Acetylcholine In Arteries Research Articles

Sex-Specific Differences in Endothelial Function Are Driven by Divergent Mitochondrial Ca2+ Handling.

BackgroundSex‐specific differences in vasodilation are mediated in part by differences in cytosolic Ca2+ handling, but how variations in mitochondrial Ca2+ contributes to this effect remains unknown. Here, we investigated the extent to which mitochondrial Ca2+ entry via the MCU (mitochondrial Ca2+ uniporter) drives sex differences in vasoreactivity in resistance arteries.Methods and ResultsEnhanced vasodilation of mesenteric resistance arteries to acetylcholine (ACh) was reduced to larger extent in female compared with male mice in 2 genetic models of endothelial MCU ablation. Ex vivo Ca2+ imaging of mesenteric arteries with Fura‐2AM confirmed higher cytosolic Ca2+ transients triggered by ACh in arteries from female mice versus male mice. MCU inhibition both strongly reduced cytosolic Ca2+ transients and blocked mitochondrial Ca2+ entry. In cultured human aortic endothelial cells, treatment with physiological concentrations of estradiol enhanced cytosolic Ca2+ transients, Ca2+ buffering capacity, and mitochondrial Ca2+ entry in response to ATP or repeat Ca2+ boluses. Further experiments to establish the mechanisms underlying these effects did not reveal significant differences in the expression of MCU subunits, at either the mRNA or protein level. However, estradiol treatment was associated with an increase in mitochondrial mass, mitochondrial fusion, and the mitochondrial membrane potential and reduced mitochondrial superoxide production.ConclusionsOur data confirm that mitochondrial function in endothelial cells differs by sex, with female mice having enhanced Ca2+ uptake capacity, and that these differences are attributable to the presence of more mitochondria and a higher mitochondrial membrane potential in female mice rather than differences in composition of the MCU complex.

Contribution of Genetic Background to Vascular Adaptation to Exercise Training Based on Critical Speed of Different Intensities

An impaired endothelial function is a fundamental component of the pathogenesis of cardiovascular disease. In mice, exercise training is well‐known to improve cardiorespiratory fitness and endothelial function. However, the post exercise training effect on endothelial function varies among different strains of mice, suggesting that genetic background contributes to the heterogeneous effect of exercise on vascular health. NZW/LacJ (NZW) mice are known as relatively low responders to exercise training compared to high responder strains such as FVB/NJ (FVB). NZW mice also have relatively poor endothelial function in thoracic aorta when compared with other mouse strains. Therefore, we aimed to determine the contribution of genetic background to endothelial adaptation to exercise training in FVB and NZW mice. Vasoreactivity was assessed in femoral arteries from female mice from both strains (NZW and FVB) after 6 weeks of training using two different critical speed (CS) treadmill training intensities. Mice from each strain were randomly assigned to one of three groups: sedentary (SED), 80% of CS training (CS80), and 90% of CS (CS90) training. After training, endothelium‐dependent vasorelaxation to acetylcholine (ACh) and endothelium‐independent vasorelaxation to sodium nitroprusside (SNP) were measured as well as the vasocontractile responses to phenylephrine (PE) and potassium chloride (KCl). Maximal relaxation responses and IC50 for ACh and SNP were similar between NZW groups (SED, CS80, and CS90). In femoral arteries from FVB mice, there were no differences for maximal relaxation to ACh, but there was significant difference in IC50 for ACh‐induced vasorelaxation between SED vs. CS90 groups (p=0.05). Maximal responses to ACh were similar between NZW and FVB; however, IC50 was significantly lower in FVB compared with NZW, indicating that sensitivity to ACh is greater in arteries from FVB. There was a significant strain‐dependent difference for IC50 for SNP despite no strain differences for maximal responses. There was no effect of exercise or strain on contractile responses to PE and KCl, although the EC50 for KCl induced vasocontraction was higher (p=0.0329) in NZW. Treadmill exercise training at 80% or 90% CS did not contribute to improved vasomotor function in NZW while higher intensity exercise improved sensitivity to ACh in arteries from FVB. Genetic background did not significantly impact vasomotor function or endothelial responses to exercise training in FVB and NZW mice.

Elevated pressure causes endothelial dysfunction in mouse carotid arteries by increasing local angiotensin signaling.

Experiments were performed to determine whether or not acute exposure to elevated pressure would disrupt endothelium-dependent dilatation by increasing local angiotensin II (ANG II) signaling. Vasomotor responses of mouse-isolated carotid arteries were analyzed in a pressure myograph at a control transmural pressure (PTM) of 80 mmHg. Acetylcholine-induced dilatation was reduced by endothelial denudation or by inhibition of nitric oxide synthase (NG-nitro-L-arginine methyl ester, 100 μM). Transient exposure to elevated PTM (150 mmHg, 180 min) inhibited dilatation to acetylcholine but did not affect responses to the nitric oxide donor diethylamine NONOate. Elevated PTM also increased endothelial reactive oxygen species, and the pressure-induced endothelial dysfunction was prevented by the direct antioxidant and NADPH oxidase inhibitor apocynin (100 μM). The increase in endothelial reactive oxygen species in response to elevated PTM was reduced by the ANG II type 1 receptor (AT1R) antagonists losartan (3 μM) or valsartan (1 μM). Indeed, elevated PTM caused marked expression of angiotensinogen, the precursor of ANG II. Inhibition of ANG II signaling, by blocking angiotensin-converting enzyme (1 μM perindoprilat or 10 μM captopril) or blocking AT1Rs prevented the impaired response to acetylcholine in arteries exposed to 150 mmHg but did not affect dilatation to the muscarinic agonist in arteries maintained at 80 mmHg. After the inhibition of ANG II, elevated pressure no longer impaired endothelial dilatation. In arteries treated with perindoprilat to inhibit endogenous formation of the peptide, exogenous ANG II (0.3 μM, 180 min) inhibited dilatation to acetylcholine. Therefore, elevated pressure rapidly impairs endothelium-dependent dilatation by causing ANG expression and enabling ANG II-dependent activation of AT1Rs. These processes may contribute to the pathogenesis of hypertension-induced vascular dysfunction and organ injury.

Uptake and protective effects of ergothioneine in human endothelial cells.

Ergothioneine is a thiourea derivative of histidine found in food, especially mushrooms. Experiments in cell-free systems and chemical assays identified this compound as a powerful antioxidant. Experiments were designed to test the ability of endothelial cells to take up ergothioneine and hence benefit from protection against oxidative stress. Reverse-transcription polymerase chain reaction and Western blotting demonstrated transcription and translation of an ergothioneine transporter in human brain microvascular endothelial cells (HBMECs). Uptake of [(3)H]ergothioneine occurred by the organic cation transporter novel type-1 (OCTN-1), was sodium-dependent, and was reduced when expression of OCTN-1 was silenced by small interfering RNA (siRNA). The effect of ergothioneine on the production of reactive oxygen species (ROS) in HBMECs was measured using dichlorodihydrofluorescein and lucigenin, and the effect on cell viability was studied using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. ROS production and cell death induced by pyrogallol, xanthine oxidase plus xanthine, and high glucose were suppressed by ergothioneine. The antioxidant and cytoprotective effects of ergothioneine were abolished when OCTN-1 was silenced using siRNA. The expression of NADPH oxidase 1 was decreased, and those of glutathione reductase, catalase, and superoxide dismutase enhanced by the compound. In isolated rat basilar arteries, ergothioneine attenuated the reduction in acetylcholine-induced relaxation caused by pyrogallol, xanthine oxidase plus xanthine, or incubation in high glucose. Chronic treatment with the compound improved the response to acetylcholine in arteries of rats with streptozotocin-induced diabetes. In summary, ergothioneine is taken up by endothelial cells via OCTN-1, where the compound then protects against oxidative stress, curtailing endothelial dysfunction.

Acute cyclooxygenase inhibition does not alter muscle sympathetic nerve activity or forearm vasodilator responsiveness in lean and obese adults.

Obesity is often characterized by chronic inflammation that may contribute to increased cardiovascular risk via sympathoexcitation and decreased vasodilator responsiveness. We hypothesized that obese individuals would have greater indices of inflammation compared with lean controls, and that cyclooxygenase inhibition using ibuprofen would reduce muscle sympathetic nerve activity (MSNA) and increase forearm blood flow in these subjects. We measured MSNA, inflammatory biomarkers (C‐reactive protein [CRP] and Interleukin‐6 [IL‐6]), and forearm vasodilator responses to brachial artery acetylcholine and sodium nitroprusside in 13 men and women (7 lean; 6 obese) on two separate study days: control (CON) and after 800 mg ibuprofen (IBU). CRP (1.7 ± 0.4 vs. 0.6 ± 0.3 mg/L; P < 0.05) and IL‐6 (4.1 ± 1.5 vs. 1.0 ± 0.1pg/mL; P < 0.05) were higher in the obese group during CON and tended to decrease with IBU (IL‐6: P < 0.05; CRP: P = 0.14). MSNA was not different between groups during CON (26 ± 4 bursts/100 heart beats (lean) versus 26 ± 4 bursts/100 heart beats (obese); P = 0.50) or IBU (25 ± 4 bursts/100 heart beats (lean) versus 30 ± 5 bursts/100 heart beats (obese); P = 0.25), and was not altered by IBU. Forearm vasodilator responses were unaffected by IBU in both groups. In summary, an acute dose of ibuprofen did not alter sympathetic nerve activity or forearm blood flow responses in healthy obese individuals, suggesting that the cyclooxygenase pathway is not a major contributor to these variables in this group.

Abstract T MP114: PPARγ in Endothelial Cells Plays an Essential Role in Protecting Against Vascular Aging: Role of Oxidative Stress and Rho Kinase

Although aging is the greatest risk factor for vascular disease and stroke, relatively little is known regarding mechanisms that regulate vascular aging. Endothelial dysfunction - a key element of carotid artery and cerebrovascular disease - progresses with age, greatly increasing the risk for ischemic stroke and cognitive impairment. The nuclear receptor peroxisome proliferator activated receptor-γ (PPARγ) is a ligand-activated transcription factor that may exert diverse effects depending on the cell type. Because little is known regarding the role of PPARγ in vascular aging, we used transgenic mice expressing a dominant negative mutation in human PPARγ (V290M) under control of the endothelial-specific vascular cadherin promoter (designated E-V290M) to examine the hypothesis that cell-specific interference with PPARγ would promote age-induced vascular dysfunction. Responses of carotid arteries from adult (11-12 mo) and old (24±1 mo) E-V290M mice and non-transgenic littermates were examined in vitro. Acetylcholine (an endothelium-dependent agonist) produced similar relaxation of arteries from adult control and E-V290M mice as well as old control mice. In contrast, responses to acetylcholine in arteries from old E-V290M mice were reduced by more than 50% in old E-V290M mice (P&lt;0.01). Endothelial function in old E-V290M mice was not altered by indomethacin but was restored to normal by tempol (a superoxide scavenger) or VAS-2870 (an inhibitor of NADPH oxidase). Reactive oxygen species can activate Rho kinase (a potential mediator of vascular disease) and inhibition of Rho kinase with Y-27632 restored endothelial function to normal in old E-V290M mice. Relaxation of arteries to nitroprusside, which acts directly on vascular muscle, was similar in all groups. These findings provide the first evidence that age-related vascular dysfunction is accelerated following cell-specific interference with endothelial PPARγ through mechanisms involving oxidative stress and Rho kinase. This novel role for endothelial PPARγ has implications for understanding vascular pathophysiology as well as therapeutic approaches for age-induced large and small vessel disease.

Interleukin-10 protects against aging-induced endothelial dysfunction

Carotid and cerebrovascular disease increase markedly with age contributing to stroke and cognitive impairment. Inflammation is a key element of vascular disease. In these studies, we tested the hypothesis that interleukin-10 (IL-10), a potent anti-inflammatory cytokine, protects against aging-induced endothelial dysfunction. Responses of carotid arteries from adult (5 ± 1 months) and old (22 ± 1 months) wild-type and IL-10-deficient mice were examined in vitro. Acetylcholine (an endothelium-dependent agonist) produced relaxation in arteries from adult wild-type that was not altered in old mice. In contrast, relaxation to acetylcholine in arteries from old IL-10-deficient mice was reduced by ∼50% (P < 0.05). Tempol, a scavenger of superoxide, did not affect responses in adult or old wild-type mice, but restored vasodilation to acetylcholine to normal in old IL-10-deficient mice. Responses of the carotid artery to nitroprusside (an endothelium-independent agonist) were not altered in any group. Vascular expression of IL-6 (a proinflammatory mediator of vascular disease) and components of NADPH oxidase (a major source of superoxide) was increased in old IL-10-deficient mice compared with wild-type (P < 0.05). These findings provide the first evidence that age-related and superoxide-mediated endothelial dysfunction occurs earlier with IL-10 deficiency. Our findings suggest a novel role for IL-10 to protect against age-related increases in expression of IL-6, oxidative stress, and endothelial dysfunction.

Curcumin ameliorates arterial dysfunction and oxidative stress with aging

We tested the hypothesis that curcumin supplementation would reverse arterial dysfunction and vascular oxidative stress with aging. Young (Y, 4–6months) and old (O, 26–28months) male C57BL6/N mice were given normal or curcumin supplemented (0.2%) chow for 4weeks (n=5–10/group/measure). Large elastic artery stiffness, assessed by aortic pulse wave velocity (aPWV), was greater in O (448±15 vs. 349±15cm/s) and associated with greater collagen I and advanced glycation end-products and less elastin (all P<0.05). In O, curcumin restored aPWV (386±15cm/s), collagen I and AGEs (AGEs) to levels not different vs. Y. Ex vivo carotid artery acetylcholine (ACh)-induced endothelial-dependent dilation (EDD, 79±3 vs. 94±2%), nitric oxide (NO) bioavailability and protein expression of endothelial NO synthase (eNOS) were lower in O (all P<0.05). In O, curcumin restored NO-mediated EDD (92±2%) to levels of Y. Acute ex vivo administration of the superoxide dismutase (SOD) mimetic TEMPOL normalized EDD in O control mice (93±3%), but had no effect in Y control or O curcumin treated animals. O had greater arterial nitrotyrosine abundance, superoxide production and NADPH oxidase p67 subunit expression, and lower manganese SOD (all P<0.05), all of which were reversed with curcumin. Curcumin had no effects on Y. Curcumin supplementation ameliorates age-associated large elastic artery stiffening, NO-mediated vascular endothelial dysfunction, oxidative stress and increases in collagen and AGEs in mice. Curcumin may be a novel therapy for treating arterial aging in humans.

Abstract 108: Cell-Specific Interference with Endothelial PPARγ Produces Oxidative Stress and Accelerates Vascular Aging

Despite the fact that aging is the greatest risk factor for vascular disease and stroke, relatively little is known regarding mechanisms that promote or protect against vascular aging in experimental models or people. Endothelial dysfunction is a key contributor to both the initiation and progression of vascular disease. Atherosclerosis in carotid arteries (carotid artery disease) greatly increases the risk for ischemic stroke and may contribute to dementia (including Alzheimer’s disease). Peroxisome proliferator activated receptor-γ (PPARγ) is a ligand-activated transcription factor that exerts diverse effects depending on the cell type. Recent work suggests that PPARγ can have beneficial effects in the vasculature including protection against oxidative stress. Relatively little is known regarding the potential role of PPARγ in aging. We used transgenic mice expressing a dominant negative mutation in human PPARγ (V290M) under control of the endothelial-specific vascular cadherin promoter (designated E-V290M) to examine the hypothesis that interference with endothelial PPARγ will promote age-induced vascular dysfunction. Responses of carotid arteries from adult (6±1 mo) and old (23±1 mo) E-V290M mice and non-transgenic littermates (controls) were examined in vitro. Acetylcholine (an endothelium-dependent agonist) produced similar relaxation of arteries from adult control and E-V290M mice as well as old control mice. In contrast, responses to acetylcholine in arteries from old E-V290M mice were markedly impaired. For example, relaxation of the carotid artery to 10 µmol/L acetylcholine was 84±3 and 40±6% in old control versus old E-V290M mice, respectively (P&lt;0.01). The impaired response to acetylcholine in old E-V290M mice could be restored to normal (81±4% relaxation) by tempol, a scavenger of superoxide. Relaxation of the carotid artery to nitroprusside (a nitric oxide donor that acts directly on vascular muscle) was similar in all groups. These findings provide the first evidence that age-related vascular dysfunction is accelerated following cell-specific interference with endothelial PPARγ function. The mechanism that accounts for this change appears to involve reactive oxygen species. Our findings suggest a major protective role for endothelial PPARγ in age-induced oxidative stress and vascular dysfunction.

Obesity Up-Regulates Intermediate Conductance Calcium-Activated Potassium Channels and Myoendothelial Gap Junctions to Maintain Endothelial Vasodilator Function

The mechanisms involved in altered endothelial function in obesity-related cardiovascular disease are poorly understood. This study investigates the effect of chronic obesity on endothelium-dependent vasodilation and the relative contribution of nitric oxide (NO), calcium-activated potassium channels (K(Ca)), and myoendothelial gap junctions (MEGJs) in the rat saphenous artery. Obesity was induced by feeding rats a cafeteria-style diet (∼30 kJ as fat) for 16 to 20 weeks, with this model reflecting human dietary obesity etiology. Age- and sex-matched controls received standard chow (∼12 kJ as fat). Endothelium-dependent vasodilation was characterized in saphenous arteries by using pressure myography with pharmacological intervention, Western blotting, immunohistochemistry, and ultrastructural techniques. In saphenous artery from control, acetylcholine (ACh)-mediated endothelium-dependent vasodilation was blocked by NO synthase and soluble guanylate cyclase inhibition, whereas in obese rats, the ACh response was less sensitive to such inhibition. Conversely, the intermediate conductance K(Ca) (IK(Ca)) blocker 1-[(2-chlorophenyl)diphenyl-methyl]-1H pyrazole attenuates ACh-mediated dilation in obese, but not control, vessels. In a similar manner, putative gap junction block with carbenoxolone increased the pEC(50) for ACh in arteries from obese, but not control, rats. IK1 protein and MEGJ expression was up-regulated in the arteries of obese rats, an observation absent in control. Addition of the small conductance K(Ca) blocker apamin had no effect on ACh-mediated dilation in either control or obese rat vessels, consistent with unaltered SK3 expression. Up-regulation of distinct IK(Ca)- and gap junction-mediated pathways at myoendothelial microdomain sites, key mechanisms for endothelial-derived hyperpolarization-type activity, maintains endothelium-dependent vasodilation in diet-induced obese rat saphenous artery. Plasticity of myoendothelial coupling mechanisms represents a significant potential target for therapeutic intervention.

Interleukin‐10 Protects Against Vascular Dysfunction with Aging

Aging produces vascular inflammation and dysfunction in blood vessels. We tested the hypothesis that interleukin‐10 (IL‐10), a potent anti‐inflammatory cytokine, protects against aging‐induced vascular dysfunction. Responses of carotid arteries from young (7‐8 mo) and old (19‐20 mo) wild‐type and IL‐10 deficient mice (IL‐10‐/‐) were examined in vitro. In arteries from wild‐type mice, acetylcholine (an endothelium‐dependent agonist) produced relaxation that was not altered at this age. In contrast, relaxation to acetylcholine in arteries from IL‐10 deficient mice was markedly impaired with age. For example, relaxation of the carotid artery to 10 μmol/L acetylcholine was 87±3 and 48±2% in old wild‐type versus old IL‐10 deficient mice, respectively (P&lt;0.01). Tempol (1 mM), a scavenger of superoxide, did not affect responses in young or old wild‐type or young IL‐10 deficient mice, but restored vasodilation to acetylcholine to normal in old IL‐10 deficient mice. Relaxation of the carotid artery to nitroprusside was not altered in any group. These findings provide the first evidence that age‐related and superoxide‐mediated endothelial dysfunction occurred much earlier and to a greater extent with IL‐10 deficiency. Our findings suggest a novel role for IL‐10 in oxidative stress and age‐related vascular dysfunction.

Greater fractalkine expression in mesenteric arteries of female spontaneously hypertensive rats compared with males

A mircoarray analysis was performed to identify novel inflammatory genes that are differentially expressed in the mesenteric arteries of male and female spontaneously hypertensive rats (SHRs). Fractalkine was found to be the inflammatory gene with the greatest differential expression in mesenteric arteries, with the expression being greater in female SHRs compared with males. Greater inflammatory mediators in female SHRs were verified by measuring urinary monocyte chemoattractant protein-1, transforming growth factor-beta, and tumor necrosis factor-alpha (TNF-alpha) excretion, all of which were greater in female SHRs compared with males. Real-time PCR, Western blot analysis, and ELISA verified greater soluble fractalkine in mesenteric arteries of female SHRs. Consistent with increased fractalkine expression, TNF-alpha-converting enzyme and TNF-alpha levels in mesenteric arteries were also greater in female SHRs. We next tested the hypothesis that mesenteric arteries from female SHRs will have greater fractalkine-induced dysfunction. Acetylcholine, sodium nitroprusside, phenylephrine, and KCl concentration-response curves were performed in third-order mesenteric arteries from male and female SHRs pretreated with either vehicle or fractalkine (1 microg/ml). Fractalkine decreased sensitivity to 1) acetylcholine in arteries from male SHRs, 2) phenylephrine in arteries from both sexes, and 3) KCl in arteries from female SHRs. In conclusion, urinary and vascular markers of inflammation are greater in female SHRs compared with males, although blood pressure and cardiovascular risk are less in females.

NO with no NOS in ischemic heart

See article by Martin et al. [5] (pages 46–55) in this issue. The seminal observation by Furchgott and Zawadzki [1] of a paradoxical vasoconstrictive effect of acetylcholine in arteries deprived of endothelium paved the way for the rapid discovery of nitric oxide (NO) and of the mechanisms of NO generation by a specific family of isoforms known as NO synthases (NOS). The role of NO in the endothelium was characterized as a predominant mechanism of vasodilation through the production of cyclic GMP (cGMP) in vascular smooth muscle cells. The rapid progress of this research in endothelial cells automatically led to the question whether the cardiac cell itself could be a source of NO. It was found that the three characterized isoforms of NOS are expressed in cardiac myocytes [2]. However, the role of NO in the myocardium, and the regulation of its production, seems much more complex than what was described before in the endothelium, and includes both cGMP-dependent and -independent mechanisms. Myocardial NO is involved in cellular processes as diverse as cardiac contractility, Ca2+ handling, mitochondrial respiration, substrate metabolism, generation of radical oxygen species, and ischemic preconditioning among others (see Fig. 1). In addition, the mechanisms of action of NO are diverse and sometimes contradictory. For example, depending on the experimental setting, blocking cardiac NOS can be either beneficial or deleterious for the ischemic heart [3,4]. Part of this complexity comes from the fact that NO production in the heart is performed by the three different isoforms (endothelial, inducible and neuronal) that differ in their properties, … *Corresponding author. Tel.: +1 973 972 3926; fax: +1 973 972 7489. Email address: deprech{at}umdnj.edu

Nitric oxide‐dependent relaxation is modulated by intermediate conductance Ca2+‐activated potassium channels in rat mesenteric resistance arteries

Endothelium-dependent relaxation of isolated rat mesenteric resistance arteries to acetylcholine (ACh) is mediated by the release of nitric oxide (NO) and by endothelium-dependent hyperpolarization. We have investigated the role of Ca2+-activated potassium channels (KCa) in NO-mediated, endothelium-dependent relaxation of mesenteric resistance arteries. Pre-incubation with either 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (Tram-34; 5 μM), an inhibitor of intermediate conductance KCa (IKCa), or L-NG-nitroarginine methylester (L-NAME; 100 μM), an inhibitor of NO synthase, caused significant inhibition of relaxation to ACh in arterial segments pre-contracted with phenylephrine (1–3 μM). The magnitude of inhibition caused by Tram-34 was not significantly different from that caused by L-NAME and the effects of the two inhibitors were not additive. Pre-incubation with apamin (50 nM), an inhibitor of small conductance KCa, did not significantly alter ACh-evoked relaxation. However, in the presence of L-NAME, apamin significantly inhibited relaxations to ACh and the combination of apamin with Tram-34 reduced the maximum relaxation to ACh by 80%. Addition of L-NAME with apamin and Tram-34 did not cause further significant inhibition. Tram-34 and apamin were without effect on relaxations evoked by the NO donor diethylamine NONOate. These data indicate that endothelial IKCa play a key role modulating NO-dependent relaxation to ACh in rat mesenteric resistance arteries. Supported by Heart & Stroke Foundation of Canada, URGC & USRP.

Protein kinase C activation increases endothelial nitric oxide release in mesenteric arteries from orchidectomized rats

The aim of the present study was to assess the effect of endogenous male sex hormones on endothelial nitric oxide synthase (eNOS) expression, release and function of the endothelial nitric oxide (NO), as well as to assess the regulatory action of protein kinase C (PKC) on acetylcholine (ACh)-induced endothelial NO release. For this purpose, superior mesenteric arteries from control and orchidectomized male Sprague-Dawley rats were used. eNOS expression and basal-and ACh-induced NO release were similar in arteries from both groups of rats. Orchidectomy decreased the vasodilator effect induced by ACh but did not alter that induced by sodium nitroprusside (SNP). The superoxide anion scavenger, superoxide dismutase (SOD), or the membrane-permeable mimetic of SOD, tempol, only enhanced ACh-induced relaxation in arteries from orchidectomized rats. ACh-induced TXA(2) formation was higher in arteries from orchidectomized than from control rats. Neither the PKC activator, phorbol 12,13-dibutyrate (PDBu), nor the non-selective PKC inhibitor, calphostin C, modified basal- or ACh-induced NO release in arteries from control rats. In arteries from orchidectomized rats, basal- and ACh-induced endothelial NO release were increased by PDBu but decreased by calphostin C. Both Gö6976, a PKC inhibitor that is partially selective for conventional PKC isoforms, as well as PKCzeta pseudosubstrate inhibitor (PKCzeta-PI) decreased both basal- and ACh-induced NO release in arteries from orchidectomized rats. Neither PDBu nor calphostin C modified the vasodilator response induced by ACh in arteries from control rats. In segments from orchidectomized rats, PDBu enhanced the ACh-induced response, but this response was not modified by calphostin C, Gö6976 or PKCzeta-PI. The vasodilator response induced by SNP was not altered by the PKC activators or inhibitors in any artery from either group. These results show that endogenous male sex hormone deprivation does not affect the eNOS expression or the endothelial NO release induced by ACh, but does decrease the vasodilator action of ACh, by increasing NO metabolism and TXA(2) formation. In addition, PKC seems to modulate eNOS activity only in mesenteric arteries from orchidectomized rats, in which conventional and PKCzeta isoforms are involved in the positive regulation of eNOS.

Developmental Regulation of GTP-CH1 in the Porcine Lung and Its Relationship to Pulmonary Vascular Relaxation

Nitric oxide (NO) plays an important role in lowering pulmonary vascular resistance after birth. However, in persistent pulmonary hypertension of the newborn (PPHN) NO-mediated dilation is dysfunctional. GTP-cyclohydrolase 1 (GTP-CH1) is the rate-limiting enzyme for the biosynthesis of 6R-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) an essential cofactor for nitric oxide synthase (NOS) activity. Suboptimal levels of BH4 may result in NOS uncoupling and the subsequent generation of harmful superoxide anions. We therefore investigated the functional effects of supplementing BH4 and/or a superoxide dismutase mimetic (MnTMPyP) in porcine intrapulmonary arteries from normal animals and from a porcine model of PPHN. We investigated whether any functional effects of BH4 could be explained by changes in GTP-CH1 expression. Supplementation of BH4 significantly improved endothelium-dependent relaxations in arteries from 3- and 14-d-old healthy animals, whereas no effect was seen in vessels from younger animals and adults. GTP-CH1 protein expression was lowest at 3 and 14 d, suggesting a rate limitation of BH4 at this time. BH4 supplementation alone did not improve the relaxant response to acetylcholine in arteries obtained in a porcine model of PPHN. Furthermore, GTP-CH1 protein expression was normal for age. However, co-treatment with both BH4 and MnTMPyP restored endothelial function. GTP-CH1 is developmentally regulated in the pulmonary vasculature of neonates and this results in a functionally significant limitation of BH4. Although GTP-CH-1/BH4 levels alone do not explain the profound endothelial dysfunction seen in PPHN, increasing NO bioavailability by supplementing BH4 and quenching superoxide may prove to be therapeutically beneficial.

Interleukin‐10 Protects Against Angiotensin II‐Induced Oxidative Stress and Endothelial Dysfunction

Angiotensin II (Ang II) produces inflammation, oxidative stress and endothelial dysfunction in blood vessels. The goal of the present study was to test the hypothesis that interleukin 10 (IL-10), a potent anti-inflammatory cytokine, protects against Ang II-induced vascular dysfunction. Responses of carotid arteries from wild-type and IL-10 deficient mice (IL-10 −/−) were examined in vitro following overnight incubation with either vehicle or a low concentration of Ang II (1 nmol/L). In arteries from wild-type mice, acetylcholine (an endothelium-dependent agonist) produced relaxation that was not affected by Ang II. In contrast, relaxation to acetylcholine in arteries from IL-10 −/− mice was markedly and selectively reduced by treatment with Ang II (e.g., 100 μmol/L acetylcholine produced 82±8 and 37±5% relaxation in vehicle- and Ang II-treated arteries, respectively). This effect of Ang II in IL-10 −/ − mice were reversed by PEG-SOD (300 U/ml). Superoxide levels increased in aorta following treatment with Ang II in IL-10 −/− mice but not in wild-type. Following systemic administration of Ang II (1000 ng/kg/min or vehicle for 10 days via osmotic minipump), Ang II produced modest impairment of endothelial function in arteries from wild-type mice but marked impairment in IL-10 −/− mice (e.g., 100 μmol/L acetylcholine produced 102±3 and 55±1% relaxation in vehicle- and Ang II-treated mice, respectively). Baseline levels and increases in arterial pressure in response to Ang II were similar in wild-type and IL-10 −/− mice. These findings provide the first direct evidence that IL-10 limits Ang II-mediated oxidative stress and impairment of endothelial function. This effect occurs both in vitro and in vivo suggesting that protective effects of IL-10 may occur within the vessel wall.

Nongenomic responses to 17β‐estradiol in male rat mesenteric arteries abolish intrinsic gender differences in vascular responses

The aim of the present study was to investigate the gender differences in the acute effects of 17beta-estradiol on the rat superior mesenteric artery. Isometric tension was measured in rings of mesenteric arteries from both male and female Sprague-Dawley rats. Relaxation to acetylcholine was not significantly different between arteries (with endothelium) from male and female rats in the absence or presence of 17beta-estradiol. After blockade of endothelium-dependent hyperpolarizations with apamin (0.3 microM) plus charybdotoxin (0.1 microM), acute exposure to 17beta-estradiol (1 nM) for 30 min resulted in enhancement of relaxation to acetylcholine in arteries from male but not female rats. After acute exposure to 17beta-estradiol, mesenteric arteries from male rats were more sensitive to sodium nitroprusside than arteries from female rats. Contractions of mesenteric arteries to phenylephrine and 9,11-dideoxy-11alpha,9alpha-epoxymethanoprostaglandin F(2alpha) (U46619) were greater in arteries from male rats than female rats. This difference was not detected after acute exposure to 17beta-estradiol. In preparations without endothelium, the enhancement of relaxation and reduction in contraction in arteries from male rats were preserved. These results suggest that there exists a gender difference in the response to the acute nongenomic modulatory effect of 17beta-estradiol in rat mesenteric arteries. Arteries from male rats seem to be more sensitive to the modulatory effects of 17beta-estradiol than arteries from female rats. The effect appears to be mainly at the level of the vascular smooth muscles.

Critical Role for CuZn-Superoxide Dismutase in Preventing Angiotensin II-Induced Endothelial Dysfunction

The goal of the present study was to test the hypothesis that the CuZn isoform of superoxide dismutase (CuZnSOD) protects against angiotensin II (Ang II)-induced endothelial dysfunction. Vascular responses of carotid arteries from control, CuZnSOD-deficient (CuZnSOD(+/-)), and CuZnSOD transgenic mice were examined in vitro after overnight incubation with either vehicle or Ang II (1 or 10 nmol/L). In control mice, acetylcholine produced concentration-dependent relaxation that was not affected by 1 nmol/L Ang II. In contrast, relaxation to acetylcholine in arteries from CuZnSOD+/- mice was markedly and selectively attenuated after incubation with 1 nmol/L Ang II (eg, 100 micromol/L acetylcholine produced 93+/-6% and 44+/-15% relaxation in vehicle- and Ang II-treated arteries, respectively). A higher concentration of Ang II (10 nmol/L) selectively impaired relaxation to acetylcholine in arteries from control mice (eg, 100 micromol/L acetylcholine produced 96+/-4% and 45+/-7% relaxation in vehicle- and Ang II-treated vessels, respectively). In contrast, 10 nmol/L Ang II had no effect on responses to acetylcholine in carotid arteries from CuZnSOD transgenic mice (or in control mice treated with the superoxide scavenger Tiron [1 mmol/L]). Superoxide levels in control mice were higher in aorta treated with Ang II than with vehicle and were markedly reduced in CuZnSOD transgenic mice. These findings provide the first direct evidence that CuZnSOD limits Ang II-mediated impairment of endothelial function and that loss of 1 copy of the CuZnSOD gene is sufficient to enhance Ang II-induced vascular dysfunction.

Effect of Acute Sublethal Endotoxaemia onIn VitroDigital Vascular Reactivity in Horses

Endotoxaemia is a syndrome linked to the development of equine laminitis; however, the relationship between them is uncertain. The aim of this experiment was to evaluate the effect of an experimental acute sublethal endotoxaemia model on in vitro equine palmar digital vascular reactivity. Rings of arteries and veins of each forelimb were obtained from 11 clinically healthy horses submitted to two surgical procedures, 3 weeks apart. Before the second surgery, 0.25 microg/kg of lipopolysaccharide from Escherichia coli O55:B5 in saline, was administered i.v. in 30 min. After 3 h, the vessels were harvested and submitted to in vitro vascular reactivity experiments and histopathology. The response to depolarizing Krebs solution (DKS, 40 mm), phenylephrine (PHE), acetylcholine (ACh) and sodium nitroprusside (SNP) were evaluated. All horses showed colic pain and watery diarrhoea, tachycardia, tachypnea, hyperthermia and leucopenia. Concentration-response curve (CRC) to PHE was shifted to the left in arteries rings from endotoxemic horses without any effect on vein rings. The CRC to ACh was shifted to the right with a reduction in the maximal response. The response to SNP and DKS was similar between groups. There was no evidence of histopathological effects. The increased response to PHE in digital arteries together with a reduction of the endothelium-dependent response to ACh in arteries and veins, confirm the existing reports where endotoxaemia was found to modify the digital vascular reactivity during the acute phase. As the digital endothelial function is impaired, there may be an increased potential to develop a digital prothrombotic state with a reduced vasodilatory capacity.