Vasorelaxation In Rat Aortic Rings Research Articles

Novel enhancers of guanylyl cyclase-A activity acting via allosteric modulation.

Guanylyl cyclase-A (GC-A), activated by endogenous atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), plays an important role in the regulation of cardiovascular and renal homeostasis and is an attractive drug target. Even though small molecule modulators allow oral administration and longer half-life, drug targeting of GC-A has so far been limited to peptides. Thus, in this study we aimed to develop small molecular activators of GC-A. Hits were identified through high-throughput screening and optimized by in silico design. Cyclic GMP was measured in QBIHEK293A cells expressing GC-A, GC-B or chimerae of the two receptors using AlphaScreen technology. Binding assays were performed in membrane preparations or whole cells using 125 I-ANP. Vasorelaxation was measured in aortic rings isolated from Wistar rats. We have identified small molecular allosteric enhancers of GC-A, which enhanced ANP or BNP effects in cellular systems and ANP-induced vasorelaxation in rat aortic rings. The mechanism of action appears novel and not mediated through previously described allosteric binding sites. In addition, the selectivity and activity depend on a single amino acid residue that differs between the two similar receptors GC-A and GC-B. We describe a novel allosteric binding site on GC-A, which can be targeted by small molecules to enhance ANP and BNP effects. These compounds will be valuable tools in further development and proof-of-concept of GC-A enhancement for the potential use in cardiovascular therapy.

Chemical characterization of Passiflora edulis extracts and their in vitro antioxidant, anti-inflammatory, anti-lipid activities, and ex-vivo vasodilation effect

Passion fruit is a dietary plant with the protective proterties against many diseases such as cardiovascular diseases. The aim of this study was to explore the biological properties (anti-oxidant, anti-inflammatory, anti-lipid, and vascular relaxation) of the P. edulis extracts obtained from seed (PSEE) and fruit (PFWE). Thin-layer chromatography (TLC) and HPLC were used to analyze the phytochemical constituents in the extracts. Antioxidant and enzyme inhibition properties were examined. Anti-inflammatory activities of PFWE and PSEE were evaluated in lipopolysaccharide (LPS)-induced macrophages and ex-vivo vascular relaxation were also determined. A high contents of piceatannol and polyphenolic stilbene were found in PSEE extracts. On the other hand, beta-carotene and gamma-tocopherol were predominant in PFWE. PFWE exhibited stronger anti-oxidant activity than PSEE. Both PSEE and PFWE showed significant inhibition of lipase and cholesterol esterase activities. However, only PFWE showed minimal inhibitory effect against HMG-CoA reductase. PSEE and PFWE caused a significant reduction of nitric oxide level in LPS-induced inflammation in RAW264.7 cells. Both extracts also showed ex-vivo vasorelaxation of rat aortic rings. It has been demonstrated that PFWE and PSEE possesses anti-oxidant activity, anti-inflammatory activity, pancreatic lipase and cholesterol esterase, and ex-vivo vasorelaxation of rat aortic rings, however, the activity of HMG-CoA reductase was inhibited by only PFWE. These findings suggest the beneficial effect of PSEE and PFWE against hyperlipidemia thus preventing the development of hypertension. However, further investigations in animal are needed to elucidate.

Effect of a new herbal composition comprised of red clover and hop extract on human endothelial cell damage and vasorelaxant activity.

Hormone replacement therapy may cause various side effects, including enhancing the risk of cardiovascular disease (CVD) in postmenopausal women. Here, we investigated the effect of red clover and hop extract combination (RHEC) on estrogen receptor (ER) binding and endothelial function of human umbilical vein endothelial cells (HUVECs) to develop an herbal agent for reducing the risk of CVDs. In ER competitor assay, RHEC showed binding affinity toward ERα and ERβ with IC50 values of 5.92µg/ml and 1.66µg/ml, respectively. In HUVECs, RHEC significantly increased the cell viability and reduced the reactive oxygen species production against oxidative stress-induced damage. We also showed that RHEC increased the NO production through upregulating the endothelial nitric oxide synthase expression via ER activation in estrogen depleted condition. In particular, RHEC showed greater efficacy with increase in NO and decrease in endothelin-1 than red clover or hop treatment alone. Additionally, 0.3-0.5mg/ml of RHEC-induced vasorelaxation of rat aortic rings precontracted by phenylephrine. PRACTICAL APPLICATIONS: Recently, a large interest has grown in the synergistic effects of phytochemicals for better therapies to treat various diseases. Red clover and hop are well-known edible plants which are widely used to help relieve postmenopausal symptoms including CVD. However, their combination has not been studied so far. For the first time, we demonstrated that RHEC, a new herbal combination comprising the extracts from red clover and hop, appeared to be effective in protection of endothelial function against oxidative stress and estrogen depletion. Therefore, RHEC could be a potent herbal agent for reducing the risk of endothelial damage.

Vasodilation of Tea Polyphenols Ex Vivo Is Mediated by Hydrogen Peroxide Under Rapid Compound Decay.

Improvement of endothelial function represents a major health effect of tea in humans. Ex vivo, tea and tea polyphenols stimulate nitric oxide (NO)-dependent vasodilation in isolated blood vessels. However, it was reported that polyphenols can generate reactive oxygen species (ROS) in vitro. We therefore aimed to elucidate the role of ROS production in tea polyphenol-induced vasodilation in explanted aortic rings. Vasorelaxation of rat aortic rings was assessed in an organ chamber model with low concentrations of epigallocatechin-3-gallate (EGCG), theaflavin-3,3’-digallate (TF3), and with green and black tea, with or without pretreatment with catalase or superoxide dismutase (SOD). The stability of EGCG and TF3 was measured by HPLC, and the levels of hydrogen peroxide (H2O2) were determined. EGCG and green tea-induced vasorelaxation was completely prevented by catalase and slightly increased by SOD. TF3 and black tea yielded similar results. Both EGCG and TF3 were rapidly degraded. This was associated with increasing H2O2 levels over time. Hydrogen peroxide concentrations produced in a time range compatible with tea polyphenol decay induced NO-dependent vasodilation in aortic rings. In conclusion, tea polyphenol-induced vasodilation in vitro is mediated by low levels of H2O2 generated during compound decay. The results could explain the apparent lack of vasodilatory effects of isolated tea polyphenols in humans.

Vasodilator effects and putative guanylyl cyclase stimulation by 2-nitro-1-phenylethanone and 2-nitro-2-phenyl-propane-1,3-diol on rat aorta

Compounds containing a nitro group may reveal vasodilator properties. Several nitro compounds have a NO2 group in a short aliphatic chain connected to an aromatic group. In this study, we evaluated in rat aorta the effects of two nitro compounds, with emphasis on a putative recruitment of the soluble guanylate cyclase (sGC) pathway to induce vasodilation. Isolated aortic rings were obtained from male Wistar rats to compare the effects induced by 2-nitro-1-phenylethanone (NPeth) or 2-nitro-2-phenyl-propane-1,3-diol (NPprop). In aortic preparations contracted with phenylephrine or KCl, NPeth and NPprop induced vasorelaxant effects that did not depend on the integrity of vascular endothelium. NPeth had a lesser vasorelaxant efficacy than NPprop and only the NPprop effects were inhibited by pretreatment with the sGC inhibitors, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) or methylene blue. In an ODQ-preventable manner, NPprop inhibited the contractile component of the phenylephrine-induced response mediated by intracellular Ca2+ release or by extracellular Ca2+ recruitment through receptor- or voltage-operated Ca2+ channels. In contrast, NPprop was inert against the transient contraction induced by caffeine in Ca2+-free medium. In an ODQ-dependent manner, NPprop inhibited the contraction induced by the protein kinase C activator phorbol 12,13-dibutyrate or by the tyrosine phosphatase inhibitor sodium orthovanadate. In silico docking analysis of a sGC homologous protein revealed preferential site for NPprop. In conclusion, the nitro compounds NPeth and NPprop induced vasorelaxation in rat aortic rings. Aliphatic chain substituents selectively interfered in the ability of these compounds to induce vasorelaxant effects, and only NPprop relaxed aortic rings via a sGC pathway.

Reduced Activity of the Aortic Gamma-Glutamyltransferase Does Not Decrease S-Nitrosoglutathione Induced Vasorelaxation of Rat Aortic Rings.

Aims: Gamma-glutamyl transferase (GGT), an enzyme present on the endothelium, is involved in the release of nitric oxide (NO) from S-nitrosoglutathione (GSNO) and in the GSNO-induced vasodilation. Endogenous GSNO is a physiological storage form of NO in tissues while exogenous GSNO is an interesting candidate for compensating for the decreased NO bioavailability occurring during cardiovascular diseases. We investigated in a rat model of human hypertension, the spontaneous hypertensive rat (SHR), submitted or not to high salt diet, whether a decreased vascular GGT activity modifies the vasorelaxant effect of GSNO.Methods: Thoracic aortic rings isolated from male SHR and Wistar Kyoto rats (WKY) aged 20–22 weeks—submitted or not for 8 weeks to a high salt diet (1% w/v NaCl in drinking water) were pre-constricted with phenylephrine then submitted to concentration-vasorelaxant response curves (maximal response: Emax; pD2) to carbachol or sodium nitroprusside to evaluate endothelial dependent or independent NO-induced vasodilation, or GSNO (exogenous NO vasodilation depending from the endothelial GGT activity). GGT activity was measured using a chromogenic substrate in aortic homogenates. Its role in GSNO-induced relaxation was assessed following inhibition of the enzyme activity (serine-borate complex). That of protein disulfide isomerase (PDI), another redox sensitive enzyme involved in GSNO metabolism, was assessed following inhibition with bacitracin.Results: Aortic GGT activity (18–23 μmol/min/mg of tissue in adult WKY) decreased by 33% in SHR and 45% in SHR with high salt diet. Emax and pD2 for sodium nitroprusside were similar in all groups. Emax for carbachol decreased by −14%, reflecting slight endothelial NO-dependent dysfunction. The GSNO curve was slightly shifted to the left in SHR and in SHR with high salt diet, showing a small enhanced sensitivity to GSNO. Involvements of GGT, as that of PDI, in the GSNO effects were similar in all groups (pD2 for GSNO −0.5 to −1.5 following enzymatic inhibition).Conclusion: Hypertension is associated with a decreased aortic GGT activity without decreasing the vasorelaxant effects of GSNO, whose bioactivity may be supplemented through the alternative enzymatic activity of PDI.

Study on the Absorbed Fingerprint-Efficacy of Yuanhu Zhitong Tablet Based on Chemical Analysis, Vasorelaxation Evaluation and Data Mining

Yuanhu Zhitong Tablet (YZT) is an example of a typical and relatively simple clinical herb formula that is widely used in clinics. It is generally believed that YZT play a therapeutical effect in vivo by the synergism of multiple constituents. Thus, it is necessary to build the relationship between the absorbed fingerprints and bioactivity so as to ensure the quality, safety and efficacy. In this study, a new combinative method, an intestinal absorption test coupled with a vasorelaxation bioactivity experiment in vitro, was a simple, sensitive, and feasible technique to study on the absorbed fingerprint-efficacy of YZT based on chemical analysis, vasorelaxation evaluation and data mining. As part of this method, an everted intestinal sac method was performed to determine the intestinal absorption of YZT solutions. YZT were dissolved in solution (n = 12), and the portion of the solution that was absorbed into intestinal sacs was analyzed using rapid-resolution liquid chromatography coupled with quadruple time-of-flight mass spectrometry (RRLC-Q-TOF/MS). Semi-quantitative analysis indicated the presence of 34 compounds. The effect of the intestinally absorbed solution on vasorelaxation of rat aortic rings with endothelium attached was then evaluated in vitro. The results showed that samples grouped by HCA from chemical profiles have similar bioactivity while samples in different groups displayed very different. Moreover, it established a relationship between the absorbed fingerprints and their bioactivity to identify important components by grey relational analysis, which could predict bioactive values based on chemical profiles and provide an evidence for the quantification of multi-constituents.

Interference in mevalonate pathway ameliorates homocysteine-induced endothelium-dysfunction

Homocysteine is a risk factor for atherosclerosis and hypertension and induces endothelium-dysfunction. Accumulation of cholesterol and reactive oxygen species plays a key role in the endothelium-dysfunction. This study investigated the hypothesis of an involvement of mevalonate pathway and oxidative pathway in homocysteine-induced endothelial damage. Homocysteine induced impairment of the endothelium-dependent vasorelaxation of rat aortic rings by isometric tension, while it also reduced the nitric oxide level and the nitric oxide synthase activity in human umbilical vein endothelial cells, followed by accumulation of superoxide anion and cholesterol. However, the level of asymmetric dimethylarginine remained unaffected by homocysteine. The adverse effect of homocysteine on endothelial function was found to be partially enhanced either by squalestatin-reducing cholesterol or by superoxide dismutase-reducing superoxide anion. Moreover, this effect of homocysteine could be completely ameliorated by simvastatin, very similar to that of cotreatment of squalestatin and superoxide dismutase. Respectively, mevalonolactone partly or squalene fully attenuated the effect of simvastatin or squalestatin on homocysteine-induced endothelial dysfunction. In conclusion, our results suggested that the mevalonate pathway mediates homocysteine-induced endothelium dysfunction besides the oxidative pathway. Interference in the mevalonate pathway and oxidative pathway provides effective protection of endothelial function.

Green and black tea are equally potent stimuli of NO production and vasodilation: new insights into tea ingredients involved

Epidemiological studies suggest that consumption of tea is associated with beneficial cardiovascular effects. Since different types of tea are consumed throughout the world, a question of much interest is whether green tea is superior to black tea in terms of cardiovascular protection. We therefore compared the effects of green and black tea on nitric oxide (NO) production and vasodilation and elucidated the tea compounds involved. We chose a highly fermented black tea and determined concentrations of individual tea compounds in both green and black tea of the same type (Assam). The fermented black tea was almost devoid of catechins. However, both teas stimulated eNOS activity and phosphorylation in bovine aortic endothelial cells (BAEC) as well as vasorelaxation in rat aortic rings to a similar extent. In green tea, only epigallocatechin-3-gallate (EGCG) resulted in pronounced NO production and NO-dependent vasorelaxation in aortic rings. During tea processing to produce black tea, the catechins are converted to theaflavins and thearubigins. Individual black tea theaflavins showed a higher potency than EGCG in NO production and vasorelaxation. The thearubigins in black tea are highly efficient stimulators of vasodilation and NO production. Green and black tea compounds induced comparable phosphorylation of eNOS and upstream signalling kinases. Whereas stimulation of eNOS activity by EGCG was only slightly affected by pretreatment of cells with various ROS scavengers, TF3(theaflavin-3',3-digallate)-induced eNOS activity was partially inhibited by PEG-catalase. These results implicate that highly fermented black tea is equally potent as green tea in promoting beneficial endothelial effects. Theaflavins and thearubigins predominantly counterbalance the lack of catechins in black tea. The findings may underline the contribution of black tea consumption in prevention of cardiovascular diseases.

Addition of milk prevents vascular protective effects of tea

Experimental and clinical studies indicate that tea exerts protection against cardiovascular diseases. However, a question of much debate is whether addition of milk modifies the biological activities of tea. We studied the vascular effects of tea, with or without milk, in humans and elucidated the impact of individual milk proteins in cell culture experiments, with isolated rat aortic rings and by HPLC analysis. A total of 16 healthy female volunteers consumed either 500 mL of freshly brewed black tea, black tea with 10% skimmed milk, or boiled water as control. Flow-mediated dilation (FMD) was measured by high-resolution vascular ultrasound before and 2 h after consumption. Black tea significantly improved FMD in humans compared with water, whereas addition of milk completely blunted the effects of tea. To support these findings, similar experiments were performed in isolated rat aortic rings and endothelial cells. Tea induced vasorelaxation in rat aortic rings and increased the activity of endothelial nitric oxide synthase by phosphorylation of the enzyme in endothelial cells. All effects were completely inhibited by the addition of milk to tea. Of the various kinds of milk proteins, the caseins accounted for these inhibiting effects of milk, probably by formation of complexes with tea catechins. Milk counteracts the favourable health effects of tea on vascular function. This finding indicates the need for particular awareness in the interpretation and design of studies comprising nutritional flavonoids.

Effect of sildenafil on the isolated rat aortic rings

Sildenafil, a highly selective inhibitor of PDE 5, is effective in the treatment of erectile dysfunction. Penile erection involves relaxation of smooth muscle of corpus cavernosum and its associated arterioles. The objective of this study was to investigate the effect of sildenafil on nitric oxide/cyclic guanosine monophosphate (NO/cGMP)-dependent relaxation of rat aortic rings. The contribution of sildenafil to the vasorelaxation of rat aortic rings was also investigated. Sildenafil produced significant potentiation of acetylcholine (ACh, 2 x 10(-6) m)-induced relaxation at concentration > or =1 x 10(-8) m. Addition of sildenafil (1 x 10(-7) m) to aortic rings failed to alter the effect of N(G)-nitro-L-arginine (l-NNA, 3 x 10(-5) m) or methylene blue (MB, 3 x 10(-5) m) on ACh response. Similarly, sildenafil (1 x 10(-7) m) augmented significantly the vasorelaxation induced by sodium nitroprusside over the range of 1 x 10(-9)-1 x 10(-8) m. When added to phenylephrine (3 x 10(-6) m)-precontracted rat aortic rings, sildenafil (1 x 10(-9)-1 x 10(-4) m) induced concentration-dependent relaxation reaching a maximum of 96.48 +/- 1.44%. These relaxations were not significantly attenuated by previous incubation with L-NNA (3 x 10(-5) m) or MB (3 x 10(-5) m). Denudation did not significantly affect the vasorelaxant effect of sildenafil. Sildenafil may act in the rat aortic rings through the amplification of NO/cGMP pathway. It may augment both basal endothelial NO function and exogenous NO-dependent vasodilatation. However, sildenafil may act by a mechanism independent of NO/cGMP pathway and this mechanism contributes to its smooth muscle relaxant effect.

The Vasorelaxing Action of Labedipinedilol-A Involves Endothelial Cell-Derived NO and eNOS Expression Caused by Calcium Influx

Labedipinedilol-A, a novel dihydropyridine-type calcium antagonist, has been shown to induce hypotension and vasorelaxation. The objective of the present study was to investigate the effect of labedipinedilol-A on vascular function of rat aortic rings and cultured human umbilical vein endothelial cells (HUVECs). Labedipinedilol-A induced vasorelaxation in rat aortic rings that had been precontracted with phenylephrine in a concentration-dependent manner. This labedipinedilol-A-induced relaxation was significantly reduced by endothelium removal and by exposure to L-NG-nitroarginine methyl ester (L-NAME), methylene blue, or 1H-[1,2,4]oxadiazolol[4,3,a]quinoxalin-1-one (ODQ). In addition, the cyclic GMP content was significantly increased by labedipinedilol-A, which was inhibited by L-NAME in aorta. In cultured HUVECs, labedipinedilol-A induced concentration-dependent formation of NO and Ca2+ influx, and it increased the abundance of endothelial NO synthase (eNOS) protein. Furthermore, labedipinedilol-A suppressed basal, 10% FBS- and thrombin-stimulated endothelin-1 production, which were reversed by pretreatment with L-NAME, demonstrating that NO was able to inhibit production of ET-1 in HUVECs. Labedipinedilol-A significantly protected cultured HUVECs against dihydroxyfumarate/iron ion-induced decrease of glutathione and cell death. Moreover, labedipinedilol-A also inhibited iron-induced lipid peroxidation in rat brain homogenate and scavenged 2,2'-azobis (2-amidinopropane) dihydrochloride-derived peroxy radicals. Labedipinedilol-A acts as lacidipine with additional antioxidant effects and can protect endothelial cells against free radical-induced lipid peroxidation and cell injury. Our results indicate that the endothelium-dependent vasorelaxation by labedipinedilol-A is mediated through Ca2+-dependent activation of NO synthase and stimulation of NO/cyclic GMP pathway.

Rutin-induced endothelium-dependent vasorelaxation in rat aortic Rings and the underlying mechanism.

The aim of the present study was to determine the possible mechanism by which rutin causes vasodilatation in isolated thoracic aorta rings from the rat. The effects of rutin on rings preconstricted with phenylephrine, with or without endothelium, were determined using an organ bath technique. The mechanism was explored by measuring the effects of the nitric oxide synthase inhibitor L-N(G)-nitroarginine methyl ester (L-NAME), the guanylyl cyclase inhibitor methylene blue, the cyclooxygenase inhibitor indomethacin, the ATP-sensitive K+channel blocker glibenclamide and the beta-adrenoceptor antagonist propranolol. Rutin at the range of 10-160 mumol/L caused dose-dependent vasorelaxation in preconstricted endothelium-intact rings, but had no effect on rings without endothelium. The maximal response calculated from the vasorelaxation curves of rutin was 44.28 plus or mines 7.48%. Pretreatment with L-NAME (0.1 mmol/L), methylene blue (10 mumol/L), glibenclamide (10 mmol/L) or indomethacin (10 mmol/L) attenuated the vasorelaxation induced by rutin in endothelium-intact rings. Glibenclamide (10 mmol/L) enhanced the vasorelaxation of rutin. Propranolol (10 mumol/L) did not block the effect of rutin. The results indicate that vasorelaxation is induced by rutin via the nitric oxide-guanylyl cyclase pathway and a prostaglandin-mediated mechanism, as well as activation of the ATP-sensitive potassium channel.

Ursodeoxycholic acid and in vitro vasoactivity of hydrophobic bile acids.

Lipophilic bile acids, such as deoxycholic acid (DCA), are nonspecific endothelium-independent vasorelaxants whose underlying basis is complex, involving membrane calcium channels blockade and receptor antagonism. The vasorelaxant action of these acids has also been linked to the generation of reactive oxygen species and an increased extent of lipid peroxidation. Ursodeoxycholic acid (UDCA) is a naturally occurring tertiary dihydroxy hydrophilic acid whose mechanism of action has been attributed to minimizing the effects of lipophilic bile acids. Hence, we considered UDCA might be a useful pharmacological tool to delineate the role of enhanced lipid peroxidation in lipophilic bile acid-induced vasorelaxation. UDCA abrogates in vitro DCA-induced vasorelaxation in rat aortic rings and can suppress DCA-initiated lipid peroxidation in vascular smooth muscle microsomal membrane fractions prepared from the rat aortae. Three different studies were performed. In study 1, the ability of UDCA to restore the DCA-blunted contractile response to the alpha1-adrenoceptor, phenylephrine in rat aortic rings, was evaluated. In study 2, the ability of UDCA to restore DCA-induced vasorelaxation in precontracted rat aortic rings was assessed. In study 3, the ability of UDCA to suppress the increased extent of lipid peroxidation effected by DCA in vascular smooth muscle microsomal membrane fractions prepared from rat aortae was measured using the thiobarbituric acid reactive substance (TBARS) assay. UDCA, at a concentration equivalent to that seen in the plasma of patients with cholestatic liver disease treated with the bile acid, partially restored DCA-induced impaired contractility, prevented DCA-induced vasorelaxation, and abolished DCA-induced increases in the extent of lipid peroxidation. In conclusion, these data suggest that DCA-induced vasorelaxation is mediated by increasing the extent of lipid peroxidation in vascular tissue.

Involvement of Ca2+ -activated K+ channels in ginsenosides-induced aortic relaxation in rats.

Ginsenosides (GS), an extract of Panax ginseng, have been reported to be effective in inducing vascular relaxation mediated by nitric oxide (NO) release. The present experiments were designed to determine whether this GS-induced vasorelaxation also involves Ca2+ -activated K+ (KCa) channels in vascular smooth muscle cells (VSMC) in addition to endothelium-derived NO. GS induced vasorelaxation in rat aortic rings, which had been precontracted with phenylephrine, in a concentration-dependent manner. This GS-induced relaxation was partially reversed by tetraethylammonium (TEA), an inhibitor of KCa channels; methylene blue (MB), an inhibitor of soluble guanylate cyclase; as well as Nomega-nitro-L-arginine (L-NNA), but not by glybenclamide. In cultured VSMC and endothelial cells, KCa channels were activated by GS. This action was abolished by TEA, but was not blocked by glybenclamide. In addition, the GS-induced activity of KCa channels was partially inhibited by MB or H-8. These results indicate that the activation of KCa channels involved, at least in part, the GS-induced vasorelaxation of rat aorta.