23-O-Acetylshengmanol 3-O-β-D-xylopyranoside (Ac-SM) isolated from Actaea racemosa L.—an herbal remedy for the treatment of mild menopausal disorders—has been recently identified as a novel efficacious modulator of GABAA receptors composed of α1-, β2-, and γ2S-subunits. In the present study, we analyzed a potential subunit-selective modulation of GABA-induced chloride currents (IGABA) at GABA concentrations eliciting 3–8% of the maximal GABA response (EC3–8) through nine GABAA receptor isoforms expressed in Xenopus laevis oocytes by Ac-SM with two-microelectrode voltage clamp and behavioral effects 30 minutes after intraperitoneal application in a mouse model. Efficacy of IGABA enhancement by Ac-SM displayed a mild α-subunit dependence with α2β2γ2S (maximal IGABA potentiation [Emax] = 1454 ± 97%) and α5β2γ2S (Emax = 1408 ± 87%) receptors being most efficaciously modulated, followed by slightly weaker IGABA enhancement through α1β2γ2S (Emax = 1187 ± 166%), α3β2γ2S (Emax = 1174 ± 218%), and α6β2γ2S (Emax = 1171 ± 274%) receptors and less pronounced effects on receptors composed of α4β2γ2S (Emax = 752 ± 53%) subunits, whereas potency was not affected by the subunit composition (EC50 values ranging from α1β2γ2S = 35.4 ± 12.3 µM to α5β2γ2S = 50.9 ± 11.8 µM). Replacing β2- with β1- or β3-subunits as well as omitting the γ2S-subunit affected neither efficacy nor potency of IGABA enhancement by Ac-SM. Ac-SM shifted the GABA concentration-response curve toward higher GABA sensitivity (about 3-fold) and significantly increased the maximal GABA response by 44 ± 13%, indicating a pharmacological profile distinct from a pure allosteric GABAA receptor modulator. In mice, Ac-SM significantly reduced anxiety-related behavior in the elevated plus maze test at a dose of 0.6 mg/kg, total ambulation in the open field test at doses ≥6 mg/kg, stress-induced hyperthermia at doses ≥0.6 mg/kg, and significantly elevated seizure threshold at doses ≥20 mg/kg body weight. High efficacy and long biologic half-life of Ac-SM suggest that potential cumulative sedative side effects upon repetitive intake of A. racemosa L. preparations might not be negligible.
GABA mediates fast inhibitory neurotransmission in the mammalian brain by activating synaptic and extrasynaptic GABAA receptors. GABAA receptors are composed of five subunits that form a chloride-conducting ion pore (Macdonald and Olsen, 1994; Sieghart, 1995; Sieghart and Sperk, 2002; Sigel and Steinmann, 2012). Nineteen different GABAA receptor subunits have been identified in the human genome (Simon et al., 2004), theoretically allowing the formation of approximately 800 subunit combinations. However, so far the existence of only 11 native GABAA receptor subtypes has been confirmed (Olsen and Sieghart, 2008). There is consensus that the mainly expressed GABAA receptor isoform consists of α1, β2, and γ2 subunits (Olsen and Sieghart, 2008) with a commonly accepted 2α:2β:1γ subunit stoichiometry (Tretter et al., 1997; Baumann et al., 2001, 2002). The subunit combination determines the pharmacologic properties of GABAA receptors (Olsen and Sieghart, 2009). GABAA receptors play a major role in controlling the excitability of the mammalian brain and are thus involved in regulating, among others, sleep, vigilance, mood, and emotions (Möhler et al., 2005; Möhler, 2006). GABAA receptors are modulated by structurally diverse compounds including clinically applied drugs, such as benzodiazepines, barbiturates, neurosteroids, and anesthetics, as well as a large number of natural products, including flavonoids, terpenoids, or polyacetlyenes (for review, see Johnston et al., 2006; Olsen and Sieghart, 2009; Hanrahan et al., 2011; Nilsson and Sterner, 2011). Recently, we have identified four cycloartane glycosides (actein, cimigenol 3-O-β-d-xylopyranoside, 25-O-acetylcimigenol 3-O-α-l-arabinopyranoside, and 23-O-acetylshengmanol 3-O-β-d-xylopyranoside) as novel efficacious GABAA modulators isolated from Actaea racemosa L. Whereas enhancement of GABA-induced chloride currents (IGABA) by actein, cimigenol 3-O-β-d-xylopyranoside, and 25-O-acetylcimigenol 3-O-α-l-arabinopyranoside averaged at about 300%, a more than 5-fold stronger IGABA potentiation was observed for cycloartane glycoside 23-O-acetylshengmanol 3-O-β-d-xylopyranoside (Ac-SM) (Cicek et al., 2010).
The aim of the present study was to obtain deeper insights into the molecular mechanism of Ac-SM action by analyzing a potential subunit-dependent modulation of nine different GABAA receptor subtypes expressed in Xenopus laevis oocytes. In addition, potential anxiolytic, stress-reducing, anticonvulsant effects as well as changes in locomotor activity upon application of Ac-SM were studied in mice to investigate the potential effects of this compound on mood, sedation, and seizure threshold.
Materials and Methods
All procedures involving animals were approved by the Austrian Animal Experimentation Ethics Board in compliance with the European Convention (Directive 2010/63/EU) for the protection of vertebrate animals used for experimental and other scientific purposes (European Treaty Series no. 123). Every effort was taken to minimize the number of animals used.
Ac-SM (Fig. 1) was isolated from A. racemosa L. as previously described (Cicek et al., 2010). Stock solutions (100 mM for in vitro experiments and 1 mg/10 µl for in vivo experiments, respectively) were prepared in dimethylsulfoxide (DMSO). All chemicals were obtained from Sigma-Aldrich (Vienna, Austria).
Due to low solubility in the bath solution, Ac-SM was used only up to a concentration of 300 µM in in vitro experiments. Equal amounts of DMSO were present in control and compound-containing solutions. The maximum DMSO concentration in the bath (0.3%) did not affect IGABA (Khom et al., 2006).
For in vivo experiments, working concentrations were adjusted by dilution with 0.9% sodium chloride; the final concentration of DMSO was fixed at 10% including control solutions. To enhance solubility of the compound, Tween 80 (3% final concentration) was added to all solutions. pH was adjusted to 7.2–7.4 with 1 M sodium hydroxide. All solutions were prepared fresh every day prior to experiments.
Expression and Functional Characterization of GABAA Receptors.
Preparation of stage V–VI oocytes from X. laevis (Nasco, Fort Atkinson, WI) and synthesis of capped off run-off poly(A+) rat cRNA transcripts from linearized cDNA templates (pCMV vector) was performed as previously described (Khom et al., 2006). Briefly, female X. laevis were anesthetized by exposing them for 15 minutes to a 0.2% solution of MS-222 (methane sulfonate salt of 3-aminobenzoic acid ethyl ester) before surgically removing parts of the ovaries. Follicle membranes from isolated oocytes were enzymatically digested with 2 mg/ml collagenase (type 1A). Oocytes were stored at 18°C in a medium containing 90 mM NaCl, 1 mM KCl, 1 mM MgCl2×6H2O, 1 mMCaCl2, and 5 mM HEPES (Methfessel et al., 1986). One day after isolation, the oocytes were injected with about 10–50 nl of diethyl pyrocarbonate–treated water containing the different rat cRNAs (150–3000 ng/μl per subunit). To ensure expression of the γ2S-subunit in the case of α1β2γ2S, α2β2γ2S, α3β2γ2S, α5β2γ2S, α6β2γ2S, and α1β3γ2S receptors, cRNAs were mixed in a ratio of 1:1:10 (Boileau et al., 2002); for α4β2γ2S receptors, a ratio of 3:1:5 was used. For α1β2 receptors, cRNAs were mixed in a ratio of 1:1. cRNAs for α1β1γ2S channels were injected in a ratio of 3:1:10 to avoid formation of β1-homo-oligomeric GABAA receptors (Krishek et al., 1996). The amount of cRNA was determined by means of a NanoDrop ND-1000 (Kisker, Steinfurt, Germany).
Electrophysiological experiments were performed using the two-microelectrode voltage clamp technique at a holding potential of −70 mV, making use of a TURBO TEC 01C amplifier (NPI Electronic, Tamm, Germany) and an Axon Digidata 1322A interface (Molecular Devices, Sunnyvale, CA). Data acquisition was carried out using pCLAMP version 9.2 (Molecular Devices). The bath solution contained 90 mM NaCl, 1 mM KCl, 1 mM MgCl2⋅6H2O, 1 mM CaCl2, and 5 mM HEPES (pH 7.4). Microelectrodes were filled with 2 M KCl and had resistances between 1 and 3 MΩ (Khom et al., 2006).
GABA and Ac-SM were applied by means of fast perfusion system (for details, see Baburin et al., 2006). Drug or control solutions were applied by means of a TECAN Miniprep 60 (NPI Electronic) permitting automation of the experiments. To elicit IGABA, the chamber was perfused with 120 µl of GABA-containing solution at a volume rate between 300 and 1000 µl/s. The IGABA rise time ranged between 100 and 250 milliseconds (Khom et al., 2006). To account for possible slow recovery from increasing levels of desensitization in the presence of high GABA or compound concentrations, the duration of washout periods was extended stepwise, i.e., 1.5 minutes (GABA EC3–8) to 3 minutes (coapplication of GABA EC3–8 in the presence of ≤1 µM Ac-SM) to 5–10 minutes (coapplication of GABA EC3–8 in the presence of ≤10 µM compound) to 15–20 minutes (coapplication of GABA EC3–8 and ≤100 µM Ac-SM) to 30 minutes (GABA EC3–8 in the presence of 300 µM Ac-SM). The duration of washout periods in experiments analyzing the effect of Ac-SM (300 µM) at different GABA concentrations was fixed at 30 minutes. Potential run-down or run-up effects were ruled out by application of GABA control at the end of each experiment. Oocytes with maximal current amplitudes >3 µA were discarded to exclude voltage-clamp errors (Khom et al., 2006).
Analyzing Concentration-Response Curves.
Stimulation of chloride currents by modulators of the GABAA receptor was measured at a GABA concentration eliciting between 3 and 8% of the maximal current amplitude (EC3–8). The EC3–8 was determined at the beginning of the experiment for each oocyte by application of 1 mM GABA followed by submaximal GABA concentrations.
Enhancement of the chloride current was defined as where I(GABA+Comp) is the current response in the presence of Ac-SM, and IGABA is the control GABA current. To measure the sensitivity of the GABAA receptor for a given compound, it was applied for an equilibration period of 1 minute before applying GABA (EC3–8). Concentration-response curves were generated and the data were fitted by nonlinear regression analysis using Origin software (OriginLab Corporation, Northampton, MA). Data were fitted to the equationwhere nH is the Hill coefficient. Each data point represents the mean ± S.E.M. from at least 3 oocytes and ≥2 oocyte batches.
Male mice (C57BL/6N) were obtained from Charles River Laboratories (Sulzfeld, Germany). For maintenance, mice were group-housed (maximum five mice per type IIL cage) with free access to food and water. At least 24 hours before the commencement of experiments, mice were transferred to the testing facility, with continued ad libitum access to food and water. The temperature in the holding and testing facilities was 22 ± 2°C, the humidity was 40–60%, and a 12-hour light/dark cycle was in operation (lights on from 7:00 AM to 7:00 PM). Male mice aged 3–6 months were tested.
Intraperitoneal injection of control or Ac-SM–containing solutions was usually done 30 minutes before the test, except for home cage (HC) activity analysis (60 minutes before test) and measurement of stress-induced hyperthermia (3 hours before test), to reduce the impact of stress on the analysis. Application of the solvent alone did not influence animal behavior. All doses are indicated as milligrams per kilogram body weight of the animal.
Open Field Test.
Exploration of a novel environment was tested over 10 minutes in a 50 × 50–cm box built from gray PVC equipped with infrared beams. Illumination intensity was set to 150 lux in the center. Animals’ explorative behavior was analyzed using ActiMot-2 equipment and software (TSE Systems, Bad Homburg, Germany). Arenas were subdivided into three fields: border (up to 8 cm from the wall), center (20 × 20 cm, i.e., 16% of the total area), and intermediate area, according to the recommendations of EMPRESS (European Mouse Phenotyping Resource of Standardized Screens; http://empress.har.mrc.ac.uk).
Home Cage Activity.
For HC activity, mice were singly placed into a type IIL cage with free access to food and water, enriched with a plastic tube. Movement of mice was monitored through an infrared detection system (InfraMot; TSE Systems). HC behavior analysis was started at 5 PM and ran for 72 hours. Arbitrary activity counts were cumulated in 60-minute intervals.
Elevated Plus Maze Test.
The animals’ behavior was tested over 5 minutes on an elevated plus maze (EPM) 1 m above ground. The EPM consisted of two closed arms (walls 20 cm in height) and two open arms, facing each other. The test instrument was built from gray PVC, each arm 50 × 5 cm. Illumination intensity was set to 180 lux at the intersection of the arms (defined as the neutral field). Animals were placed in the center, facing an open arm (OA). Analysis of open arm entries, time spent on open arms, and distance on open arms was automatically done with Video-Mot 2 equipment and software (TSE Systems; Wittmann et al., 2009).
Stress-Induced Hyperthermia Test.
Stress-induced hyperthermia was assessed as described previously (Wittmann et al., 2009). In short, a temperature probe, lubricated with glycerol, was inserted into the rectum of the mouse to a depth of up to 2 cm to measure basal temperature (T1). The temperature probe remained in the animal until a stable temperature was reached (maximum 10 seconds). Measurement of T1 served also as the handling stressor. After 15 minutes, the measurement was repeated (T2), and the rise in temperature was considered as stress-induced hyperthermia ΔT (Olivier et al., 2003).
Seizure threshold was determined by pentylenetetrazole (PTZ) tail-vein infusion on freely moving animals at a rate of 100 µl/min (10 mg/ml PTZ in saline, pH = 7.4). Infusion was stopped when animals displayed generalized clonic seizures. Animals were immediately killed by cervical displacement after onset of seizures. The seizure threshold dose was calculated from the infused volume in relation to body weight.
Statistical significance was calculated by one-way analysis of variance (ANOVA) and, where appropriate, by two-way ANOVA (followed by a post-hoc mean comparison with Bonferroni; GraphPad Software, La Jolla, CA). P values <0.05 were accepted as statistically significant. All data are given as the mean ± S.E.M.
In the present study, we demonstrate a comparable potency and efficacy of IGABA modulation by Ac-SM (for structural formula, see Fig. 1) on nine different GABAA receptor subtypes. IGABA modulation was independent of the presence of a γ2S-subunit. Ac-SM induces anxiolytic, stress-reducing, sedative, and anticonvulsant effects.
IGABA Enhancement by Ac-SM Does Not Require the Presence of a γ2S-Subunit.
Ac-SM efficaciously potentiates IGABA through GABAA receptors composed of α1β2γ2S subunits (Cicek et al., 2010). As illustrated in Fig. 2A, Ac-SM, however, also modulated GABAA receptors lacking a γ2S-subunit with similar efficacy and potency [maximal potentiation of IGABA, Emax(α1β2γ2S): 1187 ± 166% and EC50(α1β2γ2S): 47.1±20.9 µM versus Emax(α1β2): 1277 ± 243% and EC50(α1β2γ2S): 45.0 ± 11.4 µM; see also Table 1].
Ac-SM Enhances IGABA Irrespective of the GABAA Receptor Subunit Composition.
To investigate a possible α-subunit–dependent IGABA enhancement, the α1-subunit was substituted with α2-, α3-, α4-, α5-, and α6-subunits, respectively, and expressed in combination with β2- and γ2S-subunits (Fig. 2B; Table 1). The highest efficacy was observed for receptors containing either α2- or α5-subunits, with a maximal IGABA potentiation of Emax(α2β2γ2S): 1454 ± 97% (n = 7) and Emax(α5β2γ2S): 1408 ± 87% (n = 3), respectively (Table 1), followed by receptors comprising either α1- [Emax(α1β2γ2S): 1187 ± 166%; n = 6], α3- [Emax(α3β2γ2S): 1174 ± 218%; n = 4], or α6-subunits [Emax(α6β2γ2S): 1171 ± 274%; n = 3]. Ac-SM, however, displayed a lower efficacy on GABAA receptors comprising α4-subunits compared with α1β2γ2S, α3β2γ2S, α6β2γ2S (P < 0.05), α2β2γ2S, and α5β2γ2S (P < 0.001) receptors [Emax(α4β2γ2S): 752 ± 53%; n = 3; F(5,20) = 9.185; Table 1].
In contrast to this moderate α-subunit dependence, replacing the β2-subunit in α1β2γ2S receptors by either β1- or β3-subunits (Fig. 2C) did not affect the strength of IGABA enhancement by Ac-SM [Emax(α1β1γ2S): 1069 ± 214%, n = 6 versus Emax(α1β2γ2S): 1187 ± 166%, n = 6 versus Emax(α1β3γ2S): 1247 ± 222%, n = 4].
Comparison of the potencies of the respective GABAA receptor isoforms revealed no significant differences, with EC50 values ranging from 35.4±12.3 μM (α1β3γ2S) to 50.9 ± 11.8 μM (α5β2γ2S) (see Table 1). Typical current traces for concentration-dependent IGABA enhancement by Ac-SM are illustrated in Fig. 2D.
Ac-SM Shifts the GABA Concentration-Response Curves and also Enhances Saturating GABA Concentrations.
To gain further insight into the mechanism of IGABA modulation by Ac-SM, we compared GABA concentration-response curves in the presence and absence of Ac-SM on α1β3 channels. Ac-SM was applied at a saturating concentration of 300 µM. At this concentration, Ac-SM shifted the concentration-effect curve toward significantly lower GABA concentrations [EC50(GABA): 3.7 ± 0.4 μM versus EC50(GABA+Ac-SM): 1.2 ± 0.2 μM, P < 0.01] and increased the maximal GABA response by 44 ± 13% (n = 3; see Fig. 3). Ac-SM at 300 µM induced only small currents in the absence of GABA (not exceeding 1% of maximal IGABA induced by 1 mM GABA), indicating modulatory effects (see also Cicek et al., 2010).
Ac-SM Dose-Dependently Reduces Locomotor Activity.
To get first insight into the in vivo effects of Ac-SM, the explorative behavior of male C57BL/6N mice was studied 30 minutes after intraperitoneal application in the open field test (OF). As illustrated in Fig. 4A, no changes in total ambulation were observed upon application of Ac-SM at doses ≤2 mg/kg. Reduced ambulation compared with solvent-treated controls started at doses ≥6 mg/kg, reaching a maximum at a dose of 60 mg/kg by reducing the total distance by more than 60% [control: 38.0 ± 1.2 m versus Ac-SM 60 mg/kg: 14.7 ± 3.1 m; F(6,84) = 19.597, P < 0.01]. Time spent in the center of the OF did not significantly differ between the compound-treated (doses ≤20 mg/kg) and control groups. Only mice treated with Ac-SM at a dose of 60 mg/kg spent significantly more time in the center area of the OF [control: 57.7 ± 6.1 seconds versus Ac-SM 60 mg/kg: 212.5 ± 101.6 seconds; F(6,84) = 2.815, P < 0.05; see Fig. 4B), reflecting primarily the strong suppression of motor activity. The number of entries into the center area mostly paralleled the time spent in this compartment. However, mice treated with 0.2 mg/kg Ac-SM tended to visit the center area more frequently compared with the control group (control: 28.2 ± 2.1 versus Ac-SM 0.6 mg/kg: 33.1 ± 4.1). In addition, in line with reduced ambulation, the number of center entries significantly dropped at a dose of 60 mg/kg [control: 28.2 ± 2.1 versus Ac-SM 60 mg/kg: 5.9 ± 1.6; F(6,84) = 8.828, P < 0.01; see Fig. 4C]. Control mice and Ac-SM–treated mice covered a comparable percentage of total distances in the center area (see Fig. 4D). Doses higher than 60 mg/kg were not tested due to limited solubility of the compound.
To investigate the duration of in vivo Ac-SM effects, we investigated locomotor activity of mice treated with a dose inducing marked reduction of ambulation. Home cage activity analysis (Fig. 5) revealed a long-lasting reduction of motor activity at a dose of 20 mg/kg for at least 6–12 hours. No rebound effect on the subsequent time periods was measured [two-way ANOVA, drug effect: F(1,670) = 16.81, P < 0.0001; time effect: F(66,670) = 5.77, P < 0.0001].
Ac-SM Paradigm Dependently Reduces Anxiety-Related Behavior and Stress-Induced Hyperthermia.
Ac-SM slightly reduced motor activity at doses >2 mg/kg in the OF compared with vehicle (see Fig. 4A). Thus, to assess changes in anxiety-related behavior, the effect of lower doses was analyzed in the elevated plus maze test: as illustrated in Fig. 6A, Ac-SM–treated mice (0.6 mg/kg) visited the OAs of the EPM more frequently [control: 15.0 ± 1.6% of total entries versus Ac-SM 0.6 mg/kg: 28.3 ± 4.5% of total entries; F(2,37) = 4.667, P < 0.05]. In addition, a tendency toward more time spent (Fig. 6B) and longer distances covered on the OA compared with control animals was observed (Fig. 6C); these effects were, however, not statistically significant. Total motor activity was not affected at these doses (data not shown).
Studying potential anxiolytic effects at higher doses was, however, hampered by the concomitantly occurring reduction of motor activity that might easily mask anxiolytic effects, as the behavioral tests (OF and EPM) rely on exploratory behavior. Therefore, we included a locomotion-independent test of a physiologic stress response. Activation of the autonomic nervous system in response to stress, such as exposure to noise, heat, or pain, in animals can be measured by changes in body temperature—a process referred to as stress-induced hyperthermia (SIH). SIH is a short-lasting body temperature elevation (ΔT) that can be reduced or even ablated by anxiolytic drugs (Vinkers et al., 2008). Basal body temperature measured before compound application did not differ between control and compound-treated mice (data not shown).
As shown in Fig. 7, body temperature upon stress exposure (i.e., insertion of the temperature probe) was raised in saline-treated control mice by 1.2 ± 0.1°C. Three hours after application, Ac-SM at doses ≥0.6 mg/kg significantly reduced ΔT in a dose-dependent manner [0.6 mg/kg: ΔT = 0.6 ± 0.1°C; F(4,41) = 15.350, P < 0.01]. The maximal effect was reached at a dose of 6 mg/kg [ΔT = 0.1 ± 0.1°C; F(4,41) = 15.350, P < 0.01], where SIH was not traceable any more.
Ac-SM Increases the Seizure Threshold upon Pentylenetetrazole Infusion.
A common feature of most positive allosteric modulators of GABAA receptors is an increased threshold against pentylenetetrazole-induced seizures (De Deyn and Macdonald, 1989; Rudolph et al., 1999; Gerak and France, 2011; Löscher et al., 2013). Indeed, Ac-SM significantly increased the seizure threshold at doses ≥20 mg/kg [control: 40.4 ± 3.5 mg/kg PTZ versus Ac-SM 20 mg/kg: 58.5 ± 1.7 mg/kg PTZ; F(6,23) = 19.903, P < 0.001; see Fig. 8].
Black cohosh (A. racemosa L.) preparations are widely used, mainly to relieve menopausal symptoms, although their efficacy is still controversially discussed (Borrelli and Ernst, 2008; Rees, 2009; Sassarini and Lumsden, 2010; Bedell et al., 2014; Wuttke et al., 2014). Despite extensive studies on the plant’s potential active principles, its mechanism of action is still not entirely understood (Palacio et al., 2009; Bedell et al., 2014; Wuttke et al., 2014). Understanding its molecular mechanism(s) of action is, however, a conditio sine qua non for considering potential beneficial and unwanted side effects. The initially suggested estrogenic activity of lipophilic components, such as triterpenoids, was disproved by later studies (Gaube et al., 2007; Mercado-Feliciano et al., 2012; Hajirahimkhan et al., 2013). There, however, is evidence that A. racemosa L. preparations interact with distinct neurotransmitter systems in the central nervous system, including µ-opioid receptors (Rhyu et al., 2006; Reame et al., 2008), serotonin (5-HT) receptors (subtypes 5-HT1A, 5-HT1D, and 5-HT7) (Burdette et al., 2003), and dopamine subtype 2 receptors (Jarry et al., 2003). We have previously shown that A. racemosa L. extracts efficaciously modulate GABAA receptors and identified four structurally related cycloartane glycosides in A. racemosa L. extracts as a novel class of GABAA receptor ligands (Cicek et al., 2010). Although the potency of these compounds was apparently rather low, we observed a remarkably high efficacy of IGABA enhancement compared with established GABAA receptor modulators, such as benzodiazepines [e.g., maximal IGABA enhancement by the highly potent benzodiazepine site ligand triazolam Emax(α1β2γ2S) = 253 ± 12% (data taken from Khom et al., 2006) versus IGABA enhancement by Ac-SM Emax(α1β2γ2S) = 1187 ± 166% in this study (Sigel and Baur, 1988)]. Here, we analyzed potential subunit-selective effects of the most efficacious compound, Ac-SM. Ac-SM positively allosterically modulated IGABA through nine major GABAA receptor subtypes with equal potency, but slightly different efficacy (Fig. 2, A–C). Enhancement of IGABA by Ac-SM was only slightly influenced by the α-subunit isoform incorporated into the channel, but not by the β-subunit isoform, and did not depend on the presence of a γ2S-subunit. Based on these observations, we exclude an interaction of Ac-SM with the benzodiazepine binding site (Sigel and Buhr, 1997; Rudolph et al., 1999; Olsen and Sieghart, 2009; Richter et al., 2012; Sigel and Steinmann, 2012). In addition, as Ac-SM efficaciously modulates a broad variety of GABAA receptor subtypes/does not discriminate between various GABAA receptor subtypes, its profile is apparently similar to GABAA receptor modulators such as barbiturates or neurosteroids. However, whether Ac-SM also shares a common binding site with these compounds, or whether Ac-SM interacts with a novel, yet undefined binding site on GABAA receptors remains to be exploited in future studies.
Our in vivo experiments revealed a wide spectrum of effects induced by low doses of Ac-SM, which fit the profile of nonsubtype-selective GABAA agonists or positive allosteric modulators (Rudolph et al., 1999; Sieghart, 2000; Olsen and Sieghart, 2009; Möhler, 2011; Sigel and Steinmann, 2012). These effects include reduction of motor activity paralleled by reduced stress-induced hyperthermia. Due to the strong motoric inhibition at higher doses (Fig. 4A), suggesting pronounced sedative effects, potential anxiolytic effects of Ac-SM (Fig. 6) are difficult to assess; however, increased exploration of the open arms of the elevated plus maze was observed at low doses not affecting motor activity. The motor activity–independent SIH test revealed a marked dose-dependent reduction of stress response (Fig. 7), supporting the assumption of anxiolytic effects induced by Ac-SM (Olivier et al., 2003). Furthermore, we observed a dose-dependent increase in PTZ-induced seizure threshold (Fig. 8). The long-lasting reduction of activity in the home cage suggests a long biologic half-life of Ac-SM (Fig. 5).
Taken together, our data demonstrate that the recently identified novel GABAA receptor modulator Ac-SM enhances IGABA through nine receptor subtypes with similar potency but slightly different efficacies (α1/2/3/5/6β1/2/3γ2S ∼ α1β2 > α4β2γ2S). Due to the positive GABAA allosteric modulatory effects of Ac-SM (and potentially other cycloartane glycosides; Cicek et al., 2010), it is tempting to speculate that this GABAA receptor modulation leading to sedation and muscle relaxation contributes to the alleviation of mild to moderate symptoms in menopause reported in clinical studies with preparations from A. racemosa L., especially when hot flushes are associated with sleep and mood disturbances (Wuttke et al., 2003; Frei-Kleiner et al., 2005; Osmers et al., 2005; Verhoeven et al., 2005; Newton et al., 2006; Pockaj et al., 2006; Uebelhack et al., 2006; Cheema et al., 2007).
A. racemosa L. preparations are currently standardized for their 23-epi-26-deoxyactein content (Pepping, 1999), one of the five main Actaea triterpenoids (actein, 23-epi-26-deoxyactein, cimigenol 3-O-β-D-xylopyranoside, cimiracemoside C, and cimiracemoside F). Significant, although varying, amounts of Ac-SM were recently identified in A. racemosa L. plant material (ranging from 0.0137 ± 1.65% to 0.0092 ± 1.97%) as well as in four Actaea preparations (ranging from 0.004 ± 6.42% to 0.0184 ± 1.92%) that are commercially available in Austria (Cicek et al., 2011). Whether these estimated amounts of pure Ac-SM are also sufficient to induce anxiolytic, stress-reducing, anticonvulsant, and sedative effects in humans remains, however, to be clarified. Due to the efficacy of Ac-SM and its long biologic half-life (Fig. 5), cumulative sedative side effects of repetitive intake of A. racemosa L. preparations might not be neglectable. In addition, Actaea preparations are consumed in most cases without prescription. As Ac-SM as well as other hitherto unknown components/metabolites of A. racemosa L. preparations interact among others with various targets in the central nervous system, potential harmful interactions with other psychoactive drugs—in particular, central nervous system–depressant drugs—should be also carefully considered.
Participated in research design: Khom, Schwarzer, Hering.
Conducted experiments: Strommer, Khom, Kastenberger, Schwarzer.
Contributed new reagents or analytic tools: Cicek, Stuppner.
Performed data analysis: Strommer, Khom, Kastenberger, Schwarzer.
Wrote or contributed to the writing of the manuscript: Khom, Schwarzer, Hering.
- Received July 1, 2014.
- Accepted August 21, 2014.
B.S. and S.K. contributed equally to this work.
This work was supported by the Austrian Science Fund [Grants P-22395, P-21241, TRP-107, W-1232, and I-977].
- 23-O-acetylshengmanol 3-O-β-D-xylopyranoside
- analysis of variance
- elevated plus maze
- concentration eliciting between 3 and 8% of the maximal current amplitude
- maximal potentiation of IGABA
- HC, home cage
- GABA-induced chloride currents
- methane sulfonate salt of 3-aminobenzoic acid ethyl ester
- open arm
- open field test
- stress-induced hyperthermia
- body temperature elevation
- Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics