RESUMEN
Significance: Increased plasma concentrations of total homocysteine (tHcy; mild-moderate hyperhomocysteinemia: 15-50 µM tHcy) are considered an independent risk factor for the onset/progression of various diseases, but it is not known about how the increase in tHcy causes pathological conditions. Recent Advances: Reduced homocysteine (HSH â¼1% of tHcy) is presumed to be toxic, unlike homocystine (â¼9%) and mixed disulfide between homocysteine and albumin (HSS-ALB; homocysteine [Hcy]-albumin mixed disulfide, â¼90%). This and other notions make it difficult to explain the pathogenicity of Hcy because: (i) lowering tHcy does not improve pathological outcomes; (ii) damage due to HSH usually emerges at supraphysiological doses; and (iii) it is not known why tiny increments in plasma concentrations of HSH can be pathological. Critical Issues: Albumin may have a role in Hcy toxicity, because HSS-ALB could release toxic HSH via thiol-disulfide (SH/SS) exchange reactions in cells. Similarly, thiol-disulfide exchange processes of reduced albumin (albumin with free SH group of Cys34 [HS-ALB]) or N-homocysteinylated albumin are plausible alternatives for initiating Hcy pathological events. Adverse effects of albumin and other data reviewed here suggest the hypothesis of a role of albumin in Hcy toxicity. Future Directions: HSS-ALB might be involved in disruption of the antioxidant/oxidant balance in critical tissues (brain, liver, kidney). Since homocysteine-albumin mixed disulfide is a possible intermediate of thiol-disulfide exchange reactions, we suggest that homocysteinylated albumin could be a new pathological factor, and that studies on the redox role of albumin and mixed disulfide production via thiol-disulfide exchange reactions could offer new therapeutic insights for reducing Hcy toxicity.
Asunto(s)
Hiperhomocisteinemia , Compuestos de Sulfhidrilo , Humanos , Disulfuros , Homocistina , HomocisteínaRESUMEN
The toxicity risk of hyperhomocysteinemia is prevented through thiol drug administration which reduces plasma total homocysteine (tHcy) concentrations by activating thiol exchange reactions. Assuming that cysteine (Cys) is a homocysteinemia regulator, the hypothesis was verified in healthy and pathological individuals after the methionine loading test (MLT). The plasma variations of redox species of Cys, Hcy, cysteinylglycine, glutathione and albumin (reduced, HS-ALB, and at mixed disulfide, XSS-ALB) were compared in patients with cerebral small vessels disease (CSVD) (n = 11), multiple sclerosis (MS) (n = 12) and healthy controls (n = 11) at 2-4-6 h after MLT. In MLT-treated subjects, the activation of thiol exchange reactions provoked significant changes over time in redox species concentrations of Cys, Hcy, and albumin. Significant differences between controls and pathological groups were also observed. In non-methionine-treated subjects, total Cys concentrations, tHcy and thiol-protein mixed disulfides (CSS-ALB, HSS-ALB) of CSVD patients were higher than controls. After MLT, all groups displayed significant cystine (CSSC) increases and CSS-ALB decreases, that in pathological groups were significantly higher than controls. These data would confirm the Cys regulatory role on the homocysteinemia; they also explain that the Cys-Hcy mixed disulfide excretion is an important point of hyperhomocysteinemia control. Moreover, in all groups after MLT, significant increases in albumin concentrations, named total albumin (tALB) and measured as sum of HS-ALB (spectrophometric), and XSS-ALB (assayed at HPLC) were observed. tALB increases, more pronounced in healthy than in the pathological subjects, could indicate alterations of albumin equilibria between plasma and other extracellular spaces, whose toxicological consequences deserve further studies.
Asunto(s)
Trastornos Cerebrovasculares , Cisteína/sangre , Homocisteína/sangre , Hiperhomocisteinemia , Metionina/administración & dosificación , Esclerosis Múltiple , Adulto , Trastornos Cerebrovasculares/sangre , Trastornos Cerebrovasculares/fisiopatología , Femenino , Humanos , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/fisiopatología , Masculino , Metionina/farmacocinética , Persona de Mediana Edad , Esclerosis Múltiple/sangre , Esclerosis Múltiple/fisiopatología , Albúmina Sérica Humana/metabolismoRESUMEN
In hyperhomocysteinemic patients, after reaction with homocysteine-albumin mixed disulfides (HSS-ALB), mesna (MSH) forms the mixed disulfide with Hcy (HSSM) which can be removed by renal clearance, thus reducing the plasma concentration of total homocysteine (tHcy). In order to assess the HSS-ALB dethiolation via thiol exchange reactions, the distribution of redox species of cysteine, cysteinylglycine, homocysteine and glutathione was investigated in the plasma of healthy subjects: (i) in vitro, after addition of 35 µM reduced homocysteine (HSH) to plasma for 72 h, followed by MSH addition (at the concentration range 10-600 µM) for 25 min; (ii) in vivo, after oral treatment with methionine (methionine, 200 mg/kg body weight, observation time 2-6 h). In both experiments the distribution of redox species, but not the total amount of each thiol, was modified by thiol exchange reactions of albumin and cystine, with changes thermodynamically related to the pKa values of thiols in the corresponding mixed disulfides. MSH provoked a dose-response reversal of the redox state of aged plasma, and the thiol action was confirmed by in vivo experiments. Since it was observed that the dimesna production could be detrimental for the in vivo optimization of HSSM formation, we assume that the best plasma tHcy lowering can be obtained at MSH doses producing the minimum dimesna concentration in each individual.
Asunto(s)
Antioxidantes/farmacología , Hiperhomocisteinemia/tratamiento farmacológico , Mesna/farmacología , Adulto , Antioxidantes/uso terapéutico , Evaluación Preclínica de Medicamentos , Femenino , Homocisteína/sangre , Humanos , Masculino , Mesna/uso terapéutico , Metionina/sangre , Persona de Mediana Edad , Oxidación-ReducciónRESUMEN
Three different NO donors, S-nitrosoglutathione (GSNO), sodium nitroprusside (SNP) and 3-morpholino-sydnonimine hydrochloride (SIN-1) were used in order to investigate mechanisms of platelet inhibition through cGMP-dependent and -independent pathways both in human and rat. To this purpose, we also evaluated to what extent cGMP-independent pathways were related with the entity of NO release from each drug. SNP, GSNO and SIN-1 (100 µM) effects on platelet aggregation, in the presence or absence of a soluble guanylate cyclase inhibitor (ODQ), on fibrinogen receptor (α(IIb)ß(3)) binding to specific antibody (PAC-1), and on the entity of NO release from NO donors in human and rat platelet rich plasma (PRP) were measured. Inhibition of platelet aggregation (induced by ADP) resulted to be greater in human than in rat. GSNO was the most powerful inhibitor (IC(50) values, µM): (a) in human, GSNO=0.52±0.09, SNP=2.83 ± 0.53, SIN-1=2.98 ± 1.06; (b) in rat, GSNO = 28.4 ± 6.9, SNP = 265 ± 73, SIN-1=108 ± 85. GSNO action in both species was mediated by cGMP-independent mechanisms and characterized by the highest NO release in PRP. SIN-1 and SNP displayed mixed mechanisms of inhibition of platelet aggregation (cGMP-dependent and independent), except for SIN-1 in rat (cGMP-dependent), and respectively lower or nearly absent NO delivery. Conversely, all NO-donors prevalently inhibited PAC-1 binding to α(IIb)ß(3) through cGMP-dependent pathways. A modest relationship between NO release from NO donors and cGMP-independent responses was found. Interestingly, the species difference in NO release from GSNO and inhibition by cGMP-independent mechanism was respectively attributed to S-nitrosylation of non-essential and essential protein SH groups.
Asunto(s)
Plaquetas/efectos de los fármacos , GMP Cíclico/metabolismo , Molsidomina/análogos & derivados , Donantes de Óxido Nítrico/farmacología , Nitroprusiato/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , S-Nitrosoglutatión/farmacología , Animales , Plaquetas/citología , Humanos , Masculino , Molsidomina/farmacología , Agregación Plaquetaria/efectos de los fármacos , Conformación Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores Fibrinógenos/químicaRESUMEN
Understanding the structural basis of protein redox activity is still an open question. Hence, by using a structural genomics approach, different albumins have been chosen to correlate protein structural features with the corresponding reaction rates of thiol exchange between albumin and disulfide DTNB. Predicted structures of rat, porcine, and bovine albumins have been compared with the experimentally derived human albumin. High structural similarity among these four albumins can be observed, in spite of their markedly different reactivity with DTNB. Sequence alignments offered preliminary hints on the contributions of sequence-specific local environments modulating albumin reactivity. Molecular dynamics simulations performed on experimental and predicted albumin structures reveal that thiolation rates are influenced by hydrogen bonding pattern and stability of the acceptor C34 sulphur atom with donor groups of nearby residues. Atom depth evolution of albumin C34 thiol groups has been monitored during Molecular Dynamic trajectories. The most reactive albumins appeared also the ones presenting the C34 sulphur atom on the protein surface with the highest accessibility. High C34 sulphur atom reactivity in rat and porcine albumins seems to be determined by the presence of additional positively charged amino acid residues favoring both the C34 Sâ» form and the approach of DTNB.
Asunto(s)
Albúminas/química , Disulfuros/química , Ácido Ditionitrobenzoico/química , Alineación de Secuencia/métodos , Compuestos de Sulfhidrilo/química , Secuencia de Aminoácidos , Animales , Bovinos , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Terciaria de Proteína , RatasRESUMEN
Blood platelets are central to haemostasis and platelet aggregation is considered to be a direct index of platelet function. Although protein disulfides (PSSP) are structural components of most proteins, current evidence suggests that PSSP work together with protein SH groups (PSH) to activate various platelet functions in dynamic processes involving thiol/disulfide exchange reactions. Based on these assumptions, we performed experiments to demonstrate how PSH and PSSP are involved in platelet aggregation and how modifications of PSH and PSSP concentrations on the platelet surface by N-ethylmaleimide (NEM) (a PSH-blocking reagent) and dithiothreitol (DTT) (a PSSP-reducing reagent), respectively, may condition platelet susceptibility in protein rich plasma and washed platelets and integrin αIIbß3 conformation. Our data strongly suggest that the PSH blockage and the PSSP reduction of the platelet surface are deeply involved in aggregation processes evoked in protein rich plasma and washed platelets by ADP and collagen; that endogenous thiols (e.g. GSH) may interfere with NEM actions; that NEM and DTT, acting on preexisting PSH and PSSP of active platelets have opposite conformational changes on integrin αIIbß3 conformation. Although the precise mechanism and the populations of specific PSH and PSSP involved remain unresolved, our data support the notion that PSH and PSSP of the platelet surface are involved in platelet activation by thiol exchange reactions. A plausible molecular mechanism of PSH and PSSP recruitment during thiol exchange reactions is here proposed.
Asunto(s)
Plaquetas/metabolismo , Disulfuros/sangre , Agregación Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Compuestos de Sulfhidrilo/sangre , Plaquetas/efectos de los fármacos , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Etilmaleimida/farmacología , Humanos , Agregación Plaquetaria/efectos de los fármacos , Pruebas de Función Plaquetaria , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/química , Conformación Proteica , Sustancias Reductoras/farmacología , Reactivos de Sulfhidrilo/farmacologíaRESUMEN
Mixed disulfides between protein cysteines and low-molecular-weight thiol cysteine or glutathione lead to the formation of cysteinylated proteins or glutathionylated proteins. These types of posttranslational modification are of great importance in the so-called redox regulation, by which changes in the redox state of the cell regulate a number of biochemical processes. We describe the methods for quantitatively measuring the various redox states of cellular thiols including protein cysteines and these mixed disulfides. These include spectrophotometric methods, which do not distinguish between protein-cysteine and protein-glutathione disulfides, and HPLC methods that make such distinction. Finally, we report a method for labeling proteins susceptible to glutathionylation with biotin, to allow their visualization by Western blot after electrophoretic separation, which is used to identify proteins undergoing this posttranslational modification.
Asunto(s)
Técnicas de Laboratorio Clínico , Disulfuros/análisis , Glutatión/metabolismo , Proteínas/análisis , Proteínas/metabolismo , Animales , Disulfuro de Glutatión/análisis , Disulfuro de Glutatión/metabolismo , Humanos , Oxidación-Reducción , Procesamiento Proteico-Postraduccional , Proteínas/química , Compuestos de Sulfhidrilo/análisis , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Protein thiol modifications including cysteinylation (CSSP) and glutathionylation (GSSP) in erythrocytes of rat treated with diamide have been reported, but mechanism and origin of CSSP formation are unknown. Experiments were performed to relate CSSP formation to GSH hydrolysis via gamma-glutamyltranspeptidase (gamma-GT) and know whether cysteine may act as deglutathionylation factor. Time-dependent variations of redox forms of glutathione and cysteine were investigated in erythrocytes, plasma, liver and kidney of diamide-treated rats (0.4 mmol/kg by infusion for 45 min followed by 135 min of washout) in the presence and absence of acivicin (10 mg/kg administered twice 1 h before diamide) a known gamma-GT inhibitor. Diamide-treated rats showed decreased concentrations of erythrocyte GSH and increased levels of GSSP and CSSP. The rate of CSSP formation was slower than that of GSSP. Besides the entity of CSSP accumulation of erythrocytes was high and equivalent to approximately 3-fold of the normal plasma content of total cysteine. The result was paradoxically poorly related to gamma-GT activity because the gamma-GT inhibition only partially reduced erythrocyte CSSP. After gamma-GT inhibition, a large concentration fluctuation of glutathione (increased) and cysteine (decreased) was observed in plasma of diamide-treated rats, while little changes were seen in liver and kidney. There were indications from in vitro experiments that the CSSP accumulation in erythrocytes of diamide-treated rats derives from the coexistence of GSH hydrolysis via gamma-GT and production of reduced cysteine via plasma thiol exchanges. Moreover, reduced cysteine was found to be involved in deglutathionylation processes. Mechanisms of protein glutathionylation by diamide and deglutathionylation by cysteine were proposed.
Asunto(s)
Cisteína/metabolismo , Diamida/farmacología , Compuestos de Sulfhidrilo/metabolismo , gamma-Glutamiltransferasa/metabolismo , Animales , Inhibidores Enzimáticos/farmacología , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Glutatión/metabolismo , Isoxazoles/farmacología , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Oxidación-Reducción/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Reactivos de Sulfhidrilo/farmacología , Factores de Tiempo , gamma-Glutamiltransferasa/antagonistas & inhibidoresRESUMEN
Increases in plasma concentrations of total homocysteine (tHcy) have recently been reported in multiple sclerosis (MS) as the alteration of the methionine cycle for the onset of autoimmune diseases. Homocysteine (Hcy) and cysteine (Cys) are generated by the methionine cycle and transsulfuration reactions. Their plasma levels are subjected to complex redox changes by oxidation and thiol/disulfide (SH/SS) exchange reactions regulated by albumin. The methionine loading test (MLT) is a useful in vivo test to assay the functionality of the methionine cycle and transsulfuration reactions. Time courses of redox species of Cys, cysteinylglycine (CGly), Hcy, and glutathione have been investigated in plasma of MS patients versus healthy subjects after an overnight fasting, and 2, 4, and 6 h after an oral MLT (100 mg/kg body weight), to detect possible dysfunctions of the methionine cycle, transsulfuration reactions and alterations in plasma distribution of redox species. After fasting, the MS group showed a significant increase in cysteine-protein mixed disulfides (bCys) and total Cys (tCys). While plasma bCys and tCys in MS group remained elevated after methionine administration when compared to control, cystine (oxCys) increased significantly with respect to control. Although increased plasma concentrations of bCys and tCys at fasting might reflect an enhance of transsulfuration reactions in MS patients, this was not confirmed by the analysis of redox changes of thiols and total thiols after MLT. This study has also demonstrated that albumin-dependent SH/SS exchange reactions are a potent regulation system of thiol redox species in plasma.
Asunto(s)
Albúminas/metabolismo , Metionina/administración & dosificación , Esclerosis Múltiple/sangre , Compuestos de Sulfhidrilo/sangre , Adulto , Estudios de Casos y Controles , Cromatografía Líquida de Alta Presión , Cisteína/sangre , Femenino , Glutatión/sangre , Humanos , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Espectrometría de FluorescenciaRESUMEN
Previously we reported that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) shifts the cells into a state of oxidative stress that induces apoptosis and necrosis. In this study, flow cytometric analysis showed that CMI/MI induces early perturbation of calcium homeostasis, increasing cytosolic and mitochondrial calcium and depleting the intracellular endoplasmic reticulum (ER) stores. The calcium chelator BAPTA-AM reduced necrosis and secondary necrosis, the loss of DeltaPsim and S-glutathionylation induced by necrotic doses of CMI/MI, but did not protect against CMI/MI-induced apoptosis, mitochondrial calcium uptake and mitochondrial hyperpolarization. This indicates that increased cytoplasmic calcium does not have a causal role in the induction of apoptosis, while cross-talk between the ER and mitochondria could be responsible for the induction of apoptosis. GSH-OEt pretreatment, which enhances cellular GSH content, reduced S-glutathionylation and cytosolic and mitochondrial calcium levels, thus protecting against both apoptosis and necrosis shifting to apoptosis. Therefore, the degree of GSH depletion, paralleled by the levels of protein S-glutathionylation, may have a causal role in increasing calcium levels. The mitochondrial calcium increase could be responsible for apoptosis, while necrosis is associated with cytoplasmic calcium overload. These findings suggest that S-glutathionylation of specific proteins acts as a molecular linker between calcium and redox signalling.
Asunto(s)
Calcio/metabolismo , Glutatión/metabolismo , Tiazoles/toxicidad , Muerte Celular , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Citometría de Flujo , Células HL-60 , Humanos , Potencial de la Membrana Mitocondrial , Mitocondrias/metabolismoRESUMEN
We investigated the effect of extra virgin olive oil (EVOO) on platelet aggregation and plasma concentrations of homocysteine (Hcy) redox forms in rats in relation to the minor polar compound (MPC) concentration of EVOO. We used 3 olive oil samples with similar fatty acid but different MPC concentrations: refined olive oil (RF) with traces of MPC (control oil), native EVOO with low MPC concentration (LC), and EVOO with high MPC concentration (HC) enriching LC with its own MPC. Oil samples were administered to rats by gavage (1.25 mL/kg body weight) using 2 experimental designs: acute (24-h food deprivation and killed 1 h after EVOO administration) and subacute (12-d treatment, a daily dose of oil for 12 d, and killed after 24 h of food deprivation). Platelet aggregation was induced by ADP (ex vivo tests) and a reduction in platelet reactivity occurred in cells from rats given LC in the subacute study and in cells from rats administered HC in both studies as indicated by an increase in the agonist half maximal effective concentration. HC inhibited platelet aggregation induced by low ADP doses (reversible aggregation) in cells of rats in both the acute and subacute studies, whereas LC had this effect only in the subacute experiment. Moreover, in rats administered HC in both experiments, the plasma concentration of free reduced Hcy (rHcy) was lower and Hcy bound to protein by disulfide bonds (bHcy) was greater than in RF-treated rats. bHcy was also greater in rats given LC than in RF-treated rats in the subacute experiment. Plasma free-oxidized Hcy was greater in rats given LC and HC than in those administered RF only in the subacute experiment. In conclusion, these results show that MPC in EVOO inhibit platelet aggregation and reduce the plasma rHcy concentration, effects that may be associated with cardiovascular protection.
Asunto(s)
Homocisteína/sangre , Aceites de Plantas/farmacología , Agregación Plaquetaria/efectos de los fármacos , Animales , Esquema de Medicación , Ácidos Grasos/análisis , Masculino , Aceite de Oliva , Oxidación-Reducción , Aceites de Plantas/administración & dosificación , Aceites de Plantas/química , Ratas , Ratas Sprague-DawleyRESUMEN
Regulation of protein function through post-translational modifications (PTM) can be important pharmacological target, and there are drugs developed to modulate specific PTM such as protein kinase or histone deacetylase inhibitors. We are still far behind in considering protein glutathionylation as pharmacological target as the biological consequences and role of this PTM are still unclear. We discuss the possible relevance of glutathionylation in diseases and its biases compared with other PTM. In particular, we discuss the different roles of glutathionylation in the context of redox regulation as opposed to that of oxidative stress, and the difficulties arising from the overlaps of these two concepts.
Asunto(s)
Sistemas de Liberación de Medicamentos , Glutatión/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Glutatión/química , Humanos , Oxidación-Reducción , Estrés Oxidativo , Especies Reactivas de Oxígeno , Transducción de SeñalRESUMEN
Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.
Asunto(s)
Cistina/metabolismo , Disulfuros/metabolismo , Modelos Moleculares , Albúmina Sérica/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Secuencia de Aminoácidos , Animales , Bovinos , Cistina/química , Cistina/genética , Disulfuros/química , Humanos , Datos de Secuencia Molecular , Oxidación-Reducción , Albúmina Sérica/química , Albúmina Sérica/genética , Compuestos de Sulfhidrilo/químicaRESUMEN
We recently described that brief exposure of HL60 cells to a mixture of 5-chloro-2-methyl-4-isothiazolin-3-one (CMI) and 2-methyl-4-isothiazolin-3-one (MI) induces apoptosis at low concentrations (0.001-0.01%) and necrosis at higher concentrations (0.05-0.1%). In this study, we show that glutathione (GSH) depletion, reactive oxygen species generation, hyperpolarization of mitochondrial transmembrane potential (DeltaPsim) and formation of protein-GSH mixed disulphides (S-glutathionylation) are early molecular events that precede the induction of cell death by CMI/MI. When the cells exhibit common signs of apoptosis, they show activation of caspase-9, reduction of DeltaPsim and, more importantly, decreased protein S-glutathionylation. In contrast, necrosis is associated with severe mitochondrial damage and maximal protein S-glutathionylation. CMI/MI-induced cytotoxicity is also accompanied by decreased activity of GSH-related enzymes. Pre-incubation with L-buthionine-(S,R)-sulfoximine (BSO) clearly switches the mode of cell death from apoptosis to necrosis at 0.01% CMI/MI. Collectively, these results demonstrate that CMI/MI alters the redox status of HL60 cells, and the extent and kinetics of GSH depletion and S-glutathionylation appear to determine whether cells undergo apoptosis or necrosis. We hypothesize that S-glutathionylation of certain thiol groups accompanied by GSH depletion plays a critical role in the molecular mechanism of CMI/MI cytotoxicity.
Asunto(s)
Apoptosis/efectos de los fármacos , Glutatión/metabolismo , Mitocondrias/metabolismo , Conservadores Farmacéuticos/farmacología , Tiazoles/farmacología , Caspasa 9 , Caspasas/análisis , Cromatografía Líquida de Alta Presión , Disulfuros/análisis , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Glucosafosfato Deshidrogenasa/análisis , Glutatión/análisis , Glutatión/deficiencia , Glutatión Peroxidasa/análisis , Glutatión Reductasa/análisis , Glutatión Transferasa/análisis , Células HL-60 , Humanos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/fisiología , Necrosis , Especies Reactivas de Oxígeno/metabolismo , Espectrofotometría UltravioletaRESUMEN
Protein thiolation is elicited by oxidation by different mechanisms and is involved in a variety of biological processes. Thiols, protein SH (PSH) and non-protein SH groups (NPSH, namely GSH), are in competition in all biological environments in the regulation of oxidant homeostasis because oxidants thiolate proteins, whereas GSH dethiolates them (e.g., GSSG + PSH --> GSSP + GSH). Although poorly investigated, the elimination of disulfides from thiolated proteins to regenerate critical PSH is important. These aspects are poorly known in cells, where glutaredoxin and peroxiredoxin operate as enzymes or potential chaperones to accelerate dethiolation. On the contrary, studies with plasma or albumin have highlighted the importance of protein conformation in dethiolation processes and have clarified the reason why homocysteine (thiol with potential toxicity) is preferentially bound to albumin as protein-thiol mixed disulfide with respect to other NPSH. Here we provide an overview of protein thiolation/dethiolation processes, with an emphasis on recent developments and future perspectives in this field.
Asunto(s)
Células/metabolismo , Plasma/metabolismo , Proteínas/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Animales , Enzimas/metabolismo , Humanos , Conformación Proteica , Proteínas/químicaRESUMEN
We assayed the redox forms of cysteine (reduced [CSH], oxidized [CSSC], and bound to protein [CS-SP]), cysteinylglycine (CGSH; cysteinylgycine disulfide [CGSSGC] and cysteinylglycine-protein mixed disulfide [CGS-SP]), glutathione (GSH; glutathione disulfide [GSSG] and glutathione-protein mixed disulfide [GS-SP]), homocysteine (Hcy; homocystine [HcyS] and homocystine-protein mixed disulfides [bHcy]), and protein sulfhydryls in the plasma of healthy subjects (divided into 8 groups ranging in age from birth to 70 years) and patients with mild hyperhomocysteinemia associated with cardiovascular disease (heart-transplant patients) or vascular atherosclerosis, with or without renal failure. In healthy individuals, levels of disulfides and protein-mixed disulfides were more abundant than those of thiols, and those of protein-thiol mixed disulfides were higher than disulfides. Concentrations of CSH, GSH, and CGSH in the various groups had profiles characterized by a maximum over time. The concentration of Hcy was unchanged up to the age of 30 years, after which it increased. CSSC concentration increased gradually with age, whereas concentrations of the other disulfides were essentially unchanged. By contrast, the concentrations of all protein-thiol mixed disulfides, especially those with CSH, increased gradually with age. Ranks of distribution of the reduced forms changed with age (at birth, CSH > CGSH > GSH > Hcy; in 1- to 2-year-olds, CSH > GSH > CGSH > Hcy; and in 51- to 70-year-olds, CSH > CGSH = GSH > Hcy), whereas those of disulfides and protein-thiol mixed disulfides were substantially unchanged (in all age groups, CSSC > CGSSGC > GSSG = HcyS and CS-SP > CGS-SP > bHcy > GS-SP). In patients with pathologic conditions, plasma levels of disulfide forms CSSC, HcyS, CS-SP, and bHcy were significantly increased, whereas other redox forms of thiols were unchanged or showed variations opposite (increasing or decreasing) to control values. Maximal increases in disulfides and protein-thiol mixed disulfides were associated with renal failure. Our data suggest that increases in plasma bHcy concentrations in subjects with pathologic conditions were more likely the result of activation of thiol-disulfide exchange reactions between free reduced Hcy and CS-SP than of a direct action of reactive oxygen species.
Asunto(s)
Envejecimiento/sangre , Hiperhomocisteinemia/sangre , Compuestos de Sulfhidrilo/sangre , Adolescente , Adulto , Anciano , Arteriosclerosis/sangre , Proteínas Sanguíneas/metabolismo , Enfermedades Cardiovasculares/sangre , Niño , Preescolar , Cisteína/sangre , Disulfuros/sangre , Glicina/sangre , Trasplante de Corazón , Humanos , Lactante , Recién Nacido , Persona de Mediana Edad , Oxidación-Reducción , Unión Proteica , Insuficiencia Renal/sangreRESUMEN
We previously demonstrated a high susceptibility of neonatal red blood cells (RBC) to oxidative stress at birth. The aim of this study was to compare the RBC antioxidant capacity and redox cycle enzyme activities as well as glutathione (GSH) recycling in full-term and preterm infants at birth and in normal adults. GSH and GSH disulfide (GSSG) concentrations, GSH/GSSG ratio, and the activities of glucose-6-phosphate dehydrogenase (G-6-PDH), GSH peroxidase, GSH reductase (GR), catalase (CAT), superoxide dismutase (SOD), and hexokinase (HK) were measured in RBC of 25 healthy adults and 56 newborns (23 term, 33 preterm) at birth. The GSH recycling was measured in adult and newborn RBC exposed to oxidative stress (1 mM tert-butylhydroperoxide). The RBC of term and preterm babies showed higher GSH, GSSG, G-6-PDH, GR, and HK levels/activities and lower GSH/GSSG ratios and higher GSH-recycling rates than those of adults. In preterm babies significant correlations were found between G-6-PDH and CAT, GSH, GSH/GSSG ratio, and GSSG (r = -0.67, r = 0.71, r = -0.66, p < 0.01; r = 0.71, p < 0.05, respectively). In term newborns, statistically significant correlations were observed between G-6-PDH and CAT, SOD, and GSH (r = -0.65, r = -0.65, r = -0.69, p < 0.01, respectively). The results indicate the central role of the G-6-PDH activity in antioxidant defenses. We speculate that preterm babies have prompter involvement of antioxidant defenses than term babies.
Asunto(s)
Eritrocitos/enzimología , Disulfuro de Glutatión/sangre , Glutatión/sangre , Recién Nacido/sangre , Recien Nacido Prematuro/sangre , Adulto , Catalasa/sangre , Eritrocitos/metabolismo , Glucosafosfato Deshidrogenasa/sangre , Glutatión Peroxidasa/sangre , Glutatión Reductasa/sangre , Hexoquinasa/sangre , Humanos , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo/fisiología , Superóxido Dismutasa/sangreRESUMEN
In vitro, high concentrations of ethanol (EtOH) reduce platelet aggregation. Less is known about the effect of low EtOH doses on platelet function in a selected human population of long-life abstainers and low moderate-wine drinkers to avoid rebound effect of EtOH on platelet aggregation. Results of our experiments suggest that moderate-wine drinkers have higher levels of high density lipoprotein (HDL) than long-life abstainers while fibrinogen levels are unchanged. Furthermore, platelets obtained from these individuals do not differ in their response when stimulated by agonists such as AA and collagen. The effect of in vitro exposure of low doses of EtOH has been studied in PRP and in washed platelets. EtOH (0.1-10 mM) inhibits platelet aggregation induced by collagen at its ED50 while is ineffective when aggregation was triggered by U-46619 and by 1 microM adenosine diphosphate (ADP). 5-10 mM EtOH partially reduces the second wave of aggregation induced by 3 microM ADP. 0.1-10 mM EtOH dose-dependently lowers the aggregation induced by AA at its ED50 but it is less effective at ED75 of AA. The antiaggregating effect of EtOH on aggregation induced by AA is unchanged by inhibitor of nitric oxide synthase. In addition, 10 mM EtOH reduces thromboxane (Tx) formation. In washed platelets, 1-10 mM EtOH partially inhibits platelet aggregation induced by thrombin. In washed resting platelets, 10 mM EtOH does not change the resting [Ca++]i while significantly reduces the increase in [Ca++]i triggered by AA. The results of ex vivo experiments have demonstrated that wine increases the HDL. However, this observation may or may not influence the response of platelets to agonists. Results of our studies demonstrate that low doses of alcohol reduces platelet function.
Asunto(s)
Plaquetas/efectos de los fármacos , Depresores del Sistema Nervioso Central/farmacología , Etanol/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Agregación Plaquetaria/fisiología , Vino , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico/farmacología , Adenosina Difosfato/farmacología , Adulto , Consumo de Bebidas Alcohólicas , Plaquetas/fisiología , Colesterol/metabolismo , Colágeno/farmacología , Relación Dosis-Respuesta a Droga , Femenino , Fibrinógeno/metabolismo , Humanos , Lipoproteínas HDL/metabolismo , Masculino , Trombina/farmacología , Tromboxano B2/sangre , Triglicéridos/sangreRESUMEN
Hyperhomocysteinemia has recently been suggested to contribute to the progression of the so-called chronic rejection or cardiac allograft vasculopathy (CAV) in heart-transplant patients in which the major determinant of the increase in homocysteine (Hcy) was the progressive decline of renal function. The exact mechanisms of tissue injury by Hcy is unknown, but some aspects of its toxicity have been related to its capacity for altering the redox state of plasma and forming protein adducts by intermediate lactone. To study the relationships between Hcy levels and variations in the redox state governed by thiols, plasma levels of Hcy, cysteine, glutathione, cysteinylglycine, and corresponding disulfides and protein-mixed disulfides were evaluated in subjects with moderate hyperhomocysteinemia represented by heart-transplant patients with (HTRF) and without (HT) renal failure, as well as patients with renal failure of different origin (RF), and compared with those of a control group (C) of normal subjects matched for age and sex. Plasma levels of Hcy and the corresponding protein mixed disulfides increased progressively in HTs, RFs, and HTRFs with respect to control. These changes were correlated with cysteine variations (as cystine and protein-mixed disulfides) but not with glutathione or cysteinylglycine that varied only as disulfides with a similar tendency. Moreover, an alteration in the plasma redox was evidenced by the decrease in thiol/disulfide ratios of cysteine, Hcy, and cysteinylglycine. In all groups, cysteine was directly correlated with Hcy but not with glutathione or cysteinylglycine, which in turn were correlated each other. Therefore levels of plasma cysteine were more linked to Hcy than to metabolism of glutathione. The clinical meaning of cysteine changes remains undefined and requires further study.
Asunto(s)
Trasplante de Corazón , Hiperhomocisteinemia/sangre , Hiperhomocisteinemia/metabolismo , Adulto , Anciano , Progresión de la Enfermedad , Disulfuros/sangre , Femenino , Humanos , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Insuficiencia Renal/sangre , Insuficiencia Renal/metabolismo , Compuestos de Sulfhidrilo/sangreRESUMEN
BACKGROUND: The reported mean concentration of glutathione disulfide (GSSG) in human blood/erythrocytes varies widely (1 to >500 micromol/L), as does that of reduced glutathione (GSH) to a lesser extent. We have identified and investigated possible pitfalls in measurement of both GSH and GSSG. METHODS: We measured GSH and GSSG using a spectrophotometer with a modification of the GSH recycling method; the same samples were also measured by reversed-phase HPLC after derivatization of thiols (dithiothreitol was used to reduce disulfides) with monobromobimane. The thiol-bimane adduct was measured by a fluorescence detector. RESULTS: Measured GSH/GSSG concentrations were affected by the following: (a) oxidation of thiols in acidified samples; (b) oxidation after restoring neutral-alkaline pH; (c) oxidation during acid deproteinization; (d) shift in the GSH/GSSG equilibrium because of irreversible blocking of free thiols; and (e) reaction of electrophiles with amino groups. In particular, oxidation during sample deproteinization with acid influenced and produced artifacts (30-150 micromol/L GSSG was produced by this procedure); this phenomenon was directly correlated with the presence of oxygenated hemoglobin, being minimized by both oxygen deprivation and incubation in an atmosphere of 5% carbon monoxide. CONCLUSIONS: GSSG is present in healthy human blood at low concentrations (2-6 micromol/L), and most published data on GSSG may be affected by artifacts.