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Nitric oxide (NO) is considered an important signaling molecule implied in various different physiological processes, including nervous transmission, vascular regulation, and immune defence, as well as the pathogenesis of several diseases. NO reportedly also has an antiviral effect on several DNA and RNA virus families. The NO-mediated S-nitrosylation of viral and host (macro)molecules appears to be an intriguing general mechanism for the control of the virus life cycle. In this respect, NO is able to nitrosylate cysteine-containing enzymes (e.g., proteases, reverse transcriptase, and ribonucleotide reductase). Moreover, zinc-fingers and related domains present in enzymes (e.g., HIV-1-encoded integrase or herpes simplex virus type-1 heterotrimeric helicase-primase complex) or nucleocapsid proteins may be considered as NO targets. Also, NO may regulate both host (e.g., nuclear factor-kappaB) and viral-encoded (e.g., HIV-1 tat protein or Epstein-Barr virus Zta) transcriptional factors that are involved in virus replication. Finally, NO-mediated S-nitrosylation of cysteine-containing glycoproteins and hemagglutinin may also occur. Here, NO targets are summarised, and the molecular bases for the antiviral effect of NO are discussed.

Amyloid-β (Aβ) deposition, a hallmark of Alzheimer’s disease, is known to induce free radical production and oxidative stress, leading to neuronal damage. During oxidative stress, several cell types (including astrocytes) can activate the nuclear factor erythroid 2-related factor 2 (Nrf2), a regulator of several phase II detoxifying and antioxidant genes, such as the System Xc− subunit xCT. Here, we studied (i) the effect of the Aβ fragment 25-35 (Aβ25-35) on Nrf2-dependent System Xc− expression in U373 human astroglial cells and (ii) the effect of Aβ25-35-induced astrocytic response on neuronal cell viability using an in vitro co-culture system. We found that Aβ25-35 was able to activate an antioxidant response in astrocytes, by inducing both Nrf2 activation and System Xc− up-regulation. However, this astrocytic response caused an enhanced cell mortality of co-cultured SH-SY5Y cells, taken as a neuronal model. Consistently, the specific System Xc− inhibitor sulfasalazine prevented the increase of both neuronal mortality and extracellular glutamate levels, thus indicating that the neurotoxic effect was due to an augmented release of glutamate through the transporter. The involvement of NMDA receptor activation in this pathway was also demonstrated using the specific inhibitor MK801 that completely restored neuronal viability at the control levels. The present study sheds light on the Nrf2/system Xc− pathway in the toxicity induced by Aβ25-35 and may help to better understand the involvement of astrocytes in neuronal death during Alzheimer’s disease.

Nitric oxide (NO) may modulate the catalytic activity of cysteine proteases. In the present study, the inhibitory effect of NO, released by the NO-donors (+/-)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide and nitroprusside, on papain action is reported. Papain inactivation via NO-mediated nitrosylation of the Cys25 catalytic residue represents a molecular model for cysteine protease inhibition.

HIV-associated neurocognitive disorder (HAND) is a common cognitive impairment in AIDS that affects 15 to 50% of adults infected with human immunodeficiency virus (HIV). Excessive amounts of nitric oxide (NO), as produced by inducible NO synthase (iNOS) upon exposure of activated microglia and astrocytes to cytokines and/or viral proteins (e.g., HIV tat and gp120), are assumed to contribute to neuronal abnormalities in HAND. Evidence exists supporting the notion that iNOS induction takes place after an early decline in physiological NO levels (i.e., NO released by constitutive NOS). Here, we demonstrate that HIV-1 gp120 is able to inhibit neuronal NOS through a cytosolic phospholipase A(2) (cPLA(2))-dependent arachidonic acid (AA) production, this response being critical for allowing activation of the transcriptional factor NF-kappa B and subsequent iNOS and interleukin-1 beta transcription in astroglial cells. In this context, AA seems to act as an upstream proinflammatory effector. In view of the pathogenic role of cPLA(2) in HAND, a deeper insight into the molecular and cellular mechanisms of its modulation may be helpful in finding new drugs to manage cognitive impairment in HIV-1 patients.

Production of nitric oxide (NO) in the cell is catalysed by NO synthase (NOS), which using L-arginine and oxygen as substrates, synthesises NO. Normally cells express neuronal and/or endothelial NOS (nNOS and eNOS, respectively) producing nanomolar NO. Under inflammatory conditions cells express inducible NOS (iNOS), which, once expressed, produces micromolar NO. Since micromolar NO could elicit damage to cells, iNOS expression is time-spatially finely regulated. Recent evidence points out the possible interaction between n/eNOS and iNOS. Under inflammatory conditions interferon-γ, tumour necrosis factor-α and interleukin 1 -β rapidly trigger inhibition of e/nNOS activity with successive drop in the amounts of NO. Since NO normally exerts suppressive action on NF- κ B activation, drop in NO may create a favourable conditions for NF- κ B activation and successive iNOS exptression. Evidence in the literature indicates that during ageing nNOS activity gradually drops with concomitant increase in iNOS expression in the brain. However, how nNOS activity may influence iNOS expression remains to be elucidated. This review points out the possible functional link between age-dependent decrease in nNOS activity and increase in iNOS expression in the brain. Evidence for the possible scenario that often underestimated continuous infections to the brain, one of the hallmarks of ageing, may trigger apparently spontaneous iNOS expression is presented. Future treatment of aged people based on this scenario is also described.

The action of copper on the nitric oxide (NO) pathway was investigated in rat C6 glioma cells expressing both inducible and constitutive NO synthase (NOS) isoforms. The inducible NOS-II-mediated NO synthesis (i.e., nitrite production induced by LPS plus IFNgamma) was found to be increased upon copper uptake by cells, this effect being attributable to NOS-II mRNA transcriptional over-expression. On the other hand, the constitutive neuronal isoform (NOS-I) was inhibited after copper uptake, as revealed by the decrease of basal intracellular cGMP levels in C6 cells. Consistently, in vitro experiments showed that copper selectively blocked the catalytic activity of NOS-I, but not of NOS-II. The observed modulation of NOS isoforms by copper in C6 cells is in line with the previous hypothesis that selective inhibition of NOS-I leads to enhanced NO production through transcriptional activation of NOS-II.

Astroglial cells represent a target for HIV infection in the central nervous system. In astrocytes, HIV infection is poorly productive, being characterized by a persistent state of viral latency. However, activation of the nuclear factor NF-κB and its binding to HIV long terminal repeat (LTR) can induce HIV replication. Moreover, nitric oxide (NO) can affect NF-κB activation in glial cells. Therefore, we hypothesize that NO may reduce HIV replication in human astroglial cells by inhibiting HIV-1 LTR transcriptional activity. In this respect, we show that NO donors reduce viral replication in HIV-1-infected human astrocytoma T67 cells, taken as an astroglial model. Furthermore, using transfected T67 cells, we demonstrate that NO donors inhibit HIV-1 LTR transcriptional activity. These results suggest that the use of NO-releasing drugs may represent a potential, novel approach in inhibiting HIV replication in the central nervous system.

Nitric oxide (NO) may modulate the catalytic activity of cysteine-containing enzymes. HIV-1 protease action is modulated by the redox equilibrium of Cys67 and Cys95 regulatory residues. In the present study, the inhibitory effect of NO, released by the NO-donor (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (NOR-3), on the aspartyl HIV-1 protease action is reported. HIV-1 protease inactivation via NO-mediated nitrosylation of Cys regulatory residue(s) may represent a possible mechanism for inhibition of HIV-1 replication.

Evidence for the presence of a type-III nitric oxide synthase-like protein (NOS-III-like protein) in astroglial cells is reported. The mRNA of a NOS-III-like protein is constitutively expressed in human astrocytoma T67 cells, taken as an astroglial model. The nucleotide sequence of the PCR product (422 bp) shares more than 99% identity with the cDNA (from 1588 to 2009) of the human endothelial nitric oxide synthase (NOS-III). The molecular mass of the astroglial NOS-III-like protein is about 140 kDa, as observed for human NOS-III. Moreover, the astroglial NOS-III-like protein is constitutively tyrosine-phosphorylated and associated with caveolin-1. The astroglial NOS-III-like protein is apparently inactive, as reported for phosphorylated human NOS-III associated with caveolin-1.

Considering the structural similarity between gabexate mesylate (FOY), a drug for serine proteinase-mediated diseases, and L-arginine, the effect of gabexate mesylate on the nitric oxide (NO) pathway has been investigated. Gabexate mesylate inhibits competitively constitutive and inducible NO synthase (cNOS and iNOS, respectively), with Ki values of 1.0 x 10(-4) M and 5.0 x 10(-3) M, respectively, at pH 7.4 and 37.0 degrees C. However, gabexate mesylate is not an NO precursor. Moreover, like other NOS inhibitors, gabexate mesylate increases iNOS mRNA expression in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Finally, gabexate mesylate inhibits dose-dependently nitrite production (i.e. NO release) in rat C6 glioma cells, as induced by E. coli lipopolysaccharide plus interferon-gamma. Thus, this drug should be administered under careful control, since enzyme inhibition may occur also in vivo.

The effect of the HIV coating glycoprotein gp 120 on the generation of NO by human cultured T67 astrocytoma cells was investigated. Preincubation of astrocytoma cells with gp 120 (10 pM, 100 and 500 nM) produced a significant, dose-dependent increase of nitrite levels in supernatant of pretreated cells which was higher when compared to untreated cells. This effect was prevented by coincubation of cells with monoclonal antibodies directed against gp 120, or by pretreatment of cells with the selective NO synthase inhibitor L-NAME (100 μM). The rise of nitrite following pretreatment of astrocytoma cells with gp 120 was accompanied by an increase in NO synthase activity which was mainly Ca ++ -independent. Also this effect was inhibited by antibodies against gp 120, showing the specificity of the activation of the L-Arg-NO pathway subsequent to incubation of astrocytoma cells with the HIV coating protein. In conclusion, the present results are consistent with an activation of the inducible, Ca ++ -independent isoform of NO synthase in cultured astrocytoma cells following coincubation with gp 120. This may contribute to explain some of the neuropathological changes accompanying HIV-related cognitive disorders.

The amyloid peptides Abeta1-42 and Abeta25-35 strongly inhibited the activity of constitutive neuronal and endothelial nitric oxide synthases (i.e., NOS-I and NOS-III, respectively) in cell-free assays. The molecular mechanism of NOS inhibition by Ab fragments was studied in detail with Abeta25-35. The inhibitory ability was mostly NADPH-dependent and specific for the soluble form of Abeta25-35. Optical, fluorescence, and NMR spectroscopy showed that the soluble, but not aggregated, Abeta25-35 interacted with NADPH, thus suggesting that a direct recruitment of NADPH may result in diminished availability of the redox cofactor for NOS functioning. To assess the physiological relevance of our findings, rat neuronal-like PC12 and glioma C6 cell lines were used as cellular models. After Abeta25-35 internalization into cells was verified, the activity of constitutive NOS was measured using the DAF-2DA detection system and found to be severely impaired upon Abeta25-35 uptake. Consistent with previous results on the molecular cross-talk between NOS isoforms, repression of constitutive NOS by Abeta25-35 resulted in enhanced expression of inducible NOS (NOS-II) mRNA in C6 cells. Our results represent the first evidence that amyloid fragments impair constitutive NOS activity in cell-free and cellular systems, providing a possible molecular mechanism for the onset and/or maintenance of Alzheimer's disease.