RESUMO
We have previously established that 670 nm energy induces relaxation of blood vessels via an endothelium derived S-nitrosothiol (RSNO) suggested to be embedded in vesicles. Here, we confirm that red light facilitates the exocytosis of this vasodilator from cultured endothelial cells and increases ex vivo blood vessel diameter. Ex vivo pressurized and pre-constricted facial arteries from C57Bl6/J mice relaxed 14.7% of maximum diameter when immersed in the medium removed from red-light exposed Bovine Aortic Endothelial Cells. In parallel experiments, 0.49 nM RSNO equivalent species was measured in the medium over the irradiated cells vs dark control. Electron microscopy of light exposed endothelium revealed significant increases in the size of the Multi Vesicular Body (MVB), a regulator of exosome trafficking, while RSNO accumulated in the MVBs as detected with immunogold labeling electron microscopy (1.8-fold of control). Moreover, red light enhanced the presence of F-actin related stress fibers (necessary for exocytosis), and the endothelial specific marker VE-cadherin levels suggesting an endothelial origin of the extracellular vesicles. Flow cytometry coupled with DAF staining, an indirect sensor of nitric oxide (NO), indicated significant amounts of NO within the extracellular vesicles (1.4-fold increase relative to dark control). Therefore, we further define the mechanism on the 670 nm light mediated traffic of endothelial vasodilatory vesicles and plan to leverage this insight into the delivery of red-light therapies.
Assuntos
Células Endoteliais , Luz Vermelha , Animais , Bovinos , Camundongos , Modelos Animais de Doenças , Óxido Nítrico , Células Cultivadas , Exocitose , EndotélioRESUMO
Far red/near infrared (R/NIR) energy is a novel therapy, but its mechanism of action is poorly characterized. Cytochrome c oxidase (Cco) of the mitochondrial electron transport chain is considered the primary photoacceptor for R/NIR to photolyze a putative heme nitrosyl in Cco to liberate free nitric oxide (NO). We previously observed R/NIR light directly liberates NO from nitrosylated hemoglobin and myoglobin, and recently suggested S-nitrosothiols (RSNO) and dinitrosyl iron complexes (DNIC) may be primary sources of R/NIR-mediated NO. Here we indicate R/NIR light exposure induces wavelength dependent dilation of murine facial artery, with longer wavelengths (740, and 830â¯nm) exhibiting reduced potency when compared to 670â¯nm. R/NIR also stimulated NO release from pure solutions of low molecular weight RSNO (GSNO and SNAP) and glutathione dinitrosyl iron complex (GSH-DNIC) in a power- and wavelength-dependent manner, with the greatest effect at 670â¯nm. NO release from SNAP using 670 was nearly ten-fold more than GSNO or GSH-DNIC, with no substantial difference in NO production at 740â¯nm and 830â¯nm. Thermal effects of irradiation on vasodilation or NO release from S-nitrosothiols and DNIC was minimal. Our results suggest 670â¯nm is the optimal wavelength for R/NIR treatment of certain vascular-related diseases.
Assuntos
Artérias/efeitos dos fármacos , Ferro/farmacologia , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/farmacologia , S-Nitrosotióis/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Artérias/efeitos da radiação , Raios Infravermelhos , Luz , Camundongos Endogâmicos C57BL , Vasodilatação/efeitos da radiaçãoRESUMO
Dinitrosyl iron complexes (DNIC) spontaneously form in aqueous solutions of Fe(II), nitric oxide (NO), and various anions. They exist as an equilibrium between diamagnetic, dimeric (bi-DNIC) and paramagnetic, monomeric (mono-DNIC) forms. Thiolate groups (e.g., on glutathione or protein cysteine residues) are the most biologically relevant anions to coordinate to Fe(II). Low molecular weight DNIC have previously been suggested to be important mediators of NO biology in cells, and emerging literature supports their role in the control of iron-dependent cellular processes. Recently, it was shown that DNIC may be one of the most abundant NO-derived products in cells and may serve as intermediates in the cellular formation of S-nitrosothiols. In this work, we examined the stability of low molecular weight DNIC and investigated issues with their detection in the presence of other NO-dependent metabolites such as S-nitrosothiols. By using spectrophotometric, Electron Paramagnetic Resonance, ozone-based chemiluminesence, and HPLC techniques we established that at neutral pH, bi-DNIC remain stable for hours, whereas excess thiol results in decomposition to form nitrite. NO was also detected during the decomposition, but no S-nitrosothiol formation was observed. Importantly, mercury chloride accelerated the degradation of DNIC; thus, the implications of this finding for the diagnostic use of mercury chloride in the detection of S-nitrosothiols were determined in simple and complex biological systems. We conclude S-nitrosothiol levels may have been substantially overestimated in all methods where mercury chloride has been used.
Assuntos
Compostos Ferrosos/análise , S-Nitrosotióis/análise , Animais , Cisteína/análogos & derivados , Cisteína/química , Cisteína/farmacologia , Compostos Ferrosos/química , Compostos Ferrosos/metabolismo , Glutationa/análise , Glutationa/química , Humanos , Concentração de Íons de Hidrogênio , Lipopolissacarídeos/farmacologia , Luminescência , Células MCF-7 , Camundongos , Óxido Nítrico/análise , Óxido Nítrico/metabolismo , Nitritos/análise , Nitritos/síntese química , Células RAW 264.7 , S-Nitrosotióis/química , S-Nitrosotióis/metabolismo , S-Nitrosotióis/farmacologia , Espermina/análogos & derivados , Espermina/farmacologiaRESUMO
BACKGROUND: S-nitrosothiols have been recognized as biologically-relevant products of nitric oxide that are involved in many of the diverse activities of this free radical. SCOPE OF REVIEW: This review serves to discuss current methods for the detection and analysis of protein S-nitrosothiols. The major methods of S-nitrosothiol detection include chemiluminescence-based methods and switch-based methods, each of which comes in various flavors with advantages and caveats. MAJOR CONCLUSIONS: The detection of S-nitrosothiols is challenging and prone to many artifacts. Accurate measurements require an understanding of the underlying chemistry of the methods involved and the use of appropriate controls. GENERAL SIGNIFICANCE: Nothing is more important to a field of research than robust methodology that is generally trusted. The field of S-nitrosation has developed such methods but, as S-nitrosothiols are easy to introduce as artifacts, it is vital that current users learn from the lessons of the past. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
Assuntos
Medições Luminescentes/métodos , S-Nitrosotióis/análise , Animais , HumanosRESUMO
Changes in oxidative capacities and phospholipid remodeling accompany temperature acclimation in ectothermic animals. Both responses may alter redox status and membrane susceptibility to lipid peroxidation (LPO). We tested the hypothesis that phospholipid remodeling is sufficient to offset temperature-driven rates of LPO and, thus, membrane susceptibility to LPO is conserved. We also predicted that the content of LPO products is maintained over a range of physiological temperatures. To assess LPO susceptibility, rates of LPO were quantified with the fluorescent probe C11-BODIPY in mitochondria and sarcoplasmic reticulum from oxidative and glycolytic muscle of striped bass (Morone saxatilis) acclimated to 7°C and 25°C. We also measured phospholipid compositions, contents of LPO products [i.e., individual classes of phospholipid hydroperoxides (PLOOH)], and two membrane antioxidants. Despite phospholipid headgroup and acyl chain remodeling, these alterations do not counter the effect of temperature on LPO rates (i.e., LPO rates are generally not different among acclimation groups when normalized to phospholipid content and compared at a common temperature). Although absolute levels of PLOOH are higher in muscles from cold- than warm-acclimated fish, this difference is lost when PLOOH levels are normalized to total phospholipid. Contents of vitamin E and two homologs of ubiquinone are more than four times higher in mitochondria prepared from oxidative muscle of warm- than cold-acclimated fish. Collectively, our data demonstrate that although phospholipid remodeling does not provide a means for offsetting thermal effects on rates of LPO, differences in phospholipid quantity ensure a constant proportion of LPO products with temperature variation.
Assuntos
Aclimatação , Bass/metabolismo , Membrana Celular/metabolismo , Peroxidação de Lipídeos , Músculo Esquelético/metabolismo , Estresse Oxidativo , Temperatura , Animais , Membrana Celular/patologia , Glicólise , Cinética , Peróxidos Lipídicos/metabolismo , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/patologia , Oxirredução , Fosfolipídeos/metabolismo , Retículo Sarcoplasmático/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo , Vitamina E/metabolismoRESUMO
Red light (670 nm) energy controls vasodilation via the formation of a transferable endothelium-derived nitric oxide (NO)-precursor-containing substance, its intracellular traffic, and exocytosis. Here we investigated the underlying mechanistic effect of oxidative stress on light-mediated vasodilation by using pressure myography on dissected murine arteries and immunofluorescence on endothelial cells. Treatment with antioxidants Trolox and catalase decreased vessel dilation. In the presence of catalase, a lower number of exosomes were detected in the vessel bath. Light exposure resulted in increased cellular free radical levels. Mitochondrial reactive oxygen species were also more abundant but did not alter cellular ATP production. Red light enhanced the co-localization of late exosome marker CD63 and cellular S-nitrosoprotein to a greater extent than high glucose, suggesting that a mild oxidative stress favors the localization of NO precursor in late exosomes. Exocytosis regulating protein Rab11 was more abundant after irradiation. Our findings conclude that red-light-induced gentle oxidative stress facilitates the dilation of blood vessels, most likely through empowering the traffic of vasodilatory substances. Application of antioxidants disfavors this mechanism.
RESUMO
S-nitrosothiols are products of nitric oxide (NO) metabolism that have been implicated in a plethora of signalling processes. However, mechanisms of S-nitrosothiol formation in biological systems are uncertain, and no efficient protein-mediated process has been identified. Recently, we observed that ferric cytochrome c can promote S-nitrosoglutathione formation from NO and glutathione by acting as an electron acceptor under anaerobic conditions. In the present study, we show that this mechanism is also robust under oxygenated conditions, that cytochrome c can promote protein S-nitrosation via a transnitrosation reaction and that cell lysate depleted of cytochrome c exhibits a lower capacity to synthesize S-nitrosothiols. Importantly, we also demonstrate that this mechanism is functional in living cells. Lower S-nitrosothiol synthesis activity, from donor and nitric oxide synthase-generated NO, was found in cytochrome c-deficient mouse embryonic cells as compared with wild-type controls. Taken together, these data point to cytochrome c as a biological mediator of protein S-nitrosation in cells. This is the most efficient and concerted mechanism of S-nitrosothiol formation reported so far.
Assuntos
Citocromos c/metabolismo , S-Nitrosotióis/metabolismo , Aerobiose , Anaerobiose , Animais , Antimicina A/farmacologia , Células Cultivadas , Embrião de Mamíferos/metabolismo , Glutationa/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Nitric oxide dependent vasodilation is an effective mechanism for restoring blood flow to ischemic tissues. Previously, we established an ex vivo murine model whereby red light (670 nm) facilitates vasodilation via an endothelium derived vasoactive species which contains a functional group that can be reduced to nitric oxide. In the present study we investigated this vasodilator in vivo by measuring blood flow with Laser Doppler Perfusion imaging in mice. The vasodilatory nitric oxide precursor was analyzed in plasma and muscle with triiodide-dependent chemiluminescence. First, a 5-10 min irradiation of a 3 cm2 area in the hind limb at 670 nm (50 mW/cm2) produced optimal vasodilation. The nitric oxide precursor in the irradiated quadriceps tissue decreased significantly from 123 ± 18 pmol/g tissue by both intensity and duration of light treatment to an average of 90 ± 17 pmol/g tissue, while stayed steady (137 ± 21 pmol/g tissue) in unexposed control hindlimb. Second, the blood flow remained elevated 30 min after termination of the light exposure. The nitric oxide precursor content significantly increased by 50% by irradiation then depleted in plasma, while remained stable in the hindlimb muscle. Third, to mimic human peripheral artery disease, an ameroid constrictor was inserted on the proximal femoral artery of mice and caused a significant reduction of flow. Repeated light treatment for 14 days achieved steady and significant increase of perfusion in the constricted limb. Our results strongly support 670 nm light can regulate dilation of conduit vessel by releasing a vasoactive nitric oxide precursor species and may offer a simple home-based therapy in the future to individuals with impaired blood flow in the leg.
RESUMO
Preeclampsia is a serious pregnancy disorder which in extreme cases may lead to maternal and fetal injury or death. Preexisting conditions which increase oxidative stress, e.g., hypertension and diabetes, increase the mother's risk to develop preeclampsia. Previously, we established that when the extracellular matrix is exposed to oxidative stress, trophoblast function is impaired, and this may lead to improper placentation. We investigated how the oxidative ECM present in preeclampsia alters the behavior of first trimester extravillous trophoblasts. We demonstrate elevated levels of advanced glycation end products (AGE) and lipid oxidation end product 4-hydroxynonenal in preeclamptic ECM (28%, and 32% increase vs control, respectively) accompanied with 35% and 82% more 3-chlorotyrosine and 3-nitrotyrosine vs control, respectively. Furthermore, we hypothesized that 670 nm phototherapy, which has antioxidant properties, reverses the observed trophoblast dysfunction as depicted in the improved migration and reduction in apoptosis. Since NO is critical for placentation, we examined eNOS activity in preeclamptic placentas compared to healthy ones and found no differences; however, 670 nm light treatment triggered enhanced NO availability presumably by using alternative NO sources. Light exposure decreased apoptosis and restored trophoblast migration to levels in trophoblasts cultured on preeclamptic ECM. Moreover, 670 nm irradiation restored expression of Transforming Growth Factor (TGFß) and Placental Growth Factor (PLGF) to levels observed in trophoblasts cultured on healthy placental ECM. We conclude the application of 670 nm light can successfully mitigate the damaged placental microenvironment of late onset preeclampsia as depicted by the restored trophoblast behavior.
Assuntos
Pré-Eclâmpsia , Trofoblastos , Matriz Extracelular/metabolismo , Feminino , Humanos , Placenta/metabolismo , Fator de Crescimento Placentário , Placentação , Pré-Eclâmpsia/metabolismo , Gravidez , Trofoblastos/metabolismoRESUMO
Normally, cell free haemoglobin is bound by haptoglobin and efficiently cleared. However, the chronic haemolysis in sickle cell disease (SCD) overwhelms haptoglobin binding capacity and protein turnover, resulting in elevated cell free haemoglobin. Cell free haemoglobin acts as both a scavenger of vasoactive nitric oxide and a pro-oxidant. In addition, methaemoglobin (metHb) releases the haem moiety, which can bind to albumin to form methaemalbumin (metHSA). This study used electron paramagnetic resonance to detect metHSA in SCD plasma and demonstrated that haptoglobin prevents haem transfer from metHb to HSA. MetHSA may either provide a second line of defence against haemoglobin/haem-mediated oxidation or contribute to the pro-oxidant environment of SCD plasma. We demonstrated that HSA inhibited oxidative protein modification induced by metHb. Additionally, we showed that while metHb induced haem oxygenase 1 (HO-1), an indicator of oxidative stress, HSA attenuated metHb induction of this enzyme, thereby limiting the potential benefits of HO-1. Furthermore, HO-1 induction by metHSA was less than HO-1 induction by equimolar metHb not bound to albumin. Our findings confirm the presence of metHSA in SCD and suggest that haem transfer from metHb to HSA reduces the oxidative effects of free haemoglobin/haem on endothelium with both beneficial (reduced protein oxidation) and potentially harmful (reduced HO-1 induction) outcomes.
Assuntos
Anemia Falciforme/sangue , Heme Oxigenase-1/metabolismo , Metemalbumina/biossíntese , Animais , Bovinos , Células Cultivadas , Endotélio Vascular/metabolismo , Haptoglobinas/fisiologia , Heme/metabolismo , Humanos , Peroxidação de Lipídeos/fisiologia , Metemalbumina/metabolismo , Oxirredução , Ligação Proteica/fisiologia , Albumina Sérica/metabolismoRESUMO
Red light (670 nm) promotes ex vivo dilation of blood vessels in a nitric oxide (NO) dependent, but eNOS independent manner by secreting a quasi-stable and transferable vasoactive substance with the characteristics of S-nitrosothiols (RSNO) from the endothelium. In the present work we establish that 670 nm light mediated vasodilation occurs in vivo and is physiologically stable. Light exposure depletes intracellular S-nitroso protein while concomitantly increasing extracellular RNSO, suggesting vesicular pathways are involved. Furthermore, we demonstrate this RSNO vasodilator is embedded in extracellular vesicles (EV). The action of red light on vesicular trafficking appears to increase expression of endosome associated membrane protein CD63 in bovine aortic endothelial cells, enhance endosome localization in the endothelium, and induce exit of RSNO containing EVs from murine facialis arteries. We suggest a mechanism by which the concerted actions of 670 nm light initiate formation of RSNO containing EVs which exit the endothelium and trigger relaxation of smooth muscle cells.
Assuntos
Vesículas Extracelulares/metabolismo , Luz , Vasodilatação/efeitos da radiação , Animais , Bovinos , Células Cultivadas , Endotélio Vascular/citologia , Endotélio Vascular/efeitos da radiação , Camundongos , Camundongos Endogâmicos C57BL , Compostos Nitrosos/metabolismo , Compostos de Sulfidrila/metabolismoRESUMO
Nitric oxide is an important messenger in numerous biological processes, such as angiogenesis, hypoxic vasodilation, and cardioprotection. Although nitric oxide synthases (NOS) produce the bulk of NO, there is increasing interest in NOS independent generation of NO in vivo, particularly during hypoxia or anoxia, where low oxygen tensions limit NOS activity. Interventions that can increase NO bioavailability have significant therapeutic potential. The use of far red and near infrared light (R/NIR) can reduce infarct size, protect neurons from methanol toxicity, and stimulate angiogenesis. How R/NIR modulates these processes in vivo and in vitro is unknown, but it has been suggested that increases in NO levels are involved. In this study we examined if R/NIR light could facilitate the release of NO from nitrosyl heme proteins. In addition, we examined if R/NIR light could enhance the protective effects of nitrite on ischemia and reperfusion injury in the rabbit heart. We show both in purified systems and in myocardium that R/NIR light can decay nitrosyl hemes and release NO, and that this released NO may enhance the cardioprotective effects of nitrite. Thus, the photodissociation to NO and its synergistic effect with sodium nitrite may represent a noninvasive and site-specific means for increasing NO bioavailability.
Assuntos
Cardiotônicos/metabolismo , Hemoglobinas/metabolismo , Raios Infravermelhos , Mioglobina/metabolismo , Óxido Nítrico/metabolismo , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Sequestradores de Radicais Livres/farmacologia , Hemeproteínas/metabolismo , Hemodinâmica/efeitos dos fármacos , Medições Luminescentes , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Ozônio/metabolismo , Coelhos , Análise EspectralRESUMO
Nitric oxide (NO*) participates in the regulation of a wide array of biological processes and its deficit contributes to the severity of many diseases. Recently, a role of NO deficiency that occurs as a result of intravascular hemolysis and increases in levels of cell-free hemoglobin in the pathway of chronic anemic pathologies has been suggested. Experimental evidence for deoxyhemoglobin-catalyzed reduction of nitrite to NO* leads to the possibility of nitrite infusion-based therapies to correct NO* deficits. However, the presence of plasma hemoglobin also raises the possibility of deleterious free radical-mediated oxidative damage from the reaction between nitrite and oxyhemoglobin in the vasculature. We show that the conditions required for the reaction between nitrite and oxyhemoglobin to exhibit free radical-mediated autocatalytic kinetics are highly unlikely to occur in the plasma compartment, even during extensive hemolysis and with pharmacological nitrite doses. Although the presence of haptoglobin enhances the rate of the reaction between nitrite and oxyhemoglobin, common plasma antioxidants-ascorbate and urate, as well as catalase-prevent autocatalysis. Our findings suggest that pharmacological doses of nitrite are unlikely to cause free radical or ferrylhemoglobin formation in plasma originating from the reaction of nitrite with cell-free oxyhemoglobin in vivo.
Assuntos
Nitritos/metabolismo , Oxiemoglobinas/metabolismo , Plasma/metabolismo , Ácido Ascórbico/metabolismo , Catalase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres/metabolismo , Haptoglobinas/metabolismo , Hemoglobinas/metabolismo , Humanos , Metemoglobina/metabolismo , Nitritos/química , Oxiemoglobinas/química , Análise de Regressão , Ácido Úrico/metabolismoRESUMO
There is significant therapeutic advantage of nitric oxide synthase (NOS) independent nitric oxide (NO) production in maladies where endothelium, and thereby NOS, is dysfunctional. Electromagnetic radiation in the red and near infrared region has been shown to stimulate NOS-independent but NO-dependent vasodilation, and thereby has significant therapeutic potential. We have recently shown that red light induces acute vasodilatation in the pre-constricted murine facial artery via the release of an endothelium derived substance. In this study we have investigated the mechanism of vasodilatation and conclude that 670â¯nm light stimulates vasodilator release from an endothelial store, and that this vasodilator has the characteristics of an S-nitrosothiol (RSNO). This study shows that 670â¯nm irradiation can be used as a targeted and non-invasive means to release biologically relevant amounts of vasodilator from endothelial stores. This raises the possibility that these stores can be pharmacologically built-up in pathological situations to improve the efficacy of red light treatment. This strategy may overcome eNOS dysfunction in peripheral vascular pathologies for the improvement of vascular health.
Assuntos
Ácido Ascórbico/farmacologia , Luz , S-Nitrosotióis/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatação/efeitos da radiação , Vasodilatadores/farmacologia , Acetilcolina/farmacologia , Animais , Artérias/efeitos dos fármacos , Artérias/metabolismo , Artérias/efeitos da radiação , Camundongos , Modelos Biológicos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
This review discusses proteomic methods to detect and identify S-nitrosated proteins. Protein S-nitrosation, the post-translational modification of thiol residues to form S-nitrosothiols, has been suggested to be a mechanism of cellular redox signaling by which nitric oxide can alter cellular function through modification of protein thiol residues. It has become apparent that methods that will detect and identify low levels of S-nitrosated protein in complex protein mixtures are required in order to fully appreciate the range, extent and selectivity of this modification in both physiological and pathological conditions. While many advances have been made in the detection of either total cellular S-nitrosation or individual S-nitrosothiols, proteomic methods for the detection of S-nitrosation are in relative infancy. This review will discuss the major methods that have been used for the proteomic analysis of protein S-nitrosation and discuss the pros and cons of this methodology.
Assuntos
Proteômica/métodos , S-Nitrosotióis/análise , Animais , Bioensaio , Transporte Biológico , Humanos , Peso Molecular , Proteoma/análise , S-Nitrosotióis/químicaRESUMO
Peripheral artery disease (PAD) is a morbid condition whereby ischemic peripheral muscle causes pain and tissue breakdown. Interestingly, PAD risk factors, e.g. diabetes mellitus, cause endothelial dysfunction secondary to decreased nitric oxide (NO) levels, which could explain treatment failures. Previously, we demonstrated 670nm light (R/NIR) increased NO from nitrosyl-heme stores, therefore we hypothesized R/NIR can stimulate vasodilation in healthy and diabetic blood vessels. Vasodilation was tested by ex vivo pressure myography in wild type C57Bl/6, endothelial nitric oxide synthase (eNOS) knockout, and db/db mice (10mW/cm2 for 5min with 10min dark period). NOS inhibition with N-Nitroarginine methyl ester (L-NAME) or the NO scavenger Carboxy-PTIO (c-PTIO) tested the specificity of NO production. 4,5-Diaminofluorescein diacetate (DAF-2) measured NO in human dermal microvascular endothelial cells (HMVEC-d). R/NIR significantly increased vasodilation in wild type and NOS inhibited groups, however R/NIR dilation was totally abolished with c-PTIO and blood vessel denudation. Interestingly, the bath solution from intact R/NIR stimulated vessels could dilate light naïve vessels in a NO dependent manner. Characterization of the bath identified a NO generating substance suggestive of S-nitrosothiols or non heme iron nitrosyl complexes. Consistent with the finding of an endothelial source of NO, intracellular NO increased with R/NIR in HMVEC-d treated with and without L-NAME (1mM), yet c-PTIO (100µm) reduced NO production. R/NIR significantly dilated db/db blood vessels. In conclusion, R/NIR stimulates vasodilation by release of NO bound substances from the endothelium. In a diabetes model of endothelial dysfunction, R/NIR restores vasodilation, which lends the potential for new treatments for diabetic vascular disease.
Assuntos
Diabetes Mellitus Experimental/metabolismo , Endotélio Vascular/metabolismo , Fatores Relaxantes Dependentes do Endotélio/metabolismo , Luz , Animais , Diabetes Mellitus Experimental/enzimologia , Endotélio Vascular/enzimologia , Endotélio Vascular/efeitos da radiação , Humanos , Raios Infravermelhos , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismoRESUMO
The reaction of nitrite with hemoglobin has become of increasing interest due to the realization that plasma nitrite may act as an NO congener that is activated by interaction with red blood cells. Using a combination of spectrophotometry, immuno-spin trapping, and EPR, we have examined the formation of radicals during the oxidation of oxyhemoglobin (oxyHb) and oxymyoglobin (oxyMb) by inorganic nitrite. The proposed intermediacy of ferryl species during this oxidation was confirmed by spectrophotometry using multiple linear regression analysis of kinetic data. Using EPR/spin trapping, a protein radical was observed in the case of oxyMb, but not oxyHb, and was inhibited by catalase. When DMPO spin trapping was combined with Western blot analysis using an anti-DMPO-nitrone antibody, globin/DMPO adducts of both oxyHb and oxyMb were detected, and their formation was inhibited by catalase. Catalase effects confirm the intermediacy of hydrogen peroxide as a heme oxidant in this system. Spectrophotometric kinetic studies revealed that the presence of DMPO elongated the lag phase and decreased the maximal rate of oxidation of both oxyHb and oxyMb, which suggests that the globin radical plays an active role in the mechanism of autocatalysis. Interestingly, the oxidation of oxyHb or oxyMb by nitrite, but not by hydrogen peroxide, produced a diffusible radical that was able to generate spin adducts on a bystander protein. This indicates that the oxidation of oxyhemeproteins by nitrite may cause more widespread oxidative damage than the corresponding oxidation by hydrogen peroxide. The immuno-spin trapping technique represents an important new development for the study of the range and extent of protein oxidation by free radicals and oxidants.
Assuntos
Mioglobina/química , Nitritos/farmacologia , Oxiemoglobinas/química , Detecção de Spin , Animais , Catalase/farmacologia , Catálise , Bovinos , Espectroscopia de Ressonância de Spin Eletrônica , Radicais Livres , Coração , Heme/química , Cavalos , Humanos , Peróxido de Hidrogênio/farmacologia , Imunoensaio , Cinética , Mioglobina/metabolismo , Oxidantes/farmacologia , Oxirredução , Oxiemoglobinas/metabolismoRESUMO
Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H2O2-dependent intracellular oxidation of DCFH to a green fluorescent product, 2',7'-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H2O2-induced DCF fluorescence was inhibited by the monoclonal IgA-class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H2O2-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H2O2-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.
Assuntos
Endotélio Vascular/metabolismo , Peróxido de Hidrogênio/farmacologia , Ferro/metabolismo , Estresse Oxidativo/fisiologia , Receptores da Transferrina/metabolismo , Aconitato Hidratase/antagonistas & inibidores , Animais , Anticorpos Monoclonais/farmacologia , Apoptose/efeitos dos fármacos , Caspase 3 , Caspases/metabolismo , Bovinos , Células Cultivadas , Endocitose/efeitos dos fármacos , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Fluoresceínas/metabolismo , Fluoresceínas/farmacocinética , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacocinética , Glutationa/farmacologia , Ferro/farmacocinética , Quelantes de Ferro/farmacologia , Proteína 1 Reguladora do Ferro/metabolismo , Peróxidos Lipídicos/farmacologia , Metaloporfirinas/farmacologia , Oxidantes/farmacologia , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Receptores da Transferrina/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.
Assuntos
Ácido Ascórbico/química , Biotina/análise , Proteínas/química , S-Nitrosotióis/análise , Animais , Biotina/química , Células Cultivadas , Cisteína/análogos & derivados , Cisteína/farmacologia , Camundongos , Nitrosação , Oxirredução , Proteínas/imunologia , Proteínas/metabolismo , Proteômica , S-Nitrosotióis/farmacologiaRESUMO
The kinetics of the reaction between superoxide and the spin trapping agents 5,5-dimethyl-1-pyrroline N-oxide (DMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO), and 5-tert-butoxycarbonyl-5-methyl-1-pyrroline N-oxide (BMPO) were re-examined in the superoxide-generating xanthine/xanthine oxidase system, by competition with spontaneous dismutation. The approach used singular value decomposition (SVD), multiple linear regression, and spectral simulation. The experiments were carried out using a two-syringe mixing arrangement with fast scan acquisition of 100 consecutive EPR spectra. Using SVD analysis, the extraction of both temporal and spectral information could be obtained from in a single run. The superoxide spin adduct was the exclusive EPR active species in the case of DEPMPO and BMPO, and the major component when DMPO was used. In the latter case a very low concentration of hydroxyl adduct was also observed, which did not change during the decay of the DMPO-superoxide adduct. This indicates that the hydroxyl radical adduct is not formed from the spontaneous decay of the superoxide radical adduct, as has been previously suggested [correction]. It was established that in short-term studies (up to 100 s) DMPO was the superior spin trapping agent, but for reaction times longer than 100 s the other two spin traps were more advantageous. The second order rate constants for the spin trapping reaction were found to be DMPO (2.4 M(-1)s(-1)), DEPMPO (0.53 M(-1)s(-1)), and BMPO (0.24 M(-1)s(-1)) determined through competition with spontaneous dismutation of superoxide, at pH 7.4 and 20 degrees C.