Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide and nitroxyl ion

I Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre-contracted) were compared with those of endothelium-derived NO (generated with acetylcholine), NO free radical (NO; NO gas solution) and nitroxyl ion (NO-; from Angeli's salt). 2 The soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4-)oxadiazolo(4,3-a)-quinoxalin-1-one; 0.3, 1 and 10 muM), concentration-dependently inhibited responses to all agents. 10 muM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration-response curves for NO gas solution, Angeli's salt and spermine NONOate. 3 The NO scavengers, carboxy-PTIO, (2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-indazoline-1-oxyl-3-oxide; 100 muM) and hydroxocobalamin (100 muM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy-PTIO, also inhibited responses to acetylcholine. 4 The NO- inhibitor, L-cysteine (3 mm), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution. 5 The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO and NO-. Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium-derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.

Copolymerization of methyl methacrylate and allyl acetate Part I. Rate of reaction

Free radical bulk copolymerization of methyl methacrylate (MMA) and allyl acetate (AAc) has been investigated using electron spin resonance (ESR) and FT-near infrared (FTNIR) spectroscopy. Data are used to evaluate the rate constants. The mole fraction of AAc plays an important role in the copolymerization of these two monomers. AAc not only delays the Trommsdorff effect but also increases the onset of percentage total conversion at which the Trommsdorff region begins. With AAc fraction 0.5 and higher, no Trommsdorff effect was observed. Inclusion of AAc into copolymer structure mainly occurs in the Trommsdorf region or when the AAc fraction in the comonomer feed is dominant. This is associated with a drop in the concentration of propagating radicals. However, ESR spectra indicate that the MMA propagating radical is predominant during the reaction. In the comonomer mixtures where a Trommsdorff region can be observed, the addition of AAc does not produce any significant change in k(p) and k(t) in the steady state region. Major changes in k(p) and k(t) are observed after the gel point and glassy state, respectively. (C) 2001 Society of Chemical Industry.

The copolymerization of N-vinyl-2-pyrrolidone with 2-hydroxyethyl methacrylate

The bulk free radical copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N-vinyl-2-pyrrolidone (VP) was carried out to low conversions at 50 degreesC, using benzoyl peroxide (BPO) as initiator. The compositions of the copolymers; were determined using C-13 NMR spectroscopy. The conversion of monomers to polymers was studied using FT-NIR spectroscopy in order to predict the extent of conversion of monomer to polymer. From model fits to the composition data, a statistical F-test revealed that die penultimate model describes die copolymerization better than die terminal model. Reactivity ratios were calculated by using a non-linear least squares analysis (NLLS) and r(H) = 8.18 and r(V) = 0.097 were found to be the best fit values of the reactivity ratios for the terminal model and r(HH) = 12.0, r(VH) = 2.20, r(VV) = 0.12 and r(HV) = 0.03 for the penultimate model. Predictions were made for changes in compositions as a function of conversion based upon the terminal and penultimate models.

Synthesis of polymer-montmorillonite nanocomposites by in situ intercalative polymerization

Two polymer-montmorillonite (MMT) nanocomposites have been synthesized by in situ intercalative polymerization. The styrene monomer is intercalated into the interlayer space of organically modified MMT, a layered clay mineral. Upon the intercalation, the complex is subsequently polymerized in the confinement environment of the interlayer space with a free radical initiator, 2,2-azobis isobutyronitrile. The aniline monomer is also intercalated and then polymerized within the interlayer space of sodium- and copper-MMT initiated by ammonium peroxodisulphate and interlayer copper cations respectively. X-ray diffraction indicates that the MMT layers are completely dispersed in the polystyrene matrix and an exfoliated structure has been obtained. The resulting polyaniline-MMT nanocomposites show a highly ordered structure of a single polyaniline layer stacked with the MMT layers. Fourier transform infrared spectra further confirm the intercalation and formation of both polymer-MMT nanocomposites.

Exposure to automotive pollution increases plasma susceptibility to oxidation

Low-density lipoprotein oxidation is implicated in the development of atherosclerosis. Plasma susceptibility to oxidation may be used as a marker of low-density lipoprotein oxidation and thus predict atherosclerotic risk. In this study the authors investigated the relationship between plasma susceptibility to oxidation and exposure to automotive pollution in a group of automobile mechanics (n = 16) exposed to high levels of automotive pollution, vs. matched controls (n = 13). The authors induced plasma oxidation by a free radical initiator and they determined susceptibility to oxidation by (1) change in absorbance at 234 nm, (2) lag time to conjugated diene formation, and (3) linear slope of the oxidation curve. Mechanics had significantly higher values (mean standard error) for change in absorbance (1.60 +/- 0.05 vs. 1.36 +/- 0.05; p < .002), and slope (1.6 x 10(-3) +/- 0.1 x 10(-3) vs. 1.3 x 10(-3) +/- 0.1 x 10(-3); p < .001), compared with controls. These results indicate that regular exposure to automotive pollutants increases plasma susceptibility to oxidation and may, in the long term, increase the risk of developing atherosclerosis.

In vivo and in vitro models demonstrate a role for caveolin-1 in the pathogenesis of ischaemic acute renal failure

Caveolae and their proteins, the caveolins, transport macromolecules; compartmentalize signalling molecules; and are involved in various repair processes. There is little information regarding their role in the pathogenesis of significant renal syndromes such as acute renal failure (ARF). In this study, an in vivo rat model of 30 min bilateral renal ischaemia followed by reperfusion times from 4 h to 1 week was used to map the temporal and spatial association between caveolin-1 and tubular epithelial damage (desquamation, apoptosis, necrosis). An in vitro model of ischaemic ARF was also studied, where cultured renal tubular epithelial cells or arterial endothelial cells were subjected to injury initiators modelled on ischaemia-reperfusion (hypoxia, serum deprivation, free radical damage or hypoxia-hyperoxia). Expression of caveolin proteins was investigated using immunohistochemistry, immunoelectron microscopy, and immunoblots of whole cell, membrane or cytosol protein extracts. In vivo, healthy kidney had abundant caveolin-1 in vascular endothelial cells and also some expression in membrane surfaces of distal tubular epithelium. In the kidneys of ARF animals, punctate cytoplasmic localization of caveolin-1 was identified, with high intensity expression in injured proximal tubules that were losing basement membrane adhesion or were apoptotic, 24 h to 4 days after ischaemia-reperfusion. Western immunoblots indicated a marked increase in caveolin-1 expression in the cortex where some proximal tubular injury was located. In vitro, the main treatment-induced change in both cell types was translocation of caveolin-1 from the original plasma membrane site into membrane-associated sites in the cytoplasm. Overall, expression levels did not alter for whole cell extracts and the protein remained membrane-bound, as indicated by cell fractionation analyses. Caveolin-1 was also found to localize intensely within apoptotic cells. The results are indicative of a role for caveolin-1 in ARF-induced renal injury. Whether it functions for cell repair or death remains to be elucidated. Copyright (C) 2003 John Wiley Sons, Ltd.

Platelet inhibitory effects of the nitric oxide donor drug MAHMA NONOate in vivo in rats

The platelet inhibitory effects of the nitric oxide (NO) donor drug MAHMA NONOate ((Z-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino] diazen-1-ium-1,2-diolate) were examined in anaesthetised rats and compared with those of S-nitrosoglutathione (GSNO; an S-nitrosothiol). Bolus administration of the aggregating agent ADP dose-dependently reduced the number of circulating free platelets. Intravenous infusions of MAHMA NONOate (3-30 nmol/kg/min) dose-dependently inhibited the effect of 0.3 mumol/kg ADP. MAHMA NONOate was approximately 10-fold more potent than GSNO. MAHMA NONOate (0.3-10 nmol/kg/min) also reduced systemic artery pressure and was again 10-fold more potent than GSNO. Thus MAHMA NONOate has both platelet inhibitory and vasodepressor effects in vivo. The dose ranges for these two effects overlapped, although blood pressure was affected at slightly lower doses. The platelet inhibitory effects compared favourably with those of GSNO, even though NONOates generate free radical NO which, in theory, could have been scavenged by haemoglobin. Therefore platelet inhibition may be a useful therapeutic property of NONOates. (C) 2003 Elsevier B.V. All rights reserved.

Generation of aldehyde-derived protein modifications in ethanol-exposed heart

Background: Although excessive ethanol consumption is known to lead to a variety of adverse effects in the heart, the molecular mechanisms of such effects have remained poorly defined. We hypothesized that posttranslational covalent binding of reactive molecular species to proteins occurs in the heart in response to acute ethanol exposure. Methods: The generation of protein adducts with several aldehydic species was examined by using monospecific antibodies against adducts with malondialdehyde (MDA), acetaldehyde (AA), MDA-AA hybrids, and hydroxyethyl radicals. Specimens of heart tissue were obtained from rats after intraperitoneal injections with alcohol (75 mmol/kg body weight) with or without pretreatment with cyanamide (0.05 mmol/kg body weight), an aldehyde dehydrogenase inhibitor. Results: The amounts of MDA and unreduced AA adducts were found to be significantly increased in the heart of the rats treated with ethanol, cyanamide, or both, whereas no other adducts were detected in statistically significant quantities. Immunohistochemical studies for characterization of adduct distribution revealed sarcolemmal adducts of both MDA and AA in the rats treated with ethanol and cyanamide in addition to intracellular adducts, which were also present in the group treated with ethanol alone. Conclusions: These findings support the role of enhanced lipid peroxidation and the generation of protein-aldehyde condensates in vivo as a result of excessive ethanol intake. These findings may have implications in the molecular mechanisms of cardiac dysfunction in alcoholics.

Simultaneous FT-NIR and ESR analyses to yield propagation rate coefficients for polymerization of methyl methacrylate based monomers

A hyphenated instrumental approach has been used to obtain reliable values for the propagation rate coefficients as a function of conversion for polymerizations of methyl methacrylate (MMA) and a mixture of MMA and ethyleneglycol dimethacrylate (EGDMA) with a 1:1 concentration of double bonds, from near the onset of the Trommsdorf region into the glass region. ESR spectroscopy was used to measure the radical concentration while FT-NIR fibre-optic spectroscopy was employed to measure instantaneously the double-bond concentration within the temperature-controlled cavity of the ESR instrument during polymerization. The advantage of this approach to the measurement of the rate coefficient is that it is equally applicable to branching and linear polymerizations. For the polymerization of methyl methacrylate, the values of the rate coefficient at the lowest conversions at which reliable values could be obtained were in agreement with recently reported values obtained by the PLP-SEC method. For the lowest conversions, the values obtained were 403 1 mol(-1) s(-1) at 306 K for MMA and 5201 mol(-1) s(-1) at 310 K for a 1:1 mixture of MMA and EGDMA. (C) 2003 Society of Chemical Industry.

S-Nitrosocaptopril: In vitro characterization of pulmonary vascular effects in rats

1 On rat isolated pulmonary arteries, vasorelaxation by S-nitrosocaptopril (SNOcap) was compared with S-nitrosoglutathione (GSNO) and nitroprusside, and inhibition by SNOcap of contractions to angiotensin I was compared with the angiotensin converting enzyme (ACE) inhibitor, captopril. 2 SNOcap was equipotent as a vasorelaxant on main (i.d. 2-3 mm) and intralobar (i.d. 600 mum)pulmonary arteries (pIC(50) values: 5.00 and 4.85, respectively). Vasorelaxant responses reached equilibrium rapidly (2-3 min). 3 Pulmonary vasorelaxant responses to SNOcap, like GSNO, were (i) partially inhibited by the soluble guanylate cyclase inhibitor, ODQ (1H-(1,2,4) oxadiazolo(4,3-a)-quinoxalin-1-one; 3 muM) whereas responses to nitroprusside were abolished and (ii) potentiated by hydroxocobalamin (HCOB; NO. free radical scavenger; 100 muM) whereas responses to nitroprusside were inhibited. 4 The relative potencies for pulmonary vasorelaxation compared with inhibition of platelet aggregation were: SNOcap 7: 1; GSNO 25: 1; nitroprusside > 2000:1. 5 SNOcap, like captopril, concentration-dependently and time-dependently increased the EC50 for angiotensin I but not angiotensin II. The dependence on incubation time was independent of the presence of tissue but differed for SNOcap and captopril. This difference reflected the slow dissociation of SNOcap and instability of captopril, and precluded a valid comparison of the potency of the two drugs. After prolonged incubation (greater than or equal to 5.6 h) SNOcap was more effective than captopril. 6 Thus, in pulmonary arteries SNOcap (i) possesses NO donor properties characteristic of S-nitrosothiols but different from nitroprusside and (ii) inhibits ACE at least as effectively as captopril. These properties suggest that SNOcap could be valuable in the treatment of pulmonary hypertension.

The impact of carboxy nitroxide antioxidants on irradiated ataxia telangiectasia cells

Three water-soluble carboxy nitroxide antioxidants, 5-carboxy-1,1,3,3-tetramethylisoindolin-2-yloxyl, 4-carboxy-2,2,6,6-tetramethylpiperidin-1-yloxyl, and 3-carboxy-2,2,5,5-tetramethylpyrrolidin-1-yloxyl, show significant impact on the postirradiation survival rates of ataxia telangiectasia (A-T) cells compared to normal cells, an assay which represents a model for understanding the impact of ROS damage on the A-T phenotype. The effects of these antioxidants are much more significant than those of vitamin E or Trolox (a water-soluble vitamin E analog), studied using the same cell survival model. (C) 2004 Elsevier Inc. All rights reserved.

Glutamate-mediated excitotoxicity and neurodegeneration in Alzheimer's disease

Alzheimer's disease (AD) is the most common form of dementia, accounting for 60-70% of cases in subjects over 65 years of age. Several postulates have been put forward that relate AD neuropathology to intellectual and functional impairment. These range from free-radical-induced damage, through cholinergic dysfunction, to beta-amyloid-induced toxicity. However, therapeutic strategies aimed at improving the cognitive symptoms of patients via choline supplementation, cholinergic stimulation or beta-amyloid vaccination, have largely failed. A growing body of evidence suggests that perturbations in systems using the excitatory amino acid L-glutamate (L-Glu) may underlie the pathogenic mechanisms of (e.g.) hypoxia-ischemia, epilepsy, and chronic neurodegenerative disorders such as Huntington's disease and AD. Almost all neurons in the CNS carry the N-methyl-D-aspartate (NMDA) subtype of ionotropic L-glutamate receptors, which can mediate post-synaptic Ca2+ influx. Excitotoxicity resulting from excessive activation of NMDA receptors may enhance the localized vulnerability of neurons in a manner consistent with AD neuropathology, as a consequence of an altered regional distribution of NMDA receptor subtypes. This review discusses mechanisms for the involvement of the NMDA receptor complex and its interaction with polyamines in the pathogenesis of AD. NMDA receptor antagonists have potential for the therapeutic amelioration of AD. (C) 2004 Elsevier Ltd. All rights reserved.

An investigation of the nitroxide-mediated preirradiation grafting of styrene onto PFA

Simultaneous and preirradiation grafting of styrene onto fluorinated polyolefins does not enable control of the molecular weights or polydispersities of the styrene grafts. The nitroxide-mediated grafting of styrene onto PFA with TEMPO and TEISO using a preirradiation method has been investigated as a means of controlling the graft properties and especially to produce grafts with improved suitability for SPOC. The yields of graft were found to be in the range 15-20% for nitroxide concentrations between 5 x 10(-3) and 2 x 10(-2) M and were similar for the two nitroxides studied. Raman mapping was used to obtain the depth profile for the styrene grafts. The grafts were found to be principally located within the PFA substrate, and little graft was formed at the PFA surface. Fmoc loading tests were performed to assess the suitability of the grafted PFA as a support for SPOC, but these showed no significant loading was achieved, thus indicating that the graft properties are not suitable for SPOC. However, the study has important implications for the applications of PFA-grafted polymers in other areas, such as chemically resistant ion-exchange and separation membranes.

Mutation in mitochondrial DNA as a cause of presbyacusis

Much of the hearing loss that occurs in old age is likely to be due to the long-term deterioration of the mitochondria in the different structures of the cochlea. The current review surveys some of the basic information on mitochondria and mitochondrial DNA, as a background to their possible involvement in presbyacusis. It is likely that oxygen radicals damage mitochondrial DNA and other components of the mitochondria, such as their proteins and lipids. This further compromises both oxidative phosphorylation and the repair processes in mitochondria, setting up a vicious cycle of degradation. Evidence is presented from inherited point mutations on the possibly most critical sites for mutations in mitochondrial DNA associated with hearing loss. It is suggested that random sorting and clonal expansion of mutations both maintain the integrity of the pool of mitochondrial DNA molecules and give rise to the apoptosis that leads to loss of vulnerable cells, and hence to deafness. It is moreover suggested that apoptosis of the vulnerable cells of the inner ear may to some extent be preventable, or at least delayed. Copyright (C) 2004 S. Karger AG, Basel.

Transition-state-theory calculations for reactions of O(3P) with halogenated olefins

The potential energy surfaces for the reactions of atomic oxygen in its ground electronic state, O(P-3), with the olefins: CF2=CCl2 and CF2=CF - CF3, have been characterized using ab initio molecular orbital calculations. Geometry optimization and vibrational frequency calculations were performed for reactants, transition states and products at the MP2 and QCISD levels of theory using the 6-31G(d) basis set. This database was then used to calculate the rate constants by means of Transition-State-Theory. To obtain a better reference and to test the reliability of the activation barriers we have also carried out computations using the CCSD(T)(fc)/6-311Gdagger, MP4(SDQ)(fc)/CBSB4 and MP2(fc)/CBSB3 single point energy calculations at both of the above levels of theory, as well as with the composite CBS-RAD procedure ( P. M. Mayer, C. J. Parkinson, D. M. Smith and L. Radom, J. Chem. Phys., 1998, 108, 604) and a modi. cation of this approach, called: CBS-RAD( MP2, MP2). It was found that the kinetic parameters obtained in this work particularly with the CBS-RAD ( MP2, MP2) procedure are in reasonable agreement with the experimental values. For both reactions it is found that the channels leading to the olefin double-bond addition predominates with respect to any other reaction pathway. However, on account of the different substituents in the alkenes we have located, at all levels of theory, two transition states for each reaction. Moreover, we have found that, for the reactions studied, a correlation exists between the activation energies and the electronic structure of the transition states which can explain the influence of the substituent effect on the reactivity of the halo-olefins.