Curcumin Activates Vagal Afferent Neurons Through the Modulation of Ion Channels via α7 nAChR

Abstract

Curcumin, a plant polyphenol, has been previously reported to attenuate collagen-induced arthritis in rats by modulating the function of the cholinergic system, but the underlying mechanism remains to be identified. In this study, primary nodose ganglion (NG) neurons were prepared from the adult rats and the electrophysiological recording was performed using the whole-cell patch clamp technique. Curcumin was shown to reduce total potassium currents and A-type currents, without significant effect on the activation or inactivation of potassium channels. Moreover, curcumin selectively enhanced tetrodotoxin-sensitive (TTX-S) sodium channel currents. These effects could be abolished by methyllycaconitine citrate (specific antagonist of α7 nACh receptor). Interestingly, curcumin did not modulate TTX-resistant (TTX-R) or calcium channels. These results suggest that curcumin increases the excitability of NG neurons by decreasing potassium currents and increasing TTX-S sodium currents via α7 nicotinic acetylcholine receptor (nAchR).

The vagus nerve is the 10th cranial nerve, descending from the brain stem and arriving to most visceral organs. As a major branch of the parasympathetic nervous system, vagus nerve is responsible for the parasympathetic control of the heart, lungs, and digestive tract. ¹ The modulatory role of the vagus nerve is related to multiple chronic diseases, such as diabetes, chronic obstructive pulmonary disease, and rheumatoid arthritis. ² It is well known that the modulation of membrane ion channels is an important mechanism for neurotransmitters, including acetylcholine (Ach) released from the vagus nerve endings, to regulate organ activity. The generation of neuronal action potential is regulated by the ion influx/efflux through the cell membrane. The voltage-gated ion channel is activated by changing the voltage difference across the membrane. ³ The activation curve reflects the speed and difficulty of channel opening. The study of channel dynamics is important for explaining the functional characteristics of ion channels.

Curcumin has been used historically as a spice and medicinal herb in India and China. ⁴ Considerable evidence suggests that curcumin possesses diverse bioactivities. Our previous studies demonstrated that curcumin could attenuate collagen-induced arthritis in rats through the “gut-brain axis” via modulating the function of the cholinergic system. Moreover, electrophysiological studies on vagal nodose ganglion (NG) neurons have shown that curcumin could change action potentials and affect the neuronal excitability. ⁵ In the present study, we investigate the underlying mechanism by which curcumin regulates the ion channels and subsequently increases the neuronal excitability.

Materials and Methods

Reagents

Curcumin (purity: ≥98%) was obtained from Zelang Pharmaceutical Technology Co., Ltd. (Nanjing, China). Hank’s balanced saline solution (HBSS), neurobasal A, B-27, heat-inactivated horse serum, fetal calf serum, streptomycin, and l-glutamax were purchased from Invitrogen (Thermo Fisher Scientific, Waltham, United States). Papain was purchased from Worthington Biochemical (NJ, United States); collagenase, 12 mm poly-d-lysine, and laminin were purchased from Sigma (St. Louis, United States). PNU282987 and methyllycaconitine citrate (MLA) were purchased from Tocris Bioscience (Bristol, United Kingdom).

Isolation and Culture of Vagus NG Neurons

Primary NG neurons were extracted from female rats as described previously with some modifications. ⁶ Briefly, rats were anesthetized with overdose of isoflurane. The NG of the vagus nerve was carefully removed and washed by HBSS. Then, the NG was incubated on an incubator shaker (New Brunswick Scientific, NJ, United States) for 30 minutes at a speed of 135 rpm at 37°C in HBSS which contains papain (15 U/mL) and collagenase (5  ×  10⁻⁴ g/mL). After that, the NG was washed 3 times with HBSS and placed in culture medium containing Neurobasal A, 2% B-27, 2% heat-inactivated horse serum, 2% fetal calf serum, 0.2 mM l-glutamax, 100 U/mL penicillin, and 100 µg/mL streptomycin. The fragments were triturated and dissociated mechanically with a pipette. The dispersed neurons were seeded onto 12 mm poly-d-lysine and laminin-coated coverslips at a density of 3000 cells per well. The neurons were cultured at 37°C in a humidified atmosphere containing 5% CO₂.

Electrophysiological Recording

Standard whole-cell recordings were performed at room temperature using an EPC 10 amplifier and PatchMaster software (HEKA Elektronik, Lambrecht, Germany) as described previously. ⁶ Electrode resistances ranging between 3 and 6 MΩ with series resistances of 6 to 15 MΩ were compensated to the maximal current amplitude. The bath solution was Tyrode’s solution containing 140 mM NaCl, 5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM 4-(2-hydroxyerhyl)piperazine-1-erhanesulfonic acid (HEPES), and 5.6 mM glucose, and pH was adjusted to 7.36 with NaOH. The intracellular solution contained 140 mM KMeSO₄, 2 mM MgCl₂, 1 mM ethylene glycol tetraacetic acid (EGTA), 10 mM HEPES, 3 mM Na₂ATP, and 0.3 mM Na₂GTP, and pH was adjusted to 7.4 with KOH (osmolality ~292 mmol/kg). Action potentials were recorded in the current-clamp mode at a membrane potential around −65 mV. A current injection was used to evoke 2 to 3 action potentials and remained constant throughout the recording. Only 1 neuron was recorded from 1 coverslip.

Electrophysiological Data Analysis

Offline data analysis was processed using PatchMaster and Origin 8.1 software.

Statistical Analysis

Statistical analysis was performed with Origin 8.1 or SPSS statistical software (SPSS, Chicago, IL, United States), and the data were expressed as means ± standard errors of the means. The mean differences between 2 groups were compared by T test; the mean differences between multiple groups were compared by one-way analysis of variance (ANOVA) followed by post hoc Tukey’s test. P  <  0.05 was considered to represent a significant difference.

Results

Curcumin Negatively Modulated Potassium Channels in NG Neurons

The potassium currents were evoked by test pulses ranging from −60 to +80 mV for 500 ms from the holding potential of −80 mV before and after 5 minutes application of curcumin (3, 10, and 30 µM). We found that curcumin (10 and 30 µM) significantly decreased the potassium currents (Figure 1(a)), suggesting that curcumin has a negative regulation effect on potassium channels. The previous studies suggested that the voltage at +40 mV could trigger a large potassium current, and curcumin (10 and 30 µM) significantly decreased the total outward potassium current. ⁷ There are many subtypes of potassium channels, and A-type potassium current plays an important role in pain and inflammatory diseases as previously reported. ⁸ To investigate the effect of curcumin on potassium channels in NG neurons, the membrane potential was held at −80 mV, and the total potassium current was recorded by a voltage stimulation of +40 mV. Then, we recorded a continuously delayed potassium current after a −10 mV prepulse was applied, and the A-type potassium current was obtained by offline treatment. Curcumin (10 and 30 µM) treatment for 5 minutes significantly inhibited the total potassium currents and the A-type currents (Figure 1(b)), but had no significant effect on the sustained potassium currents. Moreover, curcumin had no significant effect on steady-state activation and inactivation potassium currents in NG neurons (Figure 1(c)). The data suggested that curcumin may increase the neural neuronal excitability by inhibiting the potassium channels and increasing the action potential.

Figure 1

Effect of curcumin on potassium channels in rat nodose ganglion neurons. (a) Current-voltage (I-V) relationships of curcumin-induced potassium currents. (b) Mean densities of total potassium currents, A-type currents, and sustained currents were recorded. (c) Effect of curcumin on steady-state activation and inactivation potassium currents in nodose ganglion neurons. Curcumin-induced currents were shown in red. Data were shown as means ± standard errors of the means for each group (n = 4-20). ^* P < 0.05 and ^** P < 0.01 vs Control group.

Curcumin Selectively Enhanced Tetrodotoxin-Sensitive (TTX-S) Sodium Currents in NG Neurons

The sodium channels in the NG neurons are divided into tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) channels. ⁹ First, the membrane potential was maintained at −80 mV, and curcumin (10 µM) was perfused for 5 minutes. The TTX-S sodium current (from −50 to +60 mV) was recorded and the current-voltage curve was plotted. As shown in Figure 2(a), curcumin significantly increased TTX-S sodium currents from −30 to +10 mV, and the peak current was at −20 mV. To determine whether curcumin promoted the TTX-S sodium channel in a concentration-dependent manner, the membrane potential was maintained at −80 mV, and the inward sodium current was recorded before and 5 minutes after curcumin (3, 10, and 30 µM) application at −20 mV. As shown in Figure 2(b), curcumin (3, 10, and 30 µM) increased the influx of sodium ions. To investigate the voltage dependence of curcumin on TTX-S sodium channel, the voltage was recorded at −90 or −55 mV, and a voltage pulse of −20 mV was given. As shown in Figure 2(c), curcumin (10 µM) increased the sodium current influx at both −90 and −55 mV. At −55 mV, the effect was stronger, suggesting that curcumin has a voltage-dependent regulation of the TTX-S sodium channel. As shown in Figure 2(d), curcumin had no significant effect on the activation or inactivation of the TTX-S sodium channel.

Figure 2

Effect of curcumin on sodium channels in rat nodose ganglion neurons. (a) Effect of curcumin on tetrodotoxin-sensitive voltage-gated sodium currents in nodose ganglion neurons. (b) Effect of curcumin on tetrodotoxin-sensitive voltage-gated sodium currents in a dose-independent manner. (c) Effect of curcumin on tetrodotoxin-sensitive voltage-gated sodium currents in a voltage-dependent manner. (d) Effect of curcumin on steady-state activation and inactivation tetrodotoxin-sensitive voltage-gated sodium currents. (e) Effect of curcumin on tetrodotoxin-resistant voltage-gated gated sodium currents. (f) Effect of curcumin on tetrodotoxin-resistant voltage-gated sodium currents in a voltage-independent manner. (g) Effect of curcumin on steady-state activation and inactivation on tetrodotoxin-resistant voltage-gated sodium currents. Curcumin-induced currents were shown in red. Data were shown as means ± standard errors of the means for each group (n = 5-17). ^* P < 0.05 vs Control group.

Second, the neurons were maintained at −80 mV; TTX-R sodium currents (from −50 to +60 mV) were recorded before and 5 minutes after curcumin application. As shown in Figure 2(e), curcumin (10 µM) did not affect the TTX-R sodium current. To investigate whether curcumin voltage dependently modulated TTX-R sodium channel, a voltage pulse of −20 mV was recorded at a voltage of −90 or 55 mV, and curcumin (10 µM) was administered for 5 minutes. As shown in Figure 2(f), curcumin (10 µM) failed to increase the sodium current peak at −90 or −55 mV. Furthermore, as shown in Figure 2(g), curcumin had no significant effect on the activation and inactivation of the TTX-R sodium channel, indicating that curcumin had no significant effect on the opening and closing properties of this type of sodium channel.

Curcumin Did not Affect Calcium Channels in NG Neurons

To investigate the modulation of curcumin on calcium channels in NG, neurons were maintained at a voltage of −70 mV, and a step pulse voltage (from −30 to +60 mV) was continuously administered. As shown in Figure 3(a), curcumin (10 µM) treatment for 5 minutes did not affect the peak calcium current at different voltages, suggesting that curcumin had no significant effect on calcium channels. The membrane potential was maintained at −70 mV, and the effect of curcumin (3, 10, and 30 µM) on calcium channels was recorded at +10 mV. As shown in Figure 3(b), curcumin (3, 10, and 30 µM) failed to modulate the calcium currents. Moreover, the voltage was maintained at −100 and −70 mV, respectively. The effects of curcumin on inward calcium current at +10 mV were investigated. As shown in Figure 3(c), curcumin had no effect on the activation and inactivation of calcium channels, suggesting that curcumin made no contribution to the calcium channel modulation.

Figure 3

Effect of curcumin on calcium channels in rat nodose ganglion neurons (a) Effect of curcumin on inward calcium currents in nodose ganglion neurons. (b) Effect of curcumin on calcium currents in dose- and voltage-independent manner. (c) Effect of curcumin on steady-state activation and inactivation calcium currents. Curcumin-induced currents were shown in red. Data were shown as means ± standard errors of the means for each group (n = 4-6).

The Modulation of Curcumin on Potassium and Sodium Channels Was Mediated by α7 nAChR

The increase of action potential may be caused by the decrease in outward potassium current. Anti-inflammatory action initiated by vagal nerve activation was followed by the release of Ach and the activation of the α7 nAChR. ¹⁰ To determine whether the effects of curcumin on potassium and sodium currents were mediated by nAChR, MLA (a central and peripheral neuronal nAChR antagonist) was used. The application of curcumin (10 µM) for 5 minutes significantly inhibited the outward potassium currents and the A-type potassium currents. Methyllycaconitine citrate (10 nM) alone had no significant effect on the potassium channel, but it diminished the potassium currents inhibition induced by curcumin (Figure 4(a) and (b)). The application of curcumin (10 µM) for 5 minutes significantly enhanced TTX-S sodium currents, but this effect was diminished by MLA (10 nM). Perfusion of the α7 nAChR-specific agonist PNU282987 (30 µM) for 5 minutes also significantly inhibited potassium channel currents and increased sodium channel currents (Figure 4(c)), which in turn affects the generation of action potentials and increases the neuronal excitability.

Figure 4

Effect of curcumin on potassium currents and tetrodotoxin-sensitive voltage-gated sodium currents in the nodose ganglion neurons dependent on α7 nAChR. Curcumin (10 µM), methyllycaconitine citrate (a selective α7 nAChR antagonist, 10 nM), and PNU282987 (a selective α7 nAChR agonist, 30 µM) were used. Each treatment was recorded for 5 minutes. (a) Total potassium currents change in nodose ganglion neurons. (b) A-type potassium currents. The membrane potential was held at −80 mV and sodium currents were activated by a voltage step to −20 mV. (c) Sodium currents change. Data were shown as means ± standard errors of the means for each group (n = 5-9). ^# P < 0.05 vs Control group; ^$$ P < 0.01 vs Curcumin group.

Discussion

The opening speed, openness, and internal and external diffusion of potassium, sodium, and calcium ions will cause changes in the potential difference inside and outside the cell membrane, which in turn affects the generation and characteristics of action potentials and participates in the pathogenesis of disease or drug intervention. ¹¹ The ion channel current-voltage curve (I-V curve) is an important parameter reflecting the dynamic properties of the ion channel, which can reflect the activation process of the channel, the threshold potential, the reversal potential, and the rectification characteristics.

The present study examined the modulation of curcumin on 3 types of ion channels in NG neurons and explored the possible mechanisms by which curcumin affected action potentials. The NG neurons have many types of ion channels, and each channel has multiple subtypes. We examined the current-voltage curve, concentration-dependent, voltage-dependent, and steady-state activation of curcumin on 3 major ion channels. The results showed that curcumin could inhibit the opening of potassium channels, promote the opening of TTX-S channels, promote the production of neuronal action potentials, and increase the excitability of vagus nerves. These effects were nearly abolished by α7 nAChR antagonist in NG neurons. Curcumin showed no significant effect on TTX-R sodium channel and calcium channel. These findings suggested that curcumin increased neuron excitability by inhibiting the potassium channels and promoting the opening of TTX-S sodium channel, which was mediated by α7 nAChR in NG neurons.