RESUMEN
Nitric oxide (NO) performs a central role in biological systems, binding to the heme site of soluble guanylyl cyclase (sGC), leading to enzyme activation and elevation of intracellular levels of cGMP. Organic nitrates, in particular, nitroglycerin (GTN), are clinically important nitrovasodilators that function as NO-mimetics in biological systems. Comparison of sGC activation data with electrochemically measured rates of NO release for genuine NO donors, NONOates and nitrosothiols, yields an excellent correlation between the EC(50) for sGC activation and the rate constant for NO release, k(NO). However, activation of sGC by GTN and the nitrates has very different characteristics, including the requirement for specific added thiols, for example, cysteine. The reaction of GTN with cysteine in anaerobic solution yields NO slowly, and NO release, measured by chemiluminescence detection, is quenched by added metal ion chelator. The generation of NO under aerobic conditions is 100-fold slower than the anaerobic reaction. Furthermore, NO release from the reaction of GTN with cysteine in phosphate buffer is too slow to account for sGC activation by GTN/cysteine. The slow rate of the chemical reaction to release NO suggests that nitrates can activate sGC by an NO-independent mechanism. In contrast to the genuine NO donors, GTN behaves as a partial agonist with respect to sGC activation, but in the presence of the allosteric sGC activator, YC-1, GTN exhibits full agonist activity.
Asunto(s)
Activadores de Enzimas/farmacología , Guanilato Ciclasa/metabolismo , Donantes de Óxido Nítrico/metabolismo , Óxido Nítrico/metabolismo , Animales , Aorta/enzimología , Cisteína/farmacología , Activación Enzimática/efectos de los fármacos , Activadores de Enzimas/metabolismo , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Glutatión/análogos & derivados , Glutatión/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Hidrazinas/farmacología , Indazoles/farmacología , Imitación Molecular , Nitratos/farmacología , Donantes de Óxido Nítrico/farmacología , Nitroglicerina/farmacología , Compuestos Nitrosos/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , Ratas , S-Nitrosoglutatión , SolubilidadRESUMEN
[structure: see text]. The important biological secondary messenger NO can be generated from exogenous nitrovasodilators and NO donors. Nitrate esters are nitrovasodilators and NO mimetics, believed to be biotransformed to NO in vivo. On the basis of a mechanistic hypothesis, nitrates have been synthesized that release NO at significant rates in neutral aqueous solution in the presence only of added thiol. The novel masked beta-mercaptonitrates reported (SS-nitrates), provide information on possible sulfhydryl-dependent biotransformation mechanisms for nitrates in clinical use.
Asunto(s)
Ésteres/química , Óxido Nítrico/química , Electroquímica , Modelos Químicos , Compuestos de Sulfhidrilo/químicaRESUMEN
Unraveling the biology, pharmacology, and toxicology of NO depends on accurate NO assays, two of the more common being the oxyHb (oxyhemoglobin) assay and potentiometric detection using a Clark-type NO-selective electrode. Comparison of the specificity and sensitivity of the oxyHb and potentiometric methods was carried out using a broad series of nitrovasodilators, including organic nitrates, nitrites, thionitrates, nitrosothiols, and diazenium diolates. Only with the more labile diazenium diolates was a linear relationship observed between the rates of NO release measured potentiometrically and the rate of oxyHb oxidation from the oxyHb assay. The nonlinear plots indicate that N,O-species other than NO itself are capable of oxidizing oxyHb.
Asunto(s)
Óxido Nítrico/análisis , Oxihemoglobinas/química , Vasodilatadores/química , Calibración , Colorimetría , Hidrólisis , Cinética , Oxidación-Reducción , PotenciometríaRESUMEN
The water-soluble antioxidant, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic (Trolox), (4-14C)-labelled, was used to trace its location in the aqueous and lipid phases of liposomes. Trolox was found to partition 20 to 25% into the lipid phase of multilamellar (MLV) and 38-46% into the lipid phase of unilamellar (ULV) egg lecithin liposomes. Trolox and its oxidation products partition readily (40%) into the lipid phase of dilinoleoylphosphatidylcholine (DLPC) MLV liposomes during inhibited peroxidation, thermally initiated by azo-bis(2,4-dimethylvaleronitrile) (ADVN). The time-course of the consumption of Trolox during peroxidation of DLPC liposomes, initiated by ADVN, was followed by separation and analyses of [4-14C]Trolox and its oxidation products. Such studies showed that the consumption of Trolox followed the profile of the inhibition of oxygen uptake. This indicates that Trolox can be used in quantitative studies of membrane peroxidation; for example, to measure the rate of chain initiation (Ri). The product distribution of hydroperoxides, the 9- and 13-cis,trans (c,t) and trans,trans (t,t) isomers, formed during inhibited peroxidation of linoleate, in DLPC and methyl linoleate in dimyristoyl PC (DMPC) liposomes was determined by HPLC of the derived hydroxy methyl esters. The c,t/t,t (kinetic/thermodynamic) ratios were related to the antioxidant activity of the inhibitors. Both Trolox and alpha-tocopherol (vitamin E) gave relatively high initial c,t/t,t ratios (6.6 and 7.1) during inhibited peroxidation of DLPC, initiated by water-soluble azo-bis(2-amidinopropane.HCl) (ABAP). High initial c,t/t,t ratios (6.2) were also observed for alpha-tocopherol-inhibited peroxidation of DLPC liposomes, initiated by lipid-soluble ADVN. On the other hand, the combination of Trolox with ADVN-initiated peroxidation of DLPC or of methyl linoleate in DMPC gave relatively low initial c,t/t,t ratios of 3.5 and 1.3. These results are interpreted in terms of the relative hydrogen atom donating ability of the antioxidants and the homogeneity of the system used. The 9/13 ratios of hydroperoxides were constant (0.9 to 1.0) in all experiments and did not give evidence for preferential trapping by Trolox of peroxyls at the 9-position.