The role of inducible nitric oxide synthase for interstitial remodeling of alveolar septa in surfactant protein D-deficient mice

Surfactant protein D (SP-D) modulates the lung's immune system. Its absence leads to NOS2-independent alveolar lipoproteinosis and NOS2-dependent chronic inflammation, which is critical for early emphysematous remodeling. With aging, SP-D knockout mice develop an additional interstitial fibrotic component. We hypothesize that this age-related interstitial septal wall remodeling is mediated by NOS2. Using invasive pulmonary function testing such as the forced oscillation technique and quasistatic pressure-volume perturbation and design-based stereology, we compared 29-wk-old SP-D knockout (Sftpd(-/-)) mice, SP-D/NOS2 double-knockout (DiNOS) mice, and wild-type mice (WT). Structural changes, including alveolar epithelial surface area, distribution of septal wall thickness, and volumes of septal wall components (alveolar epithelium, interstitial tissue, and endothelium) were quantified. Twenty-nine-week-old Sftpd(-/-) mice had preserved lung mechanics at the organ level, whereas elastance was increased in DiNOS. Airspace enlargement and loss of surface area of alveolar epithelium coexist with increased septal wall thickness in Sftpd(-/-) mice. These changes were reduced in DiNOS, and compared with Sftpd(-/-) mice a decrease in volumes of interstitial tissue and alveolar epithelium was found. To understand the effects of lung pathology on measured lung mechanics, structural data were used to inform a computational model, simulating lung mechanics as a function of airspace derecruitment, septal wall destruction (loss of surface area), and septal wall thickening. In conclusion, NOS2 mediates remodeling of septal walls, resulting in deposition of interstitial tissue in Sftpd(-/-). Forward modeling linking structure and lung mechanics describes the complex mechanical properties by parenchymatous destruction (emphysema), interstitial remodeling (septal wall thickening), and altered recruitability of acinar airspaces.

Nitric oxide sustains IL-1β expression in human dendritic cells enhancing their capacity to induce IL-17-producing T-cells

The role played by lung dendritic cells (DCs) which are influenced by external antigens and by their redox state in controlling inflammation is unclear. We studied the role played by nitric oxide (NO) in DC maturation and function. Human DCs were stimulated with a long-acting NO donor, DPTA NONOate, prior to exposure to lipopolysaccharide (LPS). Dose-and time-dependent experiments were performed with DCs with the aim of measuring the release and gene expression of inflammatory cytokines capable of modifying T-cell differentiation, towardsTh1, Th2 and Th17 cells. NO changed the pattern of cytokine release by LPS-matured DCs, dependent on the concentration of NO, as well as on the timing of its addition to the cells during maturation. Addition of NO before LPS-induced maturation strongly inhibited the release of IL-12, while increasing the expression and release of IL-23, IL-1β and IL-6, which are all involved in Th17 polarization. Indeed, DCs treated with NO efficiently induced the release of IL-17 by T-cells through IL-1β. Our work highlights the important role that NO may play in sustaining inflammation during an infection through the preferential differentiation of the Th17 lineage.

Nitric oxide inhalation reduces brain damage, prevents mortality, and improves neurological outcome after subarachnoid hemorrhage by resolving early pial microvasospasms.

Subarachnoid hemorrhage is a stroke subtype with particularly bad outcome. Recent findings suggest that constrictions of pial arterioles occurring early after hemorrhage may be responsible for cerebral ischemia and - subsequently - unfavorable outcome after subarachnoid hemorrhage. Since we recently hypothesized that the lack of nitric oxide may cause post-hemorrhagic microvasospasms, our aim was to investigate whether inhaled nitric oxide, a treatment paradigm selectively delivering nitric oxide to ischemic microvessels, is able to dilate post-hemorrhagic microvasospasms; thereby improving outcome after experimental subarachnoid hemorrhage. C57BL/6 mice were subjected to experimental SAH. Three hours after subarachnoid hemorrhage pial artery spasms were quantified by intravital microscopy, then mice received inhaled nitric oxide or vehicle. For induction of large artery spasms mice received an intracisternal injection of autologous blood. Inhaled nitric oxide significantly reduced number and severity of subarachnoid hemorrhage-induced post-hemorrhage microvasospasms while only having limited effect on large artery spasms. This resulted in less brain-edema-formation, less hippocampal neuronal loss, lack of mortality, and significantly improved neurological outcome after subarachnoid hemorrhage. This suggests that spasms of pial arterioles play a major role for the outcome after subarachnoid hemorrhage and that lack of nitric oxide is an important mechanism of post-hemorrhagic microvascular dysfunction. Reversing microvascular dysfunction by inhaled nitric oxide might be a promising treatment strategy for subarachnoid hemorrhage.

The role of nitric oxide in small intestinal motility in a model of acute inflammation

Motility responses of the small intestine of iNOS deficient mice (iNOS −/−) and their wildtype littermates (iNOS+/+) to the inflammatory challenge of lipopolysaccharide (LPS) were investigated. LPS administration failed to attenuate intestinal transit in iNOS−/− mice but depressed transit in their iNOS+/+ littermates. Supporting an inhibitory role for sustained nitric oxide (NO) synthesis in the regulation of intestinal motility during inflammation, iNOS immunoreactivity was upregulated in all regions of the small intestine of iNOS+/+ mice. In contrast, neuronal NOS was barely affected. Cyclooxygenase activation was determined by prostaglandin E2 (PGE2) concentration. Following LPS challenge, PGE2 levels were elevated in all intestinal segments in both animal groups. Moreover, COX-1 and COX-2 protein levels were elevated in iNOS+/+ mice in response to LPS, while COX-2 levels were similarly increased in iNOS −/− intestine. However, no apparent relationship was observed between increased prostaglandin concentrations and attenuated intestinal transit. The presence of heme oxygenase 1 (HO-1) in the murine small intestine was also investigated. In both animal groups HO-1 immunoreactivity in the proximal intestine increased in response to treatment, while the constitutive protein levels detected in the middle and distal intestine were unresponsive to LPS administration. No apparent correlation of HO-1 to the suppression of small intestinal motility induced by LPS administration was detected. The presence of S-nitrosylated contractile proteins in the small intestine was determined. γ-smooth muscle actin was basally nitrosylated as well as in response to LPS, but myosin light chain kinase and myosin regulatory chain (MLC20) were not. In conclusion, in a model of acute intestinal inflammation, iNOS-produced NO plays a significant role in suppressing small intestinal motility while nNOS, COX-1, COX-2 and HO-1 do not participate in this event. S-nitrosylation of γ-smooth muscle actin is associated with elevated levels of nitric oxide in the smooth muscle of murine small intestine. ^

Nitric Oxide Equilibrium

The explicit formulas for following the passage to chemical equilibrium are derived.

Nitric oxide contributes to LPS/IFN-gamma-induced macrophage apoptosis via JNK and BCL-2 family regulation

Lipopolysaccharide (LPS) and interferon-gamma (IFN) activate macrophages and produce nitric oxide (NO) by initiating the expression of inducible Nitric Oxide Synthase (iNOS). Prolonged LPS/IFN-activation results in the death of macrophage-like RAW 264.7 cells and wild-type murine macrophages. This study was implemented to determine how NO contributes to LPS/IFN-induced macrophage death. The iNOS-specific inhibitor L-NIL protected RAW 264.7 cells from LPS/IFN-activated death, supporting a role for NO in the death of LPS/IFN-activated macrophages. A role for iNOS in cell death was confirmed in iNOS-/- macrophages which were resistant to LPS/IFN-induced death. Cell death was accompanied by nuclear condensation, caspase 3 activation, and PARP cleavage, all of which are hallmarks of apoptosis. The involvement of NO in modulating the stress-activated protein kinase (SAPK)/c-jun N-terminal kinase (JNK) signal transduction pathway was examined as a possible mechanism of LPS/IFN-mediated apoptosis. Western analysis demonstrated that NO modifies the phosphorylation profile of JNK and promotes activation of JNK in the mitochondria in RAW 264.7 cells. Inhibition of JNK with sIRNA significantly reduced cell death in RAW 264.7 cells, indicating the participation of the JNK pathway in LPS/IFN-mediated death. JNK has been demonstrated to induce mitochondrial-mediated apoptosis through modulation of Bcl-2 family members. Therefore, the effect of NO on the balance between pro- and anti-apoptotic Bcl-2 family members was examined. In RAW 264.7 cells, Bim was upregulated and phosphorylated by LPS/IFN independently of NO. However, co-immunoprecipitation studies demonstrated that NO promotes the association of Bax with the BimL splice variant. Examination of Bax phosphorylation by metabolic labeling demonstrated that Bax is basally phosphorylated and becomes dephosphorylated upon LPS/IFN treatment. L-NIL inhibited the dephosphorylation of Bax, indicating that Bax dephosphorylation is NO-dependent. NO also mediated LPS/IFN-induced downregulation of Mcl-1, an anti-apoptotic Bcl-2 family member, as demonstrated by Western blotting for Mcl-1 protein expression. Thus, NO contributes to macrophage apoptosis via a JNK-mediated mechanism involving interaction between Bax and Bim, dephosphorylation of Bax, and downregulation of Mcl-1. ^

Does exhaled nitric oxide predict asthma exacerbation in children?

Asthma is a chronic complex disorder of the respiratory tract that affects millions of people globally, a large percentage of which are children. Triggered by a host of factors such as allergens and changes in temperature, the pathophysiologic and clinical indices vary among patients and have contributed to difficulties in overall management of asthma. Shortly after exhaled nitric oxide (eNO) was discovered in higher concentrations in asthma patients, it was shown to be superior to other markers such as PEFR, FEV1 and sputum eosinophils in screening asthma patients. Studies have also noted promising results regarding the use of eNO to predict asthma exacerbation in adults while in children, asthma symptoms have been observed to be good predictors of asthma exacerbation. Currently however, the potential of eNO as a predictor of asthma exacerbation in children is yet to be examined. The objective of this study was to assess eNO potential to predict asthma exacerbation in children by examining the relationship between eNO and changes in pulmonary function, asthma symptoms and rescue medication use.^ The primary study "Air Toxics and Asthma in Children" (ATAC), recruited children aged 9 to 14 years with labile persistent asthma diagnosed at least one year earlier. The data obtained from 30 study participants, included exhaled nitric oxide concentration, PEFR, FEV1, asthma symptoms and frequency of emergency medication use.^ Descriptive statistics, Pearson's and Spearman's correlation tests were followed by a simple linear regression in which eNO was the independent (predictor) variable while FEV1, PEFR, asthma symptoms and frequency of emergency medication use were the dependent (outcome) variables.^ Results showed that eNO was associated with percent change in FEV1, day time wheeze, night time shortness of breath, but correlated only weakly with PEFR, amplitude percent of mean PEFR, FEV1, percent change in FEV1 and asthma symptoms.^ Further research is imperative to better define the role of eNO and understand intrinsic pathologic mechanisms towards asthma management in children.^

Endogenous nitric oxide regulates p53 signaling and DNA damage-induced apoptosis in melanoma cells

Nitric oxide is involved in a multitude of processes including regulation of vascular tone, neurotransmission, immunity, and cancer. Evidence suggests that nitric oxide exhibits anti-apoptotic activity in melanoma cells. Our laboratory showed that tumor expression of inducible nitric oxide synthase correlated strongly with poor survival in stage III and IV melanoma patients, suggesting an antagonistic role for nitric oxide in melanoma response to therapy. Therefore, the hypothesis that endogenously produced nitric oxide antagonizes chemotherapy-induced apoptosis was formed. Using cisplatin as a model for DNA damage in melanoma cell lines, the capacity of nitric oxide to regulate cell growth and apoptotic responses to cisplatin treatment was examined. The depletion of endogenously generated nitric oxide resulted in changes in cell cycle regulation and enhanced cisplatin-induced apoptosis in melanoma cells. Since nitric oxide was shown to be involved in the regulation of p53 stability, conformation and DNA binding activity, whether signaling through wild-type p53 in melanoma cells is regulated by nitric oxide was tested. Cisplatin-induced p53 accumulation and p21Waf1/Cip1/Sdi1 expression in nitric oxide-depleted melanoma cells were found to be strongly suppressed. When p53 binding to the p21Waf1/Cip1/Sdi1 promoter was examined, it was found that nitric oxide depletion significantly reduced the cisplatin-induced formation of p53-DNA complexes. These results suggest that nitric oxide is required for activation of wild-type p53 after DNA damage in melanoma cells. Finally, whether signaling through p53 controls melanoma response to DNA damage was examined. Transfection of a plasmid containing a dominant negative form of mutated p53 inhibited p21 Waf1/Cip1/Sdi1 expression and concomitantly enhanced apoptosis after cisplatin treatment. These data suggest that the induction of wild-type p53 protects melanoma cells against DNA damage via the up-regulation of p21 Waf1/Cip1/Sdi1. Together, these data strongly support the model that endogenous nitric oxide is required for p53 activation and p21Waf1/Cip1/Sdi1 expression after DNA damage, which can enhance melanoma resistance to therapy. Thus, in context of melanoma cells with wild-type p53 , low levels of endogenous constitutively-produced nitric oxide appear to facilitate the activation of p53 in response to DNA damage, thereby allowing for cell cycle arrest via p21Waf1/Cip1/Sdi1 induction, adequate DNA repair, and ultimately enhanced resistance to apoptosis. ^

Stimulation of renin secretion by nitric oxide is mediated by phosphodiesterase 3

This study aimed to characterize the cellular pathways along which nitric oxide (NO) stimulates renin secretion from the kidney. Using the isolated perfused rat kidney model we found that renin secretion stimulated 4- to 8-fold by low perfusion pressure (40 mmHg), by macula densa inhibition (100 μmol/liter of bumetanide), and by adenylate cyclase activation (3 nmol/liter of isoproterenol) was markedly attenuated by the NO synthase inhibitor nitro-l-arginine methyl ester (l-Name) (1 mM) and that the inhibition by l-Name was compensated by the NO-donor sodium nitroprusside (SNP) (10 μmol/liter). Similarly, inhibition of cAMP degradation by blockade of phosphodiesterase 1 (PDE-1) (20 μmol/liter of 8-methoxymethyl-1-methyl-3-(2-methylpropyl)xanthine) or of PDE-4 (20 μmol/liter of rolipram) caused a 3- to 4-fold stimulation of renin secretion that was attenuated by l-Name and that was even overcompensated by sodium nitroprusside. Inhibition of PDE-3 by 20 μmol/liter of milrinone or by 200 nmol/liter of trequinsin caused a 5- to 6-fold stimulation of renin secretion that was slightly enhanced by NO synthase inhibition and moderately attenuated by NO donation. Because PDE-3 is a cGMP-inhibited cAMP-PDE the role of endogenous cGMP for the effects of NO was examined by the use of the specific guanylate cyclase inhibitor 1-H-(1,2,4)oxodiazolo(4,3a)quinoxalin-1-one (20 μmol). In the presence of 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-1-one the effect of NO on renin secretion was abolished, whereas PDE-3 inhibitors exerted their normal effects. These findings suggest that PDE-3 plays a major role for the cAMP control of renin secretion. Our findings are compatible with the idea that the stimulatory effects of endogenous and exogenous NO on renin secretion are mediated by a cGMP-induced inhibition of cAMP degradation.

Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia

Nitric oxide (NO) is known to have various biologic and pathophysiologic effects on organisms. The molecular mechanisms by which NO exerts harmful effects are unknown, although various O2 radicals and ions that result from reactivity of NO are presumed to be involved. Here we report that adaptive cellular response controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) in hypoxia is suppressed by NO. Induction of erythropoietin and glycolytic aldolase A mRNAs in hypoxically cultured Hep3B cells, a human hepatoma cell line, was completely and partially inhibited, respectively, by the addition of sodium nitroprusside (SNP), which spontaneously releases NO. A reporter plasmid carrying four hypoxia-response element sequences connected to the luciferase structural gene was constructed and transfected into Hep3B cells. Inducibly expressed luciferase activity in hypoxia was inhibited by the addition of SNP and two other structurally different NO donors, S-nitroso-l-glutathione and 3-morpholinosydnonimine, giving IC50 values of 7.8, 211, and 490 μM, respectively. Inhibition by SNP was also observed in Neuro 2A and HeLa cells, indicating that the inhibition was not cell-type-specific. The vascular endothelial growth factor promoter activity that is controlled by HIF-1 was also inhibited by SNP (IC50 = 6.6 μM). Induction generated by the addition of cobalt ion (this treatment mimics hypoxia) was also inhibited by SNP (IC50 = 2.5 μM). Increased luciferase activity expressed by cotransfection of effector plasmids for HIF-1α or HIF-1α-like factor in hypoxia was also inhibited by the NO donor. We also showed that the inhibition was performed by blocking an activation step of HIF-1α to a DNA-binding form.

Cloning and characterization of human inducible nitric oxide synthase splice variants: A domain, encoded by exons 8 and 9, is critical for dimerization

The inducible nitric oxide synthase (iNOS) contains an amino-terminal oxygenase domain, a carboxy-terminal reductase domain, and an intervening calmodulin-binding region. For the synthesis of nitric oxide (NO), iNOS is active as a homodimer. The human iNOS mRNA is subject to alternative splicing, including deletion of exons 8 and 9 that encode amino acids 242–335 of the oxygenase domain. In this study, iNOS8−9− and full-length iNOS (iNOSFL) were cloned from bronchial epithelial cells. Expression of iNOS8−9− in 293 cell line resulted in generation of iNOS8−9− mRNA and protein but did not lead to NO production. In contrast to iNOSFL, iNOS8−9− did not form dimers. Similar to iNOSFL, iNOS8−9− exhibited NADPH-diaphorase activity and contained tightly bound calmodulin, indicating that the reductase and calmodulin-binding domains were functional. To identify sequences in exons 8 and 9 that are critical for dimerization, iNOSFL was used to construct 12 mutants, each with deletion of eight residues in the region encoded by exons 8 and 9. In addition, two “control” iNOS deletion mutants were synthesized, lacking either residues 45–52 of the oxygenase domain or residues 1131–1138 of the reductase domain. Whereas both control deletion mutants generated NO and formed dimers, none of the 12 other mutants formed dimers or generated NO. The region encoded by exons 8 and 9 is critical for iNOS dimer formation and NO production but not for reductase activity. This region could be a potential target for therapeutic interventions aimed at inhibiting iNOS dimerization and hence NO synthesis.

Persistent inhibition of cell respiration by nitric oxide: Crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

Calcium-independent activation of endothelial nitric oxide synthase by ceramide

The endothelial isoform of NO synthase (eNOS) is targeted to sphingolipid-enriched signal-transducing microdomains in the plasma membrane termed caveolae. Among the caveolae-targeted sphingolipids are the ceramides, a class of acylated sphingosine compounds that have been implicated in diverse cellular responses. We have explored the role of ceramide analogues in eNOS signaling in cultured bovine aortic endothelial cells (BAEC). Addition of the ceramide analogue N-acetylsphingosine (C2-ceramide; 5 μM) to intact BAEC leads to a significant increase in NO synthase activity (assayed by using the fluorescent indicator 4,5-diaminofluorescein) and translocation of eNOS from the endothelial cell membrane to intracellular sites (measured by using quantitative immunofluorescence techniques); the biologically inactive ceramide N-acetyldihydrosphingosine is entirely without effect. C2-ceramide-induced eNOS activation and translocation are unaffected by the intracellular calcium chelator 1,2-bis-o-aminophenoxyethane-N,N,N′,N′-tetraacetic acid (BAPTA). Using the calcium-specific fluorescent indicator fluo-3, we also found that C2-ceramide activation of eNOS is unaccompanied by a drug-induced increase in intracellular calcium. These findings stand in sharp contrast to the mechanism by which bradykinin, estradiol, and other mediators acutely activate eNOS, in which a rapid, agonist-promoted increase in intracellular calcium is required. Finally, we show that treatment of BAEC with bradykinin causes a significant increase in cellular ceramide content; the response to bradykinin has an EC50 of 3 nM and is blocked by the bradykinin B2-receptor antagonist HOE140. Bradykinin-induced ceramide generation could represent a mechanism for longer-term regulation of eNOS activity. Our results suggest that ceramide functions independently of Ca2+-regulated pathways to promote activation and translocation of eNOS, and that this lipid mediator may represent a physiological regulator of eNOS in vascular endothelial cells.

A mechanism of paraquat toxicity involving nitric oxide synthase

Paraquat (PQ) is a well described pneumotoxicant that produces toxicity by redox cycling with cellular diaphorases, thereby elevating intracellular levels of superoxide (O2⨪). NO synthase (NOS) has been shown to participate in PQ-induced lung injury. Current theory holds that NO reacts with O2⨪ generated by PQ to produce the toxin peroxynitrite. We asked whether NOS might alternatively function as a PQ diaphorase and reexamined the question of whether NO/O2⨪ reactions were toxic or protective. Here, we show that: (i) neuronal NOS has PQ diaphorase activity that inversely correlates with NO formation; (ii) PQ-induced endothelial cell toxicity is attenuated by inhibitors of NOS that prevent NADPH oxidation, but is not attenuated by those that do not; (iii) PQ inhibits endothelium-derived, but not NO-induced, relaxations of aortic rings; and (iv) PQ-induced cytotoxicity is potentiated in cytokine-activated macrophages in a manner that correlates with its ability to block NO formation. These data indicate that NOS is a PQ diaphorase and that toxicity of such redox-active compounds involves a loss of NO-related activity.

Recombinant interleukin-1β dilates steelhead trout coronary microvessels : effect of temperature and role of the endothelium, nitric oxide and prostaglandins

© 2015. Published by The Company of Biologists Ltd. Acknowledgements We thank Wenjuan Xu and Xin Xu (Hein Lab) for their excellent instruction in microvessel techniques, Dr David Heeley (Biochemistry Department, MUN) for assistance with selecting an appropriate (non-vasoactive) protein stabilizer, Dr Zou (SFIRC, Aberdeen) for advice with regards to the use of rIL-1β and Gordon Nash (Gamperl Lab) for his assistance with the rIL-1β purification protocol. Funding This research was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant [RGPIN249926] and Accelerator Supplement [RGPAS412325-2011] to A.K.G. a National Institutes of Health Grant [EY018420] to T.W.H., and a doctoral fellowship from Fundaçã o para a Ciência e a Tecnologia, Portugal [SFRH/BD/27497/2006] to I.A.S.F.C. Deposited in PMC for release after 12 months.