±1.7 µM and decreased the EC50 of the receptor for GABA by approximately 12-fold. The potentiating effect of glabridin is flumazenil-insensitive and does not require the benzodiazepine binding site. Glabridin acts on the ß subunit of GABAA receptors by a mechanism involving the M286 residue, which is a key amino acid at the binding site for general anesthetics, such as propofol and etomidate. Our results demonstrate that GABAA receptors are strongly potentiated by one of the main flavonoid compounds from Glycyrrhiza glabra and suggest that glabridin could contribute to the reported hypnotic effect of Glycyrrhiza extracts.

Timberol® Inhibits TAAR5-Mediated Responses to Trimethylamine and Influences the Olfactory Threshold in Humans.

In mice, trace amine-associated receptors (TAARs) are interspersed in the olfactory epithelium and constitute a chemosensory subsystem that is highly specific for detecting volatile amines. Humans possess six putative functional TAAR genes. Human TAAR5 (hTAAR5) is highly expressed in the olfactory mucosa and was shown to be specifically activated by trimethylamine. In this study, we were challenged to uncover an effective blocker substance for trimethylamine-induced hTAAR5 activation. To monitor blocking effects, we recombinantly expressed hTAAR5 and employed a commonly used Cre-luciferase reporter gene assay. Among all tested potential blocker substances, Timberol®, an amber-woody fragrance, is able to inhibit the trimethylamine-induced hTAAR5 activation up to 96%. Moreover, human psychophysical data showed that the presence of Timberol® increases the olfactory detection threshold for the characteristic fishy odor of trimethylamine by almost one order of magnitude. In conclusion, our results show that among tested receptors Timberol® is a specific and potent antagonist for the hTAAR5-mediated response to trimethylamine in a heterologous system. Furthermore, our data concerning the observed shift of the olfactory detection threshold in vivo implicate that hTAAR5 or other receptors that may be inhibited by Timberol® could be involved in the high affinity olfactory perception of trimethylamine in humans.

Activation and modulation of recombinantly expressed serotonin receptor type 3A by terpenes and pungent substances.

Serotonin receptor type 3 (5-HT3 receptor) is a ligand-gated ion channel that is expressed in the central nervous system (CNS) as well as in the peripheral nervous system (PNS). The receptor plays an important role in regulating peristalsis of the gastrointestinal tract and in functions such as emesis, cognition and anxiety. Therefore, a variety of pharmacologically active substances target the 5-HT3 receptor to treat chemotherapy-induced nausea and vomiting. The 5-HT3 receptors are activated, antagonized, or modulated by a wide range of chemically different substances, such as 2-methyl-serotonin, phenylbiguanide, setrones, or cannabinoids. Whereas the action of all of these substances is well described, less is known about the effect of terpenoids or fragrances on 5-HT3A receptors. In this study, we screened a large number of natural odorous and pungent substances for their pharmacological action on recombinantly expressed human 5-HT3A receptors. The receptors were functionally expressed in Xenopus oocytes and characterized by electrophysiological recordings using the two-electrode voltage-clamp technique. A screening of two odorous mixes containing a total of 200 substances revealed that the monoterpenes, thymol and carvacrol, act as both weak partial agonists and positive modulators on the 5-HT3A receptor. In contrast, the most effective blockers were the terpenes, citronellol and geraniol, as well as the pungent substances gingerol, capsaicin and polygodial. In our study, we identified new modulators of 5-HT3A receptors out of the classes of monoterpenes and vanilloid substances that frequently occur in various plants.