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

doi:10.1016/j.bbrc.2015.09.074

Biochemical and Biophysical Research Communications

Volume 467, Issue 4, 27 November 2015, Pages 1090-1096

https://doi.org/10.1016/j.bbrc.2015.09.074 Get rights and content

Highlights

•
We examined the action of multiple fragrance and aroma substances on 5-HT₃ receptors.
•
5-HT₃ receptor are blocked or modulated by naturally occurring terpenes.
•
5-HT₃ receptor are blocked by capsaicin and other natural vanilloids.

Abstract

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.

Introduction

Serotonin receptor type 3 (5-HT₃ receptor) is a ligand-gated ion channel and belongs to the family of cys-loop receptors along with GABA, glycine or nicotinic acetylcholine receptors. Until now, research has described five different 5-HT₃ receptor subunits (5-HT_3A-5-HT_3E) in mammals that form pentameric receptors [1]. Only the 5-HT_3A subunit can form functional homomeric receptors, whereas all of the other subunits build heteromeric receptors that comprise the 5-HT_3A subunit [2], [3]. The most frequent subunit combination is the 5-HT_3A+5-HT_3B subunit composition [4]. 5-HT₃ receptors are expressed in the peripheral nervous system (PNS) as well as in the central nervous system (CNS). Within the PNS, they are mainly expressed in the sympathicus and parasympathicus neurons and the gastrointestinal tract, thereby regulating peristalsis [5]. In the CNS, 5-HT₃ receptors are primarily found in the limbic system, the brain stem and the spinal cord and are important for a variety of functions, including emesis, cognition and anxiety. Pharmacologically active substances target the 5-HT₃ receptor to treat chemotherapy-induced nausea and vomiting. The receptor is antagonized by setrones, (e.g. granisetron, ondansetron, tropisetron) the classic 5-HT3 antagonists. Additionally, receptors are inhibited by many plant derived substances like morphine, cocaine, cannabinoids, terpenes like α-thujone (reviewed by Wallstab 2010) as well as recently shown for (+)- or (−)-nicotine [6] and boldine [7]. A wide range of substances, including divalent cations, alcohols, steroids, anesthetics (propofol), and barbiturates, have been reported to modulate 5-HT₃ receptors [1]. In summary, there are several compounds that modulate 5-HT₃ receptors, and some of them may be useful as therapeutic agents. In our work, we analyzed the action of terpenes and odorant and pungent substances of various chemical structures on human 5-HT_3A receptors that were functionally expressed in Xenopus laevis oocytes.

Section snippets

Xenopus laevis oocyte expression

The plasmid containing cDNA coding for the human 5-HT_3A receptor is in the expression vector pRc/CMV [8]. cRNAs were prepared using the AmpliCap T7 high-yield message maker kit (Epicenter, Madison, WI, USA). As previously described oocytes were obtained from female X. laevis frogs by standard methods [9]. Oocytes were injected with 5–20 ng of receptor coding cRNA using the nanoliter injector 2000 (WPI, Berlin, Germany). Oocytes were measured 1–4 days after the injection.

Electrophysiology

Electrophysiological

Screening of odorant mixes on 5-HT_3A receptors

In our first experiment, we aimed to find pharmacologically active compounds from fragrance and aroma substances that act as agonists on human 5-HT_3A receptors. For this screening, we expressed the human 5-HT_3A receptor recombinantly in X. laevis oocytes and characterized the ion channels using a two-electrode voltage clamp. To determine suitable 5-HT concentrations for our screening experiments, we first determined the EC₅₀ value for 5-HT (1.85 ± 0.09 μM Hill coefficient 1.72 ± 0.12) under our

Discussion

In our study, we showed that 5-HT_3A receptors are inhibited by a variety of terpenes and pungent substances with some of them belonging to the vanilloid class. In recent years, several natural plant ingredients have been identified as 5-HT_3A receptor blockers. These include the two terpenes (−)-menthol and α-thujone [7], [23], [24]. However, effective concentrations of these blockers are typically in the high μM range. The potency of receptor block by ginsenosides from ginseng or gingerols from

Acknowledgments

This work was funded by a grant (SFB 642) from the German Research Foundation (Deutsche Forschungsgemeinschaft) to HH.

References (39)

J. Walstab et al.
5-HT(3) receptors: role in disease and target of drugs
Pharmacol. Ther.
(2010)
A.A. Jensen et al.
3B but which 3B and that's just one of the questions: the heterogeneity of human 5-HT3 receptors
Trends Pharmacol. Sci.
(2008)
B. Niesler et al.
Cloning, physical mapping and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3C, HTR3D and HTR3E
Gene
(2003)
B.S. Schreiner et al.
Direct action and modulating effect of (+)- and (−)-nicotine on ion channels expressed in trigeminal sensory neurons
Eur. J. Pharmacol.
(2014)
A. Saras et al.
Histamine action on vertebrate GABAA receptors: direct channel gating and potentiation of GABA responses
J. Biol. Chem.
(2008)
J.L. Donovan et al.
Analysis of (+)-catechin, (−)-epicatechin and their 3'- and 4'-O-methylated analogs. A comparison of sensitive methods
J. Chromatogr. B Biomed. Sci. Appl.
(1999)
O.A. Sergeeva et al.
Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors
J. Biol. Chem.
(2010)
T. Deiml et al.
Alpha-thujone reduces 5-HT3 receptor activity by an effect on the agonist-reduced desensitization
Neuropharmacology
(2004)
S. Choi et al.
Effects of ginsenoside Rg2 on the 5-HT3A receptor-mediated ion current in Xenopus oocytes
Mol. Cells
(2003)
G. Buchbauer et al.
Fragrance compounds and essential oils with sedative effects upon inhalation
J. Pharm. Sci.
(1993)

E. Ernst et al.

Efficacy of ginger for nausea and vomiting: a systematic review of randomized clinical trials

Br. J. Anaesth.

(2000)

H. Abdel-Aziz et al.

Mode of action of gingerols and shogaols on 5-HT3 receptors: binding studies, cation uptake by the receptor channel and contraction of isolated guinea-pig ileum

Eur. J. Pharmacol.

(2006)

N.A. Darmani et al.

Additive antiemetic efficacy of low-doses of the cannabinoid CB(1/2) receptor agonist Δ(9)-THC with ultralow-doses of the vanilloid TRPV1 receptor agonist resiniferatoxin in the least shrew (Cryptotis parva)

Eur. J. Pharmacol.

(2014)

B. Niesler et al.

Characterization of the novel human serotonin receptor subunits 5-HT3C,5-HT3D, and 5-HT3E

Mol. Pharmacol.

(2007)

P.A. Davies et al.

The 5-HT3B subunit is a major determinant of serotonin-receptor function

Nature

(1999)

J. Walstab et al.

Natural compounds boldine and menthol are antagonists of human 5-HT3 receptors: implications for treating gastrointestinal disorders

Neurogastroenterol. Motil. Off. J. Eur. Gastrointest. Motil. Soc.

(2014)

S. Lankiewicz et al.

Molecular cloning, functional expression, and pharmacological characterization of 5-hydroxytryptamine3 receptor cDNA and its splice variants from guinea pig

Mol. Pharmacol.

(1998)

M.A. Sherkheli et al.

Monoterpenoids induce agonist-specific desensitization of transient receptor potential vanilloid-3 (TRPV3) ion channels, Journal of pharmacy & pharmaceutical sciences a publication of the Canadian Society for Pharmaceutical Sciences

Société Can. Des. Sci. Pharm.

(2009)

J. Walstab et al.

Ginger and its pungent constituents non-competitively inhibit activation of human recombinant and native 5-HT3 receptors of enteric neurons

Neurogastroenterol. Motil. Off. J. Eur. Gastrointest. Motil. Soc.

(2013)

Cited by (19)

Monoterpenes as a perspective for the treatment of seizures: A Systematic Review
2021, Phytomedicine
Citation Excerpt :
After administration of a seizure agent (PTZ), geraniol reduces seizure signs by 50% and mortality by 100% at a dose of 200 mg/kg administered intraperitoneally (Lins et al., 2014). This effect can be explained by the ability of geraniol to block the 5-HT3A receptor (Ziemba et al., 2015) and depress the central nervous system (Medeiros et al., 2018). In addition, the complexation of geraniol in β-cyclodextrin seems to improve the anticonvulsive effect of geraniol (Lins et al., 2014).
Epilepsy affects more than 65 million people worldwide. Treatment for epileptic seizures is ineffective and has many adverse effects. For this reason, the search for new therapeutic options capable of filling these limitations is necessary.
In this sense, natural products, such as monoterpenes, have been indicated as a new option to control neurological disorders such as epilepsy.
Therefore, the objective of this study was to review the monoterpenes that have anticonvulsive activity in animal models.
The searches were performed in the PubMed, Web of Science and Scopus databases in September, 2020 and compiled studies using monoterpenes as an alternative to seizure. Two independent reviewers performed the study selection, data extraction and methodological quality assessment using the Syrcle tool.
51 articles that described the anticonvulsant activity of 35 monoterpenes were selected with action on the main pharmacological target, including GABAA receptors, glutamate, calcium channels, sodium and potassium. In addition, these compounds are capable of reducing neuronal inflammation and oxidative stress caused by seizure.
These compounds stand out as a promising alternative for acting through different pharmacological mechanisms, which may not only reduce seizure, but also promote neuroprotective effect by reducing toxicity in brain regions. However, further studies are needed to determine the mechanism of action and safety assessment of these compounds.
Orthosteric and Allosteric Activation of Human 5-HT<inf>3</inf>A Receptors
2020, Biophysical Journal
Citation Excerpt :
Thus, our macroscopic characterization indicates that the high-conductance receptor behaves similarly to the wild-type 5-HT3A receptor. Carvacrol and thymol are two phenolic monoterpenes that have been macroscopically characterized as allosteric partial agonists and positive allosteric modulators of the human 5-HT3A receptor (ago-PAMs) (31,32). We recorded macroscopic currents elicited by these ligands at a concentration range from 1 μM to 1 mM (Fig. 1, B and C).
The serotonin type 3 receptor (5-HT₃) is a ligand-gated ion channel that converts the binding of the neurotransmitter serotonin (5-HT) into a transient cation current that mediates fast excitatory responses in peripheral and central nervous systems. Information regarding the activation and modulation of the human 5-HT₃ type A receptor has been based only on macroscopic current measurements because of its low ion conductance. By constructing a high-conductance human 5-HT₃A receptor, we here revealed mechanistic information regarding the orthosteric activation by 5-HT and by the partial agonist tryptamine, and the allosteric activation by the terpenoids, carvacrol, and thymol. Terpenoids potentiated macroscopic currents elicited by the orthosteric agonist and directly elicited currents with slow-rising phases and submaximal amplitudes. At the single-channel level, activation by orthosteric and allosteric agonists appeared as openings in quick succession (bursts) that showed no ligand concentration dependence. Bursts were grouped into long-duration clusters in the presence of 5-HT and even longer in the presence of terpenoids, whereas they remained isolated in the presence of tryptamine. Kinetic analysis revealed that allosteric and orthosteric activation mechanisms can be described by the same scheme that includes transitions of the agonist-bound receptor to closed intermediate states before opening (priming). Reduced priming explained the partial agonism of tryptamine; however, equilibrium constants for gating and priming were similar for 5-HT and terpenoid activation. Thus, our kinetic analysis revealed that terpenoids are efficacious agonists for 5-HT₃A receptors. These findings not only extend our knowledge about the human 5-HT₃A molecular function but also provide novel insights into the mechanisms of action of allosteric ligands, which are of increasing interest as therapeutic drugs in all the superfamily.
Inhibition of salt appetite in sodium-depleted rats by carvacrol: Involvement of noradrenergic and serotonergic pathways
2019, European Journal of Pharmacology
Citation Excerpt :
On the other hand, serotonergic depletion induced by peripheral administration of pCPA increases sodium intake in rats (Lima et al., 2004; Walters, 1977). In a screening of 200 pungent substances, carvacrol was previously identified as an agonist and potentiator of 5HT3A receptors (Ziemba et al., 2015). It has been shown that intraperitoneal administration of oregano´s extract inhibits monoamine oxidase (MAO) and inhibits the reuptake and degradation of serotonin in the hippocampus of rodents (Mechan et al., 2011).
Carvacrol, a monoterpene phenol present in the essential oil of oregano, possesses several biological properties, such as antioxidant, anti-inflammatory, anxiolytic, anticonvulsive and antinociceptive. In vitro studies have shown that carvacrol inhibits serotonin, noradrenaline and dopamine transporters and the enzymes monoamine oxidase-A and B. Different brain functions are controlled by monoamines, including cardiovascular control, thirst and sodium appetite. In the present study we investigated the effects of intracerebroventricular (i.c.v.) injection of carvacrol on sodium appetite, and the participation of brain serotonergic and noradrenergic pathways on carvacrol effects. Neuronal activation in homeostasis-related brain areas induced by i.c.v. injection of carvacrol was also evaluated. Carvacrol dose-dependently inhibited hypertonic saline intake (1.5%) in sodium-depleted rats, and this antinatriorexigenic effect was reduced by brain serotonergic depletion and by alpha-adrenergic blockade. Furthermore, i.c.v. injections of carvacrol significantly increased the neuronal activation in brain areas involved in the control of salt appetite, such as MnPO, OVLT, PVN, SON, CeA and MeA. Taken together, our data show that carvacrol presents antinatriorexigenic activity through serotonin and noradrenaline pathways within brain circuits involved in the modulation of the body fluid homeostasis.
A randomized placebo-controlled study of aromatherapy for the treatment of postoperative nausea and vomiting
2019, Complementary Therapies in Medicine
Citation Excerpt :
It has been reported that rose oil can be a stimulator or inhibitor of the smooth muscle of the ileum24. Additionally, some of the main components of rose oil, like geraniol and citronellol, are 5HT3 antagonists19. Therefore, we hypothesized that Rosa damascena may be effective for treating PONV; however, our results failed to show such an effect.
The purpose of this study was to compare the aromatherapy treatment effects on PONV patients using ginger, lavender and rose oils and a placebo.
A randomized 4-armed placebo controlled study.
Gaziosmanpasa University, School of Medicine, Health Research and Application Center.
The total of 184 patients were randomized into 4 groups: Aromatherapy with lavender essential oil (Lavender group), with rose essential oil (Rose group), with ginger essential oil (Ginger group) or with pure water (Placebo group).
Postoperative nausea (0–3 Likert type; 0=no nausea, 1=some, 2=a lot, 3=severe) and vomiting scores (0–3 Likert type; 0=no vomiting, 1 = 1 time, 2 = 2 or 3 time, 3 = 4 times and up) and antiemetic medication requirement.
The nausea scores at 15 min were statistically significantly different between the groups (p = 0.00). The postoperative nausea scores improved in 20 (43.5%) subjects in the placebo group, 38 (82.6%) subjects in the lavender group, 22 (47.8%) subjects in the rose group and 30 (65.2%) subjects in the ginger group (p = 0.00). There were statistically significant differences between the groups with regard to the vomiting and antiemetic drug requirements (p = 0.00).
The aromatherapy can be used as an alternative or complementary method for managing PONV. Specifically, the ginger and lavender essential oils were superior to the rose oil and pure water for the aromatherapy treatments. However, further studies with larger sample sizes are necessary to confirm these results.
Delineation of the functional properties and the mechanism of action of TMPPAA, an allosteric agonist and positive allosteric modulator of 5-HT<inf>3</inf> receptors
2016, Biochemical Pharmacology
Citation Excerpt :
The complexity of Cys-loop receptor function means not only that the receptors can be modulated or activated via multiple allosteric sites but also that the modulation/activation induced through these sites can differ dramatically depending on the specific allosteric transitions affected in the receptors [4,14,16]. Several ago-PAMs have been identified for GABAARs, nAChRs and GlyRs [47–52], and two recent studies have identified the monoterpenes carvacrol and thymol as ago-PAMs of 5-HT3Rs [53,54]. Following our previous identification of a series of putative 5-HT3R ago-PAMs [20], we here present the functional properties and the molecular basis for the activity of one of these compounds, TMPPAA.
We have previously identified a novel class of 5-hydroxytryptamine type 3 receptor (5-HT₃R) agonists sharing little structural similarity with orthosteric 5-HT₃R ligands (Jørgensen et al., 2011). In the present study we have elucidated the functional characteristics and the mechanism of action of one of these compounds, trans-3-(4-methoxyphenyl)-N-(pentan-3-yl)acrylamide (TMPPAA). In electrophysiological recordings TMPPAA was found to be a highly-efficacious partial agonist equipotent with 5-HT at the 5-HT₃A receptor (5-HT₃AR) expressed in COS-7 cells and somewhat less potent at the receptor expressed in Xenopus oocytes. The desensitization kinetics of TMPPAA-evoked currents were very different from those mediated by 5-HT. Moreover, repeated TMPPAA applications resulted in progressive current run-down and persistent non-responsiveness of the receptor to TMPPAA, but not to 5-HT. In addition to its direct activation, TMPPAA potentiated 5-HT-mediated 5-HT₃AR signalling, and the allosteric link between the two binding sites was corroborated by the analogous ability of 5-HT to potentiate TMPPAA-evoked responses. The agonism and potentiation exerted by TMPPAA at a chimeric α7-nACh/5-HT₃A receptor suggested that the ligand acts through the transmembrane domain of 5-HT₃AR, a notion further substantiated by its functional properties at chimeric and mutant human/murine 5-HT₃ARs. A residue in the transmembrane helix 4 of 5-HT3A was identified as an important molecular determinant for the different agonist potencies exhibited by TMPPAA at human and murine 5-HT₃ARs. In conclusion, TMPPAA is a novel allosteric agonist and positive allosteric modulator of 5-HT₃Rs, and its aberrant signalling characteristics compared to 5-HT at the 5-HT₃AR underline the potential in Cys-loop receptor modulation and activation through allosteric sites.
Coriandrum sativum and Its Utility in Psychiatric Disorders
2023, Molecules

View all citing articles on Scopus

¹: Both authors contributed equally.

View full text

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

Highlights

Abstract

Introduction

Section snippets

Xenopus laevis oocyte expression

Electrophysiology

Screening of odorant mixes on 5-HT3A receptors

Discussion

Acknowledgments

Pharmacol. Ther.

Trends Pharmacol. Sci.

Gene

Eur. J. Pharmacol.

J. Biol. Chem.

J. Chromatogr. B Biomed. Sci. Appl.

J. Biol. Chem.

Neuropharmacology

Mol. Cells

J. Pharm. Sci.

Br. J. Anaesth.

Eur. J. Pharmacol.

Eur. J. Pharmacol.

Characterization of the novel human serotonin receptor subunits 5-HT3C,5-HT3D, and 5-HT3E

Mol. Pharmacol.

The 5-HT3B subunit is a major determinant of serotonin-receptor function

Nature

Natural compounds boldine and menthol are antagonists of human 5-HT3 receptors: implications for treating gastrointestinal disorders

Neurogastroenterol. Motil. Off. J. Eur. Gastrointest. Motil. Soc.

Molecular cloning, functional expression, and pharmacological characterization of 5-hydroxytryptamine3 receptor cDNA and its splice variants from guinea pig

Mol. Pharmacol.

Monoterpenoids induce agonist-specific desensitization of transient receptor potential vanilloid-3 (TRPV3) ion channels, Journal of pharmacy & pharmaceutical sciences a publication of the Canadian Society for Pharmaceutical Sciences

Société Can. Des. Sci. Pharm.

Ginger and its pungent constituents non-competitively inhibit activation of human recombinant and native 5-HT3 receptors of enteric neurons

Neurogastroenterol. Motil. Off. J. Eur. Gastrointest. Motil. Soc.

Screening of odorant mixes on 5-HT_3A receptors