Sodium Nitroprusside Exposure Research Articles

Metabolic adaptation of the clam Ruditapes philippinarum during air exposure and the positive effects of sodium nitroprusside pretreatment.

The Manila clam (Ruditapes philippinarum), as one of the shellfish living in the intertidal zone, is known for its strong ability to withstand air exposure. Sodium nitroprusside (SNP), a donor of nitric oxide (NO), has been shown to be useful for antioxidant and immune regulation in aquatic animals. In this study, an untargeted metabolomics (LC-MS/MS) technique was employed for the first time in Manila clam to analyze the metabolic and histological impacts after air exposure and the positive effects of SNP pretreatment. During air exposure, a significant increase in taurine, L-glutamate, and several polyunsaturated fatty acids in clams was detected, which indicates that clams may experience inflammatory reactions, oxidative stress, and an increase in blood ammonia content. When clams were exposed to SNP for 6h, arginine, spermine, L-glutamic acid, and glutathione content were all upregulated, indicating that the SNP exposure induced NO production and improved antioxidant capacity in clams. When the clams were exposed to air after SNP pretreatment, there were no significant differences in the levels of taurine, L-glutamate, or aliphatic acids between the experimental and control groups. Gill tissue was more severely damaged in clams directly exposed to air than in those that experienced air exposure after SNP pretreatment, especially in clams exposed to air for a long time (72h). Both metabolomics and tissue section structure indicated that SNP pretreatment decreased the stress responses caused by air exposure in R. philippinarum. These findings provided fresh insights and a theoretical foundation for understanding the tolerance to air exposure and physiological functions of SNP (or NO) in R. philippinarum.

DNA fragmentation of human spermatozoa: Simple assessment of single- and double-strand DNA breaks and their respective dynamic behavioral response.

Procedures to detect sperm DNA fragmentation (SDF), like the sperm chromatin dispersion (SCD) test, determine the "global" SDF without discriminating between spermatozoa with single-strand DNA breaks only (SDF-SSBs) and those containing double-strand DNA breaks (SDF-DSBs). (a) To validate a test to distinguish human spermatozoa with massive DSBs (DSB-SCD assay), (b) to study the baseline SDF-SSBs and SDF-DSBs, and (c) to assess their dynamics in vitro. (a) SDF-DSBs were determined by visualization of diffused DNA fragments from spermatozoa lysed under non-denaturing conditions. This was validated by in vitro incubation with DNase I and the comet assay. (b) Baseline SDF-DSBs and SDF-SSBs were determined in ejaculates from 95 males. (c) Their dynamic appearance was studied in samples untreated or exposed to hyperthermia, acidic pH, nitric oxide released by sodium nitroprusside (SNP), and the metabolic energy inhibitors 2-deoxy-D-glucose and antimycin A. (a) DNase I and comet assay experiments confirmed that the assay successfully determined SDF-DSBs. (b) The higher the SDF of the semen sample, the higher the frequency of SSBs, whereas DSBs behaved independently. Abnormal samples showed higher SDF than normozoospermic, the difference being only significant for SDF-SSBs. (c) During the first hours of incubation, the linear rate of increase in SDF-SSBs was 3.7 X higher than that of SDF-DSBs. All hazardous agents accelerated the SDF rate when compared to untreated spermatozoa, primarily being associated with SDF-SSBs. SNP treatment was the most damaging, rapidly inducing spermatozoa with SSBs which progressively evolved to DSBs. Remarkably, this phenomenon was also evidenced after acute SNP exposure, revealing cryptic sperm damage. The DSBs-SCD is an easy complement for SDF assessment. The dynamic study of SSBs and DSBs may improve the evaluation of sperm quality in clinical settings, particularly "unmasking" the presence of non-specific cryptic sperm damage that might otherwise go undetected.

Reactive nitrogen species induced catalases promote a novel nitrosative stress tolerance mechanism in Vibrio cholerae

Vibrio cholerae faces nitrosative stress during successful colonization in intestine. Very little information is available on the nitrosative stress protective mechanisms of V. cholerae. Reports show that NorR regulon control two genes hmpA and nnrS responsible for nitric oxide (NO) detoxification in V. cholerae. In the present study we first time report a novel role of V. cholerae catalases under nitrosative stress. Using zymogram analysis of catalase we showed that KatB and KatG activity were induced within 30 min in V. cholerae in the presence of sodium nitroprusside (SNP), a NO donor compound. Surprisingly, V. cholerae cell survival was found to be decreased under nitrosative stress if catalase activities were blocked by ATz, a catalase inhibitor. Flow cytometry study was conducted to detect reactive oxygen species (ROS) and reactive nitrogen species (RNS) using DHE and DHR123, fluorescent probes respectively. Short exposure of SNP to V. cholerae did not generate ROS but RNS was detectable within 30 min. Total glutathione content was increased in V. cholerae cells under nitrosative stress. Furthermore, Superoxide dismutase (SOD) and Glutathione reductase (GR) activities remained unchanged under nitrosative stress in V. cholerae indicated antioxidant role of NO which could produce peroxynitrite. To investigate the role of catalase induction under nitrosative stress in V. cholerae, we conducted peroxynitrite reductase assay using cell lysates. Interestingly, SNP treated V. cholerae cell lysates showed lowest DHR123 oxidation compared to the control set. The extent of DHR123 oxidation was more in V. cholerae cell lysate when catalases were blocked by ATz.

Preventive effect of oligonol on nitric oxide and reactive oxygen species production through regulation of nuclear factor kappa B signaling pathway in RAW 264.7 macrophage cells against sodium nitroprusside.

Oligonol, a low-molecular weight polyphenol isolated from lychee fruit, has been shown to possess beneficial properties, including anti-oxidative, anti-diabetic, and hepatoprotective properties in vitro and in vivo. This study was performed to investigate the anti-inflammatory effects of oligonol using sodium nitroprusside (SNP)-stimulated RAW 264.7 macrophage cells. Our results demonstrated that exposure of SNP to RAW 264.7 cells significantly decreased cell viability, and increased nitric oxide (NO) and reactive oxygen species (ROS) production. However, treatment with oligonol inhibited cell death and suppressed the over-production of NO and ROS induced by SNP in a dose-dependent manner. Consistent with these findings, oligonol significantly downregulated the mRNA levels of pro-inflammatory mediators, inducible nitric oxide synthase and cyclooxygenase-2, when compared with the SNP-treated control group. Furthermore, suppression of nuclear factor-κB (NF-κB) activation was also observed after treatment with oligonol in RAW 264.7 macrophage cells. These results suggest that oligonol attenuated SNP-induced oxidative stress and inflammatory responses via regulation of the NF-κB signalling pathway. On the basis of such potent anti-oxidant and anti-inflammatory properties, we propose that oligonol may contribute in the prevention and treatment of inflammation-related disorders.

Attenuation of Thrombin-Mediated Fibrin Formation via Changes in Fibrinogen Conformation Induced by Reaction with S-nitroso-N-acetylpenicillamine, but not S-nitrosoglutathione.

Previous work in a 4 h rabbit thrombogenicity model has shown that a nitric oxide- (NO) generating polymer extracorporeal circuits (ECC) with infusion of S-nitroso-N-acetyl-penicillamine (SNAP) preserved platelets eventhough platelets were activated as shown by an increase in the glycoprotein, p-selectin. The platelet preservation mechanism was shown to be due to a changing fibrinogen structure leading to attenuation of platelet aggregation. Understanding the effects that SNAP, another RSNO, S-nitroso-glutathione (GSNO) as well as the non-RSNO, sodium nitroprusside (SNP), may have on human fibrinogen polymerization, this in vitro study evaluated the released NO effects on the thrombin-mediated fibrin formation and fibrinogen structure. Thrombin-induced fibrin formation at 300 μM SNAP (50 + 11% of baseline) was significantly reduced compared to SNAP's parent, N-acetyl-penicillamine (NAP) (95 + 13%) after 1 h of RSNO exposure. GSNO, its parent, glutathione (GSH) and 1000 ppm NO gas did not attenuate the thrombin-mediated fibrin formation. SNAP, NAP and SNP exposure for 1 h, however, did not decrease thrombin activity by directly inhibiting thrombin itself. Changes in fibrinogen conformation as measured by intrinsic tryptophan fluorescence significantly decreased in the 300 μM SNAP (38057 + 1196 mean fluorescence intensity (MFI) and SNP (368617 + 541 MFI) groups versus the NAP control (47937 + 1196 MFI). However, infused 1000 ppm NO gas had no direct effect on the ITF after 1 h incubation at 37°C. High performance liquid chromatography (HPLC) showed that fibrinogen degradation by 0.03 U/ml thrombin was concentration-dependently reduced after 1 h with SNAP but not with NAP or SNP. Western blotting showed RSNOs, SNAP, NAP and the non-RSNO, SNP-incubated fibrinogen solutions showed that the percent level of the Aγ dimer to total Aγ dimer + γ monomer was significantly reduced in the case of the SNAP group when compared to SNP group. These results suggest that NO donors such as SNAP and SNP induce fibrinogen conformational changes by potentially nitrosating fibrinogen tyrosine residues. These NO-mediated fibrinogen changes induced via NO donors may provide another mechanism of NO for improving thromboresistance in ECC.

Peroxynitrite dominates sodium nitroprusside-induced apoptosis in human hepatocellular carcinoma cells.

This study aims to explore which radicals dominate sodium nitroprusside (SNP)-induced cytotoxicity in human hepatocellular carcinoma (HCC) cells (HepG2 and Hep3B). Exposure of SNP to cell medium produced abundant nitric oxide (NO), superoxide anion (O2·−), hydrogen peroxide (H2O2) and iron ions. SNP potently induced caspases activation, mitochondrial membrane permeabilization and apoptosis in HCC cells. In Hep3B cells, pretreatment with NO scavenger (PTIO) did not prevent SNP-induced cytotoxicity. However, in HepG2 cells, SNP-induced cytotoxicity was prevented significantly by pretreatment with PTIO and O2·− scavenger, and especially was almost completely blocked by pretreatment with FeTPPS (peroxynitrite scavenger). In contrast, although H2O2 scavenger potently scavenged SNP-induced H2O2 production, it did not prevent SNP-induced cytotoxicity in HepG2 cells. In addition, pretreatment with DFO (iron ions chelator) and iron-saturated DFO respectively completely prevented SNP-induced cytotoxicity in HepG2 cells. Collectively, peroxynitrite from the reaction between NO and O2·− elicited from SNP dominates the SNP-induced apoptosis of HepG2 cells, in which both iron ions and H2O2 are not involved.

Nitric Oxide Exerts Basal and Insulin-Dependent Anorexigenic Actions in POMC Hypothalamic Neurons

The arcuate nucleus of the hypothalamus represents a key center for the control of appetite and feeding through the regulation of 2 key neuronal populations, notably agouti-related peptide/neuropeptide Y and proopimelanocortin (POMC)/cocaine- and amphetamine-regulated transcript neurons. Altered regulation of these neuronal networks, in particular the dysfunction of POMC neurons upon high-fat consumption, is a major pathogenic mechanism involved in the development of obesity and type 2 diabetes mellitus. Efforts are underway to preserve the integrity or enhance the functionality of POMC neurons in order to prevent or treat these metabolic diseases. Here, we report for the first time that the nitric oxide (NO(-)) donor, sodium nitroprusside (SNP) mediates anorexigenic actions in both hypothalamic tissue and hypothalamic-derived cell models by mediating the up-regulation of POMC levels. SNP increased POMC mRNA in a dose-dependent manner and enhanced α-melanocortin-secreting hormone production and secretion in mHypoA-POMC/GFP-2 cells. SNP also enhanced insulin-driven POMC expression likely by inhibiting the deacetylase activity of sirtuin 1. Furthermore, SNP enhanced insulin-dependent POMC expression, likely by reducing the transcriptional repression of Foxo1 on the POMC gene. Prolonged SNP exposure prevented the development of insulin resistance. Taken together, the NO(-) donor SNP enhances the anorexigenic potential of POMC neurons by promoting its transcriptional expression independent and in cooperation with insulin. Thus, increasing cellular NO(-) levels represents a hormone-independent method of promoting anorexigenic output from the existing POMC neuronal populations and may be advantageous in the fight against these prevalent disorders.

Neuroprotective and Anti-Apoptotic Propensity of Bacopa monniera Extract Against Sodium Nitroprusside Induced Activation of iNOS, Heat Shock Proteins and Apoptotic Markers in PC12 Cells

Sodium nitroprusside (SNP) is a widely used nitric oxide (NO) donor, known to exert nitrative stress by up-regulation of inducible nitric oxide synthase (iNOS). Nω-nitro-L-arginine-methyl esther (L-NAME) is a NO inhibitor, which inhibits iNOS expression, is used as positive control. The present study was designed to assess neuroprotective propensity of Bacopa monniera extract (BME) in SNP-induced neuronal damage and oxido-nitrative stress in PC12 cells via modulation of iNOS, heat shock proteins and apoptotic markers. Our results elucidate that pre-treatment of PC12 cells with BME ameliorates the mitochondrial and plasma membrane damage induced by SNP (200 μM) as evidenced by MTT and LDH assays. BME pre-treatment inhibited NO generation by down regulating iNOS expression. BME replenished the depleted antioxidant status induced by SNP treatment. SNP-induced damage to cellular, nuclear and mitochondrial integrity was also restored by BME, which was confirmed by ROS estimation, comet assay and mitochondrial membrane potential assays respectively. BME pre-treatment efficiently attenuated the SNP-induced apoptotic protein biomarkers such as Bax, Bcl-2, cytochrome-c and caspase-3, which orchestrate the proteolytic damage of the cell. Q-PCR results further elucidated up-regulation of neuronal cell stress markers like HO-1 and iNOS and down-regulation of BDNF upon SNP exposure was attenuated by BME pre-treatment. By considering all these findings, we report that BME protects PC12 cells against SNP-induced toxicity via its free radical scavenging and neuroprotective mechanism.

Kobophenol A inhibits sodium nitroprusside-induced cardiac H9c2 cell death through suppressing activation of JNK and preserving mitochondrial anti-apoptotic Bcl-2 and Mcl-1.

Sodium nitroprusside (SNP) releases nitric oxide (NO), a powerful vasodilator, and thus widely used in intensive care unit for treating hypertension emergency. However, cardiac toxicity after SNP administration is a clinical problem. For finding a natural compound that suppressing SNP-induced cardiac toxicity, we tested the protective potential of kobophenol A (Kob A), purified from the root of Caragana sinica, against the toxic effects of SNP. The severe cardiac H9c2 cell death was induced by SNP (2 mM) treatment. Kob A ameliorated SNP-induced cardiac H9c2 cell death, and this protective effect of Kob A may be related to the inhibition of c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein (MAP) kinase activation following SNP administration. In addition, the downregulation of cellular Bcl-2 and Mcl-1 levels by SNP exposure was strongly abrogated in the presence of Kob A. These biological properties of Kob A might provide insights into developing new cardioprotectant against SNP-induced cardiac cell death.

Sodium Nitroprusside inhibits HEK293 Cell Growth by cGMP-Dependent and Independent Mechanisms

The acute and chronic effects of sodium nitroprusside (SNP) are well characterized for vascular smooth muscle cells (VSMC). Stimulation of soluble guanylyl cyclase (sGC) gives a rapid elevation of intracellular cGMP levels and relaxation of VSMC. The antiproliferative effect of SNP needs days to develop. In the present study human embryonic kidney (HEK 293) cells were used to study the growth after repeated exposure to SNP. A dose-dependent antiproliferative effect was evident and after 5 days with an IC50 value of 108 μM. Cyclic GMP was able to mimic the antiproliferative effect of SNP on HEK293 cells. When cGMP (1000 μM) was added to the cell culture medium for 5 days the cell densities were reduced with 37% below baseline and cGMPin increased from 5.3 to 195 pmol/107cells. The interaction with the non-selective PDE (cyclic nucleotide phosphodiesterase) inhibitor 3-isobutyl-1-methylxanthine (IBMX) was tested after three days. IBMX alone (1000 μM) reduced cell densities with 48% and elevated cGMPin (from 5.2 to 9.3 pmol/107cells). The effect of 10 μM SNP was reinforced on proliferation (from 13% to 90%) and elevation of cGMP levels (from 7.6 to 13.5 pmol/107cells). A corresponding effect was observed after addition of 1000 μM cGMP and 1000 μM IBMX for 3 days. The antiproliferative effect of cGMP increased from 30% to 89% and the cGMPin increased from 240 to 480 pmol/107cells. However, additional mechanisms exist for the antiproliferative effect of SNP. One of these is the intracellular oxidative effect which includes production of S-nitrosoglutathione. The fall in ratios between GSH and GSSG from 260 to 85 after 100 μM SNP exposure is compatible with such a mechanism since cGMP (1000 μM) added to the culture medium did not change the ratio. This study shows that the antiproliferative effects of SNP on HEK293 cells are mediated through cGMP-dependent and cGMP-independent mechanisms. The concentration-dependent effects develop over time. HEK293 cells had an efficient efflux system for cGMP and the use of inside-out vesicles (IOVs) showed high affinity ATP-dependent cGMP transport with a Km value of 2.3 μM. The antiproliferative effect of SNP was correlated to cGMPex/in.

Pharmacological shift of the ambiguous nitric oxide action from neurotoxicity to cyclic GMP-mediated protection

Objectives: The effect of intracellular cyclic guanosine monophosphate (GMP) increase on neuronal damage was tested using a newly developed nitric oxide-related injury model of cultured spinal cord neurons.Methods: Neuronal damage after 24-hour-exposure to sodium nitroprusside (SNP), a nitric oxide (NO) donor, was evaluated by measuring the activity of released lactate dehydrogenase from injured neurons.Results: Oxygen radical scavengers had a protective effect, indicating that the neuronal damage, elicited by 10 μM SNP, was largely due to peroxynitrite formation. Alternatively, a strong inhibition of the NO-induced damage could also be achieved by an intracellular cyclic GMP increase resulting from the addition of 100 μM 8-bromo-cyclic GMP. Propentofylline (PPF, 1–100 μM), a xanthine derivative and rather selective phosphodiesterase (PDE) inhibitor, enhanced intracellular cyclic GMP elevation induced by SNP exposure. The neuronal damage induced by 10 μM SNP exposure for 24 hours was almost completely blocked in the presence of 1 μM PPF.Discussion: These results suggest that NO has an ambiguous action, i.e. toxic by favoring the formation of, but protective by intracellular cyclic GMP elevation which can be reinforced by PDE inhibition. Therefore, PDE inhibitors, such as PPF, may be useful therapeutic drugs to limit oxidative neuronal damage in the central nervous system.

The protective activities of water-soluble C 60 derivatives against nitric oxide-induced cytotoxicity in rat pheochromocytoma cells

In this study, the protective activities of water-soluble C(60) derivatives against nitric oxide (NO) induced cytotoxicity were investigated. To overcome C(60) insolubility in water, we modified C(60) with β-alanine, valine or folacin. The compounds were characterized by FT-IR, (1)H NMR, (13)C NMR, LC-MS, elemental analysis, light scattering and TEM. Investigation of the possible NO-scavenging activities of water-soluble C(60) derivatives demonstrated that they expressed direct scavenging activity toward NO liberated within solution of sodium nitroprusside (SNP). In parallel, following exposure of cells to SNP (1 mM), a marked decrease in mitochondrial membrane potential, cell viability, activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), as well as increased levels of intracellular NO accumulation and malondialdehyde (MDA) production were observed. Moreover, SNP caused significant elevation in intracellular caspase-3 activity, and induced apoptotic death as determined by flow cytometric assay. However, pretreatment of the cells with water-soluble C(60) derivatives prior to SNP exposure blocked these NO-induced cellular events noticeably. Experiments demonstrated that the aggregation morphology could impact the NO-scavenging abilities and protective effects on apoptosis of water-soluble C(60) derivatives. The results suggest that water-soluble C(60) derivatives have the potential to prevent NO-mediated cell death without evident toxicity.

Nitric oxide‐induced biphasic mechanism of vascular relaxation via dephosphorylation of CPI‐17 and MYPT1

Nitric oxide (NO) from endothelium is a major mediator of vasodilatation through cGMP/PKG signals that lead to a decrease in Ca(2+) concentration. In addition, NO-mediated signals trigger an increase in myosin light chain phosphatase (MLCP) activity. To evaluate the mechanism of NO-induced relaxation through MLCP deinhibition, we compared time-dependent changes in Ca(2+), myosin light chain (MLC) phosphorylation and contraction to changes in phosphorylation levels of CPI-17 at Thr38, RhoA at Ser188, and MYPT1 at Ser695, Thr696 and Thr853 in response to sodium nitroprusside (SNP)-induced relaxation in denuded rabbit femoral artery. During phenylephrine (PE)-induced contraction, SNP reduced CPI-17 phosphorylation to a minimal value within 15 s, in parallel with decreases in Ca(2+) and MLC phosphorylation, followed by a reduction of contractile force having a latency period of about 15 s. MYPT1 phosphorylation at Ser695, the PKG-target site, increased concurrently with relaxation. Phosphorylation of RhoA, MYPT1 Thr696 and Thr853 differed significantly at 5 min but not within 1 min of SNP exposure. Inhibition of Ca(2+) release delayed SNP-induced relaxation while inhibition of Ca(2+) channel, BK(Ca) channel or phosphodiesterase-5 did not. Pretreatment of resting artery with SNP suppressed an increase in Ca(2+), contractile force and phosphorylation of MLC, CPI-17, MYPT1 Thr696 and Thr853 at 10 s after PE stimulation, but had no effect on phorbol ester-induced CPI-17 phosphorylation. Together, these results suggest that NO production suppresses Ca(2+) release, which causes an inactivation of PKC and rapid CPI-17 dephosphorylation as well as MLCK inactivation, resulting in rapid MLC dephosphorylation and relaxation.

Reactive Oxygen Species Mediate p53 Activation and Apoptosis Induced by Sodium Nitroprusside in SH-SY5Y Cells

Sodium nitroprusside (SNP) is a water-soluble iron nitrosyl complex clinically used as a powerful vasodilator for treatment of hypertension; and, in basic research, it has been used to mainly investigate the cytotoxic effects of nitrosative stress. Although NO is considered a pharmacologically active molecule, not all of the biological effects of SNP are dependent on its NO moiety. To elucidate the molecular executioner(s) responsible for SNP cytotoxicity, this study determines the involvement of oxidative stress in p53 activation and apoptotic induction elicited by SNP in SH-SY5Y neuroblastoma cells. We demonstrate that proapoptotic activity of SNP is independent of NO production, because SNP and its 2-day light-exhausted compound SNP(ex) trigger apoptosis to the same extent. We provide evidence for the occurrence of oxidative stress and oxidative damage during both SNP and SNP(ex) exposure and demonstrate that iron-derived reactive oxygen species (ROS) are the genuine mediators of their cytotoxicity. We show that p53 is equally activated upon both SNP and SNP(ex) treatments. Moreover, as demonstrated by small interfering RNA experiments, we indicate its primary role in the induction of apoptosis, suggesting the ineffectiveness of NO in its engagement. The attenuation of p53 levels, obtained by oxy-radical scavengers, is consistent with the recovery of cell viability and ROS decrease, demonstrate that SNP-mediated p53 activation is an event triggered by ROS and/or ROS-mediated damages. Together, our results suggest that investigations of the physiopathological effects of SNP should consider the role of ROS, other than NO, particularly in some conditions such as apoptotic induction and p53 activation.

Effect of nitration on protein tyrosine phosphatase and protein phosphatase activity in neuronal cell membranes of newborn piglets

Protein tyrosine phosphatase predominantly determines the status of protein tyrosine kinase-dependent phosphorylation of specific proteins and controls the survival and death of neurons. Previous studies have shown that protein tyrosine phosphatase activity is decreased during hypoxia in cortical membranes of the newborn piglet. We have also shown that nitric oxide (NO) free radicals are generated during hypoxia, and may result in modification of protein tyrosine phosphatase via peroxynitrite-mediated modification. The present study tests the hypothesis that the hypoxia-induced decrease in protein tyrosine phosphatase activity is NO-mediated. To test this hypothesis, in vitro experiments were conducted by measuring protein tyrosine phosphatase activity in the presence of an NO donor, sodium nitroprusside (SNP), or peroxynitrite. Since 3-nitrotyrosine is produced as a consequence of peroxynitrite reactions, we have also examined the effect of 3-nitrotyrosine on protein phophatase activity. Cerebral cortical P 2 membranes were prepared from seven normoxic newborn piglets and each sample was divided into three aliquots: a control group, a SNP group (exposed to 200 μM SNP ), and a peroxynitrite group (exposed to 100 μM peroxynitrite ). Protein tyrosine phosphatase activity was determined spectrophotometrically in the presence or absence of 2 μM bpV(phen), a highly selective inhibitor of protein tyrosine phosphatase. The protein tyrosine phosphatase activity was 198 ± 25 nmol/mg protein/h in the normoxic group, 177 ± 30 nmol/mg protein/h in the SNP group ( p = NS versus normoxic) and 77 ± 20 nmol/mg protein/h in the peroxynitrite group ( p < 0.001 versus normoxic). The results show that peroxynitrite but not SNP exposure results in decreased protein tyrosine phosphatase activity in vitro. Furthermore 3-nitrotyrosine (100 μm), a product of peroxynitrite, decreased the enzyme activity from 926 ± 102 to 200 ± 77 ( p < 0.001). We conclude that protein tyrosine phosphatase regulation is mediated by peroxynitrite. We propose that hypoxia-induced NO production leading to peroxynitrite formation is a potential mechanism of protein tyrosine phosphatase inactivation in vivo. The NO-induced decrease in protein tyrosine phosphatase and protein phosphatase activity, leading to Bcl-2 protein phosphorylation and loss of its antiapoptotic activity may be a NO-mediated mechanism of programmed cell death in the hypoxic brain.

Interstitial telomeric DNA sequences of Chinese hamster cells are hypersensitive to nitric oxide damage, and DNA‐PKcs has a specific local role in its repair

The DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) procedure was used to analyze DNA single-strand breaks (SSBs) and alkali-labile sites induced by exposure to the nitric oxide (NO) donors sodium nitroprusside (SNP) and 3-morpholinosydnomine hydrochloride (SIN-1) in the whole genome and in long interstitial telomeric repeat sequence (ITRS) blocks from Chinese hamster cells. The relative density of DNA damage generated in the ITRS by X-rays was similar to that induced in the genome overall, whereas it was 1.7 times higher when the alkylating agent MNNG was assayed. Nevertheless, after SNP or SIN-1 treatment, ITRSs proved to be 2.8 and 2.7 times relatively more damaged, respectively, than the whole genome. When the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) was not active, as in XR-C1 mutant cells, the repair kinetics in the whole genome did not differ from that in the parental cell line with X-ray or SNP exposure. However, whereas the SSBs and alkali-labile sites induced in the ITRS by X-rays exhibited rejoining kinetics similar to that of the parental cell line, the damage induced by SNP was more slowly rejoined. This implies a role for DNA-PKcs in the repair of DNA damage induced by NO, especially in ITRSs. The results demonstrated intragenomic heterogeneity of NO-induced DNA damage and repair; there was a higher density of DNA damage in the ITRS blocks, possibly because of their guanine richness. This suggests that a parallel process may occur in the terminal telomeres, which has implications for premature aging and neoplastic development by chronic NO exposure in vivo.

Increase of intracellular glutathione by low-level NO mediated by transcription factor NF-κB in RAW 264.7 cells

The mechanism underlying the elevation of intracellular glutathione (GSH) in RAW 264.7 cells exposed to low concentrations of sodium nitroprusside (SNP), a well-known nitric oxide (NO) donor, was investigated. The peak of intracellular GSH was reached at 6 h after exposure of the cells to SNP (0.1–0.5 mM), and this was preceded by the induction of mRNA for γ-glutamylcysteine synthetase (γ-GCS; the rate-limiting enzyme of de novo GSH synthesis), which peaked at 3 h. N-α-Tosyl- l-phenylalanine chloromethyl ketone (TPCK) and caffeic acid phenethyl ester (CAPE), specific inhibitors of NF-κB, significantly suppressed the SNP-induced elevation of GSH protein and γ-GCS mRNA, while curcumin, an inhibitor of AP-1, was less effective. Electrophoretic mobility shift assay (EMSA) showed that SNP exposure markedly increased the DNA binding of NF-κB, but not that of AP-1. Deletion or mutagenesis of the NF-κB site in the γ-GCS gene promoter abolished the SNP-induced up-regulation of GSH protein and γ-GCS mRNA. These results suggest that the elevation of intracellular GSH in RAW 264.7 cells exposed to low concentrations of SNP occurs through the operation of the de novo GSH pathway, and is mediated by transcriptional up-regulation of the γ-GCS gene, predominantly at the NF-κB binding site in its promoter.

Nitric oxide-induced apoptosis is mediated by Bax/Bcl-2 gene expression, transition of cytochrome c, and activation of caspase-3 in rat vascular smooth muscle cells

Background: In contrast to the anti-apoptotic action of nitric oxide (NO) on endothelial cells, NO exerts a pro-apoptotic effect on vascular smooth muscle cells (VSMCs). This study was designed to elucidate the mechanism underlying NO-induced apoptosis in rat VSMCs. Methods and results: (1) Using the terminal deoxynucleotidyl transferase-mediated digoxigenin-11-dUTP nick end labeling (TUNEL) assay and fluorescence activated cell sorter (FACS) analyses, apoptosis of rat VSMCs were confirmed after exposure to sodium nitroprusside (SNP) (0.5 to 4 mmol/l), an exogenous NO donor. The effects of SNP were blocked by hemoglobin. (2) A universal caspase inhibitor, z-VAD-fmk, dose-dependently inhibited NO-induced apoptosis. VSMCs degraded Ac-DEVD-pNA rather than Ac-WHED-pNA after exposure to SNP, which suggested that the activation of caspase 3 rather than caspase 1 was involved in the process. Immunoblot analysis confirmed the activation of caspase-3. (3) Exposure to SNP induced the release of cytochrome c from the mitochondria to the cytosol, which was detected by immunoblot analysis of mitochondrial and cytosol fractions. (4) SNP exposure increased the ratio of Bax/Bcl-2 protein expression twofold by immunoblot analysis. Conclusions: The mechanism of NO-induced apoptosis in rat VSMCs involves an increase in the ratio of Bax/Bcl-2 gene expression, which leads to the release of cytochrome c from the mitochondria to the cytosol, finally activating caspase-3 and resultant apoptosis.

Despite in vitro increase in cyclic guanosine monophosphate concentrations, intracarotid nitroprusside fails to augment cerebral blood flow of healthy baboons.

During cerebral angiography, intracarotid infusion of sodium nitroprusside (SNP), an endothelium-independent nitric oxide donor, fails to increase cerebral blood flow (CBF) of human subjects. A confounding effect of intracranial pathology or that of radiocontrast could not be ruled out in these experiments. The authors hypothesized that, if nitric oxide was a significant regulator of CBF of primates, then intracarotid SNP will augment CBF of baboons. In studies, CBF (intraarterial (133)Xe technique) was measured in healthy baboons during isoflurane anesthesia at (1) baseline and during (2) induced hypertension with intravenous phenylephrine, (3) concurrent infusions of intravenous phenylephrine and intracarotid SNP, and (4) intracarotid verapamil (positive control drug). In studies, the authors measured tissue cyclic guanosine monophosphate (cGMP) by radioimmunoassay after incubating vascular rings obtained from freshly killed baboons (1) with increasing concentrations of SNP and (2) after SNP exposure following preincubation with the radiocontrast agent, iohexhol. In the studies, coinfusion of intravenous phenylephrine and intracarotid SNP did not increase CBF. However, intracarotid verapamil significantly increased CBF (from 26 +/- 7 to 43 +/- 11 ml x 100 g(-1) x min(-1); P < 0.0001) without a change in mean arterial pressure. In the studies, incubation of intracranial arterial rings in SNP resulted in dose-dependent increases in cGMP concentrations. A similar increase in cGMP content was evident despite iohexhol preincubation. Collectively, these results suggest that, in healthy baboons, intracarotid SNP does not decrease arteriolar resistance, although SNP could affect proximal arterial tone, as demonstrated by the increase in cGMP content of these vessels.

Sodium Nitroprusside-Induced Mitochondrial Apoptotic Events in Insulin-Secreting RINm5F Cells Are Associated with MAP Kinases Activation

Exposure of insulin-secreting RINm5F cells to the chemical nitric oxide donor sodium nitroprusside (SNP) resulted in apoptotic cell death, as detected by cytochrome c release from mitochondria and caspase 3 activation. SNP exposure also leads to phosphorylation and activation of enzymes involved in cellular response to stress such as signal-regulated kinase 2 (ERK2) and c-Jun NH2-terminal kinase 46 (JNK46). Both cytochrome c release and caspase 3 activation were abrogated in cells exposed to MEK and p38 inhibitors. Treatment of cells with the NO donors SNP, DETA–NO, GEA 5024, and SNAP resulted in phosphorylation of the antiapoptotic protein Bcl-2, which was resistant to blockade of MEK, p38, and JNK pathways and sensitive to phosphoinositide 3-kinase (PI3K) inhibition. In addition, transient transfection of cells with the wild-type PI3K γ gene mimics the increased rate of Bcl-2 phosphorylation detected in NO-treated cells. The generation of phosphoinositides seems to participate in the process since Bcl-2 phosphorylation was not observed in cells overexpressing lipid-kinase-deficient PI3Kγ. The potential of SNP toxicity directly from NO was supported by our finding that the NO scavenger carboxy-PTIO prevented cell death. We found no evidence to support the contention that oxygen radicals generated during cellular SNP metabolism mediate cell toxicity in RINm5F cells, since neither addition of catalase/superoxide dismutase nor transfection with superoxide dismutase prevented SNP-induced cell death. Thus, we propose that exposure to apoptotic concentrations of NO triggers ERK- and p38-dependent cytochrome c release, caspase 3 activation, and PI3K-dependent Bcl-2 phosphorylation.