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1.
Physiol Rev ; 102(2): 859-892, 2022 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-34486392

RESUMO

Globin proteins exist in every cell type of the vasculature, from erythrocytes to endothelial cells, vascular smooth muscle cells, and peripheral nerve cells. Many globin subtypes are also expressed in muscle tissues (including cardiac and skeletal muscle), in other organ-specific cell types, and in cells of the central nervous system (CNS). The ability of each of these globins to interact with molecular oxygen (O2) and nitric oxide (NO) is preserved across these contexts. Endothelial α-globin is an example of extraerythrocytic globin expression. Other globins, including myoglobin, cytoglobin, and neuroglobin, are observed in other vascular tissues. Myoglobin is observed primarily in skeletal muscle and smooth muscle cells surrounding the aorta or other large arteries. Cytoglobin is found in vascular smooth muscle but can also be expressed in nonvascular cell types, especially in oxidative stress conditions after ischemic insult. Neuroglobin was first observed in neuronal cells, and its expression appears to be restricted mainly to the CNS and the peripheral nervous system. Brain and CNS neurons expressing neuroglobin are positioned close to many arteries within the brain parenchyma and can control smooth muscle contraction and thus tissue perfusion and vascular reactivity. Overall, reactions between NO and globin heme iron contribute to vascular homeostasis by regulating vasodilatory NO signals and scavenging reactive species in cells of the mammalian vascular system. Here, we discuss how globin proteins affect vascular physiology, with a focus on NO biology, and offer perspectives for future study of these functions.


Assuntos
Fenômenos Fisiológicos Cardiovasculares , Citoglobina/metabolismo , Células Endoteliais/metabolismo , Globinas/metabolismo , Animais , Humanos , Mioglobina/metabolismo , Neuroglobina/metabolismo
2.
Circulation ; 2024 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-38836358

RESUMO

BACKGROUND: Whether aortic valve stenosis (AS) can adversely affect systemic endothelial function independently of standard modifiable cardiovascular risk factors is unknown. METHODS: We therefore investigated endothelial and cardiac function in an experimental model of AS mice devoid of standard modifiable cardiovascular risk factors and human cohorts with AS scheduled for transcatheter aortic valve replacement. Endothelial function was determined by flow-mediated dilation using ultrasound. Extracellular hemoglobin (eHb) concentrations and NO consumption were determined in blood plasma of mice and humans by ELISA and chemiluminescence. This was complemented by measurements of aortic blood flow using 4-dimensional flow acquisition by magnetic resonance imaging and computational fluid dynamics simulations. The effects of plasma and red blood cell (RBC) suspensions on vascular function were determined in transfer experiments in a murine vasorelaxation bioassay system. RESULTS: In mice, the induction of AS caused systemic endothelial dysfunction. In the presence of normal systolic left ventricular function and mild hypertrophy, the increase in the transvalvular gradient was associated with elevated eryptosis, increased eHb and plasma NO consumption; eHb sequestration by haptoglobin restored endothelial function. Because the aortic valve orifice area in patients with AS decreased, postvalvular mechanical stress in the central ascending aorta increased. This was associated with elevated eHb, circulating RBC-derived microvesicles, eryptotic cells, lower haptoglobin levels without clinically relevant anemia, and consecutive endothelial dysfunction. Transfer experiments demonstrated that reduction of eHb by treatment with haptoglobin or elimination of fluid dynamic stress by transcatheter aortic valve replacement restored endothelial function. In patients with AS and subclinical RBC fragmentation, the remaining circulating RBCs before and after transcatheter aortic valve replacement exhibited intact membrane function, deformability, and resistance to osmotic and hypoxic stress. CONCLUSIONS: AS increases postvalvular swirling blood flow in the central ascending aorta, triggering RBC fragmentation with the accumulation of hemoglobin in the plasma. This increases NO consumption in blood, thereby limiting vascular NO bioavailability. Thus, AS itself promotes systemic endothelial dysfunction independent of other established risk factors. Transcatheter aortic valve replacement is capable of limiting NO scavenging and rescuing endothelial function by realigning postvalvular blood flow to near physiological patterns. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT05603520. URL: https://www.clinicaltrials.gov; Unique identifier: NCT01805739.

3.
Physiol Genomics ; 56(2): 113-127, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-37982169

RESUMO

Endothelial cells (ECs) adapt to the unique needs of their resident tissue and metabolic perturbations, such as obesity. We sought to understand how obesity affects EC metabolic phenotypes, specifically mitochondrial gene expression. We investigated the mesenteric and adipose endothelium because these vascular beds have distinct roles in lipid homeostasis. Initially, we performed bulk RNA sequencing on ECs from mouse adipose and mesenteric vasculatures after a normal chow (NC) diet or high-fat diet (HFD) and found higher mitochondrial gene expression in adipose ECs compared with mesenteric ECs in both NC and HFD mice. Next, we performed single-cell RNA sequencing and categorized ECs as arterial, capillary, venous, or lymphatic. We found mitochondrial genes to be enriched in adipose compared with mesentery under NC conditions in artery and capillary ECs. After HFD, these genes were decreased in adipose ECs, becoming like mesenteric ECs. Transcription factor analysis revealed that peroxisome proliferator-activated receptor-γ (PPAR-γ) had high specificity in NC adipose artery and capillary ECs. These findings were recapitulated in single-nuclei RNA-sequencing data from human visceral adipose. The sum of these findings suggests that mesenteric and adipose arterial ECs metabolize lipids differently, and the transcriptional phenotype of the vascular beds converges in obesity due to downregulation of PPAR-γ in adipose artery and capillary ECs.NEW & NOTEWORTHY Using bulk and single-cell RNA sequencing on endothelial cells from adipose and mesentery, we found that an obesogenic diet induces a reduction in adipose endothelial oxidative phosphorylation gene expression, resulting in a phenotypic convergence of mesenteric and adipose endothelial cells. Furthermore, we found evidence that PPAR-γ drives this phenotypic shift. Mining of human data sets segregated based on body mass index supported these findings. These data point to novel mechanisms by which obesity induces endothelial dysfunction.


Assuntos
Endotélio Vascular , Genes Mitocondriais , Humanos , Camundongos , Animais , Endotélio Vascular/metabolismo , Células Endoteliais/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Artérias , Obesidade/metabolismo , Dieta Hiperlipídica/efeitos adversos , Tecido Adiposo/metabolismo
4.
Circulation ; 144(11): 870-889, 2021 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-34229449

RESUMO

BACKGROUND: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. METHODS: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. RESULTS: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/inv mice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. CONCLUSIONS: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis.


Assuntos
Pressão Sanguínea/fisiologia , Células Endoteliais/metabolismo , Eritrócitos/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/metabolismo , Acetilcolina/farmacologia , Animais , Doenças da Aorta/tratamento farmacológico , Arginina/análogos & derivados , Arginina/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Contagem de Eritrócitos/métodos , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Camundongos
5.
Nitric Oxide ; 125-126: 69-77, 2022 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-35752264

RESUMO

Arginase 1 (Arg1) is a ubiquitous enzyme belonging to the urea cycle that catalyzes the conversion of l-arginine into l-ornithine and urea. In endothelial cells (ECs), Arg1 was proposed to limit the availability of l-arginine for the endothelial nitric oxide synthase (eNOS) and thereby reduce nitric oxide (NO) production, thus promoting endothelial dysfunction and vascular disease. The role of EC Arg1 under homeostatic conditions is in vivo less understood. The aim of this study was to investigate the role of EC Arg1 on the regulation of eNOS, vascular tone, and endothelial function under normal homeostatic conditions in vivo and ex vivo. By using a tamoxifen-inducible EC-specific gene-targeting approach, we generated EC Arg1 KO mice. Efficiency and specificity of the gene targeting strategy was demonstrated by DNA recombination and loss of Arg1 expression measured after tamoxifen treatment in EC only. In EC Arg1 KO mice we found a significant decrease in Arg1 expression in heart and lung ECs and in the aorta, however, vascular enzymatic activity was preserved likely due to the presence of high levels of Arg1 in smooth muscle cells. Moreover, we found a downregulation of eNOS expression in the aorta, and a fully preserved systemic l-arginine and NO bioavailability, as demonstrated by the levels of l-arginine, l-ornithine, and l-citrulline as well as nitrite, nitrate, and nitroso-species. Lung and liver tissues from EC Arg1 KO mice showed respectively increase or decrease in nitrosyl-heme species, indicating that the lack of endothelial Arg1 affects NO bioavailability in these organs. In addition, EC Arg1 KO mice showed fully preserved acetylcholine-mediated vascular relaxation in both conductance and resistant vessels but increased phenylephrine-induced vasoconstriction. Systolic, diastolic, and mean arterial pressure and cardiac performance in EC Arg1 KO mice were not different from the wild-type littermate controls. In conclusion, under normal homeostatic conditions, lack of EC Arg1 expression is associated with a down-regulation of eNOS expression but a preserved NO bioavailability and vascular endothelial function. These results suggest that a cross-talk exists between Arg1 and eNOS to control NO production in ECs, which depends on both L-Arg availability and EC Arg1-dependent eNOS expression.


Assuntos
Arginase , Óxido Nítrico Sintase Tipo III , Animais , Arginase/genética , Arginase/metabolismo , Arginina/metabolismo , Regulação para Baixo , Células Endoteliais/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Ornitina , Tamoxifeno/metabolismo , Ureia/metabolismo
6.
Biol Chem ; 402(3): 317-331, 2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33544503

RESUMO

The mechanical properties of red blood cells (RBCs) are fundamental for their physiological role as gas transporters. RBC flexibility and elasticity allow them to survive the hemodynamic changes in the different regions of the vascular tree, to dynamically contribute to the flow thereby decreasing vascular resistance, and to deform during the passage through narrower vessels. RBC mechanoproperties are conferred mainly by the structural characteristics of their cytoskeleton, which consists predominantly of a spectrin scaffold connected to the membrane via nodes of actin, ankyrin and adducin. Changes in redox state and treatment with thiol-targeting molecules decrease the deformability of RBCs and affect the structure and stability of the spectrin cytoskeleton, indicating that the spectrin cytoskeleton may contain redox switches. In this perspective review, we revise current knowledge about the structural and functional characterization of spectrin cysteine redox switches and discuss the current lines of research aiming to understand the role of redox regulation on RBC mechanical properties. These studies may provide novel functional targets to modulate RBC function, blood viscosity and flow, and tissue perfusion in disease conditions.


Assuntos
Eritrócitos/metabolismo , Cisteína/metabolismo , Humanos , Oxirredução , Espectrina/metabolismo
7.
Nitric Oxide ; 115: 44-54, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34325012

RESUMO

Diaminofluoresceins (DAFs) are fluorescent probes widely applied to measure nitric oxide (NO) formation in cells and tissues. The main advantages of these compounds are their availability and low cost, and the general availability of instruments able to detect green fluorescence in all laboratories; these include fluorimeters, flow cytometers, and fluorescent microscopes. What made these molecules particularly interesting for many scientists approaching the NO field is that they are apparently very easy to use, as compared with other techniques requiring specific instrumentation and knowledge like chemiluminescence and electron paramagnetic resonance. However, the reactivity and biological chemistry of these probes in the cellular environment is rather complex and still not fully understood. Moreover, secondary reactions with ascorbate, or interference with thiols occur in cells. Therefore, the use of DAFs requires specific experimental planning and a careful interpretation of the results obtained. In this methodological review, we described in detail what is known about the reactivity of DAFs, their application in biological assays, list some principles to help experimental planning, including the necessary controls, and list the caveats concerning result interpretation. These guiding principles will help to understand the "Method behind our DAF-madness".


Assuntos
Fluoresceína/química , Corantes Fluorescentes/química , Óxido Nítrico/análise , Estrutura Molecular
8.
Basic Res Cardiol ; 115(4): 43, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32533377

RESUMO

Anaemia is frequently present in patients with acute myocardial infarction (AMI) and contributes to an adverse prognosis. We hypothesised that, besides reduced oxygen carrying capacity, anaemia is associated with (1) red blood cell (RBC) dysfunction and a reduced circulating nitric oxide (NO) pool, (2) compensatory enhancement of vascular and cardiac endothelial nitric oxide synthase (eNOS) activity, and (3) contribution of both, RBC dysfunction and reduced circulatory NO pool to left ventricular (LV) dysfunction and fatal outcome in AMI. In mouse models of subacute and chronic anaemia from repeated mild blood loss the circulating NO pool, RBC, cardiac and vascular function were analysed at baseline and in reperfused AMI. In anaemia, RBC function resulted in profound changes in membrane properties, enhanced turnover, haemolysis, dysregulation of intra-erythrocytotic redox state, and RBC-eNOS. RBC from anaemic mice and from anaemic patients with acute coronary syndrome impaired the recovery of contractile function of isolated mouse hearts following ischaemia/reperfusion. In anaemia, the circulating NO pool was reduced. The cardiac and vascular adaptation to anaemia was characterised by increased arterial eNOS expression and activity and an eNOS-dependent increase of end-diastolic left ventricular volume. Endothelial dysfunction induced through genetic or pharmacologic reduction of eNOS-activity abrogated the anaemia-induced cardio-circulatory compensation. Superimposed AMI was associated with decreased survival. In summary, moderate blood loss anaemia is associated with severe RBC dysfunction and reduced circulating NO pool. Vascular and cardiac eNOS are crucial for the cardio-circulatory adaptation to anaemia. RBC dysfunction together with eNOS dysfunction may contribute to adverse outcomes in AMI.


Assuntos
Adaptação Fisiológica/fisiologia , Anemia/fisiopatologia , Eritrócitos/patologia , Coração/fisiopatologia , Óxido Nítrico/sangue , Síndrome Coronariana Aguda/sangue , Síndrome Coronariana Aguda/fisiopatologia , Anemia/sangue , Animais , Artérias/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/sangue , Infarto do Miocárdio/fisiopatologia , Óxido Nítrico Sintase Tipo III/metabolismo
9.
Nitric Oxide ; 96: 44-53, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31911123

RESUMO

There is accumulating evidence that biological membranes are not just homogenous lipid structures, but are highly organized in microdomains, i.e. compartmentalized areas of protein and lipid complexes, which facilitate necessary interactions for various signaling pathways. Each microdomain exhibits unique composition, membrane location and dynamics, which ultimately shape their functional characteristics. In the vasculature, microdomains are crucial for organizing and compartmentalizing vasodilatory signals that contribute to blood pressure homeostasis. In this review we aim to describe how membrane microdomains in both the endothelium and red blood cells allow context-specific regulation of the vasodilatory signal nitric oxide (NO) and its corresponding metabolic products, and how this results in tightly controlled systemic physiological responses. We will describe (1) structural characteristics of microdomains including lipid rafts and caveolae; (2) endothelial cell caveolae and how they participate in mechanosensing and NO-dependent mechanotransduction; (3) the myoendothelial junction of resistance arterial endothelial cells and how protein-protein interactions within it have profound systemic effects on blood pressure regulation, and (4) putative/proposed NO microdomains in RBCs and how they participate in control of systemic NO bioavailability. The sum of these discussions will provide a current view of NO regulation by cellular microdomains.


Assuntos
Cavéolas/metabolismo , Células Endoteliais/metabolismo , Eritrócitos/metabolismo , Óxido Nítrico/metabolismo , Transdução de Sinais/fisiologia , Animais , Mecanotransdução Celular/fisiologia
10.
Arterioscler Thromb Vasc Biol ; 38(9): 1969-1985, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30354262

RESUMO

Cardiovascular disease is a leading cause of death worldwide and accounts for >17.3 million deaths per year, with an estimated increase in incidence to 23.6 million by 2030. 1 Cardiovascular death represents 31% of all global deaths 2 -with stroke, heart attack, and ruptured aneurysms predominantly contributing to these high mortality rates. A key risk factor for cardiovascular disease is hypertension. Although treatment or reduction in hypertension can prevent the onset of cardiovascular events, existing therapies are only partially effective. A key pathological hallmark of hypertension is increased peripheral vascular resistance because of structural and functional changes in large (conductive) and small (resistance) arteries. In this review, we discuss the clinical implications of vascular remodeling, compare the differences between vascular smooth muscle cell remodeling in conductive and resistance arteries, discuss the genetic factors associated with vascular smooth muscle cell function in hypertensive patients, and provide a prospective assessment of current and future research and pharmacological targets for the treatment of hypertension.


Assuntos
Artérias/fisiopatologia , Hipertensão/fisiopatologia , Músculo Liso Vascular/fisiopatologia , Remodelação Vascular , Animais , Anti-Hipertensivos/uso terapêutico , Artérias/patologia , Doenças Cardiovasculares/fisiopatologia , Humanos , Hipertensão/tratamento farmacológico , Hipertensão/genética , Hipertensão/patologia , Inflamação/patologia , Inflamação/fisiopatologia , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/fisiologia , Fatores de Risco , Transdução de Sinais , Transmissão Sináptica/fisiologia , Resistência Vascular
11.
Proc Natl Acad Sci U S A ; 112(34): E4651-60, 2015 Aug 25.
Artigo em Inglês | MEDLINE | ID: mdl-26224837

RESUMO

Experimental evidence suggests that nitric oxide (NO) and hydrogen sulfide (H2S) signaling pathways are intimately intertwined, with mutual attenuation or potentiation of biological responses in the cardiovascular system and elsewhere. The chemical basis of this interaction is elusive. Moreover, polysulfides recently emerged as potential mediators of H2S/sulfide signaling, but their biosynthesis and relationship to NO remain enigmatic. We sought to characterize the nature, chemical biology, and bioactivity of key reaction products formed in the NO/sulfide system. At physiological pH, we find that NO and sulfide form a network of cascading chemical reactions that generate radical intermediates as well as anionic and uncharged solutes, with accumulation of three major products: nitrosopersulfide (SSNO(-)), polysulfides, and dinitrososulfite [N-nitrosohydroxylamine-N-sulfonate (SULFI/NO)], each with a distinct chemical biology and in vitro and in vivo bioactivity. SSNO(-) is resistant to thiols and cyanolysis, efficiently donates both sulfane sulfur and NO, and potently lowers blood pressure. Polysulfides are both intermediates and products of SSNO(-) synthesis/decomposition, and they also decrease blood pressure and enhance arterial compliance. SULFI/NO is a weak combined NO/nitroxyl donor that releases mainly N2O on decomposition; although it affects blood pressure only mildly, it markedly increases cardiac contractility, and formation of its precursor sulfite likely contributes to NO scavenging. Our results unveil an unexpectedly rich network of coupled chemical reactions between NO and H2S/sulfide, suggesting that the bioactivity of either transmitter is governed by concomitant formation of polysulfides and anionic S/N-hybrid species. This conceptual framework would seem to offer ample opportunities for the modulation of fundamental biological processes governed by redox switching and sulfur trafficking.


Assuntos
Sulfeto de Hidrogênio/metabolismo , Óxido Nítrico/metabolismo , Óxidos de Nitrogênio/metabolismo , Sulfetos/metabolismo , Animais , Disponibilidade Biológica , Masculino , Nitrogênio/metabolismo , Ratos Wistar , Enxofre/metabolismo
12.
Am J Physiol Cell Physiol ; 313(6): C593-C603, 2017 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-28855161

RESUMO

Red blood cell (RBC)-derived adenosine triphosphate (ATP) has been proposed as an integral component in the regulation of oxygen supply to skeletal muscle. In ex vivo settings RBCs have been shown to release ATP in response to a number of stimuli, including stimulation of adrenergic receptors. Further evidence suggested that ATP release from RBCs was dependent on activation of adenylate cyclase (AC)/cyclic adenosine monophosphate (cAMP)-dependent pathways and involved the pannexin 1 (Panx1) channel. Here we show that RBCs express Panx1 and confirm its absence in Panx1 knockout (-/-) RBCs. However, Panx1-/- mice lack any decrease in exercise performance, challenging the assumptions that Panx1 plays an essential role in increased blood perfusion to exercising skeletal muscle and therefore in ATP release from RBCs. We therefore tested the role of Panx1 in ATP release from RBCs ex vivo in RBC suspensions. We found that stimulation with hypotonic potassium gluconate buffer resulted in a significant increase in ATP in the supernatant, but this was highly correlated with RBC lysis. Next, we treated RBCs with a stable cAMP analog, which did not induce ATP release from wild-type or Panx1-/- mice. Similarly, multiple pharmacological treatments activating AC in RBCs increased intracellular cAMP levels (as measured via mass spectrometry) but did not induce ATP release. The data presented here question the importance of Panx1 for exercise performance and dispute the general assumption that ATP release from RBCs via Panx1 is regulated via cAMP.


Assuntos
Trifosfato de Adenosina/sangue , Conexinas/sangue , AMP Cíclico/sangue , Metabolismo Energético , Eritrócitos/metabolismo , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/sangue , Sistemas do Segundo Mensageiro , 1-Metil-3-Isobutilxantina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Trifosfato de Adenosina/metabolismo , Adenilil Ciclases/sangue , Adulto , Animais , Colforsina/farmacologia , Conexinas/deficiência , Conexinas/genética , Metabolismo Energético/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Tolerância ao Exercício , Feminino , Genótipo , Gluconatos/farmacologia , Hemólise , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Contração Muscular , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Fenótipo , Fatores de Tempo , Adulto Jovem
13.
Mol Cell Biochem ; 417(1-2): 155-67, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-27206740

RESUMO

Red blood cells (RBCs) enzymatically produce nitric oxide (NO) by a functional RBC-nitric oxide synthase (RBC-NOS). NO is a vascular key regulatory molecule. In RBCs its generation is complex and influenced by several factors, including insulin, acetylcholine, and calcium. NO availability is reduced in end-stage renal disease (ESRD) and associated with endothelial dysfunction. We previously demonstrated that, through increased phosphatidylserine membrane exposure, ESRD-RBCs augmented their adhesion to human cultured endothelium, in which NO bioavailability decreased. Since RBC-NOS-dependent NO production in ESRD is unknown, this study aimed to investigate RBC-NOS levels/activation, NO production/bioavailability in RBCs from healthy control subjects (C, N = 18) and ESRD patients (N = 27). Although RBC-NOS expression was lower in ESRD-RBCs, NO, cyclic guanosine monophosphate (cGMP), RBC-NOS Serine1177 phosphorylation level and eNOS/Calmodulin (CaM)/Heat Shock Protein-90 (HSP90) interaction levels were higher in ESRD-RBCs, indicating increased enzyme activation. Conversely, following RBCs stimulation with insulin or ionomycin, NO and cGMP levels were significantly lower in ESRD- than in C-RBCs, suggesting that uremia might reduce the RBC-NOS response to further stimuli. Additionally, the activity of multidrug-resistance-associated protein-4 (MRP4; cGMP-membrane transporter) was significantly lower in ESRD-RBCs, suggesting a possible compromised efflux of cGMP across the ESRD-RBCs membrane. This study for the first time showed highest basal RBC-NOS activation in ESRD-RBCs, possibly to reduce the negative impact of decreased NOS expression. It is further conceivable that high NO production only partially affects cell function of ESRD-RBCs maybe because in vivo they are unable to respond to physiologic stimuli, such as calcium and/or insulin.


Assuntos
GMP Cíclico/metabolismo , Eritrócitos/metabolismo , Falência Renal Crônica/metabolismo , Óxido Nítrico/biossíntese , Idoso , Calmodulina/metabolismo , Eritrócitos/patologia , Feminino , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Falência Renal Crônica/patologia , Falência Renal Crônica/terapia , Masculino , Pessoa de Meia-Idade , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Óxido Nítrico Sintase Tipo III/metabolismo
14.
Pharmacol Res ; 113(Pt A): 490-499, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27663261

RESUMO

Nitric oxide (NO), hydrogen sulfide and polysulfides have been proposed to contribute to redox signaling by activating the Keap-1/Nrf2 stress response system. Nitrosopersulfide (SSNO-) recently emerged as a bioactive product of the chemical interaction of NO or nitrosothiols with sulfide; upon decomposition it generates polysulfides and free NO, triggering the activation of soluble guanylate cyclase, inducing blood vessel relaxation in vitro and lowering blood pressure in vivo. Whether SSNO- itself interacts with the Keap-1/Nrf2 system is unknown. We therefore sought to investigate the ability of SSNO- to activate Nrf2-dependent processes in human vascular endothelial cells, and to compare the pharmacological effects of SSNO- with those of its precursors NO and sulfide at multiple levels of target engagement. We here demonstrate that SSNO- strongly increases nuclear levels, binding activity and transactivation activity of Nrf2, thereby increasing mRNA expression of Hmox-1, the gene encoding for heme oxygenase 1, without adversely affecting cell viability. Under all conditions, SSNO- appeared to be more potent than its parent compounds, NO and sulfide. SSNO--induced Nrf2 transactivation activity was abrogated by either NO scavenging with cPTIO or inhibition of thiol sulfuration by high concentrations of cysteine, implying a role for both persulfides/polysulfides and NO in SSNO- mediated Nrf2 activation. Taken together, our studies demonstrate that the Keap-1/Nrf2 redox system is a biological target of SSNO-, enriching the portfolio of bioactivity of this vasoactive molecule to also engage in the regulation of redox signaling processes. The latter suggests a possible role as messenger and/or mediator in cellular sensing and adaptations processes.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Nitrosaminas/farmacologia , Oxirredução/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Heme Oxigenase-1/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Sulfeto de Hidrogênio/farmacologia , Óxido Nítrico/metabolismo , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Compostos de Sulfidrila/farmacologia , Sulfetos/farmacologia , Ativação Transcricional/efeitos dos fármacos
15.
Nitric Oxide ; 46: 14-24, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25541073

RESUMO

Sulfide (H2S/HS(-)) has been demonstrated to exert an astounding breadth of biological effects, some of which resemble those of nitric oxide (NO). While the chemistry, biochemistry and potential pathophysiology of the cross-talk between sulfide and NO have received considerable attention lately, a comparable assessment of the potential biological implications of an interaction between nitrite and sulfide is lacking. This is surprising inasmuch as nitrite is not only a known bioactive oxidation product of NO, but also efficiently converted to S-nitrosothiols in vivo; the latter have been shown to rapidly react with sulfide in vitro, leading to formation of S/N-hybrid species including thionitrite (SNO(-)) and nitrosopersulfide (SSNO(-)). Moreover, nitrite is used as a potent remedy against sulfide poisoning in the clinic. The chemistry of interaction between nitrite and sulfide or related bioactive metabolites including polysulfides and elemental sulfur has been extensively studied in the past, yet much of this information appears to have been forgotten. In this review, we focus on the potential chemical biology of the interaction between nitrite and sulfide or sulfane sulfur molecules, calling attention to the fundamental chemical properties and reactivities of either species and discuss their possible contribution to the biology, pharmacology and toxicology of both nitrite and sulfide.


Assuntos
Nitritos/química , Nitritos/metabolismo , Sulfetos/química , Sulfetos/metabolismo , Animais , Bioquímica , Humanos , Óxido Nítrico/química , Óxido Nítrico/metabolismo , Especificidade de Órgãos
16.
Basic Res Cardiol ; 109(1): 398, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24346018

RESUMO

Nitric oxide (NO) derived from endothelial NO synthase (NOS3) plays a central role in myocardial ischemia/reperfusion (I/R)-injury. Subsets of circulating blood cells, including red blood cells (RBCs), carry a NOS3 and contribute to blood pressure regulation and RBC nitrite/nitrate formation. We hypothesized that the circulating blood born NOS3 also modulates the severity of myocardial infarction in disease models. We cross-transplanted bone marrow in wild-type and NOS3(-/-) mice with wild-type mice, producing chimeras expressing NOS3 only in vascular endothelium (BC-/EC+) or in both blood cells and vascular endothelium (BC+/EC+). After 60-min closed-chest coronary occlusion followed by 24 h reperfusion, cardiac function, infarct size (IS), NOx levels, RBCs NO formation, RBC deformability, and vascular reactivity were assessed. At baseline, BC-/EC+ chimera had lower nitrite levels in blood plasma (BC-/EC+: 2.13 ± 0.27 µM vs. BC+/EC+ 3.17 ± 0.29 µM; *p < 0.05), reduced DAF FM associated fluorescence within RBCs (BC-/EC+: 538.4 ± 12.8 mean fluorescence intensity (MFI) vs. BC+/EC+: 619.6 ± 6.9 MFI; ***p < 0.001) and impaired erythrocyte deformability (BC-/EC+: 0.33 ± 0.01 elongation index (EI) vs. BC+/EC+: 0.36 ± 0.06 EI; *p < 0.05), while vascular reactivity remained unaffected. Area at risk did not differ, but infarct size was higher in BC-/EC+ (BC-/EC+: 26 ± 3 %; BC+/EC+: 14 ± 2 %; **p < 0.01), resulting in decreased ejection fraction (BC-/EC+ 46 ± 2 % vs. BC+/EC+: 52 ± 2 %; *p < 0.05) and increased end-systolic volume. Application of the NOS inhibitor S-ethylisothiourea hydrobromide was associated with larger infarct size in BC+/EC+, whereas infarct size in BC-/EC+ mice remained unaffected. Reduced infarct size, preserved cardiac function, NO levels in RBC and RBC deformability suggest a modulating role of circulating NOS3 in an acute model of myocardial I/R in chimeric mice.


Assuntos
Infarto do Miocárdio/sangue , Óxido Nítrico Sintase Tipo III/sangue , Disfunção Ventricular Esquerda/sangue , Animais , Western Blotting , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Traumatismo por Reperfusão Miocárdica/sangue , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Óxido Nítrico/sangue , Quimeras de Transplante , Disfunção Ventricular Esquerda/patologia , Disfunção Ventricular Esquerda/fisiopatologia
17.
Blood ; 120(20): 4229-37, 2012 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-23007404

RESUMO

A nitric oxide synthase (NOS)-like activity has been demonstrated in human red blood cells (RBCs), but doubts about its functional significance, isoform identity and disease relevance remain. Using flow cytometry in combination with the nitric oxide (NO)-imaging probe DAF-FM we find that all blood cells form NO intracellularly, with a rank order of monocytes > neutrophils > lymphocytes > RBCs > platelets. The observation of a NO-related fluorescence within RBCs was unexpected given the abundance of the NO-scavenger oxyhemoglobin. Constitutive normoxic NO formation was abolished by NOS inhibition and intracellular NO scavenging, confirmed by laser-scanning microscopy and unequivocally validated by detection of the DAF-FM reaction product with NO using HPLC and LC-MS/MS. Using immunoprecipitation, ESI-MS/MS-based peptide sequencing and enzymatic assay we further demonstrate that human RBCs contain an endothelial NOS (eNOS) that converts L-(3)H-arginine to L-(3)H-citrulline in a Ca(2+)/calmodulin-dependent fashion. Moreover, in patients with coronary artery disease, red cell eNOS expression and activity are both lower than in age-matched healthy individuals and correlate with the degree of endothelial dysfunction. Thus, human RBCs constitutively produce NO under normoxic conditions via an active eNOS isoform, the activity of which is compromised in patients with coronary artery disease.


Assuntos
Doença da Artéria Coronariana/enzimologia , Eritrócitos/enzimologia , Óxido Nítrico Sintase Tipo III/sangue , Adulto , Sequência de Aminoácidos , Arginina/sangue , Cromatografia Líquida de Alta Pressão , Citrulina/sangue , Doença da Artéria Coronariana/sangue , Doença da Artéria Coronariana/patologia , Endotélio Vascular/patologia , Citometria de Fluxo , Fluoresceínas/análise , Corantes Fluorescentes/análise , Humanos , Imunoprecipitação , Espectrometria de Massas , Microscopia Confocal , Dados de Sequência Molecular , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Óxido Nítrico Sintase Tipo III/fisiologia , Oxiemoglobinas/metabolismo , Alinhamento de Sequência , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos
18.
Arch Biochem Biophys ; 559: 17-23, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-24717599

RESUMO

Accumulating evidence suggest that diets rich in cocoa flavanols may have beneficial effects on cardiovascular health. The major cocoa flavanol monomer, (-)-epicatechin (EC), is readily absorbed and circulates primarily as glucuronidated, sulfated, and O-methylated metabolites in human plasma. However, cellular metabolism, for example in endothelial cells, is less well defined. In the present study we detail the uptake and cellular metabolism of EC and its major in vivo metabolites, (-)-epicatechin-3'-ß-D-glucuronide (E3G), (-)-epicatechin-3'-sulfate (E3S), 3'-O-methyl-(-)-epicatechin-5-sulfate (3ME5S), and 3'-O-methyl-(-)-epicatechin-7-sulfate (3ME7S) in human endothelial (HUVEC), liver (HepG2) and intestinal epithelial cells (Caco-2 monolayer). Our results indicate that EC associates with HUVECs, leading to its intracellular metabolism to 3ME7G and 3ME7S. In contrast, none of the metabolites were taken up by the cells. The metabolic rate and pattern of metabolism in HUVECs was similar to that observed in HepG2 cells, whilst in Caco-2 cells EC was metabolized to E3G, 3ME5G, 3ME7G, 4ME5G, 4ME7G and 3ME7S. Our data support the notion that endothelial cells may contribute significantly to EC metabolism. However, major human circulating metabolites are not accounted for in these model systems underscoring that caution should be taken when drawing conclusions on in vivo flavanol metabolism from in vitro experiments.


Assuntos
Catequina/metabolismo , Células Endoteliais/metabolismo , Absorção , Transporte Biológico , Células CACO-2 , Células Hep G2 , Humanos
19.
Arterioscler Thromb Vasc Biol ; 33(8): 1861-71, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23702660

RESUMO

OBJECTIVE: Mice genetically deficient in endothelial nitric oxide synthase (eNOS(-/-)) are hypertensive with lower circulating nitrite levels, indicating the importance of constitutively produced nitric oxide (NO•) to blood pressure regulation and vascular homeostasis. Although the current paradigm holds that this bioactivity derives specifically from the expression of eNOS in endothelium, circulating blood cells also express eNOS protein. A functional red cell eNOS that modulates vascular NO• signaling has been proposed. APPROACH AND RESULTS: To test the hypothesis that blood cells contribute to mammalian blood pressure regulation via eNOS-dependent NO• generation, we cross-transplanted wild-type and eNOS(-/-) mice, producing chimeras competent or deficient for eNOS expression in circulating blood cells. Surprisingly, we observed a significant contribution of both endothelial and circulating blood cell eNOS to blood pressure and systemic nitrite levels, the latter being a major component of the circulating NO• reservoir. These effects were abolished by the NOS inhibitor L-NG-nitroarginine methyl ester and repristinated by the NOS substrate L-arginine and were independent of platelet or leukocyte depletion. Mouse erythrocytes were also found to carry an eNOS protein and convert (14)C-arginine into (14)C-citrulline in NOS-dependent fashion. CONCLUSIONS: These are the first studies to definitively establish a role for a blood-borne eNOS, using cross-transplant chimera models, that contributes to the regulation of blood pressure and nitrite homeostasis. This work provides evidence suggesting that erythrocyte eNOS may mediate this effect.


Assuntos
Pressão Sanguínea/fisiologia , Homeostase/fisiologia , Hipertensão/metabolismo , Óxido Nítrico Sintase Tipo III/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitritos/sangue , Animais , Arginina/sangue , Arginina/farmacocinética , Radioisótopos de Carbono , Citrulina/sangue , Citrulina/farmacocinética , Endotélio Vascular/enzimologia , Inibidores Enzimáticos/farmacologia , Eritrócitos/enzimologia , Hipertensão/genética , Camundongos , Camundongos Knockout , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/sangue , Óxido Nítrico Sintase Tipo III/antagonistas & inibidores , Transdução de Sinais/fisiologia
20.
Arterioscler Thromb Vasc Biol ; 33(7): 1639-46, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23685552

RESUMO

OBJECTIVE: Sister-of-Mammalian Grainyhead (SOM) is a member of the Grainyhead family of transcription factors. In humans, 3 isoforms are derived from differential first exon usage and alternative splicing and differ only in their N terminal domain. SOM2, the only variant also present in mouse, induces endothelial cell migration and protects against apoptosis. The functions of the human specific isoforms SOM1 and SOM3 have not yet been investigated. Therefore we wanted to elucidate their functions in endothelial cells. APPROACH AND RESULTS: Overexpression of SOM1 in primary human endothelial cells induced migration, phosphorylation of Akt1 and endothelial nitric oxide synthase, and protected against apoptosis, whereas SOM3 had opposite effects; isoform-specific knockdowns confirmed the disparate effects on apoptosis. After reporter assays demonstrated that both are active transcription factors, microarray analyses revealed that they induce different target genes, which could explain the different cellular effects. Overexpression of SOM3 in zebrafish embryos resulted in increased lethality and severe deformations, whereas SOM1 had no deleterious effect. CONCLUSIONS: Our data demonstrate that the splice variant-derived isoforms SOM1 and SOM3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fatores de Transcrição/metabolismo , Animais , Apoptose , Movimento Celular , Proteínas de Ligação a DNA/genética , Ativação Enzimática , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Células MCF-7 , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Isoformas de Proteínas , Proteínas Proto-Oncogênicas c-akt/metabolismo , Interferência de RNA , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Peixe-Zebra/genética , Peixe-Zebra/metabolismo
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