Ginger and Its Pungent Constituents Non-Competitively Inhibit Serotonin Currents on Visceral Afferent Neurons

Nodose neuron dissociation

Visceral afferent neurons were dissociated from nodose ganglia from two to three week old Sprague-Dawley rats (Orient Bio Inc., Seongnam, Korea) of either gender as previously described [11]. All animal procedures were conducted with the approval of the institutional Animal Care and Use Committee in Kyung Hee University, Seoul, ROK, study protocol #KHUASP(SE)-13-002 (approval date: April 2, 2013). Briefly, the rats were deeply anesthetized (Zolazepam 0.3 ml/kg, Virbac Laboratories., Carros, France), and the vagus nerve from the jugular foramen was exposed for a length of 0.5 cm caudally. After the vagus nerve was carefully freed from surrounding tissue, the attached ganglion was immediately placed into a Petri dish containing chilled (4℃) artificial cerebrospinal fluid (ACSF) solution, which was composed of the following (in mM): 125 NaCl, 3 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 10 glucose, and 2 CaCl₂, and bubbled with 95% O₂-5% CO₂. After, gently removing excess connective tissue, the bulb of the nodose ganglion was incubated for 28~40 min at 31℃ in ACSF solution containing type II-S trypsin (4~5 mg/ml at 1,310 units/mg; Sigma-Aldrich., St. Louis, MO). Following enzyme treatment, the ganglion was placed in enzyme-free ACSF for 1~3 hours at room temperature. The ganglia were then mechanically dispersed using fire-polished Pasteur pipettes in a glass bottom perfusion chamber filled with standard external recording solution containing (in mM): 150 NaCl, 5 KCl, 1 MgCl₂, 2 CaCl₂, 10 HEPES, and 10 glucose (pH was adjusted to 7.4 with Tris-base).

Electrical measurements

For voltage-clamp recording, isolated neurons were visualized using a phase contrast microscope (IX70; Olympus, Tokyo, Japan) with a 40× objective and 10× ocular lens. Recordings utilized an EPC 9 (HEKA, Lambrecht, Germany) and pClamp 9 software (Molecular Devices, Sunnyvale, California). Electrical recordings used a modified perforated patch method using a nystatin concentration of 450 µg/ml [12]. The ionic composition of the internal patch-pipette solution for nystatin perforated patch recordings was (mM): 50 KCl, 100 K gluconate, and 10 HEPES. The pH of this solution was adjusted to 7.2 with Tris-OH. The nystatin-internal solution filled patch-pipettes has 5~7 MΩ resistance. Junction potential for these internal and standard external solutions was 11.9 mV and compensated throughout experiments. The access resistance, measured at approximately 15 min after formation of a gigaohm seal, was 8.5±1.2 MΩ. Neurons were voltage clamped to -50 mV and currents were sampled at 10 KHz with 3 KHz filtering and saved to computer. All experiments were conducted at room temperature (21~22℃).

Drugs & ginger extraction

Serotonin, CPBG (1-(3-Chlorophenyl)biguanide hydrochloride), zingerone, [6]-gingerol, and [6]-shogaol were purchased from Sigma-Aldrich (St. Louis, MO). All three pungent constituents of ginger were dissolved in DMSO and then diluted to the standard external recording solution. The final concentration of DMSO was less than 0.3%, a concentration which had no effect on the cells. 5-HT₃ receptor selective antagonist ondansetron (1,2,3,9-Tetrahydro-9-methyl-3-[(2-methyl-1H-imidazol-1-yl)methyl]-4H-carbazol-4-one hydrochloride) was purchased from Tocris Cookson (Ballwin, MO). Ginger extract were prepared from freeze-dried Zingiberis officinalis roots. Finely grounded 10 g ginger was extracted with 400 mL water. The extract was filtered, freeze-dried and kept -20℃ until use. Each 10 g of dried ginger yielded 1.61 g dried power. All of the drugs were applied via a rapid application Y-tube microperfusion system that provided complete solution changes surrounding the recorded neurons within 0.1 seconds [13].

Data analysis

Data were analyzed off-line using Clampfit 9 software. All data are represented as mean±standard error of the mean (SEM). For statistical comparisons, one-way ANOVA with post-hoc multiple comparisons (Bonferroni/Dunn's correction, StatView, SAS Institute Inc., Cary, NC) was used. Differences were considered statistically significant for p values<0.05. The dose response curves for concentration-inhibition relationships were constructed according to the following equation (1) using a least-square fitting routine:

Where I is the current amplitude normalized to the control response, C is the corresponding ginger constituents or ondansetron concentrations. IC₅₀ and nH denote the half-maximum inhibition concentration and the Hill coefficient, respectively. The continuous theoretical curves for the concentration response relationships were constructed according to a Hill equation (2) using a least-square fitting routine as described elsewhere [14] :

Where I is the current amplitude normalized to the control response and C is the corresponding 5-HT concentration. EC₅₀ and nH denote the half-maximum concentration and Hill slope, respectively.

Inhibition of 5-HT-induced current by ginger extract

To characterize the base serotonin (5-HT) responses, whole-cell current recordings were made at a holding potential (V_H) of -50 mV. Serotonin evoked inward currents in 29% (n=9/31) of assayed nodose neurons in a concentration dependent manner (0.001 to 1 mM). Three micromole 5-HT was used for subsequent control responses, at which approximately 25% of the maximal current amplitude was obtained. As shown in Fig. 2A, 4 min repeated application of the 3 µM 5-HT produced constant 5-HT responses. Co-application of 5-HT₃ receptor selective antiemetic drug ondansetron (10 nM) reversibly blocked the 5-HT-evoked currents by an average of 99.5±0.4% (p=0.0006, n=7). Serotonin type 3 receptor selective agonist, CPBG (1 µM), mimicked the 5-HT effect by inducing inward current responses and ondansetron effectively inhibited CPBG responses (n=5, Fig. 2B). These results demonstrate that 5-HT-evoked current responses by activating 5-HT₃ receptors. In the continuous experiments, inhibitory effects of the ginger extract on the 3 µM 5-HT-induced control responses were tested. Before the simultaneous application with 5-HT, an external solution containing 0.2 or 2 mg/ml ginger extract was pretreated for 2 min (Fig. 2C). The 5-HT current was suppressed by 49.5±7.2% and 98.2±1.2% by 0.2 and 2 mg/ml ginger extract, respectively. As shown in Fig. 2C traces, ginger extract alone evoked 37.5±17.1 pA (n=8) and 38.8±8.9 pA (n=6) currents at 0.2 mg/ml and 2 mg/ml, respectively. Those responses are not inhibited by ondansetron (p>0.05, n=6).

In summary, the above result shows that ginger extract and ondansetron have similar modes of action to inhibit 5-HT-evoked response in nodose ganglia neurons.

Inhibition of 5-HT current by ginger constituents

To determine which of the pungent ginger constituents, [6]-gingerol, [6]-shogaol and zingerone, block 5-HT-evoked responses in nodose neurons we tested them independently. The neurons were perfused with an external solution containing each compound from 30 seconds prior until simultaneous application with 5-HT. All constituents concentration-dependently inhibited 5-HT-evoked currents (Fig. 3). Where control 5-HT responses were significantly (p<0.05) attenuated by ≥10 µM gingerol, ≥10 µM shogaol, and ≥300 µM zingerone. For comparison, ondansetron was tested in same conditions and the 5-HT responses were significantly (p<0.0001) inhibited by ≥10^-4 µM concentrations. All ginger constituents reversibly inhibited the control response in a concentration-dependent manner. The control responses were recovered by 4 to 5 min washing with ACSF. The order of inhibitory potency for these compounds were [6]-shogaol>[6]-gingerol>zingerone. The IC₅₀ of [6]-shogaol and [6]-gingerol were 128 and 39 times lower than zingerone, respectively (Table 1). Thus, [6]-shogaol and [6]-gingerol are more effective than zingerone.

Pungent ginger constituents act as non-competitive antagonists

The effects of three ginger constituents were examined at different 5-HT concentrations. Fig. 4 shows the concentration-response curves of 5-HT in the absence and presence of [6]-shogaol (10 µM), [6]-gingerol (30 µM), or zingerone (1 mM). The EC₅₀ of the 5-HT was 7.81±0.63 µM. In the presences of [6]-shogaol, [6]-gingerol, or zingerone, respective values for EC₅₀ were 7.2±1.32, 7.9±1.7 and 6.59±2.24 µM. The EC₅₀ value for 5-HT was not changed in the presence of three tested constituents (p>0.05, n=4^-7). In contrast that the maximal response induced by 1 mM 5-HT was significantly decreased (p<0.026, n=4) to 89±3.4, 85±3.6, and 79±2.7% of control values in the presence of [6]-shogaol (10 µM), [6]-gingerol (30 µM), and zingerone (1 mM), respectively. These results support a non-competitive interaction by ginger constituents at the 5-HT site.