Abstract
Nicotine, a major component of tobacco, is highly addictive and acts on nicotinic acetylcholine receptors (nAChRs) to stimulate reward-associated circuits in the brain. It is well known that nAChRs play critical roles in mediating nicotine reward and addiction. Current FDA-approved medications for smoking cessation are the antidepressant bupropion and the nicotinic partial agonist varenicline, yet both are limited by adverse side effects and moderate efficacy. Thus, development of more efficacious medications with fewer side effects for nicotine addiction and smoking cessation is urgently needed. l-Tetrahydropalmatine (l-THP) is an active ingredient of the Chinese medicinal herb Corydalis ambigua that possesses rich neuropharmacological actions on dopamine (DA) receptors in the mesocorticolimbic dopaminergic reward pathway. L-THP has been explored as anti-addiction treatments for drug abuse including nicotine. However, the targets and mechanisms of l-THP-caused anti-nicotine effects are largely unknown. In this study we address this question by elucidating the effects of l-THP on human neuronal nAChRs using patch-clamp recordings. Human neuronal α4β2-nAChRs were heterologously expressed in SH-EP1 human epithelial cells. Bath application of nicotine (0.1–100 μM) induced inward currents, co-application of l-THP (3 μM) inhibited nicotine-induced currents in the transfected cells. L-THP-caused inhibition was concentration-dependent (the EC50 values for inhibiting the peak and steady-state current were 18 and 2.1 μM, respectively) and non-competitive. Kinetic analysis of the whole-cell currents showed that l-THP slowed rising time and accelerated decay time constants. L-THP specifically modulated α4β2-nAChRs, as it did not affect α7-nAChRs or α1*-nAChRs (muscle type). Interestingly, two putative α4β2-nAChR isoforms, namely sazetidine A-activated, high-sensitive one (α42β23-nAChR) and cytisine-activated, low-sensitive one (α43β22-nAChR) were pharmacologically separated, and the low-sensitive one was more susceptible to l-THP inhibition than the high-sensitive one. In conclusion, we demonstrate that l-THP blocks neuronal α4β2-nAChR function, which may underlie its inhibition on nicotine addiction.
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Introduction
Cigarette smoking is a major public health problem, which has been identified as the leading cause of preventable death globally [1]. It has been reported that about one-quarter of the world’s population smokes [1]. Smoking is currently responsible for the death of one in ten adults worldwide, about five million deaths each year, and this number will increase to eight million by 2030 unless effective action is taken [1]. Many smokers have great difficulty quitting, and many other smokers are still becoming addicted. Nicotine, a major component of tobacco, is highly addictive. Nicotine acts on its primary molecular target, nicotine acetylcholine receptors (nAChRs), to stimulate reward-associated circuits in the brain. Accumulating lines of evidence demonstrate that nAChRs play critical roles in mediating nicotine reward, dependence, and addiction.
Tetrahydroprotoberberines (THPBs), a family of active alkaloid compounds isolated from the Chinese medicinal herb Corydalis ambigua and various species of Stephania, have been clinically used as effective analgesic and sedative agents since ancient China [2,3,4,5]. Mounting evidence from biochemical, immunohistochemical, behavioral, electrophysiological, and pharmacological studies suggest that THPBs mainly exert their neuropharmacological effects through dopamine (DA) receptors, with preferential affinity toward D1 and D2 receptors in the nigrostriatal and mesocorticolimbic DAergic pathways [2, 3, 6, 7]. However, different analogs of THPBs, classified according to the hydroxyl group in their structures, show distinct neuropharmacological profiles at DA receptors. The levo isomer of tetrahydropalmatine (l-THP) has been shown to be a major contributor to its therapeutic effects. It is the most extensively studied compound of the THPBs and has been used in clinical treatments of pain for more than 50 years in China [2, 3, 6, 8,9,10,11,12,13,14,15,16,17,18,19,20,21]. Considering that l-THP significantly modulates DA receptors, midbrain DA neuronal function, and DA-associated pathways/circuits, l-THP has been shown to exhibit a therapeutic potential on drug addiction [19]. For example, l-THP ameliorates morphine and heroin addiction in humans [22, 23], reduces amphetamine addicted behaviors [24, 25], decreases alcohol drinking [26], and attenuates cocaine self-administration and cocaine-induced reinstatement in rats [27,28,29]. More interestingly, l-THP reduces nicotine self-administration and blocks relapse, and its efficacy is similar to or even better than the current smoking cessation drugs, bupropion and varenicline [30]. However, the mechanisms of l-THP-induced anti-additive effects are still unclear. The targets and mechanisms of l-THP-induced reduction of nicotine reward and dependence behaviors are largely unknown. Based on the evidence that β2-containing nicotinic receptors (mostly α4β2-nAChRs) play a critical role in mediation of nicotine self-administration behavior [31], we hypothesize that l-THP antagonizes α4β2-nAChR function, which may underlie its reduction of nicotine addiction. Therefore, l-THP could be a candidate for development as a pharmacotherapy for treating nicotine dependence and helping smoking cessation.
Materials and methods
Expression of human neuronal nAChRs in SH-EP1 human epithelial cells
Heterologous expression of human α4β2-nAChRs was performed as previously described in detail [32,33,34,35]. Briefly, human nAChR α4 and β2 subunits, subcloned into pcDNA3.1-zeocin or -hygromycin vectors, respectively, were introduced [36, 37] into native nAChR-null SH-EP1 cells [38] to create the SH-EP1 cell line that expressed human α4β2-nAChRs. Cells were maintained as low passage number (1–26 from our frozen stocks) cultures in medium augmented with 0.5 mg/mL zeocin and 0.4 mg/mL hygromycin, and were passaged once weekly by splitting just-confluent cultures 1/10 to maintain cells in proliferative growth.
Patch-clamp whole-cell current recordings and data acquisition in SH-EP1 human epithelial cells
Conventional whole-cell current recordings were performed, combined with a fast drug application system allowing for both fast application and removal of drugs as previously described [32,33,34]. Briefly, transfected SH-EP1 cells were prepared in 35-mm culture dishes without poly(lysine) coating, then plated on the bottom of the dishes and later placed on the stage of an inverted microscope (Axiovert 200; Zeiss, Thornwood, NY, USA). SH-EP1 cells were continuously perfused with standard external solution containing (in mM): 120 NaCl, 3 KCl, 2 MgCl2, 2 CaCl2, 10 HEPES, and 25 glucose, while pH was adjusted to 7.4 with Tris-base [32, 39]. Patch pipettes (1.5 mm × 100 mm, Narishige, East Meadow, NY, USA), fashioned on a two-stage pipette puller (P-830, Narishige) with resistances of 3–5 MΩ, was filled with the “Tris electrode” solution containing (in mM): 110 Tris-phosphate dibasic, 28 Tris-base, 11 EGTA, 2 MgCl2, 0.5 CaCl2, and 4 Na-ATP, pH 7.3 [33]. Cells were perfused with standard external solution (2 mL/min). Glass microelectrodes between the pipette and extracellular solution were used to form tight seals (>2 GΩ) on the surface of the cells. Standard whole-cell current recording was initiated by suitable suction and then waiting 5–10 min to allow for exchange of the pipette solution with the cytosol. Thereafter, recorded cells were lifted up gently from the bottom of the culture dishes, which allows for improved solution exchange and more accurate evaluation of differences in the kinetics of agonist-induced whole-cell currents [32]. Before capacitance and resistance compensation, access resistance was measured and accepted for experiments if less than 20 MΩ. Whole-cell capacitance was minimized, and series resistance was compensated routinely to 80%. Recorded cells were voltage clamped at a holding potential of −40 mV, and inward currents induced by nicotine were measured (Axopatch 200B amplifier; Molecular Devices, Sunnyvale, CA, USA). Current signals were typically filtered at 2 kHz, acquired at 10 kHz, and displayed and digitized online (Digidata 1550A series A/D board; Molecular Devices). Data acquisition and analyses were performed using PCLAMP10.0 (Molecular Devices), and results were plotted using ORIGIN 5.0 (OriginLab Corp., North Hampton, MA, USA). All experiments were performed at room temperature (22 ± 1 °C).
Data acquisition and analyses were performed using pClampfit 10.2 (Molecular Devices), and results were plotted using Prism 7.0 (GraphPad Software, Inc., San Diego, CA, USA) or ORIGIN 8.0 (OriginLab Corp., North Hampton, MA, USA).
Chemicals
The levo isomer of l-THP, tetrahydroberberine (THB), and levo isomer of stepholidine (l-SPD) were the gifts from Prof Guo-zhang Jin (Shanghai Institute of Materia Materia, Shanghai, China). The l-THP and analogs were dissolved into a 10 mM stock solution using DMSO, and were diluted to final concentrations (low µM levels) using the standard external solution. (–)Nicotine, choline, and cyticine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Sazetidine A was purchased from Tocris (Ballwin, MO, USA).
Statistical analyses
All results were presented as raw mean values and percent control ± SEM. Results between groups were compared using a two-tailed unpaired t-test or ANOVA. Experiments relying on variance in time or current were analyzed using mixed models ANOVA with post hoc t-test at individual points. Statistical significance required ≥95% level of confidence (P ≤ 0.05).
Results
l-THP antagonizes human α4β2-nAChRs heterologously expressed in SH-EP1 cells
We evaluated the effect of l-THP on the function of heterologously expressed α4β2-nAChR-mediated currents in recombinant human α4β2-nAChRs expressed in a human epithelia (SH-EP1) cell line. Under patch-clamp whole-cell recording conditions at voltage-clamp mode, bath applications (via U-Tube system) of different concentrations of nicotine induced inward current responses at a holding potential (VH) of −40 mV (Fig. 1a), formed a sigmoidal-shaped concentration–response relationship curve (Fig. 1b). In nine cells tested, the averaged EC50 value was 2.9 × 10−6 M, and the Hill coefficient was 0.9. Bath applications of 10 µM l-THP (either co-application or with pre-treatment) reduced a 3 µM nicotine-induced inward current (Fig. 1c). Statistical analysis showed that, compared to co-application, 30 s pre-treatment of l-THP reduced more peak (Fig. 1d), but not steady-state current amplitude (Fig. 1e). These results suggest that l-THP antagonizes human α4β2-nAChRs.
l-THP inhibits human α4β2-nAChR-mediated currents in a concentration-dependent manner
Next, we examined the effects of varying concentrations of l-THP on 3 µM nicotine (EC50 concentration)-induced currents. As shown in Fig. 2a, pre-treatment (30 s) with l-THP inhibited nicotine-induced whole-cell currents in a concentration-dependent manner. After a 2 min wash out of l-THP, the nicotine-induced current was recovered (Fig. 2a). In Fig. 2b, we summarized the normalized average results from nine cells tested and showed that the IC50 values and Hill coefficient for the peak amplitude were 1.8 × 10−5 M and 0.7, and those for steady-state amplitude were 2.1 × 10−6 M and 0.8. These results demonstrate that l-THP reversibly suppresses human α4β2-nAChR function in a concentration-dependent manner with 30 s of pre-treatment, and the peak component is about 10-fold less sensitive to l-THP than the steady-state component.
Kinetic properties of l-THP inhibited human α4β2-nAChR-mediated currents
In this set of experiments, we analyzed the kinetic features of l-THP-induced inhibition on nicotine currents by the comparison of whole-cell current rising time and decay time (fast and slow decay time) both before and after l-THP exposure. Fig. 3a shows two typical current responses induced by nicotine (black trace) and nicotine plus l-THP (red trace). After expanded time scale, l-THP (red trace) increased the rising time of the nicotine-induced current (black trace, Fig. 3b), while accelerated the decay time constant of the nicotine-induced current (black trace, Fig. 3c). Statistical analyses showed that l-THP induced an increased nicotine current rising time (Fig. 3d), and accelerated fast decay time constant (Fig. 3e) and slow decay time constant (Fig. 3f). Fig. 3g summarizes the effects of l-THP on nicotinic current kinetics (normalized) and shows that l-THP slows nicotine current rising time and accelerates decay time constant.
l-THP inhibits human α4β2-nAChR-mediated currents in a non-competitive manner
To explore the nature of l-THP-induced inhibition on human α4β2-nAChR function, nicotine concentration–response curves were obtained alone or in the presence of l-THP (30 μM with 30 s pre-treatment) from whole-cell current traces as shown in Fig. 4a, b. As nicotine concentration (with pretreated 30 μM l-THP) was increased to maximal (10−4 M), the normalized peak amplitude was about half (49.1% ± 5.6%, n = 6) compared to that of nicotine alone (10−4 M nicotine as 100%), suggesting that nicotine up to 100 μM was unable to surmount the functional block of peak current by 30 μM l-THP (Fig. 4c). If the concentration–response curves with nicotine and nicotine plus l-THP were normalized, the EC50 values were 3.3 ± 0.5 and 2.7 ± 0.7 μM (n = 6, P > 0.05), respectively. Therefore, l-THP-induced inhibition of α4β2-nAChR-mediated currents represented a reduction of maximal nicotine peak current without significant alteration of nicotine EC50 values, suggesting a non-competitive inhibition.
Effects of l-THP analogs on human α4β2-nAChR-mediated currents
In these experiments, we compared the effects of l-THP analogs (l-THP, THB, and l-SPD, Fig. 5a) on human α4β2-nAChR function. As shown in Figs. 5b, 3 μM nicotine-induced currents were reduced by 10 μM l-THP (with the 30 s pre-treatment), 10 μM THB, and 10 μM l-SPD, respectively. Statistical analysis showed that all l-THP analogs (10 μM l-THP, 10 μM THB, and 10 μM l-SPD) show a similar inhibitory rate in 3 μM nicotine-induced whole-cell currents
Effects of l-THP on different nicotinic acetylcholine receptor subtypes
To determine whether l-THP modulation of α4β2-nAChR function is specific, we compared the effects of 10 μM l-THP on whole-cell current induced by nAChR subtypes α4β2-nAChRs, α6N/α3Cβ2β3-nAChRs, and α7-nAChRs heterologously expressed in SH-EP1 cells, and muscle type (α1β1δε2-) nAChR naturally expressed by TE cells. Fig. 6a shows the typical traces of the effects of l-THP (10 μM with 30 s pre-treatment) on NIC- or choline-induced currents at the EC50 concentrations for those agonists at the indicated nAChR subtype. Statistical analyses showed that l-THP inhibited α4β2-nAChR- and α6N/α3Cβ2β3-nAChR-mediated whole-cell currents, but did not alter α7-nAChR- and α1β1δε2-nAChR-mediated whole-cell currents (Fig. 6b).
Effects of l-THP on high-sensitive and low-sensitive α4β2-nAChR-mediated currents
To determine whether l-THP modulated different isoforms of α4β2-nAChRs, namely high-sensitive (HS) α4(2)β2(3)-nAChRs (Fig. 7a) and low-sensitive (LS) α4(3)β2(2)-nAChRs (Fig. 8a), we pharmacologically isolated high-sensitive and low-sensitive α4β2-nAChR-mediated currents in human α4β2-nAChRs (wild type). Based on a previous report, sazetidine A potently activates both stoichiometries of α4β2-nAChR; it is a full agonist on α4(2)β2(3)-nAChRs, whereas it has an efficacy of only 6% on α4(3)β2(2)-nAChRs [40]. Thus, we examined effects of l-THP on sazetidine A-induced current. As shown in Fig. 7b, sazetidine A induced the increased whole-cell current responses with corresponding increases in concentration and formed a sigmoidal-shaped dose–response curve with an EC50 value of 0.1 μM (Fig. 7c). Fig. 7d, e shows that 0.1 μM sazetidine A-induced current was reduced by l-THP (30 μM with 30 s pre-treatment), and statistical analysis demonstrated that compared to sazetidine A alone, the reduced current amplitude by the l-THP plus sazetidine A was significant (P < 0.05, n = 9, Fig. 7e). To isolate low-sensitive α4β2-nAChR from wild-type α4β2-nAChR, we used cytisine, an α4β2-nAChR partial agonist, because it has moderate efficacy at LS α4β2-nAChR but almost no efficacy at HS α4β2-nAChR [41]. The cytisine dose–response curve showed an EC50 value of 1 µM (Fig. 8b, c), and 1 µM cytisine-induced current was dramatically reduced by l-THP (30 μM with 30 s pre-treatment, Fig. 8d, e). The statistical analysis demonstrated that compared to cytisine alone, the reduced current amplitude by the l-THP plus cytisine was highly significant (P < 0.001, n = 9, Fig. 8f). To further confirm the effects of l-THP on LS α4β2-nAChR, we pretreated cells with 3 nM sazetidine for 10 min to desensitize the HS α4β2-nAChR, and then examined the effects of l-THP on LS α4β2-nAChR-mediated currents induced by 1 mM ACh, and we found the similar inhibitory rate (Supplementary Fig. S1) to cytisine-induced current through LS α4β2-nAChR. In a comparison of inhibitory rates by l-THP between sazetidine- (33.7% ± 7.8%, n = 9) and cytisine-induced currents (71.1% ± 5.3%, n = 10), a statistically significant difference was demonstrated (unpaired t-test, P < 0.001).
Discussion
The new findings of this study reveal possible targets and mechanisms for the l-THP-induced reduction of nicotine reward and dependence. l-THP is an active ingredient of the Chinese medicinal herb Corydalis ambigua that modulates the mesocorticolimbic dopaminergic reward pathway and exhibits anti-drug abuse activity in drugs of reward including morphine, amphetamine, cocaine, and alcohol [22,23,24,25,26,27,28,29]. Interestingly, l-THP reduces nicotine self-administration and blocks relapse, and its effects are similar to or even better than the current smoking cessation drugs, bupropion and varenicline [30], suggesting that l-THP is an excellent candidate as a smoking cessation aid [30]. In this study we found that l-THP inhibits nicotine-induced whole-cell currents in a dose-dependent manner and with a non-competitive mechanism in heterologously expressed human α4β2-nAChRs. Kinetic analysis shows that l-THP slows whole-cell current rising time and accelerates decay time constants. l-THP-induced inhibition of nAChR function is subunit specific, and is more sensitive to α4β2- and α6*-nAChRs, but insensitive to α7-nAChRs and muscle type nAChRs. Inhibition of α4β2-nAChR function was also shown with the other two l-THP analogs (THB and l-SPD), with a similar inhibitory rate to l-THP. Finally, we compared the effects of l-THP on high- and low-sensitive α4β2-nAChR-mediated currents and found that l-THP inhibited low-sensitive α4β2-nAChRs with more potency than high-sensitive α4β2-nAChR. Taken together, our results suggest that α4β2-nAChRs are a critical target to mediate l-THP’s anti-addictive effects, and l-THP-induced inhibition of α4β2-nAChRs may underlie its mechanism to reduce nicotine reward and dependence.
It is generally thought that nicotine acts on widely distributed nAChRs in the brain and alters brain reward-associated circuits and pathways, thus contributing to nicotine reinforcement and reward [42]. We postulate that the basis for nicotine reward and dependence exists at no less than three levels. At the receptor level, nicotine activates and/or desensitizes different nAChR subtypes in brain reward centers (e.g., the VTA) and alters DA neuronal function. At the synaptic level, nicotine acts on presynaptic nAChRs and modulates neurotransmitter release. At the neuronal network level, nicotine alters neuronal circuits, signaling pathways, and induces excitation of VTA DA neurons. In this study, we evaluated the effect of l-THP at receptor level, and our results suggest that l-THP blocks transfected human α4β2-nAChRs that heterologously expressed in an SH-EP1 cell line. Although not as physiologically relevant as native systems, recombinant expression systems have several advantages including (1) better control over which subunits are being expressed (e.g., showing the difference between different combinations of subunits); (2) better access to the cell in order to perform more accurate dose–response curves (e.g., less concern about the concentration of the drug becoming diluted as it diffuses into the tissue, etc.); (3) better kinetic profiling of the responses (i.e., rising time, decay time, total current, peak amplitudes, desensitization rates); (4) less artifact than that with slices; (5) simplified pharmacological analysis of the currents; and (6) facilitation of the study of human nAChR subunits in an expression system. We observed an antagonistic effect of l-THP on transfected human α4β2-nAChR function (Fig. 1). The peak amplitude and steady state of nicotine-activated currents were all reduced in a dose-dependent manner with l-THP (Fig. 2). In these experiments (Fig. 2a), l-THP was pretreated with 30 s, meaning that the l-THP was exposed to cell alone for 30 s, and no detectable responses were observed, suggesting that the l-THP itself cannot serve as an agonist to activate α4β2-nAChRs. The nicotine dose–response curve showed that in the presence of l-THP, the maximal nicotine dose-induced current was reduced to the half without shifting nicotine EC50 value, suggesting a non-competitive inhibition (Fig. 4). The inhibitory potency of l-THP on α4β2-nAChR-mediated currents (peak amplitudes) was dependent on how l-THP was delivered. Inhibition was stronger with pre-treatment of l-THP than that without pre-treatment, suggesting that l-THP exhibits a low association rate to interact on some configurations of α4β2-nAChRs. Further kinetic analysis showed that l-THP increased rising time to peak and accelerated steady-state components of nicotine-induced whole currents, suggesting that l-THP likely promotes nAChR desensitization rather than eliminates its activation. This idea is further supported by the different effects of l-THP on the easily desensitized nicotinic receptors (e.g., α4β2- and α6N/α3Cβ2β3-nAChRs) and the non-easily desensitized nicotinic receptors (e.g., α7- and α1*-, muscle-type nAChRs), in which, l-THP inhibited the easily desensitized nAChRs, but failed to inhibit non-easily desensitized nCAhRs (Fig. 6).
More interestingly, we used pharmacological tools to illustrate two putative α4β2-nAChR isoforms, namely sazetidine A-activated HS α4β2-nAChRs (α42β23-nAChRs) [40] and cytisine-activated LS α4β2-nAChRs (α43β22-nAChRs) [41], and then tested the effects of l-THP on these receptor-mediated currents. Our results showed that l-THP inhibited both α4β2-nAChR isoforms, but the inhibitory potency was stronger on cytisine-induced currents than on sazetidine A-induced currents (71.1% ± 5.3% vs. 33.7% ± 7.8%, P < 0.01). The pharmacological increase in potency of inhibition by l-THP on low-sensitive α4β2-nAChRs has significance for developing l-THP as a novel smoking cessation aid. Accumulating lines of evidence indicate that both native and heterologously expressed α4β2-nAChRs can exist in two isoforms with α42β23 and α43β22-nAChR subunit stoichiometries, respectively, displaying high and (predominantly) low sensitivities (HS and LS) to activation by ACh [43,44,45,46]. The existence of these α4β2-nAChR isoforms appears to be physiologically significant. For instance, multiple epilepsy-associated α4 and β2 subunit mutants alter ratios of HS to LS α4β2-nAChR isoforms [47, 48], and agonists capable of preferentially stimulating LS α4β2-nAChR produce more profound pharmacological effects [49,50,51]. In particular, the finding that interactions of different compounds at the α4(+)/(−)α4-binding site of LS α4β2-nAChR may result in dissimilar functional effects suggests the possibility to fine-tune drug effects on LS isoform efficacy [52]. In addition, it may be very important that α4β2*-nAChRs are the intended target of varenicline, the most successful smoking cessation pharmacotherapy currently available [53, 54], and several studies suggest that changing the balance of α4β2-nAChR isoform expression ratio can produce different physiological and pathological effects [47, 48, 50, 51]. For example, selective activation/enhancement LS α4β2-nAChR promotes the glutamatergic synaptic transmission [49]. Since in the VTA, addictive drugs including nicotine enhance glutamatergic transmission and plasticity, which underlies the neural adaptative mechanism for drug dependence and addiction [55, 56]. Based on these lines of evidence, we postulate that in the smokers’ brain, not only is α4β2-nAChR expression increasing, but there is also a change in the ratio of receptors from HS to LS α4β2-nAChR isoforms. Therefore, l-THPs specificity to inhibit the LS α4β2-nAChR isoform could make it a highly potent pharmacological agent for smoking cessation.
In this study, we collected data from human SH-EP1 cell line. Although this is an artificial cell model, our data suggest that l-THPs inhibits α4β2-nAChR, which may underlie its effect on the reduction of nicotine self-administration [42] or can be developed as a potential candidate for smoker cessation. Comparison of l-THP to the varenicline, an FDA-approved drug for smoker cessation, both drugs have two working targets. On the one hand, both drugs reduce α4β2-nACh function (l-THP non-competitive inhibition and varenicline desensitization and reduces nicotine’s effect [57]); on the other hand, both drugs are able to appropriately enhance DA neuronal function (l-THP blocks D2 receptor of VTA DA neurons and varenicline activates α7-nAChRs on glutamatergic terminals of VTA DA neurons). These two-target effects are well fit the “double target hypothesis” for smoker cessation [22], in which, both drugs reduces α4β2-nACh-mediated efficacy without decrease of DA level, and lead to a less withdrawal syndrome. In our in vitro cell model, we found that the concentrations of l-THP to inhibit α4β2-nACh function were between 10 and 30 µM, which is higher than clinical human use for reduction of pain (60 mg, three times per day, about 7 µM for a 70-kg person). In mouse nicotine self-administration (SA) experiments, 5 mg/kg dosage (about 14 µM) of l-THP remarkably reduced nicotine SA behavior [30] and 10 mg/kg dosage (about 28 µM) of l-THP reduced pain responses [58], suggesting that the concentrations of our in vitro experiments is within a range of in vivo l-THP concentrations that can reduce pain and nicotine addictive behavior. In addition to α4β2-nAChRs, we find that l-THP also inhibits α6Nα3Cβ2β3-nAChR (α6*-nAChR). Although this α6*-nAChR may not be expressed in brain as a native nAChR subtype, it exhibits many α6*-nAChR function and pharmacology [59], and the α6*-nAChRs play the important roles in drug addiction including nicotine [60] and alcohol [61]. The ability of l-THP blockade of the α6*-nAChR suggests that α6*-nAChR may be another target to mediate l-THP’s effects, which needs to be further investigated.
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Acknowledgements
SH-EP1 cells were provided by the Dr. Paul Whiteaker/Dr. Ron Lukas Laboratory (Barrow Neurological Institute, Phoenix, AZ, USA). This work was partially supported by the Shantou Leading Talent Project Funding.
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Both YBH. and ZGM. equally contributed to perform experiments, data analysis, and writing the manuscript. CZ, XKKM, DHT., and MG contributed to data collection and analysis, and made the figures; RJL contributed to discussions and editing of the manuscript. JW conceived the outline of experimental designs, data analysis, made figures, and wrote the manuscript. All authors contributed to critical discussions and finalizing the manuscript before submission. They have all given approval to the final form of the manuscript.
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Huang, Yb., Ma, Zg., Zheng, C. et al. Levo-tetrahydropalmatine inhibits α4β2 nicotinic receptor response to nicotine in cultured SH-EP1 cells. Acta Pharmacol Sin 43, 889–896 (2022). https://doi.org/10.1038/s41401-021-00709-1
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DOI: https://doi.org/10.1038/s41401-021-00709-1