Ginsenoside-Rd from panax notoginseng blocks Ca2+ influx through receptor- and store-operated Ca2+ channels in vascular smooth muscle cells
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.
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 KCl-induced 45Ca2+ influx and the increase of [Ca2+]i. Total saponins of panax notoginseng did not change phenylephrine-induced 45Ca2+ efflux which was owing to intracellular Ca2+ release, and the affinity of α-adrenoceptor (Guan et al., 1988, Guan et al., 1994). These results suggested that total saponins of panax notoginseng have an inhibitory effect on receptor-operated Ca2+ entry. Based on these 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-d-glucopyranosyl)-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 ginsenoside-Rd are consistent with the previously reported results of total saponins from panax notoginseng (Guan et al., 1985, Guan et al., 1988, Guan et al., 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.
Section snippets
Materials and animals
Ginsenoside-Rd with a purity of 98% was obtained from Tai-He 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
Effects on vascular contractile responses
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
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, Guan et al., 1988, Guan et al., 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
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).
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