Greater Nitric Oxide Production Research Articles

IL-6 Contributes to Impaired Nitric Oxide and Increased Mitochondrial ROS Production in Human Aortic Endothelial Cells

Background: Major Depressive disorder (MDD) is associated with chronic low-grade inflammation with elevated proinflammatory cytokines. Notably, elevated inflammation is a known contributor to cardiovascular dysfunction and disease. Interleukin (IL)-6 has been shown to be elevated in rodent models of depression. Additionally, preliminary data from our lab suggests that there is a greater proportion of IL-6 producing CD8+ T cells in adults with MDD. Although IL-6 has been reported to be increased in adults with MDD and known to have a direct effect on vascular endothelial cells, the exact mechanism by which IL-6 interacts with the endothelium is unknown. Therefore, we tested the hypothesis that IL-6 treatment will decrease nitric oxide (NO) production and increase mitochondrial ROS (mitoROS) in endothelial cells. Methods: Human aortic endothelial cells (HAECs) were used to study the effects of IL-6 on NO production. The HAECs were treated with vehicle or 10 ng/mL IL-6 for 16 hours, and then stimulated with Acetylcholine to induce NO production and the real time NO production was measured using a commercially available assay. We also measured mitoROS in HAECs treated with or without IL-6. Results are expressed as mean ± SEM (n=3-6 replicates) and group differences were assessed by one-way ANOVA followed by Tukey’s post-hoc test. Results: L-6 was found to significantly lower NO production in the cells (Control: 276.24 ± 7.01; IL-6: 215.19 ± 6.07 MFI, p=0.022). Acetylcholine stimulation resulted in greater NO production in the control cells (Control: 276.24 ± 7.01; Control + Ach: 354.28 ± 6.26 MFI, p=0.0005) whereas this increase was abolished in the IL-6 treated cells (IL-6: 215.19 ± 6.07; IL-6 + Ach: 225.21 ± 5.16 MFI, p=0.955). Additionally, we also found greater mitoROS in the IL-6 treated cells (Control: 683.02 ± 32.98; IL-6: 840.86 ± 38.63 MFI, p=0.002). Conclusion: These preliminary results suggest that IL-6 reduces NO production in endothelial cells, which may be in part due to elevated mitoROS. This work is supported by grants from NIH K01AG061271, R01AG060395 and AHA940023 (DWT). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effect of in-vitro heat stress challenge on the function of blood mononuclear cells from dairy cattle ranked as high, average and low immune responders

BackgroundThe warming climate is causing livestock to experience heat stress at an increasing frequency. Holstein cows are particularly susceptible to heat stress because of their high metabolic rate. Heat stress negatively affects immune function, particularly with respect to the cell-mediated immune response, which leads to increased susceptibility to disease. Cattle identified as having enhanced immune response have lower incidence of disease. Therefore, the objective of this study was to evaluate the impact of in vitro heat challenge on blood mononuclear cells from dairy cattle, that had previously been ranked for immune response, in terms of heat shock protein 70 concentration, nitric oxide production, and cell proliferation.ResultsBlood mononuclear cells from dairy cattle classified as high immune responders, based on their estimated breeding values for antibody and cell-mediated responses, produced a significantly greater concentration of heat shock protein 70 under most heat stress treatments compared to average and low responders, and greater cell-proliferation across all treatments. Similarly, a trend was observed where high responders displayed greater nitric oxide production compared to average and low responders across heat treatments.ConclusionOverall, these results suggest that blood mononuclear cells from high immune responder dairy cows are more thermotolerant compared to average and low immune responders.

Immunoprophylactic Potential of a New Recombinant Leishmania infantum Antigen for Canine Visceral Leishmaniasis: An In Vitro Finding.

The development and application of safe and effective immunoprophylactic/immunotherapeutic agents against canine visceral leishmaniasis (CanL) have been pointed out as the only means for the real control of the disease. Thus, this study aimed to evaluate the in vitro cellular immune response of dogs, elicited by the new recombinant proteins of Leishmania infantum, Lci10 and Lci13, in order to investigate their potential for vaccinology. Twenty-four dogs were submitted to clinical, parasitological, serological and molecular tests, and then separated into two study groups: 12 infected (InD) and 12 non-infected dogs (NInD), and six of each group were directed for Lci10 and Lci13 evaluation. Peripheral blood mononuclear cells (PBMC) were cultured and stimulated with Lci10 (10 μg/ml) or Lci13 (5 μg/ml), and with L. infantum soluble antigen (LSA) (25 μg/ml) or no stimulus (NS) as controls. Afterwards, the mRNA levels of different cytokines were quantified through qPCR, and Nitric Oxide (NO) production was assessed in the culture supernatants. Significant differences were considered when p ≤ 0.05. The comparative analysis revealed that, in the NInD group, Lci13 promoted a significant increase in the expression of IFN-γ in relation to LSA (p = 0.0362), and the expression of this cytokine in NInD was significantly higher than that presented in the InD (p = 0.0028). A negative expression for TGF-β was obtained in both groups. Lci13 also induced a greater production of NO in relation to the NS sample in the NInD group. No significant differences were observed after stimulation with Lci10. In conclusion, the results suggest a protective role of Lci13 for uninfected animals, thus with a potential for immunoprophylaxis. The results will help to direct the antigen Lci13 for further studies (pre-clinical trials), in order to determine its immunogenicity and reactogenicity effects, as a way to consolidate its real applicability for vaccinology against CanL.

Supplementation with Spirulina platensis Modulates Aortic Vascular Reactivity through Nitric Oxide and Antioxidant Activity.

The possible mechanism is involved in the effects of Spirulina platensis on vascular reactivity. Animals were divided into sedentary group (SG) and sedentary groups supplemented with S. platensis at doses of 50 (SG50), 150 (SG150), and 500 mg/kg (SG500). To evaluate reactivity, cumulative concentration-response curves were constructed for phenylephrine and acetylcholine. To evaluate the involvement of the nitric oxide (NO) pathway, aorta tissue was preincubated with L-NAME and a new curve was then obtained for phenylephrine. Biochemical analyses were performed to evaluate nitrite levels, lipid peroxidation, and antioxidant activity. To contractile reactivity, only SG500 (pD2 = 5.6 ± 0.04 vs. 6.1 ± 0.06, 6.2 ± 0.02, and 6.2 ± 0.04) showed reduction in phenylephrine contractile potency. L-NAME caused a higher contractile response to phenylephrine in SG150 and SG500. To relaxation, curves for SG150 (pD2 = 7.0 ± 0.08 vs. 6.4 ± 0.06) and SG500 (pD2 = 7.3 ± 0.02 vs. 6.4 ± 0.06) were shifted to the left, more so in SG500. Nitrite was increased in SG150 and SG500. Lipid peroxidation was reduced, and oxidation inhibition was increased in all supplemented groups, indicating enhanced antioxidant activity. Chronic supplementation with S. platensis (150/500 mg/kg) caused a decrease in contractile response and increase in relaxation and nitrite levels, indicating greater NO production, due to decreased oxidative stress and increased antioxidant activity.

Estrogen relaxes gastric muscle cells via a nitric oxide- and cyclic guanosine monophosphate-dependent mechanism: A sex-associated differential effect.

Various gastrointestinal (GI) disorders have a higher prevalence in women than in men. In addition, estrogen has been demonstrated to have an inhibitory effect on the contractility of GI smooth muscle. Although increased plasma estrogen levels have been implicated in GI disorders, the role of gastric estrogen receptor (ER) in these sex-specific differences remains to be fully elucidated. The present study was designed to investigate the sex-associated differences in the expression of the two ER isoforms, ERα and ERβ, and the effect of estrogen on gastric muscle contraction via the nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway. Experiments were performed on single gastric smooth muscle cells (GSMCs) isolated from male and female Sprague Dawley rats. The effect of acetylcholine (ACh), a muscarinic agonist, on the contraction of GSMCs was measured via scanning micrometry in the presence or absence of 1 µM 17β-estradiol (E2), an agonist to the majority of ERs, 1,3,5-tris(4-hydroxyphenyl)-4-propyl-1H-pyrazole (PPT), an ERα agonist, or diarylpropionitrile (DPN), an ERβ agonist. The protein expression levels of ER subtypes in GSMCs were measured using a specifically designed ELISA. GSMCs from female rats had a higher expression of ERα and ERβ protein compared with GSMCs from males. ACh induced less contraction in female that in male GSMCs. Pre-treatment of GSMCs with E2 reduced the contraction of GSMCs from both sexes, but to a greater extent in those from females. PPT and DPN inhibited ACh-induced contraction in GSMCs from females. Furthermore, E2 increased NO and cGMP levels in GSMCs from males and females; however, higher levels were measured in females. Of note, pre-incubation of female GSMCs with Nω-nitro-L-arginine, a NO synthase inhibitor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one, a guanylyl cyclase inhibitor, reduced the inhibitory effect of estrogen on GSMC contraction. In conclusion, estrogen relaxes GSMCs via an NO/cGMP-dependent mechanism, and the reduced contraction in GSMCs from females by estrogen may be associated with the sex-associated increased expression of ERα and ERβ, and greater production of NO and cGMP, compared with that in GSMCs from males.

The mitochondrial uncoupling protein-2 is a master regulator of both M1 and M2 microglial responses.

Microglial activation is a dynamic process, central to neuroinflammation, which can have beneficial or pathogenic effects to human health. Mitochondria are key players in neuroinflammatory and neurodegenerative processes, common to most brain diseases. To the best of our knowledge on the role of mitochondria in the modulation of neuroinflammation, we focused on the mitochondrial uncoupling protein-2 (UCP2), known to control mitochondrial functions and to be implicated in a variety of physiological and pathological processes. In primary microglial cultures, the M1 stimulus lipopolysaccharide induced an early and transitory decrease in UCP2 levels. The initial UCP2 down-regulation was paralleled by mitochondrial inner membrane potential (mMP) depolarization and increased mitochondrial reactive oxygen species production. The key role of UCP2 in controlling mMP and reactive oxygen species production was confirmed by both pharmacological inhibition and down-regulation by RNA interference. Additionally, UCP2-silenced microglia stimulated with lipopolysaccharide showed an enhanced inflammatory response, characterized by a greater production of nitric oxide and interleukin-6. UCP2 was differently regulated by M2 stimuli, as indicated by its persistent up-regulation by interleukin-4. In UCP2-silenced microglia, interleukin-4 failed to induce M2 genes (mannose receptor 1 and interleukin-10) and to reduce M1 genes (inducible nitric oxide synthase and tumour necrosis factor-α). Our findings indicate that UCP2 is central to the process of microglial activation, with opposite regulation of M1 and M2 responses, and point to UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes in several brain diseases where neuroinflammation is recognized to contribute to neurodegeneration. We show that the mitochondrial uncoupling protein-2 (UCP2) is central to the process of microglial activation, with opposite regulation of M1 and M2 responses. In UCP2-silenced microglia, lipopolysaccharide (LPS) triggers an enhanced inflammatory response characterized by a greater expression of M1 genes, whereas interleukin-4 (IL-4) fails in inducing M2 genes and reducing M1 genes. We propose UCP2 manipulation as a potential strategy for redirecting microglial response towards protective phenotypes.

Cytokine‐Induced Arginase Expression in Intestinal Epithelial Cells Reduce Nitric Oxide Production and Apoptosis

Intestinal epithelial cells are involved in disease progression of inflammatory bowel diseases such as necrotizing entercolitis in preterm neonates. Intestinal epithelial cells metabolize arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell viability, while NO production can lead to apoptosis. The hypothesis of this study is that in intestinal epithelial cells inflammation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were incubated with conditioned media (CM) from RAW 264.7 cells (a macrophage cell line) treated with LPS (E.coli 0127:B8; 1μg/ml) for 4 hours. rIEC-6 cells incubated with CM had significantly higher levels of iNOS, cleaved caspase-3, and arginase II protein levels than did control cells. Levels of arginase I were not different between controls and CM treated cells. rIEC-6 incubated in CM had significantly greater NO production than did untreated rIEC-6, and NO production in CM-treated cells was further increased when the arginase inhibitor nor-NOHA was added to the media. Viable cell number was significantly lower after 24 hours with CM than in controls, and inhibition of arginase activity with nor-NOHA resulted in a further significant decrease in viable cell numbers. These data suggest that cytokine-induced arginase expression in rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

Paraquat poisoning induces TNF-α-dependent iNOS/NO mediated hyporesponsiveness of the aorta to vasoconstrictors in rats.

Paraquat is a toxic herbicide that may induce acute lung injury, circulatory failure and death. The present work aimed at investigating whether there is systemic inflammation and vascular dysfunction after paraquat exposure and whether these parameters were related. There was neutrophilia and accumulation of neutrophils in lung and bronchoalveolar lavage of animals given paraquat. This was associated with an increase in serum levels of TNF-α. In rats given paraquat, the relaxant response of aortic rings to acetylcholine was not modified but the contractile response to phenylephrine was greatly reduced. Endothelium removal or treatment with non-selective (L-NAME) or selective (L-NIL) inhibitors of inducible nitric oxide synthase (iNOS) restored contraction of aortas. There was greater production of nitric oxide (NO), which was restored to basal level by L-NIL, and greater expression of iNOS in endothelial cells, as seen by Western blot analyses and confocal microscopy. Blockade of TNF-α reduced pulmonary and systemic inflammation and vascular dysfunction. Together, our results clearly show that paraquat causes pulmonary and systemic inflammation, and vascular dysfunction in rats. Vascular dysfunction is TNF-α dependent, associated with enhanced expression of iNOS in aortic endothelial cells and greater NO production, which accounts for the decreased responsiveness of aortas to vasoconstrictors. Blockers of TNF-α may be useful in patients with paraquat poisoning.

Genetic removal of basal nitric oxide enhances contractile activity in isolated murine collecting lymphatic vessels

The role of nitric oxide (NO) in regulating lymphatic contractile function and, consequently, lymph flow has been the subject of intense study. Despite this, the precise effects of NO on lymphatic contractile activity remain unclear. Recent hypotheses posit that basal levels of endogenous NO increase lymphatic contraction strength as a consequence of lowering frequency (i.e. positive lusitropy), whereas higher agonist-evoked concentrations of NO exert purely inhibitory effects on contractile function. We tested both hypotheses directly by isolating and cannulating collecting lymphatic vessels from genetically modified mice for ex vivo study. The effects of basal NO and agonist-evoked NO were evaluated, respectively, by exposing wild-type (WT), endothelial NO synthase (eNOS)(-/-) and inducible NO synthase (iNOS)(-/-) lymphatic vessels to controlled pressure steps followed by ACh doses. To compare with pharmacological inhibition of eNOS, we repeated both tests in the presence of l-NAME. Surprisingly, genetic removal of basal NO enhanced contraction amplitude significantly without increasing contraction frequency. Higher levels of NO production stimulated by ACh evoked dilation, decreased tone, slowed contraction frequency and reduced fractional pump flow. We conclude that basal NO specifically depresses contraction amplitude, and that greater NO production then inhibits all other aspects of contractile function. Further, this work demonstrates definitively that mouse collecting lymphatic vessels exhibit autonomous, large-amplitude contractions that respond to pressure similarly to collecting lymphatics of other mammalian species. At least in the peripheral lymphatic vasculature, NO production depresses contractile function, which influences lymph flow needed for fluid regulation, humoral immunity and cancer metastasis.

Efeito do treinamento físico moderado e intenso sobre os mecanismos de defesa de ratos adultos

OBJETIVO: Analisou-se o impacto do treinamento físico moderado (TFM) e intenso (TFI) sobre o perfil leucocitário e a atividade microbicida de macrófagos alveolares, em 29 ratos machos Wistar. MÉTODOS: Foram formados três grupos: controle-sedentário (CS), treino-moderado (TM) e treino-intenso (TI). Os TFM e TFI foram efetuados através da natação, com aumento progressivo de carga conforme o peso corporal, até um máximo de 3% para o TFM, e 5% para o TFI. As coletas de sangue para contagem total e diferencial dos leucócitos foram automatizadas através do analisador hematológico Sysmex XT- 1800i (Roche®) antes e após o treino. E ao final realizou-se o lavado broncoalveolar para determinar a taxa de fagocitose e a produção de óxido nítrico (ON) de macrófagos. RESULTADOS: O grupo TM apresentou valores maiores para o número de leucócitos (12,77±2,0 x 17,25±2,4 10³/mm³), linfócitos (8,87±1,0 x 12,5±2,1 10³/mm³) e neutrófilos (0,99±0,5 x 3,18±1,0 10³/mm³), p<0,05. Apresentou também uma maior produção de ON (15,77±4,9µmols/mL) e da taxa de fagocitose (38,6%±8,65) em relação ao CS (6,58±1,9µmols/mL e 24,4%±7,40, respectivamente). Enquanto que, o grupo TI apresentou menor taxa de fagocitose (13,1%±1,52 x 24,4%±7,40), maior produção de ON (38,40± 2,1x 6,58±1,9 µmols/mL) e aumento apenas, no valor dos neutrófilos (2,6±1,4x 0,99±0,5 10³/mm³) quando comparado ao CS, p<0,001. CONCLUSÕES: O TFM proporcionou melhora nos mecanismos de defesa dos animais adultos. Enquanto que o TFI reduziu a taxa de fagocitose o que poderá implicar em prejuízo da atividade microbicida dos animais.

Exercise and diet-induced weight loss attenuates oxidative stress related-coronary vasoconstriction in obese adolescents.

Obesity is a disease of oxidative stress (OS). Acute hyperoxia (breathing 100% O(2)) can evoke coronary vasoconstriction by the oxidative quenching of nitric oxide (NO). To examine if weight loss would alter the hyperoxia-related coronary constriction seen in obese adolescents, we measured the coronary blood flow velocity (CBV) response to hyperoxia using transthoracic Doppler echocardiography before and after a 4-week diet and exercise regimen in 6 obese male adolescents (age 13-17years, BMI 36.5±2.3kg/m(2)). Six controls of similar age and BMI were also studied. The intervention group lost 9±1% body weight, which was associated with a reduced resting heart rate (HR), reduced diastolic blood pressure (BP), and reduced RPP (all P<0.05). Before weight loss, hyperoxia reduced CBV by 33±3%. After weight loss, CBV only fell by 15±3% (P<0.05). In the control group, CBV responses to hyperoxia were unchanged during the two trials. Thus weight loss: (1) reduces HR, BP, and RPP; and (2) attenuates the OS-related coronary constrictor response seen in obese adolescents. We postulate that: (1) the high RPP before weight loss led to higher myocardial O(2) consumption, higher coronary flow and greater NO production, and in turn a large constrictor response to hyperoxia; and (2) weight loss decreased myocardial oxygen demand and NO levels. Under these circumstances, hyperoxia-induced vasoconstriction was attenuated.

In-vitro study of cellular viability and nitric oxide production by J774 macrophages stimulated by interferon gamma with ceramic, polycarbonate, and polyoxymethylene brackets

Ceramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade and release bisphenol-A and formaldehyde, respectively. More reliable tests are needed to assess the potential toxicity of these materials. In addition to traditional cytotoxicity tests, the study of nitric oxide (NO) cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating cytotoxic potential. The purpose of this study was to assess, with esthetic brackets, cellular viability by 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay (Sigma, St. Louis, Mo) in the macrophage cell line J774 stimulated with interferon gamma. Interferon gamma is a key cytokine in the activation of macrophages, plays an important role in immunologic processes, and also quantifies NO production by these macrophages. Well plates were seeded with 2 x 104 J774 cells per well, in a volume of 100 microL, resuspended in Roswell Park Memorial Institute Supplemented Medium 1640. The macrophage cell line J774 was stimulated with interferon gamma. Ceramic, polycarbonate, and polyoxymethylene brackets were added and kept in the culture for 24, 48, or 72 hours in 5% carbon dioxide at 37 degrees C; the control samples did not include brackets. At the end of each incubation period, the supernatant was collected for posterior NO quantification, and the cells were evaluated for cytotoxicity. Cellular viability in all groups was higher at 72 hours than at 24 hours. The final means in the bracket groups did not show significant differences compared with the control group. NO production was significantly greater in all groups at the final time than at the initial time. However, the brackets with the interferon gamma stimulation did not result in greater NO production than did the cells in the control group.

Decreased Venous Contraction and Enhanced Relaxation and Nitric Oxide Production in the Inferior Vena Cava of Pregnant Rat

Normal pregnancy (Norm‐Preg) is associated with increased cardiac output and plasma volume, partly due to increased nitric oxide (NO) production by endothelial cells in various arterial beds; however, the role of venous endothelium is unclear. We tested whether Norm‐Preg is associated with increases in venous relaxation and NO production. Segments of inferior vena cava (IVC) from Norm‐Preg (day 19) and virgin Sprague‐Dawley rats were used to measure isometric contraction/relaxation. The IVC was loaded with the NO‐sensitive DAF‐FM diacetate (DAF) for simultaneous measurement of NO production. The α‐adrenergic agonist phenylephrine (PHE, 10−5 M) and membrane depolarization by 96mM KCl caused less contraction in IVC of Norm‐Preg (20.64±8.75 and 230.07±31.40) than virgin rats (70.90±15.60 and 308.32±95.65 mg/mg tissue). Acetylcholine (Ach, 10−5 M) caused greater relaxation of KCl‐induced contraction in IVC of Norm‐Preg (46.85±12.95) than virgin rats (36.05±8.07%). Basal NO was slightly greater in IVC of Norm‐Preg (0.12±0.02) than virgin rats (0.10±0.02). In IVC precontracted with PHE, Ach (10−5 M) caused greater NO production in Norm‐Preg (0.37±0.08) than virgin rats (0.22±0.03). Thus Norm‐Preg is associated with decreased venous contraction and enhanced Ach‐induced relaxation and NO production, which could lead to more distensible veins and venous insufficiency during pregnancy.

Increased vascular angiotensin type 2 receptor expression and NOS-mediated mechanisms of vascular relaxation in pregnant rats

Normal pregnancy is associated with reduced blood pressure (BP) and decreased pressor response to vasoconstrictors, even though the renin-angiotensin system is upregulated. Angiotensin II (ANG II) activates both angiotensin type 1 receptors (AT(1)Rs) and angiotensin type 2 receptors (AT(2)Rs). Although the role of the AT(1)R in vascular contraction is well documented, the role of the AT(2)R in vascular relaxation, particularly during pregnancy, is less clear. It was hypothesized that the decreased BP and vasoconstriction during pregnancy was, at least in part, due to changes in AT(2)R amount, distribution, and/or postreceptor mechanisms of vascular relaxation. To test this hypothesis, systolic BP was measured in virgin and pregnant (day 19) Sprague-Dawley rats. Isometric contraction/relaxation was measured in isolated aortic rings, and nitric oxide (NO) production was measured using 4-amino-5-methylamino-2',7'-difluorescein fluorescence. AT(1)R and AT(2)R mRNA expression and protein amount were measured in tissue homogenates using real-time RT-PCR and Western blots, and their local distribution was visualized in cryosections using immunohistochemistry and immunofluorescence. BP was lower in pregnant than virgin rats. Phenylephrine (Phe) caused concentration-dependent contraction that was reduced in the aorta of pregnant compared with virgin rats. Treatment with the AT(2)R antagonist PD-123319 caused greater enhancement of Phe contraction, and the AT(2)R agonist CGP-42112A caused greater relaxation of Phe contraction in the aorta of pregnant than virgin rats. ANG II plus the AT(1)R blocker losartan induced greater NO production in the aorta of pregnant than virgin rats. RT-PCR revealed increased mRNA expression of vascular endothelial NO synthase (eNOS), little change in AT(1)Rs, and increased AT(2)Rs in pregnant compared with virgin rats. Western blots revealed an increased protein amount of activated phospho-eNOS, little change in AT(1)Rs, and increased AT(2)Rs in pregnant compared with virgin rats. Immunohistochemistry and immunofluorescence analysis in aortic sections of virgin rats revealed abundant AT(1)R staining in tunica media that largely colocalized with actin in vascular smooth muscle and less AT(2)Rs mainly in the tunica intima and endothelium. In pregnant rats, AT(1)R staining in the smooth muscle layer and adventitia was reduced, and endothelial AT(2)R staining was enhanced. These data suggest an enhanced AT(2)R-mediated vascular relaxation pathway involving increased expression/activity of endothelial AT(2)Rs and increased postreceptor activated phospho-eNOS, which may contribute to the decreased BP during pregnancy.

Nitric oxide as an early marker of human embryo metabolic cleavage in ART using fresh or thawed oocytes

Objective To study a possible role of nitric oxide (NO) as a marker of development in the early phases of human embryo cleavage during assisted reproduction. Study design 179 women having ART were included. 123 women used fresh oocytes and 56 oocyte thawing cycles in the Center of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova, between July 2005 and June 2006; 57 patients had IVF and 122 patients had ICSI. NO concentrations in IVF or ICSI embryo culture media were assessed by monitoring levels of NO stable oxidation products (nitrites/nitrates). Analysis of embryo quality was performed. Student’s t-test or Mann–Whitney and logistic regression model tests were applied to the data. Results In patients using fresh oocytes, there were greater NO production in embryos derived from ICSI than from IVF after 52 h of culture (38.64 μmol/L vs 11.2 μmol/L, p < 0.05). No correlation with embryo quality was observed. Embryos derived from fresh oocytes produce more NO than embryos from thawed oocytes both after 48 and 52 h of culture (16.12 μmol/L vs 6.83 μmol/L and 25.93 μmol/L vs 2.98 μmol/L respectively, p < 0.05). Conclusion(s) NO in embryo culture media is not a metabolic cleavage marker or a marker of embryo quality in ART. However, it could be an important parameter in the investigation of metabolism in frozen/thawed oocytes.

L-arginine Reduces Mercury Accumulation in Thymus of Mercury-exposed Mice: Role of Nitric Oxide Synthase Activity and Metallothioneins

Mercury, an occupational and environmental contaminant, is a well-recognized health hazard. The thymus is a target for inorganic mercury (Hg2+); thymic function is impaired in Hg2+ intoxication and is partially restored by simultaneous L-arginine supplementation. The nitric oxide (NO)-nitric oxide synthase (NOS) pathway and metallothioneins (MTs) were studied to investigate the role of L-arginine in thymic function restoration after mercury exposure. Mice received a higher and a lower dose of inorganic mercury, with and without L-arginine supplementation. Saline-treated mice were used as controls. Thymus weight and thymulin were measured as indices of thymic function. Mice treated with Hg2+ alone displayed an accumulation of metal in the thymus, reduced NOS activity, a lower plasma nitrite plus nitrate concentration and an increased MTs expression compared with control mice. L-arginine supplementation was associated with lower Hg2+ concentrations in the organ and partial preservation of other measures. Reduced accumulation of Hg2+ in mice dosed with L-arginine was probably related to greater NO production and NO-MTs interactions.

Production of nitric oxide by peripheral blood mononuclear cells from the Florida manatee, Trichechus manatus latirostris

Florida manatees ( Trichechus manatus latirostris) are exposed to many conditions in their habitat that may adversely impact health and impair immune function in this endangered species. In an effort to increase the current knowledge base regarding the manatee immune system, the production of an important reactive nitrogen intermediate, nitric oxide (NO), by manatee peripheral blood mononuclear cells (PBMC) was investigated. PBMC from healthy captive manatees were stimulated with LPS, IFN-γ, or TNF-α, either alone or in various combinations, with NO production assessed after 24, 48, 72, and 96 h of culture. NO production in response to LPS stimulation was significantly greater after 48, 72, or 96 h of culture compared to NO production after 24 h of culture. A specific inhibitor of inducible nitric oxide synthase (iNOS), l-NIL ( l- N 6-(1-iminoethyl)lysine), significantly decreased NO production by LPS-stimulated manatee PBMC. Manatee specific oligonucleotide primers for iNOS were designed to measure expression of relative amounts of mRNA in LPS-stimulated manatee PBMC from captive manatees. NO production by PBMC from manatees exposed to red tide toxins was analyzed, with significantly greater NO production by both unstimulated and LPS stimulated PBMC from red tide exposed compared with healthy captive or cold-stress manatees. Free-ranging manatees produced significantly lower amounts of nitric oxide compared to either captive or red tide rescued manatees. Results presented in this paper contribute to the current understanding of manatee immune function and represent the first report of nitric oxide production in the immune system of a marine mammal.

Apolipoprotein‐E Allele‐Specific Regulation of Nitric Oxide Production

Cognitive decline and dementia are key features of Alzheimer's disease (AD) that result from failure of neuronal function. Affected neurons demonstrate indices of nitrosative stress resulting from changes in nitric oxide (NO) mediated redox balance. Neurofibrillary tangles, a characteristic neuropathologic feature of AD, and dysfunctional neurons frequently display 3-nitrotyrosine or other markers of nitrosative stress and immunoreactive nitric oxide synthase (NOS), suggesting that NOS-containing neurons are affected in AD. Our previous studies showed that apolipoprotein E treatment of macrophages increased NO production. Using transgenic mouse models expressing human apoE2, apoE3, or apoE4 protein isoforms and no mouse apoE, we now report an isoform specific difference in microglial NO production. Mice expressing the apoE4 protein isoform have a greater NO production than mice expressing the apoE3 protein isoform. The supply of arginine, the sole substrate for NOS, is dependent on cationic amino acid transporters (CATs) that also demonstrate a similar pattern of apoE isoform dependency. Although arginine transport is greater in APOE4 microglia, this effect is not limited to tissue macrophages. Cortical neurons in primary culture from APOE4 transgenic mice exhibit a similar increase in arginine uptake over neurons cultured from APOE3 mice. The inappropriate levels of arginine transport and of NO in the presence of the APOE4 compared to the APOE3 gene and its products are likely to have significant impact in the CNS.

Modulation of vascular tone and reactivity by nitric oxide in porcine pulmonary arteries and veins.

Isolated porcine pulmonary vessels were studied in order to evaluate the role of nitric oxide in arteries and veins. Leukotriene C4 and noradrenaline contracted porcine pulmonary arteries but induced only negligible contractions of porcine pulmonary veins. After treatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (L-NOARG), significant contractions to leukotriene C4 and noradrenaline were uncovered in pulmonary veins. In arterial preparations, L-NOARG caused a less marked potentiation of noradrenaline-induced contractions and did not alter leukotriene C4-induced contractions. Endothelium-dependent relaxations to acetylcholine were greater in veins compared with arteries whereas the endothelium-independent relaxations to the nitric oxide donor sodium nitroprusside (SNP) and the cyclic nucleotide analogue 8-bromo-cGMP were similar in the two preparations. Taken together these data suggest that the apparent insensitivity of porcine pulmonary veins to leukotriene C4 and noradrenaline was because of release of nitric oxide. The effect of nitric oxide synthase inhibition was less pronounced in porcine pulmonary arteries, suggesting a preferential functional role of nitric oxide in porcine pulmonary veins, originating in a greater production of nitric oxide by veins as opposed to arteries.

Eicosapentaenoic acid (EPA) induces Ca 2+-independent activation and translocation of endothelial nitric oxide synthase and endothelium-dependent vasorelaxation

Eicosapentaenoic acid (EPA), but not its metabolites (docosapentaenoic acid and docosahexaenoic acid), stimulated nitric oxide (NO) production in endothelial cells in situ and induced endothelium-dependent relaxation of bovine coronary arteries precontracted with U46619. EPA induced a greater production of NO, but a much smaller and more transient elevation of intracellular Ca 2+ concentration ([Ca 2+]i), than did a Ca 2+ ionophore (ionomycin). EPA stimulated NO production even in endothelial cells in situ loaded with a cytosolic Ca 2+ chelator 1,2-bis- o-aminophenoxythamine- N′, N′, N′-tetraacetic acid, which abolished the [Ca 2+]i elevations induced by ATP and EPA. The EPA-induced vasorelaxation was inhibited by N ω -nitro- L-arginine methyl ester. Immunostaining analysis of endothelial NO synthase (eNOS) and caveolin-1 in cultured endothelial cells revealed eNOS to be colocalized with caveolin in the cell membrane at a resting state, while EPA stimulated the translocation of eNOS to the cytosol and its dissociation from caveolin, to an extent comparable to that of the eNOS translocation induced by a [Ca 2+]i-elevating agonist (10 μM bradykinin). Thus, EPA induces Ca 2+-independent activation and translocation of eNOS and endothelium-dependent vasorelaxation.