Nitric oxide signalling with a special focus on lipid-derived mediators

The ways in which cells communicate among each other concerns all aspects of biology, from developmental processes to diseases. Nitric oxide (NO) is one of the most remarkable and unusual regulatory molecules. It is a labile free radical gas that is not stored but generated on demand, and has been implicated in an extraordinarily diverse range of physiological and pathophysiological functions. The modulation of cell signalling by free radicals is an emerging area of research that provides insight into the orchestration of cell adaptation to a changing microenvironment. In a multicellular organism this serves to coordinate complex physiological responses, such as inflammation. Cell signalling is also accompanied by rapid remodelling of membrane lipids by activated lipases. The discovery that NO, which does not reversibly interact with membrane receptors like conventional hormones and growth factors, targets enzymes such as phospholipase A2, sphingomyelinases or ceramidases, has stimulated growing interest in the crosstalk between redox and lipid signalling.     

Spin trapping of lipid-derived radicals in liposomes

Electron-spin resonance-spin trapping has been used to detect lipid-derived radicals in liposomes. Using the lipid-soluble spin trap 2-methyl-nitrosopropane (MNP), we have detected both the lipid and hydrogen-atom spin adducts in liposomes composed of a fully saturated phospholipid (dimyristoylphosphatidylcholine, DMPC) with various mol fractions of unsaturated phospholipid (1-palmitoyl-2-arachidonoylphosphatidylcholine, PAPC) or fatty acid (arachidonic acid, AA). The lipid-derived spin adduct formed during autoxidation of liposomes was separated by thin-layer chromatography and found to co-migrate with the product(s) formed by direct addition of MNP to the corresponding unsaturated lipid or fatty acid. Both the MNP-PAPC and MNP-AA spin adducts showed some restriction of rotational motion when in the liposome bilayer (rotational correlation times 0.72 and 0.69.10(-9) s, respectively), and nitrogen hyperfine coupling constants (14.94-14.96 G) consistent with a hydrophobic localization. Radical versus non-radical mechanisms of spin adduct formation during liposome autoxidation were separated using alpha-tocopherol as a radical scavenger. The utility of nitroso spin traps in trapping of radicals in liposomes is discussed.     

Reactions of thrombin-serpin complexes with thrombospondin.

Activated platelets release proteins that form stable complexes with thrombin (J. J. Miller, P. C. Browne, and T. C. Detwiler, Biochem. Biophys. Res. Commun. 151, 9-15, 1988). A working model for the reaction (P. C. Browne, J. J. Miller, and T. C. Detwiler, Arch. Biochem. Biophys. 265, 534-538, 1988) includes a dissociable complex of thrombin with released platelet protease nexin, leading to formation of a nondissociable thrombin-nexin complex that then becomes disulfide linked to thrombospondin. This disulfide-linked complex is converted back to the thrombin-nexin complex by reduction of disulfide bonds. Results that allow elaboration on this model are presented. After longer periods of incubation or after incubation with higher concentrations of thrombin, the amount of thrombin complexed with thrombospondin exceeded the amount of thrombin-nexin complex recovered after reduction of disulfide bonds. When the reaction mixture included inhibitors of formation of the thrombin-nexin complex, a slow formation of the thrombin-thrombospondin complex was observed. It was concluded that there is a nexin-independent as well as the faster nexin-dependent disulfide linkage of thrombin to thrombospondin. Addition of thrombin-antithrombin III complexes to the supernatant solution of activated platelets also led to complexes with thrombospondin, demonstrating that serpins other than platelet protease nexin facilitate incorporation of thrombin into complexes with thrombospondin. By heparin affinity chromatography, it was shown that thrombin-nexin complexes dissociably associate with thrombospondin prior to formation of disulfide-linked complexes. These observations are incorporated into a more detailed model of the reaction.     

Reactions of nitrosobenzene with reduced glutathione.

Nitrosobenzene (NOB) formed acid labile conjugates with reduced glutathione (GSH) and hemoglobin within red cells. In vitro, NOB rapidly reacted with GSH with formation of phenylhydroxylamine (PH), oxidized glutathione (GSSG), and a water-soluble compound identified as glutathionesulfinanilide (GSO-AN). Free aniline (AN), aminophenols and azoxybenzene were not detected. The proportion of PH formed increased with increasing GSH concentration and at higher pH values. Spectroscopic analysis revealed the formation of a labile adduct following a second order reaction (K = 5 x 10(3) M-1 . sec-1 at pH 7.4 and 37 degrees). This reaction was reversible because nearly all NOB could be extracted with ether from the labile intermediate. On the other hand, the labile intermediate was transformed into GSO-AN (with increasing rate at lower pH values) or it was cleaved by GSH with formation of GSSG and PH. Intermediate formation of NOB and thiol radicals was ruled out by analysis of the equilibrium data. A tentative scheme is presented for the proposed reaction mechanism.     

Subpicogram determination of malondialdehyde by gas-liquid chromatography with nitrogen phosphorus detector. I. Standardization

A method for the measurement of malondialdehyde (MDA) using GLC with a nitrogen phosphorus detector (GLC/NPD) is described and evaluated. The method uses 2-hydrazinobenzothiazole (HBT) which forms condensation derivatives with MDA, acetoacetaldehyde, and acetylacetone (AA). GC/MS and 13C NMR studies of the three derivatives obtained showed that they are 2-(pyrazol-1'-yl)benzothiazoles and that they can be separated by GLC/NPD. Any one of these derivatives can be used as an internal standard for the measurement of the other two. The optimal conditions for the measurement of MDA were studied. At pH 2.5 and 70 degrees C, the condensation derivative is quantitatively formed in 30 min. Its extraction is obtained by a mixture of n-hexane/isoamyl alcohol 98/2 (v/v) containing HBT-AA as internal standard. The GLC detection limit is 0.04 pmol. Inter- and intrassay coefficients of variation were 2.9 and an average of 4.0%, respectively. The method is specific, and there was no interference from other carbonyl compounds. The artifactual formation of MDA from carbohydrates during the derivatization reaction is negligible. The method is proposed as a reference method for the standardization of working solutions of MDA or MDA-generating solutions.     

Methyl malondialdehyde as an internal standard for the determination of malondialdehyde

Methyl malondialdehyde (Me-MDA) is suggested as an internal standard for the determination of the lipid peroxidation product, malondialdehyde (MDA). A procedure for synthesising the Me-MDA sodium salt is described in detail. The purity and identity of the synthesised Me-MDA have been confirmed using nuclear magnetic resonance and UV spectroscopy, and by micellar electrokinetic chromatography. The applicability of Me-MDA as an internal standard has been demonstrated for rat brain homogenate samples. These samples were purified solely through ultrafiltration. The preferred analytical technique was capillary zone electrophoresis (CZE) with UV detection at 267 nm. The limits of detection (3 S/N) for the CZE separations of Me-MDA and MDA were 0.5 and 0.2 μM, respectively, and the total analysis time was approximately 10 min. Details of separations are also presented using high-performance liquid chromatography (HPLC) with UV detection at 245 nm, and gas chromatography, together with either electron capture or mass spectrometric detection. The GC separations require derivatisation of MDA and Me-MDA with pentafluorophenylhydrazine while the CZE and HPLC separations can be performed on the native molecules.     

Controlling herpes simplex virus-induced ocular inflammatory lesions with the lipid-derived mediator resolvin E1

Stromal keratitis (SK) is a chronic immunopathological lesion of the eye caused by HSV-1 infection and a common cause of blindness in humans. The inflammatory lesions are primarily perpetuated by neutrophils with the active participation of CD4(+) T cells. Therefore, targeting these immune cell types represents a potentially valuable form of therapy to reduce the severity of disease. Resolvin E1 (RvE1), an endogenous lipid mediator, was shown to promote resolution in several inflammatory disease models. In the current report, we determined whether RvE1 administration begun at different times after ocular infection of mice with HSV could influence the severity of SK lesions. Treatment with RvE1 significantly reduced the extent of angiogenesis and SK lesions that occurred. RvE1-treated mice had fewer numbers of inflammatory cells that included Th1 and Th17 cells as well as neutrophils in the cornea. The mechanisms by which RvE1 acts appear to be multiple. These included reducing the influx of neutrophils and pathogenic CD4(+) T cells, increasing production of the anti-inflammatory cytokine IL-10, and inhibitory effects on the production of proinflammatory mediators and molecules, such as IL-6, IFN-γ, IL-17, KC, VEGF-A, MMP-2, and MMP-9, that are involved in corneal neovascularization and SK pathogenesis. These findings are, to our knowledge, the first to show that RvE1 treatment could represent a novel approach to control lesion severity in a virally induced immunopathological disease.     

Spin adducts of superoxide,alkoxyl, and lipid-derived radicals with EMPO and its derivatives

The compound 5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide (EMPO) is a hydrophilic cyclic nitrone spin trap, which, in contrast to DMPO, forms a relatively stable superoxide adduct (t(1/2)=8.6 min) with an EPR spectrum similar to the respective DMPO adduct. In order to find the optimal degree of lipophilicity of this novel type of spin trap with respect to the detection of radicals formed during lipid peroxidation, the ethoxy group of EMPO was replaced by alkoxy substituents of increasing chain length, leading to the methoxy- (MeMPO), 1-propoxy- (PrMPO), 1-butoxy- (BuMPO), and 1-octyloxy- (OcMPO) derivatives of EMPO. The stability of their superoxide adducts was found to be strongly dependent on the size of the alkoxycarbonyl group. Increasing chain length of the alkoxyl substituent decreased the stability of alkoxyl radical adducts of MeMPO, EMPO, and PrMPO, but increased the stability of OcMPO adducts. The stability of alkoxyl radical adducts of BuMPO, on the other hand, were practically independent of the size of the alkoxyl group. Detection of lipid alkoxyl radicals formed by peroxidizing linoleic acid in a stationary system was therefore only possible with the most lipophilic spin trap, OcMPO. However, with the more hydrophilic spin traps MeMPO, EMPO, PrMPO, and BuMPO optimal EPR signal intensity could be obtained when a slow-flow system was used. Thus, within this series EMPO is the best spin trap for the detection of superoxide; OcMPO, on the other hand, is most suitable for the detection of lipid alkoxyl radicals.     

Reactions of proteins with ethyl vinyl sulfone.

Ethly vinyl sulfone (EVS) alkylates xi-amino groups of lysine side chains and imidazole groups of histidine residues in proteins. Amino acid analysis of hydrolyzates of EVS-treated polylysine shows that lysine forms two derivatives, presumably xi-N-(ethylsulfonylethyl)lysine and xi, xi, N,N-bis(ethylsulfonylethyl)lysine that are eluted as well-resolved peaks on the (long basic) physiological column of our amino acid analyzer at about 118 and 60 min, respectively. Peaks with identical elution times were also observed after EVS-treatment of BSA and wool. The postulated histidine derivative, presumably N3-im-(ethylsulfonylethyl)histidine is also eluted as a well-resolved peak on the same column at about 90 min. A peak with an identical elution time was observed in a hydrolyzate of EVS-treated polyhistidine. The described alkylation has potential utility for modifying proteins.     

Hydroxyl-radical-induced iron-catalysed degradation of 2-deoxyribose. Quantitative determination of malondialdehyde.

The degradation of 2-deoxyribose to thiobarbituric acid-reactive material was investigated with two hydroxyl-radical-generating systems: (i) a defined gamma-radiolysis method and (ii) incubation with FeSO4 in phosphate buffer. In each case the thiobarbituric acid-reactive material can be accounted for by malondialdehyde, as measured by an h.p.l.c. method for free malondialdehyde. In the radiolysis system there is a large post-irradiation increase in free malondialdehyde if iron ions are added to the samples. It is proposed that this is due to iron ions catalysing the formation of hydroxyl radicals from radiolytically generated H2O2 as well as stimulating the breakdown of an intermediate deoxyribose degradation product. A mechanism for the formation of malondialdehyde during deoxyribose degradation is proposed.     

Association of chondroadherin with collagen type II.

Chondroadherin is a cell binding, leucine-rich repeat protein found in the territorial matrix of articular cartilage. Several members of the leucine-rich repeat protein family present in the extracellular matrix of e.g. cartilage have been shown to interact with collagen and influence collagen fibrillogenesis. We show that complexes of monomeric collagen type II and chondroadherin can be released under non-denaturing conditions from articular cartilage treated with p-aminophenylmercuric acetate to activate resident matrix metalloproteinases. Purified complexes as well as complexes formed in vitro between recombinant chondroadherin and collagen type II were studied by electron microscopy. Chondroadherin was shown to bind to two sites on collagen type II. The interaction was characterized by surface plasmon resonance analysis showing K(D) values in the nanomolar range. Both chondroadherin and collagen interact with chondrocytes, partly via the same receptor, but give rise to different cellular responses. By also interacting with each other, a complex system is created which may be of functional importance for the communication between the cells and its surrounding matrix and/or in the regulation of collagen fibril assembly.     

Interaction of bilirubin with reconstituted collagen fibrils.

1. Interaction of bilirubin with collagen fibrils was explored in a two-phase system where collagen was present as an opaque rigid gel composed of striated fibrils, and bilirubin as an aqueous solution. 2. The Ka value of the binding of bilirubin to collagen fibrils is 5.4 X 10(3)M-1. The interaction of bilirubin with collagen fibrils depends on temperature. Below 5 degrees C, the binding is greatly diminished and denaturation of collagen fibril aggregates at 52--53 degrees C into a dissolution state abolishes binding of bilirubin. 3. Salicylate and sulphanilamide do not affect the binding of bilirubin to reconstituted collagen fibrils. 4. Serum albumin (40--80mM), known to reverse the binding of bilirubin to lipids, dissociates only 50% of the bilirubin bound to collagen fibrils. This suggests that sites located on collagen participate in some tight binding of bilirubin and the corresponding binding sites on albumin do not compete with them. 5. Urea (4M) abolishes more than 70% of the binding of bilirubin to collagen. Urea and thermal denaturation studies indicate the importance of conformation and organization of collagen fibrillar aggregates for the binding of bilirubin.     

Interaction of type-I collagen with phospholipid monolayer.

The effects of type-I collagen on dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) monolayer films with different compositions were studied using monolayer technique. The addition of collagen in the subphase of different monolayer films induced a considerable shift towards larger area/molecule in the compression-isotherm curves. This is either referred to the insertion of collagen into the monolayer by its hydrophobic residues or to an adsorption process causing a protein layer to be located parallel to the lipid monolayer [1]. The variation of collagen interaction with different lipid compositions was also verified through the penetration-kinetics experiment. Comparing our results to the results of Pajean et al. [2] and Pajean and Herbage [3] on the effect of collagen on the stability of lipid vesicles implies that the collagen induced stability could be explained on the basis of collagen-lipid monolayer interaction.     

Validation of methyl malondialdehyde as internal standard for malondialdehyde detection by capillary electrophoresis

The aim of this study was to validate, by capillary electrophoresis, the use of synthesized methyl malondialdehyde as the internal standard for the direct quantification of free and total (free+bound) malondialdehyde in biological samples. All analyses were performed in 20 cm x 50 microm uncoated capillaries at 20 degrees C, using 25 mmol/L borax (pH 9.3) and 5 mmol/L tetradecyltrimethylammonium bromide as running buffer. The applied voltage was -4kV (about 8 microA), the detector being set at 260 nm for a total run time of 8 min per sample. Free malondialdehyde was evaluated after acetonitrile extraction, while the samples evaluated for total malondialdehyde were, before extraction, hydrolyzed for 1h at 60 degrees C in the presence of 1 mol/L NaOH. The detection threshold was 0.2 micromol/L in microsomes and 0.4 micromol/L in plasma. As an application of the method, three pools of rat liver microsomes were quantified before (0.35+/-0.1 and 1.1+/-0.5 nmol/mg protein, free and total malondialdehyde, respectively, mean+/-SD) and after lipoperoxidation induction using systems able to generate oxygen free radicals (18.4+/-3.2 and 19.7+/-2.0 nmol/mg protein). The results were confirmed by isotopic dilution gas chromatography-mass spectrometry, used as the reference method. The feasibility of capillary electrophoresis for malondialdehyde determination in normal and pathological human plasma was also investigated.     

A decrease of lipid fluidity of the porcine intestinal brush-border membranes by treatment with malondialdehyde.

The effect of treatment of the porcine intestinal brush-border membranes with malondialdehyde (MDA) on their lipid fluidity was examined using a fluorescence probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). When the membranes were treated with MDA, the fluorescence anisotropy of DPH-labeled membranes increased and the amount of DPH molecules incorporated into the membranes decreased from 3.25 to 2.23 nmol/mg protein. In addition, the response of the fluorescence anisotropy of DPH-labeled membranes to benzyl alcohol, a well-known fluidizer, was markedly suppressed by treatment of the membranes with MDA. These results suggest that treatment of the membranes with MDA causes a decrease of the membrane lipid fluidity. This interpretation was further supported by the increase observed in the fluorescence anisotropy of DPH-labeled liposomes prepared from the extracted lipids of MDA-treated membranes. The results of SDS-polyacrylamide gel electrophoresis suggested that the formation of high-molecular-weight aggregates of the membrane proteins is not involved in the increase of the fluorescence anisotropy of DPH-labeled membranes by treatment with MDA. On the basis of these results, changes in the physical properties of the intestinal brush-border membranes by treatment with MDA are discussed.