Is there a role for neuronal nitric oxide synthase (nNOS) in cytokine toxicity to pancreatic beta cells?

Nitric oxide (NO), produced by the action of the inducible NO synthase, plays a crucial role in cytokine toxicity to pancreatic beta cells during type 1 diabetes development. It was the aim of this study to analyze the role of the neuronal NOS (nNOS) in proinflammatory cytokine-mediated beta cell toxicity. Expression of different isoforms of nitric oxide synthase in insulin-secreting INS1E cells and rat islets was analyzed by quantitative real-time PCR and Western blotting. The expression of nNOS in insulin-secreting INS1E cells was similar to that found in rat brain, while two other isoforms, namely the endothelial eNOS and inducible iNOS were not expressed in untreated cells. IL-1β alone or in combination with TNF-α and/or IFNγ induced iNOS but not eNOS expression. In contrast, nNOS expression was strongly decreased by the mixture of the three proinflammatory cytokines (IL-1β, TNF-α and IFNγ) both on the gene and protein level in INS1E cells and rat islet cells. The effects of cytokines on glucose-induced insulin-secretion followed the pattern of nNOS expression reduction and, on the other hand, of the iNOS induction. The data indicate that a low level of nitric oxide originating from the constitutive expression of nNOS in pancreatic beta cells is not deleterious. In particular since proinflammatory cytokines reduce this expression. This nNOS suppression can compensate for NO generation by low concentrations of IL-1β through iNOS induction. Thus, this basal nNOS expression level in pancreatic beta cells represents a protective element against cytokine toxicity.     

Tungstate: is it really a specific nitrate reductase inhibitor in plant nitric oxide research?

Nitrate reductase (NR) is an enzymatic source of nitric oxide (NO) in plants, and it needs Mo for the Mo-cofactor to be activated. Because NR-deficient mutants are not always available in some species, a cheap and simple pharmacological application of tungstate, which substitutes for Mo in the Mo-cofactor as a competitive antagonist, is widely used as a NR inhibitor in plant NO research. However, evidence indicates that tungstate not only inactivates NR but also inhibits other molybdate-dependent enzymes in plants. In addition, a number of investigations have shown that tungstate also inhibits root growth, affects cortical microtubule formation, and induces programmed cell death (PCD) in plants, just like other heavy metals do. Therefore, tungstate has been shown to exert many other effects that are not connected with the inhibition of NR activity. The origin and mechanism of using tungstate as a NR inhibitor in plants is reviewed here and the progress regarding tungstate toxicity to plants and the possible problems involved in using tungstate as a NR inhibitor in plant NO research are analysed. In summary, the use of tungstate as a NR inhibitor in plant NO research must be treated with caution, keeping in mind that it is not completely specific. It is necessary to search for more NR-deficient mutants and new, specific NR inhibitors. A combination of pharmacological and biochemical analysis with a genetic approach will be necessary in order to investigate the roles of NO in plants.     

Is nitric oxide luteolytic or antiluteolytic?

Nitric oxide (NO) has been reported to be luteolytic based on treatment of cows in vivo with an inhibitor of nitric oxide synthase (NOS-produces NO), which delayed the decline in progesterone by two to three days [Jaroszewki J, Hansel, W. Intraluteal administration of a nitric oxide synthase blocker stimulates progesterone, oxytocin secretion and prolongs the life span of the bovine corpus luteum. Proc Soc Exptl Biol Med 2000;224:50-5; Skarzynski D, Jaroszewki J, Bah, M, et al. Administration of nitric oxide synthase inhibitor counteracts prostaglandin F(2alpha)-induced luteolysis in cattle. Biol Reprod 2003;68:1674-81]. The objective of this experiment was to determine the effect of a long acting NO donor or a NOS inhibitor infused chronically into the interstitial tissue of the ovarian vascular pedicle adjacent to the ovary with a corpus luteum on secretion of progesterone during the ovine estrous cycle. Ewes were treated either with Vehicle (N=5); Diethylenetriamine (DETA-control for DETA-NONOate; N=5); (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl) amino]diazen-1-ium-1,2-diolate (DETA-NONOate-long acting NO donor; N=6); or l-nitro-arginine methyl ester (l-NAME-NOS inhibitor; N=6) every 6 h from 24:00 h (0 h) on day 8 through 18:00 h on day 18 of the estrous cycle. Jugular venous blood was collected every 6h for analysis for progesterone and corpora lutea were collected at 18:00 h on day 18 and weighed. Weights of corpora lutea were heavier (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes, which did not differ amongst each other (P> or =0.05). Profiles of progesterone in jugular venous blood on days 8-18 differed (P< or =0.05) in DETA-NONOate-treated ewes when compared to Vehicle, DETA, or l-NAME-treated ewes did not differ (P> or =0.05) amongst each other. It is concluded that NO is not luteolytic during the ovine estrous cycle, but may instead be antiluteolytic and prevent luteolysis.     

Is Ginkgo biloba (Ginkgoaceae) really an oviparous plant?

Germination of Ginkgo biloba seeds with intact and removed sarcotesta was compared to test the role of the seed coat in germination biology. The presence of an intact sarcotesta significantly reduced total germination percentage when compared to seeds with the sarcotesta removed. Some seeds were also cold stratified. This treatment was not necessary for germination, but it did improve total germination percentage. The seeds were collected during the period of natural abscission. Contrary to the accepted literature, we found that Ginkgo seeds contain well-developed embryos at the time of dispersal. These data demonstrate that the seed coat contributes to winter dormancy of G. biloba, and that the phenology of this species is less primitive than popularly believed.     

Nitric oxide control of cardiac function: is neuronal nitric oxide synthase a key component?

Nitric oxide (NO) has been shown to regulate cardiac function, both in physiological conditions and in disease states. However, several aspects of NO signalling in the myocardium remain poorly understood. It is becoming increasingly apparent that the disparate functions ascribed to NO result from its generation by different isoforms of the NO synthase (NOS) enzyme, the varying subcellular localization and regulation of NOS isoforms and their effector proteins. Some apparently contrasting findings may have arisen from the use of non-isoform-specific inhibitors of NOS, and from the assumption that NO donors may be able to mimic the actions of endogenously produced NO. In recent years an at least partial explanation for some of the disagreements, although by no means all, may be found from studies that have focused on the role of the neuronal NOS (nNOS) isoform. These data have shown a key role for nNOS in the control of basal and adrenergically stimulated cardiac contractility and in the autonomic control of heart rate. Whether or not the role of nNOS carries implications for cardiovascular disease remains an intriguing possibility requiring future study.     

Another activation switch for endothelial nitric oxide synthase: why does it have to be so complicated?

Regulation of the endothelial isoform of nitric oxide synthase (eNOS) appears to be much more complex in comparison to that of other NOS isoforms. A recent paper has expanded the regulation of the enzyme to the realm of sphingolipid signaling, specifically implicating that sphingosine 1-phosphate, endothelial differentiation gene (Edg) receptors and Akt kinase induce a signal transduction pathway via phosphorylation of a serine residue in eNOS. Bradykinin, a nonapeptide formed by enzymatic cleavage of a plasma protein precursor, activates eNOS by an independent pathway that does not require serine phosphorylation, suggesting a complex interplay of signals in the control of endothelial formation of nitric oxide.     

Spin trapping nitric oxide from neuronal nitric oxide synthase: A look at several iron–dithiocarbamate complexes

The free radical, nitric oxide (√NO), is responsible for a myriad of physiological functions. The ability to verify and study √NO in vivo is required to provide insight into the events taking place upon its generation and in particular the flux of √NO at relevant cellular sites. With this in mind, several iron-chelates (Fe²⁺(L)₂) have been developed, which have provided a useful tool for the study and identification of √NO through spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. However, the effectiveness of √NO detection is dependent on the Fe²⁺(L)₂ complex. The development of more efficient and stable Fe²⁺(L)₂ chelates may help to better understand the role of √NO in vivo. In this paper, we present data comparing several proline derived iron–dithiocarbamate complexes with the more commonly used spin traps for √NO, Fe²⁺-di(N-methyl-D-glutamine-dithiocarbamate) (Fe²⁺(MGD)₂) and Fe²⁺-di(N-(dithiocarboxy)sarcosine) (Fe²⁺(DTCS)₂). We evaluate the apparent rate constant (k_app) for the reaction of √NO with these Fe²⁺(L)₂ complexes and the stability of the corresponding Fe²⁺(NO)(L)₂ in presence of NOS I.     

Control of nitric oxide synthase expression by transforming growth factor-β: Implications for homeostasis

Production of nitric oxide (NO) can be stimulated by inflammatory cytokines and bacterial lipopolysaccharide (LPS) in mammalian cells via an inducible nitric oxide synthase (iNOS). Conversely, the transforming growth factor-βs (TGF-βs) suppress NO production by reducing iNOS expression. Production of NO leads to disparate consequences, some beneficial and some damaging to the host, depending on the cell and context in which iNOS is induced. The TGF-βs counter these NO-mediated processes in macrophages, cardiac myocytes, smooth muscle cells, bone marrow cells, and retinal pigment epithelial cells. Autocrine or paracrine production of TGF-β may thus serve as a physiological counterbalance for iNOS expression, a mechanism which may be subverted by pathogens and tumors for their own survival. A greater understanding of the mechanisms and consequences of NO and TGF-β production may lead to effective therapeutic strategies in various diseases.     

Are nitric oxide donors a valuable tool to study the functional role of nitric oxide in plant metabolism?

In the present work, we tested known nitric oxide (NO) modulators generating the NO+ (sodium nitroprusside, SNP) and NO˙ forms (S-nitroso-N-acetyl-D-penicillamine, SNAP and nitrosoglutathione, GSNO). This allowed us to compare downstream NO-related physiological effects on proteins found in leaves of pelargonium (Pelargonium peltatum L.). Protein modification via NO donors generally affects plant metabolism in a distinct manner, manifested by a lower thiobarbituric acid reactive substance (TBARS) content and lipoxygenase (LOX) activity in response to SNAP and GSNO. This is in contrast to the response observed for SNP treatment. Most changes in enzyme activity (GR, glutathione reductase; GST, glutathione-S-transferase; GPX, glutathione peroxidase) are most spectacular and repeatable during the first 8 h of incubation, which is explained by the half-life of the applied donors. In particular, a close dependence was found between the time-course of NO emission from the applied donors and the temporary inhibition of antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX). The observed changes were accompanied by time-dependent alterations in protein accumulation as analysed by two-dimensional gel electrophoresis (2-DE) in pelargonium leaves treated with NO donors (SNP, SNAP and GSNO). Using proteomics, different proteins were found to be down- and up-regulated. However, no new protein spots characteristic of all three donors were found. These results indicate that the form of NO emitted from the donor structure plays a key role in switching on appropriate metabolic modifications. It has been noted that several NO-affected metabolomic changes induced by the used donors were not comparable, which confirms the need to maintain caution when interpreting results obtained using the pharmacological approach with different NO modulator compounds.     

Chemical regulation of nitric oxide: a role for intracellular myoglobin?

The detailed chemistry of nitric oxide (*NO) and regulation of this potent signal molecule through interactions with cellular components are complex and not clearly understood. In the vasculature, *NO plays a crucial role in vessel dilation by activating soluble guanylyl cyclase (sGC) in vascular smooth muscle cells (VSMC). *NO is responsible for maintaining coronary blood flow and normal cardiac function. However, *NO is a highly reactive molecule and this reactivity toward a range of alternate substrates may interfere with the activation of its preferred molecular target within VSMC. Interestingly, marked changes to *NO homeostasis are linked to disease progression. Thus, the physiological concentration of *NO is carefully regulated. Myoglobin is a haem-containing protein that is present in relatively high concentration in cardiac and skeletal muscle. Recently, the presence of myoglobin has been confirmed in human smooth muscle. The role of intracellular myoglobin is generally accepted as that of a passive di-oxygen storage protein. However, oxygenated myoglobin readily reacts with *NO to yield higher order N-oxides such as nitrate, while both the ferrous and ferric forms of the protein form a stable complex with *NO. Together, these two reactions effectively eliminate *NO on the physiological time-scale and strongly support the idea that myoglobin plays a role in maintaining *NO homeostasis in tissues that contain the protein. Interestingly, human myoglobin contains a sulfhydryl group and forms an S-nitroso-adduct similar to haemoglobin. In this article we discuss the potential for human myoglobin to actively participate in the regulation of *NO by three distinct mechanisms, namely oxidation, ligand binding, and through formation of biologically active S-nitroso-myoglobin.