The nitric oxide prodrug, V-PYRRO/NO, protects against cadmium toxicity and apoptosis at the cellular level.

The liver is an important target tissue of cadmium. The compound O2-vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2 diolate (V-PYRRO/NO) is a liver-selective nitric oxide (NO) prodrug that is metabolized by hepatic P450 enzymes to release NO in hepatocytes. In vivo, V-PYRRO/NO can protect against the toxicity of various hepatotoxicants, including cadmium. Since NO is an effective vasodilator, whether this protective effect against cadmium toxicity is at the level of the hepatic vascular system or actually within the liver cells has not been defined. Thus, we studied the effects of V-PYRRO/NO pretreatment on cadmium-induced toxicity and apoptosis in cultured rat liver epithelial (TRL 1215) cells. Cells were pretreated with V-PYRRO/NO at 500 or 1000 microM for up to 24 h, then exposed to cadmium (as CdCl2) for additional 24 h and cytotoxicity was measured. Cadmium was significantly less cytotoxic in V-PYRRO/NO (1000 microM) pretreated cells (LC50=6.1+/-0.6 microM) compared to control cells (LC50=3.5+/-0.4 microM). TRL 1215 cells acted upon the prodrug to release NO, producing nitrite levels in the extracellular media after 24 h of exposure to 500 or 1000 microM V-PYRRO/NO measured at 87.0+/-4.2 and 324+/-14.8 microM, respectively, compared to basal levels of 7.70+/-0.46 microM. V-PYRRO/NO alone produced small increases in metallothionein (MT), a metal-binding protein associated with cadmium tolerance. However, V-PYRRO/NO pretreatment greatly enhanced cadmium induction of MT. V-PYRRO/NO pretreatment also markedly reduced apoptotic cell death induced by cadmium (5 microM), apparently by blocking cadmium-induced activation of the c-Jun N-terminal kinase (JNK) pathway. Thus, the prodrug, V-PYRRO/NO, protects against the adverse effects of cadmium directly within rat liver cells apparently through generation of NO and, at least in part, by facilitation of cadmium-induced MT synthesis.     

DNA damage and nitric oxide synthesis in experimentally infected Balb/c mice with Trypanosoma cruzi

This study aimed to evaluate whether experimental Chagas disease in acute phase under benznidazole therapy can cause DNA damage in peripheral blood, liver, heart, and spleen cells or induce nitric oxide synthesis in spleen cells. Twenty Balb/c mice were distributed into four groups: control (non-infected animals); Trypanosoma cruzi infected; T. cruzi infected and submitted to benznidazole therapy; and only treated with benznidazole. The results obtained with the single cell gel (comet) assay showed that T. cruzi was able induce DNA damage in heart cells of both benznidazole treated or untreated infected mice. Similarly, T. cruzi infected animals showed an increase of DNA lesions in spleen cells. Regarding nitric oxide synthesis, statistically significant differences (p<0.05) were observed in all experimental groups compared to negative control, the strongest effect observed in the T. cruzi infected group. Taken together, these results indicate that T. cruzi may increase the level of DNA damage in mice heart and spleen cells. Probably, nitric oxide plays an important role in DNA damaging whereas benznidazole was able to minimize induced T. cruzi genotoxic effects in spleen cells.     

Involvement of mtDNA damage in free fatty acid-induced apoptosis

A growing body of evidence indicates that free fatty acids (FFA) can have deleterious effects on beta-cells. It has been suggested that the beta-cell dysfunction and death observed in diabetes may involve exaggerated activation of the inducible form of nitric oxide synthase (iNOS) by FFA, with the resultant generation of excess nitric oxide (NO). However, the cellular targets with which NO interact have not been fully identified. We hypothesized that one of these targets might be mitochondrial DNA (mtDNA). Therefore, experiments were initiated to evaluate damage to mtDNA caused by exposure of INS-1 cells to FFA (2/1 oleate/palmetate). The results showed that FFA caused a dose-dependent increase in mtDNA damage. Additionally, using ligation-mediated PCR, we were able to show that the DNA damage pattern at the nucleotide level was identical to the one induced by pure NO and different from damage caused by peroxynitrite or superoxide. Following exposure to FFA, apoptosis was detected by DAPI staining and cytochrome c release. Treatment of INS-1 cells with the iNOS inhibitor aminoguanidine protected these cells from mtDNA damage and diminished the appearance of apoptosis. These studies suggest that mtDNA may be a sensitive target for NO-induced toxicity which may provoke apoptosis in beta-cells following exposure to FFA.     

Mitochondrial damage by nitric oxide is potentiated by dopamine in PC12 cells

Mitochondrial damage in PC12 cells, a model for dopaminergic cells, was examined in terms of the contribution of oxidative stress, nitric oxide (*NO), and dopamine to impairment of mitochondrial respiratory control (RC). A kinetic analysis suggested that the oxidative deamination of dopamine catalyzed by monoamine oxidase (MAO) was not a significant source of hydrogen peroxide, because of constrains imposed by the low cytosolic level of dopamine. *NO induced irreversible damage of mitochondrial complex I in PC12 cells: this damage followed a sigmoid response on *NO concentration with a well-defined threshold level. Dopamine did not elicit damage of mitochondria in PC12 cells; however, the amine potentiated the effects of *NO at or near the threshold level, thus leading to irreversible impairment of mitochondrial respiration. This synergism between *NO and dopamine was not observed at *NO concentrations below the threshold level. Depletion of dopamine from the storage vesicles by reserpine protected mitochondria from *NO damage. Dopamine oxidation by *NO increased with pH, and occurred at modest levels at pH 5.5. In spite of this, calculations showed that the oxidation of dopamine in the storage vesicles (pH 5.5) was higher than that in the cytosol (pH 7.4), due to the higher dopamine concentration in the storage vesicles (millimolar range) compared to that in the cytosol (micromolar range). It is suggested that storage vesicles may be the cellular sites where the potential for dopamine oxidation by *NO is higher.These data provide further support to the hypothesis that dopamine renders dopaminergic cells more susceptible to the mitochondrial damaging effects of *NO. In the early stages of Parkinson's disease, *NO production increases until reaching a point near the threshold level that induces neuronal damage. Dopamine stored in dopaminergic cells may cause these cells to be more susceptible to the deleterious effects of *NO, which involve irreversible impairment of mitochondrial respiration.     

The influence of nitric oxide on basal and cholecystokinin-8-induced proliferation and apoptosis in the rat pancreas

Nitric oxide (NO) is formed by different cell types in the pancreas. In this study, inhibition of endogenous nitric oxide by N(omega)-nitro-L-arginine (L-NNA) reduced the urinary excretion of NO(2)/NO(3) and raised serum L-arginine and the NO donator S-nitroso-N-acetylpenicillamine (SNAP) increased the urinary excretion of NO(2)/NO(3). The peptide cholecystokinin-8 (CCK-8) has a strong influence on exocrine pancreatic proliferation. Rat pancreas was excised and studied with regard to tissue weight, protein and DNA contents after 3 days of treatment with saline, L-NNA or SNAP given separately or combined with CCK-8. Further, proliferation of different pancreatic cells was studied with [3H]-thymidine incorporation and apoptotic activity was studied by analysing caspase-3 activity and histone-associated DNA fragments. The effects of L-NNA indicate that endogenous nitric oxide formation has a tonic inhibition on apoptosis in the pancreas during both basal condition and growth stimulation by CCK-8. In CCK-induced hyperplasia, NO inhibits the proliferation of acinar cells but stimulates ductal cells. Endogenous NO may regulate the balance between proliferation and apoptosis and in a situation of growth stimulation by CCK-8, it has a tonic inhibition on both mitogenesis and apoptosis thus slowing down the acinar cell turnover in the pancreas.     

Role of nitric oxide-induced mtDNA damage in mitochondrial dysfunction and apoptosis

An increasing body of evidence suggests that nitric oxide (NO) can be cytotoxic and induce apoptosis. NO can also be genotoxic and cause DNA damage and mutations. It has been shown that NO damages mitochondrial DNA (mtDNA) to a greater extent than nuclear DNA. Previously, we reported that conditional targeting of the DNA repair protein hOGG1 into mitochondria using a mitochondria targeting sequence (MTS) augmented mtDNA repair of oxidative damage and enhanced cellular survival. To determine whether enhanced repair resulting from augmented expression of hOGG1 could also protect against the deleterious effects of NO, we used HeLa TetOff/MTS-OGG1-transfected cells to conditionally express hOGG1 in mitochondria. The effects of additional hOGG1 expression on repair of NO-induced mtDNA damage and cell survival were evaluated. These cells, along with vector transfectants, in either the presence or absence of doxycycline (Dox), were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate). Functional studies revealed that cells expressing recombinant hOGG1 were more proficient at repairing NO-induced mtDNA damage, which led to increased cellular survival following NO exposure. Moreover, the results described here show that conditional expression of hOGG1 in mitochondria decreases NO-induced inhibition of ATP production and protects cells from NO-induced apoptosis.     

The influence of sodium nitrite on the osmoresistance of Chinese hamster cells

We have previously demonstrated that incubation of V-79 cells in the medium containing the nitric oxide donor, NaNO2, increases cell resistance to damaging effect of gamma-rays, UV radiation and hyperthermia. In the present study, we investigated the effects of the nitric oxide donor on the sensitivity of V-79 cells to changes in osmomolarity of the medium by adding different amounts of sodium chloride or water. We found that pretreatment of the cells with NaNO2 resulted in a significant increase in the number of growing cells in 48 h after the treatment. The osmomolarity-dependent morphological changes in cultured cells were also substantially diminished following NaNO2 treatment. This effect could be observed under both hyper- and hypoosmosis, and was dependent on concentration of sodium chloride in hypertonic medium (being maximal under 0.17 M NaCl) and on the amount of water in hypotonic medium (being maximal under 1.1 times the dilution with water). In the experiments with increased osmomolarity, we found that the observed increase in the number of growing cells following NaNO2 treatment was accompanied with a significant increase of the mitotic index. These findings indicate that nitric oxide increases cell resistance to the damaging effects of osmotic shock in the way which is similar to the protective effect of these molecules against radiation and hyperthermia. Similarities in the effects of NaNO2 under different conditions leading to cell damage suggest that nitric oxide might serve as the universal factor participating in recovery of damaged cells and mediating increased cellular resistance to the damaging conditions.     

Nitric oxide-induced damage to mtDNA and its subsequent repair.

Mutations in mitochondrial DNA (mtDNA) have recently been associated with a variety of human diseases. One potential DNA-damaging agent to which cells are continually exposed that could be responsible for some of these mutations is nitric oxide (NO). To date, little information has been forthcoming concerning the damage caused by this gas to mtDNA. Therefore, this study was designed to investigate damage to mtDNA induced by NO and to evaluate its subsequent repair. Normal human fibroblasts were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) and the resultant damage to mtDNA was determined by quantitative Southern blot analysis. This gas was found to cause damage to mtDNA that was alkali-sensitive. Treatment of the DNA with uracil-DNA glycosylase or 3-methyladenine DNA glycosylase failed to reveal additional damage, indicating that most of the lesions produced were caused by the deamination of guanine to xanthine. Studies using ligation-mediated PCR supported this finding. When a 200 bp sequence of mtDNA from cells exposed to NO was analyzed, guanine was found to be the predominantly damaged base. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites. The nucleotide pattern of damage induced by NO was different from that produced by either a reactive oxygen species generator or the methylating chemical, methylnitrosourea. Most of the lesions produced by NO were repaired rapidly. However, there appeared to be a subset of lesions which were repaired either slowly or not at all by the mitochondria.     

Antioxidant enzyme inhibitors enhance nitric oxide-induced cell death in U937 cells

Nitric oxide (NO), a radical species produced by many types of cells, is known to play a critical role in many regulatory processes, yet it may also participate in collateral reactions at higher concentrations, leading to cellular oxidative damage. The protective role of antioxidant enzymes against NO-induced oxidative damage in U937 cells was investigated in control and cells pre-treated with diethyldithiocarbamic acid, aminotriazole, and oxlalomalate, specific inhibitors of superoxide dismutase, catalase, and NADP(+)-dependent isocitrate dehydrogenase, respectively. Upon exposure to 1 mM S-nitroso-N-acetylpenicillamine (SNAP), the nitric oxide donor, to U937 cells, the viability was lower and the protein oxidation, lipid peroxidation and oxidative DNA damage reflected by an increase in 8-hydroxy-2'-deoxyguanosine, were higher in inhibitor-treated cells as compared to control cells. We also observed the significant increase in the endogenous production of reactive oxygen species, as measured by the oxidation of 2'7'-dichlorodihydrofluorescin as well as the significant decrease in the intracellular GSH level in inhibitor-treated U937 cells upon exposure to NO. Upon exposure to 0.2 mM SNAP, which induced apoptotic cell death, a clear inverse relationship was observed between the control and inhibitor-treated U937 cells in their susceptibility to apoptosis. These results suggest that antioxidant enzymes play an important role in cellular defense against NO-induced cell death including necrosis and apoptosis.     

Nitric oxide-induced mitochondrial dysfunction: implications for neurodegeneration

Excessive generation of nitric oxide (NO) has been implicated in the pathogenesis of several neurodegenerative disorders. Damage to the mitochondrial electron transport chain has also been implicated in these disorders. NO and its toxic metabolite peroxynitrite (ONOO(-)) can inhibit the mitochondrial respiratory chain, leading to energy failure and ultimately cell death. There appears to be a differential susceptibility of brain cell types to NO/ONOO(-), which may be influenced by factors including cellular antioxidant status and the ability to maintain energy requirements in the face of marked respiratory chain damage. Although formation of NO/ONOO(-) following cytokine exposure does not affect astrocyte survival, these molecules may diffuse out and cause mitochondrial damage to neighboring NO/ONOO(-)-sensitive cells such as neurons. Evidence suggests that NO/ONOO(-) causes release of neuronal glutamate, leading to glutamate-induced activation of neuronal NO synthase and generation of further damaging species. While neurons appear able to recover from short-term exposure to NO/ONOO(-), extending the period of exposure results in persistent damage to the respiratory chain and cell death ensues. These findings have important implications for acute infection vs. chronic neuroinflammatory disease states. The evidence for NO/ONOO(-)-mediated mitochondrial damage in neurodegenerative disorders is reviewed and potential therapeutic strategies are discussed.     

Anin VitroModel of Oligodendrocyte Destruction by Nitric Oxide and Its Relevance to Multiple Sclerosis

There is mounting evidence that nitric oxide (NO) is produced in the brains of patients with multiple sclerosis (MS) and in the experimental model of MS, experimental autoimmune encephalomyelitis, after the induction of Type II nitric oxide synthase (iNOS). Because NO can cause a variety of biological insults that compromise or even kill normal cells, we studied the effects of NO on oligodendrocytes since they are a target in MS tissue. In anin vitromodel, we have been able to demonstrate that NO causes damage to oligodendrocytes preferentially, sparing microglia almost completely and affecting some but not all astrocytic functions. This article describes the types of assays used to measure morphological changes, mitochondrial dysfunction, DNA strand breaks, and cell death brought on by NO or peroxynitrite (ONOO-) as well as a comprehensive review of the various techniques and sensitivities of NO and iNOS assays that would be applicable to similarin vitromodels.     

Effects of 6-formylpterin, a xanthine oxidase inhibitor and a superoxide scavenger, on production of nitric oxide in RAW 264.7 macrophages

As well as superoxide generated from neutrophils, nitric oxide (NO) produced by inducible nitric oxide synthase (iNOS) in macrophages plays an important role in inflammation. We previously showed that 6-formylpterin, a xanthine oxidase inhibitor, has a superoxide scavenging activity. In the present study, to elucidate other pharmacological activities of 6-formylpterin, we investigated the effects of 6-formylpterin on production of nitric oxide (NO) in the murine macrophage cell line RAW 264.7 stimulated by lipopolysaccharide (LPS) and interferon-gamma (INF-gamma). 6-Formylpterin suppressed the expression of iNOS, and it also inhibited the catalytic activity of iNOS, which collectively resulted in the inhibition of NO production in the stimulated macrophages. However, 6-formylpterin did not scavenge the released NO from an NO donor, S-nitroso-N-acetylpenicillamine (SNAP). These results indicate that 6-formylpterin inhibits pathological NO generation from macrophages during inflammation, but that it does not disturb the physiological action of NO released from other sources.     

Metabolism of a liver-selective nitric oxide-releasing agent, V-PYRRO/NO, by human microsomal cytochromes P450

Endogenously generated nitric oxide (NO) mediates a host of important physiological functions, playing roles in the vascular, immunological, and neurological systems. As a result, exogenous agents that release NO have become important therapeutic interventions and research tools. O(2)-Vinyl 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (V-PYRRO/NO) is a prodrug designed with the hypothesis that it might release nitric oxide via epoxidation of the vinyl group by cytochrome P450, followed by enzymatic and/or spontaneous epoxide hydration to release the ultimate NO-donating moiety, 1-(pyrrolidin-1-yl)diazen-1-ium-1,2-diolate (PYRRO/NO) ion. In this study, we investigated this hypothetical activation mechanism quantitatively for V-PYRRO/NO using cDNA-expressed human cytochrome P450 (CYP)2E1. Incubation with CYP2E1 and an NADPH-regenerating system resulted in a time-dependent decomposition of V-PYRRO/NO, with a turnover rate of 2.0 nmol/min/pmol CYP2E1. Nitrate and nitrite were detected in high yield as metabolites of NO. The predicted organic metabolites pyrrolidine and glycolaldehyde were also detected in near-quantitative yields. The enzymatic decomposition of V-PYRRO/NO was also catalyzed, albeit at lower rates, by CYP2A6 and CYP2B6. We conclude that the initial step in the metabolism of V-PYRRO/NO to NO in the liver is catalyzed efficiently but not exclusively by the alcohol-inducible form of cytochrome P450 (CYP2E1). The results confirm the proposed activation mechanism involving enzymatic oxidation of the vinyl group in V-PYRRO/NO followed by epoxide hydration and hydrolytic decomposition of the resulting PYRRO/NO ion to generate nitric oxide.     

Silibinin induced apoptosis of human epidermal cancer A431 cells by promoting mitochondrial NOS

The antitumor effects of silibinin are of increasing interest, though its mechanism is not yet clear. The goal of this study was to clarify the mechanism of silibinin-induced cell death in the A431 human epidermoid carcinoma cell line. We used a cell viability assay, flow cytometry, nitric oxide (NO) assay, and western blotting to examine relationships between silibinin, NO generation and apoptosis in A431 cells. Silibinin inhibited A431 cell growth in a dose-dependent manner, inducing mitochondrial damage, and apoptosis at a high dose. At the same time, high dose silibinin increased NO levels in A431 cells and the endothelial nitric oxide synthase (eNOS) inhibitor NG-nitro-L-arginine methylester (L-NAME) attenuated silibinin-induced cell growth inhibition. By western blotting, silibinin caused increased eNOS phosphorylation in the mitochondria. The AMP-activated protein kinase inhibitor compound C significantly decreased p-eNOS expression, while blocking eNOS did not affect p-AMPK levels, suggested that AMPK acted upstream of eNOS. This study showed that silibinin increased NO levels in A431 cells by activating the AMPK–eNOS pathway, leading to mitochondrial dysfunction and apoptosis. In this mechanism of action, mitochondrial eNOS played an important role. The results provided new understanding of the functions of intracellular NO.     

The role of p53 in nitric oxide-induced cardiomyocyte cell death

The role of p53 in mediating nitric oxide (NO)-induced cell death remains uncertain. The exogenous NO donor S-nitrosoglutathione (GSNO) produced a concentration-dependent reduction in cell viability in embryonic chick cardiomyocytes in culture. Western blotting and immunocytochemistry for p53 showed that p53 was increased in whole cell lysates by GSNO: 0.001 mM GSNO led to 1.3 +/- 0.5-fold increase compared to control, and significantly (p < 0.05) increased to 1.6 +/- 0.2-fold after 0.01 mM GSNO. Higher GSNO concentrations did not further increase p53 protein expression despite producing significant increases in cell death. The p53 inhibitor pifithrin did not block GSNO-induced cell death. GSNO induced morphological changes of DNA fragmentation, nuclear condensation, and cell shrinkage. Pifithrin failed to block these morphologic changes, while it antagonized the similar cellular changes induced by adriamycin, which operates in part through p53. NO induced a concentration-dependent DNA damage. When assessed by the comet assay, the damage was 2.1 +/- 0.3-fold and 2.6 +/- 0.5-fold more than the control following 0.01 mM and 1.0 mM GSNO treatments, respectively. The DNA damage was not reduced by treatment with the pifithrin, which markedly reduced DNA damage induced by adriamycin. There was no p53 translocation to mitochondria, any major cytochrome c release from mitochondria, or change in mitochondrial membrane potential. Furthermore, cyclosporin A, which inhibits mitochondrial pore opening and cytochrome c loss, did not alter NO-induced cell death. Translocation of p53 from the cytosol to the nucleus occurred with a maximal increase of 2.9-fold in the nucleus following 1.0 mM GSNO for 24 h. These data indicate that in cardiomyocytes, NO induced marked DNA damage and translocation of p53 to the nucleus, suggesting that p53 is involved in the cellular response to NO, perhaps to modulate the genomic response to NO-induced cellular toxicity. NO-induced cell death, however, operates through p53-independent pathways, including a mitochondrial apoptotic pathway.     

A novel lignan composition from Cedrus deodara induces apoptosis and early nitric oxide generation in human leukemia Molt-4 and HL-60 cells.

AP9-cd, a standardized lignan composition from Cedrus deodara consisting of (-)-wikstromal, (-)-matairesinol, and dibenzyl butyrolactol, showed cytotoxicity in several human cancer cell lines reported earlier. An attempt was made in this study to investigate the mechanism of cell death in human leukemia Molt-4 and HL-60 cells. It inhibited Molt-4 cell proliferation with 48-h IC(50) of approximately 15 microg/ml, increased sub-G0 cell fraction with no mitotic block, produced apoptotic bodies and induced DNA ladder formation. Flow cytometric analysis of annexinV-FITC/PI-stained cells showed time-related increase in apoptosis and post-apoptotic necrosis. All these biological end-points indicated cell death by apoptosis. Further, initial events involved massive nitric oxide (NO) formation within 4 h with subsequent late appearance of peroxides in cells; measured by flow cytometry using specific fluorescent probes. Persistently high levels of NO and peroxide appeared to decrease mitochondrial membrane potential (Psi(mt)) which was recovered by cyclosporin A in Molt-4 cells. AP9-cd caused 2-fold activation of caspase-3 in Molt-4 and 5-fold activation in HL-60 cells. Also caspases-8 and -9 were activated in HL-60 cells. Ascorbate suppressed the enhanced caspases activities indicating a pro-oxidant effect of AP9-cd. Further, caspase-3 activation correlated with NO generation that was partially impaired by nitric oxide synthase (NOS) inhibitors and ascorbate suggesting a role of pro-oxidant species in caspase-3 activation. AP9-cd produced no cytotoxicity in primary rat hepatocyte culture at the concentrations used. The studies indicated that AP9-cd mediated early NO formation leads to caspases activation, peroxide generation, and mitochondrial depolarization which may be responsible for mitochondrial-dependent and -independent apoptotic pathways involved in the killing of leukemia cells by AP9-cd.     

Constitutive and permissive roles of nitric oxide activity in embryonic ciliary cells

Embryos of Helisoma trivolvis exhibit cilia-driven rotation within the egg capsule during development. In this study we examined whether nitric oxide (NO) is a physiological regulator of ciliary beating in cultured ciliary cells. The NO donor S-nitroso-N-acetylpenicillamine (SNAP; 1-1,000 microM) produced a dose-dependent increase in ciliary beat frequency (CBF). In contrast, the nitric oxide synthase (NOS) inhibitor 7-nitroindazole (10 and 100 microM) inhibited the basal CBF and blocked the stimulatory effects of serotonin (100 microM). NO production in response to serotonin was investigated with 4,5-diaminofluorescein diacetate imaging. Although SNAP (100 microM) produced a rise in NO levels in all cells, only 22% of cells responded to serotonin with a moderate increase. The cGMP analog 8-bromo-cGMP (8-Br-cGMP; 0.2 and 2 mM) increased CBF, and the soluble guanylate cyclase inhibitor LY-83583 (10 microM) blocked the cilioexcitatory effects of SNAP and serotonin. These data suggest that NO has a constitutive cilioexcitatory effect in Helisoma embryos and that the stimulatory effects of serotonin and NO work through a cGMP pathway. It appears that in Helisoma cilia, NO activity is necessary, but not sufficient, to fully mediate the cilioexcitatory action of serotonin.     

Hepatitis C virus triggers mitochondrial permeability transition with production of reactive oxygen species, leading to DNA damage and STAT3 activation

Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Previously, we reported that HCV infection causes cellular DNA damage and mutations, which are mediated by nitric oxide (NO). NO often damages mitochondria, leading to induction of double-stranded DNA breaks (DSBs) and accumulation of oxidative DNA damage. Here we report that HCV infection causes production of reactive oxygen species (ROS) and lowering of mitochondrial transmembrane potential (DeltaPsi(m)) in in vitro HCV-infected cell cultures. The changes in membrane potential could be inhibited by BCL-2. Furthermore, an inhibitor of ROS production, antioxidant N-acetyl-L-cysteine (NAC), or an inhibitor of NO, 1,400W, prevented the alterations of DeltaPsi(m). The HCV-induced DSB was also abolished by a combination of NO and ROS inhibitors. These results indicated that the mitochondrial damage and DSBs in HCV-infected cells were mediated by both NO and ROS. Among the HCV proteins, core, E1, and NS3 are potent ROS inducers: their expression led to DNA damage and activation of STAT3. Correspondingly, core-protein-transgenic mice showed elevated levels of lipid peroxidation and oxidatively damaged DNA. These HCV studies thus identified ROS, along with the previously identified NO, as the primary inducers of DSBs and mitochondrial damage in HCV-infected cells.     

Nitric oxide, mitochondria and neurological disease

Damage to the mitochondrial electron transport chain has been suggested to be an important factor in the pathogenesis of a range of neurological disorders, such as Parkinson's disease, Alzheimer's disease, multiple sclerosis, stroke and amyotrophic lateral sclerosis. There is also a growing body of evidence to implicate excessive or inappropriate generation of nitric oxide (NO) in these disorders. It is now well documented that NO and its toxic metabolite, peroxynitrite (ONOO-), can inhibit components of the mitochondrial respiratory chain leading, if damage is severe enough, to a cellular energy deficiency state. Within the brain, the susceptibility of different brain cell types to NO and ONOO- exposure may be dependent on factors such as the intracellular reduced glutathione (GSH) concentration and an ability to increase glycolytic flux in the face of mitochondrial damage. Thus neurones, in contrast to astrocytes, appear particularly vulnerable to the action of these molecules. Following cytokine exposure, astrocytes can increase NO generation, due to de novo synthesis of the inducible form of nitric oxide synthase (NOS). Whilst the NO/ONOO- so formed may not affect astrocyte survival, these molecules may diffuse out to cause mitochondrial damage, and possibly cell death, to other cells, such as neurones, in close proximity. Evidence is now available to support this scenario for neurological disorders, such as multiple sclerosis. In other conditions, such as ischaemia, increased availability of glutamate may lead to an activation of a calcium-dependent nitric oxide synthase associated with neurones. Such increased/inappropriate NO formation may contribute to energy depletion and neuronal cell death. The evidence available for NO/ONOO--mediated mitochondrial damage in various neurological disorders is considered and potential therapeutic strategies are proposed.