Differential inhibitory action of nitric oxide and peroxynitrite on mitochondrial electron transport

Various authors have suggested that nitric oxide (.NO) exerts cytotoxic effects through the inhibition of cellular respiration. Indeed, in intact cells .NO inhibits glutamate-malate (complex I) as well as succinate (complex II)-supported mitochondrial electron transport, without affecting TMPD/ascorbate (complex IV)-dependent respiration. However, experiments in our lab using isolated rat heart mitochondria indicated that authentic .NO inhibited electron transport mostly by reversible binding to the terminal oxidase, cytochrome a3, having a less significant effect on complex II- and no effect on complex I-electron transport components. The inhibitory action of .NO was more profound at lower oxygen tensions and resulted in differential spectra similar to that observed in dithionite-treated mitochondria. On the other hand, continuous fluxes of .NO plus superoxide (O.(2)(-)), which lead to formation of micromolar steady-state levels of peroxynitrite anion (ONOO-), caused a strong inhibition of complex I- and complex II-dependent mitochondrial oxygen consumption and significantly inhibited the activities of succinate dehydrogenase and ATPase, without affecting complex IV-dependent respiration and cytochrome c oxidase activity. In conclusion, even though nitric oxide can directly cause a transient inhibition of electron transport, the inhibition pattern of mitochondrial respiration observed in the presence of peroxynitrite is the one that closely resembles that found secondary to .NO interactions with intact cells and strongly points to peroxynitrite as the ultimate reactive intermediate accounting for nitric oxide-dependent inactivation of electron transport components and ATPase in living cells and tissues.     

Bipotential primitive-definitive hematopoietic progenitors in the vertebrate embryo

The effects of the diatomic radical, nitric oxide (NO), on melphalan-induced cytotoxicity in Chinese hamster V79 and human MCF-7 breast cancer cells were studied using clonogenic assays. NO delivered by the NO-releasing agent (C2H5)2N[N(O)NO]- Na+ (DEA/NO; 1 mM) resulted in enhancement of melphalan-mediated toxicity in Chinese hamster V79 lung fibroblasts and human breast cancer (MCF-7) cells by 3.6- and 4.3-fold, respectively, at the IC50 level. Nitrite/nitrate and diethylamine, the ultimate end products of DEA/NO decomposition, had little effect on melphalan cytotoxicity, which suggests that NO was responsible for the sensitization. Whereas maximal sensitization of melphalan cytotoxicity by DEA/NO was observed for simultaneous exposure of DEA/NO and melphalan, cells pretreated with DEA/NO were sensitized to melphalan for several hours after NO exposure. Reversing the order of treatment also resulted in a time-dependent enhancement in melphalan cytotoxicity. To explore possible mechanisms of NO enhancement of melphalan cytotoxicity, the effects of DEA/NO on three factors that might influence melphalan toxicity were examined, namely NO-mediated cell cycle perturbations, intracellular glutathione (GSH) levels and melphalan uptake. NO pretreatment resulted in a delayed entry into S phase and a G2/M block for both V79 and MCF-7 cells; however, cell cycle redistribution for V79 cells occurred after the cells returned to a level of cell survival, consistent with treatment with melphalan alone. After 15 min exposure of V79 cells to DEA/NO (1 mM), GSH levels were reduced to 40% of control values; however, GSH levels recovered fully after 1 h and were elevated 2 h after DEA/NO incubation. In contrast, DEA/NO (1 mM) incubation did not reduce GSH levels significantly in MCF-7 cells (approximately 10%). Melphalan uptake was increased by 33% after DEA/NO exposure in V79 cells. From these results enhancement of melphalan cytotoxicity mediated by NO appears to be complex and may involve several pathways, including possibly alteration of the repair of melphalan-induced lesions. Our observations may give insights for improving tumour kill with melphalan using either exogenous or possibly endogenous sources of NO.     

Human xenomitochondrial cybrids. Cellular models of mitochondrial complex I deficiency

The subunits forming the mitochondrial oxidative phosphorylation system are coded by both nuclear and mitochondrial genes. Recently, we attempted to introduce mtDNA from non-human apes into a human cell line lacking mtDNA (rho degrees), and succeeded in producing human-common chimpanzee, human-pigmy chimpanzee, and human-gorilla xenomitochondrial cybrids (HXC). Here, we present a comprehensive characterization of oxidative phosphorylation function in these cells. Mitochondrial complexes II, III, IV, and V had activities indistinguishable from parental human or non-human primate cells. In contrast, a complex I deficiency was observed in all HXC. Kinetic studies of complex I using decylubiquinone or NADH as limiting substrates showed that the Vmax was decreased in HXC by approximately 40%, and the Km for the NADH was significantly increased (3-fold, p < 0.001). Rotenone inhibition studies of intact cell respiration and pyruvate-malate oxidation in permeabilized cells showed that 3 nM rotenone produced a mild effect in control cells (0-10% inhibition) but produced a marked inhibition of HXC respiration (50-75%). Immunoblotting analyses of three subunits of complex I (ND1, 75 and 49 kDa) showed that their relative amounts were not significantly altered in HXC cells. These results establish HXC as cellular models of complex I deficiency in humans and underscore the importance of nuclear and mitochondrial genomes co-evolution in optimizing oxidative phosphorylation function.     

A mechanistic analysis of nitric oxide-induced cellular toxicity

Nitric oxide (NO.)-induced toxicity was investigated in two different cell lines, Chinese hamster ovary (CHO-AA8) and human lymphoblastoid (TK6), over a range of NO. doses (0-9 mM) delivered for an exposure of 2 h. To determine both short-term and delayed effects leading to death, a range of assays was employed to decipher the major mechanisms of cytotoxicity. Examples of damage parameters measured in this study include inhibition of DNA synthesis, damage to mitochondria, loss of cell membrane integrity, apoptosis, changes in cell cycle distribution, and the occurrence of DNA strand breaks. Our results indicate that NO.-induced toxicity is an extremely complex process involving multiple pathways generally leading to apoptotic cell death. Results consistently demonstrate that TK6 cells are much more susceptible to NO.-induced toxicity than CHO-AA8 cells. This difference in sensitivity could be seen for all types of cellular damage examined. The earliest observable effect of NO. exposure is inhibition of DNA synthesis which is not the result of inhibition of ribonucleotide reductase but may be the result of DNA damage leading ultimately to cell cycle arrest.     

Suppression of nitric oxide-induced apoptosis by N-acetyl-L-cysteine through modulation of glutathione, bcl-2, and bax protein levels

It has been demonstrated that nitric oxide (NO) can promote apoptosis in human cancer cells. To test the protective effects of antioxidants (N-acetyl-L-cysteine (LNAC) and free-radical spin traps (5,5-dimethyl-1-pyrroline N-oxide and 2,2,6,6,-tetramethyl-1-piperidinyloxy) against NO-induced apoptosis, a human colon cancer cell line (COLO 205) was treated with NO, and its survival rate was evaluated both with and without antioxidant therapy. LNAC arrested the development of progression of apoptosis in COLO 205 cells in a dose-dependent manner, promoted long-term survival, and prevented the internucleosomal DNA cleavage induced by NO. The intracellular level of glutathione (GSH) was found to be elevated in cells after exposure to LNAC. The bax protein levels were elevated by NO treatment, and this effect was blocked by LNAC. On the other hand, the bcl-2 oncoprotein level in the LNAC-pretreated cells was significantly elevated in a time-dependent manner compared to cells that received NO pretreatment. In summary, our results suggest that the protective effect of LNAC may be linked to its inducement of increases in cellular GSH and bcl-2 protein levels and to its suppression of cellular bax protein in treated cells.     

Increased activity and sensitivity of mitochondrial respiratory enzymes to tumor necrosis factor alpha-mediated inhibition is associated with increased cytotoxicity in drug-resistant leukemic cell lines

The drug-resistant leukemic cell lines, CEM/VLB100 and K/DAU600, are more sensitive to tumor necrosis factor alpha (TNFalpha)-mediated cytotoxicity compared with their parental cell lines, CCRF-CEM and K562 cl.6. Drug-resistant leukemic cell lines have more active mitochondrial function, which is associated with a greater susceptibility to TNFalpha-induced respiratory inhibition. TNFalpha blocked electron transfer at three sites, NADH dehydrogenase (complex I), succinate dehydrogenase (complex II), and cytochrome c oxidase (complex IV). Respiratory rate and electron transport chain enzyme activities were significantly inhibited in the drug-resistant, TNF-sensitive cell lines. Respiratory inhibition preceded cell death by at least 5 to 8 hours. The respiratory failure was not compensated for by appropriate up-regulation of the glycolytic pathway. Increasing mitochondrial respiratory rate and enzyme activities by long-term culture with 2 mmol/L adenosine 5'-diphosphate (ADP) and Pi sensitized both drug-sensitive and drug-resistant cells to TNFalpha-induced cytolysis. Intramitochondrial free radicals generated by paraquat only had a limited and delayed effect on respiratory inhibition and cytolysis in comparison with the effect of TNFalpha. We conclude that TNFalpha-induced cytotoxicity in leukemic cells is, at least in part, mediated by inhibition of mitochondrial respiration. Free radical generation by TNFalpha may not directly lead to the observed inhibition of the mitochondrial electron transport and other mechanisms must be involved.     

Comparison of HCV RNA levels by branched DNA probe assay and by competitive polymerase chain reaction to predict effectiveness of interferon treatment for patients with chronic hepatitis C virus

Injury to the alveolar region is a hallmark of the adult respiratory distress syndrome (ARDS) whereas injury to the epithelium of the conducting airways is a characteristic of asthma. Reactive oxygen species have been implicated as mediators of lung injury in both of these conditions. We have investigated the relationship between intracellular nonprotein thiols (NPSH), and the release of the cytosolic enzyme lactate dehydrogenase (LDH), as an index of cell injury, following treatment of the human alveolar type II-like epithelial cell line (A549 cells) or the human bronchial epithelial cell line (16HBE140-) with hydrogen peroxide (H2O2). We have also assessed the protective effects of pre-incubation of both of these cells lines with H2O2 or enhancement of intracellular NPSH against H2O2-induced cell injury. Exposure of A549 and 16HBE140- cells to H2O2 (0.1 mM and 1 mM respectively for 16 h) produced the release of 40% of the total cellular LDH. H2O2 exposure produced an initial dose-dependent decrease in NPSH in A549 cells, with a subsequent increase to above control values. 16HBE140- cells also showed a dose-dependent decrease in NPSH following exposure to H2O2. Pretreatment of A549 cells with 0.1 mM H2O2 followed by subsequent exposure to H2O2 did not protect against H2O2-induced LDH release in this epithelial cell line. Pre-incubation with 2 mM N-acetylcysteine (NAC) increased NPSH but not intracellular reduced glutathione and resulted in total inhibition of H2O2-induced LDH release in both cell types. Pretreatment with reduced glutathione protected both cell types against the injurious effects of H2O2, whereas glutathione monethyl ester (GSHMEE) only partially protected A549 cells and had no effect in 16HBE140- cells. Intracellular cysteine levels were increased in both cell lines following NAC exposure but not sufficiently to account for the increase in NPSH levels. These observations raise the possibility that a critical concentration of nonprotein thiols may be necessary to protect pulmonary epithelial cells against hydrogen peroxide-induced injury.     

Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway

Nitric oxide (NO) has been invoked as an important pathogenic factor in a wide range of immunologically mediated diseases. The present study demonstrates that macrophage-derived NO may conversely function to fine tune T cell-mediated inflammation via reversible dephosphorylation of intracellular signaling molecules, which are involved in the control of T cell proliferation. Thus, T cells activated in the presence of alveolar macrophages are unable to proliferate despite expression of IL-2R and secretion of IL-2. This process is reproduced by the NO generator S-nitroso-N-acetylpenicillamine and is inhibitable by the NO synthase inhibitor N(G)-methyl-L-arginine. Analysis of T cell lysates by immunoprecipitation with specific Abs and subsequent immunoblotting indicated marked reduction of tyrosine phosphorylation of Jak3 and STAT5 mediated by NO. Further studies indicated that NO-mediated T cell suppression was reversible by the guanylate cyclase inhibitors methylene blue and LY-83583 and was reproduced by a cell-permeable analogue of cyclic GMP, implicating guanylate cyclase activation as a key step in the inhibition of T cell activation by NO.     

Nitric oxide mediates glutamate-induced mitochondrial depolarization in rat cortical neurons

Mitochondria have been considered to be a target for glutamate neurotoxicity. The aim of the present work was to investigate the mechanisms leading to glutamate-mediated mitochondrial deenergization, as measured by mitochondrial membrane potential and cell respiration in cultured neurons. Glutamate exposure to cells induced pronounced mitochondrial depolarization associated with an impairment in neuronal respiration, leading to neuronal ATP depletion. These effects were prevented by both the nitric oxide (. NO) synthase inhibitor Nomega-nitro-l-arginine methyl ester and by the N-methyl-d-aspartate glutamate-subtype receptor inhibitor d-(-)-2-amino-5-phosphopentanoate. Our results suggest that glutamate causes ATP depletion by collapsing mitochondrial membrane potential through a.NO-mediated mechanism.     

Nitric oxide exposure and sulfhydryl modulation alter L-arginine transport in cultured pulmonary artery endothelial cells

The effect of nitric oxide (NO) exposure and sulfhydryl-reactive chemicals on L-arginine transport in pulmonary artery endothelial cells was evaluated. Exposure of pulmonary artery endothelial cells to 7.5 ppm (0.4 microM) NO for 4 h resulted in a significant (p < 0.05) reduction of Na(+)-dependent but not Na(+)-independent L-arginine transport. More prolonged exposure for 12-24 h reduced both Na(+)-dependent and Na(+)-independent transport of L-arginine with maximal loss of transport after 18 h of exposure (p < 0.02 for both). Similarly, incubation of cells in the presence of 50-200 microM S-nitroso-acetyl-penicillamine (SNAP) (but not 500 microM each of nitrate or nitrite) for 2 h also reduced both the Na(+)-dependent and Na(+)-independent transport of L-arginine (p < 0.05 for all concentrations). The SNAP-induced reduction of L-arginine transport was blocked by the NO scavenger oxyhemoglobin. When cell monolayers were exposed to varying concentrations of the sulfhydryl reactive chemicals N-ethylmaleimide (NEM) and acrolein, a dose-dependent reduction of L-arginine transport by both Na(+)-dependent and Na(+)-independent processes was observed. Na(+)-dependent L-arginine transport was more susceptible to inhibition by exposure to NO and to sulfhydryl reactive chemicals. Incubation of cells with 0.5 mM of the thiol-containing agent N-acetyl-L-cysteine prior to and during NEM or acrolein exposure blocked NEM and acrolein-induced reduction of L-arginine transport by both Na(+)-dependent and Na(+)-independent processes. Similarly, NO-induced reductions of Na(+)-dependent and Na(+)-independent L-arginine transport were reversed to control levels 24 h after termination of NO exposure. Treatment with the disulfide reducing agent dithiothreitol after exposure to NO resulted in partial reversal of the decreases in L-arginine transport. These results demonstrate that exposure to exogenous NO is responsible for reversible reductions of plasma membrane-dependent L-arginine transport mediated by both the Na(+)-dependent (system Bo,+) and the Na(+)-independent (system y+) transport processes. Modulation of the sulfhydryl status of plasma membrane proteins involved in L-arginine transport, such as L-arginine transporters and/or Na+/K(+)-ATPase, may be responsible, at least in part, for reductions in overall L-arginine transport in pulmonary artery endothelial cells.     

Glutathione-dependent biotransformation of the alkylating drug thiotepa and transport of its metabolite monoglutathionylthiotepa in human MCF-7 breast cancer cells

In this study, the role of glutathione S-transferase (GST) P1-1, the cellular reduced glutathione (GSH) status, and ATP-dependent efflux pumps in the cellular glutathione-dependent biotransformation of thiotepa and transport of the main metabolite monoglutathionylthiotepa in relation to cytotoxicity was studied in control and GST-P1-1-transfected MCF-7 cell lines. It was demonstrated that an enhanced cellular level of GST-P1-1 leads to an enhanced formation of monoglutathionylthiotepa, which is transported out of the cell into the medium. Monoglutathionylthiotepa was able to reversibly inhibit the activity of purified GST-P1-1, but only at nonphysiological concentrations, indicating that feedback inhibition of GST by its metabolites is not a relevant process in vivo. The GST activity, cellular GSH level, and/or ATP-dependent efflux of monoglutathionylthiotepa were modulated using ethacrynic acid, D,L-buthionine-S,R-sulfoximine, probenecid, and verapamil to understand the interplay between GSTs, glutathione conjugation, and efflux of glutathione conjugates in more detail. Inhibition of the GSH biosynthesis by D,L-buthionine-R,S-sulfoximine, a specific inhibitor of gamma-glutamylcysteine synthetase, significantly reduced the glutathione conjugation of thiotepa and potentiated the cytotoxicity of thiotepa. Pretreatment of cells with ethacrynic acid resulted in decreased formation of monoglutathionylthiotepa as a result of inhibition of GST in the GST-P1-1 transfectant. In addition, the intracellular amount of monoglutathionylthiotepa increased in both of the cell lines on exposure to ethacrynic acid, indicating that transport of the glutathione conjugate was partially inhibited by the glutathione conjugate of ethacrynic acid. Transport activity of monoglutathionylthiotepa could also be inhibited by probenecid and verapamil, inhibitors of organic anion transport, without influencing the biotransformation capacity of the cells. It was demonstrated that inhibition of glutathione conjugate efflux by probenecid and verapamil leads to enhanced cytotoxicity, which indicates that besides thiotepa, monoglutathionylthiotepa is also cytotoxic for the cells. Only enhanced biotransformation and subsequent transport of the glutathione conjugate into the medium (which occurs with the GST-P1-1 transfectant) results in enhanced viability. Therefore, it was concluded that only enhanced biotransformation of thiotepa represents a real detoxification pathway when the resulting conjugate is transported out of the cells. Altogether, the results indicate that it is not the overexpression of GST per se but the interplay between GSH/GST and glutathione conjugate efflux pumps that results in increased resistance to alkylating anticancer drugs such as thiotepa.     

Role of nitric oxide and its interaction with superoxide in the suppression of cardiac muscle mitochondrial respiration. Involvement in response to hypoxia/reoxygenation

BACKGROUND: Nitric oxide (NO); superoxide anion (O2.d-); the reaction product of NO with O2.d-, peroxynitrite (ONOO-); and ischemia/reperfusion have all been reported to inhibit respiration in isolated mitochondria. However, the specific species involved in the inhibition of respiration in intact tissues are poorly understood. METHODS AND RESULTS: O2 consumption in isolated cardiac muscle from bovine calf hearts was quantified by use of a Clark-type electrode. Exogenous and endogenous sources of NO, from S-nitroso-N-acetylpenicillamine (SNAP) and bradykinin or carbachol, reversibly inhibited respiration, whereas the O2.- releasing agent, pyrogallol (PG), inhibited respiration in a manner that was only partially reversed when examined 15 minutes after the removal of PG. The generation of ONOO- with SNAP + PG caused a potentiation of the O2(-)-elicited inhibition of respiration when examined 15 minutes after the removal of the ONOO- generating system. Tiron (a scavenger of O2.-) did not alter the actions of SNAP, but it attenuated the direct inhibitory effects of PG +/- SNAP and essentially eliminated the suppression of respiration observed 15 minutes after removal of the O2.- or ONOO- generating system. Urate (a scavenger of ONOO-) antagonized only the actions of PG + SNAP. After exposure of muscle slices to a model of hypoxia (15 minutes) and reoxygenation (10 minutes), respiratory inhibition was observed. This reoxygenation-induced inhibition was potentiated by L-arginine, the substrate for NO biosynthesis, and was markedly blocked by nitro-L-arginine (an NO synthase inhibitor), Tiron, or urate. CONCLUSIONS: The potentially physiological reversible regulation of respiration in cardiac muscle by NO is converted to an effect that does not show rapid reversibility under conditions in which ONOO- forms, and this could contribute to cardiac dysfunction in situations such as hypoxia/reoxygenation.     

Energy thresholds in brain mitochondria. Potential involvement in neurodegeneration

Decreases in mitochondrial respiratory chain complex activities have been implicated in neurodegenerative disorders such as Parkinson's disease, Huntington's disease, and Alzheimer's disease. However, the extent to which these decreases cause a disturbance in oxidative phosphorylation and energy homeostasis in the brain is not known. We therefore examined the relative contribution of individual mitochondrial respiratory chain complexes to the control of NAD-linked substrate oxidative phosphorylation in synaptic mitochondria. Titration of complex I, III, and IV activities with specific inhibitors generated threshold curves that showed the extent to which a complex activity could be inhibited before causing impairment of mitochondrial energy metabolism. Complex I, III, and IV activities were decreased by approximately 25, 80, and 70%, respectively, before major changes in rates of oxygen consumption and ATP synthesis were observed. These results suggest that, in mitochondria of synaptic origin, complex I activity has a major control of oxidative phosphorylation, such that when a threshold of 25% inhibition is exceeded, energy metabolism is severely impaired, resulting in a reduced synthesis of ATP. Additionally, depletion of glutathione, which has been reported to be a primary event in idiopathic Parkinson's disease, eliminated the complex I threshold in PC12 cells, suggesting that antioxidant status is important in maintaining energy thresholds in mitochondria. The implications of these findings are discussed with respect to neurodegenerative disorders and energy metabolism in the synapse.     

Nitric oxide donors inhibit iodide transport and organification and induce morphological changes in cultured bovine thyroid cells

Nitric oxide (NO) has been proposed as an intracellular signal in the thyroid. The NO effect on function and morphology of bovine thyroid follicles in culture was analyzed by using the NO donors sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO). Both NO donors induced a concentration-dependent NO release measured by the nitrite accumulation in the culture medium. The SNP (10 to 500 micromol/L) treatment for 24 hours significantly inhibited the uptake, organification and transport of iodide in a concentration-dependent manner. When SNP (50 micromol/L) was withdrawn from the culture medium after 24 hours' incubation, iodide uptake and organification were partially recovered at 24 hours and reached the control value at 48 hours, indicating a reversible effect of SNP. A possible involvement of cyanide in the SNP inhibitory effect was excluded because incubation of follicles with potassium cyanide (KCN) at concentrations estimated to be present in the medium (40 and 80 micromol/L) for 24 hours did not modify iodide uptake and organification. The GSNO (10 to 500 micromol/L) treatment for 24 hours also reduced the iodide uptake, organification and transport in a concentration-dependent manner. A significant inhibition of iodide organification was induced after incubation with 1000 micromol/L of N2, 2'-O-dibutyrylguanosine 3':5'-cyclic monophosphate ([Bu]2cGMP). Morphological evaluation by light microscopy revealed that the incubation with NPS or GSNO (500 micromol/L) produced cellular dispersion with loss of follicular cell aggregates that was evident at 96 hours exposure. Cell viability was not altered by 10-500 micromol/L SNP or GSNO (80% to 85%). We concluded that long-term NO exposure induces functional and morphological modifications compatible with a loss of differentiation in thyroid follicles. These observations further support a role of NO in the regulation of the thyroid function.     

Growth and viability of macrophages continuously stimulated to produce nitric oxide

Deregulated production of nitric oxide (NO) has been implicated in the development of certain human diseases, including cancer. We sought to assess the damaging potential of NO produced under long-term conditions through the development of a suitable model cell culture system. In this study, we report that when murine macrophage-like RAW264.7 cells were exposed continuously to bacterial lipopolysaccharide (LPS) or mouse recombinant interferon-gamma (IFN-gamma) over periods of 21-23 days, they continued to grow, but with doubling times 2 to 4 times, respectively, longer than the doubling time of unstimulated cells. Stimulated cells produced NO at rates of 30 to 70 nmol per million cells per day throughout the stimulation period. Within 24 hr after removal of stimulant, cells resumed exponential growth. Simultaneous exposure to LPS and IFN-gamma resulted in decreased cell number, which persisted for 2 days after removal of the stimulants. Exponential growth was attained only after an additional 4 days. Addition of N-methyl-L-arginine (NMA), an NO synthase inhibitor, to the medium inhibited NO production by 90% of all stimulated cells, partially reduced doubling time of cells stimulated with LPS or IFN-gamma, and partially increased viability and growth rates in those exposed to both LPS and IFN-gamma. However, when incubated with LPS and IFN-gamma at low densities both in the presence and in the absence of NMA, cells grew at a rate slower than that of unstimulated cells, with no cell death, and they resumed exponential growth 24 hr after removal of stimulants. Results from cell density experiments suggest that macrophages are protected from intracellularly generated NO; much of the NO damaging activity occurred outside of the producer cells. Collectively, results presented in this study suggest that the type of cellular toxicity observed in macrophages is markedly influenced by rate of exposure to NO: at low rates of exposure, cells exhibit slower growth; at higher rates, cells begin to die; at even higher rates, cells undergo growth arrest or die. The ability of RAW264.7 cells to produce NO over many cell generations makes the cell line a useful system for the study of other aspects of cellular damage, including genotoxicity, resulting from exposure to NO under long-term conditions.     

Vesicular stomatitis in the horse

Inactivation of the mitochondrial respiratory chain in response to iron-induced oxidative stress has been studied in cultured cells. Iron loading resulted in malonaldehyde production, decreased levels of glutathione and reduced specific activities of both complexes I and IV of the respiratory chain. These results are discussed with respect to idiopathic Parkinson's disease, which is associated with increased iron levels and a specific decrease in complex I activity in the substantia nigra.     

Macrophages resistant to endogenously generated nitric oxide-mediated apoptosis are hypersensitive to exogenously added nitric oxide donors: dichotomous apoptotic response independent of caspase 3 and reversal by the mitogen-activated protein kinase kinase (MEK) inhibitor PD 098059

Nitric oxide (NO) induction through the inducible NO synthase has been demonstrated to cause cell death in macrophages. We demonstrate that, in macrophages that have been rendered resistant to apoptosis induced by inducible NO synthase (RES cells), exposure to exogenous NO donors results in a hypersensitive apoptosis reaction when compared with the parental RAW 264.7 cells. The apoptosis induced via exogenous NO donors was found to be caspase 3-independent. Although caspase 3 activity was stimulated in the apoptotic macrophages, inhibition of caspase 3 by the inhibitor DEVD-CHO (N-acetyl-Asp-Glu-Val-Asp-aldehyde) did not reverse the apoptosis induced by the NO donor S-nitrosoglutathione (GSNO). This suggests that although caspase 3 activity is stimulated during apoptosis in macrophages, this signal is not sufficient to induce apoptosis. Cleavage of the enzyme poly(ADP ribose) polymerase mirrors our results of the caspase activity. Interestingly, we show that exogenous NO donation results in an accumulation of cells at the G2/M-phase border. Here, we demonstrate that the mitogen activated protein kinase kinase (MEK) inhibitor PD 098059 can be used to reverse the G2/M-phase block and show that this treatment also inhibits the observed apoptosis in RES macrophages. Treatment with the MEK inhibitor also reversed both the caspase 3 activity and poly(ADP ribose) polymerase cleavage in cells treated with GSNO. This result indicates that the mitogen-activated protein kinase pathway may be involved in regulation of the caspase cascade. Alternatively, it may suggest an activity for the MEK inhibitor heretofore not observed, that of a cyclin kinase inhibitor. Our results suggest that selection of macrophages by resistance to endogenously generated NO may cause hypersensitivity to exogenous NO donors. These findings have relevant implications for the treatment of apoptotic-resistant cell populations that may occur in both cancer and atheroma.     

Amelanotic malignant melanoma

The purpose of this study was to investigate whether extracellular matrix proteins which influence human keratinocyte behaviour are capable of altering intracellular signalling systems in these cells. The effects of extracellular matrix proteins on two major signal transduction pathways, intracellular calcium and cyclic adenosine monophosphate (cyclic AMP), were investigated. The extracellular matrix proteins examined were the basement membrane preparation matrigel, collagens type I and IV, vitronectin and its active tripeptide component Arg-Gly-Asp (RGD). Acute additions of matrigel, vitronectin and RGD caused rapid transient increases in intracellular calcium and, together with collagen type I, also caused sustained elevations in basal calcium when cells were grown on these substrates. Cyclic AMP production was unaffected by acute exposure to these extracellular matrix proteins. Culture of cells on matrigel, collagen type I or IV, however, significantly reduced basal cyclic AMP accumulation and increased the response of the cells to the receptor-independent agonist forskolin. It is concluded that in vitro some extracellular matrix proteins can initiate both acute and sustained changes in intracellular signalling in human keratinocytes.     

Involvement of nitric oxide in nicotinic receptor-mediated myopathy

Previous studies have shown that nicotinic cholinergic agonists induce muscle cell degeneration. Although an involvement of calcium is well documented, subsequent intracellular steps have not been identified. The present experiments test whether nitric oxide (NO) may play such a role. Both the irreversible nitric oxide synthase inhibitor L-5N-iminoethyl ornithine and L-nitroarginine methyl ester, a reversible inhibitor, protected the muscle cells from the myopathic effects of nicotine. These results may suggest that nicotinic receptor stimulation produces an increase in NO that results in muscle cell degeneration. In line with this interpretation, exposure of the muscle cultures to the NO donor sodium nitroprusside resulted in a dose-dependent decline in myotube branch points. Neither L-5N-iminoethyl ornithine nor nitroprusside altered the binding of the nicotinic receptor agonist 125I-alpha-bungarotoxin to muscle cells in culture, which indicates that the effect of these agents was not mediated through an interaction at the nicotinic receptor recognition site. The results with agents that inhibit guanylate cyclase or modify extracellular levels of cGMP suggest an involvement of this cyclic nucleotide in the nicotinic receptor-mediated myopathy. To conclude, the present results suggest that nicotinic receptor activation causes skeletal muscle degeneration through an increase in NO production and a possible involvement of cGMP.