S-nitro-N-acetylpenicillamine Research Articles

Protection of Pacific white shrimp (Penaeus vannamei) against white spot syndrome virus infection by nitric oxide-generating compound S-nitrosoglutathione

Nitric oxide (NO), a highly reactive free radical generated endogenously by nitric oxide synthase (NOS), plays crucial roles in immune defense against a broad range of pathogens including viruses, bacteria, fungi, and protozoans. However, the regulation and effect of NO during viral infection in invertebrates remain largely unexplored. White spot syndrome virus (WSSV) is one of the most severe pathogens affecting the global shrimp farming industry. Our study shows that NO production in Pacific white shrimp Penaeus vannamei decreases during WSSV infection. Reducing NO production using the NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME) significantly promotes WSSV replication in primary culture of hemocytes isolated from P. vannamei. This effect is partically reversed by L-arginine, a native substrate for NOS, indicating that L-NAME's immunity-reducing action is NO-dependent. In vitro studies also reveal inhibition of WSSV replication in the presence of three NO donors: S-nitrosoglutathione (GSNO), S-nitro-N-acetylpenicillamine (SNAP), and diethylenetriamine nitric oxide adduct (DETA/NO). The involvement of NO in anti-WSSV protection is further demonstrated by an in vivo study where dietary supplementation with GSNO at non-toxic doses increases NO levels and reduces viral load and mortality in WSSV-challenged shrimp. Our results contribute to understanding the antiviral activity of NO in invertebrates and provide a theoretical basis for developing NO-based therapeutic strategies to control WSSV infection.

Blunted flow-mediated responses and diminished nitric oxide synthase expression in lymphatic thoracic ducts of a rat model of metabolic syndrome

Shear-dependent inhibition of lymphatic thoracic duct (TD) contractility is principally mediated by nitric oxide (NO). Endothelial dysfunction and poor NO bioavailability are hallmarks of vasculature dysfunction in states of insulin resistance and metabolic syndrome (MetSyn). We tested the hypothesis that flow-dependent regulation of lymphatic contractility is impaired under conditions of MetSyn. We utilized a 7-wk high-fructose-fed male Sprague-Dawley rat model of MetSyn and determined the stretch- and flow-dependent contractile responses in an isobaric ex vivo TD preparation. TD diameters were tracked and contractile parameters were determined in response to different transmural pressures, imposed flow, exogenous NO stimulation by S-nitro-N-acetylpenicillamine (SNAP), and inhibition of NO synthase (NOS) by l-nitro-arginine methyl ester (l-NAME) and the reactive oxygen species (ROS) scavenging molecule 4-hydroxy-tempo (tempol). Expression of endothelial NO synthase (eNOS) in TD was determined using Western blot. Approximately 25% of the normal flow-mediated inhibition of contraction frequency was lost in TDs isolated from MetSyn rats despite a comparable SNAP response. Inhibition of NOS with l-NAME abolished the differences in the shear-dependent contraction frequency regulation between control and MetSyn TDs, whereas tempol did not restore the flow responses in MetSyn TDs. We found a significant reduction in eNOS expression in MetSyn TDs suggesting that diminished NO production is partially responsible for impaired flow response. Thus our data provide the first evidence that MetSyn conditions diminish eNOS expression in TD endothelium, thereby affecting the flow-mediated changes in TD lymphatic function.

Activation of the cGMP/Protein Kinase G Pathway by Nitric Oxide Can Decrease TRPV1 Activity in Cultured Rat Dorsal Root Ganglion Neurons

Recent studies have demonstrated that nitric oxide (NO) activates transient receptor potential vanilloid subtype 1 (TRPV1) via S-nitrosylation of the channel protein. NO also modulates various cellular functions via activation of the soluble guanylyl cyclase (sGC)/protein kinase G (PKG) pathway and the direct modification of proteins. Thus, in the present study, we investigated whether NO could indirectly modulate the activity of TRPV1 via a cGMP/PKG-dependent pathway in cultured rat dorsal root ganglion (DRG) neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), decreased capsaicin-evoked currents (Icap). NO scavengers, hemoglobin and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (CPTIO), prevented the inhibitory effect of SNP on Icap. Membrane-permeable cGMP analogs, 8-bromoguanosine 3', 5'-cyclic monophosphate (8bromo-cGMP) and 8-(4chlorophenylthio)-guanosine 3',5'-cyclic monophosphate (8-pCPT-cGMP), and the guanylyl cyclase stimulator YC-1 mimicked the effect of SNP on Icap. The PKG inhibitor KT5823 prevented the inhibition of Icap by SNP. These results suggest that NO can downregulate the function of TRPV1 through activation of the cGMP/PKG pathway in peripheral sensory neurons.

The effect of nitric oxide inhibitors and s-nitrosothiols on hemodynamic parameters in an animal model

The effect of nitric oxide inhibitors and s-nitrosothiols on hemodynamic parameters in an animal model Sophia Bryan1, Ruby Alexander-Lindo1, Tara Dasgupta2, Donovan McGrowder31Department of Basic Medical Sciences, Faculty of Medical Sciences, 2Department of Chemistry, Faculty of Pure and Applied Sciences, 3Department of Pathology, Faculty of Medical Sciences, University of the West Indies, Kingston, JamaicaBackground: Nitric oxide (NO) is becoming an increasingly important signaling molecule implicated in a growing number of physiological and pathophysiological processes. We sought to test the hypothesis that co-administration of S-nitro-N-acetylpenicillamine (SNAP) or S-nitrosocaptopril (CapSNO) with NG-methyl-L-arginine ester (L-NAME) or NG-methyl-L-arginine acetate (L-NMMA) may reverse the elevated systolic, diastolic, and mean arterial pressures caused by the administration of L-NAME or L-NMMA only.Materials and methods: Blood pressure was measured using the CODA 6 machine. The hemodynamic parameters systolic blood pressure (SBP), diastolic blood pressure (DBP), mean arterial pressure (MAP) and heart rate (HR) were determined for each rat group. There was intravenous (IV) administration of the control (0.3 mL, saline) or dosage of 12.5 mg/kg body weight of SNAP or CapSNO via IV.Results: In CapSNO and L-NAME-treated rats, CapSNO significantly decreased SBP from 131.12 ± 4.29 mmHg to 121.42 ± 4.24 mmHg after 5 minutes (P < 0.0001), and then L-NAME (administered at t = 5 min) increased SBP to 165.29 ± 6.79 mmHg at 10 minutes (P < 0.05). In SNAP and L-NAME-treated rats, SNAP significantly decreased SBP from 135.86 ± 2.84 mmHg to 106.98 ± 1.99 mmHg (P < 0.0001) after 5 minutes, and L-NAME increased SBP to 146.18 ± 3.19 mmHg after 25.0 minutes (P < 0.05). In rats treated with SNAP and L-NAME, SNAP decreased DBP to 74.98 ± 2.49 mmHg (P < 0.0001) after 5 minutes while L-NAME increased DBP to 105.01 ± 2.60 mmHg after 25 minutes (P < 0.05). In SNAP and L-NAME treated group there was an increase in HR after the administration of SNAP (486.60 ± 30.82 at 0 minutes to 555.66 ± 32.37 beats min-1 at 5 minutes; P < 0.0001), followed by a decrease in HR to 336.90 ± 17.48 beats min-1 at 25 minutes (P

NO Link Between Notch and Inflammation?

Both nitric oxide (NO) and Notch signaling have been implicated in inflammation, although the details of their roles have not been determined. A study by Kim et al. presents evidence that NO may repress Notch signaling by nitrating the Notch intracellular domain (NICD, a transcription factor that is released from the membrane and translocates to the nucleus upon stimulation of the Notch pathway). Immortalized macrophages were exposed to lipopolysaccharide (LPS), a bacterial cell wall component, to induce an inflammatory response, and the effect on expression of three different NICD-responsive reporter genes was measured. The expression of each reporter decreased in cells exposed to LPS and in cells treated with the NO donor S-nitro-N-acetyl-penicillamine (SNAP). LPS-treated cells secreted NO into the culture medium, and pharmacological inhibitors of NO synthesis blocked the LPS-induced repression of the reporters. The amount of NICD bound to the reporter promoters decreased as the extracellular NO concentration increased, and high NO concentrations prevented NICD from associating with its DNA-binding partner RBP-Jk. An antibody that recognizes nitrotyrosine confirmed that nitrated NICD was present in extracts from LPS-treated cells. The fluorescence emission profile of NICD indicated that its conformation changed upon exposure to NO, so the authors postulated that nitration represses NICD activity by rendering it incapable of binding to its transcriptional coactivator(s). It had been previously reported that the glycosylation state of the Notch extracellular domain affects its ligand-binding activity, and now it appears that activity of the intracellular domain can also be modulated by covalent modification. M.-Y. Kim, J.-H. Park, J.-S. Mo, E.-J. Ann, S.-O. Han, S.-H. Baek, K.-J. Kim, S.-Y. Im, J.-W. Park, E.-J. Choi, H.-S. Park, Downregulation by lipopolysaccharide of Notch signaling, via nitric oxide. J. Cell Sci. 121 , 1466-1476 (2008). [Abstract] [Full Text]

Nitric oxide decreases ammonium release in tadpoles of the clawed frog, Xenopus laevis, Daudin

In the present study, we quantified the physiological consequences of nitric oxide (NO) on ammonium release in tadpoles of Xenopus laevis. Tadpoles exposed to S-nitro-N-acetylpenicillamine (SNAP), an NO-donor, or L: -arginine, the substrate of NO synthase (NOS), showed a reversible decrease, whereas animals exposed to the NOS inhibitor Nomega-methyl-L: -arginine (L: -NMMA) exhibited an increase in ammonium release. Release of ammonium may be of physiological relevance during stress response of the animal. Handling of tadpoles as well as exposure to hyposmotic environments increased ammonium release. To localize NO synthesizing cells, we used diaminofluorescein-diacetate (DAF-2DA), an NO-sensitive fluorescent dye, and NADPH-diaphorase histochemistry, an indicator for NOS activity. We observed a fluorescence signal as well as NADPH-diaphorase activity in small, solitary cells in the epidermis. Similarly to NADPH-diaphorase histochemistry, silver nitrate staining and rhodamine labelling, markers for mitochondria-rich cells, showed a strong reaction in these cells. These observations indicate that NO (1) inhibits ammonium release, and (2) is endogenously synthesized in mitochondria-rich cells in Xenopus tadpoles. Based on our histochemical results, we speculate that gill epithelium and epidermis work in parallel to release ammonium as epidermal tissue contains mitochondria-rich and NADPH-diaphorase positive cells.

Differential Mechanisms Underlying the Modulation of Delayed-Rectifier K+Channel in Mouse Neocortical Neurons by Nitric Oxide

The modulatory effects of nitric oxide (NO) on voltage-dependent K+ channels are intricate. In our present study, the augmentation and reduction of K+ currents by NO donor S-nitro-N-acetylpenicillamine (SNAP) and pure dissolved NO was observed in dissociated neurons from mice neocortex with both whole cell and cell-attached patch clamp. By using a specific electrochemical sensor, the critical concentrations of NO that increased or reduced the channel activities were accurately quantified. Low concentrations of SNAP (20 microM) or NO solution (0.1 microM) enhanced whole cell delayed rectifier K+ -current (IK) and left the fast inactivating A current (IA) unchanged. However, high concentrations of SNAP (100 microM) and NO (0.5 microM) reduced both IK and IA currents. In cell-attached experiments, a significant increase in channel open probability (NP0) was observed when using low concentrations of SNAP or NO. High concentrations of SNAP or NO dramatically decreased NP0. The increase in channel activities by low concentrations of SNAP was abolished in the presence of either inhibitors of soluble guaylate cyclase or inhibitors of cGMP-dependent protein kinase G, suggesting a link to the NO-cGMP signaling cascade. The reduction of channel activities by high concentrations of SNAP was reversed by the reducing agent dithiothreitol, implying a redox reaction mechanism. Thus both NO-cGMP signaling and a redox mechanism are involved in the modulation of IK channel activity for neuron excitability.

Role of nitric oxide in apoptosis of human peritoneal and adhesion fibroblasts after hypoxia

To study the modulation of the inducible nitric oxide synthase/nitric oxide (iNOS/NO) expression system in fibroblasts isolated from human peritoneum and adhesion tissues by hypoxia. Prospective experimental study. University medical center. Cultures of fibroblasts from both peritoneum and adhesion tissues of five patients. Hypoxia treatment of the primary cultured fibroblasts. We used Western and Northern blots to determine whether iNOS mRNA and its protein were present in peritoneal and adhesion fibroblasts and whether this expression is modulated by hypoxia. Multiplex reverse transcription polymerase chain reaction (RT-PCR) technique was used to quantify type I collagen mRNA in response to NG-nitro-L-arginine methyl ester (L-NAME). A terminal deoxynucleotidyl transferase mediated dUTP nick-end-labeling (TUNEL) assay was used to quantify apoptosis in response to NO donor S-nitro-N-acetyl-penicillamine (SNAP) treatment. A Griess assay was used to measure NO levels. Peritoneal fibroblasts have significantly higher NO levels than adhesion fibroblasts. Hypoxia decreased NO in peritoneal fibroblasts to levels observed for adhesion fibroblasts. In addition, hypoxia increased both mRNA and protein levels of the iNOS gene in peritoneal and adhesion fibroblasts. Augmentation of NO by SNAP treatment increased apoptosis in adhesion fibroblasts. In contrast, SNAP had no effect on apoptosis of peritoneal fibroblasts. Inhibition of NO by L-NAME treatment increased type I collagen mRNA levels in peritoneal fibroblasts. Our findings confirm that adhesion fibroblasts produce less NO than normal peritoneal fibroblasts; NO may be the mechanism responsible for the creation and persistence of the adhesion phenotype.

Nitric oxide acts on the mitochondria and protects human endothelial cells from apoptosis

Proliferation of small blood vessels in synovial tissues is one of the pathologic features of rheumatoid arthritis. In this study we tested the hypothesis that nitric oxide (NO) protects endothelial cells (ECs) against apoptogenic agents in vitro. Human umbilical-vein endothelial cells (HUVECs) were cultured with and without NO donor S -nitro- N -acetylpenicillamine (SNAP) and further incubated in the presence or absence of Z-leucine-leucine-leucine-aldehyde (LLL-CHO), etoposide, or C2-ceramide. After cultivation, apoptosis of HUVECs was quantified on the basis of disruption of mitochondrial transmembrane potential (DeltaPsim), activation of caspases, and the presence of hypodiploid DNA-positive cells. Treatment of HUVECs with LLL-CHO, etoposide, or C2-ceramide induced DeltaPsim, activation of caspase-3, caspase-8, and caspase-9 and the appearance of hypodiploid DNA-positive cells. NO production in HUVECs was clearly increased by SNAP. Apoptotic cell death in HUVECs induced by LLL-CHO, etoposide, and C2-ceramide was significantly suppressed by SNAP treatment. HUVECs in vitro expressed Bcl-2, Bcl-xL, and Bax; however, expression was not changed by SNAP treatment in the presence or absence of LLL-CHO, etoposide, or C2-ceramide. Although the molecule(s) responsible for the protective effects of NO remains to be identified, our data imply that NO protects HUVECs against mitochondrial perturbation caused by apoptogenic agents. These results suggest that NO promotes endothelial-cell proliferation and angiogenesis in the synovial tissues of patients with rheumatoid arthritis and that NO may be a therapeutic target for rheumatoid arthritis.

Dual actions of nitric oxide on angiogenesis: possible roles of PKC, ERK, and AP-1

Regulation of angiogenesis by nitric oxide (NO) is controversial since NO has been shown to have both pro- and anti-angiogenic effects. In this study, we examined the effect of the NO donor, S-nitro- N-acetyl-penicillamine (SNAP), on in vitro angiogenesis, and the mechanisms involved: PKC activity, ERK and c-Jun phosphorylation, and AP-1 DNA binding activity, in microvascular endothelial cells. SNAP, at 0.5–4 mM, significantly and dose-dependently inhibited angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 80%, 83%, and 63% and 73%, respectively. SNAP at concentrations >2 mM also abolished AP-1 binding activity. Lower concentrations of SNAP (0.1–0.3 mM) significantly increased angiogenesis, PKC activity, and ERK and c-Jun phosphorylation up to 46%, 60%, and 61% and 180%, respectively. These findings indicate that the dual pro- and anti-angiogenic actions of NO are dose-dependent and suggest that they are mediated by PKC and ERK acting on AP-1.

Nitric oxide decreases a calcium-activated potassium current via activation of phosphodiesterase 2 in Helix U-cells

In the present study, we investigated the underlaying mechanism of nitric oxide (NO) and cGMP on the decline of a Ca 2+-activated potassium (K Ca) current in U-cells of the right parietal ganglion of the pulmonate snail, Helix pomatia. Using a two-electrode voltage-clamp technique, we activated a K Ca-current either by opening of endogenous voltage-gated Ca 2+-channels during depolarizing voltage steps or by ionophoretic injection of Ca 2+ via a third electrode containing 100 mM Ca 2+. K Ca-current amplitude in U-cells was sensitive to Ba 2+, TEA, iberiotoxin, kaliotoxin and charybdotoxin (ChTX), but not to 4-aminopyridine (4-AP) (up to 30 mM) and apamin (up to 300 nM). Thus, the biophysical and pharmacological profile of the K Ca-current in U-cells shares similarities with the large-conductance K Ca channel (BK(Ca)). The NO-donor sodium nitroprusside (SNP) or S-nitro- N-acetylpenicillamine (SNAP) as well as NO-gas decreased the K Ca-current amplitude and decreased the rate of K Ca-current activation elicited by Ca 2+-injection. Decline of the current amplitude and decrease of activation of K Ca-current were qualitatively mimicked by the membrane-permeable cGMP analogue dibutyryl-cGMP (db-cGMP). NO-induced decrease of K Ca-current was blocked by methylene blue (50 μM), an inhibitor of the guanylyl-cyclase, and by erytho-9-(2-hydroxyl-3-nonyl) adenine (EHNA) (100 μM), an inhibitor of the cGMP-stimulated phosphodiesterase 2 (PDE2). These experiments suggest that the NO-mediated decrease of K Ca-current in U-cells results from synthesis of cGMP by activation of a guanylyl-cyclase and subsequent activation of PDE2.

Nitric oxide protects cultured rheumatoid synovial cells from Fas-induced apoptosis by inhibiting caspase-3.

Nitric oxide (NO) is elevated in the synovial fluids and sera of patients with rheumatoid arthritis (RA) and is thought to be an important proinflammatory mediator in the rheumatoid synovium. To test the hypothesis that NO might modulate the apoptosis-inducing signal pathway, we investigated the effects of NO on rheumatoid synovial-cell apoptosis induced by Fas ligation with anti-Fas antibody. Pretreatment of synovial cells with the NO donor S-nitro-N-acetylpenicillamine (SNAP) prevented the Fas-mediated induction of apoptosis. The activation of caspase-3 was required to mediate Fas-induced synovial cell apoptosis. The NO donor SNAP inhibited Fas-induced caspase-3 activation in rheumatoid synovial cells. However, NO did not interrupt Fas-induced caspase-8 cleavage or subsequent cytochrome c release into the cytosol in rheumatoid synovial cells. These data indicate that NO prevents apoptosis in rheumatoid synovial cells by directly inhibiting caspase-3 activation. Thus, we propose that NO interferes with cell death signal transduction and may contribute to rheumatoid synovial cell proliferation by inhibiting induction of apoptosis.

Nitric oxide increases excitability by depressing a calcium activated potassium current in snail neurons

In gastropods, the interneuronal messenger, nitric oxide (NO), modulates spike frequency and synaptic transmission. We have characterized the effect of NO on ion currents underlying neuronal excitability, using current-clamp and two-electrode voltage-clamp techniques. Identified neurons of the pulmonate snail, Helix pomatia, respond to the NO donor sodium nitroprusside (SNP) by increasing the firing frequency and decreasing the latency. Voltage-clamp experiments revealed that SNP or S-nitro- N-acetylpenicillamine (SNAP) depressed the macroscopic outward current, while the control compound N-acetylpenicillamine (NAP) had no effect. Current voltage curves generated from voltage steps to different membrane potentials ranging from −40 to +180 mV showed an N-shaped outward current. Superfusion of ganglia with Ca 2+ free Helix solution abolished the N-shape, indicating the contribution of a Ca 2+ activated K + current (I K,Ca). Exposure of neurons to SNP or SNAP diminished the N-shape, indicating that NO affects I K,Ca. The depressing effect of SNP on the outward current was slow and reached steady state in about 5 min. In conclusion, our findings indicate that NO enhances excitability in Helix nervous system by decreasing I K,Ca.

Inhibitory Effect of Nitric Oxide on Voltage-Dependent Calcium Currents in Rat Dorsal Root Ganglion Cells

The effect of nitric oxide (NO) on calcium current (ICa) and intracellular calcium concentration ([Ca2+]i) in primarily cultured dorsal root ganglion (DRG) neurons was investigated from neonatal rats. ICa and [Ca2+]i were simultaneously recorded using perforated-patch technique in combination with fluorescence measurement from single DRG neurons. NO donors, sodium nitroprusside (SNP) and S-nitro-N-acetylpenicillamine (SNAP), inhibited ICa in small-diameter neurons without significant change in voltage-dependence of activation and activation time constants. SNP and SNAP also reduced the transient [Ca2+]i peak accompanied by ICa. Inhibition by NO was reproducible, but gradually desensitized. In some DRG neurons, SNP and SNAP increased basal [Ca2+]i in concentration of 10 μM with little effect on NO-induced inhibition of ICa. 8-Br-cGMP, a permeable cGMP analog, mimicked the effects of SNP and SNAP. These results suggest that, in DRG neurons, NO has inhibitory effect on ICa, which is independent of NO-induced increase of basal [Ca2+]i, through cGMP-dependent pathway.

Cellular and Molecular Mechanisms of Nitric Oxide–Induced Heart Muscle Relaxation

1. The nitric oxide (NO) donor S-nitro-N-acetyl-penicillamine (SNAP) inhibits Helix aspersa heart activity and relaxes muscles. 2. K-free saline and ouabain both depress SNAP-induced relaxation in most experiments, but in a few preparations they either had no effect or potentiated SNAP-induced relaxation. 3. Na-K pump reactivation following preincubation in K-free saline leads to the pronounced transient relaxation of heart muscle, the magnitude of which depends on the duration of preincubation. 4. 0.1 mM SNAP inhibited the ouabain sensitive part of 86Rb uptake, which reflects Na-K pump activity. This inhibition is potentiated by phospholipase C. 5. SNAP increased cGMP levels in the heart. 6. These results indicate that SNAP-induced relaxation depends on Na and Ca gradients across the membrane, which suggests that Na:Ca exchange is involved in the mechanisms of SNAP-induced relaxation. It is postulated that SNAP elicits its inhibitory effect on the heart through a cGMP-dependent Na:Ca exchange.

Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication.

The antiviral effects of nitric oxide (NO) on Japanese encephalitis virus (JEV), a member of the family Flaviviridae, were investigated in this study. In vitro, inhibition of replication of JEV in gamma interferon-activated RAW 264.7 murine macrophages was correlated to cellular NO production. When cocultured with infected murine neuroblastoma N18 cells, gamma interferon-activated RAW 264.7 cells also efficiently hindered JEV replication in contiguous bystanders, and this anti-JEV effect could be reversed by an NO synthase (NOS) inhibitor, N-monomethyl-L-arginine acetate. In vivo, the mortality rate increased as the NOS activity of JEV-infected mice was inhibited by its competitive inhibitor, N-nitro-L-arginine methyl ester. Moreover, when an organic donor, S-nitro-N-acetylpenicillamine (SNAP), was used, the NO-mediated antiviral effect was also observed in primarily JEV-infected N18, human neuronal NT-2, and BHK-21 cells, as well as in persistently JEV-infected C2-2 cells. These data reaffirm that NO has an effective and broad-spectrum antimicrobial activity against diversified intracellular pathogens. Interestingly, the antiviral effect of NO was not enhanced by treatment of N18 cells with SNAP prior to JEV infection, a measure which has been shown to greatly increase the antiviral effect of NO in infection by vesicular stomatitis virus. From biochemical analysis of the impact of NO on JEV replication in cell culture, NO was found to profoundly inhibit viral RNA synthesis, viral protein accumulation, and virus release from infected cells. The results herein thus suggest that NO may play a crucial role in the innate immunity of the host to restrict the initial stage of JEV infection in the central nervous system.

Inhibition of vesicular stomatitis virus infection by nitric oxide

Inhibitory effects of nitric oxide (NO) on vesicular stomatitis virus (VSV) infection were investigated by using a VSV-susceptible mouse neuroblastoma cell line, NB41A3. Productive VSV infection of NB41A3 cells was significantly inhibited by an organic NO donor, S-nitro-N-acetylpenicillamine (SNAP), while the control compound N-acetylpenicillamine (NAP) had no effect. Survival rate of VSV-infected cells was greatly increased by the treatment with SNAP, while the NAP treatment did not have any effect. Adding SNAP 30 min prior to infection resulted in complete inhibition of viral production when a low multiplicity of infection (MOI) was used. Substantial inhibition of viral production was also obtained with treating cells 6 h earlier before infection with a higher MOI. Activating the neuronal NO synthase by treating cells with N-methyl-D-aspartate (NMDA) led to significant inhibition of viral production by cells infected at the three doses of virus tested (MOIs of 0.1, 1, and 5). The inhibitory effect of NMDA on viral infection was totally blocked by the NO synthase inhibitor N-methyl-L-arginine. However, adding hemoglobin, a strong NO-binding protein and thus an inactivator of NO activity, did not reverse the NMDA-induced inhibition of viral production, suggesting that NO might exert its antiviral effects inside the NO-producing cells. Collectively, these data support the anti-VSV effects of NO, which might be one of the important factors of natural immunity in controlling the initial stages of VSV infection in the central nervous system.