Inhibition of hepatocyte lipogenesis by nitric oxide donor: could nitric oxide regulate lipid synthesis?

Tissue lipogenesis is variably controlled by substrate supply and hormones. The possibility that nitric oxide (NO) might regulate lipogenesis derives from the action of NO on coenzyme A (CoA) to produce metabolically inactive S-nitrosoCoA. The effect of the nitric oxide donor S-nitrosoglutathione (GSNO) on long chain fatty acid and cholesterol synthesis was measured in isolated cultured rat hepatocytes. [1-14C] Butyrate was used as substrate to measure 14C incorporation into lipids as butyrate is twice as effective as acetate in hepatic lipogenesis and is ketogenic via the Lynen cycle. NO very significantly (P < 0.01) impaired long chain fatty acid and cholesterol synthesis an observation dependent upon time of exposure (3 h pre-incubation or 6 h continuous exposure) and concentration of GSNO (500 microM to 2.0 mM). Decrease in hepatic lipogenesis was paralleled by decrease in ketogenesis. ATP levels remained unchanged following short-term exposure to GSNO. Exposure of hepatocytes to GSNO together with 2.0 mM glutathione significantly diminished the inhibition of lipogenesis induced by GSNO alone. Impairment of lipogenesis by GSNO appears not to be limited by energy supply and now adduced, but not proven, to be operative via the degree of inactivation of cytosolic CoA. NO control of lipogenesis could be clinically important where NO production is increased as in demyelinating diseases, chronic arthritis or colitis and in wasting diseases such as AIDS.     

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.     

Killing of Bacillus spores is mediated by nitric oxide and nitric oxide synthase during glycoconjugate–enhanced phagocytosis

Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity against various pathogens. Bacillus spores can produce serious diseases, which might be attenuated if macrophages were able to kill the spores on contact. Present research was carried out to study whether glycoconjugates stimulated NO and nitric oxide synthase (NOS2) production during phagocytosis killing of Bacillus spores. Murine macrophages exposed to glycoconjugate-treated spores induced NOS2 and NO production that was correlated with high viability of macrophages and killing rate of bacterial spores. Increased levels of inducible NOS2 and NO production by macrophages in presence of glycoconjugates suggested that the latter provide an activation signal directed to macrophages. Glycoconjugates were shown to exert a protective influence, sparing macrophages from spore-induced cell death. In presence of glycoconjugates, macrophages efficiently kill the organisms. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores. These results suggest that glycoconjugates promote killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level and NO and NOS2 production. Glycoconjugates suggest novel antimicrobial approaches to prevention and treatment of infection caused by bacterial spores.     

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.     

Synthesis,nitric oxide release,and α-glucosidase inhibition of nitric oxide donating apigenin and chrysin derivatives

α-Glucosidase (AG) play crucial roles in the digestion of carbohydrates. Inhibitors of α-glucosidase (AGIs) are promising candidates for the development of anti-diabetic drugs. Here, five series of apigenin and chrysin nitric oxide (NO)-donating derivatives were synthesised and evaluated for their AG inhibitory activity and NO releasing capacity in vitro. Except for 9a–c, twelve compounds showed remarkable inhibitory activity against α-glucosidase, with potency being better than that of acarbose and 1-deoxynojirimycin. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure activity relationship studies indicated that 5-OH, hydrophobic coupling chain, and carbonyl groups of the coupling chain could enhance the inhibitory activity. Apigenin and chrysin derivatives therefore represents a new class of promising compounds that can inhibit α-glucosidase activity and supply moderate NO for preventing the development of diabetic complications.     

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.     

Why do nitric oxide synthases use tetrahydrobiopterin?

We are combining stopped-flow, stop-quench, and rapid-freezing kinetic methods to help clarify the unique redox roles of tetrahydrobiopterin (H(4)B) in NO synthesis, which occurs via the consecutive oxidation of L-arginine (Arg) and N-hydroxy-L-arginine (NOHA). In the Arg reaction, H(4)B radical formation is coupled to reduction of a heme Fe(II)O(2) intermediate. The tempo of this electron transfer is important for coupling Fe(II)O(2) formation to Arg hydroxylation. Because H(4)B provides this electron faster than can the NOS reductase domain, H(4)B appears to be a kinetically preferred source of the second electron for oxygen activation during Arg hydroxylation. A conserved Trp (W457 in mouse inducible NOS) has been shown to influence product formation by controlling the kinetics of H(4)B electron transfer to the Fe(II)O(2) intermediate. This shows that the NOS protein tunes H(4)B redox function. In the NOHA reaction the role of H(4)B is more obscure. However, existing evidence suggests that H(4)B may perform consecutive electron donor and acceptor functions to reduce the Fe(II)O(2) intermediate and then ensure that NO is produced from NOHA.     

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

Effects of temperature and concentration on the structure of ethylene oxide–propylene oxide–ethylene oxide triblock copolymer (Pluronic P65) in aqueous solution: a molecular dynamics simulation study

The effects of temperature and solution concentration on the structure of triblock polymeric surfactant (ethylene oxide)₁₉(propylene oxide)₂₉(ethylene oxide)₁₉ (Pluronic P65) have been investigated by fully atomistic molecular dynamics simulations. The Flory–Huggins interaction parameter χ, hydrogen bonding and molecular mobility in the aqueous solution of P65 were investigated covering a composition range of 0.1–0.73 (water weight fraction) and a temperature range of 273–373 K. The Flory–Huggins parameters indicated that propylene oxide (PO) segments became hydrophobic with the increase in temperature, whereas ethylene oxide (EO) segments remained hydrophilic, which caused the increase in repulsion between EO and PO segments. The intermolecular hydrogen bonds in P65 solution including water–water hydrogen bonds and water–P65 hydrogen bonds increased with the increase in solution concentration and decreased with the increase in temperature. The critical micellar temperature of Pluronic P65 predicted by Flory–Huggins interaction parameter χ and hydrogen bonding was in good agreement with experimental data.     

Nitric oxide synthase II in rat skeletal muscles

Constitutive expression of nitric oxide synthase (NOS) II was found in rat hindlimb muscles by immunohistochemistry and western blotting during development from embryonic day 21 to the adult stage of 75 days. The immunohistochemical NOS II expression pattern was related to the physiological metabolic fibre types SO (slow-oxidative), FOG I, II (fast-oxidative glycolytic; I more glycolytic, II more oxidative) and FG (fast-glycolytic) and to the myosin-based fibre types I and IIA, IIB (IIX not separated) identified in serial sections by enzyme histochemistry and immunohistochemistry. In adult muscles only the small population of FOG II fibres, which is a part of both IIA and IIB fibre population, showed NOS II immunoreactivity. This is the reason that only weak NOS II expression in adult hindlimb muscles has been detected by western blotting. Hindlimb muscles of embryonic, neonatal and young rats of 8 days expressed more NOS II as compared with adult rat hindlimb muscles. This can be explained by the findings that before the age of 21 days fast fibres were metabolically undifferentiated, all of them were NOS II positive and contribute to the NOS II expression of the muscle. In muscles of diabetic rats the NOS II expression was elevated indicating an inhibition of glucose uptake into the muscle fibres of diabetic muscles. Our findings suggest that the NOS II may be designated both as constitutive and inducible.     

Nitric oxide: a trigger for classic preconditioning?

To determine whether nitric oxide (NO) is involved in classic preconditioning (PC), the effect of NO donors as well as inhibition of the L-arginine-NO-cGMP pathway were evaluated on 1) the functional recovery during reperfusion of ischemic rat hearts and 2) cyclic nucleotides during both the PC protocol and sustained ischemia. Tissue cyclic nucleotides were manipulated with NO donors [S-nitroso-N-penicillamine (SNAP), sodium nitroprusside (SNP), or L-arginine] and inhibitors of nitric oxide synthase (N(omega)-nitro-L-arginine methyl ester or N-nitro-L-arginine) or guanylyl cyclase (1H-[1,2,4]oxadiazolol-[4,3-a]quinoxaline-1-one). Pharmacological elevation in tissue cGMP levels by SNAP or SNP before sustained ischemia elicited functional improvement during reperfusion comparable to that by PC. Administration of inhibitors before and during the PC protocol partially attenuated functional recovery, whereas they had no effect when given after the ischemic PC protocol and before sustained ischemia only, indicating a role for NO as a trigger but not as a mediator. Ischemic PC, SNAP, or SNP caused a significant increase in cGMP and a reduction in cAMP levels after 25 min of sustained ischemia that may contribute to the protection obtained. The results obtained suggest a role for NO (and cGMP) as a trigger in classic PC.     

Nitric oxide: NO apoptosis or turning it ON?

Nitric oxide (NO) is known for its diverse activities throughout biology. Among signaling qualities, NO affects cellular decisions of life and death either by turning on apoptotic pathways or by shutting them off. Although copious reports support both notions, the dichotomy of NO actions remains unsolved. Proapoptotic pathways of NO are compatible with established signaling circuits appreciated for mitochondria-dependent roads of death, with some emphasis on the involvement of the tumor suppressor p53 as a target during cell death execution. Antiapoptotic actions of NO are numerous, ranging from an immediate interference with proapoptotic signaling cascades to long-lasting effects based on expression of cell protective proteins with some interest on the ability of NO-redox species to block caspases by S-nitrosylation/S-nitrosation. Summarizing emerging concepts to understand p53 accumulation on the one hand while proposing inhibition of procaspase processing on the other may help to define the pro- versus antiapoptotic roles of NO.     

Nitric oxide: a pro-inflammatory mediator in lung disease?

Inflammatory diseases of the respiratory tract are commonly associated with elevated production of nitric oxide (NO•) and increased indices of NO• -dependent oxidative stress. Although NO• is known to have anti-microbial, anti-inflammatory and anti-oxidant properties, various lines of evidence support the contribution of NO• to lung injury in several disease models. On the basis of biochemical evidence, it is often presumed that such NO• -dependent oxidations are due to the formation of the oxidant peroxynitrite, although alternative mechanisms involving the phagocyte-derived heme proteins myeloperoxidase and eosinophil peroxidase might be operative during conditions of inflammation. Because of the overwhelming literature on NO• generation and activities in the respiratory tract, it would be beyond the scope of this commentary to review this area comprehensively. Instead, it focuses on recent evidence and concepts of the presumed contribution of NO• to inflammatory diseases of the lung.