Removal Of Endothelium Research Articles

Exercise training reverses aging-induced impairment of myogenic constriction in skeletal muscle arterioles.

To investigate whether exercise training can reverse age-related impairment of myogenic vasoconstriction in skeletal muscle arterioles, young (4 mo) and old (22 mo) male Fischer 344 rats were randomly assigned to either sedentary or exercise-trained groups. The roles of the endothelium and Kv1 channels in age- and exercise training-induced adaptations of myogenic responses were assessed through evaluation of pressure-induced constriction in endothelium-intact and denuded soleus muscle arterioles in the presence and absence of the Kv1 channel blocker, correolide. Exercise training enhanced myogenic constriction in arterioles from both old and young rats. In arterioles from old rats, exercise training restored myogenic constriction to a level similar to that of arterioles from young sedentary rats. Removal of the endothelium did not alter myogenic constriction of arterioles from young sedentary rats, but reduced myogenic constriction in arterioles from young exercise-trained rats. In contrast, endothelial removal had no effect on myogenic constriction of arterioles from old exercise-trained rats, but increased myogenic vasoconstriction in old sedentary rats. The effect of Kv1 channel blockade was also dependent on age and training status. In arterioles from young sedentary rats, Kv1 blockade had little effect on myogenic constriction, whereas in old sedentary rats Kv1 blockade increased myogenic constriction. After exercise training, Kv1 channel blockade increased myogenic constriction in arterioles from both young and old rats. Thus exercise training restores myogenic constriction of arterioles from old rats and enhances myogenic constriction from young rats through adaptations of the endothelium and smooth muscle Kv1 channels.

Nuciferine relaxes rat mesenteric arteries through endothelium-dependent and -independent mechanisms.

Nuciferine, a constituent of lotus leaf, is an aromatic ring-containing alkaloid, with antioxidative properties. We hypothesize nuciferine might affect vascular reactivity. This study aimed at determining the effects of nuciferine on vasomotor tone and the underlying mechanism Nuciferine-induced relaxations in rings of rat main mesenteric arteries were measured by wire myographs. Endothelial NOS (eNOS) was determined by immunoblotting. Intracellular NO production in HUVECs and Ca(2+) level in both HUVECs and vascular smooth muscle cells (VSMCs) from rat mesenteric arteries were assessed by fluorescence imaging. Nuciferine induced relaxations in arterial segments pre-contracted by KCl or phenylephrine. Nuciferine-elicited arterial relaxations were reduced by removal of endothelium or by pretreatment with the eNOS inhibitor L-NAME or the NO-sensitive guanylyl cyclase inhibitor ODQ. In HUVECs, the phosphorylation of eNOS at Ser(1177) and increase in cytosolic NO level induced by nuciferine were mediated by extracellular Ca(2+) influx. Under endothelium-free conditions, nuciferine attenuated CaCl2-induced contraction in Ca(2+)-free depolarizing medium. In the absence of extracellular calcium, nuciferine relieved the vasoconstriction induced by phenylephrine and the addition of CaCl2. Nuciferine also suppressed Ca(2+) influx in Ca(2+)-free K(+)-containing solution in VSMCs. Nuciferine has a vasorelaxant effect via both endothelium-dependent and -independent mechanisms. These results suggest that nuciferine may have a therapeutic effect on vascular diseases associated with aberrant vasoconstriction.

Lipoxin A4 Mediates Aortic Contraction via RHOA/RHO Kinase, Endothelial Dysfunction and Reactive Oxygen Species

Background: Lipoxin A4 (LXA₄) is a biologically active product generated from arachidonic acid by lipoxygenase action. The production of lipoxins is enhanced by aspirin through acetylation of cyclooxygenase-2, via a mechanism known as ‘aspirin-triggered lipoxin'. LXA₄ has both anti-inflammatory and proinflammatory actions, the latter being related with reocclusion and restenosis after coronary angioplasty in patients treated with aspirin. However, little is known of the actions of LXA₄ on the vasculature. We hypothesized that LXA₄ promotes contractile responses and contributes to endothelial dysfunction. Methods: We used aorta from Wistar rats to assess vascular function. Reactive oxygen species (ROS) production and contractile and regulatory proteins were investigated. Results: LXA₄ induced concentration-dependent contractions via formyl peptide receptor-2 activation and both RhoA/Rho kinase inhibitor and ROS scavenger decreased this contraction. Also, endothelium removal, and COX-2 and NAD(P)H oxidase inhibitors attenuate the LXA₄-induced contraction. LXA₄ potentiated phenylephrine-induced contraction and inhibited acetylcholine-induced relaxation. In the presence of LXA₄, ROS production was increased and protein expression of RhoA, phospho-myosin light chain, COX-2 and p67phox was higher. Conclusion: LXA₄ has a functional role in the vasculature and may contribute to further vascular damage in conditions where its production is exacerbated, such as in angioplasty-associated complications treated with aspirin.

MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways

Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 μg/100g; subsequent doses: 0.125μg/100g, intramuscular, 30days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20μg/dL) were used. Lead blood levels of treated rats attained 21.7±2.38μg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and did not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension.

Vasorelaxant and blood pressure lowering effects of alchemilla vulgaris: A comparative study of methanol and aqueous extracts.

Background:In the last decade, a growing interest particularly in determining the cardiovascular effects of herbal extracts took place among researchers. Objective: Herein, we aimed to investigate the microvascular and blood pressure lowering effects of two differently processed extracts of the same herb, Alchemilla vulgaris (Rosaceaea), which was revealed to contain high levels of vasoactive compounds.Materials and Methods:For the purpose, endothelium intact rat mesenteric arteries were mounted in a myograph system and contracted with prostaglandin F2α (PGF2α: 3 × 10−5 M) or potassium chloride (K+: 40 mM). Then, aqueous and methanol extracts were added at 0.01–10 mg/ml concentrations in a cumulative manner.Results:Both extracts produced relaxations in PGF2α (3 × 10−5 M) precontracted arteries which were insensitive to the inhibitors of endothelium derived vasoactive substances namely, LG-nitro-L-arginine (10−4 M), ODQ (10−5 M) and indomethacin (10−5 M) or removal of endothelium. Opposite vascular effects were observed when extracts were applied in K+ precontracted arteries. In addition, oral administration of the methanol extract of Alchemilla vulgaris, but not the aqueous extract, reduced blood pressure significantly in L-NAME hypertensive rats.Conclusion:Our results demonstrated that the methanol extract of Alchemilla vulgaris has more prominent and favourable vascular effects in normal and experimental hypertensive conditions reinforcing its traditional use in cardiovascular disorders, in particular hypertension. These results most likely give rise to further studies to reveal its mechanism of action and clinical value of this herb.

Combined aliskiren and L-arginine treatment has antihypertensive effects and prevents vascular endothelial dysfunction in a model of renovascular hypertension.

Angiotensin II is a key player in the pathogenesis of renovascular hypertension, a condition associated with endothelial dysfunction. We investigated aliskiren (ALSK) and L-arginine treatment both alone and in combination on blood pressure (BP), and vascular reactivity in aortic rings. Hypertension was induced in 40 male Wistar rats by clipping the left renal artery. Animals were divided into Sham, 2-kidney, 1-clip (2K1C) hypertension, 2K1C+ALSK (ALSK), 2K1C+L-arginine (L-arg), and 2K1C+ALSK+L-arginine (ALSK+L-arg) treatment groups. For 4 weeks, BP was monitored and endothelium-dependent and independent vasoconstriction and relaxation were assessed in aortic rings. ALSK+L-arg reduced BP and the contractile response to phenylephrine and improved acetylcholine relaxation. Endothelium removal and incubation with N-nitro-L-arginine methyl ester (L-NAME) increased the response to phenylephrine in all groups, but the effect was greater in the ALSK+L-arg group. Losartan reduced the contractile response in all groups, apocynin reduced the contractile response in the 2K1C, ALSK and ALSK+L-arg groups, and incubation with superoxide dismutase reduced the phenylephrine response in the 2K1C and ALSK groups. eNOS expression increased in the 2K1C and L-arg groups, and iNOS was increased significantly only in the 2K1C group compared with other groups. AT1 expression increased in the 2K1C compared with the Sham, ALSK and ALSK+L-arg groups, AT2 expression increased in the ALSK+L-arg group compared with the Sham and L-arg groups, and gp91phox decreased in the ALSK+L-arg group compared with the 2K1C and ALSK groups. In conclusion, combined ALSK+L-arg was effective in reducing BP and preventing endothelial dysfunction in aortic rings of 2K1C hypertensive rats. The responsible mechanisms appear to be related to the modulation of the local renin-angiotensin system, which is associated with a reduction in endothelial oxidative stress.

Effects of exercise training on stress-induced vascular reactivity alterations: role of nitric oxide and prostanoids.

Background:Physical exercise may modify biologic stress responses. Objective:To investigate the impact of exercise training on vascular alterations induced by acute stress, focusing on nitric oxide and cyclooxygenase pathways. Method:Wistar rats were separated into: sedentary, trained (60-min swimming, 5 days/week during 8 weeks, carrying a 5% body-weight load), stressed (2 h-immobilization), and trained/stressed. Response curves for noradrenaline, in the absence and presence of L-NAME or indomethacin, were obtained in intact and denuded aortas (n=7-10). Results:None of the procedures altered the denuded aorta reactivity. Intact aortas from stressed, trained, and trained/stressed rats showed similar reduction in noradrenaline maximal responses (sedentary 3.54±0.15, stressed 2.80±0.10*, trained 2.82±0.11*, trained/stressed 2.97± 0.21*, *P<0.05 relate to sedentary). Endothelium removal and L-NAME abolished this hyporeactivity in all experimental groups, except in trained/stressed rats that showed a partial aorta reactivity recovery in L-NAME presence (L-NAME: sedentary 5.23±0,26#, stressed 5.55±0.38#, trained 5.28±0.30#, trained/stressed 4.42±0.41, #P<0.05 related to trained/stressed). Indomethacin determined a decrease in sensitivity (EC50) in intact aortas of trained rats without abolishing the aortal hyporeactivity in trained, stressed, and trained/stressed rats. Conclusions:Exercise-induced vascular adaptive response involved an increase in endothelial vasodilator prostaglandins and nitric oxide. Stress-induced vascular adaptive response involved an increase in endothelial nitric oxide. Beside the involvement of the endothelial nitric oxide pathway, the vascular response of trained/stressed rats involved an additional mechanism yet to be elucidated. These findings advance on the understanding of the vascular processes after exercise and stress alone and in combination.

Nitric oxide-dependent vasodilation and Ca2+signalling induced by erythrodiol in rat aorta

ObjectiveTo evaluate the pharmacological property of erythrodiol, a natural triterpenoid contained in propolis, as vasodilatory agent, and to determine its mechanism of action. MethodsRats aortic rings were isolated and suspended in organ baths, and the effects of erythrodiol were studied by means of isometric tension recording experiments. Nitric oxide (NO) was detected by ozone-induced chemiluminescence. The technique used to evaluate changes in intracellular Ca2+ concentration in intact endothelium was opened aortic ring and loaded with 16 μmol Fura-2/AM for 60 min at room temperature, washed and fixed by small pins with the luminal face up. In situ, ECs were visualized by an upright epifluorescence Axiolab microscope (Carl Zeiss, Oberkochen, Germany) equipped with a Zeiss×63 Achroplan objective (water immersion, 2.0 mm working distance, 0.9 numerical apertures). ECs were excited alternately at 340 and 380 nm, and the emitted light was detected at 510 nm. ResultsIn aortic rings with intact endothelium pre-contracted with norepinephrine (10−4 mol/L), the addition of erythrodiol (10−8 −10 −4 mol/L) induced vasorelaxation in a concentration-dependent manner; in endothelium-denuded rings, the relaxant response induced by erythrodiol was almost completely abolished suggesting that vasorelaxation was endothelium-dependent. They had almost no relaxant effect on depolarised or endothelium-denuded aortic segments. The relaxation was significantly attenuated by pre-treatment with the NO synthase inhibitor Nv-nitro-L-arginine-methylester. Erythrodiol (10−4 mol/L) was able to significantly increase NOx levels. This effect was completely abolished after removal of the vascular endothelium. Erythrodiol (100 μmol/L) caused a slow, long-lasting increase in intracellular Ca2+ concentration. These results further supported the hypothesis that erythrodiol can induce activation of the NO/soluble guanylate cyclase/cyclic guanosine monophosphate pathway, as suggested by functional studies. ConclusionsThe present results suggest that the mechanism of relaxation seems to be mainly mediated by the endothelial production of NO. Such a vasorelaxation was an endothelium-dependent effect, via the NO/soluble guanylate cyclase/cyclic guanosine monophosphate pathway. This result also suggests that erythrodiol causes a slow influx of extracellular Ca2+ release from the intracellular Ca2+ stores and an inhibition of Ca2+ extruding mechanism. It can be concluded that erythrodiol may have interesting therapeutic potential as a new vasodilator drug, for protecting the cardiovascular system.

Ouabain induces nitric oxide release by a PI3K/Akt-dependent pathway in isolated aortic rings from rats with heart failure.

Ouabain occurs in nanomolar concentrations in myocardial infarction and heart failure (HF). However, the effects of ouabain in vascular function in HF conditions were not investigated yet. Therefore, we analyzed the effects of acute administration of 3 nM ouabain in isolated aortic rings from rats with HF 4 weeks after myocardial infarction. Rats were submitted to sham operation or coronary artery occlusion. In HF rats, left ventricular positive and negative derivatives of intraventricular pressure reduced and left ventricular end diastolic pressure increased. Phenylephrine responses increased in HF rings when compared with controls. Ouabain incubation for 45 minutes reduced phenylephrine-induced contraction in both groups. Endothelial removal increased more phenylephrine response in ouabain-treated rings of sham rats. Ouabain potentiated the effect of L-NAME in both groups but more in sham rats. Wortmannin increased the phenylephrine response only in HF rings. The effect of tetraethylammonium was potentiated by ouabain only in HF rings. Ouabain increased phenylephrine-stimulated nitric oxide production in rings from both groups but increased the activation of Akt only in vessels from HF rats. Results demonstrate that low ouabain concentration can decrease vascular reactivity of aortic rings from HF rats. Ouabain was able to increase nitric oxide production in HF rats by triggering a signal transduction PI3K/Akt-dependent pathway and increasing an endothelium-hyperpolarizing factor release.

Traumatic brain injury disrupts cerebrovascular tone through endothelial inducible nitric oxide synthase expression and nitric oxide gain of function.

BackgroundTraumatic brain injury (TBI) has been reported to increase the concentration of nitric oxide (NO) in the brain and can lead to loss of cerebrovascular tone; however, the sources, amounts, and consequences of excess NO on the cerebral vasculature are unknown. Our objective was to elucidate the mechanism of decreased cerebral artery tone after TBI.Methods and ResultsCerebral arteries were isolated from rats 24 hours after moderate fluid‐percussion TBI. Pressure‐induced increases in vasoconstriction (myogenic tone) and smooth muscle Ca2+ were severely blunted in cerebral arteries after TBI. However, myogenic tone and smooth muscle Ca2+ were restored by inhibition of NO synthesis or endothelium removal, suggesting that TBI increased endothelial NO levels. Live native cell NO, indexed by 4,5‐diaminofluorescein (DAF‐2 DA) fluorescence, was increased in endothelium and smooth muscle of cerebral arteries after TBI. Clamped concentrations of 20 to 30 nmol/L NO were required to simulate the loss of myogenic tone and increased (DAF‐2T) fluorescence observed following TBI. In comparison, basal NO in control arteries was estimated as 0.4 nmol/L. Consistent with TBI causing enhanced NO‐mediated vasodilation, inhibitors of guanylyl cyclase, protein kinase G, and large‐conductance Ca2+‐activated potassium (BK) channel restored function of arteries from animals with TBI. Expression of the inducible isoform of NO synthase was upregulated in cerebral arteries isolated from animals with TBI, and the inducible isoform of NO synthase inhibitor 1400W restored myogenic responses following TBI.ConclusionsThe mechanism of profound cerebral artery vasodilation after TBI is a gain of function in vascular NO production by 60‐fold over controls, resulting from upregulation of the inducible isoform of NO synthase in the endothelium.

Effects and mechanisms of hyperoside on vascular endothelium function in middle cerebral arteries of rats ex vivo

To investigate the effects and potential mechanisms of hyperoside (Hyp) on the vascular endothelium function in middle cerebral artery (MCA) ex vivo in rats. Isolated arterial segments from MCAs of rats were used for surveying vasomotoricity in a pressurized chamber. Transmembrane potential was recorded by using glass microelectrodes to evaluate hyperpolarization. Hyp (1 x 10(-6)-1 x 10(-4) mol . L-1) was utilized to observe the effect on 1 x 10(-7) mol . L-1 U46619-preconstricted MCA in rats. The results showed that 1 x 10(-6)-1 x 10(-4) mol . L-1 Hyp significantly induced concentration-dependent vasodilatation and hyperpolarization, leading to the maximal diastolic ratio of (73. 2 ± 6. 1)% and maximal changes in membrane potentials of (-13. 2 ± 2. 2) mV. Hyp still elicited vasorelaxation and hyperpolarization by removal of endothelium in MCA of rat, which was notably attenuated as compared with vascular endothelium-intact group (P <0. 01). In the MCAs preconstricted by U46619 (1 x 10(-7) mol . L-1), Hyp (1 x 10(-6)-1 x 10(-4) mol . L-1) produced concentration-dependent vasorelaxation and hyperpolarizition that were partially attenuated by 3 x 10(-5) mol . L-1 L-NAME(a NOS inhibitor) plus 1 x 10(-5) mol . L-1 PGI2 ,(a synthetase inhibitor). The residual effects were further decreased by 1 x 10(-3) mol . L-1 TEA (an inhibitor of Ca2+-activated potassium channel) or 1 x 10(-5) mol . L-1 PPG (a blocker of endogenous H2S synthese-CSE). Similarly, 1 x 10(-5)-1 x 10(-3) mol . L-1 NaHS (a donor of exogenous H2S) or 1 x 10(-5)-1 x 10(-3) mol . L-1 L-Cys (the substrate of endogenous H2S synthesis) obviously evoked dose-dependent vasodilatation and hyperpolarization of MCA in rats. These findings indicated that Hyp may induce endothelium-dependent and endothelium-independent responses. And the endothelium-dependent vasodilatation may be related to the increases of endogenous H2S that has been promoted Hyp in the endotheliocyte of MCAs, and activated Kca and opening of Kca channels, resulting in the hyperpolarization of vascular smooth muscle cell membrane and subsequent reduction of Ca2+ influx and vasodilation.

Evaluation of cardiovascular effects of edible fruits of &lt;i&gt;Syzygium cumini&lt;/i&gt; Myrtaceae (L) skeels in rats

Purpose: To evaluate the hypotensive, vasorelaxant and antihypertensive effects elicited by the hydroalcohol extract from the fruits of Syzygium cumini (EHSCF) in non-anesthetized rats. Methods: The rats were anesthetized and polyethylene catheters were inserted into the lower abdominal aorta and into the inferior vena cava for blood pressure measurements and administration of drugs. After a recovery period of 24 h, EHSCF (0.5; 1; 5; 10; 20 and 30 mg/kg, i.v.) was administered in non-anesthetized rats. The mean arterial pressure and the heart rate were recorded. To investigate the effects of extract, doses EHSCF were administered after pretreatment with L-NAME, atropine, indomethacin, and hexamethonium. For measurement of isometric tension, a concentration-response curve was obtained after Phenylephrine and KCl (80 mM) pre-contractions. The bioactive extract was analyzed via mass spectrometry (MS) fingerprinting using direct electrospray ionization mass spectrometry (ESI-MS). Results: EHSCF (0.5; 1; 5; 10; 20 and 30 mg/kg) induced hypotension (-15 ± 1, -14 ± 1, -15 ± 1, -13 ± 1, -11 ± 1 and -13 ± 2 %) and bradycardia (-6 ± 1, -5 ± 1, -6 ± 1, -14 ± 1, -8 ± 1 and -10 ± 2 %) in normotensive rats. These responses were attenuated by pre-treatment with L-NAME, indomethacin, hexamethonium or atropine. In phenylephrine, pre-contracted mesenteric rings, EHSCF-induced relaxation (Emax = 54.6 ± 4.5 % and pD2 = 2.7 ± 0.1) that were affected by endothelium removal. EHSCF caused relaxant effect of KCl (80 mM) pre-contracted rings (Emax = 100 ± 0.2 % and pD2 = 2.2 ± 0.1). This effect was not changed in denuded rings. A single oral administration of the extract reduced significant mean arterial pressure in spontaneously hypertensive rats. ESI-MS/MS analyses of EHSCF demonstrated that the major constituents of the analyzed samples coincided with the mass of the malic, gallic, caffeic and ferulic acids. Conclusion: The results suggest that EHSCF induces hypotension probably due to a decrease in peripheral resistance, mediated by the endothelium. Bradycardia may be due to indirect cardiac muscarinic activation. The extract also causes an antihypertensive effect.

Estrogen receptor subtypes mediate distinct microvascular dilation and reduction in [Ca2+]I in mesenteric microvessels of female rat.

Estrogen interacts with estrogen receptors (ERs) to induce vasodilation, but the ER subtype and post-ER relaxation pathways are unclear. We tested if ER subtypes mediate distinct vasodilator and intracellular free Ca(2+) concentration ([Ca(2+)]i) responses via specific relaxation pathways in the endothelium and vascular smooth muscle (VSM). Pressurized mesenteric microvessels from female Sprague-Dawley rats were loaded with fura-2, and the changes in diameter and [Ca(2+)]i in response to 17β-estradiol (E2) (all ERs), PPT (4,4',4''-[4-propyl-(1H)-pyrazole-1,3,5-triyl]-tris-phenol) (ERα), diarylpropionitrile (DPN) (ERβ), and G1 [(±)-1-[(3aR*,4S*,9bS*)-4-(6-bromo-1,3-benzodioxol-5-yl)-3a,4,5,9b-tetrahydro:3H-cyclopenta(c)quinolin-8-yl]-ethanon] (GPR30) were measured. In microvessels preconstricted with phenylephrine, ER agonists caused relaxation and decrease in [Ca(2+)]i that were with E2 = PPT > DPN > G1, suggesting that E2-induced vasodilation involves ERα > ERβ > GPR30. Acetylcholine caused vasodilation and decreased [Ca(2+)]i, which were abolished by endothelium removal or treatment with the nitric oxide synthase blocker Nω-nitro-l-arginine methyl ester (L-NAME) and the K(+) channel blockers tetraethylammonium (nonspecific) or apamin (small conductance Ca(2+)-activated K(+) channel) plus TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) (intermediate conductance Ca(2+)-activated K(+) channel), suggesting endothelium-derived hyperpolarizing factor-dependent activation of KCa channels. E2-, PPT-, DPN-, and G1-induced vasodilation and decreased [Ca(2+)]i were not blocked by L-NAME, TEA, apamin plus TRAM-34, iberiotoxin (large conductance Ca(2+)- and voltage-activated K(+) channel), 4-aminopyridine (voltage-dependent K(+) channel), glibenclamide (ATP-sensitive K(+) channel), or endothelium removal, suggesting an endothelium- and K(+) channel-independent mechanism. In endothelium-denuded vessels preconstricted with phenylephrine, high KCl, or the Ca(2+) channel activator Bay K 8644 (1,4-dihydro-2,6-dimethyl-5-nitro-4-[2-(trifluoromethyl)phenyl]-3-pyridinecarboxylic acid methyl ester), ER agonist-induced relaxation and decreased [Ca(2+)]i were with E2 = PPT > DPN > G1 and not inhibited by the guanylate cyclase inhibitor ODQ [1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one], and showed a similar relationship between decreased [Ca(2+)]i and vasorelaxation, supporting direct effects on Ca(2+) entry in VSM. Immunohistochemistry revealed ERα, ERβ, and GPR30 mainly in the vessel media and VSM. Thus, in mesenteric microvessels, ER subtypes mediate distinct vasodilation and decreased [Ca(2+)]i (ERα > ERβ > GPR30) through endothelium- and K(+) channel-independent inhibition of Ca(2+) entry mechanisms of VSM contraction.

Endothelin-1 contributes to endothelial dysfunction and enhanced vasoconstriction through augmented superoxide production in penile arteries from insulin-resistant obese rats: role of ET(A) and ET(B) receptors.

We assessed whether endothelin-1 (ET-1) inhibits NO and contributes to endothelial dysfunction in penile arteries in a model of insulin resistance-associated erectile dysfunction (ED). Vascular function was assessed in penile arteries, from obese (OZR) and lean (LZR) Zucker rats, mounted in microvascular myographs. Changes in basal and stimulated levels of superoxide (O2 (-) ) were detected by lucigenin-enhanced chemiluminescence and ET receptor expression was determined by immunohistochemistry. ET-1 stimulated acute O2 (-) production that was blunted by tempol and the NADPH oxidase inhibitor, apocynin, but markedly enhanced in obese animals. ET-1 inhibited the vasorelaxant effects of ACh and of the NO donor S-nitroso-N-acetyl-DL-penicillamine in arteries from both LZR and OZR. Selective ETA (BQ123) or ETB receptor (BQ788) antagonists reduced both basal and ET-1-stimulated superoxide generation and reversed ET-1-induced inhibition of NO-mediated relaxations in OZR, while only BQ-123 antagonized ET-1 actions in LZR. ET-1-induced vasoconstriction was markedly enhanced by NO synthase blockade and reduced by endothelium removal and apocynin. In endothelium-denuded penile arteries, apocynin blunted augmented ET-1-induced contractions in OZR. Both ETA and ETB receptors were expressed in smooth muscle and the endothelial layer and up-regulated in arteries from OZR. ET-1 stimulates ETA -mediated NADPH oxidase-dependent ROS generation, which inhibits endothelial NO bioavailability and contributes to ET-1-induced contraction in healthy penile arteries. Enhanced vascular expression of ETB receptors contributes to augmented ROS production, endothelial dysfunction and increased vasoconstriction in erectile tissue from insulin-resistant obese rats. Hence, antagonism of ETB receptors might improve the ED associated with insulin-resistant states.

Alpha-lipoic acid activates eNOS through activation of PI3-kinase/Akt signaling pathway

BackgroundLipoic acid (LA) exerts therapeutic effects on cardiovascular diseases. However, the mechanisms underlying these therapeutic effects remain elusive. Endothelial nitric oxide synthase (eNOS) plays a critical role in cardiovascular homeostasis. LA was shown to potently activate PI3-kinase/Akt pathway, and the latter is critical in the regulation of eNOS activity. In the present study, we test the hypothesis that LA improves endothelial function through PI3-kinase/Akt-mediated eNOS activation. Methods and ResultsWestern blot analysis showed that LA time- and dose-dependently induced phosphorylation of Akt and eNOS in human umbilical vein endothelial cells (HUVECs). Both PI3-kinase and Akt inhibitors abolished LA-induced eNOS phosphorylation, indicating that LA induces eNOS phosphorylation through the PI3-kinase/Akt pathway. This increase in eNOS phosphorylation was paralleled by an increase in NO release by HUVECs, supporting its relevance in eNOS activity regulation. Myograph analysis revealed that LA relaxed phenylephrine-induced contraction. Endothelium removal and NOS inhibition by L-NAME abolished this vasodilator action of LA, and Akt but not AMPK inhibition significantly reduced the vasodilator action of LA, indicating that it is mediated by PI3-kinase/Akt pathway-dependent activation of eNOS. Consistent with in vitro results, intraperitoneal injection with LA significantly increased plasma nitrite and nitrate levels in C57Bl/6j mice. ConclusionsLA activates eNOS through a PI3-kinase/Akt signaling pathway-dependent mechanism, offering a potential molecular basis for the therapeutic effects of LA on cardiovascular diseases.

Angiotensin-(1-7) modulates angiotensin II-induced vasoconstriction in human mammary artery.

The renin-angiotensin system plays a key role in cardiovascular pathophysiology and one of its members, angiotensin-(1-7) (ANG-(1-7)), is now recognized as a peptide with the ability to counter-regulate angiotensin II (ANGII) effects. We sought to investigate ANG-(1-7) actions in human vessels, particularly its effect on ANGII-induced vasoconstriction in human mammary arteries (HMA). Samples of HMA from patients submitted to coronary revascularization (22 patients, mean age 67 years) were cut into small rings, mounted in a myograph bath system, normalized and allowed to contract and dilate isometrically. In baseline experiments, the rings were incubated with ANG-(1-7) or vehicle, followed by increasing concentrations of ANGII. This protocol was repeated in the presence of A-779, PD123177, losartan and after mechanical endothelium removal. Western blot analysis and immunofluorescence were also performed in order to verify the presence of Mas receptor in HMA. ANG-(1-7) significantly attenuated ANGII-induced contraction, producing a maximal inhibition of approximately 65.2%. This effect was not abolished by A-779, PD123177 or endothelium removal. In the presence of losartan, ANGII response was attenuated and no differences were observed between ANG-(1-7) and vehicle treated rings. Finally, we observed, for the first time, that the Mas receptor is expressed in HMA endothelium. ANG-(1-7) significantly attenuates ANGII-induced vasoconstriction and, although the Mas receptor is expressed in HMA, this effect seems to be independent of its activation. Additionally, AT2 receptor and endothelium are not involved in this mechanism, which suggests a direct effect on smooth muscle cells.

PS-340 Age-related Changes And Effects Of Mild Hypothermia On Carotid Artery Reactivity In Newborn Rats

Introduction Therapeutic hypothermia has become a standard neuroprotective treatment in term newborn infants following perinatal asphyxia. Hypothermia-induced changes in the reactivity of the vessels supplying the brain might play a role in its therapeutic or side effects. We investigated the putative age-related changes and the effect of clinically relevant cooling (33°C) on the reactivity of the newborn rat carotid artery. Methods Carotid artery rings from 2–3 d-old and 9–10 d-old rats were mounted in myographs and studied at 33 and 37°C. Results Hypothermia did not significantly affect the contractions induced by KCl and U46619, nor the relaxations induced by acetylcholine (ACh), the nitric oxide (NO) donor sodium nitroprusside (SNP), the NO-independent stimulator of soluble guanylate cyclase (sGC) BAY 41–2272, the β-adrenoceptor agonist isoproterenol, the adenylate cyclase activator forskolin, and acute hypoxia (PO 2 3 kPa). The relaxations induced by ACh, isoproterenol, the β 2 -adrenoceptor agonist salbutamol, the β 3 -adrenoceptor agonist CL-316243 and hypoxia increased with postnatal age and were impaired by endothelium removal or by inhibition of NO synthase (L-NAME) or sGC (ODQ). By contrast, the relaxations induced by SNP, BAY 41–2272 and forskolin were endothelium-independent and did not change with age. Conclusions Mild hypothermia (33°C) does not affect the reactivity of neonatal rat carotid arteries. Our data suggest a reduced NO bioavailability in the carotid artery during the first days of life. This transient reduction in endothelium-dependent relaxation might play a role in the adaptation of the circulatory system to birth and in the neonatal vascular response to insults such as hypoxia.

Vasorelaxation induced by methyl cinnamate, the major constituent of the essential oil of Ocimum micranthum, in rat isolated aorta.

The aim of the present study was to investigate the vascular effects of the E-isomer of methyl cinnamate (E-MC) in rat isolated aortic rings and the putative mechanisms underlying these effects. At 1-3000μmol/L, E-MC concentration-dependently relaxed endothelium-intact aortic preparations that had been precontracted with phenylephrine (PHE; 1μmol/L), with an IC50 value (geometric mean) of 877.6μmol/L (95% confidence interval (CI) 784.1-982.2μmol/L). These vasorelaxant effects of E-MC remained unchanged after removal of the vascular endothelium (IC50 725.5μmol/L; 95% CI 546.4-963.6μmol/L) and pretreatment with 100μmol/L N(G) -nitro-l-arginine methyl ester (IC50 749.0μmol/L; 95% CI 557.8-1005.7μmol/L) or 10μmol/L 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (IC50 837.2μmol/L; 95% CI 511.4-1370.5μmol/L). Over the concentration range 1-3000μmol/L, E-MC relaxed K(+) -induced contractions in mesenteric artery preparations (IC50 314.5μmol/L; 95% CI 141.9-697.0μmol/L) with greater potency than in aortic preparations (IC50 1144.7μmol/L; 95% CI 823.2-1591.9μmol/L). In the presence of a saturating contractile concentration of K(+) (150mmol/L) in Ca(2+) -containing medium combined with 3μmol/L PHE, 1000μmol/L E-MC only partially reversed the contractile response. In contrast, under similar conditions, E-MC nearly fully relaxed PHE-induced contractions in aortic rings in a Ba(2+) -containing medium. In preparations that were maintained under Ca(2+) -free conditions, 600 and 1000μmol/L E-MC significantly reduced the contractions induced by exogenous Ca(2+) or Ba(2+) in KCl-precontracted preparations, but not in PHE-precontracted preparations (in the presence of 1μmol/L verapamil). In addition, E-MC (1-3000μmol/L) concentration-dependently relaxed the contractions induced by 2mmol/L sodium orthovanadate. Based on these observations, E-MC-induced endothelium-independent vasorelaxant effects appear to be preferentially mediated by inhibition of plasmalemmal Ca(2+) influx through voltage-dependent Ca(2+) channels. However, the involvement of a myogenic mechanism in the effects of E-MC is also possible.

A role for the sodium pump in H2O2-induced vasorelaxation in porcine isolated coronary arteries

Hydrogen peroxide (H2O2) has been proposed to act as a factor for endothelium-derived hyperpolarization (EDH) and EDH may act as a ‘back up’ system to compensate the loss of the NO pathway. Here, the mechanism of action of H2O2 in porcine isolated coronary arteries (PCAs) was investigated. Distal PCAs were mounted in a wire myograph and pre-contracted with U46619 (1nM–50μM), a thromboxane A2-mimetic or KCl (60mM). Concentration–response curves to H2O2(1μM–1mM), bradykinin (0.01nM–1μM), sodium nitroprusside (SNP) (10nM–10μM), verapamil (1nM–10μM), KCl (0–20mM) or Ca2+-reintroduction (1μM–10mM) were constructed in the presence of various inhibitors. Activity of the Na+/K+-pump was measured through rubidium-uptake using atomic absorption spectrophotometry. H2O2 caused concentration-dependent vasorelaxations with a maximum relaxation (Rmax) of 100±16% (mean±SEM), pEC50=4.18±0.20 (n=4) which were significantly inhibited by PEG-catalase at 0.1–1.0mM H2O2 (P<0.05). 10mM TEA significantly inhibited the relaxation up to 100μM H2O2 (P<0.05). 60mM K+ and 500nM ouabain significantly inhibited H2O2-induced vasorelaxation producing a relaxation of 40.8±8.5% (n=5) and 47.5±8.6% (n=6) respectively at 1mM H2O2 (P<0.0001). H2O2-induced vasorelaxation was unaffected by the removal of endothelium, inhibition of NO, cyclo-oxygenase, gap junctions, SKCa, IKCa, BKCa Kir, KV, KATP or cGMP. 100μM H2O2 had no effects on the KCl-induced vasorelaxation or Ca2+-reintroduction contraction. 1mM H2O2 inhibited both KCl-induced vasorelaxation and rubidium-uptake consistent with inhibition of the Na+/K+-pump activity. We have shown that the vascular actions of H2O2 are sensitive to ouabain and high concentrations of H2O2 are able to modulate the Na+/K+-pump. This may contribute towards its vascular actions.

Investigation of terpinen-4-ol effects on vascular smooth muscle relaxation

AimsThis study investigated the mechanisms underlying the vascular effects of terpinen-4-ol in isolated rat aortic ring preparations. Main methodsThe thoracic aortae of healthy rats were submitted to isometric tension recording. Membrane resting potential and input membrane resistance were measured by conventional microelectrode technique. Key findingsTerpinen-4-ol reversibly relaxed endothelium-containing preparations pre-contracted with high K+ and phenylephrine with IC50 values of 421.43μM and 802.50μM, respectively. These effects were significantly reduced by vascular endothelium removal. In Ca2+-free and high K+ (80mM) medium, the contractions produced by Ba2+ were reduced by terpinen-4-ol (100–1000μM) in a concentration-dependent manner. In aortic rings maintained under Ca2+-free conditions, terpinen-4-ol significantly reduced the contractions induced by either phenylephrine (1μM) or phorbol 12,13-dibutyrate (1μM). Terpinen-4-ol (10–1000μM) also relaxed the contractions evoked by BAYK-8644 (3μM) with an IC50 of 454.23μM. Neither membrane resting potential nor input resistance of smooth muscle cells was altered by terpinen-4-ol exposure. SignificanceThe present results suggest that terpinen-4-ol induced vascular smooth muscle relaxation that was preferentially due to the inhibition of electromechanical pathways related to calcium influx through voltage-operated calcium channels.