Expression and roles of Cl- channel ClC-5 in cell cycles of myeloid cells

This study investigated the effect of exogenous nitric oxide (NO) on endothelial glucocorticoid receptor (GR) function. The NO donor diethylenetriamine NONOate (DETA, 50-500microM) caused concentration dependent nuclear localization of transfected chimeric green fluorescent protein GFP-GR and elevated expression of secreted alkaline phosphatase (SEAP) from a glucocorticoid response element (GRE) promoter construct in bovine aortic endothelial cells. Other weaker NO donors (S-nitroso-N-acetylpenicillamine and spermine NONOate) failed to induce GFP-GR nuclear localization, but all the NO donors activated GRE-SEAP expression, a response unaffected by the antioxidant N-acetyl-L-cysteine. Overall, exogenous NO from high concentration donors can directly activate GR, suggesting a potential feedback mechanism for NO to regulate endothelial inducible nitric oxide synthase (iNOS) expression.     

Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction

Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.     

Role of cGMP-dependent protein kinase in development of tolerance to nitric oxide in pulmonary veins of newborn lambs

Continuous exposure to nitrovasodilators and nitric oxide induces tolerance to their vasodilator effects in vascular smooth muscle. This study was done to determine the role of cGMP-dependent protein kinase (PKG) in the development of tolerance to nitric oxide. Isolated fourth-generation pulmonary veins of newborn lambs were studied. Incubation of veins for 20 h with DETA NONOate (DETA NO; a stable nitric oxide donor) significantly reduced their relaxation response to the nitric oxide donor and to beta-phenyl-1,N2-etheno-8-bromo-cGMP (8-Br-PET-cGMP, a cell-permeable cGMP analog). Incubation with DETA NO significantly reduced PKG activity and protein and mRNA levels in the vessels. These effects were prevented by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (an inhibitor of soluble guanylyl cyclase) and Rp-8-Br-PET-cGMPS (an inhibitor of PKG). A decrease in PKG protein and mRNA levels was also observed after continuous exposure to cGMP analogs. The PKG inhibitor abrogated these effects. The decrease in cGMP-mediated relaxation and in PKG activity caused by continuous exposure to DETA NO was not affected by KT-5720, an inhibitor of cAMP-dependent protein kinase. Prolonged exposure to 8-Br-cAMP (a cell-permeable cAMP analog) did not affect PKG protein level in the veins. These results suggest that continuous exposure to nitric oxide or cGMP downregulates PKG by a PKG-dependent mechanism. Such a negative feedback mechanism may contribute to the development of tolerance to nitric oxide in pulmonary veins of newborn lambs.     

Mechanism of inhibition of matrix metalloproteinase-9 induction by NO in vascular smooth muscle cells.

Vascular smooth muscle (VSM) cell migration is a critical step in the development of a neointima after angioplasty. Matrix metalloproteinases (MMPs) degrade the basement membrane and extracellular matrix, facilitating VSM cell migration. Recently, we demonstrated that nitric oxide (NO) inhibits interleukin-1 beta (IL-1 beta)-stimulated MMP-9 induction in rat aortic VSM cells. In this study, we examined the hypothesis that NO inhibits MMP-9 induction by attenuating superoxide generation and extracellular signal-regulated kinase (ERK) activation. Stimulation of VSM cells with IL-1 beta significantly (P < 0.05) increased superoxide production, ERK activation, and MMP-9 induction. Pretreatment of VSM cells with the NO donor DETA NONOate significantly (P < 0.05) decreased IL-1 beta-stimulated superoxide generation. In addition, pretreatment of VSM cells with a specific ERK pathway inhibitor, PD-98059, or DETA NONOate inhibited IL-1 beta-stimulated ERK activation and MMP-9 induction. Direct exposure of VSM cells to increased superoxide levels by treatment with xanthine/xanthine oxidase increased ERK activation and MMP-9 induction, whereas pretreatment of cells with PD-98059 significantly (P < 0.05) inhibited xanthine/xanthine oxidase-stimulated ERK activation and MMP-9 induction. We conclude that NO inhibits IL-1 beta-stimulated MMP-9 induction by inhibiting superoxide generation and subsequent ERK activation.     

Reactive oxygen nitrogen species decrease cystic fibrosis transmembrane conductance regulator expression and cAMP-mediated Cl- secretion in airway epithelia

We investigated putative mechanisms by which nitric oxide modulates cystic fibrosis transmembrane conductance regulator (CFTR) expression and function in epithelial cells. Immunoprecipitation followed by Western blotting, as well as immunocytochemical and cell surface biotinylation measurements, showed that incubation of both stably transduced (HeLa) and endogenous CFTR expressing (16HBE14o-, Calu-3, and mouse tracheal epithelial) cells with 100 microm diethylenetriamine NONOate (DETA NONOate) for 24-96 h decreased both intracellular and apical CFTR levels. Calu-3 and mouse tracheal epithelial cells, incubated with DETA NONOate but not with 100 microm 8-bromo-cGMP for 96 h, exhibited reduced cAMP-activated short circuit currents when mounted in Ussing chambers. Exposure of Calu-3 cells to nitric oxide donors resulted in the nitration of a number of proteins including CFTR. Nitration was augmented by proteasome inhibition, suggesting a role for the proteasome in the degradation of nitrated proteins. Our studies demonstrate that levels of nitric oxide that are likely to be encountered in the vicinity of airway cells during inflammation may nitrate CFTR resulting in enhanced degradation and decreased function. Decreased levels and function of normal CFTR may account for some of the cystic fibrosis-like symptoms that occur in chronic inflammatory lung diseases associated with increased NO production.     

Nitric oxide-dependent killing of aerobic, anaerobic and persistent Burkholderia pseudomallei

Burkholderia pseudomallei infections are fastidious to treat with conventional antibiotic therapy, often involving a combination of drugs and long-term regimes. Bacterial genetic determinants contribute to the resistance of B. pseudomallei to many classes of antibiotics. In addition, anaerobiosis and hypoxia in abscesses typical of melioidosis select for persistent populations of B. pseudomallei refractory to a broad spectrum of antibacterials. We tested the susceptibility of B. pseudomallei to the drugs hydroxyurea, spermine NONOate and DETA NONOate that release nitric oxide (NO). Our investigations indicate that B. pseudomallei are killed by NO in a concentration and time-dependent fashion. The cytoxicity of this diatomic radical against B. pseudomallei depends on both the culture medium and growth phase of the bacteria. Rapidly growing, but not stationary phase, B. pseudomallei are readily killed upon exposure to the NO donor spermine NONOate. NO also has excellent antimicrobial activity against anaerobic B. pseudomallei. In addition, persistent bacteria highly resistant to most conventional antibiotics are remarkably susceptible to NO. Sublethal concentrations of NO inhibited the enzymatic activity of [4Fe-4S]-cofactored aconitase of aerobic and anaerobic B. pseudomallei. The strong anti-B. pseudomallei activity of NO described herein merits further studies on the application of NO-based antibiotics for the treatment of melioidosis.     

The effect of aspirin and two nitric oxide donors on the infarcted heart in situ

Nitric oxide (NO) donors are heterogeneous substances which release NO, a biologically active compound. NO released by nitric oxide donors has important effects on the circulation by causing vasodilation, diminishing myocardial contractile force, inhibiting platelet aggregation, and counteracting the effects of thromboxane A2. In the infarcted heart, activation of the inducible form of nitric oxide synthase (iNOS) and the formation of prostacyclin and thromboxane A2 by cyclooxygenase (COX) were increased. Myocardial infarction also resulted in increased myocardial NO production. Aspirin (acetylsalicylic acid. ASA) at low concentration (35 mg/kg/day) fails to change iNOS production, in contrast to higher dose (150 mg/kg/day) which, as previously shown, inhibits iNOS activity. ASA at all doses also suppresses myocardial prostanoid formation because of inhibition of COX. Recently, two NO donors have been synthesized: NCX 4016 and Diethylenetriamine/NO (DETA/NO). NCX 4016 combines an NO-releasing moiety with a carboxylic residue via an esteric bond. We describe here that NCX 4016 (65 mg/kg/day) increased prostacyclin and thromboxane A2 production in the infarcted heart muscle, overcoming the inhibitory effects of ASA. As a result of nitric oxide release, oxidation products of NO (NO2- and NO3-; NOx) in arterial blood rose following administration of NCX 4016. On oral administration, NCX 4016 did not change systemic arterial pressure. The effects of a single NO donor, DETA/NO (1.0 mg/kg/day) on the infarcted heart were also investigated On intravenous administration, the compound increased NO concentration in arterial blood slightly but to a lesser degree than NCX 4016. Like NCX 4016, it raised myocardial production of prostacyclin and thromboxane A2 in the infarcted heart. However, it caused a severe fall in blood pressure. These findings demonstrate that newly-synthesized NO donors release nitric oxide in situ and increase myocardial production of prostanoids. NCX 4016 has therapeutic potential because it can be orally administered, lacks hypotensive effects, increases blood levels of nitric oxide and myocardial prostacyclin production.     

Differential role of nitric oxide in regional sympathetic responses to stimulation of NTS A2a adenosine receptors

Our previous studies showed that preganglionic adrenal (pre-ASNA), renal (RSNA), lumbar, and postganglionic adrenal sympathetic nerve activities (post-ASNA) are inhibited after stimulation of arterial baroreceptors, nucleus of the solitary tract (NTS), and glutamatergic and P2x receptors and are activated after stimulation of adenosine A1 receptors. However, stimulation of adenosine A2a receptors inhibited RSNA and post-ASNA, whereas it activated pre-ASNA. Because the effects evoked by NTS A2a receptors may be mediated via activation of nitric oxide (NO) mechanisms in NTS neurons, we tested the hypothesis that NO synthase (NOS) inhibitors would attenuate regional sympathetic responses to NTS A2a receptor stimulation, whereas NO donors would evoke contrasting responses from pre-ASNA versus RSNA and post-ASNA. Therefore, in chloralose/urethane-anesthetized rats, we compared hemodynamic and regional sympathetic responses to microinjections of selective A2a receptor agonist (CGS-21680, 20 pmol/50 nl) after pretreatment with NOS inhibitors Nomega-nitro-L-arginine methyl ester (10 nmol/100 nl) and 1-[2-(trifluoromethyl)phenyl]imidazole (100 pmol/100 nl) versus pretreatment with vehicle (100 nl). In addition, responses to microinjections into the NTS of different NO donors [40 and 400 pmol/50 nl sodium nitroprusside (SNP); 0.5 and 5 nmol/50 nl 3,3-bis(aminoethyl)-1-hydroxy-2-oxo-1-triazene (DETA NONOate, also known as NOC-18), and 2 nmol/50 nl 3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine (PAPA NONOate, also known as NOC-15)], the NO precursor L-arginine (10-50 nmol/50 nl), and sodium glutamate (500 pmol/50 nl) were evaluated. SNP, DETA NONOate, and PAPA NONOate activated pre-ASNA and inhibited RSNA and post-ASNA, whereas l-arginine and glutamate microinjected into the same site of the NTS inhibited all these sympathetic outputs. Decreases in heart rate and depressor or biphasic responses accompanied the neural responses. Pretreatment with NOS inhibitors reversed the normal depressor and sympathoinhibitory responses to stimulation of NTS A2a receptors into pressor and sympathoactivatory responses and attenuated the heart rate decreases; however, it did not change the increases in pre-ASNA. We conclude that NTS NO mechanisms differentially affect regional sympathetic outputs and differentially contribute to the pattern of regional sympathetic responses evoked by stimulation of NTS A2a receptors.     

Physiological and pathophysiological role of ion channels and transporters in the colorectum and colorectal cancer

The incidence of colorectal cancer has increased annually, and the pathogenesis of this disease requires further investigation. In normal colorectal tissues, ion channels and transporters maintain the water‐electrolyte balance and acid/base homeostasis. However, dysfunction of these ion channels and transporters leads to the development and progression of colorectal cancer. Therefore, this review focuses on the progress in understanding the roles of ion channels and transporters in the colorectum and in colorectal cancer, including aquaporins (AQPs), Cl^? channels, Cl^?/ exchangers, Na⁺/ transporters and Na⁺/H⁺ exchangers. The goal of this review is to promote the identification of new targets for the treatment and prognosis of colorectal cancer.     

Response of mitochondrial antioxidant system and respiratory pathways to reactive nitrogen species in pea leaves

Nitric oxide (NO) has emerged as an important signaling molecule in plants, but little is known about the effects of reactive nitrogen species in plant mitochondria. In this study, the effects of DETA‐NONOate, a pure NO slow generator, and of SIN‐1 (3‐morpholinosydnonimine), a peroxynitrite producer, on the activities of respiratory pathways, enzymatic and non‐enzymatic antioxidants have been investigated in isolated mitochondria from pea leaves. No significant changes in lipid peroxidation, protein oxidation or in ascorbate and glutathione redox state were observed after DETA‐NONOate treatments whereas cytochrome pathway (CP) respiration was reversibly inhibited and alternative pathway (AP) respiration showed little inhibition. On the other hand, NO did not affect neither activities of Mn superoxide dismutase (Mn‐SOD) nor enzymes involved in the ascorbate and glutathione regeneration in mitochondria except for ascorbate peroxidase (APX), which was reversely inhibited depending on ascorbate concentration. Finally, SIN‐1 treatment of mitochondria produced a decrease in CP respiration, an increase in protein oxidation and strongly inhibited APX activity (90%), with glutathione reductase and dehydroascorbate reductase (DHAR) being moderately inhibited (30 and 20%, respectively). This treatment did not affect monodehydroascorbate reductase (MDHAR) and Mn‐SOD activities. Results showed that mitochondrial nitrosative stress was not necessarily accompanied by oxidative stress. We suggest that NO‐resistant AP and mitochondrial APX may be important components of the H₂O₂‐signaling pathways under nitrosative stress induced by NO in this organelle. Also, MDHAR and DHAR, via ascorbate regeneration, could constitute an essential antioxidant defense together with Mn‐SOD, against NO and ONOO^? stress in plant mitochondria.     

Modulation of mast cell activity by a peptide agonist of the thrombin receptor: role of nitric oxide.

The effect of a thrombin receptor agonist peptide (TRAP-6) on the release of nitric oxide (NO) and platelet activating factor (PAF) from resting and calcium-ionophore (A23187)-activated rat peritoneal mast cells (RPMC) was studied using a platelet aggregation bioassay. RPMC spontaneously released NO, which inhibited TRAP-6-, ADP-, and PAF-stimulated platelet aggregation. This effect of NO was abolished by the addition of an NO binding agent, oxyhemoglobin (oxyHb), to the platelet suspension. The RPMC-induced suppression of platelet aggregation was completely inhibited by the NO-synthase inhibitor L-NAME. TRAP-6 and its high affinity analog haTRAP stimulated the rapid release of NO from RPMC. The effect of TRAP-6 was inhibited by pretreatment of the RPMC with L-NAME or with the inhibitor of the constitutive NO-synthase isoform (cNOS) calmidazolium. TRAP-6 inhibited PAF release from A23187-activated RPMC via an NO-dependent mechanism. Platelet aggregation induced by PAF release from activated RPMC was also confirmed in experiments using the PAF receptor antagonist ginkgolide B. Thus, TRAP-6 is a rapidly acting modulator of mast cell reactivity; it stimulates NO release and inhibits PAF secretion.     

Adiponectin inhibits hyperlipidemia-induced platelet aggregation via attenuating oxidative/nitrative stress

Adiponectin acts as an endogenous antithrombotic factor. However, the mechanisms underlying the inhibition of platelet aggregation by adiponectin still remain elusive. The present study was designed to test whether adiponectin inhibits platelet aggregation by attenuation of oxidative/nitrative stress. Adult rats were fed a regular or high-fat diet for 14 weeks. The platelet was immediately separated and stimulated with recombinant full-length adiponectin (rAPN) or not. The platelet aggregation, nitric oxide (NO) and superoxide production, endothelial nitric oxide synthase (eNOS)/inducible NOS (iNOS) expression, and antioxidant capacity were determined. Treatment with rAPN inhibited hyperlipidemia-induced platelet aggregation (P<0.05). Interestingly, total NO, a crucial molecule depressing platelet aggregation and thrombus formation?was significantly reduced, rather than increased in rAPN-treated platelets. Treatment with rAPN markedly decreased superoxide production (-62 %, P<0.05) and enhanced antioxidant capacity (+38 %, P<0.05) in hyperlipidemic platelets. Hyperlipidemia-induced reduced eNOS phosphorylation and increased iNOS expression were significantly reversed following rAPN treatment (P<0.05, P<0.01, respectively). Taken together, these data suggest that adiponectin is an adipokine that suppresses platelet aggregation by enhancing eNOS activation and attenuating oxidative/nitrative stress including blocking iNOS expression and superoxide production.     

Nitric oxide modulation of voltage-gated calcium current by S-nitrosylation and cGMP pathway in cultured rat hippocampal neurons

Nitric oxide (NO) plays an important role in many physiological and pathophysiological processes in the brain. In this study, we examined the mechanistic effects of an NO donor, diethylenetriamine/nitric oxide adduct (DETA/NO) on the voltage-gated calcium currents in cultured rat hippocampal neurons. DETA/NO stimulated the calcium currents and slightly increased the channel sensitivity to depolarizing voltages. The effect of DETA/NO on the calcium current was blocked by either depleting the NO in DETA/NO or by pretreating the neurons with NEM, a thiol-specific alkylating agent, suggesting an involvement of S-nitrosylation in the current response to NO. In addition, activation of the cGMP pathway by 8-Br-cGMP inhibited the calcium current in the neurons. Also, inhibition of guanylyl cyclase by 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ) increased the current response to DETA/NO. Taken together, our results demonstrate that both S-nitrosylation and cGMP pathway are involved in the NO modulation of the hippocampal calcium current.     

Cloned murine T lymphocytes synthesize a molecule with the biological characteristics of nitric oxide

Recently, activated neutrophils and macrophages have been shown to synthesize nitric oxide (NO) exclusively from L-arginine. We searched for the presence of this pathway in murine T cell clones. Using a platelet aggregation bioassay sensitive to NO, we demonstrate that IL2-stimulated CTLL and HT2 cells inhibit platelet aggregation, whereas unstimulated lymphocytes do not. This action can be inhibited by the specific NO synthase competitor NG-mono-methyl arginine, and only the L form of arginine or its analogue L-homoarginine are capable of providing substrate for NO synthesis.     

Analysis of nitric oxide donor effectiveness in resistance vessels

Decreased nitric oxide (NO) bioavailability is associated with a number of pathological conditions. Administration of a supplemental source of NO can counter the pathological effects arising from decreased NO bioavailability. A class of NO-nucleophile adducts that spontaneously release NO (NONOates) has been developed, and its members show promise as therapeutic sources of NO. Because the NONOates release NO spontaneously, a significant portion of the NO may be consumed by the myriad of NO reactive species present in the body. Here we develop a model to analyze the efficacy of NO delivery, by membrane-impermeable NONOates, in the resistance arterioles. Our model identifies three features of blood vessels that will enhance NONOate efficacy: 1) the amount of NO delivered to the abluminal region increases with lumen radius; 2) the presence of a flow-induced red blood cell-free zone will augment NO delivery; and 3) extravasation of the NONOate into the interstitial space will increase abluminal NO delivery. These results suggest that NONOates may be more effective in larger vessels and that NONOate efficacy can be altered by modifying permeability to the interstitial space.     

Vasorelaxant and antiaggregatory actions of the nitroxyl donor isopropylamine NONOate are maintained in hypercholesterolemia

Nitroxyl (HNO) displays pharmacological and therapeutic actions distinct from those of its redox sibling nitric oxide (NO(?)). It remains unclear, however, whether the vasoprotective actions of HNO are preserved in disease. The ability of the HNO donor isopropylamine NONOate (IPA/NO) to induce vasorelaxation, its susceptibility to tolerance development, and antiaggregatory actions were compared with those of a clinically used NO(?) donor, glyceryl trinitrate (GTN), in hypercholesterolemic mice. The vasorelaxant and antiaggregatory properties of IPA/NO and GTN were examined in isolated carotid arteries and washed platelets, respectively, from male C57BL/6J mice [wild-type (WT)] maintained on either a normal diet (WT-ND) or high fat diet (WT-HFD; 7 wk) as well as apolipoprotein E-deficient mice maintained on a HFD (ApoE(-/-)-HFD; 7 wk). In WT-ND mice, IPA/NO (0.1-30 μmol/l) induced concentration-dependent vasorelaxation and inhibition of collagen (30 μg/ml)-stimulated platelet aggregation, which was predominantly soluble guanylyl cyclase/cGMP dependent. Compared with WT-HFD mice, ApoE(-/-)-HFD mice displayed an increase in total plasma cholesterol levels (P < 0.001), vascular (P < 0.05) and platelet (P < 0.05) superoxide (O(2)(·-)) production, and reduced endogenous NO(?) bioavailability (P < 0.001). Vasorelaxant responses to both IPA/NO and GTN were preserved in hypercholesterolemia, whereas vascular tolerance developed to GTN (P < 0.001) but not to IPA/NO. The ability of IPA/NO (3 μmol/l) to inhibit platelet aggregation was preserved in hypercholesterolemia, whereas the actions of GTN (100 μmol/l) were abolished. In conclusion, the vasoprotective effects of IPA/NO were maintained in hypercholesterolemia and, thus, HNO donors may represent future novel treatments for vascular diseases.     

Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRbeta tyrosine phosphorylation

Chondrocytes in arthriticcartilage respond poorly to insulin-like growth factor I (IGF-I).Studies with inducible nitric oxide synthase (iNOS) knockout micesuggest that NO is responsible for part of the cartilage insensitivityto IGF-I. These studies characterize the relationship between NO andchondrocyte responses to IGF-I in vitro, and define a mechanism bywhich NO decreases IGF-I stimulation of chondrocyte proteoglycansynthesis. Lapine cartilage slices, chondrocytes, and cartilage fromosteoarthritic (OA) human knees were exposed to NO from the donorsS-nitroso-N-acetylpenicillamine (SNAP) or(Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NOsynthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of ³⁵SO₄. IGF-Ireceptor phosphorylation was evaluated with Western analysis. SNAP,DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells,or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis incartilage and monolayer cultures of chondrocytes. OA cartilageresponded poorly to IGF-I; however, the response to IGF-I was restoredby culture withN^G-monomethyl-L-arginine(L-NMA). IGF-I receptor phosphotyrosine was diminished inchondrocytes exposed to NO. These studies show that NO is responsiblefor part of arthritic cartilage/chondrocyte insensitivity to anabolicactions of IGF-I; inhibition of receptor autophosphorylation ispotentially responsible for this effect.

     

Platelet inhibition by nitrite is dependent on erythrocytes and deoxygenation

BACKGROUND: Nitrite is a nitric oxide (NO) metabolite in tissues and blood, which can be converted to NO under hypoxia to facilitate tissue perfusion. Although nitrite is known to cause vasodilation following its reduction to NO, the effect of nitrite on platelet activity remains unclear. In this study, the effect of nitrite and nitrite+erythrocytes, with and without deoxygenation, on platelet activity was investigated. METHODOLOGY/FINDING: Platelet aggregation was studied in platelet-rich plasma (PRP) and PRP+erythrocytes by turbidimetric and impedance aggregometry, respectively. In PRP, DEANONOate inhibited platelet aggregation induced by ADP while nitrite had no effect on platelets. In PRP+erythrocytes, the inhibitory effect of DEANONOate on platelets decreased whereas nitrite at physiologic concentration (0.1 μM) inhibited platelet aggregation and ATP release. The effect of nitrite+erythrocytes on platelets was abrogated by C-PTIO (a membrane-impermeable NO scavenger), suggesting an NO-mediated action. Furthermore, deoxygenation enhanced the effect of nitrite as observed from a decrease of P-selectin expression and increase of the cGMP levels in platelets. The ADP-induced platelet aggregation in whole blood showed inverse correlations with the nitrite levels in whole blood and erythrocytes. CONCLUSION: Nitrite alone at physiological levels has no effect on platelets in plasma. Nitrite in the presence of erythrocytes inhibits platelets through its reduction to NO, which is promoted by deoxygenation. Nitrite may have role in modulating platelet activity in the circulation, especially during hypoxia.