RESUMEN
BACKGROUND AND PURPOSE: The antioxidant 5-hydroxymethylfurfural (5-HMF) exerts documented beneficial effects in several experimental pathologies and is currently tested as an antisickling drug in clinical trials. In the present study, we examined the cardiovascular effects of 5-HMF and elucidated the mode of action of the drug. EXPERIMENTAL APPROACH: The cardiovascular effects of 5-HMF were studied with pre-contracted porcine coronary arteries and rat isolated normoxic-perfused hearts. Isolated hearts subjected to ischaemia/reperfusion (I/R) injury were used to test for potential cardioprotective effects of the drug. The effects of 5-HMF on action potential and L-type Ca2+ current (ICa,L ) were studied by patch-clamping guinea pig isolated ventricular cardiomyocytes. KEY RESULTS: 5-HMF relaxed coronary arteries in a concentration-dependent manner and exerted negative inotropic, lusitropic and chronotropic effects in rat isolated perfused hearts. On the other hand, 5-HMF improved recovery of inotropic and lusitropic parameters in isolated hearts subjected to I/R. Patch clamp experiments revealed that 5-HMF inhibits L-type Ca2+ channels. Reduced ICa,L density, shift of ICa,L steady-state inactivation curves toward negative membrane potentials and slower recovery of ICa,L from inactivation in response to 5-HMF accounted for the observed cardiovascular effects. CONCLUSIONS AND IMPLICATIONS: Our data revealed a cardioprotective effect of 5-HMF in I/R that is mediated by inhibition of L-type Ca2+ channels. Thus, 5-HMF is suggested as a beneficial additive to cardioplegic solutions, but adverse effects and contraindications of Ca2+ channel blockers have to be considered in therapeutic application of the drug.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/fisiología , Cardiotónicos/farmacología , Vasos Coronarios/efectos de los fármacos , Furaldehído/análogos & derivados , Miocitos Cardíacos/efectos de los fármacos , Animales , Vasos Coronarios/fisiología , Femenino , Furaldehído/farmacología , Cobayas , Ventrículos Cardíacos/citología , Masculino , Miocitos Cardíacos/fisiología , Ratas Sprague-Dawley , Daño por Reperfusión/fisiopatología , PorcinosRESUMEN
BACKGROUND AND PURPOSE: L-gulonolactone oxidase-deficient (Gulo((-/-))) mice were used to study the effects of ascorbate deficiency on aortic relaxation by nitroglycerin (GTN) with focus on changes in the expression and activity of vascular aldehyde dehydrogenase-2 (ALDH2), which catalyses GTN bioactivation. EXPERIMENTAL APPROACH: Ascorbate deficiency was induced in Gulo((-/-)) mice by ascorbate deprivation for 4 weeks. Some of the animals were concomitantly treated with the proteasome inhibitor bortezomib and effects compared with ascorbate-supplemented Gulo((-/-)), untreated or nitrate-tolerant wild-type mice. Aortic relaxation of the experimental groups to GTN, ACh and a NO donor was studied. Changes in mRNA and protein expression of vascular ALDH2 were quantified by qPCR and immunoblotting, respectively, and aortic GTN denitration rates determined. KEY RESULTS: Like GTN treatment, ascorbate deprivation induced vascular tolerance to GTN that was associated with markedly decreased rates of GTN denitration. Ascorbate deficiency did not affect ALDH2 mRNA levels, but reduced ALDH2 protein expression and the total amount of ubiquitinated proteins to about 40% of wild-type controls. These effects were largely prevented by ascorbate supplementation or treating Gulo((-/-)) mice with the 26S proteasome inhibitor bortezomib. CONCLUSIONS AND IMPLICATIONS: Our data indicate that ascorbate deficiency results in vascular tolerance to GTN via proteasomal degradation of ALDH2. The results support the view that impaired ALDH2-catalysed metabolism of GTN contributes significantly to the development of vascular nitrate tolerance and reveal a hitherto unrecognized protective effect of ascorbate in the vasculature.
Asunto(s)
Aldehído Deshidrogenasa/genética , Aorta/metabolismo , Tolerancia a Medicamentos , Modelos Animales , Nitroglicerina/administración & dosificación , Complejo de la Endopetidasa Proteasomal/metabolismo , Aldehído Deshidrogenasa/metabolismo , Aldehído Deshidrogenasa Mitocondrial , Animales , Deficiencia de Ácido Ascórbico/genética , Deficiencia de Ácido Ascórbico/metabolismo , Ácidos Borónicos/farmacología , Bortezomib , Femenino , Regulación Enzimológica de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Pirazinas/farmacología , UbiquitinaciónRESUMEN
BACKGROUND AND PURPOSE: Adipose triglyceride lipase (ATGL) has been identified as a rate-limiting enzyme of mammalian triglyceride catabolism. Deletion of the ATGL gene in mice results in severe lipid accumulation in a variety of tissues including the heart. In the present study we investigated cardiac function in ATGL-deficient mice and the potential therapeutic effects of the PPARα and γ agonists Wy14,643 and rosiglitazone, respectively. EXPERIMENTAL APPROACH: Hearts isolated from wild-type (WT) mice and ATGL(-/-) mice treated with Wy14,643 (PPARα agonist), rosiglitazone (PPARγ agonist) or vehicle were perfused at a constant flow using the Langendorff technique. Left ventricular (LV) pressure-volume relationships were established, and the response to adrenergic stimulation was determined with noradrenaline (NA). KEY RESULTS: Hearts from ATGL(-/-) mice generated higher LV end-diastolic pressure and lower LV developed pressure as a function of intracardiac balloon volume compared to those from WT mice. Likewise, passive wall stress was increased and active wall stress decreased in ATGL(-/-) hearts. Contractile and microvascular responses to NA were substantially reduced in ATGL(-/-) hearts. Cardiac contractility was improved by treating ATGL(-/-) mice with the PPARα agonist Wy14,643 but not with the PPARγ agonist rosiglitazone. CONCLUSIONS AND IMPLICATIONS: Our results indicate that lipid accumulation in mouse hearts caused by ATGL gene deletion severely affects systolic and diastolic function, as well as the response to adrenergic stimulation. The beneficial effects of Wy14,643 suggest that the cardiac phenotype of these mice is partially due to impaired PPARα signalling.
Asunto(s)
Anticolesterolemiantes/farmacología , Corazón/efectos de los fármacos , Lipasa/fisiología , PPAR alfa/agonistas , Pirimidinas/farmacología , Animales , Peso Corporal/efectos de los fármacos , Femenino , Corazón/fisiopatología , Frecuencia Cardíaca/efectos de los fármacos , Técnicas In Vitro , Lipasa/deficiencia , Lipasa/genética , Masculino , Ratones , Ratones Noqueados , Miocardio/patología , Norepinefrina/farmacología , Tamaño de los Órganos/efectos de los fármacos , PPAR gamma/agonistas , Rosiglitazona , Tiazolidinedionas/farmacología , Disfunción Ventricular Izquierda/patología , Disfunción Ventricular Izquierda/fisiopatologíaRESUMEN
The neuronal and endothelial isoforms of nitric oxide (NO) synthase (nNOS and eNOS, respectively) both catalyze the production of NO but are regulated differently. Stably transfected HEK 293 cell lines containing nNOS, eNOS, and a soluble mutant of eNOS were therefore established to compare their activity in a common cellular environment. NOS activity was determined by measuring L-[3H]citrulline production in homogenates and intact cells, the conversion of oxyhemoglobin to methemoglobin, and the production of cGMP. The results indicate that nNOS is more active than eNOS, both in unstimulated as well as calcium-stimulated cells. Under basal conditions, the soluble mutant of eNOS appeared to be slightly more active than wild-type eNOS in terms of NO and cGMP formation, suggesting that membrane association may be crucial for inhibition of basal NO release but is not required for stimulation by Ca2+-mobilizing agents. The maximal activity of soluble guanylate cyclase was significantly reduced by transfection with wild-type eNOS due to downregulation of mRNA expression. These results demonstrate that nNOS and eNOS behave differently even in an identical cellular environment.
Asunto(s)
Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Cloruro de Calcio/farmacología , Línea Celular , Citrulina/farmacocinética , GMP Cíclico/metabolismo , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Inhibidores Enzimáticos/farmacología , Regulación Enzimológica de la Expresión Génica , Guanilato Ciclasa/metabolismo , Humanos , Ionomicina/farmacología , Ionóforos/farmacología , Riñón/citología , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo III , Solubilidad , Tapsigargina/farmacología , Transfección , TritioRESUMEN
YC-1 is a direct activator of soluble guanylyl cyclase (sGC) and sensitizes the enzyme for activation by nitric oxide (NO) and CO. Because the potentiating effect of YC-1 on NO-induced cGMP formation in platelets and smooth muscle cells has been shown to be substantially higher than observed with the purified enzyme, the synergism between heme ligands and YC-1 is apparently more pronounced in intact cells than in cell-free systems. Here, we investigated the mechanisms underlying the synergistic activation of sGC by YC-1 and NO in endothelial cells. Stimulation of the cells with YC-1 enhanced cGMP accumulation up to approximately 100-fold. The maximal effect of YC-1 was more pronounced than that of the NO donor DEA/NO (approximately 20-fold increase in cGMP accumulation) and markedly diminished in the presence of L-N(G)-nitroarginine, EGTA, or oxyhemoglobin. Because YC-1 did not activate endothelial NO synthase, the pronounced effect of YC-1 on cGMP accumulation was apparently caused by a synergistic activation of sGC by YC-1 and basal NO. The effect of YC-1 was further enhanced by addition of DEA/NO, resulting in a approximately 160-fold stimulation of cGMP accumulation. Thus, YC-1 increased the NO-induced accumulation of cGMP in intact cells by approximately 8-fold. Addition of endothelial cell homogenate increased the stimulatory effect of YC-1 on NO-activated purified sGC from 1.2- to 3.7-fold. This effect was not observed with heat-denatured homogenates, suggesting that a heat-labile factor present in endothelial cells potentiates the effect of YC-1 on NO-activated sGC.
Asunto(s)
Endotelio Vascular/efectos de los fármacos , Activadores de Enzimas/farmacología , Guanilato Ciclasa/metabolismo , Indazoles/farmacología , Óxido Nítrico/farmacología , Animales , Células Cultivadas , GMP Cíclico/metabolismo , Sinergismo Farmacológico , Endotelio Vascular/enzimología , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , PorcinosRESUMEN
To investigate the role of tetrahydrobiopterin (BH4) in the catalytic mechanism of nitric oxide synthase (NOS), we analyzed the spectral changes following addition of oxygen to the reduced oxygenase domain of endothelial nitric oxide synthase (NOS) in the presence of different pteridines at -30 degrees C. In the presence of N(G)-hydroxy-L-arginine (NOHLA) and BH4 or 5-methyl-BH4, both of which support NO synthesis, the first observable species were mixtures of high-spin ferric NOS (395 nm), ferric NO-heme (439 nm), and the oxyferrous complex (417 nm). With Arg, no clear intermediates could be observed under the same conditions. In the presence of the BH4-competitive inhibitor 7,8-dihydrobiopterin (BH2), intermediates with maxima at 417 and 425 nm were formed in the presence of Arg and NOHLA, respectively. In the presence of 4-amino-BH4, the maxima of the intermediates with Arg and NOHLA were at 431 and 423 nm, respectively. We ascribe all four spectra to oxyferrous heme complexes. The intermediates observed in this study slowly decayed to the high-spin ferric state at -30 degrees C, except for those formed in the presence of 4-amino-BH4, which required warming to room temperature for regeneration of high-spin ferric NOS; with Arg, regeneration remained incomplete. From these observations, we draw several conclusions. (1) BH4 is required for reductive oxygen activation, probably as a transient one-electron donor, not only in the reaction with Arg but also with NOHLA; (2) in the absence of redox-active pterins, reductive oxygen activation does not occur, which results in accumulation of the oxyferrous complex; (3) the spectral properties of the oxyferrous complex are affected by the presence and identity of the substrate; (4) the slow and incomplete formation of high-spin ferric heme with 4-amino-BH4 suggests a structural cause for inhibition of NOS activity by this pteridine.
Asunto(s)
Arginina/análogos & derivados , Biopterinas/análogos & derivados , Biopterinas/química , Óxido Nítrico Sintasa/química , Animales , Arginina/química , Biopterinas/metabolismo , Encéfalo , Catálisis , Ditionita/química , Glicol de Etileno/química , Congelación , Isoenzimas/química , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Óxido Nítrico Sintasa de Tipo III , Oxidación-Reducción , Oxígeno/química , Pterinas/química , Ratas , Sustancias Reductoras/química , EspectrofotometríaRESUMEN
OBJECTIVE: L-Ascorbic acid has been described to exert multiple beneficial effects in cardiovascular disorders associated with impaired nitric oxide (NO)/cGMP signalling. The aim of the present study was to investigate the effect of vitamin C on the most prominent physiological target of endogenous and exogenous NO, i.e. soluble guanylyl cyclase (sGC). METHODS: To address this issue we used a highly purified enzyme preparation from bovine lung (from the slaughterhouse). Enzymic activity was measured by a standard assay based on the conversion of [alpha-32P]GTP to [32P]cGMP and the subsequent quantification of the radiolabelled product. NO was quantified using a commercially available Clark-type electrode. RESULTS: Stimulation of sGC by the NO donor 2, 2-diethyl-1-nitroso-oxyhydrazine was inhibited by ascorbate with an IC(50) of approximately 2 microM. Maximal enzyme inhibition ( approximately 70%) was observed at 0.1-1 mM vitamin C. Stimulation of sGC by the NO-independent activator protoporphyrin-IX was also inhibited with similar potency. The effect of ascorbate on sGC was largely antagonised by reduced glutathione (1 mM) and the specific iron chelator diethylenetriaminepentaacetic acid (0.1 mM). Electrochemical experiments revealed that NO is potently scavenged by vitamin C. Consumption of NO by ascorbate was prevented by reduced glutathione (1 mM), diethylenetriaminepentaacetic acid (0.1 mM) and superoxide dismutase (500 units/ml) whereas up to 5000 units/ml superoxide dismutase failed to restore sGC activity. CONCLUSIONS: Our results suggest that physiological concentrations of L-ascorbic acid diminish cGMP accumulation via both scavenging of NO and direct inhibition of sGC.
Asunto(s)
Antioxidantes/farmacología , Ácido Ascórbico/farmacología , Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Electroquímica , Hidrazinas/farmacología , Pulmón/enzimología , Óxido Nítrico/análisis , Donantes de Óxido Nítrico/farmacología , Compuestos Nitrosos/farmacología , Oxidación-ReducciónRESUMEN
Nitric oxide synthase (NOS) catalysis results in formation of NO or superoxide (O(2)(-.)) depending on the presence or absence of the cofactor tetrahydrobiopterin (BH4). In the absence of O(2)(-.) scavengers, net NO formation cannot be detected even at saturating BH4 concentrations, which is thought to be due to O(2)(-.) production by BH4 autoxidation. Because the N-5-methylated analogue of BH4 (5-Me-BH4) sustains NOS catalysis and is autoxidation-resistant, net NO formation by the neuronal isoform of NOS (nNOS) can be observed at saturating 5-Me-BH4 concentrations. Here we compare the effects of 5-Me-BH4 on L-citrulline formation, NADPH oxidation, H(2)O(2) production and soluble guanylate cyclase (sGC) stimulation. All activities were stimulated biphasically (EC(50) approx. 0.2 microM and more than 1 mM), with an intermediate inhibitory phase at the same pterin concentration as that required for net NO generation and sGC stimulation (4 microM). Concomitantly with inhibition, the NADP(+)/L-citrulline stoichiometry decreased from 2.0 to 1.6. Inhibition occurred only at high enzyme concentrations (IC(50) approx. 10 nM nNOS) and was antagonized by oxyhaemoglobin and by BH4. We ascribe the first stimulatory phase to high-affinity binding of 5-Me-BH4. The inhibitory phase is due to low-affinity binding, resulting in fully coupled catalysis, complete inhibition of O(2)(-.) production and net NO formation. At high enzyme concentrations and thus high NO levels, this causes autoinhibition. NO scavenging by 5-Me-BH4 at concentrations above 1 mM, resulting in the antagonization of inhibition of NOS, explains the second stimulatory phase. In agreement with these assignments 5-Me-BH4 was found to stimulate formation of a haem-NO complex during NOS catalysis. The observation of inhibition with 5-Me-BH4 but not with BH4 implies that, unless O(2)(-.) scavengers are present, a physiological role for NO-induced autoinhibition is unlikely.
Asunto(s)
Biopterinas/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico/farmacología , Pteridinas/metabolismo , Animales , Arginina/metabolismo , Biopterinas/análogos & derivados , Biopterinas/farmacología , Catalasa/metabolismo , Catálisis , Bovinos , Citrulina/metabolismo , Dimerización , Depuradores de Radicales Libres/metabolismo , Guanilato Ciclasa/metabolismo , Hemo/metabolismo , Peróxido de Hidrógeno/metabolismo , NADP/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Oxidantes/metabolismo , Pteridinas/farmacología , Ratas , Superóxido Dismutasa/metabolismo , Superóxidos/metabolismoRESUMEN
We investigated the effects of salts on the properties of the neuronal, endothelial, and inducible isoforms of nitric oxide synthase (nNOS, eNOS, and iNOS), and found pronounced isoform-specific effects on NOS-catalyzed L-citrulline formation. Salts inhibited iNOS monotonously, whereas nNOS and eNOS were stimulated up to 3-fold at low, and inhibited at high (>/=0.1-0.2 M) salt concentrations. The effectivities of different ions mostly followed the Hofmeister series, indicating that the effects can for a large part be ascribed to changes in protein solvation. Km(Arg) increased in the presence of NaCl, demonstrating the importance of charge interactions for substrate binding. The coupling of NADPH oxidation to NO production was not affected by KCl. Salts (=1 M) had no major impact on the tertiary and quaternary structure, or on the state of the heme. Extrapolation of these results to commonly applied experimental conditions for in vitro activity assays suggests that true specific activities of nNOS and eNOS may, in some cases, be underestimated as much as 3-fold.
Asunto(s)
Isoenzimas/metabolismo , Óxido Nítrico Sintasa/metabolismo , Sales (Química)/farmacología , Animales , Aniones/farmacología , Arginina/metabolismo , Cationes/farmacología , Cinética , Ratones , NADP/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintasa/clasificación , Cloruro de Potasio/farmacología , Conformación Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , RatasRESUMEN
Soluble guanylyl cyclase (sGC) is the major physiological target of sydnonimine-based vasodilators such as molsidomine. Decomposition of sydnonimines results in the stoichiometric formation of nitric oxide (NO) and superoxide (O2-), which rapidly react to form peroxynitrite. Inasmuch as sGC is activated by NO but not by peroxynitrite, we investigated the mechanisms underlying sGC activation by 3-morpholinosydnonimine (SIN-1). Stimulation of purified bovine lung sGC by SIN-1 was found to be strongly dependent on glutathione (GSH). By contrast, GSH did not affect sGC activation by NO released from 2,2-diethyl-1-nitroso-oxyhydrazine, indicating that NO/O2- released from SIN-1 converted GSH to an activator of sGC. High performance liquid chromatography identified this product as the thionitrite S-nitrosoglutathione. Further, the reaction product decomposed to release NO upon addition of Cu(NO3)2 in the presence of GSH. Activation of sGC was antagonized by the Cu(I)-specific chelator neocuproine, whereas the Cu(II)-selective drug cuprizone was less potent. Carbon dioxide (delivered as NaHCO3) antagonized S-nitrosation by peroxynitrite but not by SIN-1. Thus, NO/O2- released from SIN-1 mediates a CO2-insensitive conversion of GSH to S-nitrosoglutathione, a thionitrite that activates sGC via trace metal-catalyzed release of NO. These results may provide novel insights into the molecular mechanism underlying the nitrovasodilator action of SIN-1.
Asunto(s)
Glutatión/análogos & derivados , Guanilato Ciclasa/efectos de los fármacos , Molsidomina/análogos & derivados , Compuestos Nitrosos/metabolismo , Vasodilatadores/farmacología , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Activación Enzimática , Glutatión/metabolismo , Guanilato Ciclasa/metabolismo , Molsidomina/metabolismo , Molsidomina/farmacología , Óxido Nítrico/metabolismo , S-Nitrosoglutatión , Superóxidos/metabolismo , Vasodilatadores/metabolismoRESUMEN
Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), described as a superoxide dismutase mimetic and peroxynitrite scavenger, has been used previously to investigate the cytotoxic potential of superoxide and peroxynitrite in several pathological models. Here we report on the interference of MnTMPyP with NO/cGMP signaling using cultured endothelial cells as well as purified soluble guanylyl cyclase (sGC) either activated by the NO donor 2,2-diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO) or reconstituted with nitric oxide synthase (NOS). MnTMPyP inhibited endothelial cGMP accumulation induced by A23187 (0.3 microM) with an IC50 of 75.0 +/- 10.4 microM but had no significant effect on the potency of the Ca2+ ionophore. Purified NOS was inhibited by MnTMPyP (IC50 = 5.5 +/- 0.8 microM) because of an interference of the Mn-porphyrin with the reductase domain of the enzyme. The most pronounced actions of MnTMPyP were direct inhibition of sGC and scavenging of NO. Purified sGC stimulated with either Ca2+/calmodulin-activated NOS (in the presence of GSH) or DEA/NO (in the absence of GSH) was inhibited with IC50 values of 0.8 +/- 0.09 microM and 0.6 +/- 0.2 microM, respectively. In the presence of GSH, MnTMPyP was reduced to the Mn(II) complex, resulting in efficient scavenging of NO under these conditions. Our data demonstrate that MnTMPyP (i) interferes with the reductase domain of NOS, (ii) scavenges NO in the presence of GSH, and (iii) is a potent direct inhibitor of sGC. These results cast doubt on the usefulness of MnTMPyP and related Mn-porphyrin complexes as probes to study the involvement of peroxynitrite/superoxide in biological systems.
Asunto(s)
Depuradores de Radicales Libres/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Metaloporfirinas/farmacología , Nitratos/metabolismo , Óxido Nítrico Sintasa/antagonistas & inhibidores , Óxido Nítrico/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/enzimología , Endotelio Vascular/metabolismo , Depuradores de Radicales Libres/química , Glutatión/metabolismo , Humanos , Manganeso/química , Metaloporfirinas/química , Ratas , Solubilidad , Espectrofotometría , PorcinosRESUMEN
Low-molecular-mass thiols and nitric oxide (NO) form S-nitrosothiols (thionitrites) in the presence of oxygen. Thionitrites play an integral role in a variety of NO-dependent physiological processes. This study describes a sensitive analytical method for the quantitative determination of thionitrites. The method is based on the Cu+-catalyzed homolytic cleavage of thionitrites and electrochemical detection of the released NO with a Clark-type electrode. Cu+ was generated by addition of Cu(NO3)2 to samples containing 1 mM GSH or 4 mM L-cysteine as reducing agents. The effect of Cu(NO3)2 on the release of NO from GSNO was concentration-dependent. In the presence of 1 mM GSH, the EC50 for Cu(NO3)2 was 1.34 +/- 0.08 mM. Using cysteine instead of GSH, NO release was quantitative at much lower concentrations of Cu(NO3)2 (EC50 = 8.5 +/- 2.8 microM. NO release was not significantly affected by pH (7.0-9.0) and was inhibited by the Cu+-selective chelator neocuproine, whereas the Cu2+ chelator cuprizone was approximately 16-fold less potent. Calibration of the method with GSNO, S-nitroso-N-acetyl-penicillamine, or S-nitrosated bovine serum albumin yielded linear plots of initial rates of NO release versus thionitrite concentration from 50 nM to 5 microM. This method may be useful for the quantitative determination of thionitrites in biological samples.
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Electroquímica/métodos , Electrodos , Óxido Nítrico/análisis , Compuestos Nitrosos/análisis , Compuestos de Sulfhidrilo/análisis , Animales , Bovinos , Cobre , Electroquímica/instrumentación , Glutatión/análogos & derivados , Glutatión/análisis , Penicilamina/análogos & derivados , Penicilamina/análisis , S-Nitroso-N-Acetilpenicilamina , S-Nitrosoglutatión , Albúmina Sérica Bovina/análisisRESUMEN
We investigated how pH affects rat brain neuronal nitric oxide synthase (nNOS) with regard to spin-state equilibrium and the thiolate ligand bond of the haem group, catalytic activity, and monomerleft and right arrow dimer equilibrium. At neutral pH, nNOS containing 1 equiv. of (6R)-5,6,7,8-tetrahydro-l-biopterin (BH4) per dimer was mostly high-spin (lambdamax at 398 nm), whereas the BH4-free enzyme consisted of a mixture of the high-spin and two low-spin forms (lambdamax at 418 nm, and at 376 and 456 nm respectively). With BH4-free nNOS, an appreciable high-spin fraction was only observed between pH 7 and 8; at pH 6 and 9, the 418 and 376/456 nm low-spin forms predominated respectively. With nNOS containing 1 equiv. of BH4 per dimer, similar observations were made, but these involved only half of the enzyme; the other half, presumably the BH4-containing subunits, remained high-spin. Since the spin state in the BH4-free subunit appeared little affected by the state of the other subunit, we conclude that, in dimeric nNOS, the two haem groups function independently. Low pH destabilized thiolate binding and the interaction between NOS subunits, as indicated by CO-binding studies and gel electrophoresis respectively. Formation of l-citrulline was optimal between pH 7.0 and 7.5; the decrease in NOS activity at lower pH proved to be due to uncoupling of NADPH oxidation, resulting in increased formation of H2O2. At high pH strict coupling of l-arginine and NADPH oxidation was maintained, even in the absence of exogenous BH4. The possible pathophysiological implications of the uncoupling at low pH are discussed.
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Proteínas del Tejido Nervioso/química , Óxido Nítrico Sintasa/química , Animales , Arginina/metabolismo , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Encéfalo/enzimología , Citrulina/metabolismo , Dimerización , Hemo/química , Peróxido de Hidrógeno/metabolismo , Concentración de Iones de Hidrógeno , NADP/metabolismo , Unión Proteica/fisiología , Conformación Proteica , Ratas , EspectrofotometríaRESUMEN
Nitric oxide (NO), a physiologically important activator of soluble guanylyl cyclase (sGC), is synthesized from L-arginine and O2 in a reaction catalyzed by NO synthases (NOS). Previous studies with purified NOS failed to detect formation of free NO, presumably due to a fast inactivation of NO by simultaneously produced superoxide (O-2). To characterize the products involved in NOS-induced sGC activation, we measured the formation of cyclic 3',5'-guanosine monophosphate (cGMP) by purified sGC incubated in the absence and presence of GSH (1 mM) with drugs releasing different NO-related species or with purified neuronal NOS. Basal sGC activity was 0.04 +/- 0.01 and 0.19 +/- 0.06 micromol of cGMP x mg-1 x min-1 without and with 1 mM GSH, respectively. The NO donor DEA/NO activated sGC in a GSH-independent manner. Peroxynitrite had no effect in the absence of GSH but significantly stimulated the enzyme in the presence of the thiol (3.45 +/- 0.60 micromol of cGMP x mg-1 x min-1). The NO/O-2 donor SIN-1 caused only a slight accumulation of cGMP in the absence of GSH but was almost as effective as DEA/NO in the presence of the thiol. The profile of sGC activation by Ca2+/calmodulin-activated NOS resembled that of SIN-1; at a maximally active concentration of 200 ng/0.1 ml, NOS increased sGC activity to 1.22 +/- 0.12 and 8.51 +/- 0.88 micromol of cGMP x mg-1 x min-1 in the absence and presence of GSH, respectively. The product of NOS and GSH was identified as the thionitrite GSNO, which activated sGC through Cu+-catalyzed release of free NO. In contrast to S-nitrosation by peroxynitrite, the novel NO/O-2-triggered pathway was very efficient (25-45% GSNO) and insensitive to CO2. Cu+-specific chelators inhibited bradykinin-induced cGMP release from rat isolated hearts but did not interfere with the direct activation of cardiac sGC, suggesting that thionitrites may occur as intermediates of NO/cGMP signaling in mammalian tissues.
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GMP Cíclico/metabolismo , Glutatión/análogos & derivados , Óxido Nítrico/metabolismo , Compuestos Nitrosos/metabolismo , Transducción de Señal , Animales , Activación Enzimática , Glutatión/metabolismo , Guanilato Ciclasa/metabolismo , Técnicas In Vitro , Miocardio/metabolismo , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Ratas , Proteínas Recombinantes/metabolismo , S-Nitrosoglutatión , Superóxido Dismutasa/metabolismoRESUMEN
To elucidate how thiols affect neuronal nitric oxide synthase (nNOS) we studied the binding of thiols to tetrahydrobiopterin (BH4)-free nNOS. Dithiothreitol (DTT), 2-mercaptoethanol, and L- and D-cysteine all bound to the heme with Kd values varying from 0.16 mM for DTT to 41 mM for L-cysteine. DTT, 2-mercaptoethanol, and L-cysteine yielded absorbance spectra with maxima at about 378 and 456 nm, indicative of bisthiolate complexes; the maximum at 426 nm with D-cysteine suggests binding of the neutral thiol. From the results with 2-mercaptoethanol we deduced that in 2-mercaptoethanol-free, BH4-free nNOS the sixth heme ligand is not a thiolate. DTT binding to nNOS containing one BH4 per dimer was biphasic. Apparently, the BH4-free subunit bound DTT with the same affinity as the BH4-free enzyme, whereas the BH4-containing subunit exhibited a > 100-fold lower affinity, indicative of competition between DTT and BH4 binding. Binding of DTT to the BH4-containing subunit was suppressed by L-arginine, whereas high-affinity binding was not affected, suggesting that L-arginine binds only to the BH4-containing subunit. DTT competitively inhibited L-citrulline production by nNOS containing one BH4 per dimer (Ki approximately 11 mM). Comparison of DTT binding and inhibition suggests that the heme of the BH4-free subunit is not involved in catalysis. Thermostability of nNOS was studied by preincubating the enzyme at various temperatures prior to activity determination. At nanomolar concentrations, nNOS was stable at 20 degrees C but rapidly deactivated at higher temperatures (t1/2 approximately 6 min at 37 degrees C). At micromolar concentrations, inactivation was 10 times slower. Absorbance and fluorescence measurements demonstrate that inactivation was not accompanied by major structural changes. The stabilization of nNOS by thiols was illustrated by the fact that omission of 2-mercaptoethanol during preincubation for 10 min at 30 degrees C led to an activity decrease of up to 90%.
Asunto(s)
Cisteína/metabolismo , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Animales , Arginina/metabolismo , Arginina/farmacología , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Calmodulina/farmacología , Cisteína/farmacología , Ditiotreitol/metabolismo , Ditiotreitol/farmacología , Inhibidores Enzimáticos/farmacología , Estabilidad de Enzimas , Flavina-Adenina Dinucleótido/farmacología , Hemo/metabolismo , Mercaptoetanol/metabolismo , Mercaptoetanol/farmacología , NADP/farmacología , Unión Proteica , Ratas , Espectrometría de Fluorescencia , Espectrofotometría , Compuestos de Sulfhidrilo/farmacología , TemperaturaRESUMEN
The properties of neuronal nitric oxide synthase containing one tetrahydrobiopterin (BH4) per dimer [nNOS(BH4+)] were compared to those of the BH4-free enzyme [nNOS(BH4-)]. The stimulation by BH4 of the formation of L-citrulline at the expense of H2O2 production unambiguously demonstrated that BH4 is essential in coupling reductive oxygen activation to Arg oxidation. The clear difference between the Stokes radii of nNOS(BH4-) and nNOS(BH4+) indicates that the introduction of one BH4 per dimer significantly changes the enzyme structure. Whereas the heme in nNOS(BH4+) was primarily high-spin, nNOS(BH4-) contained mainly low-spin heme. This was slowly converted into the high-spin form with Arg and/or BH4, with a rate that was independent of the concentration of either compound. Dithiothreitol inhibited the Arg/BH4-induced spin conversion by stabilizing low-spin heme. Formation of high-spin heme, with rates varying from 0.04 to 0.4 min-1, always correlated to an equally fast increase in activity. Radioligand binding studies showed the rapid association (within 20 s) of BH4 to nNOS(BH4-), but not to nNOS(BH4+), after preincubation with Arg. Complete and monophasic dissociation of radioligand occurred in the presence of excess unlabeled BH4, demonstrating the exchangeability of high-affinity bound BH4. Studies of the association of NG-nitro-L-arginine (L-NNA) to nNOS(BH4+) revealed that excess BH4 increased the amount of bound L-NNA 2-fold. Most of the binding data are explained by a model in which nNOS dimers accommodate two identical BH4- and Arg/L-NNA-binding sites, with cooperativity between Arg- and BH4-binding and anticooperativity between the BH4-binding sites.
Asunto(s)
Óxido Nítrico Sintasa/química , Animales , Sitios de Unión , Biopterinas/análogos & derivados , Biopterinas/análisis , Dimerización , Hemo/química , Técnicas In Vitro , Cinética , Estructura Molecular , Neuronas/enzimología , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/metabolismo , Oxidación-Reducción , Conformación Proteica , Pteridinas/química , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , EspectrofotometríaRESUMEN
The aim of the present study was to investigate the effect of Cu(II) ions on soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2; sGC] and to test for a possible physiological role of this putative cofactor of the enzyme [Gerzer et al., FEBS Lett. 132: 71-74, 1981]. CuSO4 was found to inhibit NO-stimulated 5GC with an IC50 of 2.2 +/- 0.3 microM. Virtually complete inhibition of guanosine-3',5'-cyclic monophosphate (cGMP) formation was observed at 10 microM of the copper salt. Presence of CuSO4 (2 microM) did not significantly affect the potency of 2,2-diethyl-1-nitroso-oxyhydrazine (DEA/NO) but did markedly decrease maximal cyclase activity from 3.71 +/- 0.2 mumol cGMP x mg-1 x min-1 to 1.75 +/- 0.2 mumol cGMP x mg-1 x min-1. The nonstimulated enzyme was also sensitive to CuSO4 (IC50 of 6.2 +/- 1.2 microM). Addition of glutathione, which potently complexes Cu(I) ions, induced a pronounced rightward shift of the concentration-response curves for inhibition by CuSO4 of both DEA/NO-stimulated and nonstimulated guanylyl cyclase. The inhibitory effect of CuSO4 was completely antagonized by the specific Cu(I) chelator neocuproine, with a half-maximal effect at 5.9 +/- 0.2 microM. In contrast, the Cu(II) chelator cuprizone and several thiols, which do not form stable Cu(I) complexes, were far less protective. Our results suggest that inhibition of soluble guanylyl cyclase by CuSO4 is unrelated to heme-mediated enzyme stimulation and may arise from the reversible high affinity binding of Cu(I) ions to a site of the protein that is critically involved in enzyme catalysis.
Asunto(s)
Cobre/farmacología , GMP Cíclico/metabolismo , Pulmón/efectos de los fármacos , Animales , Bovinos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacologíaRESUMEN
Nitric oxide (NO) binds with high affinity to the heme of soluble guanylyl cyclase (sGC), resulting in accumulation of the second messenger cGMP in many biological systems. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was recently described as potent and selective inhibitor of sGC, providing an invaluable tool with which to settle the function of the cGMP pathway in NO-mediated signal transduction [Mol. Pharmacol. 48:184-188 (1995)]. The present study investigated the mechanism of ODQ-induced inhibition of purified bovine lung sGC. The drug induced a rightward shift of the concentration-response curves recorded with two different NO donors and a reduction of maximal sGC activity, pointing to a mixed type of inhibition. The time course of NO-stimulated sGC activity determined in the presence of 0.3 microM ODQ showed that the inhibitory effect was time-dependent (half-time approximately 3 min) and virtually complete after about 10 min. The cyclase did not recover from ODQ-induced inhibition upon extensive dilution, pointing to an apparently irreversible inactivation of the enzyme by the quinoxalin. Light absorbance spectroscopy showed that ODQ (0.3 mM) induced a shift of the Soret band of the heme from 431 nm to 393 nm, indicating that ODQ oxidizes the ferrous form of the enzyme to the ferric species, which is though to exhibit only poor NO sensitivity. Together, our results suggest that inhibition of sGC by ODQ is NO-competitive and results in an apparently irreversible oxidation of the prosthetic heme group.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Guanilato Ciclasa/antagonistas & inhibidores , Hemo/metabolismo , Óxido Nítrico/metabolismo , Oxadiazoles/farmacología , Quinoxalinas/farmacología , Animales , Sitios de Unión , Bovinos , Guanilato Ciclasa/química , Cinética , Pulmón/enzimología , EspectrofotometríaRESUMEN
Peroxynitrite (ONOO-) is widely recognized as mediator of NO toxicity, but recent studies have indicated that this compound may also have physiological activity and induce vascular relaxation as well as inhibition of platelet aggregation. We found that ONOO- induced a pronounced increase in endothelial cyclic GMP levels, and that this effect was significantly attenuated by pretreatment of the cells with GSH-depleting agents. In the presence of 2 mM GSH, ONOO- stimulated purified soluble guanylyl cyclase with a half-maximally effective concentration of about 20 microM. In contrast to the NO donor 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), ONOO- was completely inactive in the absence of GSH, indicating that thiol-mediated bioactivation of ONOO- is involved in enzyme stimulation. Studies on the reaction between ONOO- and GSH revealed that about 1% of ONOO- was non-enzymatically converted to S-nitrosoglutathione. The authentic nitrosothiol was found to be stable in solution, but slowly decomposed in the presence of GSH. GSH-induced decomposition of S-nitrosoglutathione was apparently catalyzed by trace metals and was accompanied by a sustained release of NO and a 40-100-fold increase in its potency to stimulate purified soluble guanylyl cyclase. Our data suggest that the biologic activity of ONOO- involves S-nitrosation of cellular thiols resulting in NO-mediated cyclic GMP accumulation.