Interactions between nitric oxide and indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) is a heme-containing enzyme, which catalyzes the initial and rate-determining step of L-tryptophan (L-Trp) metabolism via the kynurenine pathway in nonhepatic tissues. Similar to inducible nitric oxide synthase (iNOS), IDO is induced by interferon-gamma and lipopolysaccharide in the inflammatory response. In vivo studies indicate that the nitric oxide (NO) produced by iNOS inhibits IDO activity by directly interacting with it and by promoting its degradation through the proteasome pathway. In this work, the molecular mechanisms underlying the interactions between NO and human recombinant IDO (hIDO) were systematically studied with optical absorption and resonance Raman spectroscopies. Resonance Raman data show that the heme prosthetic group in the NO-bound hIDO is situated in a unique protein environment and adopts an out-of-plane deformed geometry that is sensitive to L-Trp binding. Under mildly acidic conditions, the proximal heme iron-His bond is prone to rupture, resulting in a five-coordinate (5C) NO-bound species. The bond breakage reaction induces significant conformational changes in the protein matrix, which may account for the NO-induced inactivation of hIDO and its enhanced proteasome-linked degradation in vivo. Moreover, it was found that the NO-induced bond breakage reaction occurs more rapidly in the ferrous protein than in the ferric protein and is fully inhibited by L-Trp binding. The spectroscopic data presented here not only provide the first glimpse of the possible regulatory mechanism of hIDO by NO in the cell at the molecular level, but they also suggest that the NO-dependent regulation can be modulated by cellular factors, such as the NO abundance, pH, redox environment, and L-Trp availability.     

S-nitrosoglutathione incorporated in poly(ethylene glycol) matrix: potential use for topical nitric oxide delivery.

Incorporation of nitric oxide (NO) donors in non-toxic polymeric matrices can be a useful strategy for allowing topical NO delivery. We have incorporated the NO-donor S-nitrosoglutathione (GSNO) into a liquid poly(ethylene glycol) (PEG)/H2O matrix through the S-nitrosation of GSH by a NO/O2 gas mixture. Kinetic measurements of GSNO decomposition associated with NO release were performed at 25, 35, and 45 degrees C in the dark and under irradiation with UV/Vis light, lambda>480 nm and lambda=333 nm. NO release from the liquid matrix to the gas phase was confirmed by mass spectrometry. The PEG/H2O matrix stabilizes GSNO leading to expressive reductions in the initial rates of thermal and photochemical NO release, compared to aqueous GSNO solution. This matrix effect is assigned to diffusional constrains imposed on the escape of the NO and GS radicals formed in the solvent cage. This effect allows the storage of PEG-GSNO formulations for extended periods (more than 65 days at freezer) with negligible decomposition. PEG-GSNO formulation seems therefore to be applicable in topical NO delivery and GSNO displays potential as a percutaneous absorption enhancer. Moreover, the rate of NO release can be locally increased by irradiation with visible light.     

Interactions between angiotensin II and nitric oxide during exercise in normal and heart failure rats.

We hypothesized that nitric oxide (NO) opposes ANG II-induced increases in arterial pressure and reductions in renal, splanchnic, and skeletal muscle vascular conductance during dynamic exercise in normal and heart failure rats. Regional blood flow and vascular conductance were measured during treadmill running before (unblocked exercise) and after 1) ANG II AT(1)-receptor blockade (losartan, 20 mg/kg ia), 2) NO synthase (NOS) inhibition [N(G)-nitro-L-arginine methyl ester (L-NAME); 10 mg/kg ia], or 3) ANG II AT(1)-receptor blockade + NOS inhibition (combined blockade). Renal conductance during unblocked exercise (4.79 +/- 0.31 ml x 100 g(-1) x min(-1) x mmHg(-1)) was increased after ANG II AT(1)-receptor blockade (6.53 +/- 0.51 ml x 100 g(-1) x min(-1) x mmHg(-1)) and decreased by NOS inhibition (2.12 +/- 0.20 ml x 100 g(-1) x min(-1) x mmHg(-1)) and combined inhibition (3.96 +/- 0.57 ml x 100 g(-1) x min(-1) x mmHg(-1); all P < 0.05 vs. unblocked). In heart failure rats, renal conductance during unblocked exercise (5.50 +/- 0.66 ml x 100 g(-1) x min(-1) x mmHg(-1)) was increased by ANG II AT(1)-receptor blockade (8.48 +/- 0.83 ml x 100 g(-1) x min(-1) x mmHg(-1)) and decreased by NOS inhibition (2.68 +/- 0.22 ml x 100 g(-1) x min(-1) x mmHg(-1); both P < 0.05 vs. unblocked), but it was unaltered during combined inhibition (4.65 +/- 0.51 ml x 100 g(-1) x min(-1) x mmHg(-1)). Because our findings during combined blockade could be predicted from the independent actions of NO and ANG II, no interaction was apparent between these two substances in control or heart failure animals. In skeletal muscle, L-NAME-induced reductions in conductance, compared with unblocked exercise (P < 0.05), were abolished during combined inhibition in heart failure but not in control rats. These observations suggest that ANG II causes vasoconstriction in skeletal muscle that is masked by NO-evoked dilation in animals with heart failure. Because reductions in vascular conductance between unblocked exercise and combined inhibition were less than would be predicted from the independent actions of NO and ANG II, an interaction exists between these two substances in heart failure rats. L-NAME-induced increases in arterial pressure during treadmill running were attenuated (P < 0.05) similarly in both groups by combined inhibition. These findings indicate that NO opposes ANG II-induced increases in arterial pressure and in renal and skeletal muscle resistance during dynamic exercise.     

The redox pathway of S-nitrosoglutathione, glutathione and nitric oxide in cell to neuron communications

Recent results demonstrated that S-nitrosoglutathione (GSNO) and nitric oxide (*NO) protect brain dopamine neurons from hydroxyl radical (*OH)-induced oxidative stress in vivo because they are potent antioxidants. GSNO and *NO terminate oxidant stress in the brain by (i) inhibiting iron-stimulated hydroxyl radicals formation or the Fenton reaction, (ii) terminating lipid peroxidation, (iii) augmenting the antioxidative potency of glutathione (GSH), (iv) mediating neuroprotective action of brain-derived neurotrophin (BDNF), and (v) inhibiting cysteinyl proteases. In fact, GSNO--S-nitrosylated GSH--is approximately 100 times more potent than the classical antioxidant GSH. In addition, S-nitrosylation of cysteine residues by GSNO inactivates caspase-3 and HIV-1 protease, and prevents apoptosis and neurotoxicity. GSNO-induced antiplatelet aggregation is also mediated by S-nitrosylation of clotting factor XIII. Thus the elucidation of chemical reactions involved in this GSNO pathway (GSH GS* + *NO-->[GSNO]-->GSSG + *NO-->GSH) is necessary for understanding the biology of *NO, especially its beneficial antioxidative and neuroprotective effects in the CNS. GSNO is most likely generated in the endothelial and astroglial cells during oxidative stress because these cells contain mM GSH and nitric oxide synthase. Furthermore, the transfer of GSH and *NO to neurons via this GSNO pathway may facilitate cell to neuron communications, including not only the activation of guanylyl cyclase, but also the nitrosylation of iron complexes, iron containing enzymes, and cysteinyl proteases. GSNO annihilates free radicals and promotes neuroprotection via its c-GMP-independent nitrosylation actions. This putative pathway of GSNO/GSH/*NO may provide new molecular insights for the redox cycling of GSH and GSSG in the CNS.     

Interactions between substrates and the haem-bound nitric oxide of ferric and ferrous bacterial nitric oxide synthases

We report here the resonance Raman spectra of the FeIII-NO and FeII-NO complexes of the bacterial NOSs (nitric oxide synthases) from Staphylococcus aureus and Bacillus subtilis. The haem-NO complexes of these bacterial NOSs displayed Fe-N-O frequencies similar to those of the mammalian NOSs, in presence and absence of L-arginine, indicating that haem-bound NO and L-arginine had similar haem environments in bacterial and mammalian NOSs. The only notable difference between the two types of NOS was the lack of change in Fe-N-O frequencies of the FeIII-NO complexes upon (6R) 5,6,7,8-tetrahydro-L-biopterin binding to bacterial NOSs. We report, for the first time, the characterization of NO complexes with NOHA (N(omega)-hydroxy-L-arginine), the substrate used in the second half of the catalytic cycle of NOSs. In the FeIII-NO complexes, both L-arginine and NOHA induced the Fe-N-O bending mode at nearly the same frequency as a result of a steric interaction between the substrates and the haem-bound NO. However, in the FeII-NO complexes, the Fe-N-O bending mode was not observed and the nu(Fe-NO) mode displayed a 5 cm(-1) higher frequency in the complex with NOHA than in the complex with L-arginine as a result of direct interactions that probably involve hydrogen bonds. The different behaviour of the substrates in the FeII-NO complexes thus reveal that the interactions between haem-bound NO and the substrates are finely tuned by the geometry of the Fe-ligand structure and are relevant to the use of the FeII-NO complex as a model of the oxygenated complex of NOSs.     

Biochemical studies of the relationship between iodothyronine 5'-monodeiodinase and protein disulphide isomerase in rat liver.

The relationship between type I iodothyronine 5'-monodeiodinase (5'-MD) and protein disulphide isomerase (PDI) was investigated by using a synthetic 18-amino acid peptide (LAP475c), which corresponds to the sequence of amino acids at position 373-390 of PDI including its active site, and anti-LAP475c antibody. Western blot analysis revealed that our anti-LAP475c antibody was highly specific for 57K protein in solubilized rat liver microsomal protein (SRLMP) that corresponded to PDI. Anti-LAP475c IgG (1:100 dilution) precipitated 46% of 5'-MD. These data suggest that PDI may play a regulatory role in the 5'-monodeiodination reaction.     

Interactions between nitric oxide and plant hormones in aluminum tolerance

Nitric oxide (NO) is involved, together with plant hormones, in the adaptation to Al stress in plants. However, the mechanism by which NO and plant hormones interplay to improve Al tolerance are still unclear. We have recently shown that patterns of plant hormones alteration differ between rye and wheat under Al stress. NO may enhance Al tolerance by regulating hormonal equilibrium in plants, as a regulator of plant hormones signaling. In this paper, some unsolved issues are discussed based on recent studies and the complex network of NO and plant hormones in inducing Al tolerance of plants are proposed.     

Cotranslational glycosylation of proteins in systems depleted of protein disulphide isomerase.

The role of protein disulphide isomerase (PDI) and other resident proteins of the endoplasmic reticulum (ER) lumen in co- and post-translational modification of secretory proteins has been studied in experiments on translation in vitro. We have devised procedures for extracting the lumenal content proteins of dog pancreas microsomal vesicles by alkaline buffer, or detergent washing, and for reconstitution of the depleted membrane fraction. When microsomal membranes are depleted of content by washing at pH 9.1, they are able to co-translationally glycosylate human interferon-gamma (IFN-gamma) and yeast pro-alpha-factor and the products appear to be identical to those produced by control microsomes. However, when microsomal membranes are depleted of content by washing with saponin they are still able to co-translationally translocate and glycosylate human IFN-gamma, but the products were of higher apparent Mr than those generated by control microsomes. When saponin-washed microsomal membranes were reconstituted with homogeneous protein disulphide isomerase (PDI), the generated vesicles gave the same pattern of co-translationally glycosylated IFN-gamma as saponin-washed microsomal membranes lacking PDI. These results are discussed in relation to the roles of resident ER proteins in co-translational modification; they suggest that PDI is not an essential component of the machinery of co-translational N-glycosylation, but that detergent washing may inactivate or remove some ER glycosidases.     

A rapid and sensitive assay for protein disulphide isomerase activity

An assay procedure for the determination of protein disulphide isomerase activity is presented. The method is based on the reactivation of randomly cross-linked RNAase, the extent of RNAase reactivation being determined from the degradation of radioactively labelled RNA. The method is rapid and sensitive and allows one to test a large number of samples simultaneously.     

Interactions between epithelial nitric oxide signaling and phosphodiesterase activity in Drosophila

Signaling by nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) modulates fluid transport in Drosophila melanogaster. Expression of an inducible transgene encoding Drosophila NO synthase (dNOS) increases both NOS activity in Malpighian (renal) tubules and DNOS protein in both type I (principal) and type II (stellate) cells. However, cGMP content is increased only in principal cells. DNOS overexpression results in elevated basal rates of fluid transport in the presence of the phosphodiesterase (PDE) inhibitor, Zaprinast. Direct assay of tubule cGMP-hydrolyzing phosphodiesterase (cG-PDE) activity in wild-type and dNOS transgenic lines shows that cG-PDE activity is Zaprinast sensitive and is elevated upon dNOS induction. Zaprinast treatment increases cGMP content in tubules, particularly at the apical regions of principal cells, suggesting localization of Zaprinast-sensitive cG-PDE to these areas. Potential cross talk between activated NO/cGMP and calcium signaling was assessed in vivo with a targeted aequorin transgene. Activated DNOS signaling alone does not modify either neuropeptide (CAP_2b)- or cGMP-induced increases in cytosolic calcium levels. However, in the presence of Zaprinast, both CAP_2b-and cGMP-stimulated calcium levels are potentiated upon DNOS overexpression. Use of the calcium channel blocker, verapamil, abolishes the Zaprinast-induced transport phenotype in dNOS-overexpressing tubules. Molecular genetic intervention in the NO/cGMP signaling pathway has uncovered a pivotal role for cell-specific cG-PDE in regulating the poise of the fluid transporting Malpighian tubule via direct effects on intracellular cGMP concentration and localization and via interactions with calcium signaling mechanisms. Malpighian tubule; cGMP; calcium; aequorin; CNG channel     

A direct nitric oxide gas delivery system for bacterial and mammalian cell cultures.

Nitric oxide (NO) is the smallest known gaseous signaling molecule released by mammalian and plant cells. To investigate the pathophysiologic role of exogenous NO gas (gNO) in bacterial and mammalian cell cultures, a validated in vitro delivery method is required. The system should be able to deliver gNO directly to bacterial and/or cell cultures in a continuous, predictable, and reproducible manner over a long period of time (days). To accomplish this, a gas delivery system was designed to provide optimal growth conditions for bacteria and/or mammalian cells. Parameters for cell exposure, such as concentration of gNO, nitrogen dioxide (NO(2)), oxygen (O(2)), temperature, and relative humidity (RH) were continuously monitored and evaluated. Uptake of gNO into various media was monitored by measuring the nitrite concentration using the Griess reagent technique. A selection of standard growth media [saline, tryptic soy broth (TSB), Middlebrook 7H9 (MB 7H9), and Dulbecco's modified Eagle's medium (DMEM)] exposed to various concentrations of gNO revealed a steady and consistent transfer of gNO into the aqueous phase over a 48-h period. Validation of optimal growth conditions within the device, as compared to a conventional incubator, were accomplished by growing and observing viability of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and human fibroblast cultures in the absence of gNO. These results indicate that an optimal growth environment for the above tested cells was accomplished inside the proposed delivery system. Dose-dependent toxicological data revealed a significant bacteriostatic effect on P. aeruginosa and S. aureus with continuous exposure to 80 ppm gNO. No toxic effects were observed on dermal fibroblast proliferation at concentrations up to 400 ppm gNO for 48 h. In conclusion, the designed gNO exposure system is capable of supporting cellular viability for a representative range of prokaryote and eukaryotic cells. The exposure system is also capable of obtaining toxicological data. Therefore, the proposed device can be utilized to continuously expose cells to various levels of gNO for up to 72 h to study the in vitro effects of gNO therapy.     

Design,synthesis and evaluation of protein disulfide isomerase inhibitors with nitric oxide releasing activity

Protein disulfide isomerase (PDI), a chaperone protein mostly in endoplasmic reticulum, catalyzes disulfide bond breakage, formation, and rearrangement to promote protein folding. PDI is regarded as a new target for treatment of several disorders. Here, based on the combination principle, we report a new PDI reversible modulator 16F16A-NO by replacing the reactive group in a known PDI inhibitor 16F16 with nitric oxide (NO) donor. Using molecular docking experiment, 16F16A-NO could embed into the active cavity of PDI. From newly developed fluorescent assay, 16F16A-NO showed rapid NO release. Furthermore, it is capable to moderately inhibit activity of PDI and S-nitrosylate the protein, indicating by insulin aggregation assay and biotin-switch technique. Finally, it displayed a dose-dependent antiproliferative activity against SH-SY5Y and HeLa tumor cells. Our designed hybrid compound 16F16A-NO showed a reasonable activity and might offer a promising avenue to develop novel PDI inhibitors for disease treatments.     

The human protein disulphide isomerase family: substrate interactions and functional properties

The process of disulphide bond formation in the endoplasmic reticulum of eukaryotic cells was one of the first mechanisms of catalysed protein folding to be discovered. Protein disulphide isomerase (PDI) is now known to catalyse all of the reactions that are involved in native disulphide bond formation, but despite more than 40 years of study, its mechanism of action is still not fully understood. This review discusses recent advances in our understanding of the human PDI family of enzymes and focuses on their functional properties, substrate interactions and some recently identified family members.     

Interactions between hydrogen sulphide and nitric oxide regulate two soybean citrate transporters during the alleviation of aluminium toxicity

Hydrogen sulphide (H₂S) is emerging as an important signalling molecule involved in plant resistance to various stresses. However, the underlying mechanism of H₂S in aluminium (Al) resistance and the crosstalk between H₂S and nitric oxide (NO) in Al stress signalling remain elusive. Citrate secretion is a wide‐spread strategy for plants against Al toxicity. Here, two citrate transporter genes, GmMATE13 and GmMATE47, were identified and characterized in soybean. Functional analysis in Xenopus oocytes and transgenic Arabidopsis showed that GmMATE13 and GmMATE47 mediated citrate exudation and enhanced Al resistance. Al treatment triggered H₂S generation and citrate exudation in soybean roots. Pretreatment with an H₂S donor significantly elevated Al‐induced citrate exudation, reduced Al accumulation in root tips, and alleviated Al‐induced inhibition of root elongation, whereas application of an H₂S scavenger elicited the opposite effect. Furthermore, H₂S and NO mediated Al‐induced GmMATE expression and plasma membrane (PM) H⁺‐ATPase activity and expression. Further investigation showed that NO induced H₂S production by regulating the key enzymes involved in biosynthesis and degradation of H₂S. These findings indicate that H₂S acts downstream of NO in mediating Al‐induced citrate secretion through the upregulation of PM H⁺‐ATPase‐coupled citrate transporter cotransport systems, thereby conferring plant resistance to Al toxicity.     

Mitochondrial P5, a member of protein disulphide isomerase family, suppresses oxidative stress-induced cell death

P5, one of the protein disulphide isomerase (PDI) family members, catalyses disulphide bond formation in proteins and exhibits molecular chaperone and calcium binding activities in vitro, whereas its physiological significance remains controversial. Recently, we have reported that P5 localizes not only in the ER but also in mitochondria, although it remains unclear so far about its physiological significance(s) of its dual localization. Here we report that H(2)O(2)- or rotenone-induced cell death is suppressed in MTS-P5 cells, which stably express P5 in mitochondria. H(2)O(2)-induced cell death in Saos-2 cells occurred, in large part, through caspase-independent and poly(ADP-ribose) polymerase (PARP)-dependent manner. In MTS-P5 cells challenged with H(2)O(2) treatment, PARP was still activated, whereas release of cytochrome c or apoptosis-inducing factor and intramitochondrial superoxide generation were suppressed. We also found that mitochondrial P5 was in close contact with citrate synthase and maintained large parts of its activity under H(2)O(2) exposure. These results suggest that mitochondrial P5 may upregulate tricarboxylic acid cycle and possibly, other intramitochondrial metabolism.     

一氧化氮的传递及其对缺氧时呼吸变化的调控

一氧化氮(NO)在体内的传递并非以往认为的随机弥散,而是与血红蛋白(Hb)β亚单位中半胱氨酸的硫醇基(巯基)或其他含巯基化合物相结合,形成亚硝基硫醇(SNOs)。SNOs既具备NO的生物活性又保持其稳定性。SNOs从细胞外进入到胞质的途径是通过酶促反应,而从红细胞输出则经阴离子交换蛋白转运。在缺氧情况下,SNOs对呼吸的调控包括：Hb在肺部的氧合作用,将氧输送到组织和在脑干孤束核调控中枢性呼吸。     

Alteration in cell surface LETS protein during myogenesis.

Cell surface alterations during myogenesis have been investigated in Yaffe's myogenic cell line L8, using indirect immunofluorescence with an antibody against the large external transformation-sensitive (LETS) protein. The immunofluorescent technique reveals a susbstantial alteration in the distribution of this surface antigen. With the prefused myoblasts, LETS protein is dispersed all over the cell surface; following myoblast fusion, this pattern is markedly changed. All of the fibril-like surface LETS protein disappears, and in some myotubes, discrete clusters of LETS protein become conspicuous. By use of radioimmunological assay, the total LETS protein is quantitatively reduced upon myoblast fusion.