Nitric oxide and its decomposed derivatives decrease the binding of extracellular-superoxide dismutase to the endothelial cell surface

Extracellular-superoxide dismutase (EC-SOD) is bound to the vascular endothelial cell surface with an affinity for heparan sulfate proteoglycan. The binding of EC-SOD to the human umbilical vein endothelial cell (HUVEC) and bovine aortic endothelial cell surface proteoglycans was significantly decreased by the incubation with S-nitroso-N-acetyl-DL-penicillamine (SNAP) and +/- -N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4), potent nitric oxide (NO) donors. NO derived from lipopolysaccharide-stimulated J774 A-1 cells also decreased the binding of EC-SOD to HUVEC, and this decrease was blocked by N(G)-nitro-L-arginine, a nitric oxide synthase inhibitor. SNAP and NOR4 also decreased the binding of EC-SOD to immobilized heparin. Furthermore, the decomposed derivatives of NO donors and sodium nitrite decreased the binding of EC-SOD. These observations suggest that excess NO produced in the inflammatory conditions decreases the binding of EC-SOD to the vascular endothelial cell surface, which results in a loss of the ability to protect the endothelial cell surface from oxidative stress.     

一氧化氮的释放对海马脑片CA1区痫样放电的影响

用自制的一氧化氮（ＮＯ）敏感电极－Ｎａｆｉｏｎ－壳聚糖合镍修饰铂电极（Ｎａｆｉｏｎ－ＣＴＳ（Ｎｉ）－Ｐｔ）连续测定了青霉素致痫海马脑片ＣＡ１区锥体层神经元ＮＯ的释放,并同时观察了ＮＯ合酶抑制剂７－ｎｉｔｒｏ－ｉｎｄａｚｏｌｅ（７－ＮＩ）及Ｎ＾ω－ｎｉｔｒｏ－Ｌ－ａｒｇｉｎｉｎｅ（Ｌ－ＮＮＡ）对诱发痫波及ＮＯ释放量的影响。研究观察到：（１）在青霉素致痫脑片模型上,诱发的痫波随青霉素浓度的增加而增多,     

Nitric oxide (NO) serves as a retrograde messenger to activate neuronal NO synthase in the spinal cord via NMDA receptors.

We have recently demonstrated that nitric oxide (NO) produced by neuronal NO synthase (nNOS) in the spinal cord is involved in the maintenance of neuropathic pain. To clarify whether NO itself affected nNOS activity in the spinal cord as a retrograde messenger, we examined the involvement of the NO/cGMP signaling pathway in the regulation of nNOS activity by NADPH-diaphorase histochemistry. NO-generating agents NOR3 (t(1/2)=30min) and SNAP (t(1/2)=5h), but not NOR1 (t(1/2)=1.8min), significantly enhanced NADPH-diaphorase staining in the spinal cord. 8-Br-cGMP also enhanced it similar to that by NOR3, and 8-Br-cAMP and forskolin, an activator of adenylate cyclase, enhanced it moderately. NOR1 and NOR3 markedly increased the cGMP level in the spinal cord. The enhancement of NADPH-diaphorase staining by NOR3 was significantly inhibited by CPTIO, an NO scavenger, ODQ, a soluble guanylate cyclase inhibitor, and KT5823, an inhibitor of cGMP-dependent protein kinase. Additionally, the NOR3-enhanced nNOS activity was completely inhibited by NMDA antagonists MK-801 and d-AP5, partially by the GluRepsilon2-selective antagonist CP-101,606, and was attenuated in GluRepsilon1(-/-) and GluRepsilon1(-/-)/epsilon4(-/-) mice. These results suggest that NO may regulate nNOS activity as a retrograde messenger in the spinal cord via activation of NMDA receptor containing GluRepsilon1 and GluRepsilon2 subunits.     

Suppression of nitric oxide production on LPS/IFN-γ-stimulated RAW264.7 macrophages by a novel catechin, pilosanol N, from Agrimonia pilosa Ledeb

A novel catechin, pilosanol N (1), was isolated from Agrimonia pilosa Ledeb and its structure was determined by (1)H, (13)C NMR and HRESI-MS analyses. Compound 1 inhibited nitric oxide (NO) production in LPS/IFN-γ -induced RAW264.7 macrophages, and then the iNOS gene expression and its protein production with LPS/IFN-γ treatment cells were also suppressed in the presence of 1. In addition, compound 1 scavenged NO or nitrogen radicals generated from NOR3 (4-ethyl-2-hydroxyamino-5-nitro-3-hexenamide) as an NO donor. These results indicated that pilosanol N can decrease the level of NO through a mechanism that involved both a decrease in the NO production and NO scavenging.     

Effects of nitric oxide donors on vascular endothelial growth factor gene induction

Nitric oxide (NO) has been reported to modulate the vascular endothelial growth factor (VEGF) gene by accumulating hypoxia-inducible factor-1alpha (HIF-1alpha) protein, but there is a contradiction among effects of various NO donors. The effects of NO donors including S-nitroso-N-acetyl-penicillamine (SNAP), S-nitroso-glutathione (GSNO), 1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene (NOC18), 3-[(+/-)-(E)-ethyl-2(')-[(E)-hydroxyimino]-5-nitro-3-hexenecarbamoyl]-pyridine (NOR4), 3-morpholinosydnonimine (SIN-1), and nitroprusside (SNP) on the VEGF reporter gene were examined. SNAP, GSNO, NOC18, and NOR4 enhanced the VEGF reporter activity under normoxia and modulated the hypoxic induction. In contrast, SNP had only an inhibitory effect. An NO scavenger attenuated the reporter activation by NO donors except NOR4, but did not ameliorate the inhibitory effect of SNP. A reducing compound dithiothreitol suppressed NO-induced activation of the VEGF reporter gene. SNAP, GSNO, and NOC18 induced the accumulation of HIF-1alpha protein, while others did not. These results suggest that SNAP, GSNO, and NOC compounds are suitable for pharmacological studies in HIF-1-mediated VEGF gene activation by NO.     

Reversal of hyperdynamic response to continuous endotoxin administration by inhibition of NO synthesis.

Septic shock is characterized by an increase in cardiac output and a fall in systemic vascular resistance index and mean arterial pressure. Endotoxin alters the smooth muscle function of blood vessels, probably by means of an increased production of the potent vasodilator nitric oxide (NO). The present study was accomplished to determine how the inhibition of NO synthesis influences cardiovascular performance in an ovine model of hyperdynamic endotoxemia. Endotoxemia was induced in five range ewes (41 +/- 2 kg) by continuous infusion of Escherichia coli endotoxin (LPS, 10 ng.kg-1.min-1) over the entire study period. After 24 h of LPS infusion, cardiac output increased from 5.2 +/- 0.3 to 7.9 +/- 0.6 (SE) 1/min (P less than 0.05) and mean arterial pressure and systemic vascular resistance index fell from 92 +/- 5 to 79 +/- 6 mmHg (P = 0.08) and from 1,473 +/- 173 to 824 +/- 108 dyn.s.cm-5.m2 (P less than 0.05), respectively. The pulmonary shunt fraction increased from 0.23 +/- 0.03 to 0.32 +/- 0.03 (P less than 0.05). The intravenous administration of the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (25 mg/kg) 24 h after the start of the LPS infusion changed these values to approximately baseline levels over the subsequent 4 h. Although N omega-nitro-L-arginine methyl ester increased pulmonary arterial pressure and pulmonary vascular resistance (P less than 0.05), right and left ventricular stroke volume index showed no significant changes. It is concluded that NO has a major function in cardiovascular performance in endotoxemia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen

The respiratory nitric oxide reductase (NOR) from Paracoccus denitrificans catalyzes the two-electron reduction of NO to N(2)O (2NO + 2H(+) + 2e(-) --> N(2)O + H(2)O), which is an obligatory step in the sequential reduction of nitrate to dinitrogen known as denitrification. NOR has four redox-active cofactors, namely, two low-spin hemes c and b, one high-spin heme b(3), and a non-heme iron Fe(B), and belongs to same superfamily as the oxygen-reducing heme-copper oxidases. NOR can also use oxygen as an electron acceptor; this catalytic activity was investigated in this study. We show that the product in the steady-state reduction of oxygen is water. A single turnover of the fully reduced NOR with oxygen was initiated using the flow-flash technique, and the progress of the reaction monitored by time-resolved optical absorption spectroscopy. Two major phases with time constants of 40 micros and 25 ms (pH 7.5, 1 mM O(2)) were observed. The rate constant for the faster process was dependent on the O(2) concentration and is assigned to O(2) binding to heme b(3) at a bimolecular rate constant of 2 x 10(7) M(-)(1) s(-)(1). The second phase (tau = 25 ms) involves oxidation of the low-spin hemes b and c, and is coupled to the uptake of protons from the bulk solution. The rate constant for this phase shows a pH dependence consistent with rate limitation by proton transfer from an internal group with a pK(a) = 6.6. This group is presumably an amino acid residue that is crucial for proton transfer to the catalytic site also during NO reduction.     

Hypoxia enhances gene expression of inducible nitric oxide synthase and matrix metalloproteinase-9 in ras/myc-transformed serum-free mouse embryo cells under simulated inflammatory and infectious conditions

Ras/myc-transformed serum-free mouse embryo (ras/myc SFME) cells were treated with interferon-gamma (IFN-gamma; 100 U/ml) and/or lipopolysaccharide (LPS; 0.5 microg/ml) for 24 h to simulate inflammatory and infectious conditions and investigate their effects on the expression of inducible nitric oxide synthase (iNOS) mRNA, nitric oxide (NO) and matrix metalloproteinase-9 (MMP-9). In addition, aminoguanidine (AG; 1 mM), a NOS inhibitor, S-nitroso-N-acetyl-DL-penicillamine (SNAP; 10-200 microM), an NO donor or (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4; 10-200 microM), an NO donor, were added to analyze possible associations of NO with MMP-9. Tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 were also measured to analyze possible relationships of NO with the MMP-9/TIMP balance. Furthermore, the cells were treated with 1% O2 under the simulated inflammatory and infectious conditions and the mRNA expressions of iNOS and MMP-9 were analyzed to investigate the possible effects of hypoxia on the expression of genes involved in tumor malignant progression and distant metastasis. Co-treatment with IFN-gamma and LPS increased the expression levels of iNOS mRNA, NO and MMP-9, but NO may not be directly associated with MMP-9 or the MMP-9/TIMP balance. Treatment with 1% O2 markedly increased the gene expression levels of iNOS and MMP-9, indicating that ras/myc SFME cells alter the expression levels of tumor-associated genes and possibly enhance their malignancy as cancer cells under inflammatory, infectious and hypoxic conditions.     

A new assay for nitric oxide reductase reveals two conserved glutamate residues form the entrance to a proton-conducting channel in the bacterial enzyme

A specific amperometric assay was developed for the membrane-bound NOR [NO (nitric oxide) reductase] from the model denitrifying bacterium Paracoccus denitrificans using its natural electron donor, pseudoazurin, as a co-substrate. The method allows the rapid and specific assay of NO reduction catalysed by recombinant NOR expressed in the cytoplasmic membranes of Escherichia coli. The effect on enzyme activity of substituting alanine, aspartate or glutamine for two highly conserved glutamate residues, which lie in a periplasmic facing loop between transmembrane helices III and IV in the catalytic subunit of NOR, was determined using this method. Three of the substitutions (E122A, E125A and E125D) lead to an almost complete loss of NOR activity. Some activity is retained when either Glu122 or Glu125 is substituted with a glutamine residue, but only replacement of Glu122 with an aspartate residue retains a high level of activity. These results are interpreted in terms of these residues forming the mouth of a channel that conducts substrate protons to the active site of NOR during turnover. This channel is also likely to be that responsible in the coupling of proton movement to electron transfer during the oxidation of fully reduced NOR with oxygen [U. Flock, N. J. Watmough and P. Adelroth (2005) Biochemistry 44, 10711-10719].     

Nitric oxide induces apoptotic death of cardiomyocytes via a cyclic-GMP-dependent pathway

Recently, we have reported that excess amounts of nitric oxide (NO) produced by inducible NO synthase are involved in the development of myocardial damage in rats with induced myocarditis. However, there remain many problems to be solved concerning its mechanism of action. In this study, we examined whether NO induces apoptotic cell death in cardiomyocytes. Cultured neonatal rat cardiomyocytes were exposed to S-nitroso-N-acetylpenicillamine (SNAP) and (+/-)-E-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamine (NOR 3), as NO donors, or 8-bromo-cyclic GMP (cGMP), an analog of cGMP which functions as a second messenger in cells stimulated by NO. DNA fragmentation was confirmed by electron microscopy, by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method, and by agarose gel electrophoresis. Exogenously supplied SNAP or NOR 3 induced cardiomyocyte apoptosis in a dose- and time-dependent manner. Cardiomyocytes exposed to SNAP displayed typical features of apoptosis as demonstrated by electron microscopy. Treatment of the cells with 8-bromo-cGMP also induced apoptosis. In cardiomyocytes, SNAP-induced apoptosis was completely blocked by a PKG inhibitor (KT5823) and by a soluble guanylate cyclase inhibitor (ODQ) and was suppressed by hemoglobin and was completely blocked by ZVAD-FMK, a caspase inhibitor. These results show that NO-mediated apoptosis of cardiomyocytes is cGMP dependent and that caspases are involved in this process.     

Nitric oxide regulates actin reorganization through cGMP and Ca(2+)/calmodulin in RAW 264.7 cells

Nitric oxide (NO) has been reported to be involved in the regulation of pseudopodia formation, phagocytosis and adhesion in macrophages through the reorganization of actin. In the present study, we directly separated the globular (G) and filamentous (F) actin from quiescent or NO-stimulated macrophage-like cell line RAW 264.7 cells in order to investigate the dynamic redistribution of actin pools. We also focused on the regulatory mechanisms of actin assembly, induced by NO and its possible subsequent signaling pathway. We showed that predominant G-actin coexisted with Triton X-100-insoluble filamentous (TIF) and Triton X-100-soluble filamentous actin in resting RAW 264.7 cells. The exogenous NO produced by (+/-)-(E)-2-[(E)-hydroxyimino]-6-methoxy-4-methyl-5-nitro-3-hexenamide (NOR1), the endogenous NO induced by lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma), and dibutyryl-cGMP increased the contents of TIF-actin in dose- and time-dependent manners and altered its morphology. The increase in the TIF-actin contents induced by NOR1 or LPS plus IFNgamma was efficiently blocked by the radical scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide and the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or the arginine analogue N(G)-monomethyl-L-arginine acetate, respectively. Preincubation with the calmodulin antagonist W-7 almost completely blocked the NO-induced TIF-actin increase and morphological change. On the other hand, preincubation with C3 transferase, an inhibitor of Rho protein, efficiently prevented the change in cell morphology, but had no effect on the TIF-actin increase. We postulate that cGMP and subsequent Ca(2+)/calmodulin may be key regulators of actin reorganization in NO-stimulated RAW 264.7 cells.     

A pathway for protons in nitric oxide reductase from Paracoccus denitrificans

Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N(2)O (2NO+2e(-)+2H(+)-->N(2)O+H(2)O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O(2)-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O(2), we demonstrate that protons are indeed consumed from the 'outside'. First, multiple turnover reduction of O(2) resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O(2) shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O(2) as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O(2) show electron transfer coupled to proton uptake from outside the NOR-liposomes with a tau=15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Adelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711-10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.     

Receptor mechanisms in fish chromatophores--VI. Adenosine receptors mediate pigment dispersion in guppy and catfish melanophores

Using the guppy, Lebistes reticulatus, and the siluroid catfish, Parasilurus asotus , the effects of purine and pyrimidine derivatives on the movement of melanophores were studied. All the substances tested did not aggregate pigment within melanophores. Adenosine and adenine nucleotides were very effective in dispersing melanosomes within the cell, although adenine itself lacked such action. Derivatives of other purines than adenine and of pyrimidines did not disperse melanosomes. The pigment dispersion induced by adenine derivatives was specifically antagonized by methylxanthines. It was concluded that adenosine receptors are present on the melanophore membrane, which take part in the darkening reaction of fishes.     

Nitric oxide and endothelin in oxygen-dependent regulation of vascular tone of human umbilical vein

We investigated the possible contribution of nitric oxide (NO) and endothelin (ET) to oxygen-dependent regulation of human umbilical vein vascular tone by simultaneous registration of intracellular membrane potential and isometric tension of vessel strips with and without NO synthase inhibition [10-4 M N omega-nitro-L-arginine methyl ester (L-NAME)], ETA receptor blockade (10(-5) M BQ-123), or ETB receptor blockade (10(-7) M BQ-788) at Po2 values in the bath solution between 5 and 104 mmHg. Increasing PO2 above the physiological intrauterine range resulted in depolarization and an increase of isometric tension, whereas lowering PO2 resulted in hyperpolarization and a decrease in isometric tension. Removal of the endothelium reversed these effects. At PO2 values below 39 mmHg, intact preparations treated with either L-NAME, BQ-788, or BQ-123 were more depolarized than controls. In the case of treatment with L-NAME or BQ-123, this was accompanied by an increase in isometric tension. We conclude that it is NO that mediates the hypoxic hyperpolarization and vasodilatation of the human umbilical vein and that ET, via activation of ETB1 receptors on endothelial cells, contributes to this effect.     

Nitric oxide modulates intracellular translocation of pigment organelles in Xenopus laevis melanophores

Pigment organelles in Xenopus laevis melanophores are used by the animal to change skin color, and they provide a good model for studying intracellular organelle transport. Movement of organelles and vesicles along the cytoskeleton is essential for many processes, such as axonal transport, endocytosis, and intercompartmental trafficking. Nitric oxide (NO) is a signaling molecule that plays a role in, among other things, relaxation of blood vessels, sperm motility, and polymerization of actin. Our study focused on the effect NO exerts on cytoskeleton-mediated transport, which has previously received little attention. We found that an inhibitor of NO synthesis, N-nitro-L-arginine methyl ester (L-NAME), reduced the melatonin-induced aggregation of the pigment organelles, melanosomes. Preaggregated melanosomes dispersed after treatment with L-NAME but not after exposure to the inactive stereoisomer (D-NAME) or the substrate for NO synthesis (L-arginine). Signal transduction by NO can be mediated through the activation of soluble guanylate cyclase (sGC), which leads to increased production of cGMP and activation of cGMP-dependent kinases (PKG). We found that both the sGC inhibitor 1H-(1,2,4) oxadiazolo(4,3-a)quinoxalin-1-one (ODQ) and the cGMP analogue 8-bromoguanosine 3':5'-cyclic monophosphate (8-Br-cGMP) reduced melanosome aggregation, whereas the PKG inhibitor KT582 did not. Our results demonstrate that melanosome aggregation depends on synthesis of NO, and NO deprivation causes dispersion. It seems, thus, as if NO and cGMP are essential and can regulate melanosome translocation.     

Nitric oxide-cGMP-mediated vasoconstriction and effects of acetylcholine in the branchial circulation of the eel

Information about the presence and effects of nitric oxide (NO) in fish vasculature is scant and contradictory. We have studied the NO/cGMP system in the branchial circulation of the teleost Anguilla anguilla using a branchial basket preparation under basal conditions and cholinergic stimulation. The effects of endogenous and exogenous NO were tested with L-arginine, the nitric oxide synthase (NOS) substrate, and the NO donors 3-morpholinosydnonimine (SIN-1) and sodium nitroprusside (SNP), respectively. L-arginine (from 10(-11) to 10(-6) M) and the NO donors (starting from 10(-14) M) caused dose-dependent vasoconstriction. Conversely, in the ACh-pre-contracted preparations both donors elicited vasodilation. SIN-1-induced vasoconstriction was due to NO generation: it was increased by superoxide dismutase (SOD) and blocked by NO scavenger hemoglobin. Pre-treatment with sGC inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) inhibited the effects of SIN-1 and SNP. The stable cGMP analogue 8-bromo-guanosine 3',5'-cyclic monophosphate (8-Br cGMP) induced dose-dependent vasoconstriction. Unexpectedly, three NOS inhibitors, N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-monomethyl-L-arginine (L-NMMA), L-N(5)-(1-iminoethyl) ornithine (L-NIO), caused mild vasoconstriction. ACh caused vasoconstriction, but at pico- and nanomolar concentrations it caused mild but significant vasodilation in 40% of the preparations. Both responses, blocked by atropine and pirenzepine, required an intact endothelium. The ACh-induced vasoconstriction was substantially independent of a NO-cGMP mechanism.     

Structures of reduced and ligand‐bound nitric oxide reductase provide insights into functional differences in respiratory enzymes

Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N₂O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non‐heme Fe center. We report herein on the structures of the reduced and ligand‐bound forms of cytochrome c‐dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3–2.7 Å, to elucidate structure‐function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO‐bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH₃‐CH=N‐OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ～0.5 Å increase in the heme/non‐heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton‐pumping activity in cNOR, because redox‐coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non‐heme Fe distance even in the bulky ligand‐bound form of cNOR (～4.5 Å) than the heme/Cu distance in CCO (～5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N‐N coupling to produce a hyponitrite intermediate for the generation of N₂O. Proteins 2014; 82:1258–1271. © 2013 Wiley Periodicals, Inc.     

17beta-estradiol stimulates ascorbic acid and LHRH release from the medial basal hypothalamus in adult male rats

In the present investigation, 17beta-estradiol (E(2)) and tamoxifen, an antiestrogen, were evaluated for their effects on the release of ascorbic acid (AA) and luteinizing hormone-releasing hormone (LHRH). Medial basal hypothalami (MBH) from adult male rats were incubated with graded concentrations of E(2) (10 (-9) to 10(-6) M) or a combination of E(2) (10(-7) M) and tamoxifen (10(-7) and 10(-6) M ) in 0.5 ml of Krebs Ringer bicarbonate buffer for 1 hr. AA and LHRH in the incubation medium were measured by high-performance liquid chromatography and radioimmunoassay, respectively. E(2) significantly elevated both AA and LHRH release and the minimal effective dose was 10(-7) M. A combination of E(2) (10(-7) M) and tamoxifen (10(-6) M) totally blocked E(2)-induced AA and LHRH release. The stimulatory effect of E(2) was also suppressed in the presence of N(G)-monomethyl-L-arginine, a competitive inhibitor of nitric oxide synthase (NOS), illustrating that the release is mediated by nitric oxide (NO). To further characterize the role of NO, the tissues were incubated with E(2) or a combination of E(2) + (6 anilino-5, 8-quinolinedione) LY 83583 (10(-6) and 10(-5) M), an inhibitor of NOS. LY 83583 was effective in suppressing E(2)-induced AA and LHRH release, demonstrating that the effect was mediated by cyclic GMP. Incubation of the tissues with E(2) or a combination of E(2) + 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (O.D.Q.) (10(-5) and 10(-4) M), a specific inhibitor of soluble guanylyl cyclase failed to alter AA release but significantly suppressed LHRH release. The role of a prostaglandin synthesis blocker in E(2)-induced AA and LHRH release was tested by incubating the tissues with E(2) or a combination of E(2) + indomethacin (1.8 x 10 (-7) or 1.8 x 10(-6) M). Indomethacin produced a significant decrease in E(2)-induced AA and LHRH release, suggesting that the release process required prostaglandins as an intracellular mediator. In conclusion, E(2) stimulated both AA and LHRH release and the effect was mediated by NO and prostaglandins.     

Reaction of carbon monoxide with the reduced active site of bacterial nitric oxide reductase.

Bacterial nitric oxide reductase (NOR), a member of the superfamily of heme-copper oxidases, catalyzes the two-electron reduction of nitric oxide to nitrous oxide. The key feature that distinguishes NOR from the typical heme-copper oxidases is the elemental composition of the dinuclear center, which contains non-heme iron (FeB) rather than copper (CuB). UV-vis electronic absorption and room-temperature magnetic circular dichroism (RT-MCD) spectroscopies showed that CO binds to Fe(II) heme b3 to yield a low-spin six-coordinate species. Photolysis of the Fe(II)-CO bond is followed by CO recombination (k(on) = 1.7 x 10(8) M(-1) x s(-1)) that is approximately 3 orders of magnitude faster than CO recombination to the active site of typical heme-copper oxidases (k(on) = 7 x 10(4) M(-1)x s(-1)). This rapid rate of CO recombination suggests an unimpeded pathway to the active site that may account for the enzyme's high affinity for substrate, essential for maintaining denitrification at low concentrations of NO. In contrast, the initial binding of CO to reduced heme b3 measured by stopped-flow spectroscopy is much slower (k(on) = 1.2 x 10(5) M(-1) x s(-1)). This suggests that an existing heme distal ligand (water/OH-) may be displaced to elicit the spin-state change observed in the RT-MCD spectrum.     

NO reductase from Bacillus azotoformans is a bifunctional enzyme accepting electrons from menaquinol and a specific endogenous membrane-bound cytochrome c551

Bacillus azotoformans is a Gram-positive denitrifying soil bacterium, which is capable of respiring nitrate, nitrite, nitric oxide, and nitrous oxide under anaerobic conditions. It contains a unique menaquinol-dependent nitric oxide reductase (qCu(A)NOR) with a Cu(A) center in its small subunit. The qCu(A)NOR exhibits menaquinol-dependent NO reductase activity, whereas reduced horse heart cytochrome c was inactive. Here we describe the purification of three membrane-bound c cytochromes from B. azotoformans. Their apparent molecular masses on SDS-PAGE are approximately 11 kDa. At neutral pH, these c cytochromes are negatively charged and the E(m) for all is close to 150 mV. Only one of these c cytochromes, which exhibits an alpha-band maximum at 551 nm, acts as a direct electron donor to qCu(A)NOR. Further investigation demonstrated that this cytochrome c(551) possesses two lipoyl moieties, which presumably function to anchor it to the membrane. Steady-state kinetic studies reveal that cytochrome c(551) is a noncompetitive inhibitor of NO reduction when menaquinol is used as an electron donor. This finding points to the presence of two different electron donation pathways in qCu(A)NOR. The ability of qCu(A)NOR to accept electrons from both menaquinol and cytochrome c(551) might be related to the regulation of the rate of NO reduction especially as a defense mechanism of B. azotoformans against the toxicity of NO. Growth experiments in batch culture indeed show that B. azotoformans is highly NO tolerant, in contrast to, for example, Paracoccus denitrificans that has a monofunctional cytochrome c-dependent NOR. We propose that the menaquinol pathway, which has a 4-fold greater maximal activity than the pathway via cytochrome c(551), is used for NO detoxification, whereas electron donation via the endogenous cytochrome c involves the cytochrome b(6)f complex serving the bioenergetic needs of the organism.