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Ginsenoside-Rd from panax notoginseng blocks Ca2+ influx through receptor- and store-operated Ca2+ channels in vascular smooth muscle cells
European Journal of Pharmacology 548 (2006) 129 – 136 www.elsevier.com/locate/ejphar
Ginsenoside-Rd from panax notoginseng blocks Ca 2+ influx through receptor- and store-operated Ca 2+ channels in vascular smooth muscle cells Yong-Yuan Guan ⁎, Jia-Guo Zhou, Zheng Zhang, Guan-Lei Wang, Bing-Xiang Cai, Liang Hong, Qin-Ying Qiu, Hua He Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou, PR China Received 17 April 2006; received in revised form 21 July 2006; accepted 2 August 2006 Available online 17 August 2006
Abstract Previously, it was found that total saponins from panax notoginseng inhibited Ca2+ influx coupling to activation of α1-adrenoceptor. This study was designed to investigate the effects of ginsenoside-Rd from total saponins of panax notoginseng on receptor-operated (ROCC) and storeoperated (SOCC) Ca2+ channels in vascular smooth muscle cells using fura-2 fluorescence, whole cell patch clamp ion channel recording, radioligand-receptor binding, 45Ca2+ radio-trace and organ bath techniques. It was found that ginsenoside-Rd reduced phenylephrine-induced contractile responses and Ca2+ influx in normal media without significant effect on these responses in Ca2+-free media. Ginsenoside-Rd also decreased phenylephrine- and thapsigargin-induced inward Ca2+ currents, and attenuated thapsigargin- and 1-oleoy-2-acetyl-sn-glycerol (OAG)induced cation entries that are coupled to ROCC and SOCC respectively. Ginsenoside-Rd failed to inhibit KCl-induced contraction of rat aortal rings and Ca2+ influx, and did not alter voltage-dependent inward Ca2+ current (VDCC) which was blocked by nifedipine. Also, ginsenoside-Rd did not change binding site and affinity of [3H]-prazosin for α1-adrenoceptor in the vascular plasma membrane. These results suggest that ginsenoside-Rd, as an inhibitor, remarkably inhibits Ca2+ entry through ROCC and SOCC without effects on VDCC and Ca2+ release in vascular smooth muscle cells. © 2006 Elsevier B.V. All rights reserved. Keywords: Ginsenoside-Rd; Receptor-operated; Store-operated; Ca2+ channel; Ca2+ entry
1. Introduction Panax notoginseng [(Buck) F.H. Chen] is a famous traditional Chinese herb medicine. In China, it has long been used in clinical treatment as a hemostatic and analgetic drug. As the effective fractions of panax notoginseng, the total saponins of Panax notoginseng have been used in clinic for the treatment of cardiovascular diseases and stroke in China since 1982. However, it is not clear that the mechanisms are involved in the effect of total saponins from panax notoginseng on cardiovascular diseases.
⁎ Corresponding author. Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, 74 Zhongshan 2 Rd, Guangzhou, Guangdong, 510089, PR China. Tel.: +86 20 87331857; fax: +86 20 87331209. E-mail address: [email protected] (Y.-Y. Guan). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.08.001
In 1985, we first reported that total saponins of panax notoginseng inhibited contractive response induced by norepinephrine without an effect on the contraction induced by KCl in rabbit aortic rings, and therefore suggested that total saponins of panax notoginseng dilated the vascular muscle through inhibition of α-adrenoceptor operated Ca2+ influx (Guan et al., 1985). Later, using fura-2 fluorescence and 45Ca2+ trace techniques, we further found that total saponins of panax notoginseng reduced 45Ca2+ influx and cytoplasmic free Ca2+ level ([Ca2+]i) induced by phenylephrine, whereas these total saponins failed to change KClinduced 45Ca2+ influx and the increase of [Ca2+]i. Total saponins of panax notoginseng did not change phenylephrine-induced 45 Ca2+ efflux which was owing to intracellular Ca2+ release, and the affinity of α-adrenoceptor (Guan et al., 1988, 1994). These results suggested that total saponins of panax notoginseng have an inhibitory effect on receptor-operated Ca2+ entry. Based on these
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data, we try to find out which kind of sole purified component has an inhibitory effect on receptor-operated Ca2+ entry through screening various purified components extracted from total saponins of panax notoginseng. We firstly found that ginsenoside-Rd (Dammar-24(25)-ene-3β,12β,20(S)-triol-(20-O-β-Dglucopyranosyl)-3-O-β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranoside; Fig. 1), a purified component from total saponins of panax notoginseng, reduced phenylephrine-induced contractile response without an effect on KCl-induced contractile response, whereas other purified components do not have these unique characteristics as ginsenoside-Rd. These effects of ginsenosideRd are consistent with the previously reported results of total saponins from panax notoginseng (Guan et al., 1985, 1988, 1994). In this study, we used fura-2 fluorescence, whole cell patch clamp ion channel recording, radio-ligand-receptor binding, 45Ca2+ radio-trace and organ bath techniques to further characterize ginsenoside-Rd having an inhibitory effect on Ca2+ entry through receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC) in vascular smooth muscle cells. 2. Materials and methods 2.1. Materials and animals Ginsenoside-Rd with a purity of 98% was obtained from TaiHe Biopharmaceutical Co. Ltd. (Guangzhou, P.R. China), and was made the 10 mM stock solution with 3% tween-80. Fura-2/ AM was obtained from Boehringer Mannheim Co. EGTA, Triton X-100, tween-80, phenylephrine, 5-HT, bovine serum albumin, nifedipine, S-(−)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl) phenyl]-3-pyridinecarboxylic acid methyl ester (Bay K8644), thapsigargin and 1-oleoy-2-acetyl-sn-glycerol (OAG) were obtained from Sigma (St. Louis, MO, USA). DMEM/F12, MEM vitamin solution and amino acid solution were obtained from GIBICO. [3H]-prazosin was purchased from New England Nuclear (Boston MA, USA). A10 embryonic rat thoracic aortic smooth muscle cell line (A10 cells) was obtained from the American Type Culture Collection (Rockville, MD, USA). 45 CaCl2 (122.8 GBq/mol) was obtained from Institute of Atomic Energy, Chinese Academy of Sciences (Beijing).
Fig. 1. The structure of ginsenoside-Rd. The chemical structure formula of ginsenoside-Rd is C48H82O18·3H2O and the molecular weight is 1001.20.
All animals were supplied by the Experimental Animal Center of Sun Yat-Sen University in Guangzhou, China. All procedures complied with the standards for the care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (issued by the Ministry of Science and Technology of China, Beijing). 2.2. Vascular contractile response Wistar rats (150–200 g) were anaesthetized by methoxyflurane and decapitated. The rat thoracic aorta was removed rapidly and placed in Krebs solution of the following composition (mM): NaCl 115.5, KCl 4.6, Na2HPO4 1.16, CaCl2 2.5, MgSO4 1.16, NaHCO3 21.9 and glucose 11.1 (pH 7.4). The artery was carefully cleaned of fat and connective tissue. The endothelium was removed by gently rubbing with a cotton swab moistened with Krebs solution. Then, the vessel was cut into small rings and suspended in the organ baths (3 ml capacity) containing Krebs solution at 37 °C and aerated with 95% O2 plus 5% CO2. The rings were allowed to equilibrate for at least 120 min under the resting force of 1.5 g. During the equilibration period, the bath solution was changed every 20 min. Stable contractile response to 100 mM KCl was obtained. The contractile responses were considered reproducible if the maximal tension of 2 consecutive contractions differed by b10%. The preparations which failed to produce reproducible contractions were discarded. The preparation was washed and incubated with Ca2+-free Krebs solution of the following composition (mM): NaCl 115.5, KCl 4.6, Na2HPO4 1.16, MgSO4 1.16, NaHCO3 21.9, EGTA 0.05 and glucose 11.1 (pH 7.4). In Ca2+-free Krebs solution, the agonists produced an initial response and made the further contractile response to subsequent addition of CaCl2 into bath (The final concentration in the bath was 2.5 mM.). The tension was recorded by Powerlab system (AD Instrument, Australia). 2.3. Cell preparation and culture Fresh isolated rat aorta smooth muscle cells were prepared from rat thoracic aorta. Wistar rats (180–200 g) were anaesthetized with methoxyflurane and decapitated. Thoracic aorta was aseptically removed rapidly and immersed in physiological balanced saline (PBS, in mM) containing NaCl 138.0, KCl 5.0, Na2HPO4 0.3, KH2PO4 0.3, NaHCO3 4.0. After being cleaned of connective tissue and adhered fat tissue, isolated arteries were cut open longitudinally and the endothelium was removed by gently rubbing with a cotton swab moistened with PBS. Denuded aorta was cut into small pieces about 1–3 mm3, then was incubated in a gently shaking bath at 37 °C in 1 ml enzymatic solution containing 14 mg collagenase type IA, 4 mM ATP, 0.1% elastase, 0.1% soybean tyrosine inhibitor, 0.5% bovine serum albumin, 1 μM isoproterenol, 1% MEM vitamin solution, 1% amino acid solution and 2 mM L-glutamine in Ca2+ Krebs constantly bubbled. After 60 min incubation, the solution was replaced with second half of solution (which was kept on ice) preheated in water bath and incubated for another 60 min. Then the tissues were transferred into 1 mM Ca2+ Krebs solution and were subjected to vigorous shaking for 10 min to disperse the cells.
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A10 cells were grown in DMEM/F12 medium with 10% fetal calf serum, 100 μg/ml streptomycin and 100 U/ml penicillin. Cultures were maintained at 37 °C in a humidified incubator in a 95% O2 plus 5% CO2 atmosphere. For electrophysiological experiments, the cells were cultured in coverslips for 1–2 days. 2.4. Measurement of [Ca2+ ]i and Mn2+ quenching of fura-2 The measurement of [Ca2+]i was carried out as previously reported (Tao et al., 1997). Briefly, the freshly isolated rat aorta smooth muscle cells were incubated in DMEM/F12 medium containing 2 μM fura-2/AM for 45 min at room temperature. The extracellular fura-2/AM was washed out with physiological solution (mM: NaCl 130, KCl 5, MgCl2 1, CaCl2 1.3, HEPES 20, glucose 10 and 0.1% bovine serum albumin pH 7.4). The [Ca2+]i was monitored by a RF-5000 fluorescence Spectrofluorophotometer (Shimadzu, Japan) with dual excitation at 340 nm/380 nm and emission at 500 nm. [Ca2+]i was calculated from the following formula: [Ca2+]i = Kd × Sf380/b380 (R − Rmin) / (Rmax − R). Where, Kd is 225 nM in the cytoplasmic environment; Sf380/b380 is the ratio of the intensities of the free and bound dye forms at 380 nm; R is the fluorescence ratio (340 nm/ 380 nm) of intracellular fura-2; Rmax and Rmin are the maximal and minimal fluorescence ratio obtained by addition of Triton X100 (final concentration is 0.09%) and EGTA (final concentration is 3 mM) respectively. In the Mn2+ quenching fura-2 experiments, when the fluorescence baseline was stable, Mn2+ was added, and the final concentration of Mn2+ was 500 μM.
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Maximum rate of quenching of fura-2 fluorescence induced by Mn2+ was measured. 2.5.
45
Ca2+ efflux
New Zealand rabbits of either sex weighing 1.7 ± 0.3 kg were anaesthetized by methoxyflurane and killed by exsanguinations. Segments of thoracic aorta were cut into small rings (20–40 mg). The preparations were equilibrated at 37 °C for 90 min in HEPES buffer solution (mM: NaCl 160, KCl 4.5, MgCl2 1.0, CaCl2 1.5, glucose 10, HEPES 5, pH 7.2) and then incubated in a low Ca2+ HEPES buffer solution (45CaCl2 0.2 mM, 37 MBq/L) for 20 min. 45 Ca2+ bound on the external surface of membrane was washed away by stirring the preparation in a high CaCl2 HEPES buffer solution (mM: CaCl2 20.5 and EGTA 20) for 6 s. Then the preparations were moved from one into another in a series of tubes at 10 min intervals containing 2 ml HEPES buffer solution at 37 °C. The radioactivity in each tube and tissue determined by Beckman LS-3801 liquid scintillation counter was totaled, and the results were expressed as the loss of 45Ca2+ (pM/min) in each tube (Deth and Van Breemen, 1977). 2.6. Electrophysiological experiments Thapsigargin is a sarcoplasmic reticulum Ca-ATPase pump inhibitor, and inhibits Ca2+ uptake into sarcoplasmic reticulum followed by the depletion of Ca2+ store, leading the receptorindependent inward Ca2+ current through SOCC. The ROCC
Fig. 2. Effects of ginsenoside-Rd (Rd) on rat aortic ring contraction induced by 5-HT, phenylephrine (Phe) and KCl. A. Representative traces of contractive responses to 5-HT and phenylephrine in rat aorta (aortic rings) and the effects of ginsenoside-Rd on contractive responses in Ca2+-free media and subsequent addition of Ca2+. B. Densitometric analysis shows that pretreatment with 100 μM ginsenoside-Rd for 10 min, ginsenoside-Rd had no effect on the responses induced by both 5-HT and phenylephrine in the Ca2+-free media ( p N 0.05; n = 8 for each group), whereas it could significantly reduce the responses to subsequent addition of Ca2+ (⁎⁎P b 0.01; n = 8 for each group). C. Representative traces of effects of ginsenoside-Rd and nifedipine (nif ) on contractive responses to KCl in rat aorta. D. Densitometric analysis on the effect of ginsenoside-Rd on contractive response to KCl. 10–80 μM ginsenoside-Rd did not inhibit KCl-induced contractile response, whereas nifedipine almost completely blocked this response.
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Table 1 Effects of 100 μM ginsenoside-Rd (Rd) and 1 μM nifedipine (Nif ) on the increase in [Ca2+]i (nM) induced by 100 mM KCl and 10 μM phenylephrine (each value from 10 different experiments) KCl
Control Rd Nif
Phenylephrine
Resting
Normal sol
Resting
Ca2+-free
Addition of Ca2+
107.2 ± 14.9 99.8 ± 17.0 102 ± 13.5
267.9 ± 32.4 252.0 ± 32.0 105.0 ± 5.4a
99.9 ± 8.7 97.7 ± 11.9 98.7 ± 10.0
157.0 ± 6.2 160.0 ± 19.7 159.0 ± 8.4
239.7 ± 14.0 197.8 ± 5.9a 179.0 ± 5.6a
P b 0.01, compared with control.
a
and SOCC currents in A10 cells were evoked by phenylephrine and thapsigargin respectively, and recorded with whole-cell recording patch-clamp technique. The intracellular solution contained (mM): CsCl 140, MgCl2 2, EDTA: 10, ATP 0.3, HEPES 10, pH 7.2 with CsOH. The bath solution contained (mM): NaCl 140, MgCl2 1.2, CaCl2 1.2, glucose 10, HEPES 11.5, pH 7.4 with NaOH. Patch pipettes, made of borosilicate glass, had resistances of 3–5 MΩ, when it was filled with the intracellular solution. Cells were held at a potential of − 60 mV. In the whole-cell voltage-dependent Ca2+ current recording experiment, the pipette solution contained (mM): CsCl 120, MgCl2 3, Mg-ATP 5, EGTA 10 and Cs-HEPES 5, pH 7.4 and the bath solution contained (mM): NaCl 125, CaCl2 10, MgCl2 1, CsCl 5.4, Glucose 10 and Na-HEPES 10, pH 7.4. Currents were recorded at a room temperature (25 °C) with an Axopatch 200 B Amplifier (Axon instruments Inc, Foster City, CA, USA.), filtered at 1 kHz with four-pole Bessel filter, ROCC and SOCC currents were digitized at 25 Hz and voltage-dependent Ca2+ currents were digitized at 5 kHz. The data were stored using a Digidata 1322A analog/digital interface (Axon instruments Inc.) and the pCLAMP 8 software (Axon instruments Inc).
radio-ligand. It was freshly diluted from a stock solution with cold Mg-MOPS buffer. The incubation media contained 100 μl of MgMOPS buffer with or without Rd and 50 μl of diluted radioligand. The reaction was started by adding 100 μl of membrane suspension to make a final volume of 250 μl. Incubation was carried out in a gyratory shaker water bath at 25 °C for 25 min. The reaction was terminated by adding 2 ml of cold (4 °C) MgMOPS buffer to the entire incubation mixture. Aliquots of 2 ml from this tube were filtered rapidly over Whatman glass filter GF/C filters. Each filter was washed with cold Mg-MOPS buffer thrice (5 ml each time). Total time of filtration and washing averaged 20 s. Filters were dried overnight at 20 °C. Then filters were left to equilibrate in the scintillation cocktail at 20 °C for about 10 h and then counted in the liquid scintillation counter. In the blank tubes, the membrane was replaced by sucrose-MOPS buffer (sucrose 0.25 mM, and MOPS 10 mM, pH 7.4). Specific binding for [3H]-prazosin was calculated. 2.8. Statistical analysis All data are expressed as mean ± S.E.M. Statistical analyses were performed using Student's t test. P values less than 0.05 were considered significant.
2.7. Subcellular membrane binding experiment Plasma membrane vesicles were prepared (Kwan et al., 1979). Briefly, mongrel dogs (weighing 10–20 kg) were killed by injection of sodium pentobarbital 100 mg/kg and the aorta was removed and placed in ice-cold 250 mM sucrose solution buffer with 10 mM imidazole at pH 7.2. The vasculatures were trimmed to remove the connective tissues. Isolated arteries were homogenized in a sucrose buffer solution (10 ml/g wet weight) with a Polytron. The vascular muscle homogenate was centrifuged at 900 g for 10 min to remove connective tissues and cell debris. The supernatant was centrifuged at 10,000 g for 10 min to remove the mitochondrial membranes and then at 100,000 g for 40 min to sediment the crude microsomal membranes. The pellet was resuspended and centrifuged again at 10,000 g for 10 min to further remove the contaminating mitochondrial fragments. The refined microsomal membrane fraction was used for radio-ligand-receptor binding and Ca2+ accumulation experiments. This membrane fraction (microsome-2) is highly enriched in plasma membranes (Kwan, 1985). Radio-ligand-receptor binding was done in microsome-2. MgMOPS buffer (MOPS 50 mM and MgCl2 10 mM pH 7.2) was used in the incubation media. [3H]-prazosin was used as the
Fig. 3. Representative traces of effect of ginsenoside-Rd (Rd) on the Ca2+ influx induced by thapsigargin (TG) (A) and phenylephrine (Phe) (B) in a concentrationdependent manner. C. Densitometric analysis shows that ginsenoside-Rd had more pronounced inhibitory effect on phenylephrine-induced [Ca2+]i than that of thapsigargin (n = 10; ⁎⁎P b 0.01, compared with thapsigargin).
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3. Results
3.2. Effect on Ca2+ entry
3.1. Effects on vascular contractile responses
In Ca2+-free media, phenylephrine produced a transient rise in [Ca2+]i, which was owing to intracellular Ca2+ release. Subsequent addition of Ca2+ induced sustained increase in [Ca2+]i which was due to extracellular Ca2+ influx through the plasma membrane Ca2+ channels. Ginsenoside-Rd only reduced the sustained increase in [Ca2+]i, and did not change both transient rise in [Ca2+]i induced by phenylephrine and elevation of [Ca2+]i induced by 100 mM KCl which was completely blocked by 1 μM nifedipine (Table 1). Fig. 3 shows that, in normal media, ginsenoside-Rd inhibited the increase in [Ca2+]i induced by both 1 μM thapsigargin (Fig. 3A) and 10 μM phenylephrine (Fig. 3B) in a concentration-dependent manner. It appears that the effect of ginsenosideRd on [Ca2+]i induced by phenylephrine is stronger than that on thapsigargin-induced [Ca2+]i (Fig. 3C). The effect of ginsenoside-Rd on functional activities of ROCC and SOCC was further identified by quenching fura-2 fluorescence with Mn2+. OAG directly evokes receptor-operated cation entry, not mediated by PKC-dependent phosphorylation (Hofmann et al., 1999; Lin et al., 2004), suggesting OAG-induced cation entry is only due to ROCC. Thus, OAG- and thapsigargin-induced cation entries are only related to ROCC and SOCC, respectively. In the
At rat aortic rings, 10 μM phenylephrine and 10 μM 5-HT produced a transient contractile response in Ca2+-free Krebs solution which was owing to intracellular Ca2+ release, and induced further contractile response following subsequent addition of Ca2+ which was due to extracellular Ca2+ influx through the plasma membrane Ca2+ channels. Pretreatment with 100 μM ginsenoside-Rd for 10 min, ginsenoside-Rd significantly reduced the responses induced by subsequent addition of Ca2+ without any significant effect on the transient responses (Fig. 2A and B). Ginsenoside-Rd had no any inhibitory effect on the contractile response induced by 100 mM KCl which was nearly completely blocked by 1 μM nifedipine, a highly selective blocker of VDCC (Fig. 2C and D). When 1 μM nifedipine blocked phenylephrine-induced contractile response from 94.8 ± 3.1 to 50.9 ± 2.1 mg/mg wet tissue (n = 8; P b 0.05), which was owing to block VDCC, GinsenosideRd still produced further inhibitory effect, and the response was further decreased to 14.9 ± 9.6 mg/mg wet tissue (n = 8; P b 0.01, compared with pretreatment of ginsenoside-Rd).
Fig. 4. Effect of ginsenoside-Rd (Rd) on OAG- and thapsigargin (TG)-induced cation entry by Mn2+ quenching of fura-2 in A10 cells. A. Representative traces of control, OAG and OAG + 40 μM ginsenoside-Rd. B. Representative traces of control, TG and TG + 40 μM ginsenoside-Rd. C. and D. Densitometric analysis of effect of ginsenoside-Rd on OAG- (C; n = 6) and thapsigargin- (D; n = 6) induced cation entry.
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Fig. 5. The effect of ginsenoside-Rd (Rd) on 45Ca2+ efflux induced by phenylephrine (Phe; data from 5 different experiments). In the interval of 50 to 60 min, phenylephrine was added, and the loss of 45Ca2+ was remarkably increased, reflecting the increase in Ca2+ release. Ginsenoside-Rd had no effect on the loss of 45Ca2+ induced by phenylephrine during 50 to 60 min.
by 100 μM OAG significantly increased the maximum quenching rate to 42.1± 1.8% (from 6 different experiments, P b 0.01; Fig. 4A). Also, cation entry by depleting intracellular Ca2+ stores following exposure of cells to 1 μM thapsigargin evoked a significant increase in the maximum rate of Mn2+-induced fluorescence quenching (43.0± 1.1%; from 6 different experiments; P b 0.01; Fig. 4B). These suggest that OAG and thapsigargin evoke the opening of ROCC and SOCC, respectively. Ginsenoside-Rd significantly reduced both OAG- and thapsigargin-induced increases of quenching rate in a concentration-dependent manner. For example, at the concentration of 10, 20 and 40 μM, ginsenoside-Rd decreased OAG-induced quenching rate from 42.1 ± 1.8% to 37.1 ± 1.8, 31.6 ± 1.9 and 18.6 ± 2.0% (Fig. 4C), respectively. Similar, ginsenoside-Rd, at the concentration of 10, 20 and 40 μM, decreased thapsigargininduced quenching rate from 43.0 ± 1.1% to 37.0 ± 1.1, 30.0 ± 1.5 and 22.1 ± 1.6%, respectively (Fig. 4D). 3.3. Effect on
absence of thapsigargin and OAG, 500 μM Mn evoked a slow and weak quenching of fura-2 fluorescence (maximum quenching rate was 8.5 ± 2.2%). Activation of receptor-operated cation entry
45
Ca2+ efflux
2+
In order to further confirm the effect of ginsenoside-Rd on Ca2+ release induced by phenylephrine, we used 45Ca2+ radio-
Fig. 6. Effect of ginsenoside-Rd on the inward Ca2+ currents induced by phenylephrine (Phe) and thapsigargin (TG). A. Representative recording of the effects of ginsenoside-Rd on currents induced by phenylephrine and thapsigargin. Ginsenoside-Rd decreased inward Ca2+ current amplitudes in a concentration-dependent manner. B. Summary of the effects of ginsenoside-Rd on currents induced by phenylephrine (n = 9) and thapsigargin (n = 6). It appeared that ginsenoside-Rd had a predominant inhibitory effect on the Ca2+ current induced by phenylephrine (⁎⁎P b 0.01 compared with the effect on the Ca2+ current induced by thapsigargin). C. Representative traces of effects of ginsenoside-Rd, Bay K8644 and nifedipine on voltage-dependent Ca2+ currents. The membrane voltage was clamped at − 60 mV. The depolarization from −60 mV to 0 mV for 500 ms produced an inward Ca2+ current (a). 100 μM ginsenoside-Rd had no effect on this current (b), whereas Bay K8644 enhanced this current (c), and nifedipine inhibited it (d). D. I–V relationships for voltage-dependent Ca2+ currents following treatment with Bay K8644 (n = 5), nifedipine (n = 5) and ginsenoside-Rd (n = 6).
Y.-Y. Guan et al. / European Journal of Pharmacology 548 (2006) 129–136 Table 2 The effect of 100 μM ginsenoside-Rd (Rd) on [3H]-prazosin binding to microsome-2 from dog aorta (each value from 5 different experiments)
Control Rd
Bmax (fmol/mg)
Kd (nM)
Hill CO
C Co
344.5 ± 64.5 339.4 ± 66.9
0.125 ± 0.028 0.1555 ± 0.019
1.001 ± 0.056 0.991 ± 0.017
0.989 ± 0.004 0.993 ± 0.002
trace technique to measure phenylephrine-induced 45Ca2+ efflux, which was owing to Ca2+ release from intracellular Ca2+ stores, in rabbit aorta tissues. Fig. 5 shows that exposure of aorta tissue to 10 μM phenylephrine significantly increased 45Ca2+ efflux, and ginsenoside-Rd did not alter the increase of 45Ca2+ efflux induced by phenylephrine. 3.4. Effects on inward Ca2+ currents induced by phenylephrine and thapsigargin When the A10 cells were held at a potential of −60 mV, both 10 μM phenylephrine and 1 μM thapsigargin evoked a sustained inward Ca2+ current which was inhibited by 0Na+/Ca2+ solution and Gd3+, but not altered by nifepidine (Zhou et al., 2006). Ginsenoside-Rd could significantly inhibit these inward Ca2+ currents in a concentration-dependent manner (Fig. 6A). However, as shown in Fig. 6B, it appears that ginsenoside-Rd inhibits ROCC current more potently than SOCC current (P b 0.01). In contrast, ginsenoside-Rd did not change the inward voltagedependent Ca2+ current, which was enhanced by Bay K8644and completely blocked by nifedipine (Fig. 6C and D). 3.5. Effect on the ligand-receptor binding and affinity To test whether ginsenoside-Rd affects phenylephrine-induced Ca2+ entry by interfering with the plasma membrane receptor functions, we examined the effects of ginsenoside-Rd on α1-adrenoceptor affinity and binding amount to the [3H]prazosin ligand. The data from radio-ligand-receptor binding experiment showed that 100 μM ginsenoside-Rd did not change the Bmax and Kd values (Table 2). These results suggested that ginsenoside-Rd lacks effect on binding site and affinity of [3H]prazosin at the plasma membrane fractions from dog aorta. 4. Discussion In our previous work, we had found that total saponins of panax notoginseng had inhibitory effects on the contractile response and 45Ca influx induced by activation of α1-adrenoceptor without effect on Ca2+ release and the response to high K+ in vascular smooth muscle (Guan et al., 1985, 1988, 1994). These results suggest that total saponins of panax notoginseng may have an inhibitory effect on the Ca2+ influx through ROCC. Ginsenoside-Rd from total saponins of panax notoginseng has a molecular weight of 1001.2 and the molecular structure formula of C48H82O18·3H2O. In the present study, the results exhibit that ginsenoside-Rd inhibited the contractile responses induced by activations of α1-adrenoceptor and 5-HT receptor, and the Ca2+ influx induced by both phenylephrine and thapsigargin. It is
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known that activation of receptors in the plasmic membrane can cause Ca2+ release from intracellular Ca2+ stores followed by the depletion of intracellular Ca2+ stores and lead extracellular Ca2+ influx. Thus, logically, if ginsenoside-Rd produces an inhibition of Ca2+ release, it also can reduce receptor-activated Ca2+ entry. However, it is highly unlikely that the inhibitory effect of ginsenoside-Rd on the receptor- and Ca2+ store depletion-mediated Ca2+ influx is related to inhibition of Ca2+ release, because ginsenoside-Rd lacks the inhibitory effect on these responses to thapsigargin and phenylephrine in Ca2+-free media. In addition, ginsenoside-Rd did not alter the 45Ca2+ efflux induced by Phe which was due to Ca2+ release. We also exclude the possibility that ginsenoside-Rd produces the effect on Ca2+ entry induced by phenylephrine through the changes of α-adrenoceptor affinity and binding site, because ginsenoside-Rd lacks the effect on binding site and the affinity of [3H]-prazosin at the plasma membrane fractions of dog aorta. Present results further reveal that ginsenoside-Rd remarkably reduced both of phenylephrine- and thapsigargin-induced inward Ca2+ currents, and OAG- and thapsigargin-induced cation entries, which were considered to couple with ROCC and SOCC respectively, whereas nifedipine did not alter these responses. On the other hand, ginsenoside-Rd does not have any significant effect on KCl-induced contractile response and Ca2+ influx, which were almost completely blocked by nifedipine, a highly selective VDCC blocker. Furthermore, ginsenoside-Rd did not inhibit the voltage-dependent inward Ca2+ currents, which were enhanced by Bay K8644 and blocked by nifedipine. Taken together, our results provide compelling evidence that ginsenoside-Rd inhibits Ca2+ influx through ROCC and SOCC without effects on VDCC and Ca2+ release in vascular smooth muscle cells, suggesting ginsenoside-Rd is a major active component that is responsible for the previously reported Ca2+ influx blocking activity of total saponins from panax notoginseng (Guan et al., 1985, 1988, 1994). Ginsenoside-Rd shows a little more sensitive to the Ca2+ influx and inward Ca2+ current induced by activation of α1-adrenoceptor than to that induced by depletion of Ca2+ store, it appears, whereas ginsenoside-Rd has the same sensitivity to both OAG- and thapsigargin-induced cation entries. The possible explanation for the above difference is that receptor-dependent-Ca2+ entry is not only related to ROCC, but also to SOCC and other pathway (Shuttleworth, 2004). These results are also consistent with our previous report that both capacitative and non-capacitative Ca2+ entry pathways are involved in Ca 2+ influx induced by activation of α1-adrenoceptor in A10 cell (Zhou et al., 2006). Compared with the activation of α1-adrenoceptor, OAG only couples to ROCC and induces Ca2+ entry by directly activating TRPCs related to receptor-operated Ca2+ entry, not by protein kinase Cdependent phosphorylation. In addition of panax notoginseng, ginsenoside-Rd also can be extracted from various ginsengs such as North American ginseng and Panax ginseng. It has been found that ginsenoside-Rd from various ginsengs can produce multiple pharmacological effects, such as enhancing astrocyte differentiation from neural stem cells (Shi et al., 2005), inhibiting protein tyrosine kinase activation induced by hypoxia/reoxygenation (Dou et al., 2001), decreasing gap junction-mediated intracellular communication
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function (Zhang et al., 2001) and oxidative damage (Yokozawa et al., 2004), attenuating kainic acid-induced death in mice (Lee et al., 2003), inducing proliferation of mouse mesangial cells (Yokozawa et al., 1994), immobilizing stress-induced increase in plasma IL-6 level (Kim et al., 2003) and diminishing 3-nitropropionic acid-induced motor impairment and cell loss in the striatum (Lian et al., 2005). However, the mechanisms behind these effects of ginsenoside-Rd remain unclear. Our present study may provide an explanation for multiple pharmacological effects of ginsenoside-Rd. Both ROCC and SOCC are widely expressed in various excitable and non-excitable cells, and are also reported to be involved in many physiological functions and pathophysiological processes (Parekh and Putney, 2005). For example, both ROCC and SOCC have been suggested to play an important role in cell proliferation, apoptosis (Parekh and Penner, 1997), hypoxia/ ischemia-induced brain neuron injury (Silver and Erecinska, 1990), traumatic brain injury (Nilsson et al., 1996), hyperactivity of contractile responses in gastric fundus of diabetic (Aihara and Sakai, 1989) and inflammatory response (Li et al., 2002). It is well known that these pathophysiological processes are coupled with intracellular Ca2+ overload which is usually due to Ca2+ influx through ROCC or SOCC rather than VDCC. Therefore, searching for more specific and potent ROCC or SOCC blockers becomes desirable and extremely important for the development of new drugs for these diseases (Li et al., 2004). It has been reported that some 1,4;dihydropyridine derivatives which were structurally modified could play the inhibitory effect on SOCC (Harper et al., 2003), and the synthetic estrogen agonist, diethylstilbestrol, could inhibit SOCC and capacitative Ca2+ influx (Zakharov et al., 2004). Since ginsenoside-Rd is structurally different from current 1,4;dihydropyridine derivatives, present results might provide a bright and useful clue for the development of new Ca2+ entry blocker of ROCC and SOCC. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30472021), by Science Foundation of the Ministry of Education in China (No. 20050558072), and by China Medical Board (No. 00730). References Aihara, K., Sakai, Y., 1989. Hyperreactivity of contractile response in gastric fundus smooth muscle from rats with diabetes induced by streptozotocin. Arch. Int. Pharmacodyn. Ther. 302, 220–231. Deth, C., Van Breemen, C., 1977. Agonist induced release of intracellular Ca2+ in the rabbit aorta. J. Membr. Biol. 30, 363–380. Dou, D.Q., Zhang, Y.W., Zhang, L., Chen, Y.J., Yao, X.S., 2001. The inhibitory effects of ginsenosides on protein tyrosine kinase activated by hypoxia/ reoxygenation in cultured human umbilical vein endothelial cells. Planta Med. 67, 19–23. Guan, Y.Y., He, H., Chen, J.X., 1985. Effect of the total saponins of panax notoginseng on contraction of rabbit aortic strips. Acta Pharm. Sin. 6, 267–269. Guan, Y.Y., Kwan, C.Y., He, H., Daniel, E.E., 1988. Inhibition of norepinephrineinduced contractile responses of canine mesenteric artery by plant total saponins. Blood Vessels 25, 312–315.
Guan, Y.Y., Kwan, C.Y., He, H., Sun, J.J., Daniel, E.E., 1994. Effects of panax notoginseng saponins on receptor-operated Ca2+ channels in vascular smooth muscle. Acta Pharm. Sin. 15, 392–398. Harper, J.L., Camerini-Otero, C.S., Li, A.H., Kim, S.A., Jacobson, K.A., Daly, J.W., 2003. Dihydropyridines as inhibitors of capacitative calcium entry in leukemic HL-60 cells. Biochem. Pharmacol. 65, 329–338. Hofmann, T., Obukhov, A.G., Schaefer, M., Harteneck, C., Gudermann, T., Schultz, G., 1999. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259–263. Kim, D.H., Moon, Y.S., Lee, T.H., Jung, J.S., Suh, H.W., Song, D.K., 2003. The inhibitory effect of ginseng saponins on the stress-induced plasma interleukin-6 level in mice. Neurosci. Lett. 353, 13–16. Kwan, C.Y., 1985. Subcellular origin of the oxalate-or inorganic phosphatestimulated Ca2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin. Biochim. Biophys. Acta. 819, 148–152. Kwan, C.Y., Garfield, R.E., Daniel, E.E., 1979. An improved procedure for the isolation of plasma membranes from rat mesenteric arteries. J. Mol. Cell. Cardiol. 11, 639–650. Lee, J.K., Choi, S.S., Lee, H.K., Han, K.J., Han, E.J., Suh, H.W., 2003. Effects of ginsenoside Rd and decursinol on the neurtoxic responses induced by kainic acid in mice. Planta Med. 69, 230–234. Li, S.W., Westwick, J., Poll, CT., 2002. Receptor-operated Ca2+ influx channels in leukocytes: a therapeutic target? Trends Pharmacol. Sci. 23, 63–70. Li, S., Westwick, J., Cox, B., Poll, C.T., 2004. TRP channels as drug targets. Novartis Found. Symp. 258, 204–213. Lian, X.Y., Zhang, Z., Stringer, J.L., 2005. Protective effects of ginseng components in a rodent model of neurodegeneration. Ann. Neurol. 57, 642–648. Lin, M.J., Leung, G.P.H., Zhang, W.M., Yang, X.R., Yip, K.P., Tse, C.M., Sham, J.S.K., 2004. Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells—a novel mechanism of hypoxic pulmonary hypertension. Circ. Res. 95, 496–505. Nilsson, P., Laursen, H., Hillered, L., Hansen, A.J., 1996. Ca2+ movements in traumatic brain injury: the role of glutamine receptor-operated ion channels. J cereb Blood Flow Metab. 16, 262–270. Parekh, A.B., Penner, R., 1997. Store depletion and calcium influx. Physiol. Rev. 77, 901–930. Parekh, A.B., Putney Jr., J.W., 2005. Store-operated calcium channels. Physiol. Rev. 85, 757–810. Shi, Q., Hao, Q., Bouissac, J., Lu, J.Y., Tian, S., Luu, B., 2005. Ginsenoside-Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells. Life Sci. 76, 983–995. Silver, I.A., Erecinska, M., 1990. Intracellular and extracellular changes of [Ca2+]I in hypoxia and ischemia in rat brain in vivo. J. Gen. Physiol. 95, 837–866. Shuttleworth, T.J., 2004. Receptor-activated calcium entry channels—who does what, and when? Sci. STKE 243 (pe40). Tao, L., Guan, Y.Y., He, H., 1997. Comparison of the calcium movements induced by á1-adrenoceptor subtypes in HEK cells. Life Sci. 61, 2127–2136. Yokozawa, T., Iwano, M., Dohi, K., Hattori, M., Oura, H., 1994. Inhibitory effects of ginseng on proliferation of cultured mouse mesangial cells. Nippon Jinzo Gakkaishi 36, 13–18. Yokozawa, T., Satoh, A., Cho, E.J., 2004. Ginsenoside-Rd attenuates oxidative damage related to aging in senescence-accelerated mice. J. Pharm. Pharmacol. 56, 107–113. Zakharov, S.I., Smani, T., Dobrydneva, Y., Monje, F., Fichandler, C., Blackmore, P.F., Bolotina, V.M., 2004. Diethylstilbestrol is a potent inhibitor of storeoperated channels and capacitative Ca2+ influx. Mol. Pharmacol. 66, 702–707. Zhang, Y.W., Dou, D.Q., Zhang, L., Chen, Y.J., Yao, X.S., 2001. Effects of ginsenosides from Panax ginseng on cell-to-cell communication function mediated by gap junctions. Planta Med. 67, 417–422. Zhou, J.G., Qiu, Q.Y., Zhang, Z., Liu, Y.J., Guan, Y.Y., 2006. Evidence for capacitative and non-capacitative Ca2+ entry pathways coexist in A10 vascular smooth muscle cells. Life Sci. 78, 1558–1563.
Ginsenoside-Rd from panax notoginseng blocks Ca 2+ influx through receptor- and store-operated Ca 2+ channels in vascular smooth muscle cells Yong-Yuan Guan ⁎, Jia-Guo Zhou, Zheng Zhang, Guan-Lei Wang, Bing-Xiang Cai, Liang Hong, Qin-Ying Qiu, Hua He Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, Guangzhou, PR China Received 17 April 2006; received in revised form 21 July 2006; accepted 2 August 2006 Available online 17 August 2006
Abstract Previously, it was found that total saponins from panax notoginseng inhibited Ca2+ influx coupling to activation of α1-adrenoceptor. This study was designed to investigate the effects of ginsenoside-Rd from total saponins of panax notoginseng on receptor-operated (ROCC) and storeoperated (SOCC) Ca2+ channels in vascular smooth muscle cells using fura-2 fluorescence, whole cell patch clamp ion channel recording, radioligand-receptor binding, 45Ca2+ radio-trace and organ bath techniques. It was found that ginsenoside-Rd reduced phenylephrine-induced contractile responses and Ca2+ influx in normal media without significant effect on these responses in Ca2+-free media. Ginsenoside-Rd also decreased phenylephrine- and thapsigargin-induced inward Ca2+ currents, and attenuated thapsigargin- and 1-oleoy-2-acetyl-sn-glycerol (OAG)induced cation entries that are coupled to ROCC and SOCC respectively. Ginsenoside-Rd failed to inhibit KCl-induced contraction of rat aortal rings and Ca2+ influx, and did not alter voltage-dependent inward Ca2+ current (VDCC) which was blocked by nifedipine. Also, ginsenoside-Rd did not change binding site and affinity of [3H]-prazosin for α1-adrenoceptor in the vascular plasma membrane. These results suggest that ginsenoside-Rd, as an inhibitor, remarkably inhibits Ca2+ entry through ROCC and SOCC without effects on VDCC and Ca2+ release in vascular smooth muscle cells. © 2006 Elsevier B.V. All rights reserved. Keywords: Ginsenoside-Rd; Receptor-operated; Store-operated; Ca2+ channel; Ca2+ entry
1. Introduction Panax notoginseng [(Buck) F.H. Chen] is a famous traditional Chinese herb medicine. In China, it has long been used in clinical treatment as a hemostatic and analgetic drug. As the effective fractions of panax notoginseng, the total saponins of Panax notoginseng have been used in clinic for the treatment of cardiovascular diseases and stroke in China since 1982. However, it is not clear that the mechanisms are involved in the effect of total saponins from panax notoginseng on cardiovascular diseases.
⁎ Corresponding author. Department of Pharmacology, Zhongshan Medical College, Sun Yat-Sen University, 74 Zhongshan 2 Rd, Guangzhou, Guangdong, 510089, PR China. Tel.: +86 20 87331857; fax: +86 20 87331209. E-mail address: [email protected] (Y.-Y. Guan). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.08.001
In 1985, we first reported that total saponins of panax notoginseng inhibited contractive response induced by norepinephrine without an effect on the contraction induced by KCl in rabbit aortic rings, and therefore suggested that total saponins of panax notoginseng dilated the vascular muscle through inhibition of α-adrenoceptor operated Ca2+ influx (Guan et al., 1985). Later, using fura-2 fluorescence and 45Ca2+ trace techniques, we further found that total saponins of panax notoginseng reduced 45Ca2+ influx and cytoplasmic free Ca2+ level ([Ca2+]i) induced by phenylephrine, whereas these total saponins failed to change KClinduced 45Ca2+ influx and the increase of [Ca2+]i. Total saponins of panax notoginseng did not change phenylephrine-induced 45 Ca2+ efflux which was owing to intracellular Ca2+ release, and the affinity of α-adrenoceptor (Guan et al., 1988, 1994). These results suggested that total saponins of panax notoginseng have an inhibitory effect on receptor-operated Ca2+ entry. Based on these
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data, we try to find out which kind of sole purified component has an inhibitory effect on receptor-operated Ca2+ entry through screening various purified components extracted from total saponins of panax notoginseng. We firstly found that ginsenoside-Rd (Dammar-24(25)-ene-3β,12β,20(S)-triol-(20-O-β-Dglucopyranosyl)-3-O-β-D-glucopyranosyl-(1 → 2)-β-D-glucopyranoside; Fig. 1), a purified component from total saponins of panax notoginseng, reduced phenylephrine-induced contractile response without an effect on KCl-induced contractile response, whereas other purified components do not have these unique characteristics as ginsenoside-Rd. These effects of ginsenosideRd are consistent with the previously reported results of total saponins from panax notoginseng (Guan et al., 1985, 1988, 1994). In this study, we used fura-2 fluorescence, whole cell patch clamp ion channel recording, radio-ligand-receptor binding, 45Ca2+ radio-trace and organ bath techniques to further characterize ginsenoside-Rd having an inhibitory effect on Ca2+ entry through receptor-operated Ca2+ channel (ROCC) and store-operated Ca2+ channel (SOCC) in vascular smooth muscle cells. 2. Materials and methods 2.1. Materials and animals Ginsenoside-Rd with a purity of 98% was obtained from TaiHe Biopharmaceutical Co. Ltd. (Guangzhou, P.R. China), and was made the 10 mM stock solution with 3% tween-80. Fura-2/ AM was obtained from Boehringer Mannheim Co. EGTA, Triton X-100, tween-80, phenylephrine, 5-HT, bovine serum albumin, nifedipine, S-(−)-1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl) phenyl]-3-pyridinecarboxylic acid methyl ester (Bay K8644), thapsigargin and 1-oleoy-2-acetyl-sn-glycerol (OAG) were obtained from Sigma (St. Louis, MO, USA). DMEM/F12, MEM vitamin solution and amino acid solution were obtained from GIBICO. [3H]-prazosin was purchased from New England Nuclear (Boston MA, USA). A10 embryonic rat thoracic aortic smooth muscle cell line (A10 cells) was obtained from the American Type Culture Collection (Rockville, MD, USA). 45 CaCl2 (122.8 GBq/mol) was obtained from Institute of Atomic Energy, Chinese Academy of Sciences (Beijing).
Fig. 1. The structure of ginsenoside-Rd. The chemical structure formula of ginsenoside-Rd is C48H82O18·3H2O and the molecular weight is 1001.20.
All animals were supplied by the Experimental Animal Center of Sun Yat-Sen University in Guangzhou, China. All procedures complied with the standards for the care and use of animal subjects as stated in the Guide for the Care and Use of Laboratory Animals (issued by the Ministry of Science and Technology of China, Beijing). 2.2. Vascular contractile response Wistar rats (150–200 g) were anaesthetized by methoxyflurane and decapitated. The rat thoracic aorta was removed rapidly and placed in Krebs solution of the following composition (mM): NaCl 115.5, KCl 4.6, Na2HPO4 1.16, CaCl2 2.5, MgSO4 1.16, NaHCO3 21.9 and glucose 11.1 (pH 7.4). The artery was carefully cleaned of fat and connective tissue. The endothelium was removed by gently rubbing with a cotton swab moistened with Krebs solution. Then, the vessel was cut into small rings and suspended in the organ baths (3 ml capacity) containing Krebs solution at 37 °C and aerated with 95% O2 plus 5% CO2. The rings were allowed to equilibrate for at least 120 min under the resting force of 1.5 g. During the equilibration period, the bath solution was changed every 20 min. Stable contractile response to 100 mM KCl was obtained. The contractile responses were considered reproducible if the maximal tension of 2 consecutive contractions differed by b10%. The preparations which failed to produce reproducible contractions were discarded. The preparation was washed and incubated with Ca2+-free Krebs solution of the following composition (mM): NaCl 115.5, KCl 4.6, Na2HPO4 1.16, MgSO4 1.16, NaHCO3 21.9, EGTA 0.05 and glucose 11.1 (pH 7.4). In Ca2+-free Krebs solution, the agonists produced an initial response and made the further contractile response to subsequent addition of CaCl2 into bath (The final concentration in the bath was 2.5 mM.). The tension was recorded by Powerlab system (AD Instrument, Australia). 2.3. Cell preparation and culture Fresh isolated rat aorta smooth muscle cells were prepared from rat thoracic aorta. Wistar rats (180–200 g) were anaesthetized with methoxyflurane and decapitated. Thoracic aorta was aseptically removed rapidly and immersed in physiological balanced saline (PBS, in mM) containing NaCl 138.0, KCl 5.0, Na2HPO4 0.3, KH2PO4 0.3, NaHCO3 4.0. After being cleaned of connective tissue and adhered fat tissue, isolated arteries were cut open longitudinally and the endothelium was removed by gently rubbing with a cotton swab moistened with PBS. Denuded aorta was cut into small pieces about 1–3 mm3, then was incubated in a gently shaking bath at 37 °C in 1 ml enzymatic solution containing 14 mg collagenase type IA, 4 mM ATP, 0.1% elastase, 0.1% soybean tyrosine inhibitor, 0.5% bovine serum albumin, 1 μM isoproterenol, 1% MEM vitamin solution, 1% amino acid solution and 2 mM L-glutamine in Ca2+ Krebs constantly bubbled. After 60 min incubation, the solution was replaced with second half of solution (which was kept on ice) preheated in water bath and incubated for another 60 min. Then the tissues were transferred into 1 mM Ca2+ Krebs solution and were subjected to vigorous shaking for 10 min to disperse the cells.
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A10 cells were grown in DMEM/F12 medium with 10% fetal calf serum, 100 μg/ml streptomycin and 100 U/ml penicillin. Cultures were maintained at 37 °C in a humidified incubator in a 95% O2 plus 5% CO2 atmosphere. For electrophysiological experiments, the cells were cultured in coverslips for 1–2 days. 2.4. Measurement of [Ca2+ ]i and Mn2+ quenching of fura-2 The measurement of [Ca2+]i was carried out as previously reported (Tao et al., 1997). Briefly, the freshly isolated rat aorta smooth muscle cells were incubated in DMEM/F12 medium containing 2 μM fura-2/AM for 45 min at room temperature. The extracellular fura-2/AM was washed out with physiological solution (mM: NaCl 130, KCl 5, MgCl2 1, CaCl2 1.3, HEPES 20, glucose 10 and 0.1% bovine serum albumin pH 7.4). The [Ca2+]i was monitored by a RF-5000 fluorescence Spectrofluorophotometer (Shimadzu, Japan) with dual excitation at 340 nm/380 nm and emission at 500 nm. [Ca2+]i was calculated from the following formula: [Ca2+]i = Kd × Sf380/b380 (R − Rmin) / (Rmax − R). Where, Kd is 225 nM in the cytoplasmic environment; Sf380/b380 is the ratio of the intensities of the free and bound dye forms at 380 nm; R is the fluorescence ratio (340 nm/ 380 nm) of intracellular fura-2; Rmax and Rmin are the maximal and minimal fluorescence ratio obtained by addition of Triton X100 (final concentration is 0.09%) and EGTA (final concentration is 3 mM) respectively. In the Mn2+ quenching fura-2 experiments, when the fluorescence baseline was stable, Mn2+ was added, and the final concentration of Mn2+ was 500 μM.
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Maximum rate of quenching of fura-2 fluorescence induced by Mn2+ was measured. 2.5.
45
Ca2+ efflux
New Zealand rabbits of either sex weighing 1.7 ± 0.3 kg were anaesthetized by methoxyflurane and killed by exsanguinations. Segments of thoracic aorta were cut into small rings (20–40 mg). The preparations were equilibrated at 37 °C for 90 min in HEPES buffer solution (mM: NaCl 160, KCl 4.5, MgCl2 1.0, CaCl2 1.5, glucose 10, HEPES 5, pH 7.2) and then incubated in a low Ca2+ HEPES buffer solution (45CaCl2 0.2 mM, 37 MBq/L) for 20 min. 45 Ca2+ bound on the external surface of membrane was washed away by stirring the preparation in a high CaCl2 HEPES buffer solution (mM: CaCl2 20.5 and EGTA 20) for 6 s. Then the preparations were moved from one into another in a series of tubes at 10 min intervals containing 2 ml HEPES buffer solution at 37 °C. The radioactivity in each tube and tissue determined by Beckman LS-3801 liquid scintillation counter was totaled, and the results were expressed as the loss of 45Ca2+ (pM/min) in each tube (Deth and Van Breemen, 1977). 2.6. Electrophysiological experiments Thapsigargin is a sarcoplasmic reticulum Ca-ATPase pump inhibitor, and inhibits Ca2+ uptake into sarcoplasmic reticulum followed by the depletion of Ca2+ store, leading the receptorindependent inward Ca2+ current through SOCC. The ROCC
Fig. 2. Effects of ginsenoside-Rd (Rd) on rat aortic ring contraction induced by 5-HT, phenylephrine (Phe) and KCl. A. Representative traces of contractive responses to 5-HT and phenylephrine in rat aorta (aortic rings) and the effects of ginsenoside-Rd on contractive responses in Ca2+-free media and subsequent addition of Ca2+. B. Densitometric analysis shows that pretreatment with 100 μM ginsenoside-Rd for 10 min, ginsenoside-Rd had no effect on the responses induced by both 5-HT and phenylephrine in the Ca2+-free media ( p N 0.05; n = 8 for each group), whereas it could significantly reduce the responses to subsequent addition of Ca2+ (⁎⁎P b 0.01; n = 8 for each group). C. Representative traces of effects of ginsenoside-Rd and nifedipine (nif ) on contractive responses to KCl in rat aorta. D. Densitometric analysis on the effect of ginsenoside-Rd on contractive response to KCl. 10–80 μM ginsenoside-Rd did not inhibit KCl-induced contractile response, whereas nifedipine almost completely blocked this response.
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Table 1 Effects of 100 μM ginsenoside-Rd (Rd) and 1 μM nifedipine (Nif ) on the increase in [Ca2+]i (nM) induced by 100 mM KCl and 10 μM phenylephrine (each value from 10 different experiments) KCl
Control Rd Nif
Phenylephrine
Resting
Normal sol
Resting
Ca2+-free
Addition of Ca2+
107.2 ± 14.9 99.8 ± 17.0 102 ± 13.5
267.9 ± 32.4 252.0 ± 32.0 105.0 ± 5.4a
99.9 ± 8.7 97.7 ± 11.9 98.7 ± 10.0
157.0 ± 6.2 160.0 ± 19.7 159.0 ± 8.4
239.7 ± 14.0 197.8 ± 5.9a 179.0 ± 5.6a
P b 0.01, compared with control.
a
and SOCC currents in A10 cells were evoked by phenylephrine and thapsigargin respectively, and recorded with whole-cell recording patch-clamp technique. The intracellular solution contained (mM): CsCl 140, MgCl2 2, EDTA: 10, ATP 0.3, HEPES 10, pH 7.2 with CsOH. The bath solution contained (mM): NaCl 140, MgCl2 1.2, CaCl2 1.2, glucose 10, HEPES 11.5, pH 7.4 with NaOH. Patch pipettes, made of borosilicate glass, had resistances of 3–5 MΩ, when it was filled with the intracellular solution. Cells were held at a potential of − 60 mV. In the whole-cell voltage-dependent Ca2+ current recording experiment, the pipette solution contained (mM): CsCl 120, MgCl2 3, Mg-ATP 5, EGTA 10 and Cs-HEPES 5, pH 7.4 and the bath solution contained (mM): NaCl 125, CaCl2 10, MgCl2 1, CsCl 5.4, Glucose 10 and Na-HEPES 10, pH 7.4. Currents were recorded at a room temperature (25 °C) with an Axopatch 200 B Amplifier (Axon instruments Inc, Foster City, CA, USA.), filtered at 1 kHz with four-pole Bessel filter, ROCC and SOCC currents were digitized at 25 Hz and voltage-dependent Ca2+ currents were digitized at 5 kHz. The data were stored using a Digidata 1322A analog/digital interface (Axon instruments Inc.) and the pCLAMP 8 software (Axon instruments Inc).
radio-ligand. It was freshly diluted from a stock solution with cold Mg-MOPS buffer. The incubation media contained 100 μl of MgMOPS buffer with or without Rd and 50 μl of diluted radioligand. The reaction was started by adding 100 μl of membrane suspension to make a final volume of 250 μl. Incubation was carried out in a gyratory shaker water bath at 25 °C for 25 min. The reaction was terminated by adding 2 ml of cold (4 °C) MgMOPS buffer to the entire incubation mixture. Aliquots of 2 ml from this tube were filtered rapidly over Whatman glass filter GF/C filters. Each filter was washed with cold Mg-MOPS buffer thrice (5 ml each time). Total time of filtration and washing averaged 20 s. Filters were dried overnight at 20 °C. Then filters were left to equilibrate in the scintillation cocktail at 20 °C for about 10 h and then counted in the liquid scintillation counter. In the blank tubes, the membrane was replaced by sucrose-MOPS buffer (sucrose 0.25 mM, and MOPS 10 mM, pH 7.4). Specific binding for [3H]-prazosin was calculated. 2.8. Statistical analysis All data are expressed as mean ± S.E.M. Statistical analyses were performed using Student's t test. P values less than 0.05 were considered significant.
2.7. Subcellular membrane binding experiment Plasma membrane vesicles were prepared (Kwan et al., 1979). Briefly, mongrel dogs (weighing 10–20 kg) were killed by injection of sodium pentobarbital 100 mg/kg and the aorta was removed and placed in ice-cold 250 mM sucrose solution buffer with 10 mM imidazole at pH 7.2. The vasculatures were trimmed to remove the connective tissues. Isolated arteries were homogenized in a sucrose buffer solution (10 ml/g wet weight) with a Polytron. The vascular muscle homogenate was centrifuged at 900 g for 10 min to remove connective tissues and cell debris. The supernatant was centrifuged at 10,000 g for 10 min to remove the mitochondrial membranes and then at 100,000 g for 40 min to sediment the crude microsomal membranes. The pellet was resuspended and centrifuged again at 10,000 g for 10 min to further remove the contaminating mitochondrial fragments. The refined microsomal membrane fraction was used for radio-ligand-receptor binding and Ca2+ accumulation experiments. This membrane fraction (microsome-2) is highly enriched in plasma membranes (Kwan, 1985). Radio-ligand-receptor binding was done in microsome-2. MgMOPS buffer (MOPS 50 mM and MgCl2 10 mM pH 7.2) was used in the incubation media. [3H]-prazosin was used as the
Fig. 3. Representative traces of effect of ginsenoside-Rd (Rd) on the Ca2+ influx induced by thapsigargin (TG) (A) and phenylephrine (Phe) (B) in a concentrationdependent manner. C. Densitometric analysis shows that ginsenoside-Rd had more pronounced inhibitory effect on phenylephrine-induced [Ca2+]i than that of thapsigargin (n = 10; ⁎⁎P b 0.01, compared with thapsigargin).
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3. Results
3.2. Effect on Ca2+ entry
3.1. Effects on vascular contractile responses
In Ca2+-free media, phenylephrine produced a transient rise in [Ca2+]i, which was owing to intracellular Ca2+ release. Subsequent addition of Ca2+ induced sustained increase in [Ca2+]i which was due to extracellular Ca2+ influx through the plasma membrane Ca2+ channels. Ginsenoside-Rd only reduced the sustained increase in [Ca2+]i, and did not change both transient rise in [Ca2+]i induced by phenylephrine and elevation of [Ca2+]i induced by 100 mM KCl which was completely blocked by 1 μM nifedipine (Table 1). Fig. 3 shows that, in normal media, ginsenoside-Rd inhibited the increase in [Ca2+]i induced by both 1 μM thapsigargin (Fig. 3A) and 10 μM phenylephrine (Fig. 3B) in a concentration-dependent manner. It appears that the effect of ginsenosideRd on [Ca2+]i induced by phenylephrine is stronger than that on thapsigargin-induced [Ca2+]i (Fig. 3C). The effect of ginsenoside-Rd on functional activities of ROCC and SOCC was further identified by quenching fura-2 fluorescence with Mn2+. OAG directly evokes receptor-operated cation entry, not mediated by PKC-dependent phosphorylation (Hofmann et al., 1999; Lin et al., 2004), suggesting OAG-induced cation entry is only due to ROCC. Thus, OAG- and thapsigargin-induced cation entries are only related to ROCC and SOCC, respectively. In the
At rat aortic rings, 10 μM phenylephrine and 10 μM 5-HT produced a transient contractile response in Ca2+-free Krebs solution which was owing to intracellular Ca2+ release, and induced further contractile response following subsequent addition of Ca2+ which was due to extracellular Ca2+ influx through the plasma membrane Ca2+ channels. Pretreatment with 100 μM ginsenoside-Rd for 10 min, ginsenoside-Rd significantly reduced the responses induced by subsequent addition of Ca2+ without any significant effect on the transient responses (Fig. 2A and B). Ginsenoside-Rd had no any inhibitory effect on the contractile response induced by 100 mM KCl which was nearly completely blocked by 1 μM nifedipine, a highly selective blocker of VDCC (Fig. 2C and D). When 1 μM nifedipine blocked phenylephrine-induced contractile response from 94.8 ± 3.1 to 50.9 ± 2.1 mg/mg wet tissue (n = 8; P b 0.05), which was owing to block VDCC, GinsenosideRd still produced further inhibitory effect, and the response was further decreased to 14.9 ± 9.6 mg/mg wet tissue (n = 8; P b 0.01, compared with pretreatment of ginsenoside-Rd).
Fig. 4. Effect of ginsenoside-Rd (Rd) on OAG- and thapsigargin (TG)-induced cation entry by Mn2+ quenching of fura-2 in A10 cells. A. Representative traces of control, OAG and OAG + 40 μM ginsenoside-Rd. B. Representative traces of control, TG and TG + 40 μM ginsenoside-Rd. C. and D. Densitometric analysis of effect of ginsenoside-Rd on OAG- (C; n = 6) and thapsigargin- (D; n = 6) induced cation entry.
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Fig. 5. The effect of ginsenoside-Rd (Rd) on 45Ca2+ efflux induced by phenylephrine (Phe; data from 5 different experiments). In the interval of 50 to 60 min, phenylephrine was added, and the loss of 45Ca2+ was remarkably increased, reflecting the increase in Ca2+ release. Ginsenoside-Rd had no effect on the loss of 45Ca2+ induced by phenylephrine during 50 to 60 min.
by 100 μM OAG significantly increased the maximum quenching rate to 42.1± 1.8% (from 6 different experiments, P b 0.01; Fig. 4A). Also, cation entry by depleting intracellular Ca2+ stores following exposure of cells to 1 μM thapsigargin evoked a significant increase in the maximum rate of Mn2+-induced fluorescence quenching (43.0± 1.1%; from 6 different experiments; P b 0.01; Fig. 4B). These suggest that OAG and thapsigargin evoke the opening of ROCC and SOCC, respectively. Ginsenoside-Rd significantly reduced both OAG- and thapsigargin-induced increases of quenching rate in a concentration-dependent manner. For example, at the concentration of 10, 20 and 40 μM, ginsenoside-Rd decreased OAG-induced quenching rate from 42.1 ± 1.8% to 37.1 ± 1.8, 31.6 ± 1.9 and 18.6 ± 2.0% (Fig. 4C), respectively. Similar, ginsenoside-Rd, at the concentration of 10, 20 and 40 μM, decreased thapsigargininduced quenching rate from 43.0 ± 1.1% to 37.0 ± 1.1, 30.0 ± 1.5 and 22.1 ± 1.6%, respectively (Fig. 4D). 3.3. Effect on
absence of thapsigargin and OAG, 500 μM Mn evoked a slow and weak quenching of fura-2 fluorescence (maximum quenching rate was 8.5 ± 2.2%). Activation of receptor-operated cation entry
45
Ca2+ efflux
2+
In order to further confirm the effect of ginsenoside-Rd on Ca2+ release induced by phenylephrine, we used 45Ca2+ radio-
Fig. 6. Effect of ginsenoside-Rd on the inward Ca2+ currents induced by phenylephrine (Phe) and thapsigargin (TG). A. Representative recording of the effects of ginsenoside-Rd on currents induced by phenylephrine and thapsigargin. Ginsenoside-Rd decreased inward Ca2+ current amplitudes in a concentration-dependent manner. B. Summary of the effects of ginsenoside-Rd on currents induced by phenylephrine (n = 9) and thapsigargin (n = 6). It appeared that ginsenoside-Rd had a predominant inhibitory effect on the Ca2+ current induced by phenylephrine (⁎⁎P b 0.01 compared with the effect on the Ca2+ current induced by thapsigargin). C. Representative traces of effects of ginsenoside-Rd, Bay K8644 and nifedipine on voltage-dependent Ca2+ currents. The membrane voltage was clamped at − 60 mV. The depolarization from −60 mV to 0 mV for 500 ms produced an inward Ca2+ current (a). 100 μM ginsenoside-Rd had no effect on this current (b), whereas Bay K8644 enhanced this current (c), and nifedipine inhibited it (d). D. I–V relationships for voltage-dependent Ca2+ currents following treatment with Bay K8644 (n = 5), nifedipine (n = 5) and ginsenoside-Rd (n = 6).
Y.-Y. Guan et al. / European Journal of Pharmacology 548 (2006) 129–136 Table 2 The effect of 100 μM ginsenoside-Rd (Rd) on [3H]-prazosin binding to microsome-2 from dog aorta (each value from 5 different experiments)
Control Rd
Bmax (fmol/mg)
Kd (nM)
Hill CO
C Co
344.5 ± 64.5 339.4 ± 66.9
0.125 ± 0.028 0.1555 ± 0.019
1.001 ± 0.056 0.991 ± 0.017
0.989 ± 0.004 0.993 ± 0.002
trace technique to measure phenylephrine-induced 45Ca2+ efflux, which was owing to Ca2+ release from intracellular Ca2+ stores, in rabbit aorta tissues. Fig. 5 shows that exposure of aorta tissue to 10 μM phenylephrine significantly increased 45Ca2+ efflux, and ginsenoside-Rd did not alter the increase of 45Ca2+ efflux induced by phenylephrine. 3.4. Effects on inward Ca2+ currents induced by phenylephrine and thapsigargin When the A10 cells were held at a potential of −60 mV, both 10 μM phenylephrine and 1 μM thapsigargin evoked a sustained inward Ca2+ current which was inhibited by 0Na+/Ca2+ solution and Gd3+, but not altered by nifepidine (Zhou et al., 2006). Ginsenoside-Rd could significantly inhibit these inward Ca2+ currents in a concentration-dependent manner (Fig. 6A). However, as shown in Fig. 6B, it appears that ginsenoside-Rd inhibits ROCC current more potently than SOCC current (P b 0.01). In contrast, ginsenoside-Rd did not change the inward voltagedependent Ca2+ current, which was enhanced by Bay K8644and completely blocked by nifedipine (Fig. 6C and D). 3.5. Effect on the ligand-receptor binding and affinity To test whether ginsenoside-Rd affects phenylephrine-induced Ca2+ entry by interfering with the plasma membrane receptor functions, we examined the effects of ginsenoside-Rd on α1-adrenoceptor affinity and binding amount to the [3H]prazosin ligand. The data from radio-ligand-receptor binding experiment showed that 100 μM ginsenoside-Rd did not change the Bmax and Kd values (Table 2). These results suggested that ginsenoside-Rd lacks effect on binding site and affinity of [3H]prazosin at the plasma membrane fractions from dog aorta. 4. Discussion In our previous work, we had found that total saponins of panax notoginseng had inhibitory effects on the contractile response and 45Ca influx induced by activation of α1-adrenoceptor without effect on Ca2+ release and the response to high K+ in vascular smooth muscle (Guan et al., 1985, 1988, 1994). These results suggest that total saponins of panax notoginseng may have an inhibitory effect on the Ca2+ influx through ROCC. Ginsenoside-Rd from total saponins of panax notoginseng has a molecular weight of 1001.2 and the molecular structure formula of C48H82O18·3H2O. In the present study, the results exhibit that ginsenoside-Rd inhibited the contractile responses induced by activations of α1-adrenoceptor and 5-HT receptor, and the Ca2+ influx induced by both phenylephrine and thapsigargin. It is
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known that activation of receptors in the plasmic membrane can cause Ca2+ release from intracellular Ca2+ stores followed by the depletion of intracellular Ca2+ stores and lead extracellular Ca2+ influx. Thus, logically, if ginsenoside-Rd produces an inhibition of Ca2+ release, it also can reduce receptor-activated Ca2+ entry. However, it is highly unlikely that the inhibitory effect of ginsenoside-Rd on the receptor- and Ca2+ store depletion-mediated Ca2+ influx is related to inhibition of Ca2+ release, because ginsenoside-Rd lacks the inhibitory effect on these responses to thapsigargin and phenylephrine in Ca2+-free media. In addition, ginsenoside-Rd did not alter the 45Ca2+ efflux induced by Phe which was due to Ca2+ release. We also exclude the possibility that ginsenoside-Rd produces the effect on Ca2+ entry induced by phenylephrine through the changes of α-adrenoceptor affinity and binding site, because ginsenoside-Rd lacks the effect on binding site and the affinity of [3H]-prazosin at the plasma membrane fractions of dog aorta. Present results further reveal that ginsenoside-Rd remarkably reduced both of phenylephrine- and thapsigargin-induced inward Ca2+ currents, and OAG- and thapsigargin-induced cation entries, which were considered to couple with ROCC and SOCC respectively, whereas nifedipine did not alter these responses. On the other hand, ginsenoside-Rd does not have any significant effect on KCl-induced contractile response and Ca2+ influx, which were almost completely blocked by nifedipine, a highly selective VDCC blocker. Furthermore, ginsenoside-Rd did not inhibit the voltage-dependent inward Ca2+ currents, which were enhanced by Bay K8644 and blocked by nifedipine. Taken together, our results provide compelling evidence that ginsenoside-Rd inhibits Ca2+ influx through ROCC and SOCC without effects on VDCC and Ca2+ release in vascular smooth muscle cells, suggesting ginsenoside-Rd is a major active component that is responsible for the previously reported Ca2+ influx blocking activity of total saponins from panax notoginseng (Guan et al., 1985, 1988, 1994). Ginsenoside-Rd shows a little more sensitive to the Ca2+ influx and inward Ca2+ current induced by activation of α1-adrenoceptor than to that induced by depletion of Ca2+ store, it appears, whereas ginsenoside-Rd has the same sensitivity to both OAG- and thapsigargin-induced cation entries. The possible explanation for the above difference is that receptor-dependent-Ca2+ entry is not only related to ROCC, but also to SOCC and other pathway (Shuttleworth, 2004). These results are also consistent with our previous report that both capacitative and non-capacitative Ca2+ entry pathways are involved in Ca 2+ influx induced by activation of α1-adrenoceptor in A10 cell (Zhou et al., 2006). Compared with the activation of α1-adrenoceptor, OAG only couples to ROCC and induces Ca2+ entry by directly activating TRPCs related to receptor-operated Ca2+ entry, not by protein kinase Cdependent phosphorylation. In addition of panax notoginseng, ginsenoside-Rd also can be extracted from various ginsengs such as North American ginseng and Panax ginseng. It has been found that ginsenoside-Rd from various ginsengs can produce multiple pharmacological effects, such as enhancing astrocyte differentiation from neural stem cells (Shi et al., 2005), inhibiting protein tyrosine kinase activation induced by hypoxia/reoxygenation (Dou et al., 2001), decreasing gap junction-mediated intracellular communication
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function (Zhang et al., 2001) and oxidative damage (Yokozawa et al., 2004), attenuating kainic acid-induced death in mice (Lee et al., 2003), inducing proliferation of mouse mesangial cells (Yokozawa et al., 1994), immobilizing stress-induced increase in plasma IL-6 level (Kim et al., 2003) and diminishing 3-nitropropionic acid-induced motor impairment and cell loss in the striatum (Lian et al., 2005). However, the mechanisms behind these effects of ginsenoside-Rd remain unclear. Our present study may provide an explanation for multiple pharmacological effects of ginsenoside-Rd. Both ROCC and SOCC are widely expressed in various excitable and non-excitable cells, and are also reported to be involved in many physiological functions and pathophysiological processes (Parekh and Putney, 2005). For example, both ROCC and SOCC have been suggested to play an important role in cell proliferation, apoptosis (Parekh and Penner, 1997), hypoxia/ ischemia-induced brain neuron injury (Silver and Erecinska, 1990), traumatic brain injury (Nilsson et al., 1996), hyperactivity of contractile responses in gastric fundus of diabetic (Aihara and Sakai, 1989) and inflammatory response (Li et al., 2002). It is well known that these pathophysiological processes are coupled with intracellular Ca2+ overload which is usually due to Ca2+ influx through ROCC or SOCC rather than VDCC. Therefore, searching for more specific and potent ROCC or SOCC blockers becomes desirable and extremely important for the development of new drugs for these diseases (Li et al., 2004). It has been reported that some 1,4;dihydropyridine derivatives which were structurally modified could play the inhibitory effect on SOCC (Harper et al., 2003), and the synthetic estrogen agonist, diethylstilbestrol, could inhibit SOCC and capacitative Ca2+ influx (Zakharov et al., 2004). Since ginsenoside-Rd is structurally different from current 1,4;dihydropyridine derivatives, present results might provide a bright and useful clue for the development of new Ca2+ entry blocker of ROCC and SOCC. Acknowledgements This work was supported by the National Natural Science Foundation of China (No. 30472021), by Science Foundation of the Ministry of Education in China (No. 20050558072), and by China Medical Board (No. 00730). References Aihara, K., Sakai, Y., 1989. Hyperreactivity of contractile response in gastric fundus smooth muscle from rats with diabetes induced by streptozotocin. Arch. Int. Pharmacodyn. Ther. 302, 220–231. Deth, C., Van Breemen, C., 1977. Agonist induced release of intracellular Ca2+ in the rabbit aorta. J. Membr. Biol. 30, 363–380. Dou, D.Q., Zhang, Y.W., Zhang, L., Chen, Y.J., Yao, X.S., 2001. The inhibitory effects of ginsenosides on protein tyrosine kinase activated by hypoxia/ reoxygenation in cultured human umbilical vein endothelial cells. Planta Med. 67, 19–23. Guan, Y.Y., He, H., Chen, J.X., 1985. Effect of the total saponins of panax notoginseng on contraction of rabbit aortic strips. Acta Pharm. Sin. 6, 267–269. Guan, Y.Y., Kwan, C.Y., He, H., Daniel, E.E., 1988. Inhibition of norepinephrineinduced contractile responses of canine mesenteric artery by plant total saponins. Blood Vessels 25, 312–315.
Guan, Y.Y., Kwan, C.Y., He, H., Sun, J.J., Daniel, E.E., 1994. Effects of panax notoginseng saponins on receptor-operated Ca2+ channels in vascular smooth muscle. Acta Pharm. Sin. 15, 392–398. Harper, J.L., Camerini-Otero, C.S., Li, A.H., Kim, S.A., Jacobson, K.A., Daly, J.W., 2003. Dihydropyridines as inhibitors of capacitative calcium entry in leukemic HL-60 cells. Biochem. Pharmacol. 65, 329–338. Hofmann, T., Obukhov, A.G., Schaefer, M., Harteneck, C., Gudermann, T., Schultz, G., 1999. Direct activation of human TRPC6 and TRPC3 channels by diacylglycerol. Nature 397, 259–263. Kim, D.H., Moon, Y.S., Lee, T.H., Jung, J.S., Suh, H.W., Song, D.K., 2003. The inhibitory effect of ginseng saponins on the stress-induced plasma interleukin-6 level in mice. Neurosci. Lett. 353, 13–16. Kwan, C.Y., 1985. Subcellular origin of the oxalate-or inorganic phosphatestimulated Ca2+ transport by smooth muscle microsomes: revisitation of the old problem by a new approach using saponin. Biochim. Biophys. Acta. 819, 148–152. Kwan, C.Y., Garfield, R.E., Daniel, E.E., 1979. An improved procedure for the isolation of plasma membranes from rat mesenteric arteries. J. Mol. Cell. Cardiol. 11, 639–650. Lee, J.K., Choi, S.S., Lee, H.K., Han, K.J., Han, E.J., Suh, H.W., 2003. Effects of ginsenoside Rd and decursinol on the neurtoxic responses induced by kainic acid in mice. Planta Med. 69, 230–234. Li, S.W., Westwick, J., Poll, CT., 2002. Receptor-operated Ca2+ influx channels in leukocytes: a therapeutic target? Trends Pharmacol. Sci. 23, 63–70. Li, S., Westwick, J., Cox, B., Poll, C.T., 2004. TRP channels as drug targets. Novartis Found. Symp. 258, 204–213. Lian, X.Y., Zhang, Z., Stringer, J.L., 2005. Protective effects of ginseng components in a rodent model of neurodegeneration. Ann. Neurol. 57, 642–648. Lin, M.J., Leung, G.P.H., Zhang, W.M., Yang, X.R., Yip, K.P., Tse, C.M., Sham, J.S.K., 2004. Chronic hypoxia-induced upregulation of store-operated and receptor-operated Ca2+ channels in pulmonary arterial smooth muscle cells—a novel mechanism of hypoxic pulmonary hypertension. Circ. Res. 95, 496–505. Nilsson, P., Laursen, H., Hillered, L., Hansen, A.J., 1996. Ca2+ movements in traumatic brain injury: the role of glutamine receptor-operated ion channels. J cereb Blood Flow Metab. 16, 262–270. Parekh, A.B., Penner, R., 1997. Store depletion and calcium influx. Physiol. Rev. 77, 901–930. Parekh, A.B., Putney Jr., J.W., 2005. Store-operated calcium channels. Physiol. Rev. 85, 757–810. Shi, Q., Hao, Q., Bouissac, J., Lu, J.Y., Tian, S., Luu, B., 2005. Ginsenoside-Rd from Panax notoginseng enhances astrocyte differentiation from neural stem cells. Life Sci. 76, 983–995. Silver, I.A., Erecinska, M., 1990. Intracellular and extracellular changes of [Ca2+]I in hypoxia and ischemia in rat brain in vivo. J. Gen. Physiol. 95, 837–866. Shuttleworth, T.J., 2004. Receptor-activated calcium entry channels—who does what, and when? Sci. STKE 243 (pe40). Tao, L., Guan, Y.Y., He, H., 1997. Comparison of the calcium movements induced by á1-adrenoceptor subtypes in HEK cells. Life Sci. 61, 2127–2136. Yokozawa, T., Iwano, M., Dohi, K., Hattori, M., Oura, H., 1994. Inhibitory effects of ginseng on proliferation of cultured mouse mesangial cells. Nippon Jinzo Gakkaishi 36, 13–18. Yokozawa, T., Satoh, A., Cho, E.J., 2004. Ginsenoside-Rd attenuates oxidative damage related to aging in senescence-accelerated mice. J. Pharm. Pharmacol. 56, 107–113. Zakharov, S.I., Smani, T., Dobrydneva, Y., Monje, F., Fichandler, C., Blackmore, P.F., Bolotina, V.M., 2004. Diethylstilbestrol is a potent inhibitor of storeoperated channels and capacitative Ca2+ influx. Mol. Pharmacol. 66, 702–707. Zhang, Y.W., Dou, D.Q., Zhang, L., Chen, Y.J., Yao, X.S., 2001. Effects of ginsenosides from Panax ginseng on cell-to-cell communication function mediated by gap junctions. Planta Med. 67, 417–422. Zhou, J.G., Qiu, Q.Y., Zhang, Z., Liu, Y.J., Guan, Y.Y., 2006. Evidence for capacitative and non-capacitative Ca2+ entry pathways coexist in A10 vascular smooth muscle cells. Life Sci. 78, 1558–1563.