Hypoxia sensitizes cells to nitric oxide-induced apoptosis

Nitric oxide (NO) can induce apoptosis in a variety of cell types. A non-toxic concentration of nitric oxide under normal oxygen conditions triggered cell death under hypoxic conditions (1.5% O(2)) in fibroblasts. Nitric oxide administered during hypoxia induced the release of cytochrome c, caspase-9 activation, and the loss of mitochondrial membrane potential followed by DNA fragmentation and lactate dehydrogenase release (markers of cell death). Bcl-X(L) protected cells from nitric oxide-induced apoptosis during hypoxia by preventing the release of cytochrome c, caspase-9 activation, and by maintaining a mitochondrial membrane potential. Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice exposed to nitric oxide during hypoxia did not die, indicating that pro-apoptotic Bcl-2 family members are required for NO-induced apoptosis during hypoxia. The nitric oxide-induced cell death during hypoxia was independent of cGMP and peroxynitrite. Cells devoid of mitochondrial DNA (rho secondary-cells) lack a functional electron transport chain and were resistant to nitric oxide-induced cell death during hypoxia, suggesting that a functional electron transport chain is required for nitric oxide-induced apoptosis during hypoxia.     

Maintaining nitric oxide-induced airway relaxation with superoxide dismutase

BACKGROUND: We have previously shown that the protective effect of inhaled nitric oxide (iNO) against methacholine-induced bronchoconstriction is negated in airways subjected to hyperosmotic stress. In this study, hypothesizing that the impaired efficiency of iNO was caused by release of reactive oxygen radicals, we examined the effect of the radical scavenging enzyme superoxide dismutase (SOD). METHODS: Hemodynamic and respiratory measurements were performed on anesthetized rabbits after (1) inhalation of methacholine (MCh), (2) iNO (80ppm), followed by MCh, (3) inhalation of hypertonic saline (HS), followed by iNO and MCh and (4) pre-treatment with inhalation of SOD, followed by HS, iNO and MCh. We analyzed plasma for a marker of oxidative stress, 8-iso-prostaglandin (PG)F(2alpha) and for a marker of activation of COX-mediated inflammatory cascades, PGF(2alpha) metabolite. RESULTS: Pre-treatment with SOD restored the bronchoprotective response to iNO in hyperosmotic airways. No direct effect was seen by SOD treatment on levels of 8-iso-PGF(2alpha), but this marker of oxidative stress correlated positively with increased bronchoconstriction. Hyperosmotic challenge elevated levels of PGF(2alpha) metabolite, and pre-treatment with SOD protected against this activation of the inflammatory cascade. CONCLUSION: SOD pre-treatment restores the relaxant effects of iNO in hyperosmotically challenged airways by attenuating oxidative stress and activation of COX-mediated inflammatory cascades.     

Superoxide reactivates nitric oxide-inhibited catalase

Catalase binds nitric oxide (NO) to generate ferricatalase-NO, an inhibited form of the enzyme. Superoxide (O2-) is also an inactivator of the enzyme. We found, however, that O2- efficiently converted the inhibited ferricatalase-NO to the active ferricatalase without producing detectable intermediates. The reaction slowed down when O2- was disproportionated to H2O2 and O2 by superoxide dismutase, but H2O2 could displace the heme-bound NO slowly to regenerate ferricatalase. Reactivation was observed even under simultaneous generation of NO and O2-, suggesting that ferricatalase-NO reacts with O2- fast enough to compete with the rapid reaction of O2- and NO. Formation of peroxynitrite by the simultaneous generation of NO and O2- was only partially inhibited by ferricatalase, presumably due to slow binding of NO to catalase in comparison with the reaction of NO and O2-.     

Fumonisin blunts nitric oxide-induced and nitroprusside-induced cardiomyocyte death.

The objective of this study was to determine whether nitric oxide (NO)-induced cell death in cardiomyocytes was operative through de novo synthesis of ceramide by determining whether the ceramide synthase inhibitor fumonisin blocked NO-mediated cell death. Neonatal mouse cardiomyocytes in culture were pretreated with fumonisin B1 (FB1). FB1 is a competitive inhibitor of sphinganine N-acyl transferase, also known as ceramide synthase (EC 2.3.1.24). Cell viability was assessed by the (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay, which is based on the ability of viable cells to reduce MTT. Treatment with the NO donor nitroso-glutathione (NO-GSH) for 24h produced a significant (p<0.05) concentration-dependent reduction in OD(570) or an increase in cell death. Sodium nitroprusside (SNP) treatment for 24h produced a significant (p<0.001) concentration-dependent reduction in OD(570) and an increase in cardiomyocyte cell death but the effects of SNP were greater than those of NO-GSH. FB1 significantly (p<0.05) reduced cell death induced by either SNP or NO-GSH. The SNP (0.1mM) increase in cell death of 36.9+/-2.8% was significantly (p<0.05) reduced to 24.7+/-1.8% by FB1 (10 microM). The effect of FB1 was not mediated through inhibition of the cell death effects of H(2)O(2), which is produced by SNP, as FB1 did not prevent H(2)O(2)-induced cell death. Confirmation of the ability of ceramide to produce cell death was demonstrated by the cell-permeable ceramide analogue, C(2)-ceramide (100 and 200 microM), which induced, respectively, 23.4+/-11.3 and 78.0+/-3.7% increases in cell death. The cell death effects of SNP and NO-GSH are likely independent of cGMP signal transduction pathways, which are activated by either SNP or NO-GSH, as there was no significant concentration-dependent change in cardiomyocyte viability after treatment with the cell-permeable analogue dibutyryl-GMP. These data show that FB1 blunts SNP- and NO-induced cardiomyocyte death and raise the novel possibility of preventing some of SNP- or NO-induced cardiomyocyte cell death by ceramide synthase inhibition.     

Signaling pathway of nitric oxide-induced acrosome reaction in human spermatozoa

Nitric oxide (NO) has been recently shown to modulate in vitro motility, viability, the acrosome reaction (AR), and metabolism of spermatozoa in various mammalian species, but the mechanism or mechanisms through which it influences sperm functions has not been clarified. In human capacitated spermatozoa, both the intracellular cGMP level and the percentage of AR-positive cells were significantly increased after 4 h of incubation with the NO donor, sodium nitroprusside (SNP). SNP-induced AR was significantly reduced in the presence of the soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ; this block was bypassed by adding 8-bromo-cGMP, a cell-permeating cGMP analogue, to the incubation medium. Finally, Rp-8-Br-cGMPS and Rp-8-pCPT-cGMPS, two inhibitors of the cGMP-dependent protein kinases (PKGs), inhibited the SNP-induced AR. Furthermore, SNP-induced AR did not occur in Ca2+ -free medium or in the presence of the protein kinase C (PKC) inhibitor, calphostin C. This study suggests that the AR-inducing effect of exogenous NO on capacitated human spermatozoa is accomplished via stimulation of an NO-sensitive sGC, cGMP synthesis, and PKG activation. In this effect the activation of PKC is also involved, and the presence of extracellular Ca2+ is required.     

Cellular mechanisms underlying nitric oxide-induced vasodilation of descending vasa recta

It has been observed that vasoactivity of explanted descending vasa recta (DVR) is modulated by intrinsic nitric oxide (NO) and superoxide (O(2)(-)) production (Cao C, Edwards A, Sendeski M, Lee-Kwon W, Cui L, Cai CY, Patzak A, Pallone TL. Am J Physiol Renal Physiol 299: F1056-F1064, 2010). To elucidate the cellular mechanisms by which NO, O(2)(-) and hydrogen peroxide (H(2)O(2)) modulate DVR pericyte cytosolic Ca(2+) concentration ([Ca](cyt)) and vasoactivity, we expanded our mathematical model of Ca(2+) signaling in pericytes. We incorporated simulations of the pathways that translate an increase in [Ca](cyt) to the activation of myosin light chain (MLC) kinase and cell contraction, as well as the kinetics of NO and reactive oxygen species formation and their effects on [Ca](cyt) and MLC phosphorylation. The model reproduced experimentally observed trends of DVR vasoactivity that accompany exposure to N(ω)-nitro-L-arginine methyl ester, 8-Br-cGMP, Tempol, and H(2)O(2). Our results suggest that under resting conditions, NO-induced activation of cGMP maintains low levels of [Ca](cyt) and MLC phosphorylation to minimize basal tone. This results from stimulation of Ca(2+) uptake from the cytosol into the SR via SERCA pumps, Ca(2+) efflux into the extracellular space via plasma membrane Ca(2+) pumps, and MLC phosphatase (MLCP) activity. We predict that basal concentrations of O(2)(-) and H(2)O(2) have negligible effects on Ca(2+) signaling and MLC phosphorylation. At concentrations above 1 nM, O(2)(-) is predicted to modulate [Ca(cyt)] and MCLP activity mostly by reducing NO bioavailability. The DVR vasoconstriction that is induced by high concentrations of H(2)O(2) can be explained by H(2)O(2)-mediated downregulation of MLCP and SERCA activity. We conclude that intrinsic generation of NO by the DVR wall may be sufficient to inhibit vasoconstriction by maintaining suppression of MLC phosphorylation.     

Specific protein nitration in nitric oxide-induced apoptosis of human monocytes

The sustained overproduction of nitric oxide (NO) observed in inflammatory conditions can contribute to cell demise by affecting apoptosis. Nitration of tyrosine residues occurs in a range of diseases involving macrophage activation. Since NO induces apoptosis in monocytes/macrophages, we tested the hypothesis that nitration of specific proteins could result in apoptotic cell death. The peroxynitrite generator SIN-1 promoted apoptosis in monocytes based on oligonucleosomal DNA fragmentation, caspase-3 and -9 activation, Bcl-2 depletion and accumulation of Bax and p53 proteins. We also found that the signaling pathway triggered by SIN-1 was initiated through tyrosine kinase and Rac activation and resulted in increased JNK and p38 activities. Among the tyrosine-nitrated proteins, Rac and Lyn were identified. Using specific inhibitors for different signaling and effector molecules involved in the apoptotic process we demonstrate that NO, via protein-nitration, could play an important role in controlling the inflammatory response by regulation of monocyte homeostasis.     

Collagen IV contributes to nitric oxide-induced angiogenesis of lung endothelial cells

Nitric oxide (NO) mediates endothelial angiogenesis via inducing the expression of integrin α(v)β(3). During angiogenesis, endothelial cells adhere to and migrate into the extracellular matrix through integrins. Collagen IV binds to integrin α(v)β(3), leading to integrin activation, which affects a number of signaling processes in endothelial cells. In the present study, we evaluated the role of collagen IV in NO-induced angiogenesis. We found that NO donor 2,2'-(hydroxynitrosohydrazino)bis-ethanamine (NOC-18) causes increases in collagen IV mRNA and protein in lung endothelial cells and collagen IV release into the medium. Addition of collagen IV into the coating of endothelial culture increases endothelial monolayer wound repair, proliferation, and tube formation. Inhibition of collagen IV synthesis using gene silencing attenuates NOC-18-induced increases in monolayer wound repair, cell proliferation, and tube formation as well as in the phosphorylation of focal adhesion kinase (FAK). Integrin blocking antibody LM609 prevents NOC-18-induced increase in endothelial monolayer wound repair. Inhibition of protein kinase G (PKG) using the specific PKG inhibitor KT5823 or PKG small interfering RNA prevents NOC-18-induced increases in collagen IV protein and mRNA and endothelial angiogenesis. Together, these results indicate that NO promotes collagen IV synthesis via a PKG signaling pathway and that the increase in collagen IV synthesis contributes to NO-induced angiogenesis of lung endothelial cells through integrin-FAK signaling. Manipulation of collagen IV could be a novel approach for the prevention and treatment of diseases such as alveolar capillary dysplasia, severe pulmonary arterial hypertension, and tumor invasion.     

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.     

Inhibition of nitric oxide-induced apoptosis by nicotine in oral epithelial cells

Development of oral cancer is clearly linked to the usage of smokeless tobacco. The molecular mechanisms involved in this process are however not well understood. Toward this goal, we investigated the effect of smokeless tobacco exposure on apoptosis of oral epithelial cells. Exposure of oral epithelial cells to smokeless tobacco extract (STE) induces apoptosis in a dose-dependent manner, until a threshold level of nicotine is achieved upon which apoptosis is inhibited. 1 mM of nicotine is able to inhibit apoptosis significantly induced by STE in these oral cells. Exposure of cells to nicotine alone has no effect on apoptosis, but nicotine inhibits apoptosis induced by other agents present in STE. In this study we show that, the anti-apoptotic action of nicotine is specifically associated with down-regulation of nitric oxide (NO) production. Using specific inducers of NO, we have demonstrated that inhibition of apoptosis by nicotine is through down-regulation of NO production. Further, we observed that nicotine clearly acts as a sink of NO radicals, shown using peroxynitrite generator (SIN-1) in conjunction or absence of radical scavengers. Nicotine thus causes most damage in transformed epithelial cells as depicted by accumulation of nitrotyrosine in a 3-NT ELISA assay. Inhibition of apoptosis is a hallmark in tumor progression and propels development of cancer. It may further result in functional loss of apoptotic effector mechanisms in the transformed cells. Thus, our data clearly indicates that inhibition of NO-induced apoptosis by nicotine may lead to tobacco-induced oral carcinogenesis, and implies careful development of modalities in tobacco cessation programs. Abhijit G. Banerjee and Velliyur K. Gopalakrishnan—contributed equally.     

The mitochondrial thioredoxin system regulates nitric oxide-induced HIF-1alpha protein

Hypoxia-inducible factor-1 (HIF-1), consisting of two subunits, HIF-1alpha and HIF-1beta, is a key regulator for adaptation to low oxygen availability, i.e., hypoxia. Compared to the constitutively expressed HIF-1beta, HIF-1alpha is regulated by hypoxia but also under normoxia (21% O(2)) by several stimuli, including nitric oxide (NO). In this study, we present evidence that overexpression of mitochondrial-located thioredoxin 2 (Trx2) or thioredoxin reductase 2 (TrxR2) attenuated NO-evoked HIF-1alpha accumulation and transactivation of HIF-1 in HEK293 cells. In contrast, cytosolic-located thioredoxin 1 (Trx1) enhanced HIF-1alpha protein amount and activity under NO treatments. Taking into consideration that thioredoxins affect the synthesis of HIF-1alpha by altering Akt/mTOR signaling, we herein show that p42/44 mitogen-activated protein kinase and p70S6 kinase are involved. Moreover, intracellular ATP was increased in Trx1-overexpressing cells but reduced in cells overexpressing Trx2 or TrxR2, providing thus an understanding of how protein synthesis is regulated by thioredoxins.     

Neuronal intracellular pH directly mediates nitric oxide-induced programmed cell death.

Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pH(i)) regulation during nitric oxide (NO)-induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 microM), 3-morpholinosydnonimine (300 microM), or 6-(2-hyrdroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hex anamine (300 microM). The pH(i) was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pH(i) 7.36 +/- 0.01 to approximately 7.00 (p <.0001). Within 45 min, a biphasic alkaline response was evident, with pH(i) reaching 7.40 +/- 0.02, that was persistent for a 6-h period. To mimic the effect of NO-induced acidification, neurons were acid-loaded with ammonium ions to yield a pH(i) of 7.09 +/- 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 +/- 2% (24 h) and DNA fragmentation increased to 58 +/- 8% (24 h) (p <.0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pH(i) of 7.60 +/- 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO-induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration.     

Regulation of nitric oxide-induced apoptosis by sensitive to apoptosis gene protein

Sensitive to apoptosis gene (SAG) protein, a novel zinc RING finger protein that protects mammalian cells from apoptosis by redox reagents, is a metal chelator and a potential reactive oxygen species (ROS) scavenger, but its antioxidant properties have not been completely defined. 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 stress. In this report, we demonstrate that modulation of SAG expression in U937 cells regulates NO-induced apoptosis. When we examined the protective role of SAG against NO-induced apoptosis with U937 cells transfected with the cDNA for SAG, a clear inverse relationship was observed between the amount of SAG expressed in target cells and their susceptibility to apoptosis. We also observed the significant decrease in the endogenous production of ROS and oxidative DNA damage in SAG-overexpressed cells compared to control cells upon exposure to NO. These results suggest that SAG plays an important protective role in NO-induced apoptosis, presumably, through regulating the cellular redox status.     

Inhaled nitric oxide-induced rebound pulmonary hypertension: role for endothelin-1

Clinically significant increases in pulmonary vascular resistance have been noted on acute withdrawal of inhaled nitric oxide (NO). Endothelin (ET)-1 is a vasoactive peptide produced by the vascular endothelium that may participate in the pathophysiology of pulmonary hypertension. The objectives of this study were to determine the effects of inhaled NO on endogenous ET-1 production in vivo in the intact lamb and to determine the potential role of ET-1 in the rebound pulmonary hypertension associated with the withdrawal of inhaled NO. Seven 1-mo-old vehicle-treated control lambs and six PD-156707 (an ET(A) receptor antagonist)-treated lambs were mechanically ventilated. Inhaled NO (40 parts per million) was administered for 24 h and then acutely withdrawn. After 24 h of inhaled NO, plasma ET-1 levels increased by 119.5 +/- 42.2% (P < 0.05). Western blot analysis revealed that protein levels of preproET-1, endothelin-converting enzyme-1alpha, and ET(A) and ET(B) receptors were unchanged. On acute withdrawal of NO, pulmonary vascular resistance (PVR) increased by 77.8% (P < 0.05) in control lambs but was unchanged (-5.5%) in PD-156707-treated lambs. Inhaled NO increased plasma ET-1 concentrations but not gene expression in the intact lamb, and ET(A) receptor blockade prevented the increase in PVR after NO withdrawal. These data suggest a role for ET-1 in the rebound pulmonary hypertension noted on acute withdrawal of inhaled NO.     

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.     

NADPH: a stimulatory cofactor for nitric oxide-induced ADP-ribosylation reaction.

An endogenous ADP-ribosyltransferase is present in the cytosolic fraction of human platelets. Agents known to release nitric oxide activated this ADP-ribosylation reaction in a cGMP-independent fashion. This enzymatic activity was further enhanced by the addition of NADPH to the platelet cytosolic fraction. Interestingly, NADPH was unable to replace DTT, which has been described as an essential cofactor. Our results indicate that NADPH is a stimulatory factor of the endogenous ADP-ribosylation reaction. NADPH shifts the dose-response curve of NO to the left and possibly increases, in this way, the ADP-ribosylation reaction under physiological conditions.     

Reduced glutathione prevents nitric oxide-induced apoptosis in vascular smooth muscle cells

The control of medial and neointimal growth, in which vascular smooth muscle (VSM) plays a central role, is most important to the development of hypertension and atherosclerosis, respectively. Growth of vascular smooth muscle cells is regulated by a number of factors, including the vasodilator nitric oxide (NO). In addition, NO modulates intracellular thiol redox states and the thiol redox state of the cell influences NO production. We, therefore, examined the nature of the effect of NO on growth of VSM cells and its modulation by cellular glutathione content. Here, we report that NO, either generated by NO donors or synthesized by iNOS in VSM cells, inhibited DNA synthesis and induced apoptosis in this cell type. NO-induced apoptosis was associated with a significant decrease in the intracellular concentration of reduced glutathione and with an increase in the level of the tumor suppressor gene p53 mRNA. Moreover, addition of glutathione monoethylester to the culture restored the level of reduced glutathione in VSM cells, and prevented the NO-induced increase in p53 expression and programmed cell death. Our findings suggest a role for reduced glutathione in protecting VSM cells exposed to NO from apoptosis.