Plasma nitrite reserve and endothelial function in the human forearm circulation

Attenuation of endothelium-derived nitric oxide (NO) synthesis is a hallmark of endothelial dysfunction. Early detection of this disorder may have therapeutic and prognostic implications. Plasma nitrite mirrors acute and chronic changes in endothelial NO-synthase activity. We hypothesized that local plasma nitrite concentration increases during reactive hyperemia of the forearm, reflecting endothelial function. In healthy subjects (n = 11) plasma nitrite and nitrate were determined at baseline and during reactive hyperemia of the forearm using reductive gas-phase chemiluminescence and flow-injection analysis, respectively. Endothelium-dependent dilation of the brachial artery was measured as flow-mediated dilation (FMD) using high-resolution ultrasound. Results were compared to patients with endothelial dysfunction as defined by reduced FMD (n = 11). Reactive hyperemia of the forearm increased local plasma nitrite concentration from 68 +/- 5 to 126 +/- 13 nmol/L (p < 0.01), whereas in endothelial dysfunction nitrite remained unaffected (116 +/- 12 to 104 +/- 10 nmol/L; n.s.), corresponding to nitrite reserves of 94 +/- 21 and -8 +/- 4%. This was accompanied by a significantly greater increase in brachial artery diameter (FMD: 8.5 +/- 0.4% vs 2.9 +/- 0.5%, for healthy subjects and endothelial dysfunction, respectively; p < 0.001). This observation suggests that nitrite changes reflect endothelial function. Assessment of local plasma nitrite during reactive hyperemia may open new avenues in the diagnosis of vascular function.     

Association of renal damage and oxidative stress in a rat model of metabolic syndrome. Influence of gender

This study investigated the association between nephropathy and oxidative stress, by measurement of systolic blood pressure, lipid peroxidation, activities of catalase, manganese- and copper-zinc-superoxide dismutase and endothelial nitric oxide synthase expression and concentrations of nitrates/nitrites in kidneys from rats with Metabolic Syndrome. Weaning female or male rats had 30% sucrose to drink for 24 weeks (Metabolic Syndrome). Modulation by sex hormones was investigated by gonadectomy and hormone replacement. In Metabolic Syndrome, Castrated Metabolic Syndrome + Testosterone males and Ovariectomized Metabolic Syndrome females had increased blood pressure, proteinuria and lipid peroxidation. Nitrates/nitrites and activities of catalase, manganese and copper-zinc-superoxide dismutase decreased vs intact Control, Castrated Metabolic Syndrome males, intact Metabolic Syndrome and Ovariectomized Metabolic Syndrome + Estradiol females. The results suggest that sex hormones modulate the activity of superoxide-dismutase, catalase and endothelial nitric oxide-synthase. Ovariectomy decreased the protection against oxidative stress in females; the opposite occurred in castrated males.     

曲美他嗪对慢性心力衰竭患者血管内皮功能的影响

目的:研究曲美他嗪对慢性心力衰竭患者血管内皮功能的影响。方法:选择2013年1月~2015年12月在我院进行诊治的慢性心力衰竭患者158例为研究对象,采用奇偶数法随机分为两组,每组79例。对照组采用利尿、平衡电解质、扩血管、营养心肌等常规抗心衰治疗,观察组联合采用曲美他嗪治疗。分别于治疗前和治疗后1个月检测两组患者的血流介导血管舒张功能、内皮素-1水平、一氧化氮水平、心率、血压、白细胞计数、血肌酐和谷丙转氨酶水平。结果:治疗后,观察组的血流介导血管舒张功能明显高于治疗前(P0.05),且肱动脉内径和血流介导血管舒张功能均明显高于对照组(P0.05);两组的血浆内皮素-1均较治疗前降低(P0.05),一氧化氮升高(P0.05),且观察组的血清内皮素-1水平明显低于对照组,血清一氧化氮水平明显高于对照组(P0.05);两组的心率、血压、肾功能、肝功能和血常规结果相比差异均无统计学意义(P0.05),且两组均无明显不良反应发生。结论:曲美他嗪可显著改善慢性心力衰竭患者的血管内皮功能,可能与其降低血浆内皮素-1水平、升高一氧化氮水平有关。     

Plasma nitrite concentrations reflect the degree of endothelial dysfunction in humans

A reduced nitric oxide availability is a hallmark of endothelial dysfunction occurring early in atherosclerosis. Recently, we have shown that plasma nitrite mirrors acute changes in endothelial nitric oxide synthase activity in various mammals, including humans. Here, we examined the hypothesis that plasma nitrite levels are reduced in humans with endothelial dysfunction and the decrease is correlated with increasing numbers of cardiovascular risk factors (RF). Plasma nitrite concentrations were quantified by flow-injection analysis. The coefficient of variation for repeated measurements of plasma nitrite was <8%, and heart rate and blood pressure at the time of blood sampling had no significant effect on nitrite values measured (n=10). Baseline levels of plasma nitrite followed a normal distribution in each group studied and decreased progressively with increasing numbers of cardiovascular risk factors (n=351, p<0.001): 351+/-13 (0 RF), 261+/-10 (1 RF), 253+/-11 (2 RF), 222+/-18 (3 RF), and 171+/-29 nmol/L (4 RF). Intima media thickness (IMT) and flow-mediated dilation (FMD) were determined via ultrasound. Plasma nitrite and FMD levels were lower, whereas IMT was greater in individuals with endothelial dysfunction (n=12) compared to healthy volunteers (n=12). Nitrite correlated significantly with FMD (r=0.56, p<0.001) and inversely with IMT (r= -0.49, p<0.01). Plasma nitrite levels are reliably measurable in humans, indicate endothelial dysfunction, and correlate with cardiovascular risk factors. Future studies are necessary to identify the prognostic relevance of plasma nitrite determination in patients suffering from cardiovascular disease.     

The effect and action mechanism of resveratrol on the vascular endothelial cell by high glucose treatment

To investigate the effect and action mechanism of resveratrol on the vascular endothelial cell by high glucose treatment. Primarily cultured human umbilical vein endothelial cells (HUVECs) were pretreated by resveratrol (0.2 μmol/L) and holding for 6 h, and then cultured in Dulbecco Modified Eagle Medium (DMEM) within 0.45 mmol/L of palmimte acid and 32.8 mmol/L of glucose, which is holding for 12 h. The cells were collected to analyze the expression of E-selected element. Supernatant of cultured cells, induced by 100 nmol/L insulin for 30 min, was used to analyze the nitric oxide content. Compared with normal control cells, the secretion of nitric oxide is stimulated by insulin decrease, however, the expression of E-selected element increased in HUVEC. Resveratrol treatment increased the secretion of nitric oxide stimulated by insulin and decreased the expression of E-selected element and partly counteracts the impairment of high glucose and palmitate acid on the function of endothelial cells. Resveratrol can improve and protect the function of high glucose and fatty acid cultured endothelial cell, and therefore may be a promising medicine in the prevention or therapy of diabetic macrovascular diseases.     

Relationship between changes in brachial artery flow-mediated dilation and basal release of nitric oxide in subjects with Type 2 diabetes

Assessment of flow-mediated dilation (FMD) after forearm ischemia is widely used as a noninvasive bioassay of stimulated nitric oxide (NO)-mediated conduit artery vasodilator function in vivo. Whether this stimulated endothelial NO function reflects basal endothelial NO function is unknown. To test this hypothesis, retrospective analysis of randomized crossover studies was undertaken in 17 subjects with Type 2 diabetes; 9 subjects undertook an exercise training or control period, whereas the remaining 8 subjects were administered an angiotensin II receptor blocker or placebo. FMD was assessed by using wall tracking of high-resolution brachial artery ultrasound images in response to reactive hyperemia. Resistance vessel basal endothelium-dependent NO function was assessed by using intrabrachial administration of NG-monomethyl-L-arginine (L-NMMA) and plethysmographic assessment of forearm blood flow (FBF). FMD was higher after intervention compared with control/placebo (6.15+/-0.53 vs. 3.81+/-0.72%, P<0.001). There were no significant changes in the FBF responses to L-NMMA. Regression analysis between FMD and L-NMMA responses at entry to the study revealed an insignificant correlation (r=-0.10, P=0.7), and improvements in FMD with the interventions were not associated with changes in the L-NMMA responses (r=-0.04, P=0.9). We conclude that conduit artery-stimulated endothelial NO function (FMD) does not reflect basal resistance vessel endothelial NO function in subjects with Type 2 diabetes.     

Association of urinary N-domain Angiotensin I-converting enzyme with plasma inflammatory markers and endothelial function

The aim of this study was to investigate the association between urinary 90 kDa N-domain Angiotensin I-converting enzyme (ACE) form with C-reactive protein (CRP) and homocysteine plasma levels (Hcy), urinary nitric oxide (NOu), and endothelial function (EF) in normotensive subjects. Forty healthy subjects were evaluated through brachial Doppler US to test the response to reactive hyperemia and a panel of blood tests to determine CRP and Hcy levels, NOu, and urinary ACE. They were divided into groups according to the presence (ACE90+) or absence (ACE90-) of the 90 kDa ACE, the presence (FH+) or absence (FH-) of family history of hypertension, and the presence or absence of these two variables FH+/ACE90+ and FH-/ACE90-. We found an impaired endothelial dilatation in subjects who presented the 90 kDa N-domain ACE as follows: 11.4% +/- 5.3% in ACE90+ compared with 17.6% +/- 7.1% in ACE90- group and 12.4% +/- 5.6% in FH+/ACE90+ compared with 17.7% +/- 6.2% in FH-/ACE90- group, P < 0.05. Hcy and CRP levels were statistically significantly lower in FH+/ACE90+ than in FH-/ACE90- group, as follows: 10.0 +/- 2.3 microM compared with 12.7 +/- 1.5 microM, and 1.3 +/- 1.8 mg/L compared with 3.6 +/- 2.0 mg/L, respectively. A correlation between flow-mediated dilatation (FMD) and CRP, Hcy, and NOu levels was not found. Our study suggests a reduction in the basal NO production confirmed by NOu analysis in subjects with the 90 kDa N-domain ACE isoform alone or associated with a family history of hypertension. Our data suggest that the presence of the 90 kDa N-domain ACE itself may have a negative impact on flow-mediated dilatation stimulated by reactive hyperemia.     

17beta-estradiol prevents oxidative stress and decreases blood pressure in ovariectomized rats

In this study, we tested whether estrogen deficiency is associated with oxidative stress and decreased nitric oxide (NO) production, which could be responsible for an increased blood pressure in ovariectomized rats. Hemodynamic studies were performed on conscious, chronically instrumented rats. Chronic estrogen replacement on ovariectomized rats lowered blood pressure approximately 13 mmHg, from 119 +/- 3 mmHg in ovariectomized rats to 106 +/- 3 mmHg in ovariectomized-treated rats; it was also accompanied by an increase in cardiac index and vascular conductance, achieving hemodynamic values similar to those shown by sham-operated rats. N(G)-nitro-L-arginine methyl ester administration lowered significantly less the vascular conductance (0.14 +/- 0.01 vs. 0.22 +/- 0.03 and 0.26 +/- 0.01 ml. min(-1). mmHg(-1)/100 g; P < 0.05) in ovariectomized rats than in the sham-operated and estrogen-treated ovariectomized rats, respectively. Estrogen replacement prevented the lower plasma levels of nitrites/nitrates observed in ovariectomized rats. The lower plasma total antioxidant status and reduced thiol groups and the increase in plasma lipoperoxides presented in ovariectomized animals were reestablished with the estrogen treatment. These results show that estrogen administration decreases blood pressure and increases vascular conductance in ovariectomized rats. This effect may be related to an increase in NO synthesis and/or preventing oxidative stress, then improving endothelial function.     

Salt sensitivity of nitric oxide generation and blood pressure in mice with targeted knockout of the insulin receptor from the renal tubule

To elucidate the role of the insulin receptor (IR) on kidney nitric oxide generation and blood pressure (BP) control, we generated mice with targeted deletion of renal tubule IR using loxP recombination driven by a Ksp-cadherin promoter. Male knockout (KO) and wild-type (WT) littermates (～4 mo old) were transitioned through three 1-wk treatments: 1) low-NaCl diet (0.085%); 2) high-NaCl diet (HS; 5%); and 3) HS diet plus 3 mM tempol, a superoxide dismutase mimetic, in the drinking water. Mice were then switched to medium-NaCl (0.5%) diet for 5 days and kidneys harvested under pentobarbital anesthesia. Twenty-four-hour urinary nitrates plus nitrites were significantly higher in the WT mice under HS (2,067 ± 280 vs. 1,550 ± 230 nmol/day in WT and KO, respectively, P < 0.05). Tempol attenuated genotype differences in urinary nitrates plus nitrites. A rise in BP with HS was observed only in KO mice and not affected by tempol (mean arterial pressure, dark period, HS, 106 ± 5 vs. 119 ± 4 mmHg, for WT and KO, respectively, P < 0.05). Renal outer medullary protein levels of nitric oxide synthase (NOS) isoforms by Western blot (NOS1-3 and phosphorylated-S1177-NOS3) revealed significantly lower band density for NOS1 (130-kDa isoform) in the KO mice. A second study, when mice were euthanized under HS conditions, confirmed significantly lower NOS1 (130 kDa) in the KO, with an even more substantial (>50%) reduction of the 160-kDa NOS1 isoform. These studies suggest that the loss of renal IR signaling impairs renal nitric oxide production. This may be important in BP control, especially in insulin-resistant states, such as the metabolic syndrome.     

Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase.

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.     

Parameters of reactive oxygen species and nitric oxide metabolism in people with high arterial blood pressure

An exchange of active forms of oxygen and nitric oxide in normal conditions and under the development of oxidative stress in humans with high of arterial blood pressure was studied. The activity of NO-synthase was estimated in the human thrombocytes. The nitric oxide formations were determined by the quantity level of its final metabolites--nitrites and nitrates. The peroxynitrite formations were determined by the quantity level of 3-nitrotyrosine. An analysis of the investigation results has shown the increase of processes of oxidative stress, violation of nitric oxide formation in humans with high arterial blood pressure. Application of ascorbic acid allows to reduce the level of free radicals and to increase the formation of nitric oxide, but does not result in statistically reliable changes of the parameters describing formation of peroxynitrite and products of peroxide oxidation of lipids in humans with high arterial blood pressure. Application of ascorbic acid does not result in changes of researched parameters in the control group.     

Asymmetric dimethylarginine, oxidative stress, and vascular nitric oxide synthase in essential hypertension

We reported impaired endothelium-derived relaxation factor/nitric oxide (EDRF/NO) responses and constitutive nitric oxide synthase (cNOS) activity in subcutaneous vessels dissected from patients with essential hypertension (n = 9) compared with normal controls (n = 10). We now test the hypothesis that the patients in this study have increased circulating levels of the cNOS inhibitor, asymmetric dimethylarginine (ADMA), or the lipid peroxidation product of linoleic acid, 13-hydroxyoctadecadienoic acid (HODE), which is a marker of reactive oxygen species. Patients had significantly (P < 0.001) elevated (means +/- SD) plasma levels of ADMA (P(ADMA), 766 +/- 217 vs. 393 +/- 57 nmol/l) and symmetric dimethylarginine (P(SDMA): 644 +/- 140 vs. 399 +/- 70 nmol/l) but similar levels of L-arginine accompanied by significantly (P < 0.015) increased rates of renal ADMA excretion (21 +/- 9 vs. 14 +/- 5 nmol/mumol creatinine) and decreased rates of renal ADMA clearance (18 +/- 3 vs. 28 +/- 5 ml/min). They had significantly increased plasma levels of HODE (P(HODE): 309 +/- 30 vs. 226 +/- 24 nmol/l) and renal HODE excretion (433 +/- 93 vs. 299 +/- 67 nmol/micromol creatinine). For the combined group of normal and hypertensive subjects, the individual values for plasma levels of ADMA and HODE were both significantly (P < 0.001) and inversely correlated with microvascular EDRF/NO and positively correlated with mean blood pressure. In conclusion, elevated levels of ADMA and oxidative stress in a group of hypertensive patients could contribute to the associated microvascular endothelial dysfunction and elevated blood pressure.     

硫酸镁联合拉贝洛尔对重度子痫前期患者凝血功能、氧化应激及血管内皮功能的影响

摘要 目的：探讨硫酸镁联合拉贝洛尔对重度子痫前期患者凝血功能、氧化应激及血管内皮功能的影响。方法：选择2020年1月~2022年9月期间来安徽医科大学附属巢湖医院就诊的重度子痫前期患者94例。采用随机数字表法将患者分为对照组（硫酸镁治疗,47例）和研究组（硫酸镁联合拉贝洛尔治疗,47例）。两组均于治疗前后观察收缩压（SBP）、舒张压（DBP）、24 h尿蛋白量（24hUP）、凝血功能、氧化应激及血管内皮功能的变化情况,同时观察两组用药安全性。结果：治疗后,研究组SBP、DBP、24hUP低于对照组（P<0.05）。治疗后,研究组内皮素-1（ET-1）低于对照组,一氧化氮（NO）高于对照组（P<0.05）。治疗后,研究组丙二醛（MDA）低于对照组,超氧化物歧化酶（SOD）高于对照组（P<0.05）。治疗后,研究组纤维蛋白原（Fg）、D-二聚体（D-D）低于对照组,凝血酶原时间（PT）、凝血活酶时间（APTT）高于对照组（P<0.05）。两组不良反应发生率对比无差异（P>0.05）。结论：拉贝洛尔联合硫酸镁治疗重度子痫前期患者,可减少24hUP,有效降低血压,改善凝血功能和血管内皮功能,减轻氧化应激损伤。     

Effects of BM-573 on Endothelial Dependent Relaxation and Increased Blood Pressure at Early Stages of Atherosclerosis

Endothelial dysfunction is considered to be an early event in atherosclerosis and plays a pivotal role in the development, progression and clinical complications of atherosclerosis. Previous studies have shown the beneficial effects of combined inhibition of thromboxane synthase and antagonism of thromboxane receptors by BM-573 on atherosclerosis; however our knowledge about the beneficial effects of BM-573 on endothelial function and increased blood pressure related to early stage of atherosclerosis is limited. In the present study, we investigated the effects of short-term (3μM, 1 hour) and chronic (10mg/L, 8 weeks) treatments with BM-573 on vasodilatory function, nitric oxide (NO) bioavailability, oxidative stress and systolic blood pressure in 15 weeks old apolipoprotein E-deficient (ApoE-KO) mice. ApoE-KO mice showed a reduced endothelium-derived relaxation. In addition, NO bioavailability was reduced and oxidative stress and blood pressure were increased in ApoE-KO mice versus wild-type mice. BM-573 treatments were able to improve the relaxation profile in ApoE-KO mice. Short-term effects of BM-573 were mainly mediated by an increased phosphorylation of both eNOS and Akt, whereas BM-573 in vivo treatment also reduced oxidative stress and restored NO bioavailability. In addition, chronic administration of BM-573 reduced systolic blood pressure in ApoE-KO mice. In conclusion, pharmacological modulation of TxA2 biosynthesis and biological activities by dual TP antagonism/TxAS inhibition with BM-573, already known to prevent plaque formation, has the potential to correct vasodilatory dysfunction at the early stages of atherosclerosis.     

内皮型一氧化氮合酶基因多态性与高血压合并脑 梗塞的关系

目的:探讨内皮型一氧化氮合酶(eNOS)基因894G/T多态性与原发性高血压(EH)合并脑梗塞(CI)的关系。方法:应用聚合酶链反应限制性片段长度多态性方法检测湖北地区汉族74例健康者(NT组)、103例原发性高血压无合并症者(EH组)及70例原发性高血压合并脑梗塞者(EH-CI组)的eNOS基因型;生化技术测定其血脂、一氧化氮代谢物(NOM)水平。结果:EH组及EH-CI组患者的T等位基因频率分别为0.224和0.321,均显著高于NT组(P<0.05);且两者之间的T等位基因频率差异显著性(P<0.05);EH-CI组中,GT+TT基因型者的舒张压显著高于GG基因型者(P<0.05),而NOM显著低于GG基因型者。结论:eNOS基因894位G/T多态性可能与汉族高血压病患者伴脑梗塞有关,该位点多态性可能使T等位基因携带者NOM减少,进而参与EH-CI发病。     

Prostacyclin and thromboxane A2 production in nitric oxide-deficient hypertension in vivo. Effects of high calcium diet and angiotensin receptor blockade

The effects of chronic nitric oxide deficiency on prostacyclin and thromboxane A(2) production in vivo are unknown. Therefore, we treated rats with N(G)-nitro-L-arginine methyl ester (L-NAME), and used losartan and high calcium diet as antihypertensive treatments. Forty eight Wistar rats were divided into six groups: control; losartan (20mgkg(-1)day(-1)); high calcium diet (dietary calcium elevated from 1.1% to 3%); L-NAME (20mgkg(-1)day(-1)); losartan+L-NAME and high calcium diet+L-NAME. Prostacyclin and thromboxane A(2) production were measured after eight weeks as urinary 2,3-dinor-6-keto-PGF(1alpha) and 11-dehydro-TXB(2), respectively. Both the high calcium diet and losartan reduced blood pressure in L-NAME hypertension. Chronic nitric oxide deficiency did not modulate prostacyclin production but it nearly doubled thromboxane A(2) production in vivo. This effect was not influenced by lowering of blood pressure by blockade of angiotensin II type 1 receptors. Independent of the level of blood pressure and blockade of nitric oxide synthesis the high calcium diet decreased prostacyclin production by one third and increased thromboxane A(2) production almost two-fold in vivo.     

Mechanism Underlying the Protective Effect of Selenium on NO-Induced Oxidative Damage in Bovine Mammary Epithelial Cells

This experiment was conducted to investigate the effects and mechanism of selenium (Se) on antioxidant and immune function of bovine mammary epithelial cells (BMEC) damaged by nitric oxide (NO). The third-generation BMEC was randomly divided into eight treatments with six replicates. The BMEC in the control group was cultured in the medium without Se and diethylenetriamine/NO (DETA/NO) for 30 h. For the DETA/NO group and Se protection group BMEC were exposed to different concentrations of Se (0, 10, 20, 50, 100, 150, and 200 nmol/L) for 24 h, followed by treatment with DETA/NO (1000 μmol/L) for 6 h. Compared with the control group, DETA/NO decreased proliferation rate and activity of thioredoxin reductase (TrxR; P < 0.05). Additionally, DETA/NO decreased the gene expression of both nuclear factor-E2-related factor 2 (Nrf2) and TrxR, as well as the protein expression level of TrxR. However, the activity, and expression levels of inducible nitric oxide synthase (iNOS), as well as the concentration and gene expression level of interleukin-1β (IL-1β) and the concentration of NO significantly increased (P < 0.05). The gene expression levels of indexes related to the mitogen-activated protein kinase (MAPK) signaling pathway showed similar changes. Treatment of BMEC with Se significantly reversed DETA/NO-induced changes in a linear or quadratic dose-dependent manner (P < 0.05), with greatest benefit at 50 nmol/L. These data suggests that Se improves the antioxidant function of BMEC, and protects cells from DETA/NO-induced oxidative damage, primarily by enhancing the activity of TrxR and decreasing the concentration of NO through modulation of Nrf2 and MAPK signaling pathways.

     

The construction of endothelial cellular biosensing system for the control of blood pressure drugs

In order to assess blood pressure control drugs, the endothelial cellular biosensing system for assessing blood pressure control drugs was constructed. This system consists of human umbilical vein endothelial cells (HUVEC) on a polyion-coated gold electrode, a platinum counter electrode and an Ag/AgCl reference electrode. Nitric oxide (NO) as an indicator of blood vessel relaxation was detected with a polyion-coated electrode in the system. The NO detection limit of this electrode was 8.4 nM by differential pulse voltammetry (DPV). The drugs of blood pressure control (acetylcholine chloride (AcChCl), NOC 7 and NG-monomethyl-L-arginine (L-NMMA)) were assessed with this endothelial cellular biosensing system. One milli molar of AcChCl make NO released from HUVEC stimulated by activating endothelial nitric oxide synthase (eNOS) in HUVEC. In the case of 5 mM of L-NMMA, NO releasing was inhibited by inhibiting eNOS activation by 1 mM of AcChCl. NOC 7 immediately released NO regardless of eNOS activation in endothelial cells.     

Impaired flow-mediated dilation with age is not explained by L-arginine bioavailability or endothelial asymmetric dimethylarginine protein expression.

Aging is associated with a decline in vascular endothelial function, manifesting in part as impaired flow-mediated arterial dilation (FMD), but the underlying mechanisms are uncertain. Impaired FMD may be mediated in part by a decrease in synthesis of nitric oxide by endothelial nitric oxide synthase, and in clinical populations this has been attributed to competitive inhibition of l-arginine binding sites by asymmetric dimethylarginine (ADMA). If this mechanism is involved in the age-associated decline in FMD, increasing l-arginine concentration may swing the competitive balance in favor of l-arginine binding, restoring nitric oxide synthesis, and enhancing FMD in older humans. To test this hypothesis, we measured FMD (brachial ultrasound) in 10 younger (21 +/- 1 yr) and 12 older healthy men and women (60 +/- 2 yr) following infusion of vehicle or vehicle + l-arginine. Baseline FMD in the older subjects was only approximately 60% of that in the younger subjects (P = 0.002). l-Arginine did not significantly increase FMD in either group despite 23-fold (older) and 19-fold (younger) increases in plasma l-arginine concentrations (P < 0.0001 vs. control). Protein expression (immunofluorescence) in vascular endothelial cells showed that ADMA and the enzyme isoform that controls its degradation, dimethylarginine dimethylaminohydrolase II, were not different in the younger and older subjects. Endothelium-independent vasodilation (sublingual nitroglycerine) was not different between age groups or conditions. We conclude that acutely increasing plasma concentrations of l-arginine do not significantly improve brachial artery FMD in healthy older subjects and thus does not restore the age-associated loss of FMD. Together with the finding that endothelial cell ADMA protein expression was not increased in older adults, these findings suggest that competitive inhibition of l-arginine binding sites on endothelial nitric oxide synthase by ADMA is not an important mechanism contributing to impaired conduit artery endothelium-dependent dilation with aging in healthy humans.     

Flow-dependent regulation of endothelial nitric oxide synthase: role of protein kinases

Vascular endothelial cells are directly and continuously exposed to fluid shear stress generated by blood flow. Shear stress regulates endothelial structure and function by controlling expression of mechanosensitive genes and production of vasoactive factors such as nitric oxide (NO). Though it is well known that shear stress stimulates NO production from endothelial nitric oxide synthase (eNOS), the underlying molecular mechanisms remain unclear and controversial. Shear-induced production of NO involves Ca²⁺/calmodulin-independent mechanisms, including phosphorylation of eNOS at several sites and its interaction with other proteins, including caveolin and heat shock protein-90. There have been conflicting results as to which protein kinases—protein kinase A, protein kinase B (Akt), other Ser/Thr protein kinases, or tyrosine kinases—are responsible for shear-dependent eNOS regulation. The functional significance of each phosphorylation site is still unclear. We have attempted to summarize the current status of understanding in shear-dependent eNOS regulation. shear stress; nitric oxide; endothelial cells; protein kinases