Bacterial Lipopolysaccharide Plus Interferon-γ Elicit a Very Fast Inhibition of a Ca2+-dependent Nitric-oxide Synthase Activity in Human Astrocytoma Cells

Abstract Previous results indicate that induction of inducible nitric-oxide synthase (iNOS) expression may be kept suppressed by the endogenous NO level as produced by a constitutive NOS (cNOS) enzyme. In cell types possessing both cNOS and iNOS, this may represent an evident paradox. Here, we report that lipopolysaccharide and interferon-γ, which are able to strongly induce iNOS in astrocytoma cells, can rapidly inhibit the NO production generated by the constitutive NOS isoform, thus obtaining the best conditions for iNOS induction and resolving the apparent paradox. In fact, a 30-min treatment of T67 cells with the combination of lipopolysaccharide plus interferon-γ (MIX) strongly inhibits the cNOS activity, as determined by measuring [3H]citrulline production. In addition, the effect of MIX is also observed by measuring nitrite, the stable breakdown product of NO: a 30-min pretreatment of T67 cells with MIX is able to reduce significantly the N-methyl-D-aspartate-induced nitrite production. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a 30-min treatment of T67 cells with MIX does not affect expression of mRNA coding for the neuronal NOS-I isoform. These results suggest the novel concept of a possible role of a cNOS isoform in astrocytes as a control function on iNOS induction

Rapid inactivation of NOS-I by lipopolysaccharide plus interferon-gamma-induced tyrosine phosphorylation.

Human astrocytoma T67 cells constitutively express a neuronal NO synthase (NOS-I) and, following administration of lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma), an inducible NOS isoform (NOS-II). Previous results indicated that a treatment of T67 cells with the combination of LPS plus IFNgamma, by affecting NOS-I activity, also inhibited NO production in a very short time. Here, we report that under basal conditions, a NOS-I protein of about 150 kDa was weakly and partially tyrosine-phosphorylated, as verified by immunoprecipitation and Western blotting. Furthermore, LPS plus IFNgamma increased the tyrosine phosphorylation of NOS-I, with a concomitant inhibition of its enzyme activity. The same effect was observed in the presence of vanadate, an inhibitor of phosphotyrosine-specific phosphatases. On the contrary, genistein, an inhibitor of protein-tyrosine kinases, reduced tyrosine phosphorylation of NOS-I, enhancing its enzyme activity. Finally, using reverse transcriptase-polymerase chain reaction, we have observed that a suboptimal induction of NOS-II mRNA expression in T67 cells was enhanced by vanadate (or L-NAME) and inhibited by genistein. Because exogenous NO has been found to suppress NOS-II expression, the decrease of NO production that we have obtained from the inactivation of NOS-I by LPS/IFNgamma-induced tyrosine phosphorylation provides the best conditions for NOS-II expression in human astrocytoma T67 cells

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 alpha (TNF alpha)-inducible NO synthesis, because by affecting NF-kappa B activation it inhibits inducible Ca(2+)-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/TNF alpha-inducible iNOS gene expression; this effect was reversed by reduced hemoglobin, a trapping agent for NO. The early presence of SNP during LPS/TNF alpha induction is essential for inhibition of iNOS mRNA expression. Furthermore, SNP is capable of inhibiting LPS/TNF alpha-inducible nitrite release, as determined by Griess reaction. Finally, using electrophoretic mobility shift assay, we have shown that SNP inhibits LPS/TNF alpha-elicited NF-kappa B activation. This suggests that inhibition of iNOS gene expression by exogenous NO may be ascribed to a decreased NF-kappa B availability

Dominant mutants of ceruloplasmin impair the copper loading machinery in aceruloplasminemia.

The multicopper oxidase ceruloplasmin plays a key role in iron homeostasis, and its ferroxidase activity is required to stabilize cell surface ferroportin, the only known mammalian iron exporter. Missense mutations causing the rare autosomal neurodegenerative disease aceruloplasminemia were investigated by testing their ability to prevent ferroportin degradation in rat glioma C6 cells silenced for endogenous ceruloplasmin. Most of the mutants did not complement (i.e. did not stabilize ferroportin) because of the irreversible loss of copper binding ability. Mutant R701W, which was found in a heterozygous very young patient with severe neurological problems, was unable to complement per se but did so in the presence of copper-glutathione or when the yeast copper ATPase Ccc2p was co-expressed, indicating that the protein was structurally able to bind copper but that metal loading involving the mammalian copper ATPase ATP7B was impaired. Notably, R701W exerted a dominant negative effect on wild type, and it induced the subcellular relocalization of ATP7B. Our results constitute the first evidence of "functional silencing" of ATP7B as a novel molecular defect in aceruloplasminemia. The possibility to reverse the deleterious effects of some aceruloplasminemia mutations may disclose new possible therapeutic strategies

Nitric oxide pathway in lower metazoans

The presence of nitric oxide (NO) pathway has been well demonstrated in the main invertebrate groups, showing parallel findings on the role of NO in vertebrates and invertebrates. Noteworthy is the example of the role played by the nitrergic pathway in the sensorial functions, mainly in olfactory-like systems. On the other hand, the emerging molecular information about NOSs from lower metazoans (Porifera, cnidarians up to higher invertebrates) suggests that NO pathways might represent examples of a parallel evolution of the NOS prototypes in different animal lineages. Nevertheless, increasing evidence suggests that NO is one of the earliest and most widespread signaling molecules in living organisms. Here, we attempt to provide a survey of current knowledge of the synthesis and possible roles of NO and the related signaling pathway in lower metazoans (i.e., Porifera and Cnidaria), two phyla forming a crucial bridge spanning the evolutionary gap between the protozoans and higher metazoans. From the literature data here reported, it emerges that future research on the biological roles of NO in basal metazoans is likely to be very important for understanding the evolution of signaling systems

Cross-talk between constitutive and inducible NO synthase: an update

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-κB. 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

Clonogenic, myogenic progenitors expressing MCAM/CD146 are incorporated as adventitial reticular cells in the microvascular compartment of human post-natal skeletal muscle

Recent observation identifies subendothelial (mural) cells expressing MCAM, a specific system of clonogenic, self-renewing, osteoprogenitors (a.k.a, "mesenchymal stem cells") in the microvascular compartment of post-natal human bone marrow (BM). In this study, we used MCAM/CD146, as a marker to localize, isolate and assay subendothelial clonogenic cells from the microvasculature of postnatal human skeletal muscle. We show here that these cells share with their BM counterpart, anatomic position (subendothelial/adventitial) and ex vivo clonogenicity (CFU-Fs). When assayed under the stringent conditions, these cells display a high spontaneous myogenic potential (independent of co-culture with myoblasts or of in vivo fusion with local myoblasts), which is otherwise only attained in cultures of satellite cells. These muscle-derived mural cells activated a myogenic program in culture. Cultured CD146+ cells expressed the myogenic factors (Pax7, Pax3 and Myf5), NCAM/CD56, desmin as well as proteins characteristic of more advanced myogenic differentiation, such as myosin heavy chain. In vivo, these cells spontaneously generate myotubes and myofibrils. These data identify the anatomy and phenotype of a novel class of committed myogenic progenitor in human post-natal skeletal muscle of subendothelial cells associated with the abluminal surface of microvascular compartment distinct from satellite cells

Cross-talk Between Constitutive and Inducible NO Synthase: an Update

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-kappa B. 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