Pre-incubation Of Aortic Rings Research Articles

A new oxime synthesized from Senecio nutans SCh. Bip (chachacoma) reduces calcium influx in the vascular contractile response in rat aorta.

Senecio nutans Sch. Bip is an endemic plant commonly employed in the Andes culture to counteract the effects of mountain sickness, and its bioactive molecules could provide new drugs for treating hypertension. The purpose was to determine whether the vascular response of the plant bioactive molecules, such as (5-acetyl-6-hydroxy-2-isopropenyl-2,3-dihydrobenzofurane; Sn-I), could be improved by a simple structural modification to synthesize oximes (Ox-Sn-I). We characterized both compounds using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). We investigated vascular relaxation mechanisms in response to Sn-I and Ox-Sn-I using rat aorta and vascular smooth muscle cells (A7r5) as experimental models. Preincubation of aortic rings with Sn-I (10-5 M) significantly (p < 0.001) decreased the contractile effect in response to phenylephrine (PE) and potassium chloride (KCl). The sensitivity (EC50) to PE significantly (p < 0.01) decreased in the presence of Sn-I (10-5 M), but not with Ox-Sn-I. Sn-I significantly (p < 0.001) reduced the PE-induced contraction under calcium-free conditions. When A7r5 cells were preincubated with Sn-I and Ox-Sn-I (10-5 M), both compounds blunted the increase in intracellular Ca2+ induced by KCl. 2,3-Dihydrobenzofurane derived from S. nutans (Sn-I) reduces the contractile response probably by blocking Ca2+ entry through voltage-gated Ca2+ channels (VGCC) in vascular smooth cells. This effect also causes relaxation in rat aorta mediated by reduction of intracellular Ca2+ concentration, rather than an increase of NO generation in endothelial vascular cells.

Antihypertensive effect of the stem bark aqueous extract of Garcinia lucida Vesque (Clusiaceae) in L-NAME-treated rats: Contribution of endothelium-dependent and -independent vasorelaxation

Garcinia lucida is used in Cameroonian folk medicine to handle a variety of ailments, including arterial hypertension. This study aimed at determining the phytochemical profile and the antihypertensive effect of the stem bark aqueous extract of G. lucida (AEGL). AEGL was subjected to LC-MS analysis, and its effect (75, 150, and 300 mg/kg/day; by gavage) was evaluated against Nω-nitro-L-arginine methyl ester (L-NAME; 40 mg/kg)-induced hypertension in adult male Wistar rats for four consecutive weeks. Blood pressure and heart rate were monitored weekly using tail-cuff plethysmography. The vasorelaxant effect of cumulative concentrations (3–10–30–100–300 μg/mL) of AEGL was examined on endothelium-intact and denuded thoracic aorta rings which were precontracted with KCl (90 mM) or norepinephrine (NE; 10−5 M), and in the absence or presence of L-NAME (10−4 M), indomethacin (10−5 M), methylene blue (10−6 M), tetraethylammonium (TEA, 5 × 10−6 M), glibenclamide (10 × 10−6 M) or propranolol (5 × 10−6 M). The influence of AEGL on the response to NE, KCl, and CaCl2 was also investigated. Six compounds, including Garcinia biflavonoids GB1 and GB2, were identified. AEGL prevented the development of hypertension (p < 0.01 and p < 0.001) without affecting the heart rate. AEGL induced a concentration-dependent relaxation of aortic rings precontracted with NE (EC50 = 7.915 μg/mL) that was significantly inhibited by the removal of the endothelium, L-NAME, or methylene blue (p < 0.05–0.001). Indomethacin, propranolol, TEA, and glibenclamide did not affect AEGL-evoked vasorelaxation. Preincubation of aortic rings with AEGL reduced the magnitude of contraction elicited by CaCl2 but did not alter that of KCl or NE. AEGL possesses an antihypertensive effect that is mediated by both endothelium-dependent and endothelium-independent mechanisms. The activation of the NO/sGC/cGMP pathway accounts for the endothelium-dependent vasorelaxation. These pharmacological effects of AEGL could be attributed to the presence of the Garcinia biflavonoids GB1 and GB2.

Metabolic Remodeling with Hepatosteatosis Induced Vascular Oxidative Stress in Hepatic ERK2 Deficiency Mice with High Fat Diets.

We previously demonstrated the marked hepatosteatosis and endothelial dysfunction in hepatocyte-specific ERK2 knockout mice (LE2KO) with a high-fat/high-sucrose diet (HFHSD), but detailed metabolic changes and the characteristics in insulin-sensitive organs were not tested. This study aimed to characterize metabolic remodeling with changes in insulin-sensitive organs, which could induce endothelial dysfunction in HFHSD-LE2KO. The serum glucose and fatty acid (FA) were modestly higher in HFHSD-LE2KO than HFHSD-Control. FA synthesis genes were up-regulated, which was associated with the decreased phosphorylation of AMPK and ACC, and with the up-regulation of SREBP-1 in the liver from HFHSD-LE2KO. In FA and amino acids fraction analysis, arachidonic acid/eicosapentaenoic acid ratio, L-ornithine/arginine ratio, asymmetric dimethylarginine and homocysteine levels were elevated in HFHSD-LE2KO. Insulin-induced phosphorylation of AKT was blunted in skeletal muscle. Serum leptin and IL-1β were elevated, and serum adiponectin was decreased with the enlargement of epididymal adipocytes. Finally, the enhanced superoxide levels in the aorta, which were blunted with CCCP, apocynin, and tempol, were observed in HFHSD-LE2KO. A pre-incubation of aortic rings with tempol improved endothelial dysfunction in HFHSD-LE2KO. HFHSD-LE2KO revealed an acceleration of FA synthesis in the liver leading to insulin resistance in skeletal muscle and the enlargement of visceral adipocytes. Global metabolic remodeling such as changes in arginine metabolism, ω3/ω6 ratio, and adipocytokines, could affect the vascular oxidative stress and endothelial dysfunction in HFHSD-LE2KO.

Novel Oxime Synthesized from a Natural Product of Senecio nutans SCh. Bip. (Asteraceae) Enhances Vascular Relaxation in Rats by an Endothelium-Independent Mechanism.

Senecio nutans Sch. Bip. and its constituents are reported to have antihypertensive effects. We isolated metabolite–1, a natural compound from S. nutans (4-hydroxy-3-(isopenten-2-yl)-acetophenone), and synthesized novel oxime – 1 (4-hydroxy-3-(isopenten-2-yl)-acetophenoxime) to evaluate their effect on vascular reactivity. Compounds were purified (metabolite–1) or synthetized (oxime–1) and characterized using IR and NMR spectroscopy and Heteronuclear Multiple Quantum Coherence (HMQC). Using pharmacological agents such as phenylephrine (PE) and KCl (enhancing contraction), acetylcholine (ACh), L-NAME (nitric oxide (NO) and endothelial function), Bay K8644-induced CaV1.2 channel (calcium channel modulator), and isolated aortic rings in an organ bath setup, the possible mechanisms of vascular action were determined. Pre-incubation of aortic rings with 10−5 M oxime–1 significantly (p < 0.001) decreased the contractile response to 30 mM KCl. EC50 to KCl significantly (p < 0.01) increased in the presence of oxime–1 (37.72 ± 2.10 mM) compared to that obtained under control conditions (22.37 ± 1.40 mM). Oxime–1 significantly reduced (p < 0.001) the contractile response to different concentrations of PE (10−7 to 10−5 M) by a mechanism that decreases Cav1.2-mediated Ca2+ influx from the extracellular space and reduces Ca2+ release from intracellular stores. At a submaximal concentration (10−5 M), oxime–1 caused a significant relaxation in rat aorta even without vascular endothelium or after pre-incubate the tissue with L-NAME. Oxime–1 decreases the contractile response to PE by blunting the release of Ca2+ from intracellular stores and blocking of Ca2+ influx by channels. Metabolite–1 reduces the contractile response to KCl, apparently by reducing the plasma membrane depolarization and Ca2+ influx from the extracellular space. These acetophenone derivates from S. nutans (metabolite–1 and oxime–1) cause vasorelaxation through pathways involving an increase of the endothelial NO generation or a higher bioavailability, further highlighting that structural modification of naturally occurring metabolites can enhance their intended pharmacological functions.

Irisin relaxes rat thoracic aorta: MEK1/2 signaling pathway, KV channels, SKCa channels, and BKCa channels are involved in irisin-induced vasodilation.

This study investigated the effects of irisin on vascular smooth muscle contractility in rat thoracic aorta, and the hypothesis that mitogen-activated protein kinase kinase (MEK1/2) signaling pathway, voltage-gated potassium (KV) channels, small-conductance calcium-activated potassium (SKCa) channels, and large-conductance calcium-activated potassium (BKCa) channels may have roles in these effects. Isometric contraction-relaxation responses of isolated thoracic aorta rings were measured with an organ bath model. The steady contraction was induced with 10-5M phenylephrine (PHE), and then the concentration-dependent responses of irisin (10-9-10-6 M) were examined in endothelium-intact and -denuded rat thoracic aortas. Also, the effects of irisin incubations on PHE-mediated contraction and acetylcholine (ACh) - mediated relaxation were studied. Irisin exerted the vasorelaxant effects in both endothelium-intact and -denuded aortic rings at concentrations of 10-8, 10-7, and 10-6M compared with the control groups (p< 0.001). Besides, pre-incubation of aortic rings with irisin (10nM, 100nM, or 1µM for 30min) augmented ACh-mediated (10-9-10-5) vasodilation in PHE-precontracted thoracic aorta segments but did not modulate PHE-mediated (10-9-10-5) contraction. In addition, MEK1/2 inhibitor U0126, KV channel blocker XE-991, SKCa channel blocker apamin, and BKCa channel blocker tetraethylammonium (TEA) incubations significantly inhibited the irisin-induced relaxation responses. In conclusion, the first physiological findings were obtained regarding the functional relaxing effects of irisin in rat thoracic aorta. The findings demonstrated that irisin induces relaxation responses in endothelium-intact and (or) endothelium-denuded aortic rings in a concentration-dependent manner. Furthermore, this study is the first to report that irisin-induced relaxation responses are related to the activity of the MEK1/2 pathway, KV channels, and calcium-activated K+ (SKCa and BKCa) channels.

8-Oxo-9-Dihydromakomakine Isolated from Aristotelia chilensis Induces Vasodilation in Rat Aorta: Role of the Extracellular Calcium Influx.

8-Oxo-9-dihydromakomakine is a tetracyclic indole alkaloid extracted from leaves of the Chilean tree Aristotelia chilensis. The present study investigated the effects of this alkaloid on vascular response in tissues isolated from aortic segments obtained from normotensive rats. Our results showed that 8-oxo-9-dihydromakomakine induced a dose-dependent relaxation of aortic rings pre-contracted with phenylephrine (PE; 10−6 M). The vasorelaxation induced by 8-oxo-9-dihydromakomakine in rat aortic rings is independent of endothelium. The pre-incubation of aortic rings with 8-oxo-9-dehydromakomakine (10−4 M) significantly reduced the contractile response to KCl (p < 0.001) more than PE (p < 0.05). The highest dose of 8-oxo-9-dehydromakomakine (10−4 M) drastically reduced the contraction to KCl (6·10−2 M), but after that, PE (10−6 M) caused contraction (p < 0.05) in the same aortic rings. The addition of 8-oxo-9-dihydromakomakine (10−5 M) decreased the contractile response to tetraethylammonium (a voltage-dependent potassium channels blocker; TEA; 5 × 10−3 M; p < 0.01) and BaCl2 (a non-selective inward rectifier potassium channel blocker; 5 × 10−3 M; p < 0.001) in rat aorta. 8-oxo-9-dihydromakomakine (10−5 M) decreased the contractile response to PE in rat aorta in the presence or absence of ouabain (an inhibitor of Na,K-ATPase; 10−3 M; p < 0.05). These results could indicate that 8-oxo-9-dihydromakomakine partially reduces plasma membrane depolarization-induced contraction. In aortic rings depolarized by PE, 8-oxo-9-dihydromakomakine inhibited the contraction induced by the influx of extracellular Ca2+ in a Ca2+ free solution (p < 0.01). 8-oxo-9-dihydromakomakine reduced the contractile response to agonists of voltage-dependent calcium channels type L (Bay K6844; 10−8 M; p < 0.01), likely decreasing the influx of extracellular Ca2+ through the voltage-dependent calcium channels. This study provides the first qualitative analysis indicating that traditional folk medicine Aristotelia chilensis may be protective in the treatment of cardiovascular pathologies.

UPLC/Q-TOF-MS profiling of phenolics from Canarium pimela leaves and its vasorelaxant and antioxidant activities

Canarium pimela K.D. Koenig, Burseraceae, have a long history of use in the Chinese traditional medicine treatment of various ailments including hypertension, and our research team has reported the anti-hypertensive activity and delineated the mechanism involved in the action. The following research aims to evaluate the vasorelaxant and antioxidant activities of ethanol extract from C. pimela leaves and to analyze its chemical composition by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) that may correlate with their pharmacological activities. The results showed that pre-incubation of aortic rings with the extract (0.3, 1, 3, 10, 30 and 100mg/l) significantly inhibited the contractile response of the rings to norepinephrine-induced contraction (p<0.01or p<0.05). Crude ethanol extract and refined ethanol extract showed a highest inhibitory effect against 2,2dipheyl-2-picrylhydrazyl hydrate scavenging activity (IC50 of crude ethanol extract=15.42±0.14μg/ml and IC50 of refined ethanol extract=5.72±0.31μg/ml) and 2,2′-azinobis (3-ethyl-benzothiazoline-6-sulphonic acid ammonium salt) (ABTS (IC50 of crude ethanol extract=3.24±0.18μg/ml and IC50 of refined ethanol extract=1.88±0.07μg/ml) scavenging activity, which was considerably higher than that reported for butylated hydroxytoluene and lower of that measured for ascorbic acid. Moreover, its chemical composition was analyzed by UPLC/Q-TOF-MS. Sixteen compounds including nine flavonoids, four tannins, two phenolic acids and one dianthrone were identified for the first time as constituents of this species. And of this, six major phenolic components were simultaneous quantitative analysis by HPLC-UV, chlorogenic acid is the major compounds in C. pimela leaves. These results indicate that the phenolic-rich extract of C. pimela leaves is a promising natural pharmaceutical for combating hypertension and oxidative stress.

324 - Sodium Nitrate Attenuates the Acute Antihypertensive and Vascular Effects of Sodium Nitrite by Inhibiting Nitrite-Reductase Activity of Xanthine Oxidoreductse

The antihypertensive effects of nitrite are at least in part attributable to tissue reduction of nitrite to nitric oxide (NO) by xanthine oxidoreductase (XOR). XOR also reduces nitrate to nitrite, however, at a much lower rate than it reduces nitrite to NO. Here, we examined whether nitrate decreases the formation of NO from nitrite by XOR, and therefore attenuates its antihypertensive and vascular effects. Methods Blood pressure effects were studied in rats receiving Nω-nitro-L- arginine methyl ester (L-NAME) 100 mg/Kg orally to increase the mean of arterial pressure (MAP). Forty min later, they received nitrate (40 µmol/kg) or vehicle. Then increasing doses of sodium nitrite were infused into the femoral vein. Invasive MAP was assessed in the femoral artery. Vascular reactivity studies: The vasorelaxant effects of sodium nitrite (0.3 nMto 10 mM concentrations) were examined in aortic rings pre-contracted with phenylephrine 10-7M in the presence or absence of sodium nitrate 300 µM. In addition, mesenteric arterial bed preparations infused continuously with phenylephrine (3 µM to 8 µM). Sodium nitrate 300 µM or vehicle was added to the perfusion liquid and a concentration-response curve to nitrite (0.1 µM to 1 mM) was constructed. Nitrite reductase activity was measured by injecting XOR 100 mU/ml into a purge vessel containing nitrite 1 mM, NADPH 1 mM and NADH 1 mM in the presence or absence of sodium nitrate 30 mM, and NO was detected by chemiluminescence. Results Pretreatment with sodium nitrate attenuated nitrite-induced decreases in MAP. Pre-incubation of aortic rings with nitrate shifted the concentration-response curve in response to sodium nitrite to the right and reduced the maximum response. Adding sodium nitrate to the solution perfusing the mesenteric bed attenuated the fall in perfusion pressure induced by nitrite. Nitrate decreased nitrite-induced NO formation by XOR in the purge vessel. Conclusion Our results show that nitrate attenuates the acute blood pressure responses to nitrite by mechanisms that involve reduced nitrite-derived NO formation by XOR.

Mechanisms underlying the antihypertensive properties of Urtica dioica.

BackgroundUrtica dioica has traditionally been used in the management of cardiovascular disorders especially hypertension. The aim of this study was to explore pharmacological base of its use in hypertension.MethodsCrude methanolic extract of U. dioica (Ud.Cr) and its fractions (Ud.EtAc, Ud.nHex, Ud.Chl and Ud.Aq) were tested in vivo on normotensive and hypertensive rats under anesthesia for blood pressure lowering effect. In-vitro experiments on rat and rabbit aortae were employed to probe the vasorelaxation mechanism(s). The responses were measured using pressure and force transducers connected to PowerLab Data Acquisition System.ResultsUd.Cr and fractions were found more effective antihypertensive in hypertensive rats than normotensive with remarkable potency exhibited by the ethyl acetate fraction. The effect was same in the presence of atropine. In isolated rat aortic rings, Ud.Cr and all its fractions exhibited l-NAME sensitive endothelium-dependent vasodilator effect and also inhibit K+ (80 mM)-induced pre-contractions. In isolated rabbit thoracic aortic rings Ud.Cr and its fractions induced relaxation with more potency against K+ (80 mM) than phenylephrine (1 µM) like verapamil, showing Ud.EtAc fraction the most potent one. Pre-incubation of aortic rings with Ud.Cr and its fractions exhibited Ca2+ channel blocking activity comparable with verapamil by shifting Ca2+ concentration response curves to the right. Ud.Cr and its fractions also ablated the intracellular Ca2+ release by suppressing PE peak formation in Ca2+ free medium. When tested on basal tension, the crude extract and all fractions were devoid of any vasoconstrictor effect.ConclusionsThese data indicate that crude methanolic extract and its fractions possess antihypertensive effect. Identification of NO-mediated vasorelaxation and calcium channel blocking effects explain the antihypertensive potential of U. dioica and provide a potential pharmacological base to its medicinal use in the management of hypertension.

Vasodilatory effects of nitric oxide, hydrogen sulfide and sulfur dioxide in rats: Time-dependent interaction study

The vasodilator response of nitric oxide (NO), hydrogen sulfide (H2S) and sulfur dioxide (SO2) were studied todetermine the significance of the actions and interactions of these gasotransmitters for controlling aortic tone in rats. The isometric tension of five separate sets of experiments was recorded. Sodium nitroprusside (SNP; NO donor), sodium disulphide (Na2S; H2S donor), SO2 derivatives and their paired combinations were added to phenylephrine (PE)-induced contraction during the peak value. Then maximal relaxation rate was calculated four times at 5 min intervals. Tetraethylammonium (TEA) and Glibenclamide (GLIB) were applied for investigating the molecular mechanism of the gasses. While, in a separate set of experiments, we used either L-Arginine (L-Arg), L-Cysteine (L-Cyst) or L-nitroarginine methyl ester (L-NAME) before applying gasotransmitters. Highest and prolonged relaxation rate were recorded when SNP was combined with SO2. The combination of Na2S and SO2-induced vasorelaxation was blocked by TEA and GLIB pretreatments. L-Cyst decreased relaxation compared to SNP and vice versa to SO2 induced vasorelaxation. L-Arg markedly attenuated relaxation responses of Na2S and SO2 derivatives. Also, L-NAME delayed relaxation compared to Na2S and SO2. These results suggest that exogenous paired combinations of H2S, NO and SO2 will enhance and elongate the rate of aortic relaxation. Meanwhile, preincubation of aortic rings with precursors attenuate the dilatory effects of exogenous studied gases.

Mechanisms underlying the antihypertensive effect of Alstonia scholaris

Ethnopharmacological relevanceAlstonia scholaris has a long history of use in the Ayurveda traditional treatment of various ailments including hypertension. We have reported the blood pressure lowering activity of the extract of A. scholaris. The following research aim to delineate the pharmacological mechanism involve in the antihypertensive action. Materials and methodVasorelaxant effect of the n-butanol fraction of A. scholaris (NBF-ASME) was evaluated on rat aorta pre-contracted with phenyelphrine (PE, 1µM). Aortic rings preparation were pre-incubated with various antagonists like 1H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ 10μM), methylene blue (MB 10μM), Nω-nitro-L-arginine methyl ester hydrochloride (l-NAME 10μM), atropine (10μM), indomethacin (1μM), ML-9 and various K+ channel blockers such as glibenclamide (10μM) and tetraethyl ammonium (TEA 10μM) for mechanism study. ResultThe results showed that pre-incubation of aortic rings with the extract (0.5, 1 and 2mg/mL) significantly inhibit the contractile response of the rings to phenylephrine-induced contraction (p<0.05–0.001). Removal of endothelium, incubation with l-NAME, indomethacin, atropine and propranolol did not significantly affect the relaxation effect of NBF-ASME. Furthermore, the K+ channel blockers, TEA and glibenclamide showed no inhibitory effect. However, aortic rings pretreated with ODQ and ML-9 showed a significant suppression of the relaxation curve of NBF-ASME (p<0.01–0.001). In Ca2+-free solution, NBF-ASME inhibits the release of intracellular Ca2+ from the sarcoplasmic reticulum. NBF-ASME also inhibits calcium chloride (CaCl2)-induced contraction in endothelium-denuded aortic rings. ConclusionThe results from this study suggests that A. scholaris exerts vasodilation via calcium channels blockade, direct activation of soluble guanylate cyclase and possibly by also inhibiting the formation of inositol 1, 4, 5-triphosphate.

The antimetastatic drug NAMI-A potentiates the phenylephrine-induced contraction of aortic smooth muscle cells and induces a transient increase in systolic blood pressure

The ruthenium-based drug imidazolium trans-imidazoledimethylsulphoxidetetrachlorido ruthenate (NAMI-A) is a novel antitumour drug under clinical evaluation. In this study, NAMI-A is tested on aortic rings in vitro and on the systolic blood pressure in vivo with the aim of evaluating its effects on smooth muscle cells and, more in general, on the vascular system. Pre-incubation of aortic rings with 10 µM NAMI-A for 10 min potentiates the contraction induced by phenylephrine (PE). The reduction of the B max value of [(3)H]-prazosin bound to NAMI-A-treated aortic rings and the ability of NAMI-A to displace [(3)H]-prazosin and [(3)H]-IP3 binding by 25 and 42%, respectively, suggest the involvement of α1-adrenoceptor in mediating the effects on smooth muscle cells. NAMI-A also decreases the number of maximal sites of [(3)H]-prazosin bound to kidney membrane preparation from 34 to 24 fmol/mg proteins. A single i.p. dose (105 mg/kg) or a repeated treatment for 6 consecutive days (17 mg/kg/day) in Wistar rats increases the systolic blood pressure, respectively, 1 h and 3 days after treatment, and the responsiveness of rat aortic rings to PE. Atomic absorption spectroscopy confirms the presence of ruthenium in the aortic rings excised from the treated rats. These findings suggest monitoring the cardiovascular parameters when the drug is used in humans for treating cancer patients, particularly if the drug is associated with chemicals that are potentially active at the cardiovascular level.

Rosiglitazone via PPARγ-dependent suppression of oxidative stress attenuates endothelial dysfunction in rats fed homocysteine thiolactone.

To explore whether rosiglitazone (RSG), a selective peroxisome proliferator-activated receptor γ (PPARγ) agonist, exerts beneficial effects on endothelial dysfunction induced by homocysteine thiolactone (HTL) and to investigate the potential mechanisms. Incubation of cultured human umbilical vein endothelial cells with HTL (1 mM) for 24 hrs significantly reduced cell viabilities assayed by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide, as well as enhanced productions of reactive oxygen species, activation of nuclear factor kappa B, and increased intercellular cell adhesion molecule-1 secretion. Pre-treatment of cells with RSG (0.001–0.1 mM), pyrollidine dithiocarbamate (PDTC, 0.1 mM) or apocynin (0.1 mM) for 1 hr reversed these effects induced by HTL. Furthermore, co-incubation with GW9662 (0.01 mM) abolished the protective effects of RSG on HTL-treated cells. In ex vivo experiments, exposure of isolated aortic rings from. rats to HTL (1 mM) for 1 hr dramatically impaired acetylcholine-induced endothelium-dependent relaxation, reduced release of nitric oxide and activity of superoxide dismutase, and increased malondialdehyde content in aortic tissues. Preincubation of aortic rings with RSG (0.1, 0.3, 1 mM), PDTC or apocynin normalized the disorders induced by HTL. In vivo analysis indicated that administration of RSG (20 mg/kg/d) remarkably suppressed oxidative stress and prevented endothelial dysfunction in rats fed HTL (50 mg/kg/d) for 8 weeks. RSG improves endothelial functions in rats fed HTL, which is related to PPARγ-dependent suppression of oxidative stress.

Tongxinluo Prevents Endothelial Dysfunction Induced by Homocysteine Thiolactone In Vivo via Suppression of Oxidative Stress.

Aim. To explore whether Chinese traditional medicine, tongxinluo (TXL), exerts beneficial effects on endothelial dysfunction induced by homocysteine thiolactone (HTL) and to investigate the potential mechanisms. Methods and Results. Incubation of cultured human umbilical vein endothelial cells with HTL (1 mM) for 24 hours significantly reduced cell viabilities assayed by MTT, and enhanced productions of reactive oxygen species. Pretreatment of cells with TXL (100, 200, and 400 μg/mL) for 1 hour reversed these effects induced by HTL. Further, coincubation with GW9662 (0.01, 0.1 mM) abolished the protective effects of TXL on HTL-treated cells. In ex vivo experiments, exposure of isolated aortic rings from rats to HTL (1 mM) for 1 hour dramatically impaired acetylcholine-induced endothelium-dependent relaxation, reduced SOD activity, and increased malondialdehyde content in aortic tissues. Preincubation of aortic rings with TXL (100, 200, and 400 μg/mL) normalized the disorders induced by HTL. Importantly, all effects induced by TXL were reversed by GW9662. In vivo analysis indicated that the administration of TXL (1.0 g/kg/d) remarkably suppressed oxidative stress and prevented endothelial dysfunction in rats fed with HTL (50 mg/kg/d) for 8 weeks. Conclusions. TXL improves endothelial functions in rats fed with HTL, which is related to PPARγ-dependent suppression of oxidative stress.

Sexual dimorphism in aortic endothelial function of Zucker diabetic fatty rats: possible involvement of superoxide production (1051.8)

Little is known about the interaction between diabetes and sex in vasculature. This study was designed to investigate whether there were sex differences in rat aortic endothelium‐dependent vasodilation (EDV) in Zucker diabetic fatty (ZDF) rats, and the potential role of superoxide. EDV to acetylcholine (ACh) was measured in aortic rings pre‐contracted with phenylephrine before and after pretreatment with apocynin (100 μM), a NADPH oxidase (Nox) inhibitor. In addition, the level of Nox (a potent source of superoxide) and PKCβ mRNA expression were determined using real‐time RT‐PCR. ACh‐induced relaxations were significantly greater in female lean rats compared with male lean rats. Accordingly, male lean rats had higher PKCβI expression level than female lean rats. Diabetes significantly impaired EDV in aortic rings from female ZDF rats, however, potentiated the relaxation in males. Pre‐incubation of aortic rings with apocynin increased EDV only in diabetic female group, suggesting impairment of EDV in female ZDF aorta was partly due to an increase in the activity of superoxide. Accordingly, the levels of Nox1, Nox4, and PKCβ mRNA expression were substantially enhanced in aorta of female ZDF rats compared to those in lean animals. These data suggest that an elevation of superoxide may partially contribute to the predisposition of the female aorta to injury in type 2 diabetes.Grant Funding Source: NIH/NIDCR

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity

Received 22 July 2012; revised 29 January 2013; accepted 4 March 2013AimsAldosterone plays a crucial role in cardiovascular disease. ‘Systemic’ inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the ‘endothelial’ MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis.Methods and resultsC57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high ‘endogenous’ aldosterone) and in ‘exogenous’ aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR.ConclusionObesity-induced endothelial dysfunction depends on the ‘endothelial’ MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.

Alfacalcidol suppresses α-receptor–mediated vasoconstriction via an endothelium dependent mechanism

Vitamin D level could be related to blood pressure and cardiovascular health. However, the direct suppressor effect of vitamin D on vascular contraction has not yet been explored. Therefore, the effects of alfacalcidol (1\alpha-hydroxyvitamin D_3) and calcitriol (1\alpha,25-dihydroxyvitamin D_3) on contractions induced by phenylephrine (10^{-8} to 10^{-5} M) in mice aorta were investigated. Materials and methods: Arterial rings were suspended in isolated organ chambers filled with Krebs bicarbonate solution gassed with 95% O_2 and 5% CO_2 maintained at 37 °C. Tissue responses were recorded isometrically with a force transducer and displayed on an acquisition system. Alfacalcidol and calcitriol were applied on phenylephrine-induced active tone. Furthermore, aortic rings were preincubated with alfacalcidol and calcitriol and thereafter contracted with phenylephrine. Incubation studies were conducted by endothelium removal and in the presence of L-nitro-arginine methyl ester (L-NAME). Results: Phenylephrine-induced active tones were significantly suppressed by alfacalcidol (P < 0.05 and P < 0.01) and calcitriol (both 10^{-5} and 10^{-6} M). The pEC_{50} values of alfacalcidol and calcitriol were 7.06 ± 0.91 and 7.13 ± 0.60, respectively. Furthermore, preincubation of aortic rings with alfacalcidol (10^{-5} M) but not calcitriol (10^{-5} M) significantly inhibited phenylephrine-induced contractions (10^{-8} to 10^{-5} M, P < 0.05 and P < 0.001). The suppressive effect of alfacalcidol incubation disappeared with endothelium removal. Furthermore, alfacalcidol was unable to inhibit phenylephrine-induced contractions in the presence of L-NAME, an inhibitor of nitric oxide biosynthesis. Conclusion: In this study we report for the first time that alfacalcidol may prevent phenylephrine-induced vasoconstriction in mice aorta with an endothelium-dependent nitric oxide-mediated mechanism.

Tamoxifen and Its Metabolites Cause Acute Vasorelaxation of Aortic Rings by Inducing Vasodilator Prostanoid Synthesis

The vascular effects of tamoxifen (Tam) and its metabolites are poorly known. We compared the vasorelaxation induced by Tam and its metabolites (N-desmethyl-Tam, 4-hydroxy-Tam, and endoxifen) in aortic rings from rats using standardized organ bath procedures, and we investigated the mechanisms involved in this effect. Tam and its metabolite-induced vasorelaxation in a concentration-dependent manner. Although 4-hydroxy-Tam and Tam had similar potency (pD2 = 8.5 ± 0.1 vs. 8.8 ± 0.1, respectively) and maximum effect (Emax = 88.5% ± 1.3% vs. 92.6% ± 1.3%, respectively), N-desmethyl-Tam and endoxifen were more potent and showed higher Emax than Tam did (pD2 = 9.0 ± 0.1 and 8.9 ± 0.1; Emax = 101.1% ± 1.8% and 101.0% ± 1.8% for N-desmethyl-Tam and endoxifen, respectively). Although preincubation of aortic rings with the estrogen receptor antagonist ICI 182780 or with the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride induced no changes in the vasorelaxation induced by Tam or 4-hydroxy-Tam, both drugs significantly reduced Emax in response to N-desmethyl-Tam or to endoxifen. Inhibition of cyclooxygenase with indomethacin or the incubation with the prostaglandin D2 and E2 receptor antagonist AH6809 reduced the vasorelaxation-induced Tam and its metabolites by approximately 50%. Preincubation with Nω-nitro-L-arginine methyl ester hydrochloride combined with indomethacin abolished the vasorelaxation-induced Tam and its metabolites. These results show that Tam and its metabolites cause acute vasorelaxation by inducing vasodilator prostanoids synthesis.

Effect of pravastatin on impaired endothelium-dependent relaxation induced by lysophosphatidylcholine in rat aorta1

To investigate the effects of pravastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on impaired endothelium-dependent relaxation induced by lysophosphatidylcholine (LPC), the major component of oxidized low-density lipoprotein, in rat thoracic aorta. Both the endothelium-dependent relaxation response to acetylcholine and the endothelium-independent relaxation response to sodium nitroprusside of aortic rings were measured by recording isometric tension after the rings were exposed to LPC in the absence or presence of pravastatin to estimate the injury effect of LPC and the protective effect of pravastatin on the aortic endothelium, respectively. Exposure of aortic rings to LPC (1-10 micromol/L) for 30 min induced a significant concentration-dependent inhibition of endothelium-dependent relaxation to acetylcholine, but did not affect endothelium-independent relaxation in response to sodium nitroprusside. Pre-incubation of aortic rings with pravastatin (0.3-3 mmol/L) for 15 min and then co-incubation of the rings with LPC (3 micromol/L) for another 30 min significantly attenuated the inhibition of endothelium-dependent relaxation induced by LPC. This protective effect of pravastatin (1 mmol/L) was abolished by NG-nitro-L-arginine methyl ester (30 micromol/L), an inhibitor of nitric oxide synthase, but not by indomethacin (10 micromol/L), an inhibitor of cyclooxygenase. Moreover, protein kinase C inhibitor chelerythrine (1 micromol/L) the superoxide anion scavenger superoxide dismutase (200 kU/L), and the nitric oxide precursor L-arginine (3 mmol/L) also improved the impaired endothelium-dependent relaxation induced by LPC, similar to the effects of pravastatin. Pravastatin can protect the endothelium against functional injury induced by LPC in rat aorta, a fact which is related to increasing nitric oxide bioavailability.

Atorvastatin enhances sildenafil-induced vasodilation through nitric oxide-mediated mechanisms

Statins have cholesterol-independent effects including an increased vascular nitric oxide (NO) activity and are commonly used by patients with cardiovascular disease. Such patients frequently have erectile dysfunction, which may be treated with sildenafil, a selective inhibitor of phosphodiesterase type 5. Since statins and sildenafil can activate the NO-cGMP pathway, we investigated whether pre-treatment with atorvastatin (0, 5 and 30 mg/kg/day) for 2 weeks affects sildenafil (1 pM–100 mM)-induced relaxation of aortic rings isolated from Wistar rats. We also examined the hemodynamic consequences of this interaction in Wistar rats. Plasma nitrite/nitrate (NO x ) concentrations were determined using an ozone-based chemiluminescence assay. While pre-treatment with atorvastatin increased the potency of sildenafil-induced vasorelaxation ( P<0.01), no differences were observed in the maximum sildenafil-induced relaxation. Pre-incubation of aortic rings with N G-nitro- l-arginine methyl ester ( l-NAME) reversed atorvastatin-induced increase in the potency of sildenafil relaxation. In addition, pre-treatment with atorvastatin enhanced plasma NO x concentrations and sildenafil-induced hypotension and tachycardia (all P<0.05). These results suggest that atorvastatin increases the vascular sensitivity to sildenafil through NO-mediated mechanisms.