Coptisine, a protoberberine alkaloid, relaxes mouse airway smooth muscle via blockade of VDLCCs and NSCCs

Abstract Background/Aims: Recently, effective and purified ingredients of traditional Chinese medicine (TCM) were extracted to play crucial roles in the treatment of pulmonary diseases. Our previous research focused on TCM drug screening aimed at abnormal airway muscle contraction during respiratory diseases. Coptisine, an effective ingredient extracted from bitter herbs has shown a series of antioxidant, antibacterial, cardioprotective and neuroprotective pharmacological properties. In the current study, we questioned whether coptisine could also participate in asthma treatment through relaxing abnormal contracted mouse airway smooth muscle (ASM). The present study aimed to characterize the relaxant effects of coptisine on mouse ASM and uncover the underlying molecular mechanisms. Methods: To investigate the role of coptisine on pre-contracted mouse ASM, a series of biological techniques, including force measurement and patch-clamp experiments were employed. Results: Coptisine was found to inhibit high K+ or acetylcholine chloride (ACh)-induced pre-contracted mouse tracheal rings in a dose-dependent manner. Further research demonstrated that the coptisine-induced mouse ASM relaxation was mediated by alteration of calcium mobilization via voltage-dependent L-type Ca2+ channels (VDLCCs) and non-selective cation channels (NSCCs). Conclusion: Our data showed that mouse ASM could be relaxed by coptisine via altering the intracellular Ca2+ concentration through blocking VDLCCs and NSCCs, which suggested that this pharmacological active constituent might be classified as a potential new drug for the treatment of abnormal airway muscle contraction.


Introduction
Pulmonary diseases are a series of debilitating, life-threatening respiratory illnesses that have become severe worldwide public health problems and financial burdens [1] . According to a recent study, asthma and chronic obstructive pulmonary disease (COPD) together threatened 300 million people worldwide [2]. Airway inflammation, excessive cell matrix proliferation, especially the abnormal contraction of airway smooth muscle (ASM) are the main symptoms of pulmonary diseases [3][4][5]. The development of effective medications for pulmonary diseases and improving quality of life without side effects are urgently needed.
As well known, traditional Chinese medicine (TCM) especially a large number of herbal formulations play important roles in pulmonary diseases treatment [6]. However, rigorous Western methodologies should be employed to isolate the effective substances from the complex mixture of chemicals for scientific validation and further clinical application. Our previous studies have revealed that quite a few effective ingredients or extracts of TCM could relax abnormal smooth muscle contraction in pulmonary diseases [7,8].

Statistical analysis
All data were expressed as the means + − standard deviation (SD). For all analyses, the evaluations were performed with Student's t test using Origin 8.0 software (OriginLab, Northampton, MA, U.S.A.). P<0.05 was regarded as statistically significant.

Coptisine relaxed high K + -induced pre-contraction in a dose-dependent manner
Previous research have demonstrated that high K + -induced smooth muscle contraction was mainly due to the depolarization of cell membrane, opening of VDLCC and influx of extracellular Ca 2+ , sequentially [20,21]. To explore the potential relaxant characteristic of coptisine, the dose-response curved was first calculated under presence of high K + . Our previous study has shown that high K + could contract mouse tracheal ring gradually [16] and 80 mM K + was applied to pre-contract ASM in this experiment. As shown in Figure 1A, the pre-contraction induced by high K + (80 mM) was completely inhibited by coptisine (0.01-1000 μM) in a dose-dependent manner. According to the dose-contraction curve exhibited in Figure 1B, the maximal relaxation was calculated as 82.24 + − 4.94%. The half-maximal inhibition (IC 50 ) was 45.76 + − 8.54 μM. The IC 75 was 194.69 + − 12.38 μM (n=7/7 mice). Comparing with the relaxant characteristic of coptisine on pre-contracted mouse tracheal ring, 316 μM coptisine had no effect on resting mouse tracheal ring ( Figure 1C). As shown in Figure 1D, 10 μM nifedipine, a selective blocker of VDLCCs [22], has a similar inhibitory on high K + -induced steady state contraction in mouse tracheal rings (n=6/6 mice), which confirmed that the contraction was induced via the opening of VDLCCs. These results indicated that coptisine inhibited high K + -induced pre-contraction in a dose-dependent manner. Furthermore, the relaxant effect of nifedipine suggested that VDLCCs participated in high K + -induced contraction and also might be involved in coptisine-induced relaxation.

Coptisine blocked high K + -evoked Ca 2+ influx
The previous study on rat aortic ring indicated that coptisine could attenuate calcium release from the sarcoplasmic reticulum [23]. To further characterize the relaxant mechanism of coptisine shown in Figure 1, the following experiments were designed to explore whether calcium was also involved in coptisine-induced relaxation on mouse tracheal rings. In the Ca 2+ -free solution, high K + -induced contraction did not occur (Figure 2A), proving that calcium influx was necessary for VDLCC-induced contraction. Following Ca 2+ restoration, the contraction that immediately evoked by high K + was almost completely inhibited by 200 μM coptisine ( Figure 2A) (n=7/7 mice). Meanwhile, high K + failed to induce a pre-contraction under Ca 2+ -free conditions in the presence of 200 μM coptisine, and even after Ca 2+ restoration, the ASM contraction was not obvious (P>0.05) ( Figure 2B) (n=6/6 mice). It was supposed that VDLCCs has been blocked in coptisine pre-treated mouse tracheal ring, then high K + could not evoke extracellular Ca 2+ influx via blocked VDLCCs. As shown in Figure 2C, coptisine's solvent, DMSO was applied as a negative control (n=6/6 mice). These data suggested that blocking high K + -induced Ca 2+ influx was involved in the relaxant effects of coptisine.

Coptisine blocked VDLCC currents
To further confirm the participation of VDLCCs in the ability of coptisine to relax ASM, particular VDLCC currents were measured using the whole-cell patch-clamp technique. As shown in Figure 3A, the currents were recorded with voltage steps from −70 to +40 mV. As a positive control, the currents were eliminated by the specific blocker nifedipine, indicating that VDLCC currents were recorded ( Figure 3B, top). The currents were then inhibited by 200 μM coptisine ( Figure 3B, bottom), which indicated that the effect of coptisine on VDLCC currents is similar to nifedipine. As a type of voltage-dependent channel, the current-voltage (I-V) curve of VDLCC was calculated to examine the voltage-dependent property (n=5/5 mice, Figure 3C). The averaged current of the VDLCCs in the absence and presence of nifedipine or coptisine are shown in Figure 3D. It was suggested that coptisine could inhibit VDLCC currents.
Taken together, these results demonstrated that coptisine could relax high K + -induced ASM contraction by blocking VDLCCs and then decreasing intracellular Ca 2+ .

Coptisine relaxed ACh-induced pre-contraction in a dose-dependent manner
The relaxation of smooth muscle is a complicated electrophysiological process and the collaboration of various ion channels is indispensable [24,25]. Thus, we wonder whether any other ion channels besides VDLCCs might also participate in coptisine-induced relaxation. ACh, a known muscarinic receptor agonist, which could evoke ASM contraction through both VDLCCs and NSCCs [26,27] was employed to pre-contract ASM. In our previous study, ACh could stimulate mouse tracheal contraction gradually [16] and the concentration of ACh was determined to be 100 μM to pretreat ASM in this experiment. Coptisine (0.0316-316 μM) was able to completely relax 100 μM ACh-induced pre-contraction in a dose-dependent manner ( Figure 4A). Then the dose-response curve was calculated as shown in Figure 4B, The maximal relaxation was 100.00 + − 2.01%, and the IC 50 was 4.02 + − 2.07 μM. IC 75 was 10.51 + − 4.00 μM (n=7/7 mice). As shown in Figure 5, to isolate and identify the role of NSCCs, VDLCCs were excluded with the specific blocker nifedipine before or after ACh addition. In the presence of ACh, induced pre-contraction was partially reversed by 10 μM nifedipine (the average relaxation percentage was 36.29 + − 4.13%) ( Figure 5A). Subsequently, 100 μM coptisine almost completely relaxed the remaining tension ( Figure 5A, n=7/7 mice). In the presence of nifedipine, ACh-induced pre-contraction was also relaxed by 100 μM coptisine ( Figure 5B, n=6/6 mice). These experiments indicated that besides VDLCCs, NSCCs could also be evoked by ACh and thus might involve in coptisine-induced relaxation.

Coptisine blocked ACh-evoked Ca 2+ influx
Besides VDLCC, another possible source of Ca 2+ entry during smooth muscle contraction is the NSCC permeable to external calcium ions [28]. To further extend the role of calcium in coptisine-evoked relaxation, Ca 2+ entry through NSCCs was studied under ACh-induced pre-contraction. As shown in Figure 6A, in the presence of nifedipine (10 μM) under Ca 2+ -free condition, ACh induced a sharp contraction, indicating that ACh could transiently release Ca 2+ from intracellular Ca 2+ storage. Subsequently, the restoration of 2 mM Ca 2+ triggered a steady contraction, which was completely eliminated by 10 μM coptisine (n=7/7 mice). However, in the presence of 10 μM coptisine, ACh failed to raise intracellular calcium. Even with the restoration of 2 mM Ca 2+ , sustained contraction also did not occur ( Figure 6B). DMSO, the solution of coptisine was used as a negative control ( Figure 6C). In order to isolate NSCCs evoked by ACh, VDLCC was blocked by nifedipine. As shown in Figure 6D, in Ca 2+ -free medium, intracellular Ca 2+ transiently released after the addition of 100 μM ACh. With the restoration of 2 mM Ca 2+ , a sustained contraction was induced and subsequently reversed by 10 μM coptisine. These results indicated that Ca 2+ influx played an important role in ACh-induced contraction. These data indicated that calcium mobilization via NSCC was both involved in ACh-induced contraction and coptisine-induced relaxation.
To further identify the specific components of NSCCs involved in coptisine-blocked Ca 2+ influx, TRPC3 inhibitor Pyr3 [29,30] and gadolinium, which is a blocker of TRPC1, 3, 5, 6 and 7 [31] were employed sequentially. As shown in Figure 6E,F, in the presence of nifedipine, ACh induced a transient contraction under Ca 2+ -free conditions, which indicated that intracellular Ca 2+ was transiently released after the addition of ACh. With the restoration of 2 mM Ca 2+ , a sustained contraction was induced by ACh and was partially reduced by 30 μM Pyr3 (the average relaxation percentage was 25.07 + − 6.94%), 30 μM gadolinium (the average relaxation percentage was 20.64 + − 4.87%), and finally almost completely eliminated by 10 μM coptisine (the average relaxation percentage was 43.26 + − 8.90%). Taken together, a critical molecular candidate for NSCC blocked by coptisine seems to be TRPC channels.

Coptisine blocked NSCC currents
To further test whether coptisine has some effects on NSCC currents, whole-cell patch-clamp was employed to measure ACh-induced NSCC currents with or without coptisine (Figure 7). The NSCC current showed a ramp from −80 to +60 mV ( Figure 7A). To block currents from VDLCCs, Cl − channels and K + channels, nifedipine, NA and TEA were applied respectively. Thus, the residual current was ACh-induced NSCC current. As shown in Figure 7B, NSCC currents could be completely blocked by 10 μM coptisine (n=6/6 mice). Three representative ramp current traces at time points a, b and c are shown in Figure 7C. Taken together, these results indicate that coptisine can inhibit ACh-induced NSCC currents.

Na + /Ca 2+ exchangers did not involve in coptisine-induced relaxation
Besides VDLCC S and NSCC S , Na + /Ca 2+ exchangers (NCX) also play a critical role in the intake of Ca 2+ by cells in smooth muscle [32]. To explore the role of NCX in Ca 2+ influx blocked by coptisine, Li-PSS without sodium was applied instead of PSS to evoke Ca 2+ influx via NCX. As shown in the Figure 8B, it turned out that under the condition of Li-PSS, ACh-induced a prominent contraction with an obviously higher baseline compared with PSS condition (Figure 8A), which indicated that NCX might be switched to a 'Ca 2+ influx/Na + outflow' mode to increase intracellular Ca 2+ . Following addition of 10 μM coptisine, the contraction was potently attenuated to the base line ( Figure 8A-C). Furthermore, KB-R7943, a specific NCX blocker [33,34] was applied to identify the role of NCX in coptisine-induced relaxation. As shown in Figure 8D, KB-R7943 could reverse the ACh-induced contraction (the average relaxation percentage was 54.16 + − 3.62%) in Li-PSS solution. However, in the presence of 100 μM coptisine,  KB-R7943 could continuously relax ACh-induced contraction ever lower than the baseline. The data indicated that NCX was probably not blocked in coptisine-induced relaxation.

Discussion
Recently, various studies focused on TCM for a safer and milder treatment of pulmonary disease. A plurality of the investigated natural herbal extracts or single compound exert their relaxant effect on precontracted airway muscle [7,8]. Coptisine is a natural compound which displays a broad range of pharmacological actions [11][12][13][14][15]. Previous studies have evaluated the vasorelaxant effects of coptisine on isolated rat aortic rings [23], which shed light on the possible mechanism of coptisine's action on abnormal contracted mouse tracheal rings. In current study, we investigated the relaxant effects of coptisine in agonist-triggered ASM contraction and the underlying mechanisms. We first examined whether coptisine could relax abnormal contracted mouse tracheal rings. It turned out that coptisine could inhibit contractile effect of mouse tracheal rings induced by high K + or ACh in a concentration-dependent way. VDLCCs and NSCCs are two categories of voltage-dependent and receptor-operated channel candidates which are critical for intracellular and extracellular calcium mobilization in ASM contraction [26,27,35,36]. To investigate the relaxant effect of coptisine, calcium mobilization in ASM were further calculated. The results indicated that calcium oscillation played an important role in coptisine-induced relaxation by blocking VDLCCs and NSCCs, especially TR-PCs. However, it should be noted that except for VDLCC and NSCC, NCX did not play a role in coptisine-induced relaxation [32]. To further identified the roles of VDLCCs and NSCCs in coptisine-induced relaxation, VDLCC or NSCC currents were measured. Coptisine was found to eliminate both VDLCC and NSCC currents.
In conclusion, coptisine is an important ingredient of TCM which has shown various medical properties. Through current study, its pharmacological characteristic has been extended to relaxant effect on mouse ASM and the underlying molecular mechanism has been clarified. Further fundamental studies and clinical trials are required to explore more therapeutic properties of TCM including coptisine and explain the underlying molecular mechanism with modern scientific language.

Conclusion
In summary, our research indicated that pretreatment of mouse ASM with high K + or ACh could be relaxed by coptisine through blocking VDLCCs and NSCCs then inhibiting calcium influx. Our research work provided evidence that coptisine might have potential therapeutic value for the treatment of pulmonary disease associated with abnormal ASM contraction.