Endothelial Nitric Oxide Synthase Protein Expression Research Articles

Increase in Bioavailability of Nitric Oxide After Renal Denervation Improves Kidney Function in Sheep With Hypertensive Kidney Disease

[Figure: see text].

Calpain inhibitor I attenuates atherosclerosis and inflammation in atherosclerotic rats through eNOS/NO/NF-κB pathway.

We previously reported that calpain, the Ca2+-sensitive cysteine protease, gets involved in atherogenesis. This study aimed to investigate the effects of calpain inhibitor I (CAI, 5 mg/kg per day) with or without NG-nitro-l-arginine-methyl ester (l-NAME) (100 mg/kg per day), the inhibitor of nitric oxide synthase (NOS), on atherosclerosis and inflammation in a rat model induced by high-cholesterol diet (HCD). The results demonstrated HCD increased protein expression of calpain-1 but not calpain-2 in aortic tissue. In addition, CAI reduced the thickness of atherosclerotic intima compared with HCD group, which was weakened by the l-NAME combination. CAI with or without l-NAME decreased the activity of calpain in the aorta. Also, CAI decreased the expressions of vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1), and monocyte chemoattractant protein-1 (MCP-1) in the aorta at the levels of both mRNA and protein. Furthermore, CAI increased the activity and the protein expression of endothelial NOS (eNOS) accompanied by increased content of NO and downregulated the protein expression of nuclear factor κB (NF-κB) of the nucleus in the aorta. However, the abovementioned effects were at least partly cancelled by l-NAME except for the protein expression of eNOS. The results suggested that CAI attenuated atherosclerosis and inflammation through eNOS/NO/NF-κB pathway.

Nebivolol alleviates aortic remodeling through eNOS upregulation and inhibition of oxidative stress in l-NAME-induced hypertensive rats

ABSTRACTPurpose: To investigate the effect and mechanism of nebivolol on aortic remodeling in N-nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Methods: Male Sprague–Dawley rats were treated with equal volumes of drinking water or l-NAME (60 mg/kg/day), alone or in combination with nebivolol (8 mg/kg/day) or atenolol (80 mg/kg/day) by gavage for 8 weeks. Systolic blood pressure (SBP), aortic morphometry, plasma nitric oxide (NO) levels, nitric oxide synthase (NOS) activity, and relaxation of aorta to acetylcholine were determined. Protein expression of endothelial NOS (eNOS), Akt, and NADPH oxidase (Nox) was evaluated. Results: l-NAME-treated rats showed an elevated SBP associated with aortic remodeling. l-NAME-treated rats showed reduced plasma NO levels and NOS activity and increased reactive oxygen species (ROS). Protein expression of eNOS, eNOS phosphorylated at Ser1177 (p-eNOS), Akt, and Akt phosphorylated at Ser473 (p-Akt) decreased, whereas that of Nox2, Nox4, and p22phox increased in the aortas from l-NAME-treated rats. Nebivolol treatment reduced SBP and ameliorated aortic remodeling. The effects of nebivolol were accompanied by increasing NO levels, NOS activity, and expression of eNOS, p-eNOS, Akt, and p-Akt, as well as reduction of ROS generation and Nox2, Nox4, and p22phox expression. These effects of nebivolol were not reproduced by atenolol. Conclusion: Our data indicate a protective role of nebivolol on the high blood pressure and vascular remodeling induced by l-NAME. The beneficial vascular effect of nebivolol is mediated by the upregulation of eNOS and inhibition of oxidative stress.

Augmented Contractility to Noradrenaline in Femoral Arteries from the Otsuka Long-Evans Tokushima Fatty Rat, a Model of Type 2 Diabetes

Although vasculopathies may occur systemically, there are few reports regarding femoral arteries of type 2 diabetes. Here, we investigated whether contractile response to noradrenaline in femoral arteries would change in type 2 diabetic male Otsuka Long-Evans Tokushima Fatty (OLETF) rat at the chronic stage of disease (1 year old) versus age-matched control Long-Evans Tokushima Otsuka (LETO) rat. OLETF rat exhibited hyperglycemia, hypertension, hyperlipidemia, and hypoinsulinemia compared to age-matched LETO rat. Noradrenaline-induced contraction was increased in femoral arteries in OLETF rats compared with LETO rats whereas serotonin- or phenylephrine-induced contractions were similar between these two animals. Acetylcholine- and sodium nitroprusside-induced relaxations were similar between the two groups. Very small relaxations in femoral arteries induced by clonidine and isoprenaline were obtained in LETO but not OLETF group. Noradrenaline-induced contraction was enhanced by treatment with NG-nitro-L-arginine (L-NNA), a nitric oxide synthase (NOS) inhibitor, and the between-group difference of contraction was eliminated by such treatment. Indomethacin, a non-selective cyclooxygenase (COX) inhibitor, reduced noradrenaline-induced contraction in both groups, whereas the contraction was greater in OLETF group versus LETO. Femoral arterial protein expression of endothelial NOS, COX-1, and superoxide dismutases were similar between the two groups, whereas reduction of COX-2 expression was seen in OLETF group compared with LETO. Increased contractile responsiveness to noradrenaline is seen in OLETF rat femoral artery and this may be due to reduction of suppressive effect of NO.

Impairment of Vascular Function in a Mouse Model of Neurofibromatosis Type 1

Neurofibromatosis 1 (NF1) is an autosomal dominant disorder, characterized by multiple café‐au‐lait spots, iris harmartomas, multiple nerve sheath tumors. Patients also present with heart and cerebrovascular disease, ischemia, or aneurysm. Though vasculopathy is well documented in NF1 patients, the pathophysiology is poorly understood. The objective of this study was to investigate the function and structural integrity of the thoracic aorta in a well‐established mouse model of NF1 (6‐month old). We observed a significant increase in contraction in response to depolarization of vascular smooth muscle and endothelial cells in Nf1+/‐ mice, but diminished contraction in response to phenylephrine. Interestingly, endothelium‐dependent relaxation was significantly higher in Nf1+/‐ mice, which was completely inhibited by the nitric oxide synthase (NOS) inhibitor, suggesting the relaxation results from nitric oxide (NO) production. Consistent with increased NO production, protein expression of endothelial NOS (eNOS), phospho‐eNOS, and the phospho‐Akt were increased in Nf1+/‐ aorta. Histological examination revealed that alteration in aortic function is also associated with elastin fiber disorganization within the aortic wall. This study, for the first time, underscores the pathological events underlying vascular complications in NF1, shedding light on potential new therapeutic targets to address vascular dysfunctions in NF1 patients. The presented study was funded by the Children's Tumor Foundation and the Canadian Institutes of Health Research.

Abstract 351: Activation Of The Central Renin Angiotensin System (RAS) Causes Selective Cerebrovascular Dysfunction

Approximately 30% of hypertensive patients have low plasma renin. The deoxycorticosterone (DOCA)/salt model of hypertension exhibits low peripheral RAS activity, but activation of the central RAS. The impact of central RAS activation on the cerebrovasculature is not defined. We tested the hypothesis that activation of the central RAS would impair endothelial function in brain. We produced mild hypertension in male C57BL/6 mice using subcutaneous DOCA and 0.9% salt in their drinking water for 3 weeks (no nephrectomy was performed) followed by studies of isolated arteries in vitro . Blood pressure was elevated modestly after DOCA/salt treatment (79±2 vs 95±3 mmHg). Endothelial function was severely impaired in cerebral arteries from mice treated with DOCA/salt (e.g. 10 μM acetylcholine: 33±3% vs 4±4%) but was unaffected in similar-sized mesenteric arteries. Because microvascular dysfunction is a key feature of small vessel disease in brain that increases the risk of stroke and cognitive impairment, we examined responses of small cerebral arterioles (baseline diameter ~30 μm) in vivo using a cranial window. Endothelium-dependent dilation to acetylcholine in cerebral arterioles was reduced by ~75% following DOCA/salt treatment (e.g. 10 μM acetylcholine: 26±5% vs 6±3%). In contrast, endothelium-independent vasodilation to nitroprusside (NO donor) or nifedipine (Ca 2+ channel blocker) was similar between groups. Microvascular endothelial dysfunction was not affected by a scavenger of superoxide but was restored to normal by local inhibition of Rho kinase (ROCK) with Y27632. Activation of AT1 receptors by angiotensin II can increase ROCK signaling. Local inhibition of AT1 receptors with losartan or inhibition of mineralocorticoid receptors (MR) with eplerenone improved endothelium-dependent dilation in DOCA/salt treated mice to normal levels. RhoA and ROCK2 mRNA expression and total endothelial NO synthase protein expression was similar between groups. These findings suggest that activation of the central RAS profoundly but selectively impairs endothelial function in brain. Our findings identify AT1 receptors, MR and ROCK as key contributors to endothelial microvascular dysfunction in this model of mild DOCA/salt-induced hypertension.

Protective effects of Guanxin Shutong capsule drug-containing serum on tumor necrosis factor-α-induced endothelial dysfunction through nicotinamide adenine dinucleotide phosphate oxidase and the nitric oxide pathway.

The Chinese medicinal formula Guanxin Shutong capsule (GXSTC) has been used for almost 10 years as a clinical treatment for chest pain, depression, palpitation and cardiovascular diseases. The aim of this study was to investigate the effects of GXSTC drug-containing serum on tumor necrosis factor-α (TNF-α)-stimulated endothelial cells. Cell viability was measured by MTT assay, and nitric oxide (NO) levels and NO synthase (NOS) activity were measured as standards of endothelial dysfunction. Malondialdehyde (MDA) levels and superoxide dismutase (SOD) activity were evaluated using commercial kits. In addition, the protein expression of endothelial NOS (eNOS), AKT and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits was examined to evaluate the effect of GXSTC drug-containing serum on ECV304 cells. GXSTC significantly reversed the decrease in NO production induced by TNF-α (5 ng/ml) in ECV304 cells. The expression of NADPH oxidase subunits was increased by TNF-α treatment, but markedly inhibited by treatment with GXSTC in TNF-α-stimulated cells. In summary, GXSTC increased the production of NO in ECV304 cells and exerted a protective effect on ECV304 cells stimulated with TNF-α by upregulating the mRNA and protein expression of eNOS. This was accompanied by increased SOD activity and reduced MDA levels. These results suggested that GXSTC protects the endothelium via the NO pathway and exhibits antioxidant effects.

Regional variations and age-related changes in arginine metabolism in the rat brain stem and spinal cord

Accumulating evidence suggests that the metabolism of l-arginine, a metabolically versatile amino acid, is critically involved in the aging process. The present study compared the activity and protein expression of nitric oxide synthase (NOS) and arginase, and the levels of l-arginine and its eight down-stream metabolites in the brain stem (pons and medulla) and the cervical spinal cord in 3- (young) and 22- (aged) month-old male Sprague–Dawley rats. Total NOS activity was significantly reduced with age in the spinal cord (but not brain stem), and there were no age-related changes in arginase activity in both regions. Western blot revealed decreased protein expression of endothelial NOS, but not neuronal NOS, with age in both regions. Furthermore, there were significantly decreased l-arginine, glutamate, GABA and spermine levels and increased putrescine and spermidine levels with age in both regions. Although the absolute concentrations of l-arginine and six metabolites were significantly different between the brain stem and spinal cord in both age groups, there were similar clusters between l-arginine and its three main metabolites (l-citrulline, l-ornithine and agmatine) in both regions, which changed as a function of age. These findings, for the first time, demonstrate the regional variations and age-related changes in arginine metabolism in the rat brain stem and spinal cord. Future research is required to understand the functional significance of these changes and the underlying mechanisms.

Salvianolic acid A protects against vascular endothelial dysfunction in high-fat diet fed and streptozotocin-induced diabetic rats

Salvianolic acid A (SalA) is one of the main active ingredients of Salvia miltiorrhizae. The objective of this study was to evaluate the effect of SalA on the diabetic vascular endothelial dysfunction (VED). The rats were given a high-fat and high-sucrose diet for 1 month followed by intraperitoneal injection of streptozotocin (30 mg/kg). The diabetic rats were treated with SalA (1 mg/kg, 90% purity) orally for 10 weeks after modeling, and were given a high-fat diet. Contractile and relaxant responses of aorta rings as well as the serum indications were measured. Our results indicated that SalA treatment decreased the level of serum Von Willebrand factor and ameliorated acetylcholine-induced relaxation and KCl-induced contraction in aorta rings of the diabetic rats. SalA treatment also reduced the serum malondialdehyde, the content of aortic advanced glycation end products (AGEs), and the nitric oxide synthase (NOS) activity as well as the expression of endothelial NOS protein in the rat aorta. Exposure of EA.hy926 cells to AGEs decreased the cell viability and changed the cell morphology, whereas SalA had protective effect on AGEs-induced cellular vitality. Our data suggested that SalA could protect against vascular VED in diabetes, which might attribute to its suppressive effect on oxidative stress and AGEs-induced endothelial dysfunction.

Pre-aggregated Aβ25–35 alters arginine metabolism in the rat hippocampus and prefrontal cortex

Amyloid beta (Aβ) has been proposed to play a central and causative role in the development of Alzheimer's disease. Aβ(25-35), the neurotoxic domain of the full-length Aβ, causes learning and memory impairments in rodents. The present study investigated the effects of a single bilateral i.c.v. infusion of pre-aggregated Aβ(25-35) (30 nmol/rat) on animals' performance in the open field, and on arginine metabolic enzymes and metabolites in the CA1, CA2/3, and dentate gyrus (DG) sub-regions of the hippocampus and prefrontal cortex (PFC) at the time point of 6-8 days after Aβ infusion. Aβ(25-35) rats displayed reduced exploratory activity in the open field relative to the Aβ(35-25) (reverse peptide; 30 nmol) rats. Aβ(25-35) resulted in significantly decreased nitric oxide synthase (NOS) activity and endothelial NOS protein expression, but increased arginase activity, arginase II protein expression, and ornithine and putrescine levels, in hippocampal CA2/3. There were increased glutamate and putrescine levels in the DG, but decreased agmatine levels in the DG and PFC, in the Aβ(25-35) group relative to the Aβ(35-25) one. Cluster analyses were performed to determine if the nine related neurochemical variables (arginine, citrulline, ornithine, agmatine, putrescine, spermidine, spemine, glutamate, and GABA) formed distinct groups, and whether it changed as a function of Aβ(25-35). There were substantially different clusters between the two groups in the hippocampus and PFC. These results demonstrate that Aβ(25-35) alters arginine metabolism, which further supports the prominent role of arginine and its metabolites in Alzheimer's disease (AD) pathogenesis.

Mononuclear Phagocyte System Depletion Blocks Interstitial Tonicity-Responsive Enhancer Binding Protein/Vascular Endothelial Growth Factor C Expression and Induces Salt-Sensitive Hypertension in Rats

We showed recently that mononuclear phagocyte system (MPS) cells provide a buffering mechanism for salt-sensitive hypertension by driving interstitial lymphangiogenesis, modulating interstitial Na(+) clearance, and increasing endothelial NO synthase protein expression in response to very high dietary salt via a tonicity-responsive enhancer binding protein/vascular endothelial growth factor C regulatory mechanism. We now tested whether isotonic saline and deoxycorticosterone acetate (DOCA)-salt treatment leads to a similar regulatory response in Sprague-Dawley rats. Male rats were fed a low-salt diet and received tap water (low-salt diet LSD), 1.0% saline (high-salt diet HSD), or DOCA+1.0% saline (DOCA-HSD). To test the regulatory role of interstitial MPS cells, we further depleted MPS cells with clodronate liposomes. HSD and DOCA-HSD led to Na(+) accumulation in the skin, MPS-driven tonicity-responsive enhancer binding protein/vascular endothelial growth factor C-mediated hyperplasia of interstitial lymph capillaries, and increased endothelial NO synthase protein expression in skin interstitium. Clodronate liposome MPS cell depletion blocked MPS infiltration in the skin interstitium, resulting in unchanged tonicity-responsive enhance binding protein/vascular endothelial growth factor C levels and absent hyperplasia of the lymph capillary network. Moreover, no increased skin endothelial NO synthase protein expression occurred in either clodronate liposome-treated HSD or DOCA-salt rats. Thus, absence of the MPS-cell regulatory response converted a salt-resistant blood-pressure state to a salt-sensitive state in HSD rats. Furthermore, salt-sensitive hypertension in DOCA-salt rats was aggravated. We conclude that MPS cells act as onsite controllers of interstitial volume and blood pressure homeostasis, providing a local regulatory salt-sensitive tonicity-responsive enhancer binding protein/vascular endothelial growth factor C-mediated mechanism in the skin to maintain normal blood pressure in states of interstitial Na(+) and Cl(-) accumulation. Failure of this physiological extrarenal regulatory mechanism leads to a salt-sensitive blood pressure response.

Comparison of the effects of indapamide and captopril on the development of spontaneous hypertension

The effects of indapamide, a thiazide-like diuretic, and captopril, an angiotensin-converting enzyme inhibitor, on spontaneous hypertension and the development of left ventricular hypertrophy (LVH), nitric oxide generation and oxidant status were investigated. Six-week-old male spontaneously hypertensive rats (SHR) were treated with indapamide (1 mg/kg per day) or captopril (10 mg/kg per day) or a combination of indapamide plus captopril. After the 6-week treatment, nitric oxide synthase (NOS) activity, the expression of NOS isoform proteins, conjugated dienes concentration and relaxation responses of the femoral artery were analyzed. Indapamide and captopril partly prevented a blood pressure increase in young SHR. Captopril in contrast to indapamide reduced LVH. The effect of the combined indapamide and captopril treatment on the prevention of hypertension was additive. Combined indapamide and captopril treatment increased NOS activity and endothelial NOS protein expression in the aorta and decreased conjugated dienes concentration in the kidney compared with the indapamide monotherapy group. Indapamide and indapamide and captopril treatment increased acetylcholine-induced relaxation of the femoral artery. Whereas captopril reduced LVH, indapamide enhanced NOS activity and decreased oxidative damage in the case of the combined treatment. It is concluded that the complex protective effects of the combined indapamide plus captopril treatment on hypertension may be exerted via its effects on blood pressure, hypertrophy and vasorelaxation.

Oral supplementation of propionyl-l-carnitine reduces body weight and hyperinsulinaemia in obese Zucker rats

Propionyl-L-carnitine (PLC) is an SCFA esterified to carnitine that plays an important role in fatty acid oxidation and energy expenditure, in addition to having a protective effect on the endothelium. In order to evaluate the effect of PLC on an animal model of obesity, insulin resistance and, consequently, endothelial dysfunction, lean and obese Zucker rats (OZR) received either vehicle- or PLC-supplemented drinking water (200 mg/kg per d) for 20 weeks. Body weight, food intake, systolic blood pressure and heart rate were controlled weekly and an oral glucose tolerance test was performed. Fasting glucose, TAG, cholesterol, HDL, NEFA, adiponectin and insulin were analysed in serum. Visceral adipose tissue and liver were weighed and liver TAG liver composition was evaluated. Endothelial and vascular functions were assessed in the aorta and small mesenteric arteries by response to acetylcholine, sodium nitroprusside and phenylephrine (Phe); NO participation was evaluated after incubation with the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME) and endothelial NOS protein expression by Western blotting. PLC decreased body-weight gain, food intake, adiposity, insulin serum concentration and TAG liver content and improved insulin resistance. Aortae from OZR receiving either vehicle or PLC exhibited a lower contractile response to Phe. PLC-treated OZR showed an enhanced release of endothelial NO upon the adrenergic stimulation. The protection of vascular function found after treatment with PLC in an animal model of insulin resistance supports the necessity of clinical trials showing the effect of L-carnitine supplements on metabolic disorders.

Erectile Dysfunction in a Murine Model of Sleep Apnea

Erectile dysfunction (ED) is frequent in obstructive sleep apnea syndrome (OSAS). Chronic intermittent hypoxia (CIH), one of the hallmarks of OSAS, could mediate ED. To determine whether intermittent hypoxia during sleep affects erectile dysfunction in mice. Three groups of C57BL/6 mice were exposed to CIH for 5 or 24 weeks. Sexual function was evaluated by in vivo telemetry of corpus spongiosum pressure. Spontaneous erections, sexual activity during mating, and noncontact tests were assessed after 5 weeks of CIH and after treatment with tadalafil. Plasma testosterone was measured after 8 and 24 weeks of CIH, and the expression of nitric oxide synthase (NOS) isoforms was examined in penile tissue. Noncontact, spontaneous, and contact sexual activity in the mice was suppressed after CIH. Spontaneous erection counts decreased after the first week of CIH by 55% (P < 0.001) and remained unchanged thereafter. Recovery of erectile activity during normoxia for 6 weeks was incomplete. Compared with control mice, latencies for mounts and intromissions increased by 60- and 40-fold, respectively (P < 0.001), and the sexual activity index decreased sixfold. Tadalafil treatment significantly attenuated these effects. Immunoblot analyses of NOS proteins in the erectile tissue showed decreased expression of endothelial NOS after CIH (P < 0.01), with no changes in plasma testosterone levels after 8 and 24 weeks of CIH. CIH during sleep is associated with decreased libido in mice. The decreased expression of endothelial NOS protein in erectile tissue and the favorable response to tadalafil suggest that altered nitric oxide mechanisms underlie CIH-mediated ED. No changes in testosterone emerge after intermittent hypoxia.

15-Lipoxygenase-1 Prevents Vascular Endothelial Growth Factor A– and Placental Growth Factor–Induced Angiogenic Effects in Rabbit Skeletal Muscles via Reduction in Growth Factor mRNA Levels, NO Bioactivity, and Downregulation of VEGF Receptor 2 Expression

Human 15-lipoxygenase-1 (15-LO-1) is an oxidizing enzyme capable of producing reactive lipid hydroperoxides. 15-LO-1 and its products have been suggested to be involved in many pathological conditions, such as inflammation, atherogenesis, and carcinogenesis. We used adenovirus-mediated gene transfers to study the effects of 15-LO-1 on vascular endothelial growth factor (VEGF)-A165- and placental growth factor (PlGF)-induced angiogenesis in rabbit skeletal muscles. 15-LO-1 significantly decreased all angiogenic effects induced by these growth factors, including capillary perfusion, vascular permeability, vasodilatation, and an increase in capillary number. The effects are attributable to the reduction in the amount of VEGF-A165 and PlGF transcripts by 15-LO-1, resulting in reduced protein expression. The most likely mediator of the VEGF family-induced capillary vasodilatation is nitric oxide (NO), which is produced by NO synthases. Endothelial NO synthase protein expression and NO synthase activity were significantly induced by VEGF-A165, and these inductions were reduced by 15-LO-1. VEGF-A165 induces its angiogenic effects primarily via vascular endothelial growth factor receptor (VEGFR)2, and also PlGF mediates angiogenic signaling via VEGFR2, even though it binds to VEGFR1. VEGFR2 expression is induced by peroxisome proliferator-activating receptor . We showed by quantitative RT-PCR and immunohistochemistry that expression of endogenous rabbit peroxisome proliferator-activating receptor and VEGFR2 were significantly increased in the growth factor-transduced muscles, but these inductions were efficiently prevented by 15-LO-1. In conclusion, the results suggest that expression of 15-LO-1 has an efficient antiangiogenic effect in vivo via reduction in growth factor mRNA levels, NO bioactivity, and VEGFR2 expression.

ABNORMALITIES OF NITRIC OXIDE‐MEDIATED VASORELAXATION IN A RAT MODEL OF METABOLIC SYNDROME: INVOLVEMENT OF PEROXYNITRITE FORMATION

SUMMARY 1 We reported previously that nitric oxide (NO)-mediated relaxation is impaired, despite an increase in NO production from the endothelium, in the thoracic aorta of SHR/NDmcr-cp (SHR-cp) rats, an animal model of the metabolic syndrome. In the present study, we investigated whether telmisartan, an angiotensin II receptor antagonist, can improve abnormal vasorelaxation responses. 2 Treatment with telmisartan (10 mg/kg per day, p.o.) for 8 weeks reduced elevated blood pressure in SHR-cp rats, but did not affect the increased bodyweight, serum triglyceride and glucose levels. Telmisartan restored impaired acetylcholine-induced relaxation. Both increased endothelial NO synthase protein expression in aortas and 3-nitrotyrosine content, evidence of peroxynitrite formation, were significantly suppressed by telmisartan. Serum levels of thiobarbituric acid-reactive substances, an index of oxidized lipids, were significantly decreased. 3 These results suggest that telmisartan can reduce the decline in NO bioavailability caused by peroxynitrite formation in the aortas of SHR-cp rats. We propose that antagonism of angiotensin II action may be effective in preventing vascular endothelial dysfunction in metabolic syndrome.

The effect of N-acetylcysteine and melatonin in adult spontaneously hypertensive rats with established hypertension

The attenuated nitric oxide (NO) formation and/or elevated production of reactive oxygen species are often found in experimental and human hypertension. We aimed to determine possible effects of N-acetylcysteine (1.5 g/kg/day) and N-acetyl-5-methoxytryptamine (melatonin, 10 mg/kg/day) in adult spontaneously hypertensive rats (SHR) with established hypertension. After a six-week-treatment, blood pressure was measured and NO synthase (NOS) activity, concentration of conjugated dienes, protein expression of endothelial NOS, inducible NOS and nuclear factor-κB (NF-κB) in the left ventricle were determined. Both treatments improved the NO pathway by means of enhanced NOS activity and reduced reactive oxygen species level as indicated by decreased conjugated diene concentrations and lowered NF-κB expression. N-acetylcysteine (but not melatonin) also increased the endothelial NOS protein expression. However, only melatonin was able to reduce blood pressure significantly. Subsequent in vitro study revealed that both N-acetylcysteine and melatonin lowered the tone of phenylephrine-precontracted femoral artery via NO-dependent relaxation. Nevertheless, melatonin-induced relaxation also involved NO-independent component which was preserved even after the blockade of soluble guanylate cyclase by oxadiazolo[4,3-a]quinoxalin-1-one. In conclusion, both N-acetylcysteine and melatonin were able to improve the NO/reactive oxygen species balance in adult SHR, but blood pressure was significantly lowered by melatonin only. This implies that a partial restoration of NO/reactive oxygen species balance achieved by the antioxidants such as N-acetylcysteine has no therapeutic effect in adult rats with established hypertension. The observed antihypertensive effect of melatonin is thus mediated by additional mechanisms independent of NO pathway.

Changes in vascular reactivity following administration of isoproterenol for 1 week: a role for endothelial modulation

1. The aim of this study was to assess the effects of treatment with isoproterenol (ISO, 0.3 mg kg-1 day-1, s.c.) for 7 days on the vascular reactivity of rat-isolated aortic rings. Additionally, potential mechanisms underlying the changes that involved the endothelial modulation of contractility were investigated. 2. Treatment with ISO induced cardiac hypertrophy without changes in haemodynamic parameters. Aortic rings from ISO-treated rats showed an increase in the contraction response to phenylephrine (PHE) and serotonin, but did not change relaxations produced by acetylcholine or isoproterenol. Removal of the endothelium increased the responses to PHE in both groups. However, this procedure was less effective in ISO-treated as compared with control rats. Endothelial cell removal abolished the increase in the response to PHE in ISO-treated rats. The presence of Nomega-nitro-L-arginine methyl ester shifted the concentration-response curve to PHE to the left in both groups of rats. However, this effect was more pronounced in the ISO group. In addition, aminoguanidine (50 microM) potentiated the actions of PHE only in the ISO group. ISO treatment increased nitric oxide synthase (NOS) activity and neuronal NOS and endothelial NOS protein expression in the aorta. 3. Neither losartan (10 microM) nor indomethacin (10 microM) abolished the effects of ISO on the actions of PHE. Superoxide dismutase (SOD, 150 U ml-1) and L-arginine (5 mM), but neither catalase (300 U ml-1) nor apocynin (100 microM), blocked the effect of ISO treatment. In addition, we observed an increase in superoxide anion levels as measured by ethidium bromide fluorescence and of copper and zinc superoxide dismutase protein expression in ISO-treated rats. 4. In conclusion, our data suggest that ISO treatment alters the endothelial cell-mediated modulation of the contraction to PHE in rat aorta. The increased maximal response of PHE seems to be due to an increase in superoxide anion generation, which inactivates some of the basal NO produced and counteracts NO-mediated negative modulation even in the presence of high NO production and antioxidant defence.

Effect of spironolactone and captopril on nitric oxide and S-nitrosothiol formation in kidney of L-NAME-treated rats

Although angiotensin-converting enzyme (ACE) inhibitors are well-established drugs in the treatment of hypertension, they are not supposed to be sufficient in the inhibition of aldosterone formation. The present study analyzes the effect of aldosterone receptor antagonist, spironolactone and ACE inhibitor, captopril on nitric oxide (NO) and S-nitrosothiol formation in the kidney of N(G)-nitro-L-arginine methyl ester (L-NAME)-treated rats. Male Wistar rats were divided into six groups: (1) controls, (2) L-NAME (40 mg/kg/day), (3) spironolactone (200 mg/kg/day), (4) captopril (100 mg/kg/day), (5) L-NAME+spironolactone, and (6) L-NAME+captopril. After 4 weeks, NO synthase (NOS) activity, protein expression of endothelial NOS, inducible NOS and concentration of thiol and S-nitrosothiol groups were determined in the kidney. Besides the increase in systolic blood pressure (by 32%) and the decrease in NOS activity (by 37%), L-NAME treatment lowered the concentration of thiols (by 32%) and S-nitrosothiols (by 36%) in the renal tissue. Simultaneous treatment with spironolactone preserved NOS activity and S-nitrosothiols on the control level, whereas captopril did not affect these parameters modified by L-NAME treatment. Moreover, spironolactone increased expression of endothelial NOS protein without affecting inducible NOS protein expression. In conclusion, both captopril and spironolactone prevented L-NAME-induced hypertension and the decline of the antioxidant potential of the kidney tissue. However, only spironolactone improved NOS activity which led to the S-nitrosothiols formation. Both NO itself and S-nitrosothiols may contribute to the preventive effect of spironolactone against development of L-NAME-induced hypertension.

Antioxidant N-acetylcysteine restores systemic nitric oxide availability and corrects depressions in arterial blood pressure and heart rate in diabetic rats

Increased oxidative stress and reduced nitric oxide (NO) bioactivity are key features of diabetes mellitus that eventually result in cardiovascular abnormalities. We assessed whether N-acetylcysteine (NAC), an antioxidant and glutathione precursor, could prevent the hyperglycaemia induced increase in oxidative stress, restore NO availability and prevent depression of arterial blood pressure and heart rate in vivo in experimental diabetes. Control (C) and streptozotocin-induced diabetic (D) rats were treated or not treated with NAC in drinking water for 8 weeks, initiated 1 week after induction of diabetes. At termination, plasma levels of free 15-F2t-isoprostane, a specific marker of oxygen free radical induced lipid peroxidation, was increased while the plasma total antioxidant concentration was decreased in untreated diabetic rats as compared to control rats (P < 0.05). This was accompanied by a significant reduction of plasma levels of nitrate and nitrite, stable metabolites of NO, (P < 0.05, D vs. C) and a reduced endothelial NO synthase protein expression in the heart and in aortic and mesenteric artery tissues. Systolic, diastolic and mean arterial blood pressures (SBP, DBP and MAP) and heart rate (HR) were reduced in diabetic rats (P < 0.05 vs. C) and NAC normalised the changes that occurred in the diabetic rats. The protective effects may be attributable to restoration of NO bioavailability in the circulation.