SEARCH

SEARCH BY CITATION

Keywords:

  • 5-HT3 receptor;
  • Ca2+ influx;
  • ligand-gated ion channel;
  • radioligand binding;
  • submucous plexus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information

Background

Beneficial effects of ginger in the treatment of gastrointestinal (GI) problems and chemotherapy-induced nausea and vomiting are well accepted. In rodents, the action of ginger seems to be mediated by the inhibition of 5-HT3 receptors, which are established targets to combat emesis and irritable bowel syndrome.

Methods

Heterologously expressed human 5-HT3A or 5-HT3AB receptors were characterized by means of Ca2+influx studies using HEK293 cells. Complementing Ca2+ measurements in Fluo-4-AM-stained whole-mount preparations of the human submucous plexus were carried out. Furthermore, [3H]GR65630 binding assays were performed to reveal the mode of action of ginger and its pungent compounds.

Key Results

We show for the first time that ginger extracts and its pungent arylalkane constituents concentration-dependently inhibit activation of human 5-HT3 receptors. Ginger extracts inhibited both receptors with increasing content of pungent compounds, confirming that these are part of ginger's active principle. Inhibition potencies of the arylalkanes 6-gingerol and 6-shogaol on both receptors were in the low micromolar range. A lipophilic ginger extract and 6-gingerol had no influence on 5-HT potency, but reduced the 5-HT maximum effect, indicating non-competitive inhibition. The non-competitive action was confirmed by [3H]GR65630 binding, showing that the ginger extract did not displace the radioligand from 5-HT3A and 5-HT3AB receptors. The potential relevance of the inhibitory action of ginger on native 5-HT3 receptors in the gut was confirmed in whole-mount preparations of the human submucous plexus. While a general neurotoxic effect of 6-gingerol was ruled out, it inhibited the 2-methyl-5-HT-mediated activation of 5-HT3 receptors residing on enteric neurons.

Conclusions & Inferences

Our findings may encourage the use of ginger extracts to alleviate nausea in cancer patients receiving chemotherapy and to treat functional GI disorders.


Abbreviations
[Ca2+]i

cytosolic Ca2+ concentration

2-Me-5-HT

2-methyl-5-HT

CINV

chemotherapy-induced nausea and vomiting

GI

gastrointestinal

IBS

irritable bowel syndrome

PONV

postoperative nausea and vomiting

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information

The rhizome of ginger (Zingiber officinale Roscoe) has beneficial effects in the treatment of nausea and emesis, and gastrointestinal (GI) problems. It has been widely used in herbal medicines since ancient times.[1] According to the draft monograph of the European Medicines Agency, ginger drug powder is traditionally used for symptomatic relief of travel sickness and symptomatic treatment of mild, spasmodic GI complaints.[2] Clinical studies have demonstrated the effectiveness of ginger in the therapy of nausea/vomiting during pregnancy, and postoperative and chemotherapy-induced nausea/vomiting (PONV, CINV).[3-6] It is also effective to combat pregnancy-induced nausea and vomiting (PNV), however, the clinical studies were limited to a duration of 3 weeks and can therefore provide no evidence on long-term safety and effectivity.[7] The ability of ginger to alleviate symptoms of functional GI disorders such as the irritable bowel syndrome (IBS) and dyspepsia is widely accepted, but only few systematic studies have been conducted.[8, 9]

Recent studies imply that the antiemetic effect of ginger may be to some extent mediated by targeting 5-HT3 receptors.[10, 11] This is conceivable as 5-HT3 receptors play a major role in CINV and PONV, and antagonists of these receptors, the ‘setrons’, are part of the standard therapy to combat CINV.[12] Ginger extracts concentration-dependently inhibited 5-HT3 receptors in a murine neuroblastoma cell line.[10] Pungent arylalkanes (gingerols and shogaols) seem to be part of the active principle as they blocked these receptors in the low micromolar range.[11] This is corroborated by studies in which the antiemetic effects of gingerols and shogaols were demonstrated in animal models.[13-15] On the basis of these facts, we investigated the mode of action of ginger on human 5-HT3 receptors as rodents considerably differ from humans with regard to the 5-HT3 receptor system.[16, 17] The ligand-gated 5-HT3 receptors are composed of five subunits which surround a central cation-permeable (Na+, Ca2+, K+) channel pore.[18] In humans, five 5-HT3 subunit-encoding genes exist: 5-HT3A, B, C, D, and E.[17, 19, 20] However, the composition and function of native 5-HT3 receptors and the role of the subunits 5-HT3C, D, and E has still to be determined. Herein, we analyze the effect of ginger extracts and its pungent constituents on the two best characterized human recombinant 5-HT3 receptor subtypes, i.e., homomeric 5-HT3A and heteromeric 5-HT3AB receptors. These receptors can be discriminated by their channel properties and the agonistic potency of the physiological agonist 5-HT.[19, 21] We provide details on the mode of inhibitory action of ginger on these receptors. Furthermore, we demonstrate that the inhibition of 5-HT3 receptors by ginger may play a role in the human gut; we show a direct effect of its pungent constituent 6-gingerol on human enteric neurons which have previously been demonstrated to co-express 5-HT3A and B subunits.[22]

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information

Chemicals and drugs

Coelenterazine h was from PJK (Kleinblittersdorf, Germany). Fluo-4-AM was from Invitrogen (Karlsruhe, Germany). 5-Hydroxytryptamine creatinine sulfate (5-HT, serotonin) and ondansetron hydrochloride were from Sigma-Aldrich (Munich, Germany), and 2-methyl-5-HT (2-Me-5-HT) was from Tocris (Ellisville, MO, USA). 6-Gingerol and 6-shogaol for experiments using transfected cells were from PhytoLab (Vestenbergsgreuth, Germany). 6-Gingerol for the analysis of human tissue preparations was isolated as described in the supplementary data. Self-extracted 6-gingerol and the compound from Phytolab were directly compared in human embryonic kidney (HEK) 293 cells expressing 5-HT3A receptors via aequorin Ca2+ influx recording, revealing no significant differences in the measured IC50 (data not shown). Three ginger dry extracts from Finzelberg (Andernach, Germany) were used: an aqueous extract (TPA 23–06), an ethanolic extract (ethanol 96% w/w; UB 2005-93 III), and a supercritical CO2 extract (UB 2007-47). [3H]GR65630 ([3H]-3-(5-methyl-1H-imidazol-4-yl)-1-(1-methyl-1H-indol-3-yl)-1-propanone, specific activity 83.8 Ci/mmol), was from PerkinElmer (Boston, MA, USA).

Expression constructs

Human 5-HT3A and 5-HT3B subunit-encoding cDNAs (GenBank numbers: AJ003079 and AF080582) were cloned into the expression vector pcDNA3 (Invitrogen). To establish a cell line stably expressing 5-HT3A and 5-HT3B subunits, the 5-HT3B cDNA was subcloned into the expression vector pcDNA3.1/zeo(+) (Invitrogen). The apoaequorin cDNA (GenBank accession number L29571) was cloned into pcDNA3.1/zeo(+).[23]

Cell culture and transfection

Human embryonic kidney 293 cells (ATCC; Manassas, VA, USA) were grown in DMEM supplemented with 10% fetal calf serum, 100 U mL−1 penicillin, and 100 μg mL−1 streptomycin in a humidified atmosphere containing 5% CO2 at 37 °C. Transfection was performed with TransIT-293 transfection reagent (Mobitec; Göttingen, Germany).

A cell line stably expressing 5-HT3A receptors and apoaequorin was established by transfection of a stable HEK293 5-HT3A receptor cell line[24] (gift from Heinz Bönisch, University of Bonn) with the apoaequorin cDNA and the following clonal selection with zeocin (500 μg mL−1) for 2 weeks. Cell clones were tested for their co-expression of apoaequorin and the 5-HT3A receptor by luminometric determination of 5-HT-induced Ca2+ influx (see below). The clone with the highest receptor and apoaequorin expression was used for further experiments. To create a cell line stably expressing both the 5-HT3A and the 5-HT3B subunit, the stable HEK293 5-HT3A receptor cell line was transfected with 5-HT3B in pcDNA3.1/zeo(+) and selected with zeocin (500 μg mL−1) for 2 weeks. Following transfection with apoaequorin cDNA, cell clones were tested for their expression of heteromeric 5-HT3AB receptors as stated above. The clone with the highest receptor expression displaying definite characteristics of heteromeric 5-HT3AB receptors was used for further experiments. Stably transfected HEK293 5-HT3A/apoaequorin cells and HEK293 5-HT3AB cells were cultured in the above-stated medium containing the selection antibiotics geneticine (600 μg mL−1) and zeocin (500 μg mL−1).

For characterizing 5-HT3AB receptors by Ca2+ influx, HEK293 5-HT3AB cells were transiently transfected with the apoaequorin cDNA. For radioligand binding, HEK293 cells were transiently transfected using 5-HT3A and 5-HT3B cDNAs in a ratio of 1 : 4 to promote the predominant formation of heteromeric 5-HT3AB receptors. Cells were used 48 h post-transfection.

Human tissue samples

Human tissue samples of the small and large intestine, which were taken from macroscopically normal areas, were obtained from 10 patients undergoing abdominal surgery at the Klinikum Rechts der Isar of the Technical University Munich. Diagnoses that led to surgery were: Carcinoma of the upper GI tract (two patients), carcinoma of the lower GI tract (four patients), pancreatic carcinoma (two patients), ovarian carcinoma (one patient), diverticulitis (one patient). Procedures were approved by the ethics committee of the Technical University of Munich (1748/07 and 2595/09).

Radioligand binding

Preparation of crude membranes and [3H]GR65630 binding was carried out on HEK293 5-HT3A/apoaequorin cells and HEK293 cells transiently expressing 5-HT3AB receptors as previously described.[25, 26]

Membranes were diluted in binding assay buffer to give a final protein amount of 2 or 15 μg per reaction for 5-HT3A and 5-HT3AB receptors, respectively. In competition experiments, membranes were incubated in duplicates with 0.2 nmol L−1 [3H]GR65630 in the presence or absence of the drug under study at room temperature for 90 min. Reaction mixes were filtered through GF/B filters, presoaked with 0.5% polyethylenimine, using a Brandel cell harvester, followed by three washes with ice-cold buffer. Radioactivity was measured in a liquid scintillation counter (Beckman Coulter; Fullerton, CA, USA). Non-specific binding was determined at mock-transfected cells.

In saturation experiments, membranes were incubated with five increasing concentrations of [3H]GR65630 (0.02–1.5 nmol L−1) under the conditions described above. Radioligand concentrations were corrected for depletion that amounted to about 30% for the lowest concentration.

Aequorin luminescence assay

The aequorin assay was performed as previously described.[23] Harvested cells were loaded with 5 μmol L−1 coelenterazine h for 2.5 h at room temperature. Suspensions of cells in assay buffer were used for luminometric determination of increases in the cytosolic Ca2+ concentration ([Ca2+]i) in a Centro LB 960 luminometer (Berthold; Bad Wildbad, Germany). Luminescence was recorded from 5 s prior until 20 s after autoinjection of 5-HT at a sampling rate of 2 Hz. For determination of antagonist inhibition potencies, cells were preincubated in triplicates with the antagonist for 10 min. To exclude receptor-independent effects of the antagonists on [Ca2+]i, cells were lysed with Triton X-100 0.1% (v/v) and CaCl2 50 mmol L−1 following the experiment. Under these conditions, remaining aequorin luminescence was recorded to obtain the maximum possible Ca2+ response.

Recordings of [Ca2+]i in human tissue preparations

The method to detect changes in [Ca2+]i has been previously described in detail.[27] Briefly, whole-mount preparations of the human submucous plexus were stained with Fluo-4-AM (10 μmol L−1) in carbogenated Krebs solution for 45 min followed by a 20-min wash-out period in 100 mL carbogenated Krebs solution at room temperature. The Krebs solution contained (in mmol): 117 NaCl, 4.7 KCl, 1.2 MgCl2, 1.2 NaH2PO4, 20 NaHCO3, 2.5 CaCl2, and 11 glucose. The preparation was transferred to a recording chamber (volume ~0.5 mL) and mounted on a microscope (Axio Observer A1, Axio Cam HSm, Zeiss, Jena, Germany). To equilibrate the tissue, the recording chamber was continuously perfused with carbogenated Krebs solution at 37 °C for 20 min. [Ca2+]i recordings were performed with a FITC filterset (excitation: HC475/35, dichroic: BS499, emission: HC530/43, AHF Analysentechnik, Tübingen, Germany) and a LED light source (exposure 200 ms, frame rate 2 Hz). Recording and analysis were performed with the Axio Vision Imaging System 4.8 (Zeiss). For electrical stimulation, a teflon-coated platinum electrode (25 μm diameter) was placed onto interganglionic nerve strands and a pulse train (4V, pulse duration 0.5 ms, 10 Hz for 1 s) was generated from a SD9 stimulator (Grass, Quincy, MA, USA.) The 5-HT3 receptor agonist 2-Me-5-HT was locally applied onto the ganglia by pressure pulse application via a glass pipette (spritz application). Time between two subsequent electrical or pharmacological stimulations was 30 min.

Data analysis

Relative light units for 5-HT-induced increases in [Ca2+]i in the aequorin assay were obtained by subtracting baseline luminescence from the 5-HT-induced peak maximum luminescence. Similar results were obtained in previous studies when calculating the peak integral instead of the peak amplitude (Walstab J and Niesler B, unpublished results). Concentration-response curves and corresponding pharmacological parameters were calculated with GraphPad Prism 5.0 (GraphPad Software Inc.; San Diego, CA, USA). Concentration-response data were fitted using one- and two-component sigmoidal equations. The best fitting model was determined using the extra sum-of-squares F-test. The pIC50 values were converted to pKi values using the equation of Cheng and Prusoff.[28] Data are presented as means ± SEM. Statistical analysis was performed with Student's t-test and, when appropriate, one-way anova followed by Dunnett's or Tukey's post-test. Differences were considered significant at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information

Serotonin-induced Ca2+ influx through human 5-HT3A and 5-HT3AB receptors

Serotonin induced concentration-dependent increases in [Ca2+]i on coelenterazine h-loaded HEK293 5-HT3A/apoaequorin cells, and HEK293 5-HT3AB cells transiently expressing apoaequorin (supporting Figure S1). The derived pharmacological parameters (see 5-HT control in Table 2) are comparable to those previously obtained.[21] As we have previously shown, the concentration-response curve of 5-HT on 5-HT3AB receptors is biphasic. We demonstrated that this is presumably due to a co-expression of homomeric 5-HT3A (high affinity component) next to heteromeric 5-HT3AB receptors (low affinity component) in these cells.[21]

Inhibition of 5-HT-induced Ca2+ influx through 5-HT3 receptors by ginger dry extracts and pungent constituents

Ca2+ influx through 5-HT3A and 5-HT3AB receptors, induced by an approximately twofold EC50 5-HT concentration, that is, 3 μmol L−1 for 5-HT3A and 200 μmol L−1 for 5-HT3AB receptors, was concentration-dependently inhibited by all tested ginger dry extracts (Fig. 1A). Inhibition potencies increased with increasing lipophilicity of the extracting agent; the rank order was CO2 extract > ethanolic extract > aqueous extract and correlated with the content of pungent compounds and volatile oil (Table 1). As the CO2 extract was most potent in inhibiting 5-HT3 receptors, it was used for following experiments.

Table 1. Inhibitory effects of ginger dry extracts on human 5-HT3 receptors and correlation to the content of pungent constituents and volatile oil
 Aqueous extractEthanolic extractCO2 extract
  1. Inhibition potencies derived from three to five experiments shown in Fig. 1A.

Drug extract ratio native4–8 : 18–16 : 120–30 : 1
Pungent compounds (%)0.83.25.0
Volatile oil (%)0.14.87.1
pIC50 ± SEM [IC50 (μg mL−1)]
5-HT3A1.13 ± 0.09 (74.99)2.47 ± 0.06 (3.40)2.97 ± 0.11 (1.06)
5-HT3AB1.11 ± 0.14 (77.27)2.54 ± 0.11 (2.90)2.65 ± 0.12 (2.22)
image

Figure 1. Concentration-dependent inhibition of 5-HT-induced Ca2+ influx through 5-HT3A and 5-HT3AB receptors by ginger dry extracts (A) and pungent constituents (B). Serotonin-induced aequorin luminescence as a measure of an increased cytosolic Ca2+ concentration ([Ca2+]i) was recorded in coelenterazine h-loaded HEK293 cells heterologously expressing apoaequorin and human 5-HT3 receptors. For receptor activation, 3 μmol L−1 (HT3A) or 200 μmol L−1 (HT3AB) 5-HT were applied. Ginger dry extracts or pungent constituents were present 10 min before and during 5-HT application. Data are expressed as percentages of the response to 5-HT in the absence of the respective antagonist (means ± SEM). (A) Ginger dry extracts: Shown are the mean curves from three to five experiments. Half-maximal inhibition values are shown in Table 1. (B) Pungent constituents of ginger: Shown are the mean curves and the mean pIC50 ± SEM (corresponding mean IC50) from four to six experiments. Lower right: Dehydration of 6-gingerol to 6-shogaol during desiccation and storage.

Download figure to PowerPoint

6-Gingerol, the main pungent constituent of ginger, and its dehydration product 6-shogaol concentration-dependently inhibited 5-HT-induced Ca2+ influx through both receptors (Fig. 1B). Inhibition potencies were in the low micromolar range and were slightly higher for 6-shogaol.

Influence of ginger on the concentration-response relationship of serotonin

To get insights into the mode of inhibitory action of ginger and its pungent constituents on human 5-HT3 receptors, 5-HT concentration-response experiments in the presence of a defined concentration of either the CO2 extract or 6-gingerol were performed. Both compounds did not alter the potency of 5-HT, but reduced the 5-HT-induced maximum effect on 5-HT3A and 5-HT3AB receptors, indicating a non-competitive inhibition (Fig. 2).

image

Figure 2. Concentration-dependent 5-HT-induced Ca2+ influx through 5-HT3A (A) and 5-HT3AB receptors (B) in the absence or presence of 6-gingerol or the CO2 ginger extract. A defined concentration of the respective antagonist was present 10 min before and during 5-HT application. Data are expressed as percentages of the response to 10 μmol L−1 (5-HT3A) or 300 μmol L−1 (5-HT3AB) 5-HT in the absence of the antagonist (means ± SEM of four to seven experiments). Parameters derived from these curves are summarized in Table 2. [Ca2+]i, cytosolic Ca2+ concentration.

Download figure to PowerPoint

Table 2. Parameters derived from 5-HT concentration-response curves in the absence or presence of 6-gingerol or the CO2 extract
 5-HT3A5-HT3AB
pEC50 [EC50 (μmol L−1)]Hill slopeEmax (% of 10 μmol L−1 response) n pEC50 [EC50 (μmol L−1)]*Hill slopeEmax (% of 300 μmol L−1 response) n
  1. *Biphasic curve; shown are pEC50H/pEC50L, -log EC50 of the high (H) or low (L) affinity part of the curve, top of biphasic curve was constrained to this value as this yielded the best fit, significant difference compared to control: P < 0.01 (one-way anova followed by Dunnett's post-test).

5-HT control5.75 ± 0.06 (1.78)2.65 ± 0.29105.4 ± 4.57

5.56 ± 0.07 (2.78)

3.99 ± 0.15 (103.5)

1.50 ± 0.25

1.58 ± 0.52

1087
+ 6-Gingerol 5 μmol L−15.80 ± 0.08 (1.59)2.79 ± 0.6969.9 ± 8.35

5.63 ± 0.25 (2.34)

4.23 ± 0.55 (58.61)

1.64 ± 1.04

1.69 ± 1.96

726
+ CO2 extract 1 μg mL−15.79 ± 0.10 (1.62)2.39 ± 0.1367.9 ± 2.84

5.46 ± 0.10 (3.44)

3.87 ± 0.26 (135.8)

1.31 ± 0.28

1.99 ± 1.64

724

Effect of ginger on [3H]GR65630 binding

The extent of expression of 5-HT3A receptors in HEK293 5-HT3A/apoaequorin cells and of 5-HT3AB receptors in transiently transfected cells was determined by saturation binding on membranes with the 5-HT3 receptor antagonist [3H]GR65630. The binding curves revealed Bmax values of 23.29 ± 2.44 pmol mg−1 protein and 1.63 ± 0.27 pmol mg−1 protein, respectively. Calculated Kd values of the radioligand of 0.08 ± 0.01 and 0.13 ± 0.04 nmol L−1 were similar to those determined previously.[21] For competition experiments, the twofold Kd concentration of 0.2 nmol L−1 [3H]GR65630 was applied. The expression of heteromeric 5-HT3AB receptors in transiently transfected HEK293 cells was examined by competition binding with the discriminating agonist 5-HT. The resulting inhibition curves revealed a significantly decreased pKi (6.54 ± 0.06; n = 5; P < 0.001) and a reduced Hill slope (nH) (−1.19 ± 0.03; P < 0.05) for 5-HT on membranes of cells transiently expressing 5-HT3A/B subunits compared to the pKi (7.06 ± 0.05; n = 4) and nH (−2.78 ± 0.31) determined in the stable 5-HT3A receptor-expressing cell line; this confirms the expression of heteromeric 5-HT3AB receptors.

The ginger CO2 extract did not displace the radioligand from its binding site up to a concentration of 0.5 mg mL−1, confirming ginger's non-competitive mode of action on human 5-HT3A and 5-HT3AB receptors. In contrast, the competitive antagonist ondansetron concentration-dependently displaced the radioligand from 5-HT3A and 5-HT3AB receptors with pIC50 values of 8.70 ± 0.05 (n = 4) and 8.60 ± 0.02 (n = 3), respectively. The calculated pKi (Ki) was about 9.12 (0.75 nmol L−1) and thus comparable to values determined previously.[25, 29]

Influence of a preincubation on ginger's inhibition of 5-HT-induced Ca2+ influx through 5-HT3 receptors

To further elucidate the mode of action of ginger, especially with regard to the accessibility of its binding position on the receptor, we measured the increase in [Ca2+]i, induced by 3 μmol L−1 (5-HT3A) or 100 μmol L−1 (5-HT3AB) 5-HT, upon application of either 6-gingerol or the CO2 extract using different application modes. Antagonist concentrations that caused more than 50% inhibition of 5-HT3A and 5-HT3AB receptors, i.e., 20 μmol L−1 6-gingerol and 3 μg mL−1 CO2 extract, respectively (see Fig. 1), were either exclusively co-applied with 5-HT or 1 or 10 min before and during stimulation with 5-HT. For both compounds, a preincubation time of 1 min led to a further significant reduction in the 5-HT-induced Ca2+ influx through both receptors compared with the effect after exclusive co-application of the antagonist with 5-HT. The prolongation of the preincubation with 6-gingerol to 10 min yielded a further significant reduction in Ca2+ influx whereas with the CO2 extract only a slight but non-significant further reduction was found (Fig. 3).

image

Figure 3. Magnitude of inhibition of 5-HT-induced Ca2+ influx through 5-HT3A (A) and 5-HT3AB receptors (B) by 6-gingerol and the CO2 ginger extract according to their mode of application. Concentrations of the compounds causing >50% inhibition of 5-HT3 receptors (see Fig. 1A) were applied at different modes: exclusive application together with 5-HT (co-application) or application 1 min/10 min before and during stimulation with 5-HT (preincubation 1 min/10 min). Bars represent percentages of the response to 5-HT in the absence of the antagonist (means ± SEM of six experiments). Significant differences compared to the co-application mode are shown (*P < 0.05, ***P < 0.001; repeated measures anova followed by Tukey's post-test). [Ca2+]i, cytosolic Ca2+ concentration.

Download figure to PowerPoint

Impact of 6-gingerol on the activation of 5-HT3 receptors in the submucous plexus of human colon tissue

Spritz application of the 5-HT3 receptor agonist 2-Me-5-HT (100 μmol L−1 in the pipette, final concentration at the ganglion previously calculated to be 10 μmol L−1[30]) for 400 ms evoked an increase in [Ca2+]i of 25.0 ± 4.2% ΔF/F (3 patients, 4 ganglia, 10 neurons) in human submucous plexus preparations. Responses were stable in that, Ca2+ transients evoked by a second and third application of 2-Me-5-HT were not significantly different from that after the first application (n = 10, P = 0.2) (Fig. 4B). We used the same protocol to test the influence of 6-gingerol on 5-HT3 receptor agonist-evoked increases in [Ca2+]i (3 patients, 3 ganglia, 9 neurons). After two control applications of 2-Me-5-HT which evoked comparable responses of ~20% ΔF/F, the recording chamber was perfused with 6-gingerol (50 μmol L−1) for 30 min. The following spritz application of 2-Me-5-HT induced a significantly smaller increase in [Ca2+]i of 8.8 ± 1.4% ΔF/F (Fig. 4B).

image

Figure 4. 6-Gingerol reduces increase in cytosolic Ca2+ evoked by the 5-HT3 receptor agonist 2-methyl-5-HT (2-Me-5-HT) in the human submucous plexus. Increases in cytosolic Ca2+ ([Ca2+]i) were measured in Fluo-4-AM-stained whole-mount preparations of the human submucous plexus. (A) Raw data traces of Ca2+ transients evoked by pressure pulse application of 2-Me-5-HT (400 ms) before (black trace) and during 6-gingerol (50 μmol L−1) perfusion (gray trace) in the same neuron. (B) Reproducible Ca2+ responses after three control applications of 2-Me-5-HT in ten neurons (open triangles). Responses after two control applications (open circles) and after application of 2-Me-5-HT 30 min following the addition of 50 μmol L−1 6-gingerol (closed circle) in a separate set of nine neurons. (C) Reproducible Ca2+ responses after three subsequent electrical stimulations of interganglionic nerve strands in nine neurons (open triangles). Responses after two control stimulations (open circles) and after electrical stimulation 30 min following the addition of 50 μmol L−1 6-gingerol (closed circle) in a separate set of ten neurons. Increases in [Ca2+]i are shown as means ±SEM. Significant differences compared to the control responses in the same set of neurons are shown (*P < 0.05, repeated measures anova followed by Tukey's post-test).

Download figure to PowerPoint

To rule out a non-specific decrease in neuronal excitability caused by 6-gingerol, we studied its effect on synaptically evoked Ca2+ responses. Three subsequent fiber tract stimulations (3 patients, 3 ganglia, 9 neurons) led to comparable increases in [Ca2+]i (n = 9, P = 0.26), demonstrating that responses were stable (Fig. 4C). The same protocol was used to study a possible influence of 6-gingerol on neuronal excitability (3 patients, 3 ganglia, 10 neurons). The perfusion of the tissue preparations with 6-gingerol (50 μmol L−1) after the second nerve stimulation did not cause changes in synaptically evoked Ca2+ transients (n = 10, P = 0.13) (Fig. 4C).

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information

In this study, we show for the first time that ginger extracts and isolated pungent constituents inhibit the activation of human 5-HT3 receptors. In particular, recombinant 5-HT3A and 5-HT3AB receptors expressed in HEK293 cells and native receptors residing on enteric neurons from human gut preparations were analyzed.

For the determination of the mode of action, we performed radioligand binding and an aequorin luminescence-based Ca2+ assay on HEK293 cells heterologously expressing human 5-HT3A or 5-HT3AB receptors. The suitability of the aequorin assay for characterizing 5-HT3 receptors has been demonstrated previously.[21, 23] Ginger dry extracts concentration-dependently inhibited the 5-HT-induced activation of 5-HT3A and 5-HT3AB receptors and the determined inhibition potencies increased in correlation with their content of pungent constituents and volatile oil. We showed that 1–2 μg mL−1 CO2 extract, the most lipophilic of the tested extracts containing 5% pungent compounds and 7.1% volatile oil, inhibited 5-HT3 receptor activation by 50%. With regard to the clinically demonstrated antiemetic and antinauseous efficacy of ginger,[4, 31] the relevance of 5-HT3 receptor inhibition in vivo, caused by oral application of the recommended maximum daily dose of 0.5–2 g ginger drug powder,[2] seems likely. Our further analyses revealed that the pungent arylalkanes 6-gingerol and 6-shogaol are involved in this inhibitory effect. Consequently, we assume that the findings of earlier studies in rodent models, showing that the pungent constituents[10, 11, 32] and to a lesser extent the volatile oil[33] contribute to ginger's 5-HT3 receptor inhibition, also apply to the herein studied human 5-HT3 receptors. The determined inhibition potencies of 6-gingerol and 6-shogaol in the low micromolar range were comparable to those found at murine 5-HT3 receptors.[11] With regard to the fact that the CO2 ginger dry extract contains 2.9% 6-gingerol (own HPLC analysis), we can calculate that ~0.1–0.2 μmol L−1 of 6-gingerol (Mw 294 g mol−1) should inhibit the 5-HT response. The much higher IC50 of ~10 μmol L−1 determined here indicates that further components are involved in 5-HT3 receptor blockade including 8- and 10-gingerol, and possibly the volatile oil.

Our concentration-response experiments of 5-HT in the presence of the ginger extract or 6-gingerol revealed a reduced maximum effect, but an unaltered EC50 of the endogenous agonist on 5-HT3A and 5-HT3AB receptors. Thus, we demonstrated that ginger acts by non-competitively blocking 5-HT3 receptor activation as was previously postulated for murine 5-HT3 receptors.[11] The non-competitive action was confirmed by our radioligand binding experiments; here the ginger extract was not able to displace the 5-HT3 receptor antagonist [3H]GR65630 from its binding site on 5-HT3A and 5-HT3AB receptors. Due to the binding of the active principle of ginger to a site different from the orthosteric binding site of competitive antagonists such as ondansetron, the combination of a ‘setron’ with ginger would exert an additive inhibitory effect on 5-HT3 receptors. This is in line with the clinical trials involving cancer patients receiving chemotherapy and standard treatment against CINV including a ‘setron’, which demonstrated a decreased incidence of nausea in patients that additionally received ginger.[6, 31]

Our experiments hint to the fact that at least one of the 5-HT3 receptor binding sites of the active principle(s) of ginger is not easily accessible. We showed that a preincubation of the receptor with 6-gingerol or the ginger extract significantly enhanced the inhibitory effect. Thus, a binding position in the lumen of the ion pore resulting in an open channel block is unlikely as open channel blockers only affect the activated receptor via entrance through the open pore. We showed for 6-gingerol that an equilibration time of 10 min with both receptor subtypes prior to the application of the agonist further increases its inhibitory effect compared with an equilibration time of only 1 min before agonist application. Consequently, the slow component of the blocking action of 6-gingerol may involve drug access via the hydrophobic membrane which would be consistent with the lipophilicity of the pungent compound. A binding site in the transmembrane domain or in its proximity as was also proposed for other compounds such as cannabinoids[34] is conceivable. Increasing the equilibration time from 1 to 10 min did, however, not significantly enhance the inhibitory effect of the ginger extract. This finding demonstrates that 6-gingerol is only one part of the active principle and that other ingredients of the ginger extract add to the faster component of blocking.

We did not find differential effects of ginger or its pungent constituents on 5-HT3A and 5-HT3AB receptors. Nonetheless, a differential action on native receptors of diverse composition and incorporating further 5-HT3 receptor subunits such as 5-HT3C, D, and E cannot be ruled out.

The efficacy of ginger to alleviate symptoms of functional GI disorders such as IBS can be explained at least partially by the herein detected inhibitory action of 6-gingerol on enteric neurons of the human submucous plexus. This inhibitory effect was shown to be mediated by the blockade of 5-HT3 receptors expressed in this tissue; a non-specific neurotoxic effect of 6-gingerol that would lead to a reduced neuronal excitability of enteric neurons was ruled out. Our results are of functional relevance as 6-gingerol was demonstrated to be rapidly absorbed into the systemic circulation after oral administration in rats; even higher concentrations were observed in intestinal tissues.[35] In healthy volunteers, conjugate metabolites of 6-gingerol were detected in the blood after oral intake of ginger, confirming the resorption of the compound in the GI tract.[36] These data strongly suggest that the submucous plexus layer is exposed to 6-gingerol after ingestion of ginger. However, free 6-gingerol was not detectable in the plasma indicating a high first-pass effect of the compound.[36]

The activation of 5-HT3 receptors is involved in secretional processes and gut motility in the guinea pig gut[37, 38], but their role in the human gut is not yet fully understood. Nevertheless, the impact of 5-HT3 receptors on peristaltic reflexes in the human gut is corroborated by the fact that 5-HT3 receptor antagonists alter GI motility.[39] The expression of functional 5-HT3 receptors in the human submucous plexus, likely consisting of 5-HT3A and 5-HT3B subunits, has been described previously.[22] Recent expression studies further revealed the co-expression of 5-HT3A and 5-HT3D subunits in these enteric neurons.[40] Thus, other 5-HT3 receptor subtypes besides 5-HT3A or 5-HT3AB might be involved in the responses of compounds acting in the GI tract including ginger. The human 5-HT3 receptor system is more complex than that of rodents, in which only 5-HT3A and 5-HT3B subunits seem to exist.[16] Thus, the molecular make-up of 5-HT3 receptors in different body regions including the gut has to be sorted out in future studies to allow a more precise prediction of the action of drugs from in vitro experiments.

In conclusion, we showed that ginger and its pungent constituents non-competitively inhibit the activation of human 5-HT3 receptors. We demonstrated that this effect may be relevant in the human gut as the main pungent constituent 6-gingerol inhibited the activation of enteric neurons in tissue preparations of the human submucous plexus. Our findings may help to further explain the widespread use of ginger in GI disorders. The study may encourage the initiation of longer randomized trials assessing the safety of ginger extracts in pregnant women. It may further encourage the stronger use of ginger extracts that contain high amounts of pungent arylalkanes and volatile oil to alleviate symptoms of IBS and nausea in cancer patients receiving chemotherapy.

Acknowledgment

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information

We thank Prof. Heinz Bönisch from the University of Bonn for kindly providing us with the stable HEK293 5-HT3A receptor cell line.

Funding

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information

This work was supported by the German Cancer Aid (108710 and 109226 to BN) and the Prof. Karl und Gerhard Schiller-Stiftung (BN).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information
  • 1
    Ali BH, Blunden G, Tanira MO, Nemmar A. Some phytochemical, pharmacological and toxicological properties of ginger (Zingiber officinale Roscoe): a review of recent research. Food Chem Toxicol 2008; 46: 40920.
  • 2
    Committee on Herbal Medicinal Products (HMPC): community herbal monograph on Zingiber officinale Roscoe, rhizoma. 04 June 2012; EMA/HMPC/749154/2010.
  • 3
    Bryer E. A literature review of the effectiveness of ginger in alleviating mild-to-moderate nausea and vomiting of pregnancy. J Midwifery Womens Health 2005; 50: e13.
  • 4
    Chaiyakunapruk N, Kitikannakorn N, Nathisuwan S, Leeprakobboon K, Leelasettagool C. The efficacy of ginger for the prevention of postoperative nausea and vomiting: a meta-analysis. Am J Obstet Gynecol 2006; 194: 959.
  • 5
    Pillai AK, Sharma KK, Gupta YK, Bakhshi S. Anti-emetic effect of ginger powder versus placebo as an add-on therapy in children and young adults receiving high emetogenic chemotherapy. Pediatr Blood Cancer 2011; 56: 2348.
  • 6
    Ryan JL, Heckler C, Dakhil SR et al. Ginger for chemotherapy-related nausea in cancer patients: a URCC CCOP randomized, double-blind, placebo-controlled clinical trial of 644 cancer patients. J Clin Oncol 2009; 27: 15s. (suppl; abstr 9511).
  • 7
    Ding M, Leach M, Bradley H. The effectiveness and safety of ginger for pregnancy-induced nausea and vomiting: a systematic review. Women Birth 2012; Aug 27 [Epub ahead of print].
  • 8
    Hu ML, Rayner CK, Wu KL et al. Effect of ginger on gastric motility and symptoms of functional dyspepsia. World J Gastroenterol 2011; 17: 10510.
  • 9
    Wu KL, Rayner CK, Chuah SK et al. Effects of ginger on gastric emptying and motility in healthy humans. Eur J Gastroenterol Hepatol 2008; 20: 43640.
  • 10
    Abdel-Aziz H, Nahrstedt A, Petereit F, Windeck T, Ploch M, Verspohl EJ. 5-HT3 receptor blocking activity of arylalkanes isolated from the rhizome of Zingiber officinale. Planta Med 2005; 71: 60916.
  • 11
    Abdel-Aziz H, Windeck T, Ploch M, Verspohl EJ. 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; 530: 13643.
  • 12
    Walstab J, Rappold G, Niesler B. 5-HT(3) receptors: role in disease and target of drugs. Pharmacol Ther 2010; 128: 14669.
  • 13
    Qian QH, Yue W, Wang YX, Yang ZH, Liu ZT, Chen WH. Gingerol inhibits cisplatin-induced vomiting by down regulating 5-hydroxytryptamine, dopamine and substance P expression in minks. Arch Pharm Res 2009; 32: 56573.
  • 14
    Kawai T, Kinoshita K, Koyama K, Takahashi K. Anti-emetic principles of Magnolia obovata bark and Zingiber officinale rhizome. Planta Med 1994; 60: 1720.
  • 15
    Yamahara J, Rong HQ, Naitoh Y, Kitani T, Fujimura H. Inhibition of cytotoxic drug-induced vomiting in suncus by a ginger constituent. J Ethnopharmacol 1989; 27: 3535.
  • 16
    Karnovsky AM, Gotow LF, McKinley DD et al. A cluster of novel serotonin receptor 3-like genes on human chromosome 3. Gene 2003; 319: 13748.
  • 17
    Niesler B, Frank B, Kapeller J, Rappold GA. Cloning, physical mapping and expression analysis of the human 5-HT3 serotonin receptor-like genes HTR3C, HTR3D and HTR3E. Gene 2003; 310: 10111.
  • 18
    Boess FG, Beroukhim R, Martin IL. Ultrastructure of the 5-hydroxytryptamine3 receptor. J Neurochem 1995; 64: 14015.
  • 19
    Davies PA, Pistis M, Hanna MC et al. The 5-HT3B subunit is a major determinant of serotonin-receptor function. Nature 1999; 397: 35963.
  • 20
    Maricq AV, Peterson AS, Brake AJ, Myers RM, Julius D. Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel. Science 1991; 254: 4327.
  • 21
    Walstab J, Hammer C, Bönisch H, Rappold G, Niesler B. Naturally occurring variants in the HTR3B gene significantly alter properties of human heteromeric 5-hydroxytryptamine-3A/B receptors. Pharmacogenet Genomics 2008; 18: 793802.
  • 22
    Michel K, Zeller F, Langer R et al. Serotonin excites neurons in the human submucous plexus via 5-HT3 receptors. Gastroenterology 2005; 128: 131726.
  • 23
    Walstab J, Combrink S, Brüss M, Göthert M, Niesler B, Bönisch H. Aequorin luminescence-based assay for 5-hydroxytryptamine (serotonin) type 3 receptor characterization. Anal Biochem 2007; 368: 18592.
  • 24
    Barann M, Meder W, Dorner Z et al. Recombinant human 5-HT3A receptors in outside-out patches of HEK 293 cells: basic properties and barbiturate effects. Naunyn Schmiedebergs Arch Pharmacol 2000; 362: 25565.
  • 25
    Combrink S, Kostanian A, Walstab J et al. Characterization of the naturally occurring Arg344His variant of the human 5-HT 3A receptor. Pharmacol Rep 2009; 61: 78597.
  • 26
    Niesler B, Walstab J, Combrink S et al. Characterization of the novel human serotonin receptor subunits 5-HT3C,5-HT3D, and 5-HT3E. Mol Pharmacol 2007; 72: 817.
  • 27
    Michel K, Michaelis M, Mazzuoli G, Mueller K, Vanden Berghe P, Schemann M. Fast calcium and voltage-sensitive dye imaging in enteric neurones reveal calcium peaks associated with single action potential discharge. J Physiol 2011; 589: 59417.
  • 28
    Cheng Y, Prusoff WH. Relationship between the inhibition constant (K1) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem Pharmacol 1973; 22: 3099108.
  • 29
    Brady CA, Stanford IM, Ali I et al. Pharmacological comparison of human homomeric 5-HT3A receptors versus heteromeric 5-HT3A/3B receptors. Neuropharmacology 2001; 41: 2824.
  • 30
    Breunig E, Michel K, Zeller F, Seidl S, Weyhern CW, Schemann M. Histamine excites neurones in the human submucous plexus through activation of H1, H2, H3 and H4 receptors. J Physiol 2007; 583: 73142.
  • 31
    Panahi Y, Saadat A, Sahebkar A, Hashemian F, Taghikhani M, Abolhasani E. Effect of ginger on acute and delayed chemotherapy-induced nausea and vomiting: a pilot, randomized, open-label clinical trial. Integr Cancer Ther 2012; 11: 20411.
  • 32
    Pertz HH, Lehmann J, Roth-Ehrang R, Elz S. Effects of ginger constituents on the gastrointestinal tract: role of cholinergic M3 and serotonergic 5-HT3 and 5-HT4 receptors. Planta Med 2011; 77: 9738.
  • 33
    Riyazi A, Hensel A, Bauer K, Geissler N, Schaaf S, Verspohl EJ. The effect of the volatile oil from ginger rhizomes (Zingiber officinale), its fractions and isolated compounds on the 5-HT3 receptor complex and the serotoninergic system of the rat ileum. Planta Med 2007; 73: 35562.
  • 34
    Barann M, Molderings G, Brüss M, Bönisch H, Urban BW, Göthert M. Direct inhibition by cannabinoids of human 5-HT3A receptors: probable involvement of an allosteric modulatory site. Br J Pharmacol 2002; 137: 58996.
  • 35
    Jiang SZ, Wang NS, Mi SQ. Plasma pharmacokinetics and tissue distribution of [6]-gingerol in rats. Biopharm Drug Dispos 2008; 29: 52937.
  • 36
    Yu Y, Zick S, Li X, Zou P, Wright B, Sun D. Examination of the pharmacokinetics of active ingredients of ginger in humans. Aaps J 2011; 13: 41726.
  • 37
    Johnson PJ, Bornstein JC, Furness JB, Woollard DJ, Orrman-Rossiter SL. Characterization of 5-hydroxytryptamine receptors mediating mucosal secretion in guinea-pig ileum. Br J Pharmacol 1994; 111: 12404.
  • 38
    Fox A, Morton IK. An examination of the 5-HT3 receptor mediating contraction and evoked [3H]-acetylcholine release in the guinea-pig ileum. Br J Pharmacol 1990; 101: 5538.
  • 39
    Gershon MD, Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 2007; 132: 397414.
  • 40
    Kapeller J, Möller D, Lasitschka F et al. Serotonin receptor diversity in the human colon: expression of serotonin type 3 receptor subunits 5-HT3C, 5-HT3D, and 5-HT3E. J Comp Neurol 2011; 519: 42032.

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgment
  8. Funding
  9. Disclosures
  10. References
  11. Supporting Information
FilenameFormatSizeDescription
nmo12107-sup-0001-SupportingInformation.docWord document107KFigure S1. Concentration-dependent 5-HT-induced Ca2+ influx through 5-HT3A and 5-HT3AB receptors.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.