Marco Colasanti

The dual personality of NO

In the body, nitric oxide (NO) is an important physiological regulator of functions such as vasodilatation and neurotransmission. Under pathological conditions, high concentrations of NO can be either beneficial(e.g. anti-bacterial, anti-parasitic and anti-viral) or detrimental; NO can therefore be considered a double-edged sword. When manipulating NO levels clinically, attention should be paid to minimize the negative effects and maximize the beneficial effects of NO. This article highlights recent evidence that supports the complexity of the regulatory mechanisms that lead to sophisticated endogenous NO production.

Induction of nitric oxide synthase mRNA expression. Suppression by exogenous nitric oxide.

The reactive nitrogen species, nitric oxide (NO), plays an important role in the pathogenesis of neurodegenerative diseases. The suppression of NO production may be fundamental for survival of neurons. Here, we report that pretreatment of human ramified microglial cells with nearly physiological levels of exogenous NO prevents lipopolysaccharide (LPS)/tumor necrosis factor α (TNFα)-inducible NO synthesis, because by affecting NF-κB activation it inhibits inducible Ca-independent NO synthase isoform (iNOS) mRNA expression. Using reverse transcriptase polymerase chain reaction, we have found that both NO donor sodium nitroprusside (SNP) and authentic NO solution are able to inhibit LPS/TNFα-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNFα induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNFα-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNFα-elicited NF-κB activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-κB availability.

Nitric oxide mediates anti‐inflammatory action of extracorporeal shock waves

Here, we show that extracorporeal shock waves (ESW), at a low energy density value, quickly increase neuronal nitric oxide synthase (nNOS) activity and basal nitric oxide (NO) production in the rat glioma cell line C6. In addition, the treatment of C6 cells with ESW reverts the decrease of nNOS activity and NO production induced by a mixture of lipopolysaccharides (LPS), interferon‐γ (IFN‐γ) plus tumour necrosis factor‐α (TNF‐α). Finally, ESW treatment efficiently downregulates NF‐κB activation and NF‐κB‐dependent gene expression, including inducible NOS and TNF‐α. The present report suggests a possible molecular mechanism of the anti‐inflammatory action of ESW treatment.

Nitric oxide: an inhibitor of NF-κB/Rel system in glial cells

Abstract Nitric oxide (NO) has been reported to regulate NF-κB, one of the best-characterized transcription factors playing important roles in many cellular responses to a large variety of stimuli. NO has been suggested to induce or inhibit the activation of NF-κB, its effect depending, among others, on the cell type considered. In this review, the inhibitory effect of NO on NF-κB (and subsequent suppression of NF-κB-dependent gene expression) in glial cells is reported. In particular, exogenous and endogenous NO has been observed to keep NF-κB suppressed, thus preventing the expression of NF-κB-induced genes, such as inducible NO synthase itself or HIV-1 long terminal repeat. Furthermore, the possible molecular mechanisms of NO-mediated NF-κB inhibition are discussed. More specifically, NO has been reported to suppress NF-κB activation inducing and stabilizing the NF-κB inhibitor, IκB-α. On the other hand, NO may inhibit NF-κB DNA binding through S-nitrosylation of cysteine residue (i.e., Cys62) of the p50 subunit. As a whole, a novel concept that the balance of intracellular NO levels may control the induction of NF-κB in glial cells has been hypothesized.

Mitochondrial type I nitric oxide synthase physically interacts with cytochrome c oxidase

Nitric oxide (NO) regulates key aspects of cell metabolism through reversible inhibition of cytochrome c oxidase (CcOX), the terminal electron acceptor (complex IV) of the mitochondrial respiratory chain, in competition with oxygen. Recently, a constitutive mitochondrial NOS corresponding to a neuronal NOS-I isoform (mtNOS-I) has been identified in several tissues. The role of this enzyme might be to generate NO close enough to its target without a significant overall increase in cellular NO concentrations. An effective, selective, and specific NO action might be guaranteed further by a physical interaction between mtNOS-I and CcOX. This possibility has never been investigated. Here we demonstrate that mtNOS-I is associated with CcOX, as proven by electron microscopic immunolocalization and co-immunoprecipitation studies. By affinity chromatography, we found that association is due to physical interaction of mtNOS-I with the C-terminal peptide of the Va subunit of CcOX, which displays a consensus sequence for binding to the PDZ domain of mtNOS-I previously unreported for CcOX. The molecular details of the interaction have been analyzed by means of molecular modeling and molecular dynamics simulations. This is the first evidence of a protein-protein interaction mediated by PDZ domains involving CcOX.

Re-Evaluation of Amino Acid Sequence and Structural Consensus Rules for Cysteine-Nitric Oxide Reactivity

Abstract Nitric oxide (NO), produced in different cell types through the conversion of Larginine into Lcitrulline by the enzyme NO synthase, has been proposed to exert its action in several physiological and pathological events. The great propensity for nitrosothiol formation and breakdown represents a mechanism which modulates the action of macromolecules containing NOreactive Cys residues at their active centre and/or allosteric sites. Based on the human haemoglobin (Hb) structure and accounting for the known acidbase catalysed Cys?93-nitrosylation and Cys?93NOdenitrosylation processes, the putative amino acid sequence (Lys/Arg/His/Asp/Glu)Cys(Asp/Glu) (sites 1, 0, and + 1, respectively) has been proposed as the minimum consensus motif for CysNO reactivity. Although not found in human Hb, the presence of a polar amino acid residue (Gly/Ser/Thr/Cys/Tyr/Asn/Gln) at the 2 position has been observed in some NOreactive protein sequences (e.g., NMDA receptors). However, the most important component of the tri or tetrapeptide consensus motif has been recognised as the Cys(Asp/Glu) pair [Stamler et al., Neuron (1997) 18, 691 696]. Here, we analyse the threedimensional structure of several proteins containing NOreactive Cys residues, and show that their nitrosylation and denitrosylation processes may depend on the CysS? atomic structural microenvironment rather than on the tri or tetrapeptide sequence consensus motif.

Human ramified microglial cells produce nitric oxide upon Escherichia coli lipopolysaccharide and tumor necrosis factor α stimulation

This study shows that human ramified microglial cells derived from fetal brain primary cultures, are able to produce nitric oxide (NO). In fact, stimulation with Escherichia coli lipopolysaccharide (LPS) (1 microgram ml-1) or tumor necrosis factor alpha (TNF alpha) (500 U ml-1) enhances nitrite release in cell supernatants, as determined by the Griess reaction. A synergistic effect is achieved following treatment with LPS plus TNF alpha, this effect being inhibited by pretreating cells with NOS inhibitor N omega-nitro-L-arginine methyl ester (L-NAME). Using reverse transcriptase-polymerase chain reaction (RT-PCR) and Southern blot analysis, we also found that LPS/TNF alpha produce an increase of inducible NO synthase (iNOS) mRNA expression.

Nitric oxide in invertebrates.

Nitric oxide (NO) is considered an important signaling molecule implied in different physiological processes, including nervous transmission, vascular regulation, immune defense, and in the pathogenesis of several diseases. The presence of NO is well demonstrated in all vertebrates. The recent data on the presence and roles of NO in the main invertebrate groups are reviewed here, showing the widespread diffusion of this signaling molecule throughout the animal kingdom, from higher invertebrates down to coelenterates and even to prokaryotic cells. In invertebrates, the main functional roles described for mammals have been demonstrated, whereas experimental evidence suggests the presence of new NOS isoforms different from those known for higher organisms. Noteworthy is the early appearance of NO throughout evolution and striking is the role played by the nitrergic pathway in the sensorial functions, from coelenterates up to mammals, mainly in olfactory-like systems. All literature data here reported suggest that future research on the biological roles of early signaling molecules in lower living forms could be important for the understanding of the nervous-system evolution.

Inhibition of Cysteine Protease Activity by NO-donors

Cysteine proteases represent a broad class of proteolytic enzymes widely distributed among living organisms. Although well known as typical lysosomal enzymes, cysteine proteases are actually recognized as multi-function enzymes, being involved in antigen processing and presentation, in membrane-bound protein cleavage, as well as in degradation of the cellular matrix and in processes of tissue remodeling. Very recently, it has been shown that the NO(-donor)-mediated chemical modification of the Cys catalytic residue of cysteine proteases, including Coxsackievirus and Rhinovirus cysteine proteases, cruzain, Leishmania infantum cysteine protease, falcipain, papain, as well as mammalian caspases, cathepsins and calpain, blocks the enzyme activity in vitro and in vivo. Here, inhibition of representative cysteine proteases by NO(-donors) is reviewed.

S-nitrosylation of viral proteins: Molecular bases for antiviral effect of nitric oxide

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 beconsidered 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.