Relaxation Of Coronary Artery Rings Research Articles

Carissa edulis Vahl (Apocynaceae) extract, a medicinal plant of Benin pharmacopoeia, induces potent endothelium-dependent relaxation of coronary artery rings involving nitric oxide

BackgroundHypertension is a major cardiovascular risk factor that affects most countries including those of Africa. Although Carissa edulis Vahl, Diodia scandens Sw. and Cleome gynandra L. are traditionally used in Benin as antihypertensive treatments with some efficacy mentioned by the local population, their biological activity on the cardiovascular system remains poorly studied. AimThe study investigated the vasoreactivity of the plants and assessed the underlying mechanisms using isolated arteries. Study designAqueous-ethanolic extracts of aerial parts of C. edulis, D. scandens and C. gynandra were prepared by maceration before being subjected to multi-step liquid-liquid fractionation with solvents of increasing polarity. The vasoreactivity of the extracts and fractions were assessed on isolated porcine coronary artery and rat aorta using organ chambers, the role of nitric oxide (NO) using NG-nitro-L-arginine (NO synthase inhibitor), prostanoids using indomethacin (cyclooxygenases inhibitor) and endothelium-dependent hyperpolarization using TRAM-34 plus UCL 1684 (inhibitors of calcium-dependent K+ channels), and the vascular uptake of polyphenols using Neu reagent. ResultsThe aqueous-ethanolic crude extract of C. edulis (CECE) induced potent relaxations that were exclusively endothelium-dependent and more pronounced than those to D. scandens and C. gynandra. The n-butanolic fraction of C. edulis (CEBF) was more active than the cyclohexane, dichloromethane, and ethyl acetate fractions. The relaxation induced by CECE and CEBF were inhibited by NG-nitro-L-arginine and affected neither by TRAM-34 plus UCL 1684 nor by indomethacin. CEBF induced sustained endothelium-dependent relaxations for at least 60 min, and inhibited, in a concentration-dependent manner, contractions to KCl, CaCl2, U46619 and serotonin in rings with endothelium. Analysis of CEBF by LCHRMS indicated the presence of polyphenols, terpenes, and alkaloids. Exposure of coronary artery and aorta rings to CEBF caused the accumulation of polyphenols predominantly in the endothelium. ConclusionC. edulis leaf extract induced pronounced endothelium-dependent relaxations and inhibited contractile responses by stimulating the endothelial formation of NO. LCHRMS analysis of the most active fraction, the butanolic fraction, revealed the presence of numerous compounds including polyphenols, terpenes, and alkaloids. The polyphenols of CEBF accumulated preferentially in the endothelium of the arterial wall. Thus, these observations support the folkloric use of C. edulis in hypertension.

Taxifolin as a Major Bioactive Compound in the Vasorelaxant Effect of Different Pigmented Rice Bran Extracts.

Cardiovascular disease is one of the leading causes of morbidity and mortality in recent years. The intake of polyphenol rich diets has been associated with improved cardiovascular function and reduced cardiovascular risks. Oryza sativa L. is one of the most common cereals worldwide. Rice bran, a byproduct of the rice milling process, contains many bioactive ingredients, including polyphenols, polysaccharides, proteins, and micronutrients. It is also consumed as a healthy diet in the form of rice bran oil and powder in many Asian countries like Japan, South Korea, and India for its several health benefits as a natural antioxidant. Thus, this study evaluated the vasorelaxant effect of ethanolic extracts of brown, green, red, and black rice bran and investigated its underlying vasorelaxant mechanism. Among the four rice bran extracts (RBEs) examined, the red rice bran extract (RRBE) had a strong endothelium-dependent vasorelaxant effect, which was markedly prevented by N-ω-nitro-L-arginine [endothelial nitric oxide synthase (eNOS) inhibitor], wortmannin [phosphoinositide-3 kinase (PI3K) inhibitor], and 1H-[1,2,4]oxadiazole[4,3-alpha]quinoxalin-1-one (inhibitor of guanylate cyclase). Likewise, RRBE induced the phosphorylation of eNOS and Src in cultured endothelial cells, thereby stimulating NO formation. Altogether, these findings propose that RRBE induces endothelium-dependent relaxation, involving at least in part, NO-mediated signaling through the PI3K/eNOS pathway. Further, LC-PDA analysis conducted on the four RBEs also revealed that RRBE highly contained taxifolin, which is an active flavanonol that induces endothelium-dependent vasorelaxation, compared to other RBEs. Subsequently, the underlying mechanism of taxifolin was assessed through vascular reactivity studies with pharmacological inhibitors similar to that of RRBE. These findings deciphered a distinct difference in vasorelaxant effects between RRBE and the other RBEs. We also observed that RRBE induced a potent endothelium-dependent NO-mediated relaxation in coronary artery rings, which involved the Src/PI3K pathway that activates eNOS. Additionally, taxifolin exhibited, at least in part, similar vasoprotective effects of RRBE. Therefore, we propose that RRBE may serve as natural sources of functional phytochemicals that improve cardiovascular diseases associated with disturbed NO production and endothelial dysfunction.

Nanoencapsulation of the omega-3 EPA:DHA 6:1 formulation enhances and sustains NO-mediated endothelium-dependent relaxations in coronary artery rings and NO formation in endothelial cells

• Omega-3fatty acids stimulate the endothelial formation of vasoprotective nitric oxide. • EPA:DHA 6:1 nanoparticles caused greater sustained endothelium-dependent relaxation. • EPA:DHA 6:1 nanoparticles caused sustained formation of NO in endothelial cells. • EPA:DHA 6:1 nanoparticles-treated endothelial cells had greater anti-platelet effect. The eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) formulation with a ratio of 6:1 is a potent stimulator of the endothelial formation of nitric oxide (NO). The aim of the study was to investigate whether nanoencapsulation of EPA:DHA 6:1 followed by coating with gum increases its biological activity. Vascular reactivity was assessed using porcine coronary artery rings, the formation of NO in cultured endothelial cells (ECs) using DAF-FM and indirectly by platelet aggregation studies. Coated EPA:DHA 6:1 nanoparticles induced sustained relaxations of coronary artery rings that were greater in rings with than in those without endothelium, and more pronounced than with the native form. Treatment of ECs with coated EPA:DHA 6:1 nanoparticles caused greater and more sustained formation of NO and enhanced their anti-aggregatory effects. Thus, nanoencapsulation of EPA:DHA 6:1 is an attractive strategy to enhance the beneficial effect at the vascular endothelium.

Experimental Periodontal Disease Triggers Coronary Endothelial Dysfunction in Middle-Aged Rats: Preventive Effect of a Prebiotic β-Glucan.

This study was aimed to verify the hypothesis that periodontal disease contributes to endothelial dysfunction in the coronary arteries of middle-aged rats. Besides we evaluated the effects of a prebiotic (β-glucan isolated from Saccharomyces cerevisiae) in preventing vascular dysfunction. The sample comprised young (sham and induced to periodontal disease) and middle-aged rats (sham, periodontal disease, sham-treated and periodontal disease-treated), at 12 and 57 weeks, respectively. The treated-groups received daily doses of β-glucan (50 mg/kg) orally (gavage) for 4 weeks, and periodontal disease was induced in the last 2 weeks by ligature. A myograph system assessed vascular reactivity. The expression of endothelial nitric oxide synthase (eNOS), cyclooxygenase 1 (COX-1), COX-2, p47phox, gp91phox, NF-KB p65, p53, p21, and p16 was quantified by western blotting. Serum hydroperoxide production was measured by the ferrous oxidation-xylenol orange (FOX-2) assay method. Interleukin-1 beta (IL-1β), IL-10, and tumor necrosis factor-alpha (TNF-α) levels were evaluated by spectroscopic ultraviolet-visible analysis. Periodontal disease in middle-aged rats was associated with reduced acetylcholine-induced relaxations of coronary artery rings affecting the endothelium-dependent hyperpolarization- and the nitric oxide-mediated relaxations. The endothelial dysfunction was related to eNOS downregulation, pronounced impairment of the EDH-mediated relaxation, increased IL-1β and TNF-α proinflammatory cytokines, and also upregulation of NADPH oxidase and COXs, starting accumulate aging markers such as p53/p21 and the p16. Treatment with β-glucan effectively reduced bone loss in periodontal disease and delayed endothelial dysfunction in the coronary artery. Our data show that yeast β-glucan ingestion prevented oxidative stress and synthesis of proinflammatory marker and prevented eNOS reduction induced by periodontal disease in middle-aged rats. These results suggest that β-glucan has a beneficial effect on the coronary vascular bed.

Nanoencapsulation of EPA:DHA 6:1 potentiates the endothelium-dependent relaxation of coronary artery rings compared to native form: role of NO, endothelium-dependent hyperpolarization and prostanoids

The omega 3 polyunsaturated fatty acid (PUFAs) formulation containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) with a 6:1 ratio is a potent inducer of endothelium-dependent nitric oxide (NO)-mediated vasorelaxation that also improves ageing-related and angiotensin II-induced endothelial dysfunction in rats. Since PUFAs are unstable, the possibility that nanoencapsulation of EPA:DHA 6:1 followed by coating potentiates the bioactivity was evaluated. EPA:DHA 6:1 was emulsified in water phase and coated with proteins and gum derivatives or used in native form. Changes in isometric tension of porcine coronary artery rings were determined using organ chambers. The role of NO was assessed using Nw-nitro-L-arginine (L-NA, NO synthase inhibitor), prostanoids using indomethacin (Indo, COX inhibitor) and endothelium-derived hyperpolarization (EDH) using TRAM-34 and UCL-1684 (Ca2 + -dependent potassium channel inhibitors). Coated EPA:DHA 6:1 nanoparticles (NP) caused in a concentration-dependent manner endothelium-dependent relaxations that were more sustained than those of the native form. At 0.3%, coated and native forms induced 56.6 ± 2.8% and 51.1 ± 1.5%relaxations at 10 min, and 93.8 ± 2.1% and 35.1 ± 1.7% at 60 min, respectively. The response to the coated EPA:DHA 6:1 NP was inhibited by L-NA and not affected by Indo, TRAM-34 plus UCL-1684, and by the previous incubation of rings with another oil (corn oil 0.3%). In contrast, L-NA, Indo, and TRAM-34 plus UCL-1684 inhibited the relaxation to EPA:DHA 6:1. Nanoencapsulation of EPA:DHA 6:1 followed by coating perpetuated their ability to cause endothelium-dependent relaxation of coronary artery rings that is exclusively mediated by NO. In contrast, the relaxing activity of the native form involved NO, EDH and vasorelaxant prostanoids. Therefore, coated EPA:DHA 6:1 NP appear to be an attractive approach to enhance the vasoprotective effect of omega 3 PUFAs.

Significance of neutrophil microparticles in ischaemia-reperfusion: Pro-inflammatory effectors of endothelial senescence and vascular dysfunction.

Endothelial senescence is an emerging cause of vascular dysfunction. Because microparticles are effectors of endothelial inflammation and vascular injury after ischaemia‐reperfusion, we examined leucocyte‐derived microparticles of spleen origin as possible contributors. Microparticles were generated from primary rat splenocytes by either lipopolysaccharide or phorbol‐myristate‐acetate/calcium ionophore, under conditions mimicking innate and adaptive immune responses. Incubation of primary porcine coronary endothelial cells with either type of microparticles, but not with those from unstimulated splenocytes, leads to a similar threefold raise in senescence‐associated β‐galactosidase activity within 48 hours, indicating accelerated senescence, to endothelial oxidative stress, and a fivefold and threefold increase in p21 and p16 senescence markers after 24 hours. After 12‐hour incubation, the endothelial‐dependent relaxation of coronary artery rings was reduced by 50%, at distinct optimal microparticle concentration. In vitro, microparticles were pro‐thrombotic by up‐regulating the local angiotensin system, by prompting tissue factor activity and a secondary generation of pro‐coagulant endothelial microparticles. They initiated an early pro‐inflammatory response by inducing phosphorylation of NF‐κB, MAP kinases and Akt after 1 hour, and up‐regulated VCAM‐1 and ICAM‐1 at 24 hours. Accordingly, VCAM‐1 and COX‐2 were also up‐regulated in the coronary artery endothelium and eNOS down‐regulated. Lipopolysaccharide specifically favoured the shedding of neutrophil‐ and monocyte‐derived microparticles. A 80% immuno‐depletion of neutrophil microparticles reduced endothelial senescence by 55%, indicating a key role. Altogether, data suggest that microparticles from activated splenocytes prompt early pro‐inflammatory, pro‐coagulant and pro‐senescent responses in endothelial cells through redox‐sensitive pathways. The control of neutrophil shedding could preserve the endothelium at site of ischaemia‐reperfusion–driven inflammation and delay its dysfunction.

P1837Coated EPA:DHA 6:1 nanoparticles cause greater endothelium-dependent relaxations of coronary artery rings than the native form: role of NO, endothelium-dependent hyperpolarization and prostanoids

P1837Coated EPA:DHA 6:1 nanoparticles cause greater endothelium-dependent relaxations of coronary artery rings than the native form: role of NO, endothelium-dependent hyperpolarization and prostanoids

Mechanisms underlying vasorelaxation induced in the porcine coronary arteries by Thymus linearis, Benth

Ethnopharmacological relevanceThymus linearis, Benth indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. Aim of the studyPresent study aims to investigate vasorelaxant effect of Thymus linearis and its underlying vasorelaxation mechanisms in porcine coronary artery rings. Materials and methodsAqueous-methanolic extract of aerial parts of Thymus linearis was prepared by maceration process and then bio-guided fractionation was carried out using different solvents. The effects of extract and subsequent fractions were assessed on coronary artery rings with intact and denuded endothelium. The mechanisms of vasorelaxant effect were investigated using different pharmacological tools. The in-vitro inhibitory effects of the test fractions were also assessed on purified phophodiestrases using radioenzymatic assay. Phytochemical studies were carried out using GCMS. ResultsThe aqueous-methanolic extract elicited similar relaxations in coronary artery rings with and without endothelium in dose dependent fashion and removal of endothelium did not alter this response. Further, n-butanolic fraction of Thymus liniaris (TLB) was found to be the most potent among other derived fractions. TLB did not alter the relaxation produced by endothelium dependent vasodilators in rings with intact endothelium. However, TLB significantly potentiated the relaxation elicited by cyclic AMP and cyclic GMP elevating drugs but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41–2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with TLB inhibited in a concentration-dependent manner contractions to KCl, CaCl2 and U46619 in coronary artery rings without endothelium. Further, TLB was found to non-selectively inhibit the PDE activity in concentration manner. Conclusionn-Butanolic fraction of Thymus linearis possesses endothelium independent vasorelaxant effects in coronary artery by direct acting on the smooth muscles. These effects involve the elevation of the cyclic AMP and cyclic GMP possibly through the inhibition of various PDEs. GCMS analysis revel presence of thymole and carvacrol as major constituents. Furthermore, these investigations also support the folklore use of Thymus linearis in hypertension.

O34 Endothelium-independent vasorelaxant effect of Thymus linearis via inhibition of phosphodiesterases in porcine coronary artery

Thymus linearis Benth. indigenous to Pakistan has been traditionally used for the treatment of various diseases including hypertension. The aim of the study was to investigate the vasorelaxant effect of Thymus linearis in porcine coronary artery rings. Aqueous-methanolic extract and various fractions (butanol, ethyacetate and aqueous) of Thymus linearis were prepared. The effects of extract and fractions were assessed using coronary artery rings and various pharmacological tools were applied to characterize the underlying mechanism of active fraction (butanol). The inhibitory effect of butanolic fraction was also studied on phosphodiesterases using radio enzymatic assay. Phytochemical studies were also carried out using GCMS. The aqueous-methanolic extract of Thymus linearis elicited similar relaxations in coronary artery rings with and without endothelium in a dose-dependent manner. Among the fractions butanolic fraction of Thymus linearis (TLB) was found to be the most potent. TLB significantly potentiated the relaxation induced by forskolin, isoproterenol, sodium nitroprusside and atrial natriuretic peptide whereas the relaxation induced by YC-1, BAY 41-2272, levcromakalim and 1-EBIO was unaffected by TLB. Pretreatment with TLB inhibited the contraction induced by KCl, CaCl2 and U46619 in endothelium denuded coronary artery rings. Furthermore, TLB significantly inhibited the PDE activity (PDE1CaM activated PDE2, PDE3, PDE4 AND PDE5) in a concentration-dependent manner. GCMS analysis revealed the presence of thymol and Carvacrolas as major constituents. In conclusion, TLB induced endothelium-independent relaxations in porcine coronary artery rings by acting directly at the vascular smooth muscle to increase concentrations of both cyclic AMP and cyclic GMP via inhibition of various PDEs.

O11 Endothelium-dependent relaxation by a hydroethanolic extract of Adansonia digitata leaves in porcine coronary artery rings and rat thoracic aorta, mesenteric, carotid artery rings: Role of NO and EDH

Introduction Adansonia digitata leaves are used for the traditional treatment of hypertension in Senegal. This study evaluated the relaxant effect of a hydroethanolic extract of leaves of Adansonia digitata (ADF) in artery rings, and investigated the underlying mechanism. Methods Vascular reactivity was assessed in organ chambers. Rings were either untreated or incubated with a pharmacological tool for 30 min before contraction of coronary artery rings with either U46619 or phenylephrine, and the subsequent addition of ADF. In some experiments, the endothelium was removed mechanically. The phosphorylation level of target proteins was assessed by Western blot analysis. Results ADF induced concentration-dependent relaxations in coronary artery rings with endothelium, which reached 100% at a concentration as low as 10 μg/ml. The relaxation to ADF was significantly reduced by NLA (NO synthase inhibitor) and by the combination of Tram-34 and apamin (inhibitors of intermediate and small conductance calcium-dependent K+ channels, respectively), not affected by indomethacin (cyclooxygenase inhibitor), and abolished by wortmannin (PI3-kinase inhibitor) and MnTMPyP (membrane permeant superoxide dismutase mimetic). ADF caused relaxations in the rat superior mesenteric artery, carotid artery and aortic rings but not in the secondary mesenteric artery and femoral artery rings. ADF induced a sustained phosphorylation of Akt and eNOS at Ser1177 in cultured porcine coronary artery endothelial cells. Conclusion The Adansonia digitata leave extract induced potent endothelium-dependent relaxations, involving predominantly NO and endothelium-dependent hyperpolarization. Such vasorelaxant effects may explain the antihypertensive use of this plant in traditional African medicine.

O35 An anthocyanin-rich blackcurrant extract induced NO-mediated relaxation in coronary artery rings and eNOS phosphorylation in cultured endothelial cells: Role of sodium-glucose cotransporters 1 and 2

Introduction The beneficial cardiovascular effect of polyphenol-rich food and beverages has been attributed, at least in part, to an improved vascular function through an increased endothelial formation of nitric oxide (NO) and endothelium-dependent hyperpolarization (EDH), two major vasoprotective mechanisms. Black currant is a rich source of anthocyanins, and, in particular, of glucoside- and rutinoside-conjugated delphinidin and cyanidin. This study evaluated the role of sodium-glucose cotransporters (SGLT) 1 and 2 in the blackcurrant extract (BCE)-induced NO-mediated endothelium-dependent relaxation and activation of endothelial NO synthase (eNOS). Methods The reactivity of porcine coronary artery rings was assessed in organ chambers, and the expression and phosphorylation level of proteins in cultured porcine coronary artery endothelial cells by Western blot analysis. Results BCE (310.0 ± 0.1 mg gallic acid equivalent/g) was prepared from a blackcurrant juice containing 2.7 g gallic acid equivalent/L of polyphenols using a Sephadex LH-20 column. BCE caused potent concentration-dependent relaxations of coronary artery rings with endothelium, which were significantly reduced by the dual SGLT1 and SGLT2 inhibitor LX4211, and also to some extent by the SGLT2 inhibitor canagliflozin but not dapagliflozin. In contrast, LX4211 did not affect relaxations to bradykinin, sodium nitroprusside and epigallocatechin gallate. BCE induced the phosphorylation of eNOS at the activator site Ser1177 in cultured endothelial cells, which was significantly prevented by LX4211, dapagliflozin and canagliflozin. Conclusion These observations indicate that BCE is a potent activator of eNOS and inducer of NO-mediated vasorelaxation possibly subsequent to the entry of conjugated anthocyanins via SGLT1/2 into endothelial cells.

Endothelium-independent vasorelaxant effect of a Berberis orthobotrys root extract via inhibition of phosphodiesterases in the porcine coronary artery

BackgroundBerberis orthobotrys Bien ex Aitch. (Berberidaceae) is a plant indigenous of Pakistan that is locally used for the treatment of hypertension. HypothesisThis study evaluated the vasoactive properties of a Berberis orthobotrys root extract and its fractions, and investigated the role of the endothelium and the underlying mechanism. Study designAn aqueous methanolic extract of Berberis orthobotrys roots was prepared and submitted to a multi-step liquid-liquid fractionation with solvents of increasing polarity. Vascular reactivity of the different fractions was assessed using porcine coronary artery rings either with or without endothelium, and in the presence or absence of specific pharmacological tools. The ability of Berberis orthobotrys extracts to affect phosphodiesterase (PDE) activity was evaluated using a radioenzymatic method and purified phosphodiesterases. ResultsThe aqueous methanol extract induced similar relaxations in coronary artery rings with and without endothelium, and, amongst the three derived preparations, the butanol fraction (BFBO) was slightly but significantly more effective than the ethyl acetate fraction and the aqueous residue in rings without endothelium. Analysis of the butanol fraction (BFBO) by LC-ELSD-MS indicated the presence of four major isoquinoline alkaloids including berberine. BFBO significantly potentiated the relaxations induced by cyclic GMP- and cyclic AMP-dependent relaxing agonists, and inhibited contractions to KCl, CaCl2, and U46619 in endothelium denuded rings. In contrast, BFBO did not affect relaxations to endothelium-dependent vasodilators. BFBO concentration-dependently inhibited the cyclic GMP-hydrolyzing activity of basal PDE1, calmodulin-activated PDE1 and PDE5, and of cyclic AMP-hydrolyzing activity of PDE3 and PDE4 with IC50 values ranging from 40 to 130µg/ml. ConclusionThe butanol fraction of the aqueous methanol extract of Berberis orthobotrys roots induced pronounced endothelium-independent relaxations and inhibited contractile responses by acting directly at the vascular smooth muscle in the coronary artery. Moreover, BFBO potentiated relaxations induced by both cyclic GMP- and cyclic AMP-dependent vasodilators most likely due to its ability to inhibit several vascular PDEs, and in particular PDE4 and PDE5.

KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine.

Hydrogen peroxide (H2O2) and voltage-dependent K(+) (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli.

THE EPA:DHA 6:1-EVOKED ENDOTHELIUM-DEPENDENT NO-MEDIATED RELAXATION IN THE CORONARY ARTERY INVOLVES A COPPER-DEPENDENT PRO-OXIDANT RESPONSE TRIGGERING THE PI3-KINASE/AKT-MEDIATED ACTIVATION OF ENOS

Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to reduce the risk of cardiovascular disease, in part, by stimulating the endothelial formation of nitric oxide (NO), a potent vasoprotective factor. This study determined the mechanism leading to endothelial NO synthase (eNOS) activation in response to the highly active EPA:DHA 6:1 product. Vascular reactivity was assessed using porcine coronary artery rings suspended in organ chambers, the level of oxidative stress in coronary artery sections using the redox-sensitive probe, dihydroethidine, and the phosphorylation level of target proteins in cultured coronary artery endothelial cells by Western blot analysis. EPA:DHA 6:1 caused pronounced endothelium-dependent relaxations in porcine coronary artery rings. Relaxations to EPA:DHA 6:1 were slightly but significantly reduced by an eNOS inhibitor, not affected by inhibition of endothelium-dependent hyperpolarization and abolished by both treatments. Relaxations to EPA:DHA 6:1 were reduced by inhibitors of oxidative stress (MnTMPyP, PEG-catalase), an inhibitor of either Src kinase (PP2) or PI3-kinase (wortmannin), and intracellular copper chelating agents (neocuproine, tetrathiomolybdate) and were insensitive to cyclooxygenase inhibition (indomethacin), chelating agents for iron (desferroxamine), zinc (histidine), extracellular copper (bathocuproine). EPA:DHA 6:1 induced phosphorylation of Src, Akt and eNOS at Ser 1177; these effects were inhibited by MnTMPyP and PEG-catalase. EPA:DHA 6:1 induced the endothelial formation of ROS in coronary artery sections, this effect was inhibited by MnTMPyP, PEG-catalase, and intracellular copper chelating agents. EPA:DHA 6:1 causes endothelium-dependent NO-mediated relaxations in coronary artery rings, and this effect involves an intracellular copper-dependent event triggering the redox-sensitive PI3-kinase/Akt pathway to activate eNOS by phosphorylation at Ser 1177.

Redox-sensitive induction of Src/PI3-kinase/Akt and MAPKs pathways activate eNOS in response to EPA:DHA 6:1.

AimsOmega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have vasoprotective effects, in part, by stimulating the endothelial formation of nitric oxide (NO). This study determined the role of the EPA:DHA ratio and amount, and characterized the mechanism leading to endothelial NO synthase (eNOS) activation.Methods and ResultsEPA:DHA 6∶1 and 9∶1 caused significantly greater endothelium-dependent relaxations in porcine coronary artery rings than EPA:DHA 3∶1, 1∶1, 1∶3, 1∶6, 1∶9, EPA and DHA alone, and EPA:DHA 6∶1 with a reduced EPA + DHA amount, which were inhibited by an eNOS inhibitor. Relaxations to EPA:DHA 6∶1 were insensitive to cyclooxygenase inhibition, and reduced by inhibitors of either oxidative stress, Src kinase, PI3-kinase, p38 MAPK, MEK, or JNK. EPA:DHA 6∶1 induced phosphorylation of Src, Akt, p38 MAPK, ERK, JNK and eNOS; these effects were inhibited by MnTMPyP. EPA:DHA 6∶1 induced the endothelial formation of ROS in coronary artery sections as assessed by dihydroethidium, and of superoxide anions and hydrogen peroxide in cultured endothelial cells as assessed by electron spin resonance with the spin probe CMH, and the Amplex Red based assay, respectively.ConclusionOmega-3 fatty acids cause endothelium-dependent NO-mediated relaxations in coronary artery rings, which are dependent on the EPA:DHA ratio and amount, and involve an intracellular activation of the redox-sensitive PI3-kinase/Akt and MAPKs pathways to activate eNOS.

0040: The EPA: DHA 6:1-evoked endothelium-dependent NO-mediated relaxation in the coronary artery involves a copper-dependent pro-oxidant response triggering the PI3-kinase/Akt-mediated activation of eNOS

Omega-3 fatty acid products containing eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to reduce the risk of cardiovascular disease, in part, by stimulating the endothelial formation of nitric oxide (NO), a potent vasoprotective factor. This study determined the mechanism leading to endothelial NO synthase (eNOS) activation in response to the highly active EPA:DHA 6:1 product. Vascular reactivity was assessed using porcine coronary artery rings suspended in organ chambers, the level of oxidative stress in coronary artery sections using the redox-sensitive probe, dihydroethidine, and the phosphorylation level of target proteins in cultured coronary artery endothelial cells by Western blot analysis. EPA:DHA 6:1 caused pronounced endothelium-dependent relaxations in porcine coronary artery rings. Relaxations to EPA:DHA 6:1 were slightly but significantly reduced by an eNOS inhibitor, not affected by inhibition of endotheliumdependent hyperpolarization and abolished by both treatments. Relaxations to EPA:DHA 6:1 were reduced by inhibitors of oxidative stress (MnTMPyP, PEG-catalase), an inhibitor of either Src kinase (PP2) or PI3-kinase (wortmannin), and intracellular copper chelating agents (neocuproine, tetrathiomolybdate) and were insensitive to cyclooxygenase inhibition (indomethacin), chelating agents for iron (desferroxamine), zinc (histidine), extracellular copper (bathocuproine). EPA:DHA 6:1 induced phosphorylation of Src, Akt and eNOS at Ser 1177; these effects were inhibited by MnTMPyP and PEGcatalase. EPA:DHA 6:1 induced the endothelial formation of ROS in coronary artery sections, this effect was inhibited by MnTMPyP, PEG-catalase, and intracellular copper chelating agents. EPA:DHA 6:1 causes endotheliumdependent NO-mediated relaxations in coronary artery rings, and this effect involves an intracellular copper-dependent event triggering the redox-sensitive PI3-kinase/Akt pathway to activate eNOS by phosphorylation at Ser 1177.

Hydroxylation of (−)-epigallocatechin-3-O-gallate at 3′′, but not 4′′, is essential for the PI3-kinase/Akt-dependent phosphorylation of endothelial NO synthase in endothelial cells and relaxation of coronary artery rings

(-)-Epigallocatechin-3-O-gallate (EGCg) has been shown to induce endothelium-dependent nitric oxide (NO)-mediated relaxation via the redox-sensitive Src/PI3-kinase/Akt-dependent phosphorylation of endothelial NO synthase (eNOS). Although the presence of 8 hydroxyl functions, mainly on B and D rings, is essential for the EGCg-induced activation of eNOS, the relative role of each individual hydroxyl function still remains unclear. This study examined the effect of selective replacement of hydroxyl functions by methoxy moieties on either the B or D ring on the EGCg-induced phosphorylation of Akt and eNOS, formation of reactive oxygen species (ROS) and NO in cultured coronary artery endothelial cells, and endothelium-dependent relaxation of coronary artery rings. Replacement of a single hydroxyl by the methoxy group on position 3', 4' or 4'' affected little the EGCg-induced phosphorylation of Akt and eNOS, formation of ROS and NO in endothelial cells, and induction of endothelium-dependent relaxations. In contrast, the single methylation at position 3'' and the double methylation at both positions 3' and 4' reduced markedly the phosphorylation of Akt and eNOS, the formation of ROS and NO in endothelial cells and the relaxation of artery rings. These findings suggest that the hydroxyl group at the 3'' position of the gallate ring is essential and, also, to some extent, the two hydroxyl groups at positions 3' and 4', for the EGCg-induced redox-sensitive activation of eNOS leading to the subsequent NO-mediated vascular relaxation.

Role of endothelial cell membrane transport in red wine polyphenols-induced coronary vasorelaxation: involvement of bilitranslocase

Red wine polyphenols (RWP) induce nitric oxide (NO) and endothelium-derived hyperpolarization (EDH)-mediated coronary vasodilatation involving the redox-sensitive PI3-kinase/Akt-dependent pathway in the endothelium. However, there is a gap of knowledge in explaining how bioactive polyphenols initialize their signalling pathway in endothelial cells. Here, we investigated the hypothesis that flavonoids act subsequently to their entry into the endothelium via the flavonoid membrane transporter bilitranslocase (TC 2.A.65.1.1). Thus, vascular reactivity studies were performed using isolated porcine coronary artery rings. We separately determined the NO- and EDH-mediated components of the relaxation in the presence of specific inhibitors. In either case, bilitranslocase antibodies significantly reduced the relaxations of coronary artery rings induced by RWP. Furthermore, bilitranslocase antibodies significantly reduced RWP-induced phosphorylation levels of Akt and eNOS, assessed in cultured endothelial cells from porcine coronary arteries by Western blot analysis. The present findings indicate that bilitranslocase-mediated membrane transport substantially contributes to the initial step of RWP-induced coronary vasodilatation.

Natural Product Extract of Dicksonia sellowiana Induces Endothelium-Dependent Relaxations by a Redox-Sensitive Src- and Akt-Dependent Activation of eNOS in Porcine Coronary Arteries

Background/Aims: The consumption of polyphenol-rich food is associated with a decreased mortality from coronary diseases. This study examined whether a standardized hydroalcoholic extract of Dicksonia sellowiana (HEDS) triggered endothelium-dependent relaxations in porcine coronary artery rings and characterized the underlying mechanism. Methods: The phosphorylation level of Src, Akt and eNOS was assessed by Western blot analysis, the formation of reactive oxygen species by dihydroethidine staining and the level of eNOS Ser1177 phosphorylation by immunohistochemical staining in sections of coronary arteries. Results: HEDS-induced endothelium-dependent relaxations were strongly reduced by Nω-nitro-L-arginine, an eNOS inhibitor, and by its combination with charybdotoxin plus apamin, inhibitors of endothelium-derived hyperpolarizing factor-mediated responses. These relaxations were markedly reduced by MnTMPyP (a membrane-permeant mimetic of superoxide dismutase), polyethylene glycol catalase (PEG-catalase; a membrane-permeant analog of catalase), and by wortmannin (an inhibitor of PI3-kinase). HEDS-induced sustained phosphorylation of Akt and eNOS in endothelial cells was abolished by MnTMPyP, PEG-catalase and wortmannin. Oral administration of HEDS induced a significant decrease of mean arterial pressure in spontaneously hypertensive rats. Conclusion: These findings indicate that HEDS caused endothelium-dependent relaxations of coronary artery rings through the redox-sensitive activation of the endothelial PI3-kinase/Akt pathway leading to the subsequent activation of eNOS by phosphorylation. HEDS also has antihypertensive properties.

Urocortin‐2 induces vasorelaxation of coronary arteries isolated from patients with heart failure

1. Urocortin-2 (Ucn2) is a vasoactive peptide belonging to the corticotrophin-releasing factor (CRF) family that has potent cardiovascular actions. It has been suggested that Ucn2 participates in the pathophysiology of heart failure. However, little is known about the mechanisms underlying the action of Ucn2 in human coronary arteries. The aim of the present study was to assess the effects of Ucn2 on the vascular tone of human coronary arteries dissected from heart failure patients. 2. Human coronary arteries were dissected from the hearts of patients subjected to orthotopic heart transplantation. Coronary arteries were obtained from 17 patients with heart failure due to dilated cardiomyopathy of ischaemic origin in Stage III-IV of the New York Heart Association classification. Changes in tone were measured in arterial rings using force transducers. 3. Application of increasing concentrations of Ucn2 (5-20 nmol/L) to arterial rings precontracted with agonists induced dose-dependent relaxation of the coronary artery, which was independent of endothelial cell activation. Furthermore, the inhibition of the adenylyl cyclase by MDL-12 (100 nmol/L) and protein kinase A (PKA) by H89 (1 μmol/L) prevented Ucn2-mediated relaxation of coronary artery rings. 4. The results of the present study suggest that, in heart failure patients, Ucn2 could be useful in modulating coronary artery circulation independent of endothelial integrity through mechanisms that involve adenylyl cyclase activation and PKA stimulation. The findings warrant further investigation of the role of Ucn2 in circulatory regulation and its potential therapeutic application in heart disease.