
This study was designed to investigate the effects of the aqueous ethanol extract of Astragalus membranaceus BUNGE (Leguminosae) on rat thoracic aorta. Isometric tension was recorded in response to drugs in organ bath. In endothelium-intact aortic rings, A. membranaceus extract induced a significant dose-dependent relaxation of the rings precontracted by phenylephrine, which could be inhibited by preincubation with L-N(w w)-nitro-arginine methyl ester or methylthioninium chloride. In endothelium-denuded ones, the extract could dose-dependently relax the rings contracted by phenylephrine, not by KCl; and it could also attenuate contractile response to phenylephrine, not to caffeine or phorbol-12,13-diacetate in Ca Dawley Rats Rats were euthanized by decapitation using small animal guillotine. The thoracic aorta was immediately isolated and immersed in oxygenated K-H solution 4°C of the following composition (in mM): NaCl 118.3; KCl 4.7; MgSO 4 1.2; KH 2 PO 4 1.2; NaHCO 3 25.0; CaCl 2 2.5; Glucose 11.0. Then the adherent connective tissue was cleaned and blood vessel was cut into 3-4 mm rings, with the special care of avoiding damage of endothelium. In some preparations, endothelium was mechanically removed by gently rubbing the lumen. The rings were mounted horizontally between two stirrups in organ bath filled with 10 ml K-H solution at 37°C, ventilated continuously with 95% O 2 and 5% CO 2 . The isometric tension was recorded with a force transducer (JZ101, metrical range: 0-5.0 g) and MedLab 5.0v recording system (Nanjing Medease Science and Technology Co. Ltd).
Experimental Procedure of Isometric Tension Rings were equilibrated for 60 adamcampos@example.com g resting tension, and then challenged with KCl (6.0ϫ10
Ϫ2 M) at least 3 times until a reproducible maximal contractile response was obtained. After a further equilibration period of 30 min, the integrity of the endothelium was assessed in all preparations by determining the ability of ACh (1.0ϫ10
Ϫ5 M) to induce more than 80% relaxation of rings pre-contracted with PE (1.0ϫ10 Ϫ6 M). The endothelium was considered to be removed when there was less than 10% relaxation response to ACh. (an activator of protein kinase C, PKC), 13) or caffeine (2.5ϫ10 Ϫ2 M) (an activator of ryanodine receptor) 14) was used to contract the denuded rings. In order to explore the probable effect of extracellular Ca 2ϩ inflow, 2.5 mM CaCl 2 was added after PE stimulated the rings. Then the effect of A. membranaceus (6.0ϫ10 Ϫ3 g/l) was tested. A time control (same protocol as above except for the omission of A. membranaceus to the second application of PE, PD or caffeine) was always run in parallel. Following the initial control response in Ca 2ϩ -free medium and before the second test contraction (to PE or caffeine), a 6.0ϫ10
Ϫ2 M KCl-induced contraction in K-H solution was obtained to refill the Ca 2ϩ stores in the vascular tissues.
Protocol 3: Endothelium-Dependent Vasomotor Response by A. membranaceus: In the third series of experiments, the potential influence of A. membranaceus on PE-induced vasomotor function in the intact rings was examined. For pretreatment, endothelium-intact rings were incubated with L-NAME (1.0ϫ10 Ϫ4 M) (a specific inhibitor of nitric oxide synthase), MC (1.0ϫ10 Ϫ5 M) (an inhibitor of guanylate cyclase) for 15 min 15) and phosphoramidon (5.0ϫ10 Ϫ6 M) (an inhibitor of endothelin converting enzyme) for 20 min 16) before administration of PE. Then the response curves of A. membranaceus were recorded. The rings with or without the treatment of inhibitors were always tested in parallel.
Statistical Analysis Relaxant responses were expressed as the percentage decreases of the magnitude of the contraction induced by PE or KCl (ϭ100%) before the application of vasodilators, and the contractile responses as the ratio of the responses following and before the application of A. membranaceus (second response/first response). All results are expressed as meanϮS.E.M. Statistical analysis was performed with t-test or ANOVA followed by Newman-Keuls test. Differences were accepted as statistically significant at p values Ͻ0.05 (GraphPad Prism).
RESULTS

PE (1.0ϫ10
Ϫ6 M) induced a similar sustained contraction of aortic rings in each group with a peak tension of about 3.11Ϯ0.27 g in intact aortic rings and 3.02Ϯ0.19 g in denuded ones.
Effects of A. membranaceus on the Vasotension All doses (2.0ϫ10 Ϫ5 -2.0ϫ10 Ϫ1 g/l) of A. membranaceus exhibited negligible vasomotor actions on aortic rings with or without endothelium at resting tension (Fig. 1) . A. membranaceus (2.0ϫ10
Ϫ1 g/l) produced dose-dependent relaxation in endothelium-denuded rings (Fig. 2) , after the plateau tension induced by PE. However, in PE-precontracted endothelium-intact rings, low doses (2.0ϫ10 Ϫ4 -6.0ϫ10
Ϫ3 g/l) of A. membranaceus showed a more significant relaxation than that in denuded ones (Fig. 2) , while high dose (2.0ϫ10 Ϫ2 -2.0ϫ10 Ϫ1 g/l) of the extract induced a transient contraction (Figs. 2, 3) . Endothelium-Independent Vasodilatation by A. membranaceus In contrast to the effect on PE-contracted endothelium-denuded rings in K-H solution, A. membranaceus
Ϫ1 g/l) had no dilatation-effect on rings stimulated by KCl (3.0ϫ10 Ϫ2 M) (Fig. 4) (Fig. 5) . Endothelium-Dependent Vasomotor Effects by A. membranaceus Pre-incubation of the intact rings with L-NAME (1.0ϫ10 Ϫ4 M) markedly, incompletely inhibited A. membranaceus (6.0ϫ10 Ϫ3 g/l)-induced relaxation from 36.0 to 7.3% (Fig. 6) . Similarly, MC (1.0ϫ10 Ϫ5 M) also showed an inhibition of endothelium-dependent vasodilatation from 36.0 to 8.0% (Fig. 6) . Thus, NO signaling might play a major, but not full role in the vasodilatation effect of A. membranaceus.
When the dose of A. membranaceus in the organ bath was stepwise raised to 2.0ϫ10 Ϫ2 -2.0ϫ10 Ϫ1 g/l, a transient contraction could be observed in endothelium-intact rings. Pretreatment of endothelium-intact rings with phosphoramidon (5.0ϫ10 Ϫ6 M) significantly attenuated the transient contraction response (Fig. 3) .
DISCUSSION
The present study first and detailedly explored the in vitro vascular effects of A. membranaceus in aortic rings isolated from rats. In the course of the study, a number of novel observations had been made, which contribute to better scien- tific understanding of their therapeutic use for antihypertension associated with Qi deficiency caused by the disorder of spleen and kidney in China.
First, although neurohumoral factors may contribute to antihypertension for the crude extract of A. membranaceus observed previously, 8) it is apparent that direct endothelium-dependent and independent vasorelaxation should be prominently taken into account according to this study.
Second, A. membranaceus dose-dependently dilated endothelium-denuded rings contracted with PE. However, the agent had no effect on the vessel contracted by KCl. The cellular mechanism of contraction involved in the response to KCl and PE is different. KCl induce Ca 2ϩ influx via voltagedependent Ca 2ϩ channel, which further activate Ca 2ϩ -induced Ca 2ϩ release through ryanodine-receptor. PE increase intracellular Ca 2ϩ concentration with two mechanisms, 1) through receptor-gated Ca 2ϩ channels; 2) mobilizes Ca 2ϩ from intracellular stores via the inositol-1,4,5-trisphosphate (IP 3 ) receptor or induces secondly myosin light chain phosphorylation via activating PKC. 17, 18) That is, the vasorelaxant action of A. membranaceus seems to occur in a receptor-dependent manner in SMVC of rat aortas. However, the result that A. membranaceus failed to inhibit the contraction caused by addition of CaCl 2 in Ca 2ϩ free solution was probably only due to its effects on the intracellular pathways. This hypothesis was confirmed in the further experiments that the contractions induced by PD and caffeine in Ca 2ϩ -free medium were not affected by A. membranaceus equally. Thus, it appears that the IP 3 -induced Ca 2ϩ release channels might be the site of action for A. membranaceus on endothelium-removed aortas.
Third, NO is a potent vasodilator synthesized in the endothelium 19) by NO synthase, and causes VSMC relaxation through the activation of soluble guanylate cyclase.
20) The present study demonstrated that the extract of A. membranaceus dose-dependently inhibited the contraction induced by PE in intact aorta isolated from rats. This vasorelaxant action was mostly inhibited by treatment with L-NAME or MC. Endothelium-dependent relaxation of A. membranaceus seemed to be associated with NO signaling via guanylate cyclase activation since both L-NAME and MC could attenuate this response. This result was supported by other studies which had shown that A. membranaceus could increase the plasma level of NO 21) and induce the release of NO from VSMC in vitro by NO-soluble guanylate cyclasecyclic guanosine monophosphate pathway. 22) Finally, it was also observed that high dose (2.0ϫ10 Ϫ2 -2.0ϫ10 Ϫ1 g/l) A. membranaceus induced a transient contraction in arterial rings pre-constricted by PE. This result was identical with that obtained in another report where treatment with high dose of A. membranaceus reversibly increased blood pressure of patients without hypertension previously.
23)
The mechanism underlying is under investigation. Blockage of the contraction by endothelin-converting enzyme phosphoramidon may suggest the involvement of an endothelin receptor mediated signaling.
In summary, the findings indicated that A. membranaceus have diphasic effects of relaxation and contraction on the aorta rings. The mechanisms of relaxation may include inhibition of intracellular Ca 2ϩ release in vascular smooth muscle cells, and endothelium dependent relaxation mediated by the NO-GC pathway, whilst the effect of transient contraction of A. membranaceus at a high dose was related with endothelin release.

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