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NEUROPHARMACOLOGY

Phytoestrogen Cimicifugoside-Mediated Inhibition of Catecholamine Secretion by Blocking Nicotinic Acetylcholine Receptor in Bovine Adrenal Chromaffin Cells

Department of Life Science (K.C.W., Y.S.P., D.J.J., K.T.K.), Division of Molecular and Life Science, Pohang University of Science and Technology, Pohang, South Korea; Department of Life Science and Food Engineering (K.C.W., B.S.S.), Handong Global University, Pohang, South Korea; Medical Research Institute (J.O.L.), School of Medicine, Kyungpook National University, Daegu, South Korea; Department of Anesthesiology (W.Y.B.), School of Medicine, Kyungpook National University, Daegu, South Korea

Received for publication November 3, 2003
Accepted February 2, 2004.

	Abstract

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We investigated the effect of the phytoestrogen cimicifugoside, one of the pharmacologically active ingredients of the medicinal plant Cimicifuga racemosa (black cohosh) that has been used to treat many kinds of neuronal and menopausal symptoms, such as arthritis, menopausal depression, and nerve pain. Cimicifugoside inhibited calcium increase induced by 1,1-dimethyl-4-phenylpiperazinium iodide (DMPP), a nicotinic acetylcholine receptor (nAChR) agonist in bovine adrenal chromaffin cells with a half-maximal inhibitory concentration (IC₅₀) of 18 ± 2 µM. In contrast, cimicifugoside did not affect the calcium increases evoked by high K⁺, veratridine, and bradykinin. The DMPP-induced sodium increase was also inhibited by cimicifugoside with an IC₅₀ of 2 ± 0.3 µM, suggesting that the activity of nAChRs is inhibited by cimicifugoside. Cimicifugoside did not affect the KCl-induced secretion but markedly inhibited the DMPP-induced catecholamine secretion that was monitored by carbon-fiber amperometry in real time and high-performance liquid chromatography through electrochemical detection. The results suggest that cimicifugoside selectively inhibits nAChR-mediated response in bovine chromaffin cells.

Bovine adrenal chromaffin cells are neuroendocrine cells that have been widely used as a model system for the study of catecholamine secretion (Kilpatrik et al., 1980, 1982). When acetylcholine secreted from splanchnic nerve terminal binds to acetylcholine receptors on adrenal chromaffin cells, an influx of extracellular cation such as sodium and calcium through acetylcholine receptors occurs, and the cell membrane is depolarized. Then the depolarization activates voltage-sensitive calcium channels that increase intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Finally, increased intracellular Ca²⁺ triggers exocytotic machineries to evoke catecholamine secretion. Catecholamines such as dopamine, norepinephrine, and epinephrine are synthesized in the brain, chromaffin cells, sympathetic nerves, and sympathetic ganglia and play important roles in stress and emotional behavior (Cooper et al., 1991). Many psychotropic drugs are known to act in catecholamine-containing neurons (Seeman and Van Tol, 1994). Therefore, compounds that modulate catecholamine secretion may be used as potential therapeutic drugs for the treatment of affective disorders.

Since cimicifugoside (CF), a triterpene glycoside whose biological function has not been thoroughly studied, is the main ingredient of C. racemosa (black cohosh), we investigated the effect of cimicifugoside in the catecholamine secretion from bovine adrenal chromaffin cells. Due to its steroid backbone structure, cimicifugoside has been called phytoestrogen on some occasions (Fig. 1). We found that cimicifugoside, a member of phytoestrogens, specifically and non-genomically inhibits nAChR-mediated effects evoked by DMPP, thereby leading to the inhibition of nAChR-mediated sodium, calcium increase, and catecholamine secretion.

View larger version (15K): [in this window] [in a new window]   Fig. 1. Structure of CF.

	Materials and Methods

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Materials. Cimicifugoside was purchased from ChromaDex Inc. (Santa Ana, CA). Fura-2/AM, SBFI/AM, and Pluronic F-127 were purchased from Molecular Probes (Eugene, OR) [³H]nicotine was purchased from PerkinElmer Life and Analytical Sciences (Boston, MA). UB-165 and 5-iodo-A-85380 were purchased from Tocris Cookson Inc. (Ellisville, MO). DMPP, bradykinin, veratridine, mecamylamine, and other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and Merck (Whitehouse Station, NJ).

Preparation of Chromaffin Cells. Chromaffin cells were isolated from bovine adrenal medulla by two-step collagenase digestion as previously described (Kilpatrik et al., 1980). For the measurement of catecholamine secretion and the [³H]nicotine binding assay, cells were plated in 24-well plates at a density of 5 x 10⁵ cells/well. Chromaffin cells transferred to 100-mm culture dishes (1 x 10⁷ cells per dish) were used to measure cytosolic free calcium and sodium concentrations. The cells were maintained in DMEM/F-12 (Invitrogen, Carlsbad, CA) containing 10% bovine calf serum (Hyclone Laboratories, Logan, UT) and 1% antibiotics (Invitrogen). Chromaffin cells were incubated in a humidified atmosphere of 5% CO₂/95% air at 37°C for 3 to 5 days before use.

Measurement of Catecholamine Secretion by HPLC. Catecholamine secretion from chromaffin cells was measured in 24-well plates following the method previously reported (Cheng et al., 1992, 1993; Eaton et al., 2000). Briefly, cells were rinsed two times with Ca²⁺-containing Locke's solution containing 157.4 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgCl₂, 5.6 mM D-glucose, 5 mM HEPES, 3.6 mM NaHCO₃, pH 7.4 titrated by NaOH and incubated at 37°C for 5 min in each case. The cells were subsequently stimulated with the drugs under test conditions. After incubation, the medium was removed from each well and transferred to a test tube containing (10% v/v) 0.1 N HCl. A 20-µl aliquot of each 500-µl sample was injected onto the HPLC (BAS-480; BAS Bioanalytical Systems, West Lafayette, IN) C18 column (150 x 1 mm) with electrochemical detection. The potential used was +770 mV versus Ag/AgCl, with a classic 3-mm glassy carbon electrode. The ranges of sensitivity for the electrode were 100 and 50 nA with a flow rate of 1 ml/min. The 2 liters of mobile phase included 0.55 g of heptanesulfonic acid, 0.2 g of EDTA, 80 ml of acetonitrile, 12 ml of 85% phosphoric acid, and 16 ml of triethylamine, with the pH adjusted to 2.5 with H₃PO₄ and filtered with a 0.45 micron membrane filter. Stock catecholamine (norepinephrine, epinephrine, and dopamine) solutions were used as standards.

Amperometric Measurement of Exocytosis. Recordings were performed at room temperature as described previously (Kim et al., 2000a). Chromaffin cells were buffered with amine-free solution containing 137.5 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM D-glucose, 10 mM HEPES, pH 7.3 titrated by NaOH. Carbon-fiber electrodes were fabricated from 5 to 11-µm carbon fibers (PAN T650 or P25; Amoco Performance Products, Inc., Atlanta, GA) and polypropylene 10-µl micropipettor tips. A carbon-fiber electrode, back-filled with 3M KCl to connect to the head stage, was attached to a single cell. Measurements were begun after this electrode current fell below 10 pA. The amperometric current, generated by oxidation of catecholamines, was measured using an Axopatch 200B amplifier (Axon Instruments Inc., Union City, CA) and operated in the voltage-clamp mode at a holding potential of +650 mV. Amperometric signals were low-pass filtered at 1 kHz, then sampled at 0.5 kHz. For data acquisition and analysis, pCLAMP 8 (Axon Instruments Inc.) and IGOR software (WaveMetrics, Lake Oswego, OR) were used, especially for visualizing large amounts of numeric data. Solutions were exchanged by a local perfusion system that allows complete exchange of medium bathing the cells within 2 s.

[Ca²⁺]_i Measurement and Calcium Imaging. Cytosolic free calcium concentration ([Ca²⁺]_i) was determined with the help of the fluorescent Ca²⁺ indicator Fura-2 as reported previously (Park et al., 1998). Briefly, the chromaffin cell suspension was incubated with fresh serum-free DMEM/F-12 medium containing Fura-2/AM (3 µM) for 40 min at 37°C with continuous stirring. The cells were then washed with Locke's solution containing 157.4 mM NaCl, 5.6 mM KCl, 2.2 mM CaCl₂, 1.2 mM MgCl₂, 5.6 mM D-glucose, 5 mM HEPES, 3.6 mM NaHCO₃, pH 7.4 titrated by NaOH and left at room temperature until use. Sulfinpyrazone (250 µM) was added to all solutions to prevent dye leakage. Fluorescence ratios were measured by an alternative wavelength time scanning method (dual excitation at 340 and 380 nm; emission at 500 nm). Calibration of the fluorescent signal in terms of [Ca²⁺]_i was performed as described previously (Grynkiewicz et al., 1985), according to the formula [Ca²⁺]_i = [(R - R_min)/(R_max - R)] x (Sf2/Sb2) x K_d). R_max and R_min are the ratios obtained when Fura-2/AM is saturated with Ca²⁺ and when EGTA is used to remove Ca²⁺, respectively. Sf2 and Sb2 are the proportionality coefficients of Ca²⁺-free and Ca²⁺-saturated Fura-2/AM, respectively. For multiphoton confocal microscopic calcium imaging, chromaffin cells plated on poly-D-lysine-coated cover slips were pre-loaded with 5 µM Fluo-4/AM dye. After incubation for 30 min at 37°C, the cells were washed two times with Locke's solution to remove excess dye and examined under the confocal microscope. Groups of chromaffin cells were selected under the microscope. Measurements of intracellular calcium were performed with the Bio-Rad Radiance 2100 confocal microscope (Bio-Rad, Hemel Hempstead, UK) equipped with a 40x objective (0.75 numerical apertures). The calcium-sensitive Fluo-4 dye was excited by 488 nm from an argon laser, and the emission fluorescence monitored at 515/30 nm was selected by a band-pass filter. During fluorescence data collection, each scan of a 512 x 512 pixel image took 0.35 s, and the interval between each image scan was 2 s. Images were stored and processed with laser pix software (Bio-Rad). The regions of interest distributed across the image provided an intensity versus time graphic output.

[Na⁺]_i Measurement and Whole-Cell Patch Clamp. Cytosolic free Na⁺ concentration ([Na⁺]_i) was measured using the fluorescent Na⁺ indicator SBFI as previously described (Park et al., 1999b). Briefly, the chromaffin cell suspension was incubated in fresh DMEM/F-12 medium containing 15 µM SBFI/AM, 10% bovine calf serum, and 0.2% Pluronic F-127 for 2 h at 37°C with continuous stirring. The cells were then washed twice with fresh DMEM/F-12 medium and left at room temperature until use. Sulfinpyrazone (250 µM) was added to all solutions to prevent dye leakage. Fluorescence ratios were measured by an alternative dual excitation at 340 and 380 nm and emission at 530 nm. Changes in [Na⁺]_i are presented as fluorescence ratios. Whole-cell patch-clamp recordings were performed to measure inward sodium current through nAChRs with an Axopatch 200B amplifier and Digidata 1200 interface (Axon Instruments). Isolated chromaffin cells were plated on a poly-D-lysine-coated glass chip in a 35-mm culture dish and cultured for 2 to 3 days at 37°C under a 5% CO₂-containing atmosphere. The pipettes were fire-polished and had a typical resistance of 5 to 6 M. The bath solution contained 137.5 mM NaCl, 2.5 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 10 mM glucose, and 10 mM HEPES titrated to pH 7.3 with NaOH. The intracellular solution contained 140 mM CsCl, 3 mM EGTA, 1 mM MgCl₂, and 10 mM HEPES titrated to pH 7.3 with CsOH. Currents were filtered at 1 kHz and then sampled at 5 kHz. Step pulses were applied from 0 to -120 mV for 360 ms with an interpulse interval of 1 s, and voltage ramp was performed from -120 to +50 mV for 250 ms to show the voltage-current relationship of nAChRs. For data acquisition and analysis, pCLAMP 8 software (Axon Instruments) was used. Solutions were exchanged by a local perfusion system that allows complete exchange of medium bathing the cells within 2 s.

[³H]Nicotine Binding Analysis. Binding of [³H]nicotine to intact cells was measured as previously described (Kilpatrik et al., 1982). Intact chromaffin cells in 24-well plate (5 x 10⁵ cells/well) were washed twice with Locke's solution. They were incubated with 40 nM [³H]nicotine and various concentrations of cimicifugoside for 40 min at 25°C. Then the cells were washed once with 2 ml of Ca²⁺-free Locke's solution containing 100 µM EGTA. Finally, the cells were lysed and scraped in 0.5 ml of ice-cold 5% trichloroacetic acid, and radioactivity was measured by a liquid scintillation counter. Nonspecific binding, determined by coincubation with 1 mM nicotine, amounted to less than 20% of the total binding and was routinely subtracted from the total binding. The binding data were analyzed and expressed as a percentage of total binding.

Statistical Analysis. All quantitative data were expressed as means ± S.E.M. Half-maximal inhibitory concentration (IC₅₀) values were calculated with the MicroCal Origin for Windows program.

	Results

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Inhibitory Effect of Cimicifugoside on nAChR-Mediated Exocytosis. To study the effect of cimicifugoside on DMPP-evoked catecholamine secretion, we treated chromaffin cells with cimicifugoside (from 1–100 µM), and the quantification was performed by HPLC (Fig. 2). Cimicifugoside by itself did not induce catecholamine secretion (data not shown). Values of basal catecholamine (norepinephrine) release in the absence of 10 µM DMPP were 3.8 ± 0.4 µM per 5 x 10⁵ cells. DMPP (10 µM) evoked norepinephrine secretion up to 27.9 ± 0.1 µM. In the case of preincubation with cimicifugoside for 10 min, the induction values were decreased in a concentration-dependent manner (Fig. 2A), but not in the case of 60 mM KCl-induced secretion (Fig. 2B). In addition, we observed the same inhibition phenomenon in 5 min of preincubation (data not shown). To better understand how cimicifugoside inhibits secretory response evoked by nicotinic stimulation, catecholamines secreted from single bovine chromaffin cells were measured using the amperometric method (Michael and Wightman, 1999; Venton et al., 2002). When a brief pulse (20 s) of 10 µM DMPP was applied to a single chromaffin cell, a fast and transient increase in current occurred (Fig. 3A). The addition of 60 µM cimicifugoside for 5 min before and during the DMPP pulse reduced the catecholamine secretion to 32 ± 5% (Fig. 3, A and C). The cimicifugoside-induced inhibitory effect was partially reversed after the washout of cimicifugoside (Fig. 3A). In contrast, cimicifugoside did not inhibit 60 mM KCl-induced catecholamine secretion (Fig. 3, B and D).

View larger version (11K): [in this window] [in a new window]   Fig. 2. Inhibitory effect of cimicifugoside on catecholamine secretion in bovine chromaffin cells. Bovine chromaffin cells were treated with 10 µM DMPP (A) or 60 mM KCl (B) in the presence of various concentrations of CF (filled boxes) for 10 min. Secretion of catecholamines (NE) induced by DMPP in the absence of cimicifugoside is presented (open box). Open triangles represent no stimulation by agonists. The secreted catecholamines were measured as described under Materials and Methods. The experiments were performed three times independently, and the results were reproducible. Data are the means ± S.E.M. (n = 3) values.

View larger version (28K): [in this window] [in a new window]   Fig. 3. Inhibitory effect on catecholamine secretion in single bovine chromaffin cells. A and B, chromaffin cells stimulated with 10 µM DMPP or 60 mM KCl for 20 s in the absence or presence of 60 µM CF, respectively. C and D, total amperometric currents induced by the 20-s DMPP or KCl pulse in A and B, respectively, integrated and represented as a percentage of the average currents by DMPP or KCl pulses. The experiments were performed three times independently, and the results were reproducible. Data are the means ± S.E.M. (n = 8) values.

Inhibitory Effect of Cimicifugoside on nAChR-Mediated Calcium Increase. Since the increase in [Ca²⁺]_i is an essential step in the catecholamine secretion process, we investigated the effect of cimicifugoside on the [Ca²⁺]_i increase. Cimicifugoside (up to 300 µM) by itself had no effect on [Ca²⁺]_i (data not shown), whereas 10 µM DMPP induced a prominent rise in [Ca²⁺]_i (Fig. 4A, trace a), and the calcium increase was inhibited by cimicifugoside (Fig. 4A, trace b and c) in a concentration-dependent manner with a half-maximal inhibitory concentration (IC₅₀) of 18 ± 2 µM (Fig. 4B). We have observed the inhibitory effect of cimicifugoside on calcium increase evoked by nicotine with similar potency (Fig. 10B). Incubation of cells with 300 µM cimicifugoside resulted in the complete inhibition of DMPP-induced calcium increase. To assess the mechanism of cimicifugoside action, we investigated the effects of different concentrations of DMPP in the presence of 20 µM cimicifugoside (Fig. 4C). We observed a half-maximal inhibitory effect of cimicifugoside in each concentration of DMPP. We then examined the time course of the cimicifugoside effect on the 10 µM DMPP-induced [Ca²⁺]_i elevation (Fig. 4D). When the cells were treated with 60 µM cimicifugoside and 10 µM DMPP simultaneously, there was no inhibition on [Ca²⁺]_i rise. In addition, this result shows that the inhibition effect of cimicifugoside was influenced by the preincubation time and that at least 3 min of incubation appears to be necessary to exert the maximal cimicifugoside effect. We also monitored calcium increase in single chromaffin cells using multiphoton confocal microscopic calcium imaging (Fig. 5). In the confocal image of cells treated with 60 µM cimicifugoside for 5 min in the presence of 10 µM DMPP, the fluorescence intensity of the confocal image was remarkably reduced when 10 µM DMPP was applied (Fig. 5A). In contrast, cimicifugoside did not affect KCl-evoked calcium entry (Fig. 5B).

View larger version (23K): [in this window] [in a new window]   Fig. 4. Inhibitory effect of cimicifugoside in [Ca2+]i elevation in bovine chromaffin cells. A, trace of intracellular calcium increase by DMPP. The intracellular [Ca2+]i rise induced by 10 µM DMPP was measured in the absence (trace a) or presence (trace b and c) of cimicifugoside. Cells were incubated with CF for 5 min before stimulation with DMPP. The experiments were performed five times independently, and the typical Ca2+ traces were presented. B, calcium increase induced by 10 µM DMPP measured 5 min after preincubation with the indicated concentration of cimicifugoside (filled boxes). The peak height of respective stimulation was compared with that of the control calcium increase caused by DMPP alone (open box). Data are the means ± S.E.M. (n = 5) values. C, calcium increase induced by the indicated concentration of DMPP measured in the absence (open boxes) or presence (filled boxes) of 30 µM cimicifugoside. Data are the means ± S.E.M. (n = 4) values. D, chromaffin cells preincubated for the indicated time with 60 µM cimicifugoside and then stimulated with 10 µM DMPP (filled boxes). Incubation with zero time means that cimicifugoside and DMPP were treated simultaneously. The peak height of each stimulation was compared with that of the control calcium increase caused by DMPP alone (open box). Data are the means ± S.E.M. (n = 3) values.

View larger version (15K): [in this window] [in a new window]   Fig. 10. Comparison of the inhibitory effects of cimicifugoside on calcium increase by nAChR subtype selective agonists. A, calcium increase induced by indicated concentrations of subtype selective agonists UB-165 (rectangle) and 5-iodo-A-85380 (triangle) for 32 and 42 subtype, respectively. The peak height of respective stimulation was compared in each concentration. B, maximum induction of calcium by each agonist inhibited by 5 min of pretreatment of cimicifugoside with the indicated concentration. 1 µM UB-165 (rectangle); 1 µM 5-iodo-A-85380 (triangle); 10 DMPP (diamond); 10 µM nicotine (circle).

View larger version (39K): [in this window] [in a new window]   Fig. 5. Inhibitory effect of cimicifugoside in [Ca2+]i elevation in bovine chromaffin cells. The intracellular [Ca2+]i rise induced by 10 µM DMPP (A) or 60 mM KCl (B) was measured via multiphoton confocal microscope using calcium sensitive dye Fluo-4/AM. Cells were stimulated by 10 µM DMPP or 60 mM KCl in the absence (ii) or presence (iv, 5 min of preincubation) of 60 µM cimicifugoside. Left bottom pictures (iii) show cells conditioned by 60 µM cimicifugoside alone. In the bar graphs of each panel, filled bars represent induction by DMPP or KCl alone (i and ii), and open bars represent induction by each stimulant in the 5 min of preincubation of 60 µM cimicifugoside (iii and iv), respectively. The experiments were performed three times independently, and the results were reproducible. Typical sets of pictures are presented. Values represent average fluorescence intensity (region of interest) ± S.E.M. in selected, circled three areas.

Receptor Specificity of Cimicifugoside. The specificity of cimicifugoside-induced inhibition was examined by testing the effects of cimicifugoside on VSCCs, voltage-sensitive sodium channels, and phospholipase C (PLC)-linked B2 bradykinin receptor signaling. As shown in Fig. 6, calcium increase induced by 50 mM KCl was not inhibited by pretreatment with 20 µM cimicifugoside, suggesting that voltage-sensitive calcium channels were not affected by cimicifugoside. Veratridine is a plant alkaloid that opens voltage-sensitive sodium channels by binding to pharmacological site 2 on sodium channels and slowing its inactivation (Catterall, 1980). In bovine chromaffin cells, veratridine-induced activation of sodium channels was known to cause membrane depolarization (Friedman et al., 1985; Kitayama et al., 1990), thereby leading to slow and weak calcium increase through voltage-sensitive calcium channels (Heldman et al., 1996). The calcium increase by veratridine was not affected by cimicifugoside either. Bradykinin is known to activate PLC-linked B2 bradykinin receptors in bovine chromaffin cells (McMillian et al., 1992; Park et al., 1999a). A half-maximal inhibitory concentration of cimicifugoside in the DMPP-induced [Ca²⁺]_i elevation had no effect on the bradykinin-evoked calcium increase. Together, the data suggested that cimicifugoside has no significant inhibitory effect on calcium channels, sodium channels, and PLC-linked receptors. Therefore, it seems that the effect of cimicifugoside on nAChR is highly specific.

View larger version (14K): [in this window] [in a new window]   Fig. 6. The effect of cimicifugoside on [Ca2+]i rises induced by other reagents. Chromaffin cells were incubated with 20 µM cimicifugoside for 5 min, then the cells were stimulated with 50 mM KCl, 5 µM bradykinin (BK), and 100 µM veratridine (VT). Net increase in [Ca2+]i was obtained by subtracting the basal level of [Ca2+]i from the peak height after stimulation in each case. The experiments were performed three times independently, and the results were reproducible. Data are the means ± S.E.M. (n = 3) values.

Inhibitory Effect of Cimicifugoside on the Sodium Influx through the nAChR. Since both calcium channels and nAChRs were activated by nicotinic stimulation, inhibition of DMPP-induced calcium increase can result from the inhibition of nAChRs or calcium channels. To verify whether nAChRs are inhibited by cimicifugoside, we investigated the effect of cimicifugoside on DMPP-induced sodium increase that occurs only through nAChRs. As shown in Fig. 7, DMPP induced an increase in cytosolic sodium. Cimicifugoside inhibited the DMPP-induced sodium increase in a concentration-dependent manner with an IC₅₀ of 2 ± 0.3 µM (Fig. 7B), and 60 µM cimicifugoside completely inhibited the DMPP effect (Fig. 7A, trace b). The inhibitory effect of cimicifugoside on the sodium current through nAChRs was also measured by whole-cell patch clamp recording (Fig. 7, C, D, and E). The sodium currents induced by 10 µM DMPP were remarkably reduced when the cells were costimulated with 3 or 60 µM cimicifugoside. The inhibition concentration of sodium influx by cimicifugoside was similar to those by 17-estradiol but less effective than by the prototype antagonist mecamylamine (data not shown). The results suggested that the inhibition of DMPP-induced sodium increase by cimicifugoside results from the direct inhibition of nAChRs.

View larger version (22K): [in this window] [in a new window]   Fig. 7. Inhibitory effect of cimicifugoside on sodium increase in bovine chromaffin cells. A, intracellular sodium increase induced by 10 µM DMPP measured in the absence (trace a) or presence (trace b) of 60 µM cimicifugoside. The experiments were performed three times independently, and the results were reproducible. Typical Na+ traces are presented. B, sodium increase induced by 10 µM DMPP measured 5 min after preincubation with the indicated concentration of cimicifugoside (filled boxes). The peak height of each stimulation was compared with that of the control sodium increase caused by DMPP alone (open box). C, intracellular sodium current induced by 10 µM DMPP recorded through whole-cell patch clamp in the absence or presence of indicated cimicifugoside concentrations. Inductions by DMPP were performed after 5 min of preincubation of cimicifugoside and washed out for 3 min to get back the control response. D, total peak currents induced by DMPP integrated and represented as a percentage of the average currents by DMPP. Data are the means ± S.E.M. (n = 6) values. E, traces by voltage ramp application from -120 to +50 mV presented in each case of DMPP stimulation with or without cimicifugoside.

Nicotine Binding. Since nicotine works on nAChR as a ligand, we tested whether the binding of [³H]nicotine to nAChRs was inhibited by cimicifugoside. We previously demonstrated that clozapine showed concentration-dependent competition with [³H]nicotine in the same condition (Park et al., 2001). As shown in Fig. 8, cimicifugoside did not significantly compete for binding with [³H]nicotine, suggesting that its binding site is distinct from that of the agonist including nicotine and acetylcholine.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Effect of cimicifugoside on [3H]nicotine binding. Chromaffin cells were incubated with 40 nM [3H]nicotine and various concentrations of cimicifugoside (filled box) for 40 min at 25°C. Specific binding of [3H]nicotine is presented. Total binding is presented by an open box. Nonspecific binding was determined in the presence of 1 mM unlabeled nicotine. The experiments were performed three times independently, and the results were reproducible. Data are the means ± S.E.M. (n = 3) values.

Comparison of Inhibitory Effects among Cimicifugoside and Other Antagonists. It has been reported that local anesthetics such as lidocaine, procaine, and QX-222 (2-[(2,6-dimethylphenyl)amine]-N,N,N, triethyl-2-oxoethanaminium chloride) inhibit the function of nAChRs in a noncompetitive manner (Gentry and Lukas, 2001). Therefore, we compared the inhibitory potency of cimicifugoside with that of lidocaine and the prototype antagonist mecamylamine under experimental conditions. As shown in Fig. 9, all of them inhibited DMPP-induced calcium increase, in which the most potent agent was mecamylamine and the less potent agent was lidocaine. We determined whether cimicifugoside had subtype specificity. Intracellular calcium was evoked by 5-iodo-A-85380 and UB-165, which are selective agonists to 42 and 32, respectively (Fig. 10A). Because UB-165 had a more potent effect on calcium increase (EC₅₀, 40 nM) than A-85380 (EC₅₀, 90 nM), 32 type nAChRs may presumably be more expressed than 42 types in bovine adrenal chromaffin cells. The maximum calcium induction by the selective agonists that we tested, as well as DMPP and nicotine, were inhibited by cimicifugoside with similar potency (Fig. 10B). The results suggested that CF is broad spectrum nAChR inhibitor.

View larger version (21K): [in this window] [in a new window]   Fig. 9. Comparison of the inhibitory effects of cimicifugoside and other antagonists. The calcium increase induced by 10 µM DMPP (open box) was measured 5 min after preincubation with the indicated concentrations of cimicifugoside, lidocaine, or mecamylamine. The peak height of respective stimulation was compared with that of the control calcium increase caused by DMPP alone. The experiments were performed three times independently, and the results were reproducible. Data are the means ± S.E.M. (n = 3) values.

	Discussion

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C. racemosa was historically used by American Indians to assist and ease the pain of childbirth and as an antidote against snake bites. It is an antispasmodic herb that can relieve cramps in the pelvic area while increasing blood supply to this area. It is used to treat painful periods with cramps and any inflammatory condition in the pelvic area associated with spasm, tension, or uterine discharge. It is also used in the treatment of nerve pain, headaches, arthritic pain, and inflammation (British Herbal Medicine Association Scientific Committee, 1983). Recently, the extract of C. racemosa containing phytoestrogen has been used to treat menopausal women. Phytoestrogens such as isoflavone extracted from soybeans have been highlighted mostly as agents for hormone replacement therapy of estrogen deficiency in menopausal women. Most women experience menopausal symptoms caused by estrogen deficiency, which results in hyperactivity of neuronal conduction such as hot flushes, the most prominent neurovegetative symptom (Ginsburg et al., 1981). It was reported in a recent study that the ethanol extract CR BNO 1055 of C. racemosa was more effective in releasing menopausal symptoms than other phytoestrogen-containing candidates (Jarry et al., 2003; Wuttke et al., 2003). Although cimicifugoside is an important phytoestrogen ingredient of C. Racemosa, little work has been conducted so far to elucidate its mechanism of action.

Our data clearly suggest that catecholamine secretion induced by nicotinic acetylcholine receptor-mediated stimulation is specifically inhibited by cimicifugoside and that these inhibitory effects are caused by the inhibition of sodium influx through ionotropic receptors (nAChRs). The reason why the IC₅₀ of cimicifugoside was considerably lower on sodium increase (2 µM) than that on calcium rise (18 µM) may be that electrophysiologically higher concentrations of the antagonist are needed to suppress the activation of voltage-sensitive calcium channels compared with that needed to suppress the sodium current. In other words, the amount of cimicifugoside sufficient to inhibit intracellular sodium increase can be insufficient to inhibit calcium rise. In addition, we observed that cimicifugoside inhibits cytosolic calcium increase, a downstream phenomenon of membrane depolarization caused by the activation of nAChRs, and those other direct pathways that could activate voltage-sensitive calcium channels are not significantly influenced by cimicifugoside. Although calcium channels are activated in the activation process of nAChRs, both the inhibition of DMPP-induced sodium increase and the absence of inhibitory effect on high K⁺-induced calcium increase clearly indicated that nAChRs, but not calcium channels, are the specific target of cimicifugoside. In bovine chromaffin cells, 5-iodo-A-85380 and UB-165, which are selective agonists of 42 and 32, respectively, evoked intracellular calcium increase. Both 5-iodo-A-85380- and UB-165-induced calcium rise was inhibited by cimicifugoside to similar extents, suggesting that cimicifugoside did not have specificity for a certain type of nAChR. Even though we used quite a high concentration of cimicifugoside (>50 µM) to visualize its inhibitory effect [high considering the amount (2.5% triterpene glycosides of total extract) among ingredients of C. racemosa], this concentration may be reached in clinical reality. In addition, the applicable dose for local anesthetics, such as lidocaine, can be converted to a micromolar range for it to work effectively.

In contrast to rapid onset inhibitors such as borneol (Park et al., 2003), cimicifugoside exhibits a rather slow onset, requiring at least 3 min of preincubation, suggesting that cimicifugoside does not interact directly with the extracellular domain of nAChRs. According to the inhibition of calcium induction by a selective agonist, it seems to lack receptor subtype selectivity but functions as a broad spectrum inhibitory modulator. Furthermore, the lack of inhibition by cimicifugoside in [³H]nicotine binding to nAChRs indicates that cimicifugoside acts as a noncompetitive inhibitor of nAChR. A variety of pharmacological agents are known to modulate the transitions between conformations by directly binding to several sites on nAChRs that are topographically distinct from the acetylcholine binding sites (Lena and Changeux, 1993). Many physiological or pharmacological effectors modify the properties of nAChRs even though they do not significantly affect the binding of agonists such as acetylcholine and nicotine. These molecules, known as noncompetitive blockers, inhibit the ion channel gating activity of the nAChRs through mechanisms that differ from those of the competitive blockers (Lowenick et al., 2001). Our data suggest that cimicifugoside is likely to act as a noncompetitive blocker of nAChRs. It has been reported previously that a synthetic glucocorticoid, dexamethasone, binds to the specific site located on the outer cell membrane and suppresses the I_Ach level in a noncompetitive manner in chromaffin cells, and that steroids may change the lipid environment of nAChRs, thus leading to the suppression of I_Ach. In other words, it is possible that the structure of steroids, which is changed by the cleavage of the cholesterol side chain, thus contributes to the direct inhibition of nAChRs by steroids (Inoue and Kuriyama, 1989; Uki et al., 1999). On the same principle, cimicifugoside seems to act like other steroids because it has an inhibitory potency similar to 17-estradiol against intracellular sodium increase induced by DMPP in our conditions (data not shown).

The mechanism of action of steroid hormones can be classified as genomic and nongenomic depending on whether the hormone binds to intracellular receptors and causes transcriptional activation. Nongenomic effects do not conclude gene expression but result in modulation of cellular events such as exocytosis (Machado et al., 2002). Whereas at least 30 min are required for the genomic response to estrogen to occur, the inhibition of the stimulant-induced [Ca²⁺]_i rise following cimicifugoside treatment occurs within 5 min. In consideration of effective incubation time, the action of cimicifugoside to nAChRs could be the nongenomic action of phytoestrogen. In several reports, nongenomic effects of estrogen were investigated, for example, on VSCCs, purinergic receptors, ionotropic receptors, and Maxi-K⁺ channels, but in the case of phytoestrogens, little attention has been paid to their role as bioactive materials in the cellular signal transduction system.

The specific inhibition on nAChR-mediated effects by cimicifugoside may contribute to the understanding of the basic mechanism of phytoestrogen effects as medicine and may be classified as the general action of steroids on membrane receptors reported previously. Furthermore, its specific action implicates cimicifugoside as a potential candidate for therapeutic agents. Clinical agents such as local anesthetics, products that alleviate symptoms of nicotine withdrawal, or agents that release hyperactivation of sympathetic nerves were shown to inhibit functions of nAChRs, although the correlation between the inhibitory effect and its clinical function is not yet clear. In addition, the relationship between nAChR inhibition of cimicifugoside and the alleviation of climacteric/menopausal symptoms is still unclear. The determination of cimicifugoside's mechanism of inhibition on nAChR and its potential clinical application are subjects of interest for further study and experimentation.

	Acknowledgements

 
We thank Byung-Soon Kang of Kyung-Buk Packers Company Inc. (Pohang, South Korea) for kindly providing the bovine adrenal gland. We are also grateful to Yeoul Kang for critically editing the manuscript.

	Footnotes

 
This work was supported by the IMT-2000 Program of the Korea Ministry of Information and Communication, the Korea Ministry of Health and Welfare, and the Brain Korea 21 Program of the Korea Ministry of Education.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

DOI: 10.1124/jpet.103.062331.

ABBREVIATIONS: VSCC, voltage-sensitive calcium channel; nAChR, nicotinic acetylcholine receptor; CF, cimicifugoside; DMPP, 1,1-dimethyl-4-phenylpiperazinium iodide; AM, acetoxymethyl ester; UB-165, (2-chloro-5-pyridyl)-9-azabicyclo[4.2.1]non-2-ene; DMEM, Dulbecco's modified Eagle's medium; 5-iodo-A-85380, 5-iodo-3(2(S)-azetidinylmethoxy)pyridine.

Address correspondence to: Dr. Kyong-Tai Kim, Department of Life Science, POSTECH, San 31, Hyoja Dong, Pohang, 790-784, Korea. E-mail: ktk{at}postech.ac.kr

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