The mechanism of vasorelaxation induced by Schisandra chinensis extract in rat thoracic aorta

Aim of the study

Schisandra chinensis (SC) is a known medical herb for the treatment of cardiovascular symptoms associated with menopausal symptoms in Korea. However, the pharmacological action mechanisms involved have not been well studied. This study was aimed to investigate the vascular effects of SC in rat thoracic aorta.

Materials and methods

We isolated the hexane, chloroform, and methanol extracts from SC and evaluated their vasodilatory effects in the rat thoracic aorta.

Results

Hexane extracts of SC (SCHE, 5 × 10⁻⁵ to 10⁻³ g/L) caused a concentration-dependent relaxation in both endothelium-intact and -denuded aortas. The relaxant effect of SCHE on the endothelium-intact aorta was more prominent than on the endothelium-denuded aorta. The former was significantly attenuated by L-NAME, a nitric oxide synthase inhibitor, and ODQ, a soluble guanyl cyclase inhibitor, but not by tetraethylammonium, a nonselective blocker of K⁺ channels, and indomethacin, a cyclooxygenase inhibitor. Furthermore, SCHE caused nitrite production as well as eNOS activation in aortic segments, suggesting implication of NO signal pathway in SCHE-induced relaxation. In endothelium-denuded aorta, SCHE-induced vasorelaxation was also attenuated by calyculin A, an inhibitor of myosin light chain (MLC) phosphatase, but not by ML-9, a MLC kinase inhibitor, suggestive of implication of MLC phosphatase activation. Phenylephrine-enhanced MLC phosphorylation ratio was significantly attenuated by SCHE, which was recovered to the control level by pretreatment with calyculin A.

Conclusions

Taken collectively, these findings suggest that the vascular relaxation evoked by SCHE was mediated by not only endothelium dependent NO pathway but also direct effect on vascular smooth muscle cell via dephosphorylation of MLC.     

Kudzu root: traditional uses and potential medicinal benefits in diabetes and cardiovascular diseases

Kudzu root (Gegen in Chinese) is the dried root of Pueraria lobata (Willd.) Ohwi, a semi-woody, perennial and leguminous vine native to South East Asia. It is often used interchangeably in traditional Chinese medicine with thomson kudzu root (Fengen in Chinese), the dried root of P. thomsonii, although the Chinese Pharmacopoeia has separated them into two monographs since the 2005 edition. For more than 2000 years, kudzu root has been used as a herbal medicine for the treatment of fever, acute dysentery, diarrhoea, diabetes and cardiovascular diseases. Both English and Chinese literatures on the traditional applications, phytochemistry, pharmacological activities, toxicology, quality control and potential interactions with conventional drugs of both species have been included in the present review. Over seventy phytochemicals have been identified in kudzu root, with isoflavonoids and triterpenoids as the major constituents. Isoflavonoids, in particular puerarin, have been used in most of the pharmacological studies. Animal and cellular studies have provided support for the traditional uses of kudzu root on cardiovascular, cerebrovascular and endocrine systems, including diabetes and its complications. Further studies to define the active phytochemical compositions, quality standards and clinical efficacy are warranted. Strong interdisciplinary collaboration to bridge the gap between traditional medicine and modern biomedical medicine is therefore needed for the development of kudzu root as an effective medicine for the management of diabetes and cardiovascular diseases.     

Anxiolytic effects induced by inhibition of the nitric oxide–cGMP pathway in the rat dorsal hippocampus

Rationale Conflicting results have been reported regarding the role of the nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway in the hippocampus on anxiety modulation. Objectives To investigate the effects of intrahippocampal injections of drugs that modify the NO–cGMP pathway in rats submitted to two animal models that are sensitive to anxiolytic drugs, the elevated plus-maze and the Vogel punished licking test. Materials and methods Male Wistar rats with cannulae aimed at the dentate gyrus of the dorsal hippocampus received microinjections of the NO synthase (NOS) inhibitors N ^G-nitro-L-arginine methyl ester (LNAME, 15–300 nmol/0.2 μl), N ^G-nitro-L-arginine (LNOARG, 50–300 nmol/0.2 μl), 7-nitroindazole (7NI, 10–100 nmol/0.2 μl), or the soluble guanylate cyclase inhibitor 1H-oxadiazolo-quinoxalin-1 one (ODQ, 10–100 nmol/0.2 μl), and were submitted to the elevated plus-maze. In a second group, the animals received 7NI, LNAME, or ODQ and were submitted to the Vogel punished licking test. To control for drug-induced changes in locomotor behavior, the animals were submitted to an open arena or to the Rota-rod test. Results All drugs increased the exploration of the open arms of the elevated plus-maze. They also increased the number of punished licks in the Vogel test, indicating an anxiolytic effect. The anxiolytic effect of LNAME was prevented by previous treatment with L-arginine (300 nmol/0.2 μl). Except for the lower dose of LNAME (15 nmol), administration of the NOS inhibitors or ODQ did not change exploratory activity in the open field nor cause any gross locomotor impairment in the Rota-rod test. Conclusion The results suggest that NO plays an anxiogenic role in the dentate gyrus of the dorsal hippocampus.     

小鼠十二指肠运动中NO对NA作用的影响及机制

目的观察去甲肾上腺素(noradrenaline ,NA)对离体小鼠十二指肠肌条收缩幅度及一氧化氮(nitricoxide ,NO)对NA作用的影响;探讨NO和NA之间的关系及影响机制。方法用器官浴槽孵育离体小鼠十二指肠肌条,以肌张力收缩幅度抑制比为指标观察NA对肌条收缩幅度及NO对NA作用的影响。结果NA对肌条的收缩幅度有明显的抑制作用,在浓度1.2×10 -4～1.2×10 -8mol/L范围内,呈剂量-效应关系,其中1.2×10 -8mol/L组与对照组之间无显著性差异(P >0 .0 5 )。L Arg对肌条的收缩幅度有明显的抑制作用,在浓度2×10 -2 ～2×10 -6mol/L范围内,呈剂量-效应关系。除2×10 -6mol/L组外,其余各浓度的L Arg与对照组均有显著性差异(P <0 .0 0 1)。用无反应剂量2×10 -6和2×10 -3 mol/L的L Arg孵育标本后加入NA ,浓度为1.2×10 -5,1.2×10 -6,1.2×10 -7,1.2×10 -8mol/L的NA对肌条收缩幅度的抑制作用明显增强,与单独NA组的作用相比差异有显著性(P <0 .0 0 1) ;而1.2×10 -4mol/LNA组与单独NA组的作用相比差异无显著性(P >0 .0 5 )。分别用一氧化氮合酶(NOS)的抑制剂L NNA、可溶性鸟苷酸环化酶(sGC)抑制剂ODQ、α肾上腺素能受体阻断剂酚妥拉明(phentolamine)均使NA对肌条收缩幅度抑制效应明显减弱,与对照组比较有显著性差异(P <0 .0 0     

Angiotensin AT2 receptor activates the cyclic-AMP signaling pathway in eel

A unique angiotensin type 2 receptor (AT2) that induces a cAMP signaling pathway was cloned and characterized for the first time in fish, Anguilla japonica. Phylogeny and synteny results showed that the AT2s among fishes and tetrapods share the same origin despite a sub-cluster formation among eel, salmon, and zebrafish. The eel AT2 was expressed abundantly in the spleen and localized at straight arterioles and ellipsoid regions prior to the sinusoid, suggesting a role in the regulation of microcirculation and/or immune response. Various angiotensin (Ang) peptides, including Ang II, Ang III, and Ang IV, were detected in the spleen by a radioimmunoassay coupled with HPLC separation, and these endogenous peptides stimulated a cAMP signaling, which has no crosstalk with cGMP pathway. The common and contrasting features of AT2 between fishes and mammals imply some ancestral characters of AT2, which are important information for receptor binding and evolutionary studies.     

Characterization of NS 2028 as a specific inhibitor of soluble guanylyl cyclase

1. The haeme-containing soluble guanylyl cyclase (α₁β₁-heterodimer) is a major intracellular receptor and effector for nitric oxide (NO) and carbon monoxide (CO) and mediates many of their biological actions by increasing cyclic GMP. We have synthesized new oxadiazolo-benz-oxazins and have assessed their inhibitory actions on guanylyl cyclase activity in vitro, on the formation of cyclic GMP in cultured cells and on the NO-dependent relaxation of vascular and non-vascular smooth muscle.
2. Soluble guanylyl cyclase, purified to homogeneity from bovine lung, was inhibited by 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028) in a concentration-dependent and irreversible manner (IC₅₀ 30 nM for basal and 200 nM for NO-stimulated enzyme activity). Evaluation of the inhibition kinetics according to Kitz & Wilson yielded a value of 8 nM for K_i, the equilibrium constant describing the initial reversible reaction between inhibitor and enzyme, and 0.2 min⁻¹ for the rate constant k3 of the subsequent irreversible inhibition. Inhibition was accompanied by a shift in the soret absorption maximum of the enzyme''s haem cofactor from 430 to 390 nm.
3. S-nitroso-glutathione-enhanced soluble guanylyl cyclase activity in homogenates of mouse cerebellum was inhibited by NS 2028 (IC₅₀ 17 nM) and by 17 structural analogues in a similar manner, albeit with different potency, depending on the type of substitution at positions 1, 7 and 8 of the benzoxazin structure. Small electronegative ligands such as Br and Cl at position 7 or 8 increased and substitution of the oxygen at position 1 by -S-,- NH- or -CH₂- decreased the inhibition.
4. In tissue slices prepared from mouse cerebellum, neuronal NO synthase-dependent activation of soluble guanylyl cyclase by the glutamate receptor agonist N-methyl-D-aspartate was inhibited by NS 2028 (IC₅₀ 20 nM) and by two of its analogues. Similarly, 3-morpholino-sydnonimine (SIN-1)-elicited formation of cyclic GMP in human cultured umbilical vein endothelial cells was inhibited by NS 2028 (IC₅₀ 30 nM).
5. In prostaglandin F_2α-constricted, endothelium-intact porcine coronary arteries NS 2028 elicited a concentration-dependent increase (65%) in contractile tone (EC₅₀ 170 nM), which was abolished by removal of the endothelium. NS 2028 (1 μM) suppressed the relaxant response to nitroglycerin from 88.3±2.1 to 26.8±6.4% and induced a 9 fold rightward shift (EC₅₀ 15 μM) of the concentration-relaxation response curve to nitroglycerin. It abolished the relaxation to sodium nitroprusside (1 μM), but did not affect the vasorelaxation to the K_ATP channel opener cromakalim. Approximately 50% of the relaxant response to sodium nitroprusside was recovered after 2 h washout of NS 2028.
6. In phenylephrine-preconstricted, endothelium-denuded aorta of the rabbit NS 2028 (1 μM) did not affect relaxant responses to atrial natriuretic factor, an activator of particulate guanylyl cyclase, or forskolin, an activator of adenylyl cyclase.
7. NO-dependent relaxant responses in non-vascular smooth muscle were also inhibited by NS 2028. The nitroglycerin-induced relaxation of guinea-pig trachea preconstricted by histamine was fully inhibited by NS 2028 (1 μM), whereas the relaxations to terbutaline, theophylline and vasoactive intestinal polypeptide (VIP) were not affected. The relaxant responses to electrical field stimulation of non-adrenergic, non-cholinergic nerves in the same tissue were attenuated by 50% in the presence of NS 2028 (1 μM).
8. NS 2028 and its analogues, one of which is the previously characterized 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ), appear to be potent and specific inhibitors of soluble guanylyl cyclase present in various cell types. Oxidation and/or a change in the coordination of the haeme-iron of guanylyl cyclase is a likely inhibitory mechanism.

     

Nitrite‐derived nitric oxide: a possible mediator of ‘acidic–metabolic’ vasodilation

The fundamental, yet poorly understood, physiological mechanism known as ‘acidic–metabolic’ vasodilation, contributes to local blood flow regulation during hypoxia/ischaemia and increased metabolic activity. The vasodilator nitric oxide (NO) has been suggested to be involved in this event. Besides enzymatic production by NO synthases, a novel mechanism for generation of this gas in vivo was recently described. This involves non‐enzymatic reduction of inorganic nitrite to NO, a reaction that takes place predominantly during acidic/reducing conditions. We have studied the effects of physiological amounts of nitrite on NO generation and relaxation of rat aorta in vitro in a situation where environmental pH was reduced to levels seen in tissues during hypoxia/ischaemia. The relaxatory effect of nitrite was increased in an acidic buffer solution (pH 6.6) compared with neutral pH; EC50 for nitrite was reduced from 200 to 40 μM . Nitrite‐evoked relaxation was effectively prevented by coadministration of an inhibitor of soluble guanylyl cyclase. The relaxation was further potentiated by the addition of ascorbic acid. In parallel, NO was generated from nitrite in a pH dependent manner with even larger amounts seen after addition of ascorbic acid. NO generation from nitrite correlated to the the degree of relaxation of rat aorta. These results illustrate non‐enzymatic release of NO from nitrite at physiological concentrations. This may be an important auto‐regulated physiological mechanism involved in the regulation of vascular tone during hypoxia/ischaemia.     

Effects of nitric oxide donors and inhibitors of nitric oxide signalling on endothelin‐ and serotonin‐induced contractions in human placental arteries

In order to explore the role of nitric oxide (NO) in the control of fetoplacental vascular tone in normal pregnancy we have examined the effects of NO donors on uteroplacental arteries pre‐contracted with the vasoconstrictor endothelin‐1 (ET‐1) or serotonin (5‐HT). We have furthermore examined the effects of guanylate cyclase inhibitors on the NO‐induced relaxation. Segments of placental arteries (n=102) obtained from 39 placentas immediately after delivery were mounted in organ baths and superfused with Krebs–Ringer solution at 37 °C. The vessel segments were exposed to drugs for various intervals and the tension was recorded isometrically and registered on a polygraph. Cyclic guanosine monophosphate (cGMP) analysis was performed after extraction of vessel segments using a specific radioimmunoassay. The placental artery segments responded to ET‐1 and 5‐HT with a dose‐dependent vasoconstriction. After pre‐contraction with ET‐1 (10^–7 M ) or 5‐HT (10^–6 M ), the vessels relaxed in response to the NO donors glyceryltrinitrate (GTN) (10^–6 M ) and S‐nitroso‐N‐acetyl‐penicillamine (SNAP) (10^–5 M ). In the presence of the non‐specific guanylate cyclase inhibitor LY 83583 (10^–6 M ), the vessels responded with a small contraction. In the presence of the soluble guanylate cyclase (sGC) inhibitor 1H[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ) the non‐treated vessels responded with a relaxation. 1H[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one gave no obvious relaxation in pre‐contracted vessels. Addition of 8‐Br‐cGMP, the cell‐permeant analogue of cGMP, with or without pre‐contraction had no effect on the vessels. Cyclic guanosine monophosphate analysis showed that GTN treatment caused an increase in cGMP after 12 min. Our results indicate that NO acts as a vasodilator in placental vessels. The cGMP‐dependent mechanisms may be involved in NO‐induced relaxation but cGMP‐independent mechanisms appear also to be involved.     

基于在校大学生在线24小时膳食问卷调查数据有效性研究

目的 在接受高等教育的人群中验证在线24h膳食调查问卷(ODQ)相较于面对面24h膳食调查(FDR)膳食数据的有效性。方法 采用整群随机抽样的方法,抽取四川大学142名在校大学生,在同一天之内分别进行ODQ及FDR调查。通过计算ODQ与FDR间膳食摄入的个体平均差异、Spearman相关系数、交叉表一致性检验,Bland-Altman图等方法,评价两种方法之间膳食能量及常见营养素摄入量的相关性和一致性。结果 ODQ法估计的膳食能量及营养素摄入量均低于FDR(P<0.05)。 两方法各膳食摄入量间的秩相关系数在0.54~0.74之间(P<0.05),两方法间膳食能量及营养素摄入被分类到相同或相邻的四分位数组的比例从维生素A的82.4%到维生素PP的93%,而被错误分类的比例均在5%以下,加权kappa系数分布在0.41~0.64间,呈现中度到高度的一致性。Bland-Altman图表明两方法间膳食能量及各营养素摄入呈现良好的一致性。结论 本研究表明在线24h膳食问卷在群体水平上调查大学生膳食能量和营养素摄入时具有良好的有效性。