Nitric Oxide Equilibrium

The explicit formulas for following the passage to chemical equilibrium are derived.

Time-resolved gas-phase kinetic and quantum chemical studies of the reaction of silylene with nitric oxide

Time-resolved kinetic studies of the reaction of silylene, SiH2, generated by laser flash photolysis of phenylsilane, have been carried out to obtain rate constants for its bimolecular reaction with NO. The reaction was studied in the gas phase over the pressure range 1-100 Torr in SF6 bath gas at five temperatures in the range 299-592 K. The second-order rate constants at 10 Torr fitted the Arrhenius equation log(k/cm(3) molecule(-1) s(-1)) = (- 11.66 +/- 0.01) + (6.20 +/- 0.10 kJ mol(-1))IRT In 10 The rate constants showed a variation with pressure of a factor of ca. 2 over the available range, almost independent of temperature. The data could not be fitted by RRKM calculations to a simple third body assisted association reaction alone. However, a mechanistic model with an additional (pressure independent) side channel gave a reasonable fit to the data. Ab initio calculations at the G3 level supported a mechanism in which the initial adduct, bent H2SiNO, can ring close to form cyclo-H2SiNO, which is partially collisionally stabilized. In addition, bent H2SiNO can undergo a low barrier isomerization reaction leading, via a sequence of steps, ultimately to dissociation products of which the lowest energy pair are NH2 + SiO. The rate controlling barrier for this latter pathway is only 16 kJ mol(-1) below the energy of SiH2 + NO. This is consistent with the kinetic findings. A particular outcome of this work is that, despite the pressure dependence and the effects of the secondary barrier (in the side reaction), the initial encounter of SiH2 with NO occurs at the collision rate. Thus, silylene can be as reactive with odd electron molecules as with many even electron species. Some comparisons are drawn with the reactions of CH2 + NO and SiCl2 + NO.

Chemical mechanism of controlled nitric oxide release from trans-[RuCl([15]aneN(4))NO](PF6)(2) as a vasorelaxant agent

The nitrosyl ruthenium complex, trans-[RuCl([15]aneN(4))NO](PF6)(2), ([15]aneN(4) = 1,4,8,12-tetraazacyclopentadecane), exhibits vasorelaxation characteristics attributed to its nitric oxide release properties. The observed in vitro and in vivo vasodilation is dependent on noradrenaline concentration. We report here the chemical mechanism of the reaction between noradrenaline and trans-[RuCl([15]aneN(4))NO](PF6)(2) in aqueous phosphate buffer solution at pH 7.40. NO measurement by NO-sensor electrode, cyclic voltammetry, (PNMR)-P-31 and HPLC analysis were used to investigate the reduction process as the fundamental step for NO release characteristic of trans-[RuCl([15]aneN(4))NO](PF6)(2). A supramolecular species containing HPO4 (2-) as a bridging group between noradrenaline and trans-[RuCl([15]aneN(4))NO](PF6)(2) is suggested as an intermediate prior to the reduction of the nitrosyl ruthenium complex.

Cyclic nitroxides inhibit the toxicity of nitric oxide-derived oxidants: mechanisms and implications

The substantial therapeutic potential of tempol (4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy) and related cyclic nitroxides as antioxidants has stimulated innumerous studies of their reactions with reactive oxygen species. In comparison, reactions of nitroxides with nitric oxide-derived oxidants have been less frequently investigated. Nevertheless, this is relevant because tempol has also been shown to protect animals from injuries associated with inflammatory conditions, which are characterized by the increased production of nitric oxide and its derived oxidants. Here, we review recent studies addressing the mechanisms by which cyclic nitroxides attenuate the toxicity of nitric oxidederived oxidants. As an example, we present data showing that tempol protects mice from acetaminophen-induced hepatotoxicity and discuss the possible protection mechanism. In view of the summarized studies, it is proposed that nitroxides attenuate tissue injury under inflammatory conditions mainly because of their ability to react rapidly with nitrogen dioxide and carbonate radical. In the process the nitroxides are oxidized to the corresponding oxammonium cation, which, in turn, can be recycled back to the nitroxides by reacting with upstream species, such as peroxynitrite and hydrogen peroxide, or with cellular reductants. An auxiliary protection mechanism may be down-regulation of inducible nitric oxide synthase expression. The possible therapeutic implications of these mechanisms are addressed.

Sensitive detection of nitric oxide using seeded parametric four-wave mixing

A sensitive near-resonant four-wave mixing technique based on two-photon parametric four-wave mixing has been developed. Seeded parametric four-wave mixing requires only a single laser as an additional phase matched seeder field is generated via parametric four-wave mixing of the pump beam in a high gain cell. The seeder field travels collinearly with the pump beam providing efficient nondegenerate four-wave mixing in a second medium. This simple arrangement facilitates the detection of complex molecular spectra by simply scanning the pump laser. Seeded parametric four-wave mixing is demonstrated in both a low pressure cell and an air/acetylene flame with detection of the two-photon C (2) Pi(upsilon'=0)<--X (2) Pi(upsilon =0) spectrum of nitric oxide. From the cell data a detection limit of 10(12) molecules/cm(3) is established. A theoretical model of seeded parametric four-wave mixing is developed from existing parametric four-wave mixing theory. The addition of the seeder field significantly modifies the parametric four-wave mixing behaviour such that in the small signal regime, the signal intensity can readily be made to scale as the cube of the laser pump power while the density dependence follows a more familiar square law dependence, In general, we find excellent agreement between theory and experiment. Limitations to the process result from an ac Stark shift of the two-photon resonance in the high pressure seeder cell caused by the generation of a strong seeder field, as well as a reduction in phase matching efficiency due to the presence of certain buffer species. Various optimizations are suggested which should overcome these limitations, providing even greater detection sensitivity. (C) 1998 American Institute of Physics, [S0021-9606(98)01014-9].

trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3)center dot H(2)O encapsulated in PLGA microparticles for delivery of nitric oxide to B16-F10 cells: Cytotoxicity and phototoxicity

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py = pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600 nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2 h of incubation. The complex with concentrations lower than 1 x 10(-4) M did not show toxicity in B16-F 10 murine cells. The complex in solution is toxic at higher concentrations (> 1 x 10(-3) M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by radiation with light only. (c) 2007 Elsevier Inc. All rights reserved.

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.

Low-Spin Heme b3 in the Catalytic Center of Nitric Oxide Reductase from Pseudomonas nautica

Biochemistry, 2011, 50 (20), pp 4251–4262 DOI: 10.1021/bi101605p

Camelid nanobodies raised against an integral membrane enzyme, nitric oxide reductase

Protein Sci. 2009 Mar;18(3):619-28. doi: 10.1002/pro.69.

Plasmodium falciparum: nitric oxide modulates heme speciation in isolated food vacuoles.

Nitric oxide (NO) and NO-derived reactive nitrogen species (RNS) are present in the food vacuole (FV) of Plasmodium falciparum trophozoites. The product of PFL1555w, a putative cytochrome b(5), localizes in the FV membrane, similar to what was previously observed for the product of PF13_0353, a putative cytochrome b(5) reductase. These two gene products may contribute to NO generation by denitrification chemistry from nitrate and/or nitrite present in the erythrocyte cytosol. The possible coordination of NO to heme species present in the food vacuole was probed by resonance Raman spectroscopy. The spectroscopic data revealed that in situ generated NO interacts with heme inside the intact FVs to form ferrous heme nitrosyl complexes that influence intra-vacuolar heme solubility. The formation of heme nitrosyl complexes within the FV is a previously unrecognized factor that could affect the equilibrium between soluble and crystallized heme within the FV in vivo.

Nitric oxide reduces Cl- absorption in the mouse cortical collecting duct through an ENaC-dependent mechanism.

Since nitric oxide (NO) participates in the renal regulation of blood pressure, in part, by modulating transport of Na(+) and Cl(-) in the kidney, we asked whether NO regulates net Cl(-) flux (JCl) in the cortical collecting duct (CCD) and determined the transporter(s) that mediate NO-sensitive Cl(-) absorption. Cl(-) absorption was measured in CCDs perfused in vitro that were taken from aldosterone-treated mice. Administration of an NO donor (10 μM MAHMA NONOate) reduced JCl and transepithelial voltage (VT) both in the presence or absence of angiotensin II. However, reducing endogenous NO production by inhibiting NO synthase (100 μM N(G)-nitro-l-arginine methyl ester) increased JCl only in the presence of angiotensin II, suggesting that angiotensin II stimulates NO synthase activity. To determine the transport process that mediates NO-sensitive changes in JCl, we examined the effect of NO on JCl following either genetic ablation or chemical inhibition of transporters in the CCD. Since the application of hydrochlorothiazide (100 μM) or bafilomycin (5 nM) to the perfusate or ablation of the gene encoding pendrin did not alter NO-sensitive JCl, NO modulates JCl independent of the Na(+)-dependent Cl(-)/HCO3(-) exchanger (NDCBE, Slc4a8), the A cell apical plasma membrane H(+)-ATPase and pendrin. In contrast, both total and NO-sensitive JCl and VT were abolished with application of an epithelial Na(+) channel (ENaC) inhibitor (3 μM benzamil) to the perfusate. We conclude that NO reduces Cl(-) absorption in the CCD through a mechanism that is ENaC-dependent.

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.

Involvement of a transforming-growth-factor-beta-like molecule in tumor-cell-derived inhibition of nitric-oxide synthesis in cerebral endothelial cells.

Nitric oxide (NO) has been shown to exert cytotoxic effects on tumor cells. We have reported that EC219 cells, a rat-brain-microvessel-derived endothelial cell line, produced NO through cytokine-inducible NO synthase (iNOS), the induction of which was significantly decreased by (a) soluble factor(s) secreted by DHD/PROb, an invasive sub-clone of a rat colon-carcinoma cell line. In this study, the DHD/PROb cell-derived NO-inhibitory factor was characterized. Northern-blot analysis demonstrated that the induction of iNOS mRNA in cytokine-activated EC219 cells was decreased by PROb-cell-conditioned medium. When DHD/PROb cell supernatant was fractionated by affinity chromatography using Con A-Sepharose or heparin-Sepharose, the NO-inhibitory activity was found only in Con A-unbound or heparin-unbound fractions, respectively, indicating that the PROb-derived inhibitory factor was likely to be a non-glycosylated and non-heparin-binding molecule. Pre-incubation of DHD/PROb-cell supernatant with anti-TGF-beta neutralizing antibody completely blocked the DHD/PROb-derived inhibition of NO production by EC219 cells. Addition of exogenous TGF-beta 1 dose-dependently inhibited NO release by EC219 cells. The presence of active TGF-beta in the DHD/PROb cell supernatant was demonstrated using a growth-inhibition assay. Moreover, heat treatment of medium conditioned by the less invasive DHD/REGb cells, which constitutively secreted very low levels of active TGF-beta, increased both TGF-beta activity and the ability to inhibit NO production in EC219 cells. Thus, DHD/PROb colon-carcinoma cells inhibited NO production in EC219 cells by secreting a factor identical or very similar to TGF-beta.

Role of non-nitric oxide non-prostaglandin endothelium-derived relaxing factor(s) in bradykinin vasodilation

The most conspicuous effect of bradykinin following its administration into the systemic circulation is a transient hypotension due to vasodilation. In the present study most of the available evidence regarding the mechanisms involved in bradykinin-induced arterial vasodilation is reviewed. It has become firmly established that in most species vasodilation in response to bradykinin is mediated by the release of endothelial relaxing factors following the activation of B2-receptors. Although in some cases the action of bradykinin is entirely mediated by the endothelial release of nitric oxide (NO) and/or prostacyclin (PGI2), a large amount of evidence has been accumulated during the last 10 years indicating that a non-NO/PGI2 factor accounts for bradykinin-induced vasodilation in a wide variety of perfused vascular beds and isolated small arteries from several species including humans. Since the effect of the non-NO/PGI2 endothelium-derived relaxing factor is practically abolished by disrupting the K+ electrochemical gradient together with the fact that bradykinin causes endothelium-dependent hyperpolarization of vascular smooth muscle cells, the action of such factor has been attributed to the opening of K+ channels in these cells. The pharmacological characteristics of these channels are not uniform among the different blood vessels in which they have been examined. Although there is some evidence indicating a role for KCa or KV channels, our findings in the mesenteric bed together with other reports indicate that the K+ channels involved do not correspond exactly to any of those already described. In addition, the chemical identity of such hyperpolarizing factor is still a matter of controversy. The postulated main contenders are epoxyeicosatrienoic acids or endocannabinoid agonists for the CB1-receptors. Based on the available reports and on data from our laboratory in the rat mesenteric bed, we conclude that the NO/PGI2-independent endothelium-dependent vasodilation induced by BK is unlikely to involve a cytochrome P450 arachidonic acid metabolite or an endocannabinoid agonist.

Targeting and translocation of endothelial nitric oxide synthase

This review explores advances in our understanding of the intracellular regulation of the endothelial isoform of nitric oxide synthase (eNOS) in the context of its dynamically regulated subcellular targeting. Nitric oxide (NO) is a labile molecule, and may play important biological roles both within the cell in which it is synthesized and in its interactions with nearby cells and molecules. The localization of eNOS within the cell importantly influences the biological role and chemical fate of the NO produced by the enzyme. eNOS, a Ca2+/calmodulin-dependent enzyme, is subject to a complex pattern of intracellular regulation, including co- and post-translational modifications and interactions with other proteins and ligands. In endothelial cells and cardiac myocytes eNOS is localized in specialized plasmalemmal signal-transducing domains termed caveolae; acylation of the enzyme by the fatty acids myristate and palmitate is required for targeting of the protein to caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation. In resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. However, following agonist activation, eNOS dissociates from caveolin, and nearly all the eNOS translocates to structures within the cell cytosol; following more protracted incubations with agonists, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The agonist-induced internalization of eNOS is completely abrogated by chelation of intracellular Ca2+. These rapid receptor-mediated effects are seen not only for "classic" eNOS agonists such as bradykinin, but also for estradiol, indicating a novel non-genomic role for estrogen in eNOS activation. eNOS targeting to the membrane is labile, and is subject to receptor-regulated Ca2+-dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.