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.

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.

Nonivamide, a capsaicin analogue, exhibits anti-inflammatory properties in peripheral blood mononuclear cells and U-937 macrophages.

SCOPE: Inflammation-related diseases are a worldwide problem. The counteraction of inflammation with compounds activating the trigeminal nerve is one strategy to fight these diseases. Known trigeminally active compounds found in black or red pepper are the tingling t-pellitorine, the pungent capsaicin, and the less pungent nonivamide. The presented study compares the anti-inflammatory potential of nonivamide to the two known anti-inflammatory compounds, elucidating the mechanism of action and the role of transient receptor protein (TRP) channels. METHODS AND RESULTS: Primary peripheral blood mononuclear cells (PBMCs) and U-937 macrophages were stimulated with 1 µg/mL LPS from Escherichia coli (EC-LPS) to induce inflammation. Nonivamide attenuated the EC-LPS induced release of IL-6 and TNF-α in PBMCs and U-937 macrophages determined by magnetic bead kit analysis. This anti-inflammatory mechanism was independent from nuclear factor-kappa B pathway but mitogen-activated protein kinase (MAPK) pathway may be involved. In addition, cotreatment of U-937 with the trigeminally active compound and an antagonist of TRPV1 or TRPA1 abolished the anti-inflammatory activity. CONCLUSIONS: Nonivamide possessed similar anti-inflammatory potential as capsaicin and t-pellitorine. In U-937 macrophages, the tested compounds exploited an anti-inflammatory effect by inhibiting the EC-LPS induced activation of the MAPK pathway. In addition, the TRP channel activation plays a role in the anti-inflammatory capacity of capsaicin and nonivamide.

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.

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.

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.

Identification of Glycyrrhiza as the rikkunshito constituent with the highest antagonistic potential on heterologously expressed 5-HT3A receptors due to the action of flavonoids.

The traditional Japanese phytomedicine rikkunshito is traditionally used for the treatment of gastrointestinal motility disorders, cachexia and nausea. These effects indicate 5-HT3 receptor antagonism, due to the involvement of these receptors in such pathophysiological processes. E.g., setrons, specific 5-HT3 receptor antagonists are the strongest antiemetics, developed so far. Therefore, the antagonistic effects of the eight rikkunshito constituents at heterologously expressed 5-HT3Areceptors were analyzed using the two-electrode voltage-clamp technique. The results indicate that tinctures from Aurantii, Ginseng, Zingiberis, Atractylodis and Glycyrrhiza inhibited the 5-HT3A receptor response, whereas the tinctures of Poria cocos, Jujubae and Pinellia exhibited no effect. Surprisingly, the strongest antagonism was found for Glycyrrhiza, whereas the Zingiberis tincture, which is considered to be primarily responsible for the effect of rikkunshito, exhibited the weakest antagonism of 5-HT3A receptors. Rikkunshito contains various vanilloids, ginsenosides and flavonoids, a portion of which show an antagonistic effect on 5-HT3 receptors. A screening of the established ingredients of the active rikkunshito constituents and related substances lead to the identification of new antagonists within the class of flavonoids. The flavonoids (-)-liquiritigenin, glabridin and licochalcone A from Glycyrrhiza species were found to be the most effective inhibitors of the 5-HT-induced currents in the screening. The flavonoids (-)-liquiritigenin and hesperetin from Aurantii inhibited the receptor response in a non-competitive manner, whereas glabridin and licochalcone A exhibited a potential competitive antagonism. Furthermore, licochalcone A acts as a partial antagonist of 5-HT3A receptors. Thus, this study reveals new 5-HT3A receptor antagonists with the aid of increasing the comprehension of the complex effects of rikkunshito.

Direct action and modulating effect of (+)- and (-)-nicotine on ion channels expressed in trigeminal sensory neurons.

Nicotine sensory perception is generally thought to be mediated by nicotinic acetylcholine (nACh) receptors. However, recent data strongly support the idea that other receptors (e.g., transient receptor potential A1 channel, TRPA1) and other pathways contribute to the detection mechanisms underlying the olfactory and trigeminal cell response to nicotine flavor. This is in accordance with the reported ability of humans to discriminate between (+)- and (-)- nicotine enantiomers. To get a more detailed understanding of the molecular and cellular basis underlying the sensory perception of nicotine, we studied the activity of (+)- and (-)-nicotine on cultured murine trigeminal sensory neurons and on a range of heterologously expressed receptors. The human TRPA1 channel is activated by (-)-nicotine. In this work, we show that (+)-nicotine is also an activator of this channel. Pharmacological experiments using nicotinic acetylcholine receptors and transient receptor potential blockers revealed that trigeminal neurons express one or more unidentified receptors that are sensitive to (+)- and/or (-)-nicotine. Results also indicate that the presence of extracellular calcium ions is required to elicit trigeminal neuron responses to (+)- and (-)-nicotine. Results also show that both (+)-nicotine and (-)-nicotine can block 5-hydroxytryptamine type 3 (5-HT3) receptor-mediated responses in recombinant expression systems and in cultured trigeminal neurons expressing 5-HT3 receptors. Our investigations broaden the spectra of receptors that are targets for nicotine enantiomers and give new insights into the physiological role of nicotine.

Amiloride derivatives are effective blockers of insect odorant receptors.

Heteromeric insect odorant receptors (ORs) form ligand-activated nonselective cation channels in recombinant expression systems. We performed a pharmacological characterization of Drosophila melanogaster and Bombyx mori ORs expressed in the Xenopus laevis oocyte expression system and characterized them using the 2-electrode voltage clamp. We identified amiloride derivatives as high-affinity blockers, which inhibit the ion current through the channel in a low micromolar range. For the heteromeric Drosophila Or47a + DmelOrco receptor, the potency sequence (IC(50)) is HMA [5-(N,N-hexamethylene)amiloride] (3.9 µM), MIA [5-(N-methyl-N-isobutyl)amiloride] (11.0 µM), and DMA [5-(N,N-dimethyl)amiloride] (113.3 µM). Amiloride itself is nearly ineffective. Other tested insect ORs (Drosophila Or49b + DmelOrco, B. mori BmorOr1 + BmorOrco) were blocked in a similar fashion suggesting that the amiloride derivatives were potential general blockers of all receptor combinations. Our results suggest that pyrazine derivatives of amiloride are useful probes to study the mechanism of chemosensory transduction in insects in more detail.