Cytotoxic Effects Of Nitric Oxide Research Articles

INCREASED NITRIC OXIDE SYNTHESIS IN PERITONEAL MACROPHAGES UNDER THE ACTION OF ASCORBIC ACID

The effect of ascorbic acid (AA) on the formation of nitric oxide (NO) in peritoneal macrophages of SHK line mice was studied. ESR and UV spectroscopy revealed a significant increase in oxide formation in a suspension of macrophages with AA incubated at room temperature. In ESR studies, hemoglobin added to a suspension of macrophages was used as a NO trap, and the resulting nitric oxide was detected by the formation of hemoglobin complexes Heme-NO. The quantitative assessment showed that under these conditions, under the action of AA, 1.5x109 molecules of NO per cell are produced in macrophages. The increase in nitric oxide synthesis under the action of AA was also established by UV spectroscopy in the supernatant after incubation of MP with AA. Considering the known data on the cytotoxic effect of NO on viruses and bacteria, it is assumed that the effect of increasing nitric oxide synthesis in peritoneal, tissue MP and leukocytes under the action of AA is the main in the preventive and therapeutic effect of AA in a number of diseases, such as a sick of cold.

AN INDIRECT PATHWAY FOR GENETIC INFLUENCE ON THE FORMATION OF THE CEREBRAL PALSY PHENOTYPE: GENOME AND HYPOXIA TOLERANCE

The incidence of cerebral palsy is increasing. In 70% of cases, the disease develops as a result of exposure to known pathogenesis factors, such as hypoxia-ischemia, infectious, toxic, and traumatic damage to the fetus. In 30% of cases of the disease, the effect of pathogenetic factors is absent. This determines the relevance of the search for genetic anomalies responsible for the formation of this phenotype. Many genome disorders associated with the development of the cerebral palsy phenotype have been identified. Of particular interest is the indirect influence of genetic factors of pathogenesis, affecting the formation of the disease indirectly, through the mechanisms of formation of hypoxic-ischemic brain damage. Such factors affect almost all stages of the “ischemic cascade”: the formation of a “glutamate shock”, the realization of a “glutamate shock” by cytokines, the defect of the antioxidant defense of a neuron, the realization of the cytotoxic effect of nitric oxide (NO), and the stimulation of apoptosis of neurons and microglia. This makes it possible to identify genome variants that determine the tolerance of the child’s brain to hypoxic-ischemic damage. The combination of carriers of such anomalies into risk groups will make it possible to differentiate the tactics of their observation in the natal and postnatal periods in order to reduce the likelihood of the formation of severe hypoxic-ischemic lesions of the central nervous system and cerebral palsy. Today, a large amount of scientific data has been accumulated on the molecular genetic mechanisms of the pathogenesis of perinatal brain lesions. Such as epigenetic, transcriptome gene expression control mechanisms via micro-RNA. The available information allows us to experimentally develop new methods for protecting the fetal brain from hypoxia-ischemia and stopping the effects of hypoxia-ischemia in the neonatal period.

Combination of Amino Acid/Dipeptide with Nitric Oxide Donating Oleanolic Acid Derivatives as PepT1 Targeting Antitumor Prodrugs

By taking advantage of the cytotoxic effect of nitric oxide (NO) and PepT1 for molecule-targeted drug delivery, a series of amino acid/dipeptide diester prodrugs of NO-donating oleanolic acid derivatives were designed and synthesized. Two prodrugs 6a and 8a showed potent cytotoxcity, which is probably due to their high PepT1 affinity and NO-releasing ability. Furthermore, the aqueous solubility of the prodrugs was also significantly enhanced because of the hydrophilic amino acid/dipeptide promoiety.

Desulfovibrio gigas Flavodiiron Protein Affords Protection against Nitrosative Stress In Vivo

Desulfovibrio gigas flavodiiron protein (FDP), rubredoxin:oxygen oxidoreductase (ROO), was proposed to be the terminal oxidase of a soluble electron transfer chain coupling NADH oxidation to oxygen reduction. However, several members from the FDP family, to which ROO belongs, revealed nitric oxide (NO) reductase activity. Therefore, the protection afforded by ROO against the cytotoxic effects of NO was here investigated. The NO and oxygen reductase activities of recombinant ROO in vitro were tested by amperometric methods, and the enzyme was shown to effectively reduce NO and O(2). Functional complementation studies of an Escherichia coli mutant strain lacking the ROO homologue flavorubredoxin, an NO reductase, showed that ROO restores the anaerobic growth phenotype of cultures exposed to otherwise-toxic levels of exogenous NO. Additional studies in vivo using a D. gigas roo-deleted strain confirmed an increased sensitivity to NO of the mutant strain in comparison to the wild type. This effect is more pronounced when using the nitrosating agent S-nitrosoglutathione (GSNO), which effectively impairs the growth of the D. gigas Deltaroo strain. roo is constitutively expressed in D. gigas under all conditions tested. However, real-time reverse transcription-PCR analysis revealed a twofold induction of mRNA levels upon exposure to GSNO, suggesting regulation at the transcription level by NO. The newly proposed role of D. gigas ROO as an NO reductase combined with the O(2) reductase activity reveals a versatility which appears to afford protection to D. gigas at the onset of both oxidative and nitrosative stresses.

The mechanism of action of methylprednisolone in the treatment of multiple sclerosis

Methylprednisolone plays an important role in the current treatment of multiple sclerosis (MS), particularly in the acute phase of relapse. It acts in various ways to decrease the inflammatory cycle including: dampening the inflammatory cytokine cascade, inhibiting the activation of T cells, decreasing the extravasation of immune cells into the central nervous system, facilitating the apoptosis of activated immune cells, and indirectly decreasing the cytotoxic effects of nitric oxide and tumor necrosis factor alpha. This paper reviews the most recent observations on these mechanisms both to understand the disease mechanism and its treatment. As more becomes known about these mechanisms, it may become possible to design treatment regimes that are more specific towards both the individual and the disease state.

Involvement of NF-κB and glutathione in cytotoxic effects of nitric oxide and taxol on human leukemia cells

Nitric oxide (NO) has been shown to be cytotoxic for normal and transformed cell lines. One of the intracellular targets for NO action is glutathione (GSH). GSH determinates cellular redox potential and modulates several biological events. During oxidative and nitrosative stress, glutathione system imbalance is associated with the upregulation of γ-glutamylcysteine synthetase (γ-GCS) expression, which is mediated by nuclear factor κB (NF-κB). Our previous studies demonstrated a cytotoxic effect of NO and taxol on human lymphoblastic leukemia cells triggered by inhibition of NF-κB activity. In this study, we have demonstrated the involvement of GSH in taxol- and NO-induced cytotoxic effects on human CEM leukemia cells. NO- and taxol-induced a depletion of GSH levels in CEM cells, which was potentialized by l-buthionine- S, R-sulfoximine (BSO), an inhibitor of γ-GCS. BSO induced an increase in nuclear translocation of NF-κB. However, when cells were treated with NO or taxol in association with BSO, these compounds inhibited the constitutive activity of NF-κB. These results suggest that oxidative and nitrosative damage in lymphoblastic leukemia cells shall be mediated by NO- and taxol-induced GSH depletion as a consequence of preventing GSH synthesis.

The cytotoxic anti-tumor effect of MTH-68/H, a live attenuated Newcastle disease virus is mediated by the induction of nitric oxide synthesis in rat peritoneal macrophages in vitro

Rat peritoneal macrophages were induced to produce high amounts of nitric oxide (NO) when rats were challenged by MTH68/H, (a live attenuated oncolytic Newcastle disease virus strain). The increase in NO production was observed to be viral particle dose dependent. The higher NO production measured could be due to the enhanced expression of NO synthase II enzyme. In addition, viral administration caused a higher macrophage cell count in the peritoneal cavity of treated rats. Interleukin-1 and granulocyte–monocyte colony stimulating factors were also produced by the induced macrophages. COS 7, a transformed cell line was killed by both NO donors and activated macrophages; the latter effect was markedly decreased in the presence of the inhibitors of NO production. Cytotoxic effect of NO was evidenced by the decrease of cell viability and proliferation of COS 7 cells. Excessive NO production may also be cytotoxic for macrophages themselves as proved by the addition of exogenous NO donors. These results strongly suggested the participation of induced NO synthesis of macrophages in the anti-tumor effect of MTH-68/H vaccine treatment.

Transcriptional Responses of Escherichia coli to S-Nitrosoglutathione under Defined Chemostat Conditions Reveal Major Changes in Methionine Biosynthesis

Nitric oxide and nitrosating agents exert powerful antimicrobial effects and are central to host defense and signal transduction. Nitric oxide and S-nitrosothiols can be metabolized by bacteria, but only a few enzymes have been shown to be important in responses to such stresses. Glycerol-limited chemostat cultures in defined medium of Escherichia coli MG1655 were used to provide bacteria in defined physiological states before applying nitrosative stress by addition of S-nitrosoglutathione (GSNO). Exposure to 200 microm GSNO for 5 min was sufficient to elicit an adaptive response as judged by the development of NO-insensitive respiration. Transcriptome profiling experiments were used to investigate the transcriptional basis of the observed adaptation to the presence of GSNO. In aerobic cultures, only 17 genes were significantly up-regulated, including genes known to be involved in NO tolerance, particularly hmp (encoding the NO-consuming flavohemoglobin Hmp) and norV (encoding flavorubredoxin). Significantly, none of the up-regulated genes were members of the Fur regulon. Six genes involved in methionine biosynthesis or regulation were significantly up-regulated; metN, metI, and metR were shown to be important for GSNO tolerance, because mutants in these genes exhibited GSNO growth sensitivity. Furthermore, exogenous methionine abrogated the toxicity of GSNO supporting the hypothesis that GSNO nitrosates homocysteine, thereby withdrawing this intermediate from the methionine biosynthetic pathway. Anaerobically, 10 genes showed significant up-regulation, of which norV, hcp, metR, and metB were also up-regulated aerobically. The data presented here reveal new genes important for nitrosative stress tolerance and demonstrate that methionine biosynthesis is a casualty of nitrosative stress.

New Genes Implicated in the Protection of Anaerobically Grown Escherichia coli against Nitric Oxide

Nitric oxide produced by activated macrophages plays a key role as one of the immune system's weapons against pathogens. Because the lifetime of nitric oxide is short in aerobic conditions, whereas in anaerobic conditions the cytotoxic effects of nitric oxide are greatly increased as in the infection/inflammation processes, it is important to establish which systems are able to detoxify nitric oxide under anaerobic conditions. In the present work a new set of Escherichia coli K-12 genes conferring anaerobic resistance to nitric oxide is presented, namely the gene product of YtfE and a potential transcriptional regulator of the helix-turn-helix LysR-type (YidZ). The crucial role of flavohemoglobin for anaerobic nitric oxide protection is also demonstrated. Furthermore, nitric oxide is shown to cause a significant alteration of the global E. coli gene transcription profile that includes the increase of the transcript level of genes encoding for detoxification enzymes, iron-sulfur cluster assembly systems, DNA-repairing enzymes, and stress response regulators.

Antioxidant Properties of Nitric Oxide in Cellular Physiological and Pathophysiological Mechanisms. The Implications of Biological Balance between NO and Oxidative Stress

The function of nitric oxide ( NO) in pathophysiology remains confounding as both protective and cytotoxic effects of NO have been demonstrated in many disease processes. Nitric oxide chemistry culminating in the generation of oxidative as well as nitrosative intermediates have generally been proposed as mediators of pathophysiology and have overshadowed the antioxidant capabilities of NO. However, the counteracting role of NO in providing a balance under conditions of oxidative and nitrosative stress has been underappreciated. The purpose of this review is the discussion of the role of NO as an antioxidant and interceptor of more potent reactive intermediates in normal physiology and disease.

Nitric Oxide Inhibits Ultraviolet B-induced Murine Keratinocyte Apoptosis by Regulating Apoptotic Signaling Cascades

Cytotoxic effects of nitric oxide (NO) derived from inducible nitric oxide synthase (iNOS) are considered to be one of the major causes of inflammatory diseases. On the other hand, protective effects of NO on toxic insults-induced cellular damage/apoptosis have been demonstrated recently. Ultraviolet B (UVB)-induced apoptosis of epidermal keratinocytes leads to skin inflammation and photoageing. However, it has not been elucidated what kind of effects NO has on UVB-induced keratinocyte apoptosis. Thus, in the present study, we investigated the problem and demonstrated that NO from NO donor suppressed UVB-induced apoptosis of murine keratinocytes. In addition, NO significantly suppressed activities of caspase 3, caspase 8 and caspase 9 that had been upregulated by UVB radiation. NO also suppressed p53 expression that had been upregulated by UVB radiation and upregulated Bcl-2 expression that had been downregulated by UVB radiation. These findings suggested that NO might suppress UVB-induced keratinocyte apoptosis by regulating apoptotic signaling cascades in p53, Bcl-2, caspase3, caspase 8 and caspase 9.

Long-term treatment of anterior pituitary cells with nitric oxide induces programmed cell death.

Nitric oxide (NO) plays a complex role in modulating programmed cell death. It can either protect the cell from apoptotic death or mediate apoptosis, depending on its concentration and the cell type and/or status. In this study, we demonstrate that long-term exposition to NO induces cell death of anterior pituitary cells from Wistar female rats. DETA NONOate (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate, 1 mm], a NO donor that releases NO for an extended period of time, decreased cellular viability and prolactin release from primary cultures of anterior pituitary cells. Morphological studies showed an increase in the number of cells with chromatin condensation and nuclear fragmentation at 24 and 48 h after DETA/NO exposure. DNA internucleosomal fragmentation was also observed at the same time. Reversibility of the NO effect on cellular viability and prolactin release was observed only when the cells were incubated with DETA/NO for less than 6 h. Most apoptotic cells were immunopositive for prolactin, suggesting a high susceptibility of lactotrophs to the effect of NO. The cytotoxic effect of NO is dependent of caspase-9 and caspase-3, but seems to be independent of oxidative stress or nitrosative stress. Our results show that the exposition of anterior pituitary cells to NO for long periods induces programmed cell death of anterior pituitary cells.

Cytotoxicity of nitric oxide donors in smooth muscle cells is dependent on phenotype, and mainly due to apoptosis

Two phenotypes of rat carotid arterial smooth muscle cells (SMC) have been isolated in our laboratory, and their proteolytic and anti-proteolytic activities have been investigated in the presence or absence of various stimulating agents. We report here a comparative study of the cytotoxic effects of nitric oxide (NO) donors, sodium nitroprusside (SNP) and S-nitroso- N-acetylpenicillamine (SNAP), towards the swirling-type and the epithelioid-type SMCs. The concentration- and time-dependence of NO donors’ capacity to induce cell deaths was measured by an intracellular acid phosphatase activity assay and cell counting. The typical morphological features of apoptosis, such as cell blebbing and cytoplasm condensation, were observed by phase contrast microscopy and with a flourescent DNA-binding dye. Apoptotic cell deaths were confirmed using DNA fragmentation and terminal deoxyribonucelotidyl transferase-mediated dUTP nick end labelling (TUNEL) methods. Western blots were used to investigate the protein expression of several known mediators of apoptosis. It was found that both NO donors induced cell deaths in the SMC phenotypes. Compared to the swirling SMCs, the epithelioid SMCs were much more sensitive to these agents. A time- and dose-dependent decrease of cell viability was observed at NO donor concentrations higher than 0.2 mmol/l. Microscopic methods revealed cell morphology of apoptotic cell deaths. The 180-bp DNA multimers typical of apoptosis were shown by DNA fragmentation. TUNEL technique confirmed that apoptosis occurred most readily in the epithelioid SMCs than the swirling SMCs. When epithelioid SMCs were treated with SNP, changes in p53, p21 WAF1, Bcl-2, caspase 3 and PARP protein expression were found. These protein levels were unchanged when swirling SMCs were similarly treated.

Cytotoxic effect of nitric oxide on human hematological malignant cells.

We investigated the cytotoxic effect of nitric oxide (NO) on primary culture of human hematological malignant cells. Sodium nitroprusside (SNP), an NO donor, had cytotoxic effects on the cells of some patients with malignant lymphoma (ML), acute myelocytic leukemia (AML) or chronic myelomonocytic leukemia (CMMoL), but not with multiple myeloma. Cultured cells from the ML patient remained sensitive to SNP after the cells became resistant to anti-cancer drugs. In contrast, the cells from the patients with AML and CMMoL became resistant to SNP while anti-cancer drugs remained effective. In samples of the cells of the patients with ML and AML, the number of CD3 positive lymphoma cell was decreased by SNP and the number of CD33 negative cells and normal B lymphocytes (CD19 positive cells) were increased. In the cells of the patient with ML, apoptosis was induced by SNP. SNP had no effect on lymphocytes of healthy volunteers. These results suggest that SNP had an anti-tumor effect on human hematological malignant cells.

Nitric oxide damages neuronal mitochondria and induces apoptosis in neurons

The cytotoxic effect of nitric oxide on primarily cultured rat cerebellar granule cells was studied, and the mechanisms were discussed. The results showed that nitric oxide donor S-nitroso-N-acetyl-penicillamine (SNAP; 500 μmol/L) could induce apoptosis in immature cultures of cerebellar granule cells. Flow cytometry and HPLC analyses revealed that after treatment with SNAP, the mitochondrial transmembrane potential and the cellular ATP content decreased significantly. Nitric oxide scavenger hemoglobin could effectively prevent the neuronal mitochondria from dysfunction and attenuate apoptosis. The results suggested that nitric oxide activated the apoptotic program by inhibiting the activity of mitochondrial respiratory chain and thus decreasing the cellular ATP content.

The Inflammatory and Cytotoxic Effects of a Nitric Oxide Releasing Cream on Normal Skin

We describe the pro-inflammatory and cytotoxic effects of nitric oxide in vivo in human skin. Nitrite and ascorbic acid were mixed on the skin of 12 normal volunteers, three times daily, to release nitric oxide. Exposure to nitric oxide was varied by randomizing the concentration of nitrite and duration of application. Nitric oxide treated skin showed significant increases in cells expressing CD3, CD4, CD8, CD68, neutrophil elastase, ICAM-1, VCAM-1, nitrosotyrosine, p53, and apoptotic cells compared with skin treated with ascorbic acid alone. There was no significant increase in mast cells. Following application of nitric oxide there were significantly fewer CD1a positive Langerhans cells in the epidermis. These appeared to lose dendritic morphology and migrate from the epidermis. There was no significant difference in staining for Ki-67, a marker related to proliferating cell nuclear antigen, between active and control skin but staining was greater after exposure to higher dose nitric oxide than the low dose. Apoptosis, cytotoxicity, and p53 staining were relatively greater after 48 h exposure than after 24 h. These results suggest that nitric oxide is pro-inflammatory and is toxic to DNA, leading to the accumulation of p53 and subsequent apoptosis. High-dose nitric oxide paradoxically led to a smaller increase in macrophages and T cells than low dose suggesting an immunosuppressive effect of higher levels.

Ultrastructural changes in endothelial cells of cerebral microvessels after exposure to nitric oxide donor

It has been largely accepted that nitric oxide (NO) plays an important role in cerebral vasomotor control. NO has also been proposed to be a significant chemical in the process of endothelial cell injuries in various pathological conditions. Although the toxic effect of high concentrations of NO has been shown, only a few ultrastructural studies have been reported. The purpose of this study is to demonstrate physiological and pathological changes in cerebral microvessels after exposure to a high dose of NO donor. Glyceryl trinitrate, a NO donating chemical, was administered intravenously (10 mg/kg) to experimental rats. The vasomotor response was monitored by in vivo fluorescein videomicroscopy and ultrastructural changes were studied using transmission electron microscopy. The result showed long‐lasting vasodilatation of pial microvessels. The greatest response was observed in microvessels with baseline diameter of less than 10 μm compared with microvessels of a larger diameter (P = 0.003). Exposure to NO donor produced considerable changes in the ultrastructure of cerebral microvessels. Such morphological changes were characterized by increased pinocytosis, increased microvillous formation, mitochondrial swelling, doming of the endothelial surface, swelling of the perivascular astrocytic foot plate and partial separation of endothelial cells from the adjacent brain tissue. These results confirm the cytotoxic effect of NO on cerebral endothelial cell and further elaborate the role of this substance in endothelial cell injury observed in various conditions.

Tumor necrosis factor-alpha and phorbol 12-myristate 13-acetate differentially modulate cytotoxic effect of nitric oxide generated by serum deprivation in neuronal PC12 cells.

Nitric oxide (NO) is a signaling molecule that mediates several physiological processes in a range of cell and tissue types. Here we investigated the effect of serum deprivation in the absence or presence of phorbol 12-myristate 1 3-acetate (PMA) or tumor necrosis factor-alpha (TNFalpha) on cell viability, NO formation, inducible NO synthase (iNOS) induction, and activation of mitogen-activated protein kinase in neuronal PC12 cells. Within 24 h of serum deprivation, apoptosis occurred in up to 65-70% of the cells, and significant levels of NO were generated. When PMA was added in serum-free medium, NO formation and cell death were decreased. In contrast, addition of TNFalpha in serum-free medium increased the levels of NO formation and apoptosis compared with those in serum-deprived cells. We have demonstrated that differential generation of NO levels by PMA or TNFalpha under conditions of serum deprivation is mediated by the same pattern of iNOS induction. NO formation via iNOS induction resulted in the activation of c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. From this study it is suggested that the differential formation of cytotoxic NO by serum deprivation plus PMA or TNFalpha is primarily mediated by the induction of iNOS enzymes in neuronal PC12 cells and that its action is mediated by the activation of JNK.

The role of prostaglandin E 2 and nitric oxide in cell death in J774 murine macrophages

We investigated the role of prostaglandin E 2 (PGE 2) and its interactions with nitric oxide (NO) on cell death and NO-mediated cytotoxicity in the murine macrophage cell line J774. Stimulation of the J774 cells with lipopolysaccharide together with interferon- γ resulted in a dose-dependent cytotoxicity and production of PGE 2 and NO, measured as nitrite. Our results showed a linear correlation between PGE 2 release and cytotoxicity. The cyclooxygenase (COX) inhibitor indomethacin completely inhibited PGE 2 biosynthesis, without affecting NO production or cell death. This supports previous reports suggesting that overproduction of endogenous PGE 2 is mainly the consequence of cell death and does not cause it. In contrast, the NO synthase inhibitor N ω -monomethyl- l-arginine ( l-NMMA) gave a significant, though incomplete suppression of NO release and cell death. This points to the presence of other cytotoxic factors besides NO. To evaluate the toxic effect solely due to NO, macrophages were exposed to the NO donor S-nitroso- N-acetyl- d, l-penicillamine (SNAP). Incubation with SNAP also resulted in a concentration-dependent cell injury and PGE 2 production. When exogenously added, PGE 2 protected against SNAP-mediated cytotoxicity and simultaneously increased PGE 2 release into the medium, without inducing COX-2. The cytoprotection and the stimulation of PGE 2 release were both reversed by indomethacin. In conclusion, PGE 2 biosynthesis may represent a mechanism by which inflammatory macrophages protect themselves against the cytotoxic effects of NO.

Role of peroxynitrite and activation of poly (ADP-ribose) synthase in the vascular failure induced by zymosan-activated plasma.

1. Zymosan is a wall component of the yeast Saccharomyces Cerevisiae. Injection of zymosan into experimental animals is known to produce an intense inflammatory response. Recent studies demonstrated that the zymosan-induced inflammatory response in vivo can be ameliorated by inhibitors of nitric oxide (NO) biosynthesis. The cytotoxic effects of NO are, in part, mediated by the oxidant preoxynitrite and subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). In the present in vitro study, we have investigated the cellular mechanisms of vascular failure elicited by zymosan-activated plasma and the contribution of peroxynitrite production and activation of PARS to the changes. 2. Incubation of rat aortic smooth muscle cells with zymosan-activated plasma (ZAP) induced the production of nitrite, the breakdown product of NO, due to the expression of the inducible isoform of NO synthase (iNOS) over 6 24 h. In addition, ZAP triggered the production of peroxynitrite in these cells, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123 and by nitrotyrosine Western blotting. 3. Incubation of the smooth muscle cells with ZAP induced DNA single strand breakage and PARS activation. These effects were reduced by inhibition of NOS with NG-methyl-L-arginine (L-NMA, 3 mM), and by glutathione (3 mM), a scavenger of peroxynitrite. The PARS inhibitor 3-aminobenzamide (1 mM) inhibited the ZAP-induced activation of PARS. 4. Incubation of thoracic aortae with ZAP in vitro caused a reduction of the contractions of the blood vessels to noradrenaline (vascular hyporeactivity) and elicited a reduced responsiveness to the endothelium-dependent vasodilator acetylcholine (endothelial dysfunction). 5. Preincubation of the thoracic aortae with L-NMA (1 mM), glutathione (3 mM) or by the PARS inhibitor 3-aminobenzamide (1 mM) prevented the development of vascular hyporeactivity in response to ZAP. Moreover, glutathione and 3-aminobenzamide treatment protected against the ZAP-induced development of endothelial dysfunction. The PARS-related loss of the vascular contractility was evident at 30 min after incubation in endothelium-intact, but not in endothelium-denuded vessels and also manifested at 6 h after incubation with ZAP in endothelium-denuded rings. The acute response is probably related, therefore, to peroxynitrite formation (involving the endothelial NO synthase), whereas the delayed response may be related to the expression of iNOS in the smooth muscle. 6. The data obtained suggest that zymosan-activated plasma causes vascular dysfunction by inducing the simultaneous formation of superoxide and NO. These radicals combine to form peroxynitrite, which, in turn causes DNA injury and PARS activation. The protective effect of 3-aminobenzamide demonstrates that PARS activation contributes both to the development of vascular hyporeactivity and endothelial dysfunction during the vascular failure induced by ZAP.