Nitric oxide and cell signaling pathways in mitochondrial-dependent apoptosis

Nitric oxide, generated by endogenous nitric oxide synthases or nitric oxide donors, can promote or prevent apoptosis induced by diverse pro-apoptotic stimuli in cell culture models. Both mitochondrial-dependent and -independent apoptotic signaling pathways mediate this dichotomous cellular response to nitric oxide. The molecular mechanisms behind these effects are complex and involve a number of nitrogen oxide-related species that are more reactive than nitric oxide itself. The local cellular environment plays a dynamic role in determining the nature and concentration of these species. Important components of the microenvironment include: the cellular redox state, glutathione, transition metals and the presence of other oxygen- and nitrogen-centered radicals. In particular, redox-sensitive nitrosating species are favorably generated under physiological conditions and capable of modifying multiple cell signaling pathways through reversible S-nitrosation reactions. Cytochrome c release from mitochondria is an important mechanism for the activation of caspase-3 and the initiation of cell death in response to 'intrinsic' pro-apoptotic stimuli, including oxidative and nitrosative stress. In turn, caspases and mitogen associated protein kinases may modulate cytochrome c release through their effects on the Bcl-2 family of proteins. This review will focus on (i) the importance of the cellular environment in determining the fate of nitric oxide and (ii) the ability of S-nitrosation to regulate mitochondrial-dependent apoptosis at the level of mitochondrial bioenergetics, cytochrome c release, caspases, mitogen associated protein kinases, and the Bcl-2 family of proteins.     

Bcl-2-linked apoptosis due to increase in NO synthase in brain of SAMP10

We examined the linkage of nitric oxide (NO)-induced apoptosis to acceleration of brain aging of senescence-accelerated mouse prone 10 (SAMP10). The expression of neuronal nitric oxide synthase (nNOS) increased in the cerebral cortex of the brain of SAMP10 in an age-dependent manner and significantly higher levels of neuronal nitric oxide synthase (nNOS) were observed in both young and old SAMP10 as compared to age-matched controls. Moreover, a lower level of anti-apoptotic protein Bcl-2 and a higher level of pro-apoptotic protein cytochrome c in cytosol were observed in SAMP10 compared to the control. However, there was no significant difference in the expression of pro-apoptotic protein p53 between SAMP10 and the control. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive apoptotic cells were more abundant in the cerebral cortex of aged SAMP10 than in the control. The present results suggest that an age-dependent increase of NO by up-regulation of nNOS promotes the Bcl-2-linked apoptosis in the cerebral cortex of SAMP10 and this may cause the acceleration of brain aging of SAMP10.     

LPS/IFN-γ-Induced RAW 264.7 Apoptosis is Regulated by Both Nitric Oxide–Dependent and –Independent Pathways Involving JNK and the Bcl-2 Family

Lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) stimulate macrophages to produce nitric oxide (NO) via inducible nitric oxide synthase (iNOS) and activate stress signaling cascades including the c-jun-N-terminal kinase (JNK) pathway. These events trigger an apoptotic cascade that ultimately results in death. Since JNK regulates pro-apoptotic and anti-apoptotic Bcl-2 family members, the role of NO in LPS/IFN-γ-induced activation of JNK and its effects on the Bcl-2 family was examined in RAW 264.7 macrophage-like cells. Inhibition of JNK by siRNA verified a role for JNK in LPS/IFN-γ-induced apoptosis. Suppression of NO production by a pharmacologic agent, i.e. iNOS inhibitor L-NIL, altered the kinetics of JNK activation by LPS/IFN-γ. Examination of mitochondrial and nuclear compartments of RAW 264.7 cells demonstrated NO-dependent activation of mitochondrial JNK by LPS/IFN-γ, but NO-independent, cytokine-induced phosphorylation of Bim. NO did not affect phosphorylation, but did inhibit Bax phosphorylation. These results suggest a novel mechanism of LPS/IFN-γ-induced apoptosis in macrophages involving NO-independent phosphorylation of Bim and NO-dependent dephosphorylation of Bax.     

Role of nitric oxide, reactive oxygen species, and p38 MAP kinase in the regulation of human chondrocyte apoptosis

This study addresses mechanisms by which interleukin-1beta (IL-1beta) regulates human chondrocyte apoptosis induced by a combination of the anti-CD95 antibody CH-11 and the proteasome inhibitor (PSI). The effect of IL-1beta on apoptosis varied among tissue samples. IL-1beta either enhanced (16/22 samples) or inhibited (6/22 samples) DNA fragmentation and caspase-3 processing. The protective effect of IL-1beta was abrogated by the nitric oxide (NO) synthesis inhibitor N-monomethyl-l-arginine (L-NMMA) while apoptosis stimulation was not affected. The NO-donors sodium nitroprusside (SNP) and S-nitroso-N-acetyl penicillamine (SNAP) blocked DNA fragmentation, and this was associated with partial inhibition of caspase-3 processing. Pyrrolidine dithiocarbamate (PDTC), a scavenger of reactive oxygen species (ROS) blocked apoptosis induction by CH-11/PSI as well as the enhancement by IL-1beta. The pro-apoptotic effects of IL-1beta were also abrogated by the p38 inhibitor SB 202190. In conclusion, IL-1beta augments CH-11/PSI induced apoptosis in the majority of chondrocyte samples. The pro-apoptotic effect of IL-1beta is not dependent on NO. In contrast, the anti-apoptotic effect of IL-1beta observed in a minority of samples is partially NO-dependent.     

P53-dependent miRNAs mediate nitric oxide-induced apoptosis in colonic carcinogenesis

Both miRNAs and nitric oxide (NO) play important roles in colonic inflammation and tumorigenesis. Resistance of colonic epithelial cells to apoptosis may contribute to tumor development. We hypothesized that some miRNAs could increase the resistance of colonic cancer cells to nitric oxide-induced apoptotic cell death. Here we show that NO induced apoptosis and stimulated expression of some miRNAs. Loss of p53 not only blocked NO-induced apoptosis but also dramatically inhibited the expression of NO-related miRNAs, such as miR-34, miR-203, and miR-1301. In addition, blockage of p53-dependent miRNAs significantly reduced NO-induced apoptosis. Furthermore, forced expression of these miRNAs rendered HT-29 cells, which are resistant to apoptosis with mutant p53, more sensitive to NO-induced apoptotic cell death. Most interestingly, in a colitis-associated colon cancer mouse model, the level of miRNAs dropped significantly, accompanied by downregulation of p21, which is a key target gene of p53. In human colorectal cancer samples, the expression of miR-34 significantly correlated with the level of inducible nitric oxide synthase (iNOS). We contend that increased NO production may select cells with low levels of p53-dependent miRNAs which contributes to human colonic carcinogenesis and tumor progression.     

Protective effect of panaxydol and panaxynol on sodium nitroprusside-induced apoptosis in cortical neurons

An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. The protective effect of panaxydol (PND) and panaxynol (PNN) on sodium nitroprusside (SNP)-induced neuronal apoptosis and potential mechanism were investigated in primary cultured rat cortical neurons. Pretreatment of the cells with PND or PNN for 24 h following 1 mM SNP, an exogenous NO donor, exposure for 1 h, resulted significantly in reduction of cell death induced by SNP determined by MTT assay, LDH release and Hoechst staining. 5 μM PND and PNN also reduced the up-regulation of the pro-apoptotic gene, Bax, down-regulation of the anti-apoptotic gene, Bcl-2. The observations demonstrated that PND and PNN protect neurons against SNP-induced apoptosis via regulating the apoptotic related genes. The results raise the possibility that PND and PNN reduce neurodegeneration in the Alzheimer's brain.     

An apoptotic model for nitrosative stress

Nitric oxide overproduction has been implicated in the pathogenesis of many disorders, including artherosclerosis, neurodegenerative diseases, inflammatory and autoimmune diseases, and cancer. The common view holds that nitric oxide-induced cellular injury is caused by oxidative stress. This theory predicts that interactions between reactive nitrogen species and reactive oxygen species produce powerful oxidants that initiate cell death programs. Cytokine-treated murine macrophages are the prototype of this form of cellular injury. Here we report that generation of reactive nitrogen species upon lipopolysacharide/interferon-gamma stimulation of RAW 264.7 cells is largely divorced from production of reactive oxygen species, and that oxidative stress is not principally responsible for cell death (in this model). Rather, the death program is induced mainly by a nitrosative challenge, characterized by the accrual of nitrosylated proteins without a major alteration in cellular redox state. Moreover, interactions between reactive oxygen and nitrogen species may alter the balance between pathways that yield nitrite and nitrate, without impacting the level of S-nitrosylation or extent of cell death. Our results thus (1) provide new insights into NO-related metabolic pathways, (2) demonstrate that apoptotic injury can be caused by nitrosative mechanisms, and (3) establish a model for nitrosative stress in mammalian cells.     

Pretreatment with 2-(4-Methoxyphenyl)ethyl-2-acetamido-2-deoxy-β-D-pyranoside Attenuates Cerebral Ischemia/Reperfusion-Induced Injury In Vitro and In Vivo

Salidroside, extracted from the root of Rhodiola rosea L, is known for its pharmacological properties, in particular its neuroprotective effects. 2-(4-Methoxyphenyl) ethyl-2-acetamido-2-deoxy-β-D- pyranoside (GlcNAc-Sal), an analog of salidroside, was recently synthesized and shown to possess neuroprotective properties. The purpose of the current study was to investigate the neuroprotective effects of GlcNAc-Sal against oxygen–glucose deprivation-reperfusion (OGD-R)-induced neurotoxicity in vitro and global cerebral ischemia-reperfusion (GCI-R) injury in vivo. Cell viability tests and Hoechst 33342 staining confirmed that GlcNAc-Sal pretreatment markedly attenuated OGD-R induced apoptotic cell death in immortalized mouse hippocampal HT22 cells. Western blot, immunofluorescence and PCR analyses revealed that GlcNAc-Sal pretreatment restored the balance of pro- and anti-apoptotic proteins and inhibited the activation of caspase-3 and PARP induced by OGD-R treatment. Further analyses showed that GlcNAc-Sal pretreatment antagonized reactive oxygen species (ROS) generation, iNOS-derived NO production and NO-related apoptotic cell death during OGD-R stimulation. GCI-R was induced by bilateral common carotid artery occlusion (BCCAO) and reperfusion in mice in vivo. Western blot analysis showed that GlcNAc-Sal pretreatment decreased the expression of caspase-3 and increased the expression of Bcl-2 (B-cell lymphoma 2)/Bax (Bcl-2-associated X protein) induced by GCI-R treatment. Our findings suggest that GlcNAc-Sal pretreatment prevents brain ischemia reperfusion injury by the direct or indirect suppression of cell apoptosis and GlcNAc-Sal could be developed as a broad-spectrum agent for the prevention and/or treatment of cerebral ischemic injury.     

Circulating NO pool: assessment of nitrite and nitroso species in blood and tissues

The formation of nitric oxide (NO) has been linked to many regulatory functions in mammalian cells. With the appreciation that NO-mediated nitrosation reactions are involved in cell signaling and pathology there is a need to elucidate and better characterize the different biochemical pathways of NO in vivo. Despite significant methodological advances over the years one major obstacle in assessing the significance of nitrosated species and other NO-related metabolites remains: their reliable measurement in complex biological matrices. In this review we briefly discuss the major routes of NO metabolism and transport in the mammalian circulation, considering plasma, red blood cell, and tissue compartments separately. In addition, we attempt to give a recommendation as to the most appropriate analytical technique and sample processing procedures for the reliable quantification of either species.     

Nitric oxide has dual opposite roles during early and late phases of apoptosis in cerebellar granule neurons

The involvement and the role of nitric oxide (NO) as a signaling molecule in the course of neuronal apoptosis, whether unique or modulated during the progression of the apoptotic program, has been investigated in a cellular system consisting of cerebellar granule cells (CGCs) where apoptosis can be induced by lowering extracellular potassium. Several parameters involved in NO signaling pathway, such as NO production, neuronal nitric oxide synthase (nNOS) expression, and cyclic GMP (cGMP) production were examined in the presence or absence of different inhibitors. We provide evidence that nitric oxide has dual and opposite effects depending on time after induction of apoptosis. In an early phase, up to 3 h of apoptosis, nitric oxide supports survival of CGCs through a cGMP-dependent mechanism. After 3 h, nNOS expression and activity decreased resulting in shut down of NO and cGMP production. Residual NO then contributes to the apoptotic process by reacting with rising superoxide anions leading to peroxynitrite production and protein inactivation. We conclude that whilst NO over-production protects neurons from death in the early phase of neuronal damage, its subsequent reduction may contribute to neuronal degeneration and ultimate cell death.     

Hypothesis: human serum-borne albumin bound lipids promote cellular survival after apoptosis induction by a variety of stimuli

A tight control of proliferation and cell death is required to maintain homeostasis in multicellular organisms. Several specific pro- and anti-apoptotic regulators and pathways have been deciphered being responsible for these complex tasks. Here we describe a human serum-borne activity promoting cellular fitness and inhibiting apoptosis after a plethora of different cell death stimuli. The factor(s) do not inhibit a specific death pathway, instead it/they can be considered as general pro-survival factor(s) for cultured cells. The activity is heat stable (30 min, 96°C), co-migrates with albumin in size exclusion chromatography, and is sensitive to chemical delipidation. A similar activity is observed in native, non-delipidated preparations of human albumin, while delipidated albumin is not effective. These properties point to heat stable factors that exert anti-apoptotic activities, most likely albumin bound bioactive lipids. The activity prevented Akt dephosphorylation and degradation, after apoptosis induction by staurosporine and the production of reactive oxygen species after UV-B irradiation. In conclusion human serum-enriched bioactive lipids promote survival of cultured cells overriding the pro-apoptotic effects of a variety of apoptosis inducing agents.     

Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation

Apoptosis is thought to play an important regulatory role in placental development and inappropriate trophoblast apoptosis has been implicated in complications of pregnancy such as pre-eclampsia. Here we show that apoptosis of a human extravillous trophoblast-derived cell line (SGHPL-4) can be regulated by nitric oxide (NO). Nitric oxide produced exogenously by the addition of NO donors was able to delay or inhibit apoptosis induced by a combination of tumour necrosis factor alpha and actinomycin D and to suppress the activity of caspase 3. Treatment with hepatocyte growth factor (HGF) stimulated expression of the inducible isoform of NO synthase and was also able to protect SGHPL-4 cells from caspase 3 activation and apoptosis. The inhibition of basal NO production with NO synthase inhibitors was shown to sensitise cells to apoptotic stimuli and to reduce the level of endogenous caspase 3 nitrosylation. The anti-apoptotic effects of NO in these extravillous trophoblast cells appear to be mediated through the production of cyclic GMP as inhibitors of soluble guanylate cyclase inhibited the protective effect of both HGF and NO donors.     

Role of mitogen-activated protein kinases in S-nitrosoglutathione-induced macrophage apoptosis

The inflammatory mediator nitric oxide (NO*) promotes apoptotic cell death based on morphological evidence, accumulation of the tumor suppressor p53, caspase-3 activation, and DNA fragmentation in RAW 264.7 macrophages. Since nitrosothiols may actually be the predominant form of biologically active NO* in vivo, we used S-nitrosoglutathione (GSNO) to study activation of extracellular signal-regulated protein kinases1/2 (ERK1/2), c-Jun N-terminal kinases/stress-activated protein kinases (JNK1/2), and p38 kinases. Moreover, we determined the role of mitogen-activated protein kinase signaling in the apoptotic transducing ability of GSNO. ERK1/2 became activated in response to GSNO after 4 h and remained active for the next 20 h. Blocking the ERK1/2 pathway by the mitogen-activated protein kinase kinase inhibitor PD 98059 enhanced GSNO-elicited apoptosis. p38 was activated as well, but inhibition of p38 with SB 203580 left apoptosis unaltered. Activation of JNK1/2 by GSNO showed maximal kinase activities between 2 and 8 h. Attenuating JNK1/2 by antisense-depletion eliminated the pro-apoptotic action of low GSNO concentrations (250 microM), whereas apoptosis proceeded independently of JNK1/2 at higher doses of the NO donor (500 microM). Decreased apoptosis by JNK1/2 depletion prevented p53 accumulation after the addition of GSNO, which positions JNK1/2 upstream of the p53 response at low agonist concentrations. In line, JNK1/2 activation proceeded unaltered in p53-antisense transfected macrophages. However, with higher GSNO concentrations apoptotic transducing pathways, including p53 accumulation, were JNK1/2 unrelated. The regulation of mitogen-activated protein kinases by GSNO may help to define cell protective and destructive actions of reactive nitrogen species.     

Pre-conditioning induced by carbon monoxide provides neuronal protection against apoptosis

Carbon monoxide (CO) is an endogenous product of mammalian cells generated by heme-oxygenase, presenting anti-apoptotic properties in several tissues. The present work demonstrates the ability of small amounts of exogenous CO to prevent neuronal apoptosis induced by excitotoxicity and oxidative stress in mice primary culture of cerebellar granule cells. Additionally, our data show that endogenous CO is a heme-oxygenase product critical for its anti-apoptotic activity. Despite being neuroprotective, CO also induces reactive oxygen species generation in neurons. These two phenomena suggest that CO induces pre-conditioning (PC) to prevent cell death. The role of several PC mediators, namely soluble guanylyl cyclase, nitric oxide (NO) synthase, and ATP-dependent mitochondrial K channel (mitoK(ATP)) was addressed. Inhibition of soluble guanylyl cyclase or NO synthase activity, or closing of mitoK(ATP) abolishes the protective effect conferred by CO. In addition, CO treatment triggers cGMP and NO production in neurons. Opening of mitoK(ATP), which appears to be critical for CO prevention of apoptosis, might be a later event. We also demonstrated that reactive oxygen species generation and de novo protein synthesis are necessary for CO PC effect and neuroprotection. In conclusion, CO induces PC and prevents neuronal apoptosis, therefore constituting a novel and promising candidate for neuroprotective therapies.     

Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway

Ventilator strategies allowing for increases in carbon dioxide (CO(2)) tensions (hypercapnia) are being emphasized to ameliorate the consequences of inflammatory-mediated lung injury. Inflammatory responses lead to the generation of reactive species including superoxide (O(2)(-)), nitric oxide (.NO), and their product peroxynitrite (ONOO(-)). The reaction of CO(2) and ONOO(-) can yield the nitrosoperoxocarbonate adduct ONOOCO(2)(-), a more potent nitrating species than ONOO(-). Based on these premises, monolayers of fetal rat alveolar epithelial cells were utilized to investigate whether hypercapnia would modify pathways of.NO production and reactivity that impact pulmonary metabolism and function. Stimulated cells exposed to 15% CO(2) (hypercapnia) revealed a significant increase in.NO production and nitric oxide synthase (NOS) activity. Cell 3-nitrotyrosine content as measured by both HPLC and immunofluorescence staining also increased when exposed to these same conditions. Hypercapnia significantly enhanced cell injury as evidenced by impairment of monolayer barrier function and increased induction of apoptosis. These results were attenuated by the NOS inhibitor N-monomethyl-L-arginine. Our studies reveal that hypercapnia modifies.NO-dependent pathways to amplify cell injury. These results affirm the underlying role of.NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.     

Elevated levels of NO are localized to distal airways in asthma

The contribution of nitric oxide (NO) to the pathophysiology of asthma remains incompletely defined despite its established pro- and anti-inflammatory effects. Induction of the inducible nitric oxide synthase (iNOS), arginase, and superoxide pathways is correlated with increased airway hyperresponsiveness in asthmatic subjects. To determine the contributions of these pathways in proximal and distal airways, we compared bronchial wash (BW) to traditional bronchoalveolar lavage (BAL) for measurements of reactive nitrogen/oxygen species, arginase activation, and cytokine/chemokine levels in asthmatic and normal subjects. Levels of NO were preferentially elevated in the BAL, demonstrating higher level NOS activation in the distal airway compartment of asthmatic subjects. In contrast, DHE(+) cells, which have the potential to generate reactive oxygen species, were increased in both proximal and distal airway compartments of asthmatics compared to controls. Different patterns of cytokines and chemokines were observed, with a predominance of epithelial cell-associated mediators in the BW compared to macrophage/monocyte-derived mediators in the BAL of asthmatic subjects. Our study demonstrates differential production of reactive species and soluble mediators within the distal airways compared to the proximal airways in asthma. These results indicate that cellular mechanisms are activated in the distal airways of asthmatics and must be considered in the development of therapeutic strategies for this chronic inflammatory disorder.     

Akt pathway mediates a cGMP-dependent survival role of nitric oxide in cerebellar granule neurones

Apoptotic death results from disrupting the balance between anti-apoptotic and pro-apoptotic cellular signals. The inter- and intracellular messenger nitric oxide is known to mediate either death or survival of neurones. In the present work, cerebellar granule cells were used as a model to assess the survival role of nitric oxide and to find novel signal transduction pathways related to this role. It is reported that sustained inhibition of nitric oxide production induces apoptosis in differentiated cerebellar granule neurones and that compounds that slowly release nitric oxide significantly revert this effect. Neuronal death was also reverted by a caspase-3-like inhibitor and by a cyclic GMP analogue, thus suggesting that nitric oxide-induced activation of guanylate cyclase is essential for the survival of these neurones. We also report that the Akt/GSK-3 kinase system is a transduction pathway related to the survival action of nitric oxide, as apoptosis caused by nitric oxide deprivation is accompanied by down-regulation of this, but not of other, kinase systems. Conversely, treatments able to rescue neurones from apoptosis also counteracted this down-regulation. Furthermore, in transfection experiments, overexpression of the Akt gene significantly decreased nitric oxide deprivation-related apoptosis. These results are the first evidence for a mechanism where endogenous nitric oxide promotes neuronal survival via Akt/GSK-3 pathway.     

N-acetyl cysteine enhances imatinib-induced apoptosis of Bcr-Abl+ cells by endothelial nitric oxide synthase-mediated production of nitric oxide

Introduction  Imatinib, a small-molecule inhibitor of the Bcr-Abl kinase, is a successful drug for treating chronic myeloid leukemia (CML). Bcr-Abl kinase stimulates the production of H₂O₂, which in turn activates Abl kinase. We therefore evaluated whether N-acetyl cysteine (NAC), a ROS scavenger improves imatinib efficacy. Materials and methods  Effects of imatinib and NAC either alone or in combination were assessed on Bcr-Abl⁺ cells to measure apoptosis. Role of nitric oxide (NO) in NAC-induced enhanced cytotoxicity was assessed using pharmacological inhibitors and siRNAs of nitric oxide synthase isoforms. We report that imatinib-induced apoptosis of imatinib-resistant and imatinib-sensitive Bcr-Abl⁺ CML cell lines and primary cells from CML patients is significantly enhanced by co-treatment with NAC compared to imatinib treatment alone. In contrast, another ROS scavenger glutathione reversed imatinib-mediated killing. NAC-mediated enhanced killing correlated with cleavage of caspases, PARP and up-regulation and down regulation of pro- and anti-apoptotic family of proteins, respectively. Co-treatment with NAC leads to enhanced production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). Involvement of eNOS dependent NO in NAC-mediated enhancement of imatinib-induced cell death was confirmed by nitric oxide synthase (NOS) specific pharmacological inhibitors and siRNAs. Indeed, NO donor sodium nitroprusside (SNP) also enhanced imatinib-mediated apoptosis of Bcr-Abl⁺ cells. Conclusion  NAC enhances imatinib-induced apoptosis of Bcr-Abl⁺ cells by endothelial nitric oxide synthase-mediated production of nitric oxide.