Concentration-relaxation Curves Research Articles

Electrophysiology, molecular modelling, and functional analysis of the effects of dietary quercetin and flavonoid analogues on Kir6.1 channels in rat stomach fundus smooth muscle

Flavonoids, ubiquitously distributed in the plant world, are regularly ingested with diets rich in fruit, vegetables, wine, and tea. During digestion, they are partially absorbed in the stomach. The present work aimed to assess the in vitro effects of quercetin and ten structurally related flavonoids on the rat gastric fundus smooth muscle, focussing on ATP-dependent K+ (Kir6.1) channels, which play a central role in the regulation of resting membrane potential, membrane excitability and, consequently, of gastric motility. Whole-cell currents through Kir6.1 channels (IKir6.1) were recorded with the patch-clamp technique and the mechanical activity of gastric fundus smooth muscle strips was studied under isometric conditions. Galangin ≈ tamarixetin > quercetin > kaempferol > isorhamnetin ≈ luteolin ≈ fisetin > (±)-taxifolin inhibited pinacidil-evoked, glibenclamide-sensitive IKir6.1 in a concentration-dependent manner. Morin, rutin, and myricetin were ineffective. The steric hindrance of the molecule and the number and position of hydroxyl groups on the B ring played an important role in the activity of the molecule. Molecular docking simulations revealed a possible binding site for flavonoids in the C-terminal domain of the Kir6.1 channel subunit SUR2B, in a flexible loop formed by residues 251 to 254 of chains C and D. Galangin and tamarixetin, but not rutin relaxed both high K+- and carbachol-induced contraction of fundus strips in a concentration-dependent manner. Furthermore, both flavonoids shifted to the right the concentration-relaxation curves to either pinacidil or L-cysteine constructed in strips pre-contracted by high K+, rutin being ineffective. In conclusion, IKir6.1 inhibition exerted by dietary flavonoids might counterbalance their myorelaxant activity, affect gastric accommodation or, at least, some stages of digestion.

Conditional over-expression of heme-oxygenase 1 in myelomonocytic cells reduces hypertension and vascular inflammation

Abstract Background Arterial hypertension is one of the most important modifiable risk factors for all-cause morbidity and mortality worldwide. Angiotensin II (Ang II) plays a pathogenic role in the development of hypertension, vascular dysfunction, inflammation and tissue damage. In the context of hypertension, heme-oxygenase 1 (HO-1) gene expression is upregulated as an antioxidant defense system in response to AngII through its action on heme catabolism, which generates carbon monoxide (CO), ferritin and biliverdin, which is reduced to bilirubin by biliverdin reductase A (BLVRA). Previous studies have shown the important role of HO-1 in the maturation and migration of immune cells. Myeloid HO-1 modulates macrophage polarization and protects against ischemia-reperfusion damage. However, the role of myeloid cell specific HO-1 in the detrimental effects of AngII induced vascular dysfunction has not yet been explored. Objectives To investigate the potential vascular protection of myeloid cell specific overexpression of HO-1 in AngII-induced arterial hypertension. Methods Hypertension was induced in 8–13 weeks old male mice with selective over-expression of HO-1 (HO-1indLysMcre) in myelomonocytic cells versus LysMCre/wt by AngII infusion (1mg/kg/d). Blood pressure was recorded by tail-cuff. Bilirubin levels were quantified in plasma by High Performance Liquid Chromatography (HPLC). The quantification of adherent and rolling leukocytes in carotid arteries was detected by intravital video microscopy (IVM). Endogenuos Thrombin potential (ETP) was measured in platelet rich plasma (PRP) and platelets poor plasma (PPP) by Calibrated Automated Thrombogram (CAT) assay. Endothelium-dependent vasodilation was assessed in isolated aortic rings by concentration-relaxation curves in response to acetylcholine (ACh). Results AngII-infused HO-1indLysMcre had decreased blood pressure values and improved endothelial function as compared to LysMcre controls. IVM revealed reduced leukocyte rolling and adhesion to the vascular endothelium in AngII infused HO-1indLysMcre mice compared to controls, paralleled by reduced expression of NOX-2 mediated oxidative stress and vascular inflammation in AngII-induced arterial hypertension by decreasing VCAM-1, CCR2 and MCP-1 expression. mRNA analysis revealed an increased expression of BLVRA in liver, spleen, heart and aortic tissues in response to AngII, which was higher in HO-1indLysMcre mice than in controls. By CAT assay we registered a decrease ETP in PPP and PRP in HO-1ind mice infused with AngII compatible with less abundance of inflammatory platelets, potentially regulated by BLVRA activity. This was supported by increased in bilirubin levels in response to AngII, which was higher in HO-1indLysMcre mice than in controls. Conclusion Myeloid cell specific overexpression of HO-1 confers anti-inflammatory protection to the vasculature in AngII induced hypertension. This effect is, at least in part, mediated by BLVRA. Funding Acknowledgement Type of funding sources: None.

Antispastic Effect of Fasudil and Cocktail of Fasudil and Nitroglycerin in Internal Thoracic Artery

BackgroundSpasm of arterial grafts in coronary artery bypass grafting is a clinical problem and can occasionally be lethal. Perioperative spasm in the internal thoracic artery (ITA) and coronary arteries occurs in 0.43% of patients. This study aimed to investigate the antispastic effect of a RhoA/Rho-kinase (Rho-associated coiled-coil-containing protein kinase [ROCK]) inhibitor (fasudil) with and without nitroglycerin in combination in the ITA. MethodsIsolated human ITA rings taken from 68 patients who were undergoing coronary bypass were studied in a myograph. Cumulative concentration-relaxation curves for fasudil (−9 to −3.5 log M) were established in the ITA, which was precontracted with potassium chloride or U46619. The inhibitory effect of fasudil (−6.3 or −5.3 log M) or fasudil in combination with nitroglycerin were also tested. The ROCK2 protein was measured by Western blot. ResultsFasudil caused similar relaxation in ITA rings contracted by potassium chloride or U46619. Pretreatment with −5.3 log M fasudil significantly depressed contraction induced by potassium chloride (P = .004 vs control; P = .017 vs −6.3 log M) and U46619 (P = .010 vs control; P = .041 vs. −6.3 log M). Fasudil in combination with nitroglycerin had more effect and more rapid and sustained relaxation than either vasodilator alone. Fasudil caused a decrease of ROCK2 protein content (P = .014). ConclusionsFasudil fully relaxes some vasoconstrictor-induced contraction and decreases ROCK2 protein content in the ITA. The combination of fasudil and nitroglycerin has a superior effect than either vasodilator alone. The new cocktail solution composed of fasudil and nitroglycerin (pH 7.4) has effective antispastic action and may prove to be a new antispastic method for arterial conduits during coronary bypass surgery.

Effect of RhoA/Rho‐kinase inhibitor in Human Internal Mammary Artery and Clinical Implications

ObjectivesCoronary artery bypass grafting surgery (CABG) is the treatment of choice for severe coronary artery disease (CAD). Graft failure is a common complication after CABG. Different biological mechanisms contribute to the pathophysiology of vascular graft failure including vasospasm. RhoA/Rho‐kinase (ROCK) pathway plays an important role in cell contraction of cardiovascular disease. ROCK is a downstream effector of small GTP‐binding protein RhoA and has two isoforms, ROCK1 and ROCK2. This study aimed to investigate the inhibitory effect of ROCK inhibitor fasudil on the vasoconstriction in human internal mammary artery (IMA) from patients undergoing CABG and to explore whether the combination of fasudil and nitroglycerin (NTG) is superior than being used alone.MethodsIsolated human IMA rings (n=8 in each experimental group) taken from 58 patients undergoing CABG were studied in myograph. Cumulative concentration‐relaxation curves for fasudil (−9 to −3.5 log M) were established in IMA precontracted with KCl (30 mM) and U46619 (−8 log M). Cumulative concentration‐contraction curves for KCl (5 to 120 mM) and U46619 (−9 to −4.5 log M) were also established in two ways: (1) in three groups pretreated with the plasma concentration (−6.3 log M) and 10‐fold higher concentration of fasudil (−5.3 log M) or vehicle to estimate the depression effect of fasudil; (2) in four groups pretreated with fasudil (−6.3 log M), NTG (0.1 μM), the combination, and vehicle for 40 minutes. Western Blot was used to measure the change of the ROCK2 protein.ResultsFasudil caused similar cumulative relaxation in KCl‐ or U46619‐ contracted (p > 0.05) rings. Pretreatment with −5.3 log M fasudil significantly depressed subsequent contraction by KCl (P < 0.01 vs. control, P < 0.05 vs. −6.3 log M) and U46619 (P < 0.05 vs. control or −6.3 log M). Pretreatment with the combination of NTG and fasudil significantly depressed the cumulative contraction by KCl (P < 0.01 vs. control; P < 0.05 vs. −5.3 log M; P < 0.01 vs. NTG). Similarly, in U46619‐induced contraction, the combination also significantly depressed the contraction (P < 0.001 vs. control; P < 0.05 vs. −5.3 log M fasudil; P < 0.01 vs. NTG). Fasudil also caused a decrease of ROCK2 protein content (p < 0.05).ConclusionsFasudil has a potent inhibitory effect on the vasoconstriction mediated by KCl and U46619 in human conduit arteries. Combination of fasudil and NTG is more effective than fasudil or NTG alone. These findings provide a new antispastic method in CABG and may be beneficial to long‐term patency of the grafts.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China [81870288 & 81870227]; the Non‐profit Central Research Institute Fund of Chinese Academy of Medical Sciences [2019XK310001 & 2018TX31002]; Tianjin Science and Technology Committee[18PTZWHZ00060];Binhai New Area Health Bureau (2016BWKY007 & 2018BWKZ005).

Erythromycin relaxes BALB/c mouse airway smooth muscle

AimsBitter taste receptor (TAS2R) agonists have bronchodilatory potentials. Erythromycin is a ligand of TAS2R10, but its relaxant profile is unknown. This study was performed to understand the relaxant effects of erythromycin and its potential mechanism. Main methodsAirway resistance was tested by the whole body plethysmography in the ovalbumin-aluminum hydroxide induced asthma model mice. Tracheal ring segment myography was used to investigate the isometric tension of the smooth muscle. The cyclic adenosine monophosphate (cAMP) concentration was measured by enzyme immunoassay kit. Changes in the calcium influx in airway smooth muscle cells (ASMCs) were surveyed using a real-time confocal microscopy. Key findingsErythromycin significantly relieved airway hyperreactivity in asthma model mice. Erythromycin relaxed mouse tracheal segments precontracted with carbachol, KCl, 5-hydroxytryptamine and U46619, and further dilated the tracheal rings relaxed by isoprenaline or atropine. Epithelium removal, indomethacin or NS-398 partially reduced the relaxation. U73122, 2-APB, iberiotoxin or ouabain did not change the concentration-relaxation curves of erythromycin on tracheal segments. Erythromycin didn't elevate cAMP level. CaCl2-induced contraction in the K+-rich solution was impaired by erythromycin in the Ca2+-free solution. The intercellular Ca2+ level in the ASMCs was decreased by erythromycin, which was partly inhibited by Bay K8644 but not gallein. SignificanceErythromycin had marked bronchodilatory effect. The relaxation might be related to the L-type voltage-dependent calcium channel, but not the gustducin-associated βγ/phospholipase-Cβ/inositol 1,4,5-tri-phosphate receptor/large conductance Ca2+-activated K+ channel pathway or a cAMP-dependent way.

Antiasthmatic effect of Curcuma aeruginosa extract on isolated organ of the trachea

Background:Asthma is a major health problem worldwide. Antiasthma drugs have side effects and can be expensive. It is important to develop antiasthma drugs from medicinal plants that have fewer side effects and are cheaper. One of the medicinal plants used for antiasthma treatment comes fromCurcuma aeruginosa(Zingiberaceaefamily). The aim of the research is to examine spasmolytic activity of ethanol extract ofC. aeruginosaon isolated guinea pig tracheas to determine the antiasthma effects.Methods:The spasmolytic activity ofC. aeruginosaextracts was tested in separated organs of guinea pig trachea. Guinea pig was sacrificed and its trachea rings were suspended in L-shaped wire loops in organ baths containing the Krebs solution aerated with carbogen. Isometric contractions of tracheal rings were measured by the transducer coupled to the amplifier. The trachea rings were exposed to DMSO as negative control, aminophylline as positive control andC. aeruginosaextracts. The single concentration-relaxation curve was obtained in every preparation.Results:The result showed that the decrease of the spasmolytic activity in the guinea pig tracheal tone due toC. aeruginosaextract was significantly better (p=0.022) when compared to the negative control. Meanwhile, the EC50value of aminophylline (0.019 ± 0.05) was not significantly different (p=0.454) withC. aeruginosa(0.024 ± 0.05).Conclusion:It could be concluded thatC. aeruginosaextracts have the potency to be further developed as a new natural source of the antiasthma agents.

Endothelial nitric oxide synthase enhancer AVE3085 reverses endothelial dysfunction induced by homocysteine in human internal mammary arteries

Homocysteine (Hcy) is an independent risk factor for endothelial dysfunction in cardiovascular diseases. We hypothesized that the eNOS transcription enhancer AVE3085 may protect the endothelial function damaged by Hcy in the human internal mammary artery (IMA). Cumulative concentration-relaxation curves to acetylcholine (−10 to −4.5 log mol/L) or sodium nitroprusside were established in IMA from patients undergoing coronary artery surgery precontracted by U46619 (−8 log mol/L) in the absence/presence of Hcy (100 μmol/L) with/without AVE3085 (30 μmol/L) in vitro in a myograph. RT-qPCR and ELISA were used to quantify the mRNA and protein levels of eNOS. Colorimetric assay method was used to detect the production of nitric oxide (NO). Maximal relaxation was significantly attenuated by Hcy in human IMA. Co-incubation with AVE3085 protected endothelium from the impairment by Hcy and increased the production of NO. Exposure to Hcy for 24 h downregulated eNOS protein expression (P < 0.05) whereas it upregulated the expression of eNOS at mRNA levels (P < 0.05). The presence of AVE3085 in addition to Hcy significantly increased the eNOS protein (P < 0.05) and slightly decreased the mRNA level. The study for the first time revealed that in the human blood vessels (IMA) the clinically-relevant high concentration of Hcy directly causes endothelial dysfunction by downregulating eNOS protein that may be reversed by AVE3085. These findings not only provide new direction for protecting endothelium during coronary artery bypass grafting and improving long-term patency of the grafts, but also provide evidence to the use of eNOS enhancer in the patients with endothelial dysfunction in various pathological conditions.

Tambulin is a major active compound of a methanolic extract of fruits of Zanthoxylum armatum DC causing endothelium-independent relaxations in porcine coronary artery rings via the cyclic AMP and cyclic GMP relaxing pathways

BackgroundZanthoxylum armatum DC (Z. armatum), belonging to Rutaceae family, has been traditionally used for the treatment of various diseases such as hypertension, abdominal pain, headache, fever, high altitude sickness, diarrhea, dysentery, and as a tonic, condiment, and an anthelmintic treatment. HypothesisThe present study aims to evaluate the vasorelaxant effect of a methanolic extract of the fruits of Z. armatum, isolate the active components and characterize the underlying mechanism. Study designA methanolic extract of fruits of Z. armatum was prepared and its vasorelaxant effect was studied using porcine coronary artery rings. Thereafter, the methanolic extract was analyzed, and a major compound was isolated and its structure elucidated (tambulin). Different pharmacological tools were used to characterize the vasorelaxant effect of tambulin. ResultsThe methanolic extract and the isolated tambulin caused similar endothelium-independent relaxations of porcine coronary artery rings with and without endothelium indicating a direct relaxing effect at the vascular smooth muscle. Tambulin did not affect the relaxation curves to the endothelium-dependent vasodilators, bradykinin and the calcium ionophore A23187 in rings with endothelium. Tambulin (1 µM) slightly but significantly shifted leftwards the concentration-relaxation curve to the endothelium-independent vasodilators, sodium nitroprusside (SNP), forskolin (FC) and isoproterenol but not those to soluble guanylyl cyclase activators (YC-1 and BAY 41-2272) and K+ channel openers (levcromakalim and 1-EBIO). Pretreatment with tambulin inhibited, in a concentration-dependent manner, contractions to KCl, serotonin (5-HT), CaCl2 and U46619 in coronary artery rings without endothelium. Both the protein kinase A (H-89, 10 µM) and the protein kinase G (Rp-8-br-cyclic GMPS, 30 µM) inhibitors significantly reduced relaxations to tambulin in coronary artery rings without endothelium. ConclusionThe present findings indicate that tambulin isolated from Z. armatum (fruits) is a major active principle inducing vasorelaxation through a direct effect at the vascular smooth muscle and involving both the cyclic AMP and/or cyclic GMP relaxing pathways.

The Extracellular cAMP-Adenosine Pathway in Airway Smooth Muscle.

In the respiratory tract, intracellular cAMP has a key role in the smooth muscle relaxation induced by the β2-adrenoceptor/Gs protein/adenylyl cyclase axis. In other tissues, cAMP also works as an extracellular messenger, after its efflux and interstitial conversion to adenosine by ectoenzymes. The aim of this study was to identify cAMP efflux and the "extracellular cAMP-adenosine pathway" in the airway smooth muscle. First, we tested the ability of β2-adrenoceptor agonists formoterol or fenoterol to increase the extracellular cAMP in isolated tracheal rings from adult male Wistar rats. The effects of adenosine, cAMP, 8-Br-cAMP, fenoterol, or formoterol were also evaluated in the isometric contraction of control or carbachol (CCh) precontracted tracheas, normalized as the percentage of CCh-induced response. Fenoterol and formoterol induced 70%-80% relaxation and increased extracellular cAMP levels by up to 280%-450%. Although exogenous cAMP or adenosine evoked phasic contractions, the membrane-permeable cAMP analog 8-Br-cAMP induced relaxation of CCh-precontracted tracheas. The simultaneous inhibition of adenosine degradation/uptake with EHNA [erythro-9-(2-hydroxy-3-nonyl) adenine hydrochloride] plus uridine increased by 3-fold the maximum cAMP-induced contraction, whereas it was significantly reduced by AMPCP [adenosine 5'-(α,β-methylene)diphosphate; an ecto-5'-nucleotidase inhibitor], and by adenosine receptor antagonists CGS-15943 (nonselective) or DPCPX (8-cyclopentyl-1,3-dipropylxanthine) (A1 selective). Finally, CGS-15943 shifted to the left the concentration-relaxation curve for fenoterol. In conclusion, our results show that airway smooth muscle expresses the extracellular cAMP-adenosine pathway associated with contracting effects mediated by A1 receptors. The cAMP efflux triggered by fenoterol/formoterol indicates that the extracellular cAMP-adenosine pathway may play a role in balancing the relaxant effects of β2-adrenoceptor agonists in airways, which may impact their bronchodilation effects.

The effect of urapidil, an alpha-1 adrenoceptor antagonist and a 5-HT1A agonist, on the vascular tone of the porcine coronary and pulmonary arteries, the rat aorta and the human pulmonary artery

Urapidil (Eupressyl®) an antihypertensive drug acting as an α1 antagonist and a 5-HT1A agonist, may be of special interest in the treatment of hypertension associated with preeclamptic toxaemia and hypoxia-induced pulmonary arterial vasoconstriction. However, the effect of urapidil on vascular tone has been poorly investigated. Vascular reactivity was evaluated using pulmonary and coronary arteries from 36 pigs, aortae from 22 rats and 9 human pulmonary artery samples suspended in organ chambers. Concentration-relaxation curves either to urapidil, 5-HT, or the 5-HT1A receptor agonist 8-OH-DPAT were constructed after pre-contraction of rings. Pig pulmonary and coronary artery rings were contracted with U46619, a thromboxane mimetic, rat aortic rings with either endothelin-1 or phenylephrine, and human pulmonary artery rings with U46619 or phenylephrine. Urapidil markedly inhibited phenylephrine-induced contractions in rat aortic rings with and without endothelium with a more pronounced effect observed in rings without endothelium. Both 5-HT and 8-OH-DPAT failed to induce relaxation in rat aortic rings with an intact endothelium. 5-HT, but not urapidil and 8-OH-DPAT, induced a concentration-dependent relaxation in the porcine coronary and pulmonary artery rings with an intact endothelium (P<0.05). 5-HT and phenylephrine but not urapidil caused concentration-dependent contractions in human pulmonary artery rings. The present findings, while confirming that urapidil is a potent inhibitor of α1-adrenoceptor-induced contraction, do not support the role of 5-HT1A receptor activation in the control of the vascular tone of the different types of arteries tested in response to urapidil. In addition, they indicate that urapidil seems to preferentially target arteries with endothelial dysfunction.

Platelet-Localized FXI Promotes a Glycoprotein Ib Alpha Dependent Feedback Loop in Arterial Hypertension and Vascular Inflammation

Backgroud: Vascular inflammation is a hallmark of atherosclerosis and arterial and venous thromboembolic disease. Arterial hypertension is a highly significant risk factor for death and cardiovascular disease. High levels of angiotensin II (ATII) cause arterial hypertension by a complex inflammatory pathway requiring leukocyte recruitment and reactive oxygen species production within the vessel wall. Interactions of platelets, leukocytes and the vessel wall play pivotal roles in activating coagulation and precipitating thrombosis. Platelets provide a pro-coagulant surface for amplified thrombin generation in hemostasis and thrombosis, and play important roles in vascular inflammation by preserving vascular integrity. They also promote leukocyte recruitment in wire injury and angiogenesis models through glycoprotein Ib alpha (GPIbalpha) interacting with the integrin alphaMbeta2 (CD11b/CD18 or Mac-1) on leukocytes and regulate monocyte and neutrophil activation. How platelets, coagulation factors, leukocytes and the vessel wall cooperate to promote vascular inflammation in arterial hypertension is unclear.Objective: The aim of this work was to explore the roles of TF, FXI, FXII, thrombin and platelet GPIbalpha on inflammatory monocyte-driven vascular dysfunction and arterial hypertension in ATII infused mice and rats as well as in 5/6 nephrectomized (5/6Nx) rats.Methods: FXII-/-, FXI-/-, and hIL-4R/Ibalpha mice and 5/6NX rats were used for this study. Mice and rats where treated with ATII (1 mg×kg-1 ×d-1 for 7 days) using osmotic minipumps. Blood pressure was recorded using tail cuff measurement and telemetry carotid implants. To assess vasodilator properties, isolated aortic segments were mounted to force transducers in organ chambers to test their response to acetylcholine (ACh) and glyceryl trinitrate (GTN). ROS production was quantified within the aorta using dihydroethidium-derived fluorescence. Thrombin generation in platelet rich plasma (PRP) was measured using calibrated automated thrombography.Results: ATII induces an upregulation of tissue factor, thrombin-dependent endothelial cell VCAM-1 expression and integrin alpha4- and platelet-dependent leukocyte adhesion to arterial conductance vessels. Depletion of platelets using an anti-GPIbalpha antibody as well as injection of anti-Mac-1 directed against the CD11b/CD18 integrin on leukocytes similarly prevented leukocyte rolling and adhesion. Reduced vascular recruitment of leukocytes was paralleled by diminished vascular ROS production assessed by the superoxide-sensitive dye dihydroethidium. ATII-induced vascular dysfunction unexpectedly involved the activation of FXI but not FXII. Moreover, inhibition of FXI synthesis attenuates blood pressure increase in response to ATII. The platelet FXI receptor GPIbalpha supports the upregulation of thrombin feedback activation in ATII-treated mice. Blockade of TF during ATII administration attenuated both endothelial dysfunction (acetylcholine concentration-relaxation curves) and smooth muscle dysfunction as demonstrated by improved vascular relaxation in response to the endothelium independent vasodilator glyceryl trinitrate and reduced ROS within the vessel wall. Five/6Nx rats had endothelial dysfunction that was prevented by FXI Antisense oligonucleotide. Thrombin-induced thrombin generation was increased in PRP of 5/6 Nx rats and markedly diminished by FXI Antisense oligonucleotide, mimicking the results from ATII-infused mice.Conclusion: Our results reveal a critical role of platelet GPIbalpha to promote localized thrombin amplification and a FXI-thrombin feedback loop in ATII-induced vascular inflammation. Importantly, pharmacologic inhibition of FXI synthesis is sufficient to prevent thrombin propagation on platelets, to reduce vessel wall leukocyte infiltration, and to diminish ATII-induced endothelial dysfunction and arterial hypertension in mice and rats.Targeting FXI could be a novel therapeutic possibility to interrupt this heterotypic cellular coagulation-inflammatory circuit. DisclosuresMonia:Isis Pharmaceuticals: Employment.

Vasoactive Properties of an Extract of Allium Sativum in the Rat Aorta

The aim of this study was to determine whether an hydro-alcoolic extract of cloves of Allium sativum is able to induce a relaxant effect in the rat aorta and so the mechanism underlying this effect. Rat aortic rings were suspended in organ chambers for recording of changes in isometric forces. Rings with endothelium were incubated or not with L-Nitro Arginine Methyl Ester (L-NAME) to block NO synthase, before contraction with adrenalin and a concentration relaxation curve to the hydro-alcoolic extract. In some experiments, endothelium was removed before contraction with adrenalin and concentration relaxation to the extract. Prevent effect of the extract was determine with contraction curve to adrenaline in rings with and without endothelium incubated with the extract of Allium sativum. The hydro-alcoolic extract of Allium sativum induces a vasodilatatory effect in the rat aorta contracted with adrenalin. This effect is not endothelium-dependent and is not mediated by NO. Furthermore the extract incubated with aorta rings, is able to prevent the contraction induced by adrenalin. Allium sativum induces vascular relaxation which can explain the benefic effect of this plant in treatment of high blood pressure in Senegal.

Antisense versus proopiomelanocortin mRNA reduces vascular risk in a murine model of type-2 diabetes following stress exposure in early post-natal life

Mechanisms of vascular complications in type-2 diabetes patients and animal models are matter of debate. We previously demonstrated that a double-stress model applied to male mice during nursing period produces enduring hyperfunction of endogenous opioid and adrenocorticotropin (ACTH)-corticosteroid systems, accompanied by type-2 diabetes-like alterations in adult animals. Administration of the opioid receptor antagonist naloxone, or of an antisense oligodeoxynucleotide versus proopiomelanocortin mRNA, capable to block the pro-opiomelanocortin-derived peptides β-endorphin and ACTH, selectively prevent these alterations. Here, we investigated alterations produced by our stress model on aorta endothelium-dependent relaxation and contractile responses. Mice, stressed during nursing period, showed in the adulthood hormonal and metabolic type-2 diabetes-like alterations, including hyperglycemia, increased body weight and increased plasma ACTH and corticosterone levels. Ex vivo isolated aorta rings, gathered from stressed mice, were less sensitive to noradrenaline-induced contractions versus controls. This effect was blocked by nitric-oxide synthase-inhibitor l-NG-nitroarginine added to bath organ solution. Aorta rings relaxation caused by acetylcholine was enhanced in stressed mice versus controls, but following treatment with the nitric-oxide donor sodium nitroprusside, concentration-relaxation curves in aorta from stressed groups were similar to controls. Therefore, vascular response alterations to physiologic-pharmacologic stimuli were apparently due to nitric-oxide hyperfunction-dependent mechanisms. Aorta functional alterations, and plasma stress hormones enhancement, were prevented in mice stressed and treated with antisense oligodeoxinucleotide, addressed to reduce ACTH- and corticosteroid-mediated hyperfunction. This study demonstrates the key role of ACTH-corticosteroid axis hyperfunction for the triggering of vascular conditions in male adult rodents following postnatal stress in a type-2 diabetes model.

Butylidenephthalide blocks potassium channels and enhances basal tension in isolated guinea-pig trachea.

Butylidenephthalide (Bdph, 30~300 μM), a constituent of Ligusticum chuanxiong Hort., significantly enhanced tension in isolated guinea-pig trachea. In this study, we investigate the mechanism(s) of Bdph-induced contraction in the tissue. Isolated trachea was bathed in 5 mL of Krebs solution containing indomethacin (3 μM), and its tension changes were isometrically recorded. Cromakalim (3 μM), an ATP-dependent K+ channel opener, significantly antagonized the Bdph-induced enhancement of baseline tension. Bdph (300 μM) also significantly antagonized cromakalim-induced relaxation. Bdph (300 μM) did not significantly influence the antagonistic effects of glibenclamide (GBC, 1 μM) and tetraethylammonium (TEA, 8 mM) against the cromakalim-induced relaxation. However, Bdph (300 μM) and 4-aminopiridine (4-AP, 5 mM), a blocker of Kv1 family of K+ channels, in combination significantly rightward shifted the log concentration-relaxation curve of cromakalim. The antagonistic effect of the combination almost equals the sum of the individual effects of Bdph and 4-AP, suggesting that the antagonistic mechanism of Bdph may be similar to that of 4-AP. All calcium channel blockers influenced neither the baseline tension nor antagonistic effect of Bdph against cromakalim. In conclusion, Bdph may be similar to 4-AP, a blocker of Kv1 family of K+ channels, to enhance the baseline tension of guinea-pig trachea.

GW24-e2914 Involvement of vascular peroxidase 1 in the endothelial dysfunction in hypertension

ObjectivesVascular peroxidase 1 (VPO1) is a new heme-containing peroxidase, which can utilise hydrogen peroxide (H2O2) generated from co-expressed NADPH oxidase (NOX) to produce hypochlorous acid (HOCl) and catalyse peroxidative reactions....

Paeonol induces vasodilatation in rat mesenteric artery via inhibiting extracellular Ca2+ influx and intracellular Ca2+ release

To investigate the vasodilative effect of paeonol in rat mesenteric artery and the mechanisms responsible for it. Rats were anaesthetized and sacrificed. The superior mesenteric artery was removed, dissected free of adherent tissue and cut into 2.0 mm long cylindrical segments. Isometric tension of artery rings was recorded by a myograph system in vitro. Concentration-relaxation curves of paeonol (17.8 μ mol/L to 3.16 mmol/L) were recorded on artery rings precontracted by potassium chloride (KCl) and concentration-contraction curves of KCl, 5-hydroxytryptamine (5-HT), noradrenaline (NA) or calcium chloride (CaCl2) were recorded in the presence of paeonol (10(-4.5), 10(-3.8), 10(-3.5) mol/L) respectively. And also, concentration-relaxation curves of paeonol were recorded in the presence of different potassium channel inhibitors and propranolol on rings precontracted with KCl respectively. To investigate the role of intracellular Ca(2+) release from Ca(2+) store, the contraction induced by NA (100 μ mol/L) and CaCl2 (2 mmol/L) in Ca(2+) free medium was observed in the presence of paeonol respectively. Paeonol relaxed artery rings precontracted by KCl in a concentration-dependent manner and the vasodilatation effect was not affected by endothelium denudation. Paeonol significant decreased the maximum contractions (Emax) induced by KCl, CaCl2, NA and 5-HT, as well as Emax induced by NA and CaCl2 in Ca(2+) -free medium, suggesting that paeonol dilated the artery via inhibiting the extracellular Ca(2+) influx mediated by voltage-dependent calcium channel, and receptor-mediated Ca(2+)-influx and release. Moreover, none of glibenclamide, tetraethylammonium, barium chlorded and propranolol affected the paeonol-induced vasodilatation, indicating that the vasodilatation was not contributed to ATP sensitive potassium channel, calcium-activated potassium channel, inwardly rectifying potassium channel, and β-adrenoceptor. Paeonol induces non-endothelium dependent-vasodilatation in rat mesenteric artery via inhibiting voltage-dependent calcium channel-mediated extracellular Ca(2+) influx and receptor-mediated Ca(2+) influx and release.

Heparinized blood better preserves cellular energy charge and vascular functions of intraoperatively stored saphenous vein grafts in comparison to isotonic sodium-chloride-solution

Several studies have addressed the optimal storage conditions for vascular grafts during surgery. The results remain contradictionary. This may be attributed to the fact, that the various vascular beds have a different sensitivity to storage. We analyzed the impact of storage in isotonic saline solution (NaCl) or heparinized blood the vascular functions of human saphenous vein grafts. Special care was taken to choose storage conditions which are relevant for intraoperative storage of a saphenous vein graft in a setting of coronary artery bypass grafting with vein and internal mammary artery as grafts. Intraoperatively isolated V. saphena-segments (n = 36) were stored in NaCl or heparinized blood for approximately 30 minutes at room temperature. Subsequently, the segments were examined in a Mulvany-myograph. Following preconstriction with norepinephrine, concentration-relaxation curves were assessed for bradykinin and sodium-nitroprusside to assess developed vessel-wall tension as well as endothelium- and smooth-muscle-cell dependent vasorelaxation. The availability of adenosintriphosphate (energy charge) was determined based on liquid chromatography measurements of nucleotide tissue levels. Mean storage time was 27.4 ± 2.4 min in NaCl- and 26.3 ± 2.7 min in blood-group, respectively. After this period, receptor-dependent and-independent maximum of developed vessel wall tension was significantly reduced in NaCl-group (p = 0.05 and p = 0.045, respectively). Furthermore, the energy charge was significantly (p = 0.046) better preserved after blood storage (74 ± 1%) in comparison to NaCl-group (68 ± 2%). Endothelium-induced vasodilatation in response to bradykinin reached only 12.3 ± 2.5% in NaCl-group, but 19.3 ± 5.2% in blood-group (p = 0.033). Alike, EC50-concentration of bradykinin for half-maximal relaxation was significantly lower in blood- than in NaCl-group (log EC50 -7.08 ± 0.3 and -5.91 ± 0.4; respectively; p = 0.046). Endothelium-independent smooth muscle relaxation in response to sodium-nitroprusside was not different between both groups. Heparinized blood better preserves vascular contractile and endothelial functions of the saphenous vein graft. Storage in NaCl rapidly compromises vascular functions and impaires cellular energy. NaCl should no longer be recommended for intraoperative storage of harvested V. saphena grafts.

A-FABP and Oxidative Stress Underlie the Impairment of Endothelium-Dependent Relaxations to Serotonin and the Intima-Medial Thickening in the Porcine Coronary Artery with Regenerated Endothelium

Experiments were designed to determine the cause of the selective dysfunction of G(i) proteins, characterized by a reduced endothelium-dependent relaxation to serotonin (5-hydroxytryptamine), in coronary arteries lined with regenerated endothelial cells. Part of the endothelium of the left anterior descending coronary artery of female pigs was removed in vivo to induce regeneration. The animals were treated chronically with vehicle (control), apocynin (antioxidant), or BMS309403 (A-FABP inhibitor) for 28 days before functional examination and histological analysis of segments of coronary arteries with native or regenerated endothelium of the same hearts. Isometric tension was recorded in organ chambers and cumulative concentration-relaxation curves obtained in response to endothelium-dependent [serotonin (G(i) protein mediated activation of eNOS) and bradykinin (G(q) protein mediated activation of eNOS)] and independent [detaNONOate (cGMP-mediated), isoproterenol (cAMP-mediated)] vasodilators. The two inhibitors tested did not acutely affect relaxations of preparations with either native or regenerated endothelium. In the chronically treated groups, however, both apocynin and BMS309403 abolished the reduction in relaxation to serotonin in segments covered with regenerated endothelium and prevented the intima-medial thickening caused by endothelial regeneration, without affecting responses to bradykinin or endothelium-independent agonists (detaNONOate and isoproterenol). Thus, inhibition of either oxidative stress or A-FABP likely prevents both the selective dysfunction of G(i) protein mediated relaxation to serotonin and the neointimal thickening resulting from endothelial regeneration.

Role of TRPC3 Channel in Human Internal Mammary Artery

Intracellular calcium regulation in endothelial cells depends on transient receptor potential channels (TRPs). Canonical TRPs (TRPCs) are now recognized as the most important Ca(2+)-permeable cation channels in vascular endothelium and TRPC3 channel is reported to play a role in vasodilation in animal vessels. However, little is known about the role of TRPCs in human arteries. We therefore tested the hypothesis that TRPCs play a role in human arteries. Cumulative concentration-relaxation curves to acetylcholine (-11 to -4.5 log M) were established in the human internal mammary artery (IMA) rings (n=42) taken from 28 patients undergoing coronary artery bypass grafting in precontraction induced by U46619 (-8 log M) in the absence or presence of SKF96365 (10 μmol/L) or Pyr3 (3μmol/L). Protein expressions of TRPC3 were determined by Western blot and immunohistochemistry staining. The maximal relaxation induced by acetylcholine was significantly attenuated by the nonspecific cation channels inhibitor, SKF96365 (48.2±3.7 vs. 66.0±0.9% in control, p <0.01) or the selective TRPC3 blocker, Pyr3 (58.4±2.3% vs. 67.7±1.1% in control, p <0.01). Protein expression of TRPC3 was detected in human IMA. TRPC3 exists and plays a role in the acetylcholine-induced endothelium-dependent relaxation in the human IMA. This study suggests that TRPC3 may be a potential new target in endothelial protection in patients with endothelial dysfunction such as in patients with coronary artery disease in order to improve the long-term patency of the grafting vessels.

Magnesium and diltiazem relaxes phenylephrine-precontracted rat aortic rings

Perioperative vasospasm during cardiovascular surgery is a challenging problem. Several vasodilator agents are frequently utilized for its prevention in surgical practice. Magnesium and diltiazem both have known potential vasorelaxant effects. We planned to compare the efficacy of diltiazem and magnesium in relieving phenylephrine-precontracted rat aortic rings. Ten young adult female Wistar albino rats weighing 230-260 g were used in this study. The aortic rings in the organ bath equilibrated and reached their baseline tension. Precontraction was induced by 0.001 mmol/l phenylephrine and cumulative concentration-relaxation curves were obtained by consecutively increasing the addition of either diltiazem (10(-6)-0.1 mmol/l) or magnesium (0.1-10 mmol/l). The mean maximal relaxation responses observed by diltiazem and magnesium on separate aortic rings were 90 ± 3 and 53 ± 2%, respectively. The calculated EC50 of diltiazem was 0.01035 mmol/l, whereas the EC50 of magnesium was 4.064 mmol/l (P < 0.05). Both magnesium and diltiazem produced vasorelaxation on phenylephrine-precontracted rat aortic rings in this study, but the potency of diltiazem regarding the EC50 value was significantly higher than that of magnesium. Magnesium could be a candidate together with diltiazem to inhibit vasospasm on arterial grafts during coronary bypass surgery.