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.     

Nitric oxide synthesis in the CNS endothelium and macrophages differs in its sensitivity to inhibition by arginine analogues.

Inhibition of nitric oxide production by arginine analogues was examined in three cell systems; macrophages, CNS tissue and endothelial cells. Nitric oxide production was assessed indirectly using in vitro assays measuring nitrite production (macrophages), cGMP elevation (CNS) and acetylcholine-induced relaxation of aortic ring segments (endothelium). NG-monomethyl-L-arginine and NG-amino-L-arginine possessed similar inhibitory activity in all three assays, while NG-nitro-L-arginine displayed a striking selectivity for inhibition of brain and endothelial cell nitric oxide synthesis, with IC50 values of 0.05 microM in the CNS versus 200 microM in macrophages. These results suggest that distinct enzymes are responsible for nitric oxide synthesis in different cell types, and indicate that it may be possible to selectively modulate nitric oxide production in vivo.     

Regulation of microglial activities by glial cell line derived neurotrophic factor

Much attention has been paid to the ability of glial cell line-derived neurotrophic factor (GDNF) to protect neurons from neurotoxic insults in the central nervous system (CNS). However, little is known about GDNF action on CNS glia that also can express GDNF receptor systems. In this study, we examined the effects of GDNF on primary rat microglia that function as resident macrophages in the CNS and as the source of proinflammatory mediators upon activation. We found that treatment of primary rat microglia with GDNF had no effect on the secretion of the proinflammatory cytokines, tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta), but it increased the nitric oxide (NO) production to some extent. In addition, GDNF increased the enzymatic activity of superoxide dismutase (SOD), the gene expression of surface antigen intercellular adhesion molecule-1 (ICAM-1), the production of the integrin alpha5 subunit, and the phagocytotic capability in primary rat microglia. Furthermore, inhibition of mitogen-activated protein kinase (Erk-MAPK) in the mouse microglial cell line BV2 by U0126 indicated that the MAP kinase signaling pathway may be involved in the regulation of NO and integrin alpha5 production by GDNF. In vivo evidence also showed that amoeboid cells with integrin alpha5 or with ED1 immunoreactivity appeared in GDNF-treated spinal cord tissues at the lesion site 1 week post spinal cord injury (SCI). Furthermore, inhibition of Erk-MAPK in the mouse microglial cell line BV2 by U0126 indicated that the MAP kinase signaling pathway may be involved in the regulation of NO and integrin alpha5 production by GDNF. Taken together, our results indicate that GDNF has a positive regulatory effect on microglial activities, such as phagocytosis and the upregulation of adhesion molecules.     

Nitric oxide: a potential inducer of adhesion-related apoptosis--anoikis.

Among the many initiating events that lead to apoptosis or programmed cell death, loss of contact between the cell and the extracellular matrix has been extensively studied. Adhesion-related apoptosis referred to as anoikis is initiated by the action of anti-adhesive substances. Nitric oxide is one of these anti-adhesive substances that have the capacity to signal and trigger pro-apoptotic events in a variety of cell types. Nitric oxide can inhibit cell adhesion, interfere with the assembly of focal adhesion complexes, and disrupt the cell-extracellular matrix interactions. These actions occur in cell that exhibit a dissociation of growth factor signals from alterations in the cytoskeleton, ultimately leading to apoptosis. Since this involves anti-adhesive events, nitric oxide can be considered as causing anoikis. This review article summarizes the available evidence of how nitric oxide participates in apoptosis induced by loss of anchorage (anoikis).     

Nebivolol inhibits vascular smooth muscle cell proliferation by mechanisms involving nitric oxide but not cyclic GMP.

The objective of this study was to elucidate the mechanisms by which nebivolol, a cardio-selective beta-adrenergic receptor antagonist, inhibits rat aortic smooth muscle cell (RASMC) proliferation. Nebivolol was compared with DETA-NO and S-nitroso-N-acetylpenicillamine (SNAP), two nitric oxide (NO) donor agents, and alpha-difluoromethylornithine (DFMO), a known inhibitor of ornithine decarboxylase (ODC). All four test agents inhibited RASMC proliferation in a concentration-dependent manner, with nebivolol being the most potent (IC(50) = 4.5 microM), whereas atenolol, another relatively selective beta(1)-blocker, was inactive. DFMO, nebivolol, and DETA-NO interfered with cell proliferation in a cell-density-dependent manner, the lower the cell density the greater the inhibition of cell proliferation. The cytostatic effects of nebivolol and DETA-NO were completely independent of cyclic GMP, as neither ODQ (cytosolic guanylyl cyclase inhibitor) nor zaprinast (cyclic GMP phosphodiesterase inhibitor) affected the antiproliferative action of nebivolol or DETA-NO. The cytostatic effects of nebivolol, SNAP, and DFMO were largely prevented by the addition of excess putrescine, but not ornithine, to cell cultures. Moreover, nebivolol caused a marked reduction in the intracellular levels of putrescine, spermidine, and spermine. Like DFMO, nebivolol and DETA-NO interfered with the G(1)-phase to S-phase cell cycle transition in RASMC. These observations confirm previous findings that DFMO and NO interfere with RASMC proliferation by inhibiting ODC and polyamine production and provide evidence that nebivolol works by the same mechanism.     

The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (*NO) protect brain dopamine neurons from hydroxyl radical (*OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and *NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO--S-nitrosylated GSH--is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH GS* + *NO-->[GSNO]-->GSSG + *NO-->GSH) is necessary for understanding the biology of *NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and *NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/*NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

Formation of nitric oxide hemoglobin in erythrocytes co-cultured with alveolar macrophages taken from bleomycin treated rats.

Alveolar macrophages, taken from rats treated with a single intratracheal dose of bleomycin, release reactive nitrogen intermediates in the form of nitric oxide which are cytostatic to murine leukemia L1210 cells. When cultured in the presence of erythrocytes the cytostatic activity of alveolar macrophages was inhibited which corresponded with an increase in nitrosylated hemoglobin content when compared with erythrocytes cultured alone. These results suggest that erythrocytes inhibit alveolar macrophage cytostatic activity by preventing reactive nitrogen intermediates from reaching target cells because the hemoglobin serves as a sink for reactive nitrogen intermediates in the form of nitric oxide.     

Nitric oxide in the central nervous system: neuroprotection versus neurotoxicity

At the end of the 1980s, it was clearly demonstrated that cells produce nitric oxide and that this gaseous molecule is involved in the regulation of the cardiovascular, immune and nervous systems, rather than simply being a toxic pollutant. In the CNS, nitric oxide has an array of functions, such as the regulation of synaptic plasticity, the sleep-wake cycle and hormone secretion. Particularly interesting is the role of nitric oxide as a Janus molecule in the cell death or survival mechanisms in brain cells. In fact, physiological amounts of this gas are neuroprotective, whereas higher concentrations are clearly neurotoxic.     

Effect of nitric oxide on expression of apoptotic genes and HSP70 in drosophila

Data on involvement of nitric oxide in apoptosis are contradictory. The balance between anti- and proapoptotic activities of nitric oxide depends on many factors, including its concentration in a tissue and interactions with other regulators of apoptosis. This paper describes the results of a series of experiments on the effect of nitric oxide donors and inhibitors as well as dNOS1 and dNOS4 transgenes on the apoptosis on drosophila Lobe ^RSV mutant strain and wild-type strain Oregon R. It has been shown that a high nitric oxide content in cells is able to inhibit antiapoptotic effect of HSP70 and stimulate apoptosis, possibly, via the grim-mediated apoptotic pathway. Moreover, long-term action of a high nitric oxide concentration during the entire development more efficiently stimulates the proapoptotic genes as compared with short-term action of this agent.     

Role of peroxynitrite in macrophage microbicidal mechanisms in vivo revealed by protein nitration and hydroxylation

The cytotoxins produced by phagocytic cells lacking peroxidases such as macrophages remain elusive. To elucidate macrophage microbicidal mechanisms in vivo, we compared the lesion tissue responses of resistant (C57Bl/6) and susceptible (BALB/c) mice to Leishmania amazonensis infection. This comparison demonstrated that parasite control relied on lesion macrophage activation with inducible nitric oxide synthase expression (iNOS), nitric oxide synthesis, and extensive nitration of parasites inside macrophage phagolysosomes at an early infection stage. Nitration and iNOS expression were monitored by confocal microscopy; nitric oxide synthesis was monitored by EPR. The main macrophage nitrating agent was shown to be peroxynitrite derived because parasite nitration occurred in the virtual absence of polymorphonuclear cells (monitored as peroxidase activity) and was accompanied by protein hydroxylation (monitored as 3-hydroxytyrosine levels). In vitro studies confirmed that peroxynitrite is cytotoxic to parasites whereas nitric oxide is cytostatic. The results indicate that peroxynitrite is likely to be produced close to the parasites and most of it reacts with carbon dioxide to produce carbonate radical anion and nitrogen dioxide whose concerted action leads to parasite nitration. In parallel, some peroxynitrite decomposition to the hydroxyl radical should occur due to the detection of hydroxylated proteins in the healing tissues. Consequently, peroxynitrite and derived radicals are likely to be important macrophage-derived cytotoxins.     

Hydrogen sulfide induces nitric oxide release from nitrite

Hydrogen sulfide has recently been considered to have an important role as a gasotransmitter in the cardiovascular system as well as in the central nervous system, but its action seems directly related to the presence of nitric oxide/nitric oxide-derivatives. We report here chemical evidence that emphasizes a prominent role of the hydrogen sulfide as cofactor of NO-derivatives in inducing nitric oxide release.     

Ethanol and Excitotoxicity in Cultured Cortical Neurons: Differential Sensitivity of N-Methyl-D-Aspartate and Sodium Nitroprusside Toxicity

Neural injury due to ischemia and related insults is thought to involve the action of excitatory amino acids at N-methyl-D-aspartate receptors, which results in the influx of extracellular Ca2+ and the generation of nitric oxide. Because ethanol inhibits physiologic responses to excitatory amino acids, we examined its effect on toxicity induced by N-methyl-D-aspartate and by the nitric oxide donor sodium nitroprusside in neuron-enriched cultures prepared from rat cerebral cortex. Both N-methyl-D-aspartate and sodium nitroprusside were cytotoxic, as measured by the release of lactate dehydrogenase and by microfluorescent determination of cell viability. Ethanol (3-1,000 mM) protected cultures from N-methyl-D-aspartate but not sodium nitroprusside toxicity, and the ability of a series of n-alkanols to reproduce the effect of ethanol was related to carbon-chain length. Neuroprotection by ethanol was accompanied by a decrease in the N-methyl-D-aspartate-evoked elevation of free intracellular Ca2+ and did not appear to involve gamma-aminobutyric acid- or cyclic GMP-mediated mechanisms. These findings suggest that ethanol inhibits excitotoxicity at an early step in the N-methyl-D-aspartate signaling pathway, probably by reducing Ca2+ influx, and not by interfering with the action of nitric oxide.     

Requirements for nitric oxide generation from isoniazid activation in vitro and inhibition of mycobacterial respiration in vivo

Isoniazid (INH), a front-line antituberculosis agent, is activated by mycobacterial catalase-peroxidase KatG, converting INH into bactericidal reactive species. Here we investigated the requirements and the pathway of nitric oxide (NO*) generation during oxidative activation of INH by Mycobacterium tuberculosis KatG in vitro. We also provide in vivo evidence that INH-derived NO* can inhibit key mycobacterial respiratory enzymes, which may contribute to the overall antimycobacterial action of INH.     

A Calcium/Calmodulin-Dependent Nitric Oxide Synthase, NMDAR2/3 Receptor Subunits, and Glutamate in the CNS of the Cuttlefish Sepia officinalis

Chemical, biochemical, and immunohistochemical evidence is reported demonstrating the presence in the brain of the cuttlefish Sepia officinalis of a Ca2+-dependent nitric oxide synthase, NMDAR2/3 receptor subunits, and glutamate, occurring in neurons and fibers functionally related to the inking system. Nitric oxide synthase activity was concentrated for the most part in the cytosolic fraction and was masked by other citrulline-forming enzyme(s). The labile nitric oxide synthase could be partially purified by ammonium sulfate precipitation of tissue extracts, followed by affinity chromatography on 2',5'-ADP-agarose and calmodulin-agarose. The resulting activity, immunolabeled at 150 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by antibodies to rat neuronal nitric oxide synthase, depended on NADPH and tetrahydro-L-biopterin, and was inhibited by N(G)-nitro-L-arginine. NMDAR2/3 subunit-immunoreactive proteins migrating at 170 kDa could also be detected in brain extracts, along with glutamate (whole brain: 0.32 +/- 0.03 micromol of glutamate/mg of protein; optic lobes: 0.22 +/- 0.04; vertical complex: 0.65 +/- 0.06; basal lobes: 0.58 +/- 0.04; brachial lobe: 0.77 +/- 0.06; pedal lobe: 1.04 +/- 0.08; palliovisceral lobe: 0.86 +/- 0.05). Incubation of intact brains with 1.5 mM glutamate or NMDA or the nitric oxide donor 2-(N,N-diethylamino)diazenolate-2-oxide caused a fivefold rise in the levels of cyclic GMP, indicating operation of the glutamate-nitric oxide-cyclic GMP signaling pathway. Immunohistochemical mapping of Sepia CNS showed specific localization of nitric oxide synthase-like and NMDAR2/3-like immunoreactivities in the lateroventral palliovisceral lobe, the visceral lobe, and the pallial and visceral nerves, as well as in the sphincters and wall of the ink sac.     

Bistability Analysis of an Apoptosis Model in the Presence of Nitric Oxide

Bistability in apoptosis, or programmed cell death, is crucial for the healthy functioning of multicellular organisms. The aim in this study is to show the presence of bistability in a mitochondria-dependent apoptosis model under nitric oxide effects using chemical reaction network theory. The model equations are a set of coupled ordinary differential equations arising from the assumed mass action kinetics. Whether these equations have a capacity for bistability (cell survival and apoptosis) is determined using a modular approach in which the model is decomposed into modules. Each module contains only a subset of the whole model and is analyzed separately. It is seen that bistability in a module is preserved throughout the whole model after adding the remaining reactions in the pathway on these modules. It is also found that inhibitor effect of some proteins and the appearance of a reacting protein in a later stage as a product is a desired feature but not sufficient for bistability (in the absence of cooperativity effects). On the whole model, two apoptotic and two cell survival states are obtained depending on the initial cell conditions. The results suggest that the antiapoptotic effects of nitric oxide species are responsible for the bistable character of the apoptotic pathway when cooperativity is not assumed in the apoptosome formation.     

Methylene blue blocks cGMP production and disrupts directed migration of microglia to nerve lesions in the leech CNS

Migration and accumulation of microglial cells at sites of injury are important for nerve repair. Recent studies on the leech central nervous system (CNS), in which synapse regeneration is successful, have shown that nitric oxide (NO) generated immediately after injury by endothelial nitric oxide synthase (eNOS) stops migrating microglia at the lesion. The present study obtained results indicating that NO may act earlier, on microglia migration, and aimed to determine mechanisms underlying NO's effects. Injury induced cGMP immunoreactivity at the lesion in a pattern similar to that of eNOS activity, immunoreactivity, and microglial cell accumulation, which were all focused there. The soluble guanylate cyclase (sGC) inhibitor methylene blue (MB) at 60 microM abolished cGMP immunoreactivity at lesions and blocked microglial cell migration and accumulation without interfering with axon conduction. Time-lapse video microscopy of microglia in living nerve cords showed MB did not reduce cell movement but reduced directed movement, with significantly more cells moving away from the lesion or reversing direction and fewer cells moving toward the lesion. The results indicate a new role for NO, directing the microglial cell migration as well as stopping it, and show that NO's action may be mediated by cGMP.     

Macrophage arginine metabolism and the inhibition or stimulation of cancer.

The potential of the immune system to inhibit or stimulate tumor growth is a vivid example of the "two-edged sword" nature of immune responses. Our results provide evidence that this dual capacity can be attributed, in part, to the dual pathways of arginine metabolism exhibited by intratumor macrophages. Specifically, i.p. tumor rejection in P815-preimmunized mice is accompanied by an upshift in intratumor macrophage arginine metabolism to the nitric oxide (NO) synthase pathway that yields citrulline and NO. A rapid and marked local increase in IFN-gamma (both mRNA and protein) in preimmunized mice during tumor rejection suggests that this cytokine plays a role in up-regulating nitric oxide production in vivo. Unlike tumor rejection, progressive i.p. P815 tumor growth in naive mice is associated with a marked decline in the production of citruline/NO by intratumor macrophages. Examination of macrophage arginine metabolism via arginase revealed a pattern opposite that of NO synthase. The local production of ornithine/urea markedly increases during progressive tumor growth whereas arginase activity decreases during tumor rejection. Inasmuch as nitric oxide inhibits tumor cell replication whereas ornithine is the precursor of polyamines required for cell replication, these results are consistent with the conclusion that the pathway macrophages use to metabolize arginine can influence the type of host immune responses against cancer and other conditions.