Nuciferine Relaxes Tracheal Rings via the Blockade of VDLCC and NSCC Channels

Abstract

This study aimed to clarify how nuciferine, a primary aporphine alkaloid found in lotus leaf extract, causes relaxation in contracted tracheal rings. In conditions without calcium and with 2 mM calcium, nuciferine did not affect the resting muscle tone of tracheal rings. However, nuciferine relaxed tracheal rings from mice that were contracted with high potassium in a manner that depended on the dose. It also inhibited both the entry of calcium into the cells and the electrical currents of voltage-dependent L-type calcium channels that were triggered by high potassium. Similarly, nuciferine also inhibited contractions in mouse tracheal rings that were induced by acetylcholine, and this inhibition also depended on the dose. Furthermore, nuciferine blocked both the influx of calcium into the cells caused by acetylcholine and the whole-cell currents of nonselective cation channels. Taken together, these findings suggest that the relaxation in tracheal rings caused by nuciferine mainly occurred because it prevented calcium from entering the cells by blocking voltage-dependent L-type calcium channels and/or nonselective cation channels. These results indicate that nuciferine might be useful in treating respiratory diseases that involve abnormal contraction of airway smooth muscles and/or bronchospasm.

Introduction

Asthma, a chronic inflammatory condition, is the most common disease that obstructs the airways globally, affecting an estimated 300 million people. It is characterized by symptoms that vary and recur, as well as airflow obstruction and bronchospasm. Common symptoms of asthma include wheezing, coughing, and shortness of breath. Excessive contraction of airway smooth muscles narrows the airway passage, which in turn restricts gas exchange and impacts the daily lives of individuals with asthma. To improve the quality of life for these patients, many medications have been used to treat asthma. Traditional treatments, including inhaled corticosteroids, β2 agonists, and leukotriene receptor antagonists, can significantly improve asthma control, but they are not effective for all patients. Therefore, there is a pressing need to develop new, effective, and safe bronchodilators for asthma therapy.

The leaf of Nelumbo nucifera Gaertn., a plant in the Nelumbonaceae family, is a traditional Chinese medicine that has been widely used to treat diarrhea, fever, and bleeding. Lotus leaf extract contains three main types of alkaloids: O-nornuciferine, N-nornuciferine, and nuciferine. Nuciferine, an alkaloid containing an aromatic ring, has shown beneficial pharmacological effects, such as widening blood vessels, stimulating insulin secretion, improving nonalcoholic fatty liver disease and atherosclerosis, as well as having anti-HIV, antioxidant, anti-obesity, antihyperlipidemic, and antitumor effects. Recently, we reported that lotus leaf extract significantly relaxed tracheal rings contracted by high potassium or acetylcholine. However, the active components of the lotus leaf extract and their mechanisms of relaxation are still unclear.

In this study, we focused on understanding the underlying mechanisms of nuciferine, a major component of lotus leaf extract, that can induce relaxation in tracheal rings. We found that nuciferine relaxed tracheal rings that were pre-contracted with high potassium by inhibiting the influx of calcium and blocking voltage-dependent L-type calcium channels. In tracheal rings contracted by acetylcholine, nuciferine also inhibited nonselective cation channels.

Results

To determine if nuciferine affects the tone of airway smooth muscles, we first examined its effects on tracheal rings. As shown, contraction was induced in a mouse tracheal ring by high potassium. Once the contraction reached a stable level, nuciferine (at concentrations from 0.1 to 300 µM) was added cumulatively to the solution surrounding the tissue, resulting in relaxation that increased with the dose of nuciferine. However, nuciferine had no effect on the resting tone of tracheal rings. Meanwhile, the contraction caused by high potassium was completely blocked by 10 µM nifedipine, a selective inhibitor of voltage-dependent L-type calcium channels, suggesting that the contractions induced by high potassium were due to the activation of these channels. These data indicate that nuciferine can relax tracheal rings contracted by high potassium by inhibiting voltage-dependent L-type calcium channels.

Calcium plays a central role in regulating the contraction of airway smooth muscles. Therefore, to understand how nuciferine causes relaxation in tracheal rings, we investigated its effect on calcium influx. Under conditions without calcium, high potassium did not cause contractions in tracheal rings. When 2 mM calcium was restored in the solution, a large and sustained contraction occurred and was completely inhibited by the addition of 100 µM nuciferine. Similarly, nuciferine did not cause a contraction under calcium-free conditions. Following the addition of high potassium, no contractions were observed. However, restoring 2 mM calcium in the solution resulted in a small contraction in the tracheal rings.

These results suggest that nuciferine can inhibit the influx of extracellular calcium into airway smooth muscles. Voltage-dependent L-type calcium channels are crucial in regulating the tone of airway smooth muscles. Therefore, to explore the mechanism of nuciferine-induced relaxation in tracheal rings, we studied the effects of nuciferine on the currents of these channels in airway smooth muscle cells. The results demonstrate that the recorded currents were completely blocked by either nifedipine (10 µM), a selective inhibitor of voltage-dependent L-type calcium channels, or nuciferine (31.6 µM).

These data suggest that nuciferine can block voltage-dependent L-type calcium channels. Acetylcholine caused contraction in tracheal rings by activating muscarinic receptors and their downstream signaling pathways, which are different from the pathway activated by high potassium. To further understand the mechanisms of nuciferine-induced relaxation, we examined the effects of nuciferine on tracheal rings contracted by acetylcholine. The results indicate that nuciferine relaxed these acetylcholine-contracted tracheal rings in a manner that depended on the dose. Meanwhile, the contraction in tracheal rings induced by acetylcholine that was not sensitive to nifedipine was also mostly relaxed by nuciferine. These data indicate that the relaxation induced by nuciferine can be mediated by a signaling pathway other than those related to voltage-dependent L-type calcium channels. To identify the nifedipine-insensitive signaling pathway involved in nuciferine-induced relaxation in tracheal rings, we investigated the effects of nuciferine on calcium influx. Under calcium-free conditions, nuciferine had no effect on the resting tone of tracheal rings. Restoring 2 mM calcium induced a small transient contraction.

Following the addition of 100 µM acetylcholine, a large and sustained contraction occurred and was mostly inhibited by the addition of 300 µM nuciferine. These data demonstrate that nuciferine can inhibit acetylcholine-induced calcium influx. To further explore the mechanism by which nuciferine relaxes acetylcholine-contracted tracheal rings, we then studied the effects of nuciferine on nonselective cation channels using a patch-clamping technique. The results indicate that nuciferine significantly inhibited the currents of nonselective cation channels activated by acetylcholine in single mouse airway smooth muscle cells.

These data suggest that nuciferine can block nonselective cation channel currents. To further confirm the inhibitory effect of nuciferine on calcium influx, we investigated its effects on the plateau phase of intracellular calcium concentration. The results show that 100 µM nuciferine significantly inhibited the increase in the plateau phase of intracellular calcium concentration induced by 80 mM potassium. Meanwhile, in single airway smooth muscle cells pretreated with 100 µM nuciferine, the sustained increase in intracellular calcium was significantly inhibited. Similarly, the increase in the plateau phase of intracellular calcium concentration induced by acetylcholine was inhibited by nuciferine at 300 µM, but not at 100 µM.

Discussion

Our findings demonstrated that nuciferine could significantly relax the contraction of tracheal rings induced by either high potassium or acetylcholine. Nuciferine-induced relaxation in tracheal rings contracted by high potassium occurred due to the inhibition of calcium influx and the electrical currents of voltage-dependent L-type calcium channels. Meanwhile, in tracheal rings contracted by acetylcholine, nuciferine inhibited both calcium influx and the currents of nonselective cation channels. Our previous study showed that lotus leaf extract significantly relaxed tracheal rings contracted by high potassium or acetylcholine.

However, the specific bioactive components in the lotus leaf extract and the underlying mechanisms of this relaxation were not known. In this study, we aimed to identify the functional ingredients and the associated mechanisms responsible for tracheal ring relaxation. Nuciferine is a major component of the total alkaloids extracted from lotus leaves. Therefore, we used nuciferine instead of lotus leaf extract and investigated the mechanism underlying its effects. We first studied the relaxing effects of nuciferine on tracheal rings contracted by high potassium. The results show that nuciferine relaxed the contracted tracheal rings in a manner dependent on the dose and had no effect on resting tracheal rings.

This result aligns with our previous findings using lotus leaf extract, suggesting that nuciferine present in lotus leaf extract plays an important role in relaxing airway smooth muscles. Calcium is crucial in regulating the tone of airway smooth muscles. In the present study, under conditions without calcium, nuciferine did not affect the tone in resting tracheal rings. In contrast, the contraction induced by calcium influx in tracheal rings was mostly inhibited by nuciferine, suggesting that nuciferine blocks calcium influx.

Moreover, the contraction induced by high potassium after restoring 2 mM calcium was significantly inhibited by nuciferine, also suggesting that calcium influx could be inhibited by nuciferine. Similarly, nuciferine also showed an inhibitory effect on calcium influx in tracheal rings contracted by acetylcholine. These findings are supported by a recent study reporting that nuciferine had a vasorelaxation effect by inhibiting extracellular calcium influx in rat mesenteric arteries precontracted by high potassium. These data demonstrated that nuciferine can inhibit calcium influx, which leads to a decrease in the intracellular calcium concentration and subsequently the relaxation of contracted tracheal rings.

As is well known, high potassium induces depolarization in airway smooth muscle cells, which in turn leads to the activation of voltage-dependent L-type calcium channels, resulting in extracellular calcium influx and increased intracellular calcium concentration. The increased intracellular calcium then triggers a contraction, which was completely inhibited by nifedipine, a specific inhibitor of voltage-dependent L-type calcium channels. Therefore, nuciferine-induced relaxation in tracheal rings contracted by high potassium occurred due to the inhibition of voltage-dependent L-type calcium channels.

This hypothesis was confirmed by patch-clamping experiments in single airway smooth muscle cells and calcium imaging. The blockade of voltage-dependent L-type calcium channels by nuciferine then prevented extracellular calcium influx, which resulted in a decrease in the plateau phase of intracellular calcium concentration and relaxation. Together, the results showed that nuciferine inhibited calcium influx through voltage-dependent L-type calcium channels, which resulted in the relaxation of contracted tracheal rings.

Acetylcholine, an agonist of muscarinic receptors, can activate both voltage-dependent L-type calcium channels and nonselective cation channels, which induce calcium influx and cause airway smooth muscle contraction. Our results demonstrated that nuciferine also relaxed airway smooth muscles contracted by acetylcholine in a dose-dependent manner, suggesting that nuciferine could also inhibit all nonselective cation channels except voltage-dependent L-type calcium channels. This hypothesis was supported by an experiment in which acetylcholine-induced contraction that was insensitive to nifedipine was significantly relaxed by nuciferine. Moreover, the results of patch clamping directly demonstrated that nuciferine could block nonselective cation channels.
Meanwhile, the inhibitory effects of nuciferine on intracellular calcium concentration induced by acetylcholine were confirmed by the results of calcium imaging. However, the concentration of nuciferine used to inhibit an increase in acetylcholine-induced intracellular calcium concentration was 300 µM, which was much higher than that used to inhibit a high potassium-induced increase in the plateau phase of intracellular calcium concentration. This difference may be due to the fact that the increase in the plateau phase of intracellular calcium concentration induced by high potassium and acetylcholine was activated by different signaling pathways.

The high potassium-induced increase in intracellular calcium occurred via activation of voltage-dependent L-type calcium channels, while acetylcholine induced the opening of both voltage-dependent L-type calcium channels and nonselective cation channels. In addition, nuciferine inhibited both voltage-dependent L-type calcium channels and nonselective cation channels, which reduced the increase in the plateau phase of intracellular calcium concentration and relaxed airway smooth muscles.

However, further investigations are needed to elucidate the effects of nuciferine on nonselective cation channels and other signaling pathways involved in airway smooth muscle contraction, such as calcium sensitization. Until now, no substances or plant extracts, with the exception of testosterone, have been identified as being able to block both channels in airway smooth muscle to induce relaxation. However, our data demonstrate that nuciferine can induce relaxation in contracted tracheal rings through the inactivation of voltage-dependent L-type calcium channels and nonselective cation channels. Therefore, nuciferine could be an excellent alternative for asthmatic patients with severe exacerbation who do not respond to conventional drugs used in the treatment of asthma.

Materials and Methods

Reagents and chemicals

Nuciferine (with a purity of 98% or greater based on HPLC) was purchased from Shanghai Yuanye Biotechnology Co. and dissolved in DMSO to create a stock solution. Acetylcholine (with a purity of 99% or greater), nifedipine (with a purity of 98% or greater), noradrenaline (with a purity of 98% or greater), and tetraethylammonium (with a purity of 98% or greater) were purchased from Sigma Chemical Co. Other chemicals were purchased from Sinopharm Chemical Reagent Co.

Experimental animals

Healthy male BALB/c mice, 8 weeks old and weighing 20–22 g, were purchased from the Hubei Provincial Center for Disease Control and Prevention. The animals were housed in a room with controlled temperature and humidity and had free access to water and standard laboratory food. All animal experiments were approved by the Animal Ethics Committee of South-Central University for Nationalities.

Solutions

The physiological saline solution comprised the following reagents in mM: 135 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 HEPES, and 10 glucose, with the pH adjusted to 7.4 using NaOH. To prepare the high potassium solution, the NaCl in the physiological saline solution was replaced with 75 mM KCl. All experiments were conducted at room temperature, between 22 and 25 °C.

Isometric tension in tracheal rings

Force measurement in tracheal rings was performed as previously described. Briefly, the mice were euthanized with an intraperitoneal injection of sodium pentobarbital (150 mg/kg, with a purity of 98% or greater from Sigma). The tracheae were dissected and placed in ice-cold physiological saline solution. After removing connective tissues, the tracheal rings were mounted vertically in a 10-mL organ bath with a 0.3-g preload. The tracheal rings were then perfused with an oxygenated physiological saline solution at 37 °C. After a 60-minute equilibration period, the tracheal rings were contracted with 100 µM acetylcholine, washed, and allowed to rest three times. After an additional 30-minute resting period, experiments were conducted.

Isolation of single airway smooth muscle cells

Single mouse airway smooth muscle cells were enzymatically isolated as previously described. Briefly, the mice were euthanized with an intraperitoneal injection of sodium pentobarbital (150 mg/kg). After removing the epithelium, the airway smooth muscle strips were isolated and immersed in airway smooth muscle isolation solution containing (in mM) 136 NaCl, 5.36 KCl, 0.44 KH2PO4, 4.16 NaHCO3, 0.34 Na2HPO4·12H2O, 10 glucose, and 20 HEPES (pH 7.1). The airway smooth muscle strips were minced and treated with airway smooth muscle isolation solution containing 3 mg/mL papain, 0.15 mg/mL dithioerythritol, and 1 mg/mL BSA at 35 °C for 22 minutes. Then, the strips were further digested in airway smooth muscle isolation solution containing 1 mg/mL collagenase H and 1 mg/mL BSA at 35 °C for 8 minutes. Finally, the muscle strips were triturated in airway smooth muscle isolation solution to yield single airway smooth muscle cells. These single cells were stored on ice and used within 4 hours.

Patch-clamping technique

The currents of voltage-dependent L-type calcium channels were recorded using barium as the charge carrier with an EPC-10 patch-clamp amplifier (HEKA) as previously reported. Briefly, the composition of the pipette solution was as follows (in mM): 130 CsCl, 4 MgCl2, 4 Mg-ATP, 10 HEPES, and 10 EGTA (pH 7.2). The composition of the bath solution was as follows (in mM): 107 NaCl, 1 MgCl2, 27.5 BaCl2, 11 glucose, 10 HEPES, and 10 tetraethylammonium (pH 7.4). Single airway smooth muscle cells were patched and held at −70 mV. The currents of voltage-dependent L-type calcium channels were recorded following depolarization for 500 ms from −70 to +40 mV in 10-mV increments every 1 second. The currents of nonselective cation channels were measured as previously described. Briefly, the acetylcholine-induced currents of nonselective cation channels were recorded with a ramp protocol using a whole-cell configuration. The holding potential was −60 mV. The ramp was performed over 500 ms from −80 to +60 mV. The values at −70 mV were used to represent the currents of nonselective cation channels. The composition of the pipette solution was as follows (in mM): 126 CsCl, 1.2 MgCl2, 1 CaCl2, 3 EGTA, and 10 HEPES (pH 7.2). The composition of the bath solution was as follows (in mM): 126 NaCl, 1.5 CaCl2, 10 HEPES, and 11 glucose (pH 7.2). To further isolate the currents of nonselective cation channels, 10 mM tetraethylammonium, 100 µM noradrenaline, and 10 µM nifedipine were added to block the currents of potassium, chloride, and voltage-dependent L-type calcium channels, respectively.

Calcium imaging

Intracellular calcium concentration was measured as previously described. Briefly, cells were loaded with fura-2/AM (2.5 µM) for 15 minutes and then perfused with physiological saline solution for 10 minutes. Paired 340/380 fluorescence images were acquired using a TILL Polychrome V monochromator (TILL), and ratios were calculated using the MetaFluor for Olympus software (TILL) to represent relative changes in intracellular calcium concentration.

Statistical analysis

The data are expressed as means ± standard deviation. Statistical analyses were performed using Student’s t-test or a one-way analysis of variance followed by Tukey’s multiple comparison tests, with a significance level of p < 0.05, using Origin 9.0 software (OriginLab). A p-value less than 0.05 was considered statistically significant.