Biomedical subjects
Publications and source records attributed to Tiziana Persichini.
We present here a brief description of the relationships among metals, nitric oxide metabolism, and ageing. In particular, we will discuss the interactions occurring between redox (copper, iron) and non-redox (zinc) metals and nitric oxide, the metal- and nitric oxide-catalyzed formation of thiol adducts (nitrosothiols, mixed disulphides) and the possible involvement of these species in the ageing process.
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-gamma (IFN-gamma) plus tumour necrosis factor-alpha (TNF-alpha). Finally, ESW treatment efficiently downregulates NF-kappaB activation and NF-kappaB-dependent gene expression, including inducible NOS and TNF-alpha. The present report suggests a possible molecular mechanism of the anti-inflammatory action of ESW treatment.
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.
A number of pathologies, including neurodegeneration and inflammation, have been associated with iron dysmetabolism in the brain. Hence, systems involved in iron homeostasis at the cellular level have aroused considerable interest in recent years. The iron exporter ferroportin-1 (FP) and the multicopper oxidase ceruloplasmin (CP) are essential for iron efflux from cells. By using RT-PCR, we demonstrate that FP and CP gene expression is up-regulated by treatment with the pro-inflammatory cytokine IL-1beta in rat C6 cells, taken as a glial cellular model. Following stimulation with IL-1beta, a higher expression level of CP and FP was also confirmed by Western blotting. Moreover, IL-1beta has been found to increase iron efflux from C6 cells, suggesting that both proteins may play a crucial role in iron homeostasis in pathological brain conditions, such as inflammatory and/or neurodegenerative diseases.
Previous results have indicated that lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) inhibits nitric-oxide synthase (NOS)-I activity in glial cells. We report here that arachidonic acid (AA) plays a pivotal role in this response, which was consistently reproduced in different glial cell lines and in primary rat astrocytes. This notion was established using pharmacological inhibitors of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2) antisense oligonucleotides, and AA add-back experiments. This approach not only allowed the demonstration that AA promotes inhibition of NOS-I activity but also produced novel experimental evidence that LPS/IFNgamma itself is a potential stimulus for NOS-I. Indeed, LPS/IFNgamma fails to generate nitric oxide (NO) via NOS-I activation simply because it activates the AA-dependent signal that impedes NOS-I activity. Otherwise, LPS/IFNgamma promotes NO formation, sensitive to exogenous AA, in cells in which cPLA2 is pharmacologically inhibited or genetically depleted. Because NO suppresses the NFkappaB-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFNgamma-induced AA pathway provides optimal conditions for NFkappaB activation and subsequent NOS-II expression. Inhibition of cPLA2 activity, while reducing the availability of AA, consistently inhibited NFkappaB activation and NOS-II mRNA induction and delayed NO formation. These responses were promptly reestablished by addition of exogenous AA. Finally, we have demonstrated that the LPS/IFNgamma-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.
Exposure of PC12 cells to A23187 or thapsigargin caused a concentration-dependent release of arachidonic acid (AA) mediated by cytosolic phospholipase A2 (PLA2). Under the same conditions, however, analysis of nitric oxide (NO) formation revealed that activation of NO synthase (NOS) is best described by a bell-shaped curve. Reduced detection of NO observed at increasing A23187 or thapsigargin concentrations was not due to formation of peroxynitrite or to activation of NO-consuming processes, but rather to AA-dependent inhibition of NOS activity. Furthermore, NO formation observed under optimal conditions for NOS activity was suppressed by AA as well as by the PLA2 activator melittin. Finally, the effects of AA were not the consequence of direct enzyme inhibition, because this lipid messenger failed to inhibit formation of NO by purified neuronal NOS, but were mediated by an AA-dependent signaling and not by downstream products of the cyclooxygenase and lipoxygenase pathways. In conclusion, the present study underscores a novel mechanism whereby endogenous, or exogenous, AA promotes inhibition of NOS activity. Because AA is generated in response to various agonists acting on membrane receptors and extensively released in inflammatory conditions, these findings have important physiopathological implications.
Intravenous administration of copper (up to a final concentration of ca. 35 micromol/l in the plasma) led to a progressive, dramatic fall of mean arterial pressure in rats. Copper-induced pressure changes were comparable to those elicited by 2 mg/kg LPS, and were greatly prevented by previous infusion of the inducible NOS (NOS-II) inhibitors aminoguanidine or l-N(6)-(L-imino-ethyl)lysine. RT-PCR analysis showed a significant transcriptional induction of NOS-II in a number of tissues, including aorta, liver, and lungs. Immunohistochemistry revealed that NOS-II was massively synthesized in these tissues upon copper or LPS treatment. The protein was active, as revealed by enzymatic assays on lung homogenates and by the large increase of nitrite/nitrate levels in the plasma. Copper-challenged rats displayed elevated plasma levels of TNFalpha. Extensive formation of nitrotyrosines, indicative of peroxynitrite production, was accompanied by marked morphological changes in examined tissues. Our results clearly show that copper can act as a proinflammatory agent through activation of the nitric oxide pathway, leading to the same pathological frame induced by bacterial lipopolysaccharide.
The activity of NCX 972, a new molecule obtained by adding a nitric oxide (NO) moiety to metronidazole, was tested against six isolates of Entamoeba histolytica in xenic cultures. NCX 972 released NO into trophozoite cells and enhanced killing of amoebas in a dose- and time-dependent manner compared to metronidazole.
In the present study, we have analyzed the expression of nitric oxide synthase (NOS) in the preoptic-hypothalamo-hypophyseal system of the teleost Oreochromis niloticus. The assay for enzyme activity demonstrated that a constitutive NOS activity is present both in soluble and particulate fractions of the homogenates of diencephalons. Western blot analysis using an antibody against the N-terminus of human nNOS revealed two bands both in the supernatant and in the pellet. One band co-migrates at approximately 150 kDa with that detected in the rat cerebellum homogenates and presumably corresponds to neuronal NOS (nNOS) of mammals. The additional band, which migrates at approximately 180 kDa, might be attributed to an alternatively spliced nNOS isoform. Using NADPH diaphorase (NADPHd) histochemistry in combination with NOS immunohistochemistry, nNOS expression has been detected in preoptic nuclei, hypophysiotrophic nuclei of the ventral hypothalamus, and the pituitary gland. Various degrees of dissociation of NADPHd activity and nNOS immunoreactivity have been detected that could be attributed to the expression of different subtypes of nNOS in the preoptic/hypothalamo/hypophysial system of tilapia. In this paper, we also investigated the colocalization of nNOS with arginine-vasotocin (AVT) by means of immunolabeling of consecutive sections. Results suggest that NO may be colocalized with AVT in a subpopulation of neurosecretory neurons. Present findings suggest that nitric oxide (NO) is implicated in the modulation of hormone release in teleosts in a similar way to mammals.
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.
Nitric oxide (NO) has emerged as an important cytotoxic and cytostatic effector for a number of pathogens, including viruses, bacteria, fungi, and parasites. When the microbicidal effect of NO occurs, the NO-mediated S-nitrosylation of cysteine containing proteins (e.g., cysteine proteases) appears to be a common and widespread mechanism. This overview concerns parasitic cysteine proteases as NO targets, providing molecular bases for the parasiticidal effect of NO.
Previous results indicated that intravenous injection of copper in the form of a copper-histidine complex in rats triggers the transcriptional induction of the inducible form of nitric oxide synthase (NOS-II). Here, the authors demonstrate that copper activates the transcription factor NF-kappaB in the liver and lung tissues of rats, and that this effect is mediated by oxidative stress, since all copper-induced changes, which include histological alterations, formation of nitrotyrosines, vascular pressure drop, production of tumor necrosis factor-alpha (TNF-alpha), induction of NOS-II and nitrites, are readily prevented by pretreatment of the animals with the antioxidant tempol. By using electrophoretic mobility shift assays, the p50/p65 dimer and higher molecular weight aggregates have been found to be involved in the copper-induced NF-kappaB activation. COX-2, a NF-kappaBdependent gene involved in the inflammatory response, was also transcriptionally induced by copper, this effect being reduced in the presence of tempol. These results suggest that a physiopathological status, characterized by hypercupremic situations, may lead to the onset of inflammation through production of ROS and activation of NF-kappaB.
Inducible nitric oxide synthase (iNOS) is expressed upon exposure of some cell types to bacterial lipopolysaccharides (LPS) and/or a variety of proinflammatory cytokines. The authors present an overview of some of the recent findings further supporting the notion that this response takes place after an early decline in constitutive nitric oxide (NO) levels (i.e., NO released by constitutive NOS, cNOS). This response is indeed critical for allowing activation of the transcription factor NF-kappaB. Thus, generation of NO by cNOS represents a limiting factor for iNOS expression. Some of the physiological and pathological implications of the cross-talk between these two NOS isoforms are discussed. In addition, the results of recent studies are summarized, suggesting possible mechanisms whereby LPS and/or proinflammatory cytokines may cause inhibition of cNOS.