Contraction Of Aortic Rings Research Articles

Acute Exposure to Fructose Impairs Endothelium-Dependent Relaxation via Oxidative Stress in Isolated Rat Aortic Rings

Introduction: Although both glucose and fructose are hexoses, their catabolism is quite different: the catabolism of fructose is initiated by ketohexokinase and is not regulated by negative feedback, which results in oxidative stress. Objective: We hypothesized that fructose impairs endothelium-dependent relaxation via oxidative stress in rat aortic rings. Methods: Sprague-Dawley rats were offered 20% fructose solution or tap water for 2 weeks, after which vascular reactivity was measured in isolated aortic rings. In a separate experiment, vascular reactivity was measured after acute exposure to ∼10 mM fructose in isolated aortic rings from untreated rats. Results: Although high-fructose intake statistically significantly increased blood pressure and body weight, it did not affect contraction and relaxation in aortic rings. The substitution of fructose for glucose in Krebs solution inhibited vascular relaxation in aortic rings, which was abolished by pretreatment with antioxidants. Decreasing the glucose concentration in Krebs solution inhibited vascular relaxation, whereas decreasing the fructose concentration in Krebs solution improved vascular relaxation in the aortic rings. Pretreatment with antioxidants improved the vascular relaxation in Krebs solution with fructose substituted for glucose. Conclusions: These results indicate that fructose impairs endothelium-dependent relaxation via oxidative stress in isolated rat aortic rings.

Endothelium modulates electrical field stimulation-induced contractions of Chelonoidis carbonaria aortic rings

The role of endothelium in the electrical-field stimulation (EFS)-induced contractions of Chelonoidis carbonaria aorta was investigated. Contractions were evaluated in the presence and absence of L-NAME (100 μM), tetrodotoxin (1 μM), phentolamine (10 and 100 μM), phenoxybenzamine (1 and 10 μM), prazosin (100 μM), idazoxan (100 μM), atropine (10 μM), D-tubocurarine (10 μM) or indomethacin (10 μM). EFS-induced contraction was also carried out in endothelium-denuded rings. EFS-induced contraction was investigated by the sandwich assay. Concentration curves to endothelin-1 (0.1–100 nM) and U46619 (0.001–100 μM) were also constructed to calculate both Emax and EC50. EFS at 16 Hz contracted Chelonoidis aorta, which was almost abolished by the endothelium removal. The addition of L-NAME increased the EFS response (2.0 ± 0.4 and 8.3 ± 1.9 mN). In L-NAME treated aortic rings, tetrodotoxin did not change the EFS-response (5.1 ± 1.8 and 4.9 ± 1.7 mN). Indomethacin, atropine and d-tubucurarine also did not affect the EFS-response. Phentolamine at 10 μM did not change the EFS-induced contraction; however, at 100 μM, reduced it (3.9 ± 1 and 1.9 ± 0.3 mN). Prazosin and idazoxan did not change EFS-induced contractions. Phenoxybenzamine at 1 μM reduced by 76% (9.6 ± 3.4 and 2.3 ± 0.8 mN) and at 10 μM by 90% the EFS response. Immunohistochemistry identified tyrosine hydroxylase in the endothelium and brain, whereas S100 protein was found only in brain. In conclusion, endothelium modulates EFS-induced contractions in Chelonoidis aortic rings and this modulation may be due to endothelium-derived catecholamines, possibly dopamine.

Seven pairs of new enantiomeric sesquiterpenoids from Curcuma phaeocaulis.

Seven pairs of new enantiomeric sesquiterpenoids, (+)/(-)-phaeocauline A-G [(+)/(-)-1-7], were isolated from the rhizomes of Curcuma phaeocaulis by chiral HPLC separation. Their structures, including absolute configurations, were determined by spectroscopic analyses and ECD data. The isolates were assessed for vasorelaxant, anti-platelet aggregative, and neuroprotective activities. Enantiomers (+)-1 and (-)-1 showed similar activity against abnormal platelet aggregation induced by arachidonic acid, while their C-4 epimers (+)-2 and (-)-2 were inactive, which indicated that those effects were stereoselective, but not enantioselective. Compounds (+)/(-)-3-5 exhibited vasorelaxant effects against KCl-induced contraction of rat aortic rings.

Vaso‐relaxant Effect of Zingiber officinale Extract Involves Release of Nitric Oxide from the Endothelium

IntroductionZingiber officinale (ZO) is an important plant with several medicinal, nutritional and ethnomedical values. The blood pressure lowering effect of ZO has been reported in experimental animals and human and some mechanisms have been postulated, including; blockage of voltage dependent calcium channel, stimulation of muscarinic receptors and serotonergic antagonistic property. The aim of this study is to investigate the vaso‐relaxant effect of the extract of ZO and the mechanism by which it is produced.MethodRhizomes of ZO were extracted in 80% methanol. Thoracic aorta isolated from male rabbits were cut into rings of 3–5 mm, mounted in organ baths containing 25 ml Krebs physiological solution maintained at 37 °C and bubbled continuously with a mixture of 95% O2 and 5% CO2. The aortic rings were equilibrated for 30 min at a resting tension of 1 g. Responses were recorded isometrically via a force displacement transducer connected to a recording system. The presence of functional endothelium was checked by the ability of acetylcholine (1μM) to induce relaxation on phenylepherine (PE, 1μM) contracted rings. The relaxant responses to ZO was recorded by adding cumulative concentrations (10, 20, 40, 80, 100, 500, 1000 μg/ml) of ZO. To investigate the involvement of endothelium in the vaso‐relaxant action, endothelium was removed by gently rubbing the luminal surface of the ring with a polyethylene tube. To investigate which endothelium‐derived vasorelaxant factors were involved, the rings were pre‐incubated with Nitro‐L‐arginine methyl ester (L‐NAME, 10 μM) or indomethacin (10 μM) for 30 min before addition of the spasmogen and ZO. Relaxant responses were expressed as percentage relaxation from PE‐precontraction levels. The EC50 (the concentration required to cause half‐maximal relaxation) was calculated from the concentration‐response curve. Results were expressed as means ± s.e.m. Student t‐test was used to assess the significant differences at (p < 0.05)ResultsZO produced a vaso‐relaxation effect on rings pre‐contracted with PE (1μM) at concentrations of 20–100 μg/ml in endothelium intact rings with an EC50 of 14.8±1.2 μg/ml (Figure 1). This relaxation was not however seen at 500–1000 μg/ml but rather a vaso‐contraction was observed. In endothelium denuded rings, the vaso‐relaxation effect of ZO at 20–100 μg/ml was abolished while vaso‐relaxation was observed with 500–1000 μg/ml, the concentrations that otherwise produced contraction in endothelium intact aortic rings. Pre‐incubation of the tissues with L‐NAME (10μM) abolished the relaxant effect of ZO at 20–100μg/ml but caused relaxation effect with 500–1000 μg/ml similar to that observed with endothelium denuded rings. Pretreatment with indomethacin abolished the relaxant effect of ZO at 20μg/ml but not at 40 μg/ml and above (Figure 2).ConclusionVaso‐relaxant effect of ZO is seen at lower concentrations, endothelium dependent and may involve the release of nitric oxide from the endothelium. There may be interplay between contractile and relaxant compounds in crude extract of ZO at concentrations above 500 μg/ml.Support or Funding InformationThe PhD study grant awarded to I.M Adebisi by Usmanu Danfodiyo University, Sokoto, Nigeria is acknowledgedRelaxation effect of methanol extract of Zingiber officinale (ZO) on phenylephrine‐precontracted aortic rings with intact endothelium. Data represent the mean ± s.e.m. of four individual experiments. * is p<0.05Figure 1Relaxation effect of methanol extract of Zingiber officinale (ZO) on phenylephrine‐precontracted aortic rings with endothelium, without endothelium and effect of L‐NAME (10 μM), and Indomethacin (10 μM) on ZO induced vasorelaxation on phenylephrine‐precontracted aortic rings with endothelium. Data represent the mean ± s.e.m. of four individual experiments. * is p<0.05Figure 2

Statin-mediated cholesterol depletion exerts coordinated effects on the alterations in rat vascular smooth muscle cell biomechanics and migration.

This study demonstrates and evaluates the changes in rat vascular smooth muscle cell biomechanics following statin-mediated cholesterol depletion. Evidence is presented to show correlated changes in migration and adhesion of vascular smooth muscle cells to extracellular matrix proteins fibronectin and collagen. Concurrently, integrin α5 expression was enhanced but not integrin α2. Atomic force microscopy analysis provides compelling evidence of coordinated reduction in vascular smooth muscle cell stiffness and actin cytoskeletal orientation in response to statin-mediated cholesterol depletion. Proof is provided that statin-mediated cholesterol depletion remodels total vascular smooth muscle cell cytoskeletal orientation that may additionally participate in altering ex vivo aortic vessel function. It is concluded that statin-mediated cholesterol depletion may coordinate vascular smooth muscle cell migration and adhesion to different extracellular matrix proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell. Not only does cholesterol induce an inflammatory response and deposits in foam cells at the atherosclerotic plaque, it also regulates cellular mechanics, proliferation and migration in atherosclerosis progression. Statins are HMG-CoA reductase inhibitors that are known to inhibit cellular cholesterol biosynthesis and are clinically prescribed to patients with hypercholesterolemia or related cardiovascular conditions. Nonetheless, the effect of statin-mediated cholesterol management on cellular biomechanics is not fully understood. In this study, we aimed to assess the effect of fluvastatin-mediated cholesterol management on primary rat vascular smooth muscle cell (VSMC) biomechanics. Real-time measurement of cell adhesion, stiffness, and imaging were performed using atomic force microscopy (AFM). Cellular migration on extra cellular matrix (ECM) protein surfaces was studied by time-lapse imaging. The effect of changes in VSMC biomechanics on aortic function was assessed using an ex vivo myograph system. Fluvastatin-mediated cholesterol depletion (-27.8%) lowered VSMC migration distance on a fibronectin (FN)-coated surface (-14.8%) but not on a type 1 collagen (COL1)-coated surface. VSMC adhesion force to FN (+33%) and integrin α5 expression were enhanced but COL1 adhesion and integrin α2 expression were unchanged upon cholesterol depletion. In addition, VSMC stiffness (-46.6%) and ex vivo aortic ring contraction force (-40.1%) were lowered and VSMC actin cytoskeletal orientation was reduced (-24.5%) following statin-mediated cholesterol depletion. Altogether, it is concluded that statin-mediated cholesterol depletion may coordinate VSMC migration and adhesion to different ECM proteins and regulate cellular stiffness and cytoskeletal orientation, thus impacting the biomechanics of the cell and aortic function.

Allergic sensitization increases contractile responses to 5-HT in guinea pig aorta.

Epidemiological and clinical studies suggest that asthma is associated with adverse cardiovascular outcomes, but its mechanism is uncertain. 5-Hydroxytryptamine (5-HT) is a mediator involved in asthma and in cardiovascular functioning. Thus, in the present study, we explored whether allergic sensitization in guinea pigs modifies 5-HT-induced contractile responses and 5-HT2A receptor expression in thoracic aorta rings. We found that sensitization produced a significant increase of 100 microM 5-HT-induced contractions of aorta rings (~27 % greater contraction than in non-sensitized animals, p<0.05). Preincubation with 10 nM ketanserin (a 5-HT2A receptor antagonist) reduced by ~30 % (p=0.003) and ~36 % (p=0.005) the area under the curve of 5-HT-induced contractions in aortas from non-sensitized and sensitized animals, respectively. There were no differences between sensitized and non-sensitized animals with respect to mRNA (qPCR) and protein (Western blot) expression of 5-HT2A receptor in thoracic aortas. We concluded that in this guinea pig model of asthma, allergic sensitization is not confined to airways, but also affects arterial contractile responses to 5-HT; changes in the expression of the 5-HT2A receptor appear not to be involved in this phenomenon.

The basal release of endothelium-derived catecholamines regulates the contractions of Chelonoidis carbonaria aorta caused by electrical-field stimulation.

ABSTRACTThe contractions of Chelonoidis carbonaria aortic rings induced by electrical field stimulation (EFS) are not inhibited by blockade of the voltage-gated sodium channels by tetrodotoxin but almost abolished by the α1/α2-adrenoceptor antagonist phentolamine. The objective of this study was to identify the mediator(s) responsible for the EFS-induced contractions of Chelonoidis carbonaria aortic rings. Each ring was suspended between two wire hooks and mounted in isolated 10 ml organ chambers filled with oxygenated and heated Krebs-Henseleit's solution. Dopamine, noradrenaline and adrenaline concentrations were analysed by liquid chromatography coupled to tandem mass spectrometry. The contractions caused by dopamine and EFS were done in absence and presence of the nitric oxide (NO) synthesis inhibitor L-NAME, the NO-sensitive guanylyl cyclase inhibitor ODQ, the D1-like receptor antagonist SCH-23390, the D2-like receptor antagonists risperidone, quetiapine, haloperidol, and the tyrosine hydroxylase inhibitors salsolinol and 3-iodo-L-tyrosine. Basal concentrations of dopamine, noradrenaline and adrenaline were detected in Krebs-Henseleit solution containing the aortic rings. The catecholamine concentrations were significantly reduced in endothelium-denuded aortic rings. L-NAME and ODQ significantly potentiated the dopamine-induced contractions. The D2-like receptor antagonists inhibited the EFS-induced contractions of the aortic rings treated with L-NAME, whereas SCH 23390 had no effect. Similar results were observed in the contractions induced by dopamine in L-NAME treated aortic rings. These results indicate that catecholamines released by endothelium regulate the EFS-induced contractions. This may constitute a suitable mechanism by which reptilia modulate specific organ blood flow distribution.This paper has an associated First Person interview with the first author of the article.

Evidence of Cardiovascular Calcification and Fibrosis in Pseudoxanthoma Elasticum Mouse Models Subjected to DOCA-Salt Hypertension

Pseudoxanthoma Elasticum (PXE) is a rare disorder characterized by fragmentation and progressive calcification of elastic fibres in connective tissues. Although arterial hypertension (AHT) has been reported in PXE patients, its impact on pathological manifestations has as yet been unexplored. We investigated the consequences of experimental AHT on Abcc6−/− PXE mouse models. Experimental AHT was induced by deoxycorticosterone acetate (DOCA-salt) in uni-nephrectomised mice. Blood pressure (BP) and vascular reactivity were monitored using tail-cuff plethysmography and myography respectively. Calcium content and fibrosis were assessed using colorimetry, Von Kossa and Sirius red staining respectively. The gene expression implicated in vascular biology was measured using quantitative polymerase chain reaction. DOCA-salt induced a matching rise in BP in Abcc6−/− and WT mice. Aortic ring contraction and relaxation in vitro were comparable. Calcium accumulated in the hearts of hypertensive Abcc6−/− mice along with significant fibrosis in the myocardium and aorta by contrast with the WT mice. In hypertensive Abcc6−/− mouse aortas, these results were corroborated by gene expression patterns favouring calcification, fibrosis and extracellular matrix remodelling. Abcc6 loss-of-function is associated with greater cardiovascular calcification and fibrosis in mice subjected to DOCA-Salt hypertension. These results suggest likely cardiovascular deterioration in PXE patients with AHT, necessitating diligent BP monitoring.

Curcumane C and (±)-curcumane D, an unusual seco-cadinane sesquiterpenoid and a pair of unusual nor-bisabolane enantiomers with significant vasorelaxant activity from Curcuma longa

A new seco-cadinane sesquiterpenoid (curcumane C, 1) and a pair of new nor-bisabolene enantiomers [(+)- and (−)-curcumane D, 2a and 2b] were isolated from C. longa. Compound 1 possesses an unusual 4,5-seco-cadinane skeleton with a tetrahydrophthalide moiety, while 2a and 2b contain an unusual 15-nor-bisabolene skeleton with a chromone core. All compounds exhibited significant vasorelaxant effects against KCl-induced contraction of rat aortic rings. Compound 1 also exhibited a vasorelaxant effect against phenylephrine-induced contraction of rat aortic rings. Meanwhile, compound 1 showed a stronger vasorelaxant effect in endothelium-intact rat aortic rings compared with endothelium-denuded rat aortic rings, indicating that vasodilation by 1 involved both endothelium-dependent and endothelium-independent pathways. Furthermore, compound 1 increased the NO content in human umbilical vein endothelial cells and its vasorelaxant effect could be attenuated by treatment with L-NAME, an endothelium NO synthase inhibitor. Thus, the underlying vasodilatory mechanisms of 1 may be mediated via abrogation of extracellular Ca2+ influx and regulation of NO release in vascular endothelial cells.

Chronic administration of sildenafil improves endothelial function in spontaneously hypertensive rats by decreasing COX-2 expression and oxidative stress

AimsSpontaneously hypertensive rats (SHR) exhibit impaired endothelial vasodilation and enhanced vasoconstriction. The phosphodiesterase 5 (PDE5) inhibitor sildenafil (Sild) potentiates the nitric oxide (NO)-mediated effects exerting antioxidative and anti-inflammatory actions. In the present study, we hypothesized that Sild could improve endothelial function in SHR. Materials and methodsMale rats were treated daily for 60 days by oral gavage with Sild (45 mg/kg) before the onset of the hypertensive state (pre-hypertensive protocol). The aortic relaxation to acetylcholine (ACh), sodium nitroprusside (SNP) and the phenylephrine (Phe)-induced contraction was evaluated in SHR. Protein expression of eNOS, p-eNOS, caveolin, COX-1, COX-2, ERK and p-ERK was measured by Western blot. Key findingsResting blood pressure was not modified by Sild administration. Treatment with Sild did not alter the relaxation response to SNP but improved the ACh-induced relaxation and reduced Phe-induced contraction in aortic rings from SHR. This protective effect of Sild could be attributed to reduced superoxide anions (O2−) generation, cyclooxygenase type 2 (COX-2) protein downregulation and increased NO bioavailability. SignificanceSild improves endothelial function in SHR aorta without affecting resting blood pressure values. These results indicate that PDE5 inhibition has a potential role in the improvement of vascular function and could be an adjuvant in the treatment of essential hypertension.

Biotin improves relaxation of rat aortic rings in combination with antihypertensive drugs

Biotin at pharmacological concentrations has beneficial effects on hypertriglyceridaemia, hyperglycaemia and hypertension. We recently reported in a model of metabolic syndrome that biotin has an antihypertensive effect and decreases arterial contraction. In this study, we investigated the effect of biotin (10 nM) on arterial contraction in rat aortic rings with or without an endothelium induced by phenylephrine (1 × 10−9 to 1 × 10−5 M) or calcium (1 × 10−6 to 1 × 10−2.5 M) in the presence of antihypertensive drugs (BMY 7378, 100 nM; captopril, 1 μM; or nitrendipine, 100 nM). Biotin suppressed phenylephrine-induced vasoconstriction, which is partly modulated via endothelium-independent mechanisms. Biotin incubation with BMY 7378, captopril or nitrendipine improved the vasorelaxant effects of these drugs on aortic rings contracted by phenylephrine or calcium. Moreover, biotin decreased arterial contraction induced by calcium, suggesting the inhibition of extracellular influx and intracellular release of calcium. Our data demonstrate for the first time that biotin enhanced the vasorelaxant effect of some antihypertensive drug classes in rat aortic rings. Therefore, biotin could be used as a therapeutic strategy together with antihypertensive drugs for the treatment of hypertension.

Trimebutine maleate relaxes the isolated rat thoracic aorta: The role of nitric oxide and L-type calcium channels.

Trimebutine maleate (TMB), a widely prescribed drug for functional gastrointestinal disorders, has been reported to regulate smooth muscle contractility by modulating multiple ion channel activities in the gastrointestinal tract. However, its action on isolated aorta has not yet been reported. The aim of the present study was to evaluate in vitro vasorelaxant properties and the underlying pharmacological mechanisms of TMB in isolated rat thoracic aortic rings. Vascular activity experiments were performed on thoracic aorta isolated from Sprague-Dawley rats in vitro, including endothelium-intact and endothelium-denuded aortic rings. TMB (10-10 -10-5 mol/L) induced relaxation in endothelium-intact aortic rings precontracted by phenylephrine with a potency similar to that of carbachol. TMB-induced relaxation was not altered by glibenclamide and atropine in endothelium-intact aortic rings. However, L-NAME and endothelium denudation significantly reduced but not completely reversed the vasorelaxant effect of TMB. Also, TMB-induced relaxation wasn't affected by diclofenac in endothelium-intact aortic rings. TMB at 10-5 mol/L significantly reduced the CaCl2 -induced contractions in endothelium-intact aortic rings stimulated with KCl, but not stimulated with phenylephrine under Ca2+ free conditions. Moreover, TMB at 10-5 mol/L effectively attenuated Bay-K8644-induced contractions in aortic rings. These results suggest that TMB-induced relaxation was mediated by both endothelium-dependent and endothelium-independent manner in isolated rat thoracic aorta. The mechanism of TMB-induced relaxation at low concentrations is partially related to NO- and endothelium-dependent but unrelated to prostanoids formation. However, inhibition of Ca2+ influx through voltage-operated calcium channels and L-type Ca2+ channel blocking effect appears to be involved in the mechanism of vasorelaxant effect of TMB at high concentrations.

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.

Prunetin Relaxed Isolated Rat Aortic Rings by Blocking Calcium Channels.

Prunetin, a component of herbal medicines and various foods, such as pea, peach, cherry, and Prunus yedoensis, is a useful pharmacological compound. We previously reported the potent vasorelaxant effect of the bark of P. yedoensis. Therefore, we investigated the vasorelaxant activities of prunetin on isolated rat aortic rings and hypotensive activity on spontaneously hypertensive rats (SHR) in this study. In the present study, prunetin (1–30 μg/mL) relaxed isolated rat aortic rings pre-contracted by phenylephrine (PE) in a concentration-dependent manner. Pre-incubation with prunetin (3 and 10 μg/mL) inhibited vasoconstriction induced by the supply of Ca2+ in rat aortic rings pre-contracted with PE or KCl in a Ca2+-free Krebs–Henseleit (KH) buffer. Prunetin (10 μg/mL) pre-treatment also inhibited caffeine-induced contraction of aortic rings in a Ca2+-free KH buffer. To investigate the hypotensive effect of prunetin, the systolic blood pressure (SBP) of the SHR was measured by using a tail cuff assay. The SBP of SHR was significantly lower in the prunetin (25 mg/kg)-treated group. These results suggested that prunetin decreased blood pressure and relaxed blood vessels by blocking receptor-operated calcium channels, voltage-dependent calcium channels, and ryanodine receptor channels.

Discovery of New Allosteric Modulators of the Urotensinergic System through Substitution of the Urotensin II-Related Peptide (URP) Phenylalanine Residue.

Urotensin II (UII) and urotensin II-related peptide (URP) are functionally selective, suggesting that these two hormones might play distinct physiological role through different interactions with their cognate receptor UT. Hypothesizing that the Phe3 residue of URP, which is also present in UII, is a key-element of its specific UT activation, we evaluated the impact of its replacement by non-natural amino acids in URP. Each compound was evaluated for its ability to bind UT, induce rat aortic ring contraction, and activate Gq, G12, and β-arrestin 1 signaling pathways. Such modifications impaired contractile efficacy, reflected by a reduced aptitude to activate G12 in URP but not in the truncated but equipotent UII4-11. Moreover, we have identified two structurally different UT modulators: [d-Phe(pI)3]URP and [Bip3]URP, which exert a probe-dependent action against UII and URP. These compounds should help us understand the specific roles of these hormones as well as guide further therapeutic development.

Aging related functional and structural changes in the heart and aorta: MitoTEMPO improves aged-cardiovascular performance.

Aging in humans represents declining in cardio-protective systems, however its mechanisms are not known yet. We aimed to analyse how aging affects key mechanisms responsible for contractile dysfunction via comparing the improperly synchrony between electrical and mechanical activities in male aged-rats (24-month old) comparison to those of adult-rats (6-month old). We determined significantly increased systemic oxidative stress with decreased antioxidant capacity, clear insulin resistance and hypertrophy in aged-rats with normal fasting blood glucose. We also determined significantly high level of reactive oxygen species, ROS production in fluorescent dye chloromethyl-2',7'-dichlorodihydrofluoroscein diacetate (DCFDA) loaded isolated cardiomyocytes from aged-rats, confirming the increased oxidative stress in these hearts. In situ electrocardiograms, ECGs presented significant prolongations in RR- and QT-intervals in the aged-rats. Invasive hemodynamic measurements demonstrated marked increases in the heart rate and mean arterial pressure and decreases in the ejection-fraction and preload-recruitable stroke-work, together with depressed contraction and relaxation activities in aortic rings. In light and electron microscopy examinations in aged-rats, significant increases in muscle fibre radius and amount of collagen fibres were detected in the heart as well as markedly flattened and partial local splitting in elastic lamellas in the aorta, besides irregularly clustered mitochondria and lysosomes around the myofilaments in cardiomyocytes. MitoTEMPO treatment of tissue samples and cardiomyocytes from aged-rats for 1-h induced significant structural improvements. In the second part of our study, we have shown that mitochondria-targeted antioxidant MitoTEMPO antagonized all alterations in the heart samples as well as penylephrine-induced contractile and acetylcholine-induced relaxation responses of aged-rat aortic rings. Overall, the present data strongly support the important role of mitochondrial oxidative stress in the development of aged-related insufficiencies and that antioxidant strategies specifically targeting this organelle could have therapeutic benefit in aging-associated complications.

A MINIMAL ROLE FOR ENDOTHELIAL NITRIC OXIDE IN VASOCONSTRICTOR AND VASODILATOR RESPONSES OF GUINEA-PIG AORTA

Objective: Acetylcholine (Ach) dilates rat isolated aorta by releasing nitric oxide (NO) from the vascular endothelium. The vasoconstrictors, U46619 (thromboxane mimetic), phenylephrine (α-adrenoceptor agonist) and β-phenylethylamine (PEA) (trace amine associated receptor (TAAR) agonist), also release NO causing opposing vasodilatation. The roles of NO and endothelium in these vasoconstrictor responses were examined in guinea-pig aorta, along with the status of the endothelium. Design and method: Isometric contractions of guinea-pig aortic rings immersed in Kreb's solution gassed with 5% CO2 in O2 at 37 ± 0.5oC were recorded. Aortae were either intact or denuded of endothelium by rubbing. Cumulative concentration-response curves (CRC) for acetylcholine chloride, β-phenylethylamine hydrochloride (PEA) or (-)-phenylephrine HCl were obtained. Contractions were expressed as a percentage of the contraction to KCl (60 mM). Responses were compared by Student's paired t-test, P less than 0.05 indicating significance. Results: Contractions to phenylephrine and PEA were not significantly different in endothelium intact and denuded aortae. The maxima for phenylephrine were 80.4 ± 3.0 (n = 11) and 77.1 ± 8.1% (n = 6), respectively and the maxima for PEA were 71.7 ± 3.6 (n = 22) and 60.2 ± 3.4% (n = 17), respectively. Contractions to U46619 (1 μM) were also not significantly different in intact (1.32 ± 0.14 g n = 4) and denuded tissues (1.24 ± 0.38 (n = 12). We tested whether endothelium was present in intact tissues by adding Ach to U46619-pre-contracted aortae (1 or 0.5 μM). Of 16 intact tissues, only 6 responded with small falls in tension (0.07 ± 0.02 g), representing only 7.11 ± 3.04% of the contraction to U46619 (1.33 ± 0.27 g). Sodium nitroprusside, however, relaxed U46619-precontracted (1 μM) aorta reaching a maximum of 97.3 ± 5.1% of the U46619 contraction (1.58 ± 0.21 g, n = 4). No denuded preparations responded to Ach. Ach in non-contracted intact aortae produced small dose-related contractions. After washout (x2), tissues were raised from the bath and the endothelium removed before returning to the bath for a second CRC, which was potentiated, the maximum increasing from 25.1 ± 10.5% to 48.9 ± 21.5% (n = 4). Phenylephrine contractions also increased after rubbing to remove endothelium. Conclusions: Intact guinea-pig aortae displayed poor relaxations to Ach suggesting a minimal role of endothelial muscarinic responses. In uncontracted aortae, Ach caused constriction which was potentiated when repeated after endothelium removal, indicating a minor opposing endothelium-dependent vasodilatation.

Nebivolol prevents vascular oxidative stress and hypertension in rats chronically treated with ethanol

Background and aimsChronic ethanol consumption is associated with hypertension and atherosclerosis. Vascular oxidative stress is described as an important mechanism whereby ethanol predisposes to atherosclerosis. We hypothesized that nebivolol would prevent ethanol-induced hypertension and vascular oxidative stress. MethodsMale Wistar rats were treated with ethanol 20% (vol./vol.) or nebivolol (10 mg/kg/day, p. o., gavage), a selective β1-adrenergic receptor antagonist. ResultsEthanol-induced increase in blood pressure and in the circulating levels of adrenaline and noradrenaline was prevented by nebivolol. Similarly, nebivolol prevented ethanol-induced increase in plasma levels of renin, angiotensin I and II. Chronic ethanol consumption increased the aortic levels of superoxide anion (O2−), thiobarbituric acid reactive species (TBARS) as well as the expression of Nox1 and nitrotyrosine immunostaining in the rat aorta. Treatment with nebivolol prevented these responses. The decrease in aortic levels of nitrate/nitrite (NOx) induced by ethanol was prevented by the treatment with nebivolol. Finally, nebivolol attenuated ethanol-induced increase in phenylephrine- and noradrenaline-induced contraction of endothelium-intact and endothelium-denuded aortic rings. ConclusionsThe novelty of our study is that nebivolol prevented ethanol-induced hypertension and vascular oxidative stress. Additionally, we showed that the sympathetic nervous system (SNS) and the renin-angiotensin system (RAS) are important endogenous mediators of the cardiovascular effects of ethanol.

Electrical field stimulation-induced contractions on Pantherophis guttatus corpora cavernosa and aortae.

A tetrodotoxin (TTX)-resistant mechanism is responsible for the electrical field stimulation (EFS)-induced contractions and relaxations of Crotalus durissus terrificus corpora cavernosa. Here it was investigated whether this mechanism also occurs in corpora cavernosa and aortae of the non-venomous snake Pantherophis guttatus corpora cavernosa and aortae. Corpora cavernosa and aortic rings isolated from Pantherophis guttatus snake were mounted in organ bath system for isometric tension recording. EFS-induced contractions in both tissues were performed in the presence and absence of guanethidine (30 μM), phentolamine (10 μM) and tetrodotoxin (1 μM). In another set of experiments, the endothelium was removed from aortic rings and EFS-induced contractions were performed in the denuded rings. Electrical field stimulation-induced contractions were frequency-dependent in Pantherophis guttatus corpora cavernosa and aortic rings. The contractions were significantly reduced in the presence of guanethidine (30 μM) or phentolamine (10 μM). Pre-treatment with tetrodotoxin had no effect on the EFS-induced contractions of either corpora cavernosa or aortic rings. Surprisingly, the EFS-induced contractions of aortic rings denuded of endothelium were almost abolished. These results indicate that the TTX-resistant mechanism is present in EFS-induced contractions of Pantherophis guttatus corpora cavernosa and aortae. The experiments performed in the aorta indicate that the endothelium is the main source for the release of catecholamines induced by EFS.

Roles and mechanism of protein kinase D in vasoconstriction

Protein kinase D (PKD) is known to be implicated in a variety of cellular processes and physiological functions, including cell motility, cardiac contraction, angiogenesis and smooth muscle contraction through regulation of multiple molecules. Although these regulations are presumed to be closely associated with vasomotor activity, role of PKD in vascular function is unknown. Then, we investigated the role of PKD in systemic circulation and contraction of aorta in in rats. We also investigated precise role s of PKD isoforms using human aortic smooth muscle cells (HASMCs) with RNA interference.Material and methodsSystemic blood pressure (SBP) through femoral artery, cardiac output (CO) and systemic vascular resistances (SVR) were measured in Sprague‐Dawley rats. The hemodynamic changes in response to intravenous injections of PKD inhibitor, CRT0066101 were recorded. Isometric tension of aorta ring samples was measured to examine inhibitory effect of CRT0066101 on norepinephrine (NE)‐induced contraction. To explore mechanisms of PKD in vasomotor activity, phosphorylations of PKD, myosin targeting subunit‐1 (MYPT1), CPI‐17, regulatory myosin light chain (RLC) and heat shock protein 27 (HSP27) stimulated with NE were measured as an index of activation of the pathways in aorta rings. In HASMCs, simultaneous measurements of intracellular calcium concentrations and cell shortening with NE stimulation were performed. PKD1, 2, and 3 were knocked down by their specific siRNA respectively in HASMCs for measurements of phosphorylation of PKD, MYPT1, CPI‐17 and RLC and actin polymerization.ResultsInjection of CRT0066101 decreased SBP and SVR. CRT0066101 also inhibited NE‐induced contraction in aorta rings dose‐dependently. In the rings, NE increased phosphorylations of PKD, MYPT1, RLC and CPI‐17, which were attenuated by pretreatment of CRT0066101 except CPI‐17. NE did not increase HSP27 phosphorylation in the rings. In HASMCs, CRT0066101 inhibited NE‐induced cell shortening without affecting increased calcium concentrations. In HASMCs, increased phosphorylations of MYPT1 and RLC with NE were significantly inhibited by PKD1 but not PKD2, 3 knock down. NE did not affect actin polymerization in the cells. Collectively, attenuation of NE‐induced contraction of aorta rings with CRT0066101 was associated with decreased phosphorylations of MYPT1 and RLC, but not CPI‐17 and HSP27. In HASMCs, PKD1 knocked down or PKD inhibition attenuated NE‐induced phosphorylation of MYPT1 and RLC without affecting actin polymerization and intracellular calcium regulation.ConclusionsPKD1 may play roles in contraction of aorta and systemic circulation. The regulation of vasomotor activity by PKD1 may be associated with phosphorylation of MYPT1. Understanding of PKD may lead to novel regulation of circulation.Support or Funding InformationThis work was supported by Grant‐in‐Aid for Scientific Research (C) 17K11058 from Japan Society for the Promotion of ScienceThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.