Dysregulation Of Nitric Oxide Synthase Research Articles

Alterations in the phosphodiesterase type 5 pathway and oxidative stress correlate with erectile function in spontaneously hypertensive rats.

To explore how alterations in the phosphodiesterase type 5 (PDE5) signalling pathway and oxidative stress correlate with changes in the expression of relaxation and contraction molecules and erectile dysfunction (ED) in the corpus cavernosum smooth muscle (CCSM) of spontaneously hypertensive rats (SHR). In this study, SHR and Wistar‐Kyoto (WKY) rats were used. Erectile function was determined by apomorphine test and electrical stimulation (ES) of cavernous nerve. Masson's trichrome staining and confocal microscopy were performed. Nitric oxide synthase (NOS), PDE5, phosphorylated‐PDE5 and α1‐adrenergic receptor (α1AR) were determined by RT‐PCR and Western blotting while oxidative stress in CC was determined by colorimetric analysis. SHR exhibited obvious ED. CC of SHR showed less SM but more collagen fibres. The expression of NOS isoforms in SHR was significantly decreased while all α1AR isoforms were increased. In addition, PDE5 and phosphorylated‐PDE5 were down‐regulated and its activity attenuated in the hypertensive rats. Meanwhile, the SHR group suffered oxidative stress, which may be modulated by endoplasmic reticulum stress and NADPH oxidase up‐regulation. Dysregulation of NOS and α1AR, histological changes and oxidative stress in CC may be associated with the pathophysiology of hypertension‐induced ED. In addition, PDE5 down‐regulation may lead to the decreased efficacy of PDE5 inhibitors in some hypertensive ED patients and treatment of oxidative stress could be used as a new therapeutic target for this type of ED.

EFFECTS OF EXERCISE ON PLASMA NITRITE/NITRATE LEVELS, AND NITRIC OXIDE SYNTHASE ACTIVITY IN ELDERLY AFRICAN AMERICANS WITH MILD COGNITIVE IMPAIRMENT

Studies have shown a positive correlation between exercise and vasodilatation, and this may be beneficial to brain perfusion and possibly memory. Proposed mediators for this observed effects of exercise include increases in nitric oxide (NO) via up-regulation of the rate controlling enzymes nitric oxide synthase (NOS) isoform (eNOS. Because endothelial dysfunction is an early marker of atherosclerosis, and hypoxia, impaired capacity of the vascular endothelium to release NO and NOS, and consequent vascular dysregulations have been implicated in cognitive impairment. This study seeks to establish whether mild cognitively impaired older adults exhibit NO and NOS dysregulation while on a supervised exercise program. African American older adult's ≥55 year, determined to have mild cognitive impairment, were enrolled and randomized into a six-month program of either treadmill exercise or stretch exercise. Both groups engaged in 40 minutes of supervised training three times per week. Aerobic exercise consisted of treadmill walking or jogging at an initial intensity that correlated with 50% VO2max and gradually increased to 70% VO2max. Stretch exercise consisted of a variety of dynamic stretch positions that were held for 15–30 second intervals. Fasting blood samples were taken at baseline, 3, 6, and 12 months of exercise training. Enzyme-linked photometric assay was used to measure nitrite/nitrate (NOx) levels in plasma and urine. Descriptive statistics were computed to assess patient baseline demographics for differences in exercise groups (Aerobic vs. Stretch). Significance level was set at P< 0.05 for all tests, and completed all analyses using the Statistical Analysis System (SAS) software 9.3. Six months of exercise resulted in decrease plasma NOx levels in both groups. Percent change in plasma NOx was significantly different from baseline to 12 months (p=0.05), but percent change in urine levels did not reach significance level. Plasma or urine NOx levels did not significantly correlate with age, MMSE, NO3 gene or VO2max. These results, suggests that exercise training-induced changes in NOx levels may be independent of eNOS gene in older African American MCI participants. Consequent vasodilatory effects of NOx may be beneficial to brain perfusion at the prodromal stage of AD.

Interleukin-6 displays lung anti-inflammatory properties and exerts protective hemodynamic effects in a double-hit murine acute lung injury

BackgroundInterleukin 6 (IL-6) is a predictive factor of poor prognosis in patients with acute respiratory distress syndrome (ARDS). However, its acute pulmonary hemodynamic effects and role in lung injury have not been investigated in a clinically relevant murine model of ARDS.MethodsWe used adult C57Bl6 wild-type (WT) and IL-6 knock-out (IL-6KO) mice. The animals received intravenous recombinant human IL-6 (rHuIL-6) or vehicle followed by intratracheal lipopolysaccharide (LPS) or saline before undergoing low tidal volume mechanical ventilation (MV) for 5 h. Before sacrifice, right ventricular systolic pressure and cardiac output were measured and total pulmonary resistance was calculated. After sacrifice, lung inflammation, edema and injury were assessed with bronchoalveolar lavage (BAL) and histology. In other experiments, right ventricular systolic pressure was recorded during hypoxic challenges in uninjured WT mice pretreated with rHuIL-6 or rHuIL-6 + non-selective nitric oxide synthase inhibitor L-NAME or vehicle.ResultsIL-6KO(LPS+MV) mice showed a faster deterioration of lung elastic properties and more severe bronchoalveolar cellular inflammation as compared to WT(LPS+MV). Treatment with rHuIL-6 partially prevented this lung deterioration. Total pulmonary resistance was higher in IL-6KO(LPS+MV) mice and this increase was abolished in rHuIL-6-treated IL-6KO mice. Finally, rHuIL-6 reduced hypoxic pulmonary vasoconstriction in uninjured WT mice, an effect that was abolished by co-treatment with L-NAME.ConclusionsIn a double-hit murine model of ARDS, IL-6 deficient mice experienced more severe bronchoalveolar cellular inflammation as compared to wild-type littermates. Furthermore, IL-6 deficiency caused marked acute pulmonary hypertension, which may be, at least partially, due to vasoactive mechanisms. A dysregulation of nitric oxide synthase may account for this observation, a hypothesis that will need to be investigated in future studies.

Exposure to High or Low Glucose Levels Accelerates the Appearance of Markers of Endothelial Cell Senescence and Induces Dysregulation of Nitric Oxide Synthase

To test the hypothesis that aging impairs endothelial cell response to glucose stress, we utilized a human umbilical vein endothelial cell in vitro model in which clinically relevant concentrations of normal (5.5 mM), high (25 mM), and low (1.5mM) glucose were tested. With advancing population doubling, exposure to normal glucose gradually decreased endothelial nitric oxide synthase expression and activity, resulting in slow, progressive development of markers of cell senescence (by population doubling level [PDL] 44). High or low glucose treatment accelerated the appearance of markers of senescence (by ~PDL 35) along with declines in endothelial nitric oxide synthase expression and activity. Human umbilical vein endothelial cells exposed to alternating low and high glucose gave even more rapid acceleration in the appearance of markers of senescence (by ~PDL 18) and reduction in endothelial nitric oxide synthase levels. Thus, exposure to low and high glucose induces earlier appearance of markers of endothelial cell senescence and dysregulation of the nitric oxide synthase gene and protein expression and function. These findings will help to elucidate endothelial dysfunction associated with glucose intolerance and improve future therapy for diabetic seniors.

Involvement of the endothelin and nitric oxide systems in the pathogenesis of renal ischemic damage in an experimental diabetic model

AimsIschemic acute kidney injury (iAKI) in experimental diabetes mellitus (DM) is associated with a rapid kidney dysfunction more than in non-diabetic rats. We hypothesize that this vulnerability is due to excessive endothelin-1 (ET-1) expression along with dysregulation of nitric oxide synthase (NOS) isoforms. The aim of the present study was to assess the impact of ischemia on renal function in diabetic rats as compared with non-diabetic rats, and to investigate the involvement of ET-1 and NO systems in the susceptibility of diabetic kidney to ischemic damage. Main methodsDM was induced by Streptozotocin. iAKI was induced by clamping of left renal artery for 30min. Right intact kidney served as control. 48h following ischemia, clearance protocols were applied to assess glomerular filtration rate (GFR), urinary flow (V) and sodium excretion (UNaV) in both kidneys. The renal effects of ABT-627, ETA antagonist; A192621.1, ETB antagonist; L-NAME, NOS non-selective inhibitor; 1400W, inducible NOS (iNOS) inhibitor; and NPLA, neuronal NOS (nNOS) inhibitor, were assessed following ischemic renal injury in diabetic rats. Key findingsInduction of iAKI in diabetic and non-diabetic rats caused significant reductions in GFR, V, and UNaV, which were greater in diabetic than non-diabetic rats. While, treatment with ABT-627 decreased V and UNaV, and increased GFR, A192621.1 decreased all these parameters. L-NAME, 1400W, and NPLA improved GFR in the ischemic diabetic kidney. SignificanceExcessive vasoconstrictive effects of ET-1 via ETA and upregulation of iNOS, are partly responsible for the impaired recovery of renal function following ischemia in diabetic rats.

Abstract 4062: Nitric Oxide synthase dysregulation in solid tumors: The therapeutic potential of tetrahydrobiopterin

Abstract Nitric Oxide (NO), in terms of cancer therapeutics, can be viewed as a double edge sword. Both anti-NO and NO-based anticancer treatments appear effective in several preclinical models. NO can promote apoptosis in some cells, whereas it inhibits apoptosis in others. This complexity is an indication of the importance of NO source (NO donors vs. endogenous), location of that source, NO concentration and cell type in understanding the role of NO. Current experiments examine NO-dependent pathways involved in tumor cell survival from a new perspective of Nitric Oxide synthase (NOS) dysregulation. Tetrahydrobiopterin (BH4) is a necessary cofactor of NOS and can be readily oxidized to BH2 in an inflammatory environment, such as that found in solid tumors. When BH4 levels are low, electron transfer in the active site of the enzyme becomes uncoupled from L-arginine oxidation resulting in the production of O2- instead of NO. The uncoupled enzyme therefore becomes a generator of peroxynitrite (ONOO-), which is produced rapidly by the reaction of O2- with NO, and is a highly reactive species that can modify proteins, lipids and DNA. In normal tissue the ratio of BH4 to BH2 (which when utilized by NOS the enzyme is uncoupled) is 40-70:1 and sometimes greater. We examined BH4 levels by HPLC in MCF7, Fadu, and A431 cells in vitro and Fadu and A431 cells in flank tumor xenografts. Our experiments showed that the BH4:BH2 ratio in tissue culture and flank tumor xenografts is less than 1. These findings show that NOS in tumor cells as compared to normal cells is uncoupled. Subsequent experiments focused on the ability to manipulate BH4 levels through exogenous sepiapterin (SP), converted to BH4 through sepiapterin reductase and dihydrofolate reductase, or through overexpression of GTP cyclohydrolase I, the rate-limiting enzyme in BH4 production. Both tissue culture cells and flank tumor xenografts showed a 10-15-fold increase in BH4:BH2 when given SP in the tissue culture media or in the drinking water of mice. Increasing the BH4:BH2 ratio leads to enhanced NO production as evidenced by an increase in cGMP formation. To directly examine the effects of manipulating the BH4:BH2 ratio, MCF7 and MDA cells were treated with varying concentrations of SP. MCF7 cells when exposed to SP showed a 2-fold decrease in colony formation and after 3 days of treatment had no viable cells, which was assessed by the MTT assay. Effects of SP on MDA 231 cells were not as dramatic, but by using the MTT assay we were able to show that the growth rate of the cells was cut in half. Current experiments focus on SP's effects in vivo and elucidating the mechanism of cell death and the signaling pathways involved. These results represent a potential new therapeutic strategy in the treatment of solid tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4062. doi:10.1158/1538-7445.AM2011-4062

Prenatal Lipopolysaccharide Increases Postnatal Intestinal Injury in a Rat Model of Necrotizing Enterocolitis

An increased incidence of necrotizing enterocolitis (NEC) has been noted in infants who are born to mothers with chorioamnionitis. Our objective was to test the hypothesis that newborn rat pups born to mothers exposed to prenatal lipopolysaccharide during pregnancy would be more susceptible to intestinal injury in a rat model of NEC and that the increased intestinal injury is mediated by dysregulation of inducible nitric oxide synthase. Time-dated pregnant Sprague-Dawley dams were given an intraperitoneal injection of either 2 mg/kg of lipopolysaccharide or vehicle. Rat pups from each group of dams were delivered at term and placed in a rat NEC model. A subset of pups was given either vehicle or aminoguanidine. Intestines were harvested and graded for degree of intestinal injury. Maternal prenatal lipopolysaccharide exposure increased the frequency and severity of intestinal injury in the neonatal rat NEC model. Treatment with aminoguanidine significantly decreased plasma nitric oxide levels. Additionally, aminoguanidine significantly decreased intestinal injury. Intestinal injury observed may be mediated via nitric oxide synthase dysregulation.

Sickle Cell Pulmonary Hypertension and Dysregulated NO Axis in a Mouse Model Are Modulated by Apolipoprotein a-1 Availability

We have recently shown that the pulmonary hypertension and pulmonary vascular response impairment in models of sickle cell disease are associated with a dysregulation of nitric oxide synthase (NOS) coupling and cGMP signaling. However, the pathophysiologic mechanisms and exacerbating factors leading to pulmonary vasculopathy remain unclear. In the present study, we investigated the effect of sickle cell disease in the context of ApoE deficient mice (ApoEKO) that express advanced atherosclerosis and mice that express the protective ApoA-1 protein (TgApoA1) that received bone marrow transplant with sickle cell marrow.HYPOTHESES:Mice with low ApoA-1 and sickle red blood cells will have higher oxidant stress than sickle cell mice, increasing the severity of development of pulmonary vasculopathy.High levels of ApoAI should protect mice with sickle red blood cells by reducing the oxidant stress and pulmonary hypertension.METHODS: Bone marrow harvested from “Berkeley” sickle cell mouse donors were transplanted into 3 groups of myeloablatively irradiated recipients: Tg ApoA1 mice with high HDL levels, ApoE knockout (ApoEKO) mice with high VLDL/IDL plasma levels, and wild-type mice. Mice received routine rodent chow. Half of the recipients were studied by closed-chest cardiac catheterization soon after marrow engraftment (11 wks after BMT) and the other half studied 3 months later. Blood samples were obtained for measurement of CBC/reticulocyte count, LDH, plasma hemoglobin, and total cholesterol. Tissue samples were assayed for NOS activity and dimerization.RESULTS: Mice transplanted with SS marrow showed significant pulmonary hypertension, profound pulmonary and systemic endothelial dysfunction, and vascular instability characterized by diminished responses to authentic nitric oxide (NO), NO donors, and endothelium-dependent vasodilators and enhanced responses to vasoconstrictors. The baseline pulmonary hypertension and endothelial dysfunction was augmented in ApoEKO+SS mice, but this added impairment was abrogated in TgApoA1+SS mice. However, endothelium-independent vasodilation in all mice was normal. Mechanisms of this vasculopathy in sickle mice involve global dysregulation of the NO axis: impaired constitutive nitric oxide synthase activity (NOS) with loss of endothelial NOS (eNOS) dimerization, increased NO scavenging by plasma hemoglobin and superoxide all of which were markedly augmented in ApoEKO-SS and improved in TgApoA1-SS mice when compared to ApoEKO-SS mice. In addition, vascular arginase levels were markedly higher in ApoEKO-SS mice (~50%, P<0.05 vs ApoEKO controls and ~40% vs SS mice) suggesting an increased reaction to sickle cell/hemolysis in ApoE deficient conditions.CONCLUSION: Adjusting chronic apolipoprotein A-1 levels can modulate the pulmonary hypertension and endothelial dysfunction in this sickle cell animal model. Depleting ApoA-1 may serve as a mechanism for further inhibition of NOS activity in SS disease. These animal model data provide a mechanistic basis for our epidemiologic observations that low ApoA-1 and low HDL are associated with more severe pulmonary hypertension in sickle cell patients. Conversely, these data extend the observations of Ou and Pritchard that acute administration of LF4, an ApoA-1 mimetic, improves arterial vaso-reactivity in a similar sickle cell mouse model. Taken together, these preclinical data indicate that ApoA-1 deserves further study as a potential therapy for the global dysregulation of the NO axis in SCD.

Is there a relationship between the AT‐1 receptor and O2‐ in Renal Wrap Hypertension?

Angiotensin II (Ang II) has been implicated as a mediator of oxidative stress that contributes to hypertension via AT-1 receptor activation and subsequent superoxide (O2-) production. We have previously shown plasma renin activity (PRA) to be increased at 7 days post-wrap, but not elevated at 28 days. Furthermore, a dysregulation of nitric oxide synthase, nitrotyrosine, and superoxide dismutase expression in the kidney has been shown supporting the role of Ang II in this context. Thus, we hypothesized that there was an up-regulation of AT-1 receptor expression and O2− in the kidney in renal wrap hypertension. Rats were uninephrectomized and subjected to wrap/sham surgery and studied at 7 or 28 days. Kidneys from hypertensive wrap and normotensive sham rats were isolated; cortex and medulla were separated for protein isolation for western analysis or chemiluminescent O2− measurements (Mean Arterial Pressure; 7 day: Sham = 92 ± 4 mm Hg, Wrap = 139 ± 10 mm Hg; 28 day: Sham = 92 ± 6 mm Hg, Wrap =152 ± 7 mm Hg). At 7 days, there was a significant reduction in wrap medullary AT-1 expression compared to sham (704 ± 252 vs. 2090 ± 476 arbitrary densitometry units), as well as medullary O2− production (10 ± 2 vs. 20 ± 4 pmol/min*mg). At 28 days, there were no changes in AT-1 expression, but medullary O2− production was increased compared to sham (31 ± 3 vs. 19 ± 2 pmol/min*mg). No changes were found in cortical AT-1 expression or O2− production at 7 or 28 days. The data suggest that during the onset of hypertension the elevated PRA may cause a down-regulation of AT-1 receptors and O2− in the renal medulla. During chronic hypertension, mechanisms other than Ang II are related to the increased O2−.