Reduced Nitric Oxide Release Research Articles

The capacity of red blood cells to reduce nitrite determines nitric oxide generation under hypoxic conditions.

Nitric oxide (NO) is a key regulator of vascular tone. Endothelial nitric oxide synthase (eNOS) is responsible for NO generation under normoxic conditions. Under hypoxia however, eNOS is inactive and red blood cells (RBC) provide an alternative NO generation pathway from nitrite to regulate hypoxic vasodilation. While nitrite reductase activity of hemoglobin is well acknowledged, little is known about generation of NO by intact RBC with physiological hemoglobin concentrations. We aimed to develop and apply a new approach to provide insights in the ability of RBC to convert nitrite into NO under hypoxic conditions. We established a novel experimental setup to evaluate nitrite uptake and the release of NO from RBC into the gas-phase under different conditions. NO measurements were similar to well-established clinical measurements of exhaled NO. Nitrite uptake was rapid, and after an initial lag phase NO release from RBC was constant in time under hypoxic conditions. The presence of oxygen greatly reduced NO release, whereas inhibition of eNOS and xanthine oxidoreductase (XOR) did not affect NO release. A decreased pH increased NO release under hypoxic conditions. Hypothermia lowered NO release, while hyperthermia increased NO release. Whereas fetal hemoglobin did not alter NO release compared to adult hemoglobin, sickle RBC showed an increased ability to release NO. Under all conditions nitrite uptake by RBC was similar. This study shows that nitrite uptake into RBC is rapid and release of NO into the gas-phase continues for prolonged periods of time under hypoxic conditions. Changes in the RBC environment such as pH, temperature or hemoglobin type, affect NO release.

Differences in Vascular Nitric Oxide and Endothelium-Derived Hyperpolarizing Factor Bioavailability in Blacks and Whites

Abnormalities in nitric oxide (NO) bioavailability have been reported in blacks. Whether there are differences in endothelium-derived hyperpolarizing factor (EDHF) in addition to NO between blacks and whites and how these affect physiological vasodilation remain unknown. We hypothesized that the bioavailability of vascular NO and EDHF, at rest and with pharmacological and physiological vasodilation, varies between whites and blacks. In 74 white and 86 black subjects without known cardiovascular disease risk factors, forearm blood flow was measured using plethysmography at rest and during inhibition of NO with N(G)-monomethyl-L-arginine and of K(+) Ca channels (EDHF) with tetraethylammonium. The reduction in resting forearm blood flow was greater with N(G)-monomethyl-L-arginine (P=0.019) and similar with tetraethylammonium in whites compared with blacks. Vasodilation with bradykinin, acetylcholine, and sodium nitroprusside was lower in blacks compared with whites (all P<0.0001). Inhibition with N(G)-monomethyl-L-arginine was greater in whites compared with blacks with bradykinin, acetylcholine, and exercise. Inhibition with tetraethylammonium was lower in blacks with bradykinin, but greater during exercise and with acetylcholine. The contribution to both resting and stimulus-mediated vasodilator tone of NO is greater in whites compared with blacks. EDHF partly compensates for the reduced NO release in exercise and acetylcholine-mediated vasodilation in blacks. Preserved EDHF but reduced NO bioavailability and sensitivity characterizes the vasculature in healthy blacks. http://clinicaltrials.gov/. Unique identifier: NCT00166166.

Protective effect of the methanol extract from Cryptotaenia japonica Hassk. against lipopolysaccharide-induced inflammation in vitro and in vivo

BackgroundIn folk medicine, the aerial part of Crytotaenia japonica Hassk. (CJ), is applied for treatment of the common cold, cough, urinary problems, pneumonia, and skin rashes. In this paper, the in vitro and in vivo anti-inflammatory activity of CJ methanol extract was tested using lipopolysaccharide (LPS)-induced inflammatory models.MethodsWe measured nitric oxide (NO), inducible NO synthase (iNOS), and inflammatory cytokine levels from LPS-stimulated mouse peritoneal macrophages. Also, several cellular signaling molecules which regulate the expressions of these inflammatory markers were examined. Finally, we tested whether oral administration of CJ methanol extract might affect the serum cytokine levels in LPS-injected mice.ResultsCJ methanol extract reduced NO release via iNOS protein inhibition. The extract was also shown to decrease the secretions of tumor necrosis factor (TNF)-α, interleukin (IL)-6, and IL-12. Analysis of signaling molecules showed that CJ inhibited the phosphorylation of STAT1, p38, JNK and ERK1/2 as well as IκBα degradation. Finally, CJ decreased the serum levels of TNF-α and IL-6 in LPS-injected mice.ConclusionsOur results demonstrated the anti-inflammatory activity of CJ methanol extract and its possible underlying mechanisms that involve modulation of IκBα, MAPK, and STAT1 activities.

109 Overexpression of endothelial insulin-like growth factor-1 receptors (IGF-1R): a novel role for IGF-1R in endothelial function and repair

IntroductionEndothelium-derived nitric oxide (NO) is a critical regulator of vascular homeostasis, repair and regeneration. We recently demonstrated that reducing insulin-like growth factor-1 receptor (IGF-1R) numbers in the endothelium, thereby decreasing...

Paclitaxel-Induced Endothelial Dysfunction in Living Rats Is Prevented by Nicorandil via Reduction of Oxidative Stress

Paclitaxel-eluting stents dramatically reduce rates of in-stent restenosis; however, paclitaxel is known to lead to endothelial dysfunction. Protective effects of nicorandil on paclitaxel-induced endothelial dysfunction by examining flow-mediated dilation (FMD) were investigated in anesthetized rats. After 7-day osmotic infusion of paclitaxel (5 mg/kg per day), FMD was measured by high-resolution ultrasound in the femoral artery of living rats. Paclitaxel significantly reduced FMD (21.6% ± 3.2% to 7.1% ± 1.7%); this reduction was prevented by co-treatment with nicorandil (15 mg/kg per day), while paclitaxel did not affect nitroglycerin-induced vasodilation. Diazoxide and tempol, but not isosorbide dinitrate, had an effect similar to nicorandil in preventing paclitaxel-induced decrease in FMD. Nicorandil significantly prevented paclitaxel-induced reduction in acetylcholine-induced vasodilation. On the underling mechanisms, paclitaxel increased reactive oxygen species (ROS) production (dihydrorhodamine 123, DCF fluorescence intensity) and NADPH oxidase (p47phox, gp91phox mRNA) in arteries and human coronary artery endothelial cells (HCAECs), while paclitaxel reduced nitric oxide (NO) release (DAF-2 fluorescence intensity), but not endothelial NO synthase (eNOS) phosphorylation in HCAECs. Nicorandil prevented the increased ROS production in arteries and HCAECs, which was 5-hydroxydecanoate (5-HD)-sensitive but 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ)-resistant, without significant effect on the reduced NO release. In conclusion, nicorandil prevents paclitaxel-induced endothelial dysfunction, which may be brought by improved NO bioavailability due to the reduction of oxidative stress via KATP channel activation.

Inhibitory effects of palm α-, γ- and δ-tocotrienol on lipopolysaccharide-induced nitric oxide production in BV2 microglia

Anti-inflammatory actions of the vitamin E fragment tocotrienol have not been described for microglia. Here, we screened palm α-, γ- and δ-tocotrienol isoforms and Tocomin® 50% (contains spectrum of tocotrienols and tocopherols) for their ability to limit nitric oxide (NO) production by BV2 microglia. Microglia were treated with varying doses of tocotrienols for 24 h and stimulated with 1 μg/ml lipopolysaccharide (LPS). All tocotrienol isoforms reduced NO release by LPS-stimulated microglia, with 50 μM being the most potent tocotrienol dose. Of the isoforms tested, δ-tocotrienol lowered NO levels the most, reducing NO by approximately 50% at 48 h post-LPS treatment ( p < .05). None of the tocotrienol doses tested affected microglia viability.

Anti-inflammatory effects of crocin and crocetin in rat brain microglial cells

Microglial cells play critical roles in the immune and inflammatory responses of the central nervous system (CNS). Under pathological conditions, the activation of microglia helps in restoring CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that observed in Alzheimer's and Parkinson's diseases. Crocin and crocetin, found in the fruits of gardenia and in the stigmas of saffron, have been considered for the treatment of various disorders in traditional oriental medicine. Crocin and crocetin have been reported to have diverse pharmacological functions, such as anti-hyperlipidemic, anti-atherosclerotic, and anti-cancer effects. Specifically, the neuroprotective potential of crocetin derivatives has previously been demonstrated. The specific aim of this study was to examine whether crocin or crocetin represses microglial activation. Crocin and crocetin were shown to be effective in the inhibition of LPS-induced nitric oxide (NO) release from cultured rat brain microglial cells. These compounds reduced the LPS-stimulated productions of tumor necrosis factor-α, interleukin-1β, and intracellular reactive oxygen species. The compounds also effectively reduced LPS-elicited NF-κB activation. In addition, crocin reduced NO release from microglia stimulated with interferon-γ and amyloid-β. In organotypic hippocampal slice cultures, both crocin and crocetin blocked the effect of LPS on hippocampal cell death. These results suggest that crocin and crocetin provide neuroprotection by reducing the production of various neurotoxic molecules from activated microglia.

Palmitate induces C-reactive protein expression in human aortic endothelial cells. Relevance to fatty acid–induced endothelial dysfunction

Circulating levels of free fatty acids are commonly elevated in patients with the metabolic syndrome and exert, through activating proinflammatory pathways, harmful effects of the vascular endothelium. In this study, we examined the effect of palmitate (PA) on endothelial C-reactive protein (CRP) expression and the role of CRP in PA-induced nitric oxide (NO) inhibition. Palmitate increased, in a dose-dependent manner, CRP protein expression and production in human aortic endothelial cells (HAECs). Induction of CRP protein was mimicked by ceramide, whereas bromopalmitate and other common free fatty acids such as oleate or linoleate were ineffective. Palmitate also elicited reactive oxygen species production in HAECs, an effect prevented by protein kinase C (PKC) inhibition and adenosine monophosphate–activated kinase (AMPK) activation. Palmitate-treated HAECs showed increased CRP messenger RNA expression and nuclear factor (NF)– κB activation. Induction of CRP expression by PA was prevented by antioxidants and normalized by PKC and mitogen-activated protein kinase inhibitors. Disrupting NF- κB and Janus kinase/signal transducers and activators of transcription pathways or inducing AMPK activation also suppressed the stimulatory effect of PA on CRP messenger RNA expression. Finally, in HAECs, PA reduced NO release, an effect reversed by anti-CRP antibody. These data demonstrate that PA-induced endothelial CRP expression involves PKC-driven oxidative stress, possibly through AMPK inhibition, and activation of downstream redox-sensitive signaling pathways, including NF- κB. They further support a role for endothelial cell–derived CRP as mediator of the suppressive effect of PA on NO production.

Cellular resistance to a nitric oxide releasing glutathione S-transferase P-activated prodrug, PABA/NO

PABA/NO is a diazeniumdiolate selectively activated by glutathione S-transferase P (GSTP) to release nitric oxide (NO) and is a potent inducer of protein S-glutathionylation, a redox-sensitive post-translational modification of cysteine residues. Using a procedure that incrementally increased exposure of cells to PABA/NO, an acquired drug resistant human promyelocytic leukemia HL60 cell line (HL60(PABA)) that exhibited 1.9-fold resistance to the drug (IC(50) 15 μM vs ~8 μM for wild-type) was created. HL60(PABA) cells had a decreased growth rate attributable to altered cellular differentiation, as measured by increased expression of CD11b; decreased expression of CD14; decreased nuclear to cytoplasmic ratios and a condensation of nuclear chromatin. This was accompanied by alterations in both plasma and mitochondrial membrane potentials. Both GSTP expression and nitric oxide release were reduced two-fold, while increased expression levels of genes involved in the unfolded protein response (UPR) were evident in HL60(PABA) cells. Wild type cells treated with PABA/NO had increased levels of protein S-glutathionylation and JNK activation, while JNK was constitutively active in HL60(PABA) cells and these cells had reduced levels of S-glutathionylation. By removing PABA/NO from the growth medium, HL60(PABA) cells reverted to sensitivity within 21 days suggesting that resistance was not genetically stable. Mechanistically, PABA/NO resistance is mediated through reduced levels of GSTP resulting in reduced NO release and its subsequent alterations in cellular response to nitrosative stress.

Genipin inhibits the inflammatory response of rat brain microglial cells

Microglia are the prime effectors in immune and inflammatory responses of the central nervous system (CNS). Under pathological conditions, the activation of these cells helps restore CNS homeostasis. However, chronic microglial activation endangers neuronal survival through the release of various proinflammatory and neurotoxic factors. Thus, negative regulators of microglial activation have been considered as potential therapeutic candidates to target neurodegeneration, such as that in Alzheimer's and Parkinson's diseases. Genipin, the aglycon of geniposide found in gardenia fruit has long been considered for treatment of various disorders in traditional oriental medicine. Genipin has recently been reported to have diverse pharmacological functions, such as antimicrobial, antitumor, and anti-inflammatory effects. The specific aim of this study was to examine whether genipin represses brain microglial activation. Genipin was effective at inhibiting LPS-induced nitric oxide (NO) release from cultured rat brain microglial cells. Genipin reduced the LPS-stimulated production of tumor necrosis factor-α, interleukin-1β, prostaglandin E 2, intracellular reactive oxygen species, and NF-κB activation. In addition, genipin reduced NO release from microglia stimulated with interferon-γ and amyloid-β. Both pretreatment and post-treatment of genipin to LPS-stimulated microglia were effective at decreasing NO release. Furthermore, genipin effectively inhibited microglial activation in a mouse model of brain inflammation. These results suggest that genipin provide neuroprotection by reducing the production of various neurotoxic molecules from activated microglia.

The selective serotonin reuptake inhibitor fluoxetine reduces striatal in vivo levels of voltammetric nitric oxide (NO): A feature of its antidepressant activity?

Voltammetric (electrochemical) methodologies such as differential pulse voltammetry and amperometry used together with electrically and chemically treated carbon fibre micro-electrodes (mCFE) allow selective monitoring of nitric oxide (NO). Preliminary in vitro studies have shown that the selective serotonin reuptake inhibitor (SSRI) antidepressant paroxetine inhibits constitutive nitric oxide synthase (cNOS) activity in animals and humans and that another SSRI such as fluoxetine reduced NO release in the media of synovial cells. The aim of this work was to verify by means of amperometry and specifically treated mCFE the capability of fluoxetine to alter the in vivo release of central NO, in the attempt to further clarify such putative antidepressant mechanism of action of this SSRI compound. The in vivo results support the chemical NO related nature of the endogenous amperometric signal evoked by NMDA injection in the striatum of anaesthetised rats, as pre-treatment with NOS inhibitor l-NAME prevented its appearance. Subsequently fluoxetine treatment resulted in decreased striatal NO, further supporting in vitro studies proposing a link between the serotonergic system and the NO system.

Advanced glycation endproducts increase EPC apoptosis and decrease nitric oxide release via MAPK pathways

Objective Previous studies have shown that advanced glycation endproducts (AGE) can induce endothelial progenitor cells (EPC) apoptosis, which contributes to the pathogenesis of diabetes mellitus. Nitric oxide (NO) signaling is closely associated with apoptosis. We therefore investigated the effects of AGE on human EPC apoptosis, NO release and related signal transduction pathways. Methods EPC isolated from healthy human subjects were cultured with various concentrations of AGE (0, 2, 20 and 200 mg/L) for 0, 24, 48 and 72 h in the presence or absence of various MAPK (ERK/P38/JNK) inhibitors, respectively. EPC apoptosis (detected by flow cytometric analyses) and NO concentration in culture supernatant were determined. The mRNA levels of eNOS, COX-2, Bcl-2 and Bax were assessed by RT-PCR and the protein expressions of NF-κB and Caspase-3 assessed by Western blot. Results Increased EPC apoptosis and reduced NO release were induced by 200 mg/L AGE, accompanied by a downregulation of eNOS and Bcl-2 expressions as well as an elevation in COX-2, Bax, NF-κB and Caspase-3 expressions in a time-dependent manner (all P < 0.05). These changes were significantly attenuated by pretreatment with various MAPK (ERK/P38/JNK) inhibitors ( P < 0.05). Conclusions AGE can promote EPC apoptosis and decrease NO release via MAPK pathways.

Antinociceptive and anti-inflammatory activities of Aquilaria sinensis (Lour.) Gilg. Leaves extract

Aim of the study The analgesic and anti-inflammatory activities of the ethanol extract of Aquilaria sinensis (Lour.) Gilg. Leaves were observed in various experimental models related to nociception and inflammation, so as to provide some evidence for its traditional use. Materials and methods Acetic acid-induced writhing and a hot plate test in mice were used to evaluate its analgesic activity. On the other hand, its anti-inflammatory activity was observed in xylene or carrageenan-induced edema, carboxymethylcellulose sodium (CMC-Na)-induced leukocyte migration in mice and lipopolysaccharide (LPS)-induced nitric oxide (NO) release from mouse peritoneal macrophages in vitro. Results The ethanol extract significantly inhibited acetic acid-induced writhing after single oral administration at doses of 424 and 848 mg extract/kg, and the response to the thermal stimulus in mice at the dose of 848 mg/kg. Meanwhile, the ethanol extract also remarkably lessened xylene-induced ear swelling, carrageenan-induced paw edema, and CMC-Na-induced leukocyte migration. Furthermore, the extract considerably reduced NO release from LPS-stimulated macrophages with IC 50 of 80.4 mg/ml. Conclusion These findings suggest that Aquilaria sinensis (Lour.) Gilg. Leaves extract present notable analgesic and anti-inflammatory activities, which support its folkloric use for some diseases related with painful and inflammatory conditions such as trauma etc.

The methionine connection: Homocysteine and hydrogen sulfide exert opposite effects on hepatic microcirculation in rats

Increased intrahepatic resistance in cirrhotic livers is caused by endothelial dysfunction and impaired formation of two gaseous vasodilators, nitric oxide (NO) and hydrogen sulfide (H(2)S). Homocysteine, a sulfur-containing amino acid and H(2)S precursor, is formed from hepatic methionine metabolism. In the systemic circulation, hyperhomocystenemia impairs vasodilation and NO production from endothelial cells. Increased blood levels of homocysteine are common in patients with liver cirrhosis. In this study, we demonstrate that acute liver perfusion with homocysteine impairs NO formation and intrahepatic vascular relaxation induced by acetylcholine in methoxamine-precontracted normal livers (7.3% +/- 3.0% versus 26% +/- 2.7%; P < 0.0001). In rats with mild, diet-induced hyperhomocystenemia, the vasodilating activity of acetylcholine was markedly attenuated, and incremental increases in flow induced a greater percentage of increases in perfusion pressure than in control livers. Compared with normal rats, animals rendered cirrhotic by 12 weeks' administration of carbon tetrachloride exhibited a greater percentage of increments in perfusion pressure in response to shear stress (P < 0.05), and intrahepatic resistance to incremental increases in flow was further enhanced by homocysteine (P < 0.05). In normal hyperhomocysteinemic and cirrhotic rat livers, endothelial dysfunction caused by homocysteine was reversed by perfusion of the livers with sodium sulfide. Homocysteine reduced NO release from sinusoidal endothelial cells and also caused hepatic stellate cell contraction; this suggests a dual mechanism of action, with the latter effect being counteracted by H(2)S. Impaired vasodilation and hepatic stellate cell contraction caused by homocysteine contribute to the dynamic component of portal hypertension.

Antioxidant, anti-inflammatory and anticancer activities of methanolic extracts from Ledum groenlandicum Retzius

Labrador tea ( Ledum groenlandicum Retzius) is an ericaceae widely distributed in North America. The leaves and twigs were used in Native American traditional medicine to treat several inflammatory pathologies such as asthma, rheumatisms and burns. Reactive oxygen species as well as reactive nitrogen species such as nitric oxide (NO) contribute significantly to these pathologies. In this study, the antioxidant, anti-inflammatory and anticancer activities of crude methanol extracts of leaves and twigs from Ledum groenlandicum were investigated. Both extracts showed a strong antioxidant activity using the ORAC method and a cell based-assay. Moreover, the twig and leaf extracts showed significant anti-inflammatory activity, inhibiting NO release, respectively, by 28 and 17% at 25 μg/ml in LPS-stimulated RAW 264.7 macrophages. In comparison, N(G)-nitro- l-arginine methyl ester (L-NAME), a nitric oxide synthase inhibitor, reduced NO release by 24% at 25 μg/ml. The twig extract was also found to be active against DLD-1 colon carcinoma and A-549 lung carcinoma cells, with IC 50 values of 43 ± 1 and 65 ± 8 μg/ml, respectively. The bioguided study of the twig extract resulted in the isolation and identification of ursolic acid, a known triterpene. Ursolic acid was active against DLD-1 (IC 50: 9.3 ± 0.3 μM) and A-549 (IC 50: 8.9 ± 0.2 μM), suggesting it is, in part, responsible of the anticancer activity of the twig extract.

Intracellular location regulates calcium-calmodulin-dependent activation of organelle-restricted eNOS

Mislocalization of endothelial nitric oxide (NO) synthase (eNOS) in response to oxidized low-density lipoprotein, cholesterol depletion, elevated blood pressure, and bound eNOS interacting protein/NOS traffic inducer is associated with reduced NO release via unknown mechanisms. The proper targeting of eNOS to the plasma membrane or intracellular organelles is an important regulatory step controlling enzyme activity. Previous studies have shown that plasma membrane eNOS is constitutively phosphorylated on serine 1179 and highly active. In contrast, the activity of eNOS targeted to intracellular organelles is more complex. The cis-Golgi eNOS is fully activated by Akt-dependent phosphorylation. However, eNOS targeted to the trans-Golgi is decidedly less active in response to all modes of activation, including mutation to the phosphomimetic aspartic acid. In this study, we establish that when expressed within other intracellular organelles, such as the mitochondria and nucleus, the activity of eNOS is also greatly reduced. To address the mechanisms underlying the impaired catalytic activity of eNOS within these locations, we generated subcellular-targeted constructs that express a calcium-independent NOS isoform, iNOS. With the use of organelle specific (plasma membrane, cis- vs. trans-Golgi, plasma membrane, and Golgi, nucleus, and mitochondria) targeting motifs fused to the wild-type iNOS, we measured NO release from intact cells. With the exception of the Golgi lumen, our results showed no impairment in the ability of targeted iNOS to synthesize NO. Confirmation of correct targeting was obtained through confocal microscopy using identical constructs fused to the green fluorescent protein. We conclude that the reduced activation of eNOS within discrete cytoplasmic regions of the Golgi, the mitochondria and the nucleus is primarily due to insufficient access to calcium-calmodulin.

Inhibition of inducible nitric oxide synthase expression by yuccaol C from Yucca schidigera roezl

Yucca schidigera extract finds wide commercial application in foods, cosmetics and pharmaceuticals. In a previous paper we have found as the main constituents of yucca bark, yuccaol A, B and C, new and very unusual spiro-derivatives made up of a C 15 unit and a stilbenic portion closely related to resveratrol. This study was performed to examine whether yuccaol A, B or C (0.01–100 μM) could affect cytosolic inducible nitric oxide synthase (iNOS) protein expression and nitric oxide (NO) generation in vitro in Escherichia coli lipopolysaccharide (LPS)-activated J774.A1 macrophage cell line. NO production, detected as NO 2 −, increased significantly in LPS treated J774.A1 cells from 0.05 ± 0.03 μM to 16.64 ± 0.58 μM (P < 0.001). Yuccaol C (0.01–100 μM), added to the culture medium 1 h before LPS-stimulation, significantly (P < 0.001) and in a concentration related manner reduced NO release (P < 0.001) and iNOS protein expression (P < 0.05). In contrast, no inhibitory effect either on iNOS protein expression or on NO release was observed when yuccaol C was added after LPS stimulation. In contrast yuccaol A inhibited significantly (P < 0.001) only NO release at the highest concentration tested (100 μM) while yuccaol B did not reduce either NO release or iNOS expression. Yuccaol C was demonstrated to reduce iNOS protein expression via the transcription factor NF-κB. These results indicated that the empirical use of Y. schidigera as anti-inflammatory remedy could be addressed not only to the resveratrol content but also to the presence of yuccaol C.

Food restriction attenuates age-related increase in the sensitivity of endothelial cells to oxidized lipids.

Age-related endothelial dysfunction has been suggested to play a role in atherogenesis. Food restriction (FR) has been shown to retard the development of atherosclerosis. The goal of this report is to assess the effect of aging and FR on endothelial functions, including the release of endothelial nitric oxide (NO) and the adhesion of mononuclear cells (MNCs) to endothelial cells (ECs). ECs were obtained from the aorta of young mice fed ad libitum (Y-AL), old mice fed ad libitum (O-AL), or a food-restricted diet (O-FR). When compared with those obtained from Y-AL and O-FR mice, ECs obtained from O-AL mice decreased the basal level of NO release and increased the basal level of peroxynitrite, superoxide, and hydrogen peroxide. In addition, ECs obtained from O-AL elevated the response to CuSO4-oxidized low-density lipoprotein (oxLDL). For example, incubation with oxLDL reduced NO release approximately 52% in ECs obtained from O-AL mice. In contrast, the same dose of oxLDL reduced NO release only approximately 40% in ECs obtained from Y-AL and O-FR mice. Moreover, the level of oxLDL-induced adhesion of MNCs and oxLDL-induced expression of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 was markedly higher in ECs obtained from O-AL mice as compared with those obtained from Y-AL and O-FR mice. These results suggest that aging increases the sensitivity of ECs in response to oxLDL-reduced endothelial NO release and oxLDL-increased adhesion of MNCs to ECs. FR attenuates age-related increase in the sensitivity of ECs to oxLDL, which might be responsible, at least in part, for the antiatherogenic action of FR.

Phytoestrogen α-Zearalanol Antagonizes Oxidized LDL-Induced Inhibition of Nitric Oxide Production and Stimulation of Endothelin-1 Release in Human Umbilical Vein Endothelial Cells

Oxidative modification of low-density lipoprotein (LDL) leads to formation of the atherogenic molecule oxidized LDL (oxLDL), which is considered to be an important mediator for vascular endothelial dysfunction and atherosclerosis. It is speculated that reduced nitric oxide (NO) release/bioavailability and enhanced release of endothelin-1 (ET-1) may contribute to oxLDL-induced endothelial dysfunction. Estrogen may improve lipid profile and inhibit oxLDL-induced endothelial damage. However, estrogen replacement therapy has been suspended due to uncertainty in benefits versus risk (such as cancer progression) in postmenopausal women. This study was designed to evaluate the effect of a novel phytoestrogen, alpha-zearalanol (alpha-ZAL), on oxLDL-induced effect on NO and ET-1 production in human umbilical vein endothelial cells (HUVEC). HUVEC were incubated with oxLDL (50 microg/mL) for 24 h in the absence or presence of alpha-ZAL (0-1000 nM), 17beta-estradiol (E2, 10 nM), or the E2 receptor antagonist ICI182780 (1 microM). Levels of NO and ET-1 were measured by spectrophotometry and enzymatic immunoassay, respectively. NOS activity was evaluated by conversion of 3H-arginine to 3H-citrulline. Protein and mRNA expression of NOS and ET-1 were measured by Western blot and RT-PCR. Our results indicated that oxLDL significantly reduced NO release and NOS activity, and enhanced ET-1 pro-duction associated with reduced NOS3 (but not NOS2) expression and enhanced ET-1 mRNA expression. All these oxLDL-induced alterations were significantly attenuated or abolished by co-incubation with alpha-ZAL or E2, both through an E2 receptor-dependent mechanism. alpha-ZAL, E2, and ICI182780 had no effect on NO/ET-1 release, NOS activity, or expression of NOS and ET-1. These data suggested that the phytoestrogen alpha-ZAL, like E2, may effectively antagonize oxLDL-induced decrease in NO and increase in ET-1, which may be protective for endothelial function.

The dual effects of nitric oxide synthase inhibitors on ischemia-reperfusion injury in rat hearts.

Nitric oxide (NO) is known to act as a mediator of tissue injury as well as being a potent endogenous vasodilator. The functional and metabolic effects of NO on ischemia-reperfusion injury are still controversial. The aim of this study was to clarify the relationship between the degree of NO synthase (NOS) inhibition and the effects on ischemia-reperfusion injury. Langendorff-perfused rat hearts were subjected to 30 minutes of global ischemia followed by 30 minutes of reperfusion. The recovery of left ventricular developed pressure (LVDP), creatine kinase (CK) release, and myocardial high energy phosphates were measured in hearts perfused with or without NOS inhibitors, L-N(G)-monomethyl arginine (L-NMMA) or N(G)nitro-L-arginine methylester (L-NAME). NOS inhibitors exerted different effects on the recovery of LVDP and CK release depending on the concentration. The low dose of L-NMMA improved the recovery of LVDP, decreased the CK release during reperfusion, and preserved the myocardial adenosine triphosphate content after reperfusion. In contrast, the high dose of L-NMMA had adverse effects. L-NMMA reduced NO release in coronary effluent in a dose-dependent fashion. Both effects of L-NMMA were abolished by excessive co-administration of L-arginine and the same doses of D-N(G)-monomethyl arginine (D-NMMA) showed no effect on ischemia-reperfusion injury. Therefore, both effects were due to NOS inhibition. In addition, L-NMMA suppressed the myocardial malondialdehyde accumulation, an indicator of oxidative stress, which might be attributed to the beneficial effects by partial NOS inhibition. On the other hand, the high dose L-NMMA significantly decreased coronary fl ow during aerobic perfusion and reperfusion. Therefore, it is conceivable that the vasoactive NOS inhibition contributes to the harmful effects, which might exceed the beneficial effects due to a decrease in oxidative stress. The present results showed that NO inhibitors had dual effects on mechanical function and energy metabolism depending on the concentration. Non-vasoactive inhibition of NOS had beneficial effects due to the suppression of oxidative injury. However, strong vasoactive inhibition of NOS exacerbated the ischemia-reperfusion injury.