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1.
Adv Exp Med Biol ; 1131: 183-213, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646511

RESUMO

Ca2+ binding proteins (CBP) are of key importance for calcium to play its role as a pivotal second messenger. CBP bind Ca2+ in specific domains, contributing to the regulation of its concentration at the cytosol and intracellular stores. They also participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e. decoding Ca2+ signals. Since CBP are integral to normal physiological processes, possible roles for them in a variety of diseases has attracted growing interest in recent years. In addition, research on CBP has been reinforced with advances in the structural characterization of new CBP family members. In this chapter we have updated a previous review on CBP, covering in more depth potential participation in physiopathological processes and candidacy for pharmacological targets in many diseases. We review intracellular CBP that contain the structural EF-hand domain: parvalbumin, calmodulin, S100 proteins, calcineurin and neuronal Ca2+ sensor proteins (NCS). We also address intracellular CBP lacking the EF-hand domain: annexins, CBP within intracellular Ca2+ stores (paying special attention to calreticulin and calsequestrin), proteins that contain a C2 domain (such as protein kinase C (PKC) or synaptotagmin) and other proteins of interest, such as regucalcin or proprotein convertase subtisilin kexins (PCSK). Finally, we summarise the latest findings on extracellular CBP, classified according to their Ca2+ binding structures: (i) EF-hand domains; (ii) EGF-like domains; (iii) ɣ-carboxyl glutamic acid (GLA)-rich domains; (iv) cadherin domains; (v) Ca2+-dependent (C)-type lectin-like domains; (vi) Ca2+-binding pockets of family C G-protein-coupled receptors.


Assuntos
Proteínas de Ligação ao Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Humanos , Espaço Intracelular/metabolismo
2.
Biochem Pharmacol ; : 113677, 2019 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-31647926

RESUMO

The EMPA-REG OUTCOME (Empagliflozin, Cardiovascular Outcome Event Trial in patients with Type 2 Diabetes Mellitus (T2DM)) trial made evident the potentiality of pharmacological sodium-glucose cotransporter 2 (SGLT2) inhibition for treating patients with diabetes and cardiovascular disease. Since the effect of empagliflozin or other SGLT2 inhibitors on the whole cardiac metabolic profile was never analysed before, and with the purpose to contribute to elucidate the benefits at cardiac level of the use of empagliflozin, we explored the effect of the treatment with empagliflozin for six weeks on the cardiac metabolomic profile of Zucker diabetic fatty rats, a model of early stage T2DM, using untargeted metabolomics approach. Empagliflozin reduced significantly the cardiac content of sphingolipids (ceramides and sphingomyelins) and glycerophospholipids (major bioactive contributing factors linking insulin resistance to cardiac damage) and decreased the cardiac content of the fatty acid transporter cluster of differentiation 36 (CD36); induced significant decreases of the cardiac levels of essential glycolysis intermediaries 2,3-bisphosphoglycerate and phosphoenolpyruvate, and regulated the abundance of several amino acids of relevance as tricarboxylic acid suppliers and/or in the metabolic control of the cardiac function as glutamic acid, gamma-aminobutyric acid and sarcosine. Empagliflozin treatment activated the cardioprotective master regulator of cellular energyhomeostasis AMP-activatedproteinkinase (AMPK) and enhanced autophagy at cardiac level, while it decreased significantly the cardiac mRNA levels of the pro-inflammatory cytokines interleukin-6 (IL-6), chemerin, TNF-α and MCP-1, reinforcing the hypothesis of a direct role for empagliflozin in attenuating cardiac inflammation. Our results provide an advancement on the knowledge of the mechanisms linking the therapy with empagliflozin with protective effects on the development of cardiometabolic diseases whose course is associated with remarkable cardiac bioenergetics dysregulation and disarrangement in cardiac metabolome and lipidome.

3.
Oxid Med Cell Longev ; 2019: 9042526, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31281596

RESUMO

Background: Excessive consumption of soft drinks (SD) has become a health problem worldwide due to its association with related cardiovascular diseases. We investigated the possible impacts associated with the consumption of Brazilian guarana (normal and zero) SD in dyslipidemic mice, thus mitigating potential clinical confounders such as poor-quality diet, lifestyle, body composition, and/or comorbidities. Methods: Sixteen-month-old LDLr-/- mice were divided into the following groups: (1) control; (2) GSD: normal guarana SD; and (3) Z-GSD: zero guarana SD. All were fed ad libitum, and blood pressure was measured noninvasively. After 8 weeks, aorta, blood, liver, and stomach samples were collected for histological and biochemical analyses. Results: Guarana soft drinks increased atherosclerosis (~60%) and were associated with hypercholesterolemia, hypertension, oxidative stress, DNA fragmentation, and apoptosis (~2-fold) of blood cells, besides presenting an increase in liver and gastric damage even in normoglycemia. Interestingly, Z-GSD did not cause the aforementioned changes, except in hemodynamic and renal parameters. Conclusions: Chronic administration of GSD is prooxidative, compromising the cardiovascular, gastric, and hepatic systems; the effects are due at least in part to free sugar consumption but not to guarana extract per se.

4.
Oxid Med Cell Longev ; 2019: 3086270, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31205584

RESUMO

The gut microbiota, the ecosystem formed by a wide symbiotic community of nonpathogenic microorganisms that are present in the distal part of the human gut, plays a prominent role in the normal physiology of the organism. The gut microbiota's imbalance, gut dysbiosis, is directly related to the origin of various processes of acute or chronic dysfunction in the host. Therefore, the ability to intervene in the gut microbiota is now emerging as a possible tactic for therapeutic intervention in various diseases. From this perspective, evidence is growing that a functional dietary intervention with probiotics, which maintain or restore beneficial bacteria of the digestive tract, represents a promising therapeutic strategy for interventions in cardiovascular diseases and also reduces the risk of their occurrence. In the present work, we review the importance of maintaining the balance of the intestinal microbiota to prevent or combat such processes as arterial hypertension or endothelial dysfunction, which underlie many cardiovascular disorders. We also review how the consumption of probiotics can improve autonomic control of cardiovascular function and provide beneficial effects in patients with heart failure. Among the known effects of probiotics is their ability to decrease the generation of reactive oxygen species and, therefore, reduce oxidative stress. Therefore, in this review, we specifically focus on this antioxidant capacity and its relationship with the beneficial cardiovascular effects described for probiotics.

5.
J Chemother ; 31(1): 49-57, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30580667

RESUMO

An observational retrospective study in patients treated with voriconazole was made to evaluate outcomes, safety, drug interactions and characteristics of the treatment. A total of 96 patients were included. In 78.12%, at least one inducer or enzyme inhibitor was detected. The most frequently observed potential interaction was the simultaneous administration of omeprazole. A large number of patients were concurrently treated with corticosteroids. The simultaneous administration of drugs acting as CYP450 enzyme inhibitors was associated with a higher risk of toxicity while concomitant administration of corticosteroids seemed a protective factor. Our study is one of the few ones, which evaluate the use of voriconazole in a real life clinical setting. We demonstrate the wide variety of strategies in the voriconazole using and the large number of dugs that are susceptible to pharmacokinetic interactions. This study reinforces the need to implement voriconazole pharmacokinetic monitoring in order to optimize antifungal treatment.


Assuntos
Antifúngicos/efeitos adversos , Micoses/tratamento farmacológico , Voriconazol/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Antifúngicos/farmacocinética , Interações de Medicamentos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Voriconazol/farmacocinética , Adulto Jovem
6.
Pancreas ; 47(10): 1304-1311, 2018 Nov/Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30286014

RESUMO

OBJECTIVES: The aim of this study was to comparatively analyze the effects of different concentrations of cigarette smoke condensate (CSC, a standardized tobacco extract) and ethanol on intracellular enzyme activation, cell necrosis, alteration of cytosolic calcium concentration ([Ca]c), and amylase secretion in pancreatic acinar cells. METHODS: The effects of CSC (1 µg/mL to 0.4 mg/mL) and ethanol (10-100 mM) on intracellular enzyme activity, cell necrosis, and [Ca]c were measured by fluorescence assays in isolated pancreatic acinar cells. Amylase secretion was evaluated by spectrophotometry. Supramaximal concentrations of cholecystokinin (10-100 nM) were used as positive control. RESULTS: Neither CSC nor ethanol induced trypsin or elastase activation. Both CSC (0.1-0.4 mg/mL) and ethanol (10-75 mM) significantly increased [Ca]c. Amylase secretion was increased only in CSC-treated cells (0.3 and 0.4 mg/mL). After 60 minutes, CSC (0.3 and 0.4 mg/mL) significantly increased acinar cell necrosis at a similar percentage to that induced by cholecystokinin. Ethanol did not induce any significant cell necrosis. CONCLUSIONS: Cigarette smoke condensate induces acinar cell injury and increases [Ca]c and amylase secretion, independently of intracellular enzyme activation, suggesting that tobacco could induce several main early events of pancreatitis in pancreatic acinar cells. However, ethanol only induces increases [Ca]c, having no effect on cell injury, amylase secretion, or intracellular enzyme activation.

7.
Cell Physiol Biochem ; 48(5): 1901-1914, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30092577

RESUMO

The gut microbiota maintains a complex mutual interaction with different organs of the host. Whereas in normal conditions this natural community of trillions of microorganisms greatly contributes to the human health, gut dysbiosis is related with onset or worsening of diverse chronic systemic diseases. Thus, the reestablishment of gut microbiota homeostasis with consumption of prebiotics and probiotics may be a relevant strategy to prevent or attenuate several cardiovascular and metabolic complications. Among these functional foods, the synbiotic kefir, which is a fermented milk composed of a mixture of bacteria and yeasts, is currently the most used and has attracted the attention of health care professionals. The present review is focused on reports describing the feasibility of kefir consumption to provide benefits in cardiometabolic diseases, including hypertension, vascular endothelial dysfunction, dyslipidemia and insulin resistance. Interestingly, recent studies show that mechanisms of actions of kefir in cardiometabolic diseases include recruitment of endothelial progenitor cells, improvement of the balance vagal/sympathetic nervous system, diminution of excessive generation of reactive oxygen species, angiotensin converting enzyme inhibition, anti-inflammatory cytokines profile and alteration of the intestinal microbiota. These findings provide a better understanding about the mechanisms of the beneficial actions of kefir and motivate further investigations to determine whether the use of this synbiotic could also be translated into clinical improvements in cardiometabolic diseases.


Assuntos
Doenças Cardiovasculares/patologia , Kefir/microbiologia , Doenças Metabólicas/patologia , Doenças Cardiovasculares/metabolismo , Microbioma Gastrointestinal , Glucose/metabolismo , Humanos , Kefir/análise , Doenças Metabólicas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fatores de Necrose Tumoral/metabolismo
8.
Curr Pharm Biotechnol ; 19(6): 483-494, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29938618

RESUMO

BACKGROUND: By acting on multiple targets and promoting diverse actions, angiotensin II (Ang II) plays a pivotal role in vascular function. Recent studies suggested that phosphodiesterase-5 (PDE-5) inhibitors exhibit therapeutic effects in cardiovascular diseases. Here, the effects of sildenafil on vascular disturbances were analyzed in a mouse model of Ang II-induced hypertension. METHODS AND RESULTS: Male C57BL/6 mice were used as untreated animals (control) or infused with Ang II (1000 ηg/kg/min) for 28 days and treated with sildenafil (40 mg/kg/min) or vehicle (Ang II) during the last two weeks. After 4 weeks, the Ang II animals exhibited a high systolic blood pressure (186±3 mmHg vs. 127±3 mmHg for control mice), which was attenuated by sildenafil (163±7 mmHg). The mesenteric vessels from the Ang II animals revealed damage to the endothelial layer, an increase in the cross-section area (1.9-fold) and vascular cell production of peroxynitrite (512±13 a.u.), which was ameliorated in the Ang II-Sil group (1.2-fold and 400±17 a.u.). Analysis of the vascular responsiveness showed an increased contractility response to norepinephrine in Ang II animals (Rmax: 70%), which was abolished by sildenafil through increased nitric oxide (NO) bioavailability and decreased reactive oxygen species (ROS) and vasoconstrictor prostanoids. CONCLUSION: Sildenafil attenuates the morphofunctional deleterious effects of Ang II on resistance vessels. The benefits of sildenafil seem to occur through restoring the balance of ROS/NO/eicosanoids. Therefore, this study opened new avenues for further clinical targeting of the treatment of cardiovascular diseases related to activation of the renin-angiotensin system.

9.
Cell Physiol Biochem ; 44(5): 1796-1809, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29216624

RESUMO

BACKGROUND/AIMS: The atherosclerotic apolipoprotein E-deficient (apoE-/-) mouse exhibits impaired vasodilation and enhanced vasoconstriction responsiveness. The objectives of this study were: a) to determine the relative contribution of cyclooxygenases (Cox-1 and Cox-2), thromboxane A2 (TXA2) and endothelin-1 (ET-1) to enhancing vascular hyperresponsiveness in this model of atherosclerosis and b) to investigate the beneficial effects of the phosphodiesterase 5 inhibitor sildenafil on this endothelial dysfunction. METHODS: Adult male apoE-/- mice were treated with sildenafil (40 mg/kg/day, for 3 weeks) and compared with non-treated ApoE-/- and wild-type mice. The beneficial effects of sildenafil on vascular contractile response to phenylephrine (PE) in aortic rings were evaluated before and after incubation with Cox-1 (SC-560) or Cox-2 (NS-398) inhibitors or the TP antagonist SQ-29548, and on contractile responsiveness to ET-1. RESULTS: ApoE-/- mice exhibited enhanced vasoconstriction to PE (Rmax ∼35%, p<0.01), which was prevented by treatment with sildenafil. The enhanced PE-induced contractions were abolished by both Cox-1 inhibition and TP antagonist, but were not modified by Cox-2 inhibition. Aortic rings from ApoE-/- mice also exhibited enhanced contractions to ET-1 (Rmax ∼30%, p<0.01), which were attenuated in sildenafil-treated ApoE-/- mice. In addition, we observed augmented levels of vascular proinflammatory cytokines in ApoE-/- mice, which were partially corrected by treatment with sildenafil (IL-6, IL-10/IL-6 ratio and MCP-1). CONCLUSION: The present data show that the Cox-1/TXA2 pathway prevails over the Cox-2 isoform in the mediation of vascular hypercontractility observed in apoE-/-mice. The results also show a beneficial effect of sildenafil on this endothelial dysfunction and on the proinflammatory cytokines in atherosclerotic animals, opening new perspectives for the treatment of other endothelium-related cardiovascular abnormalities.


Assuntos
Apolipoproteínas E/genética , Ciclo-Oxigenase 1/metabolismo , Citrato de Sildenafila/farmacologia , Tromboxano A2/metabolismo , Vasoconstrição/efeitos dos fármacos , Animais , Apolipoproteínas E/deficiência , Compostos Bicíclicos Heterocíclicos com Pontes , Ciclo-Oxigenase 1/química , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/metabolismo , Inibidores de Ciclo-Oxigenase 2/farmacologia , Inibidores de Ciclo-Oxigenase/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Ácidos Graxos Insaturados , Hidrazinas/farmacologia , Interleucina-10/análise , Interleucina-6/análise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nitrobenzenos/farmacologia , Fenilefrina/farmacologia , Pirazóis/farmacologia , Receptores de Tromboxanos/antagonistas & inibidores , Receptores de Tromboxanos/metabolismo , Sulfonamidas/farmacologia
10.
Biochem Pharmacol ; 145: 94-101, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-28912066

RESUMO

3',5'-Cyclic adenosine monophosphate (cAMP) exerts an endothelium-dependent vasorelaxant action by stimulating endothelial NO synthase (eNOS) activity, and the subsequent NO release, through cAMP protein kinase (PKA) and exchange protein directly activated by cAMP (Epac) activation in endothelial cells. Here, we have investigated the mechanism by which the cAMP-Epac/PKA pathway activates eNOS. cAMP-elevating agents (forskolin and dibutyryl-cAMP) and the joint activation of PKA (6-Bnz-cAMP) and Epac (8-pCPT-2'-O-Me-cAMP) increased cytoplasmic Ca2+ concentration ([Ca2+]c) in ≤30% of fura-2-loaded isolated human umbilical vein endothelial cells (HUVEC). However, these drugs did not modify [Ca2+]c in fluo-4-loaded HUVEC monolayers. In DAF-2-loaded HUVEC monolayers, forskolin, PKA and Epac activators significantly increased NO release, and the forskolin effect was reduced by inhibition of PKA (Rp-cAMPs), Epac (ESI-09), eNOS (L-NAME) or phosphoinositide 3-kinase (PI3K; LY-294,002). On the other hand, inhibition of CaMKII (KN-93), AMPK (Compound C), or total absence of Ca2+, was without effect. In Western blot experiments, Serine 1177 phosphorylated-eNOS was significantly increased in HUVEC by cAMP-elevating agents and PKA or Epac activators. In isolated rat aortic rings LY-294,002, but not KN-93 or Compound C, significantly reduced the vasorelaxant effects of forskolin in the presence of endothelium. Our results suggest that Epac and PKA activate eNOS via Ser 1177 phosphorylation by activating the PI3K/Akt pathway, and independently of AMPK or CaMKII activation or [Ca2+]c increase. This action explains, in part, the endothelium-dependent vasorelaxant effect of cAMP.


Assuntos
AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Sinalização do Cálcio , Colforsina/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Masculino , Óxido Nítrico/metabolismo , Fosfatidilinositol 3-Quinases/genética , Proteínas Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Endogâmicos WKY , Vasodilatação/efeitos dos fármacos
11.
Acta Diabetol ; 54(6): 581-591, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28353063

RESUMO

AIMS: To investigate the consequences of oxidative stress and hypoxia on EPAC-1 expression during retinopathy. METHODS: Oxygen-induced retinopathy was induced in mice and EPAC-1 expression investigated by immunofluorescence. In silico analyses were used to identify a link between EPAC-1 expression and microRNA-7-5p in endothelial cells and confirmed by western blot analyses on cells expressing microRNA-7-5p. In vitro, endothelial cells were either incubated at 2% oxygen or transfected with microRNA-7-5p, and the effects of these treatments on EPAC-1 expression, endothelial hyperpermeability and NO production were assessed. In the Ins2Akita mouse model, levels of EPAC-1 expression as well as microRNA-7-5p were assessed by qPCR. Endothelial nitric oxide synthase was assessed by immunoblotting in the Ins2Akita model. RESULTS: Hypoxia induces the expression of microRNA-7-5p that translationally inhibits the expression of EPAC-1 in endothelial cells, resulting in hyperpermeability and the loss of eNOS activity. Activation of EPAC-1 by the cAMP analogue 8-pCPT-2'-O-Me-cAMP reduced the sensitivity of EPAC-1 to oxidative stress and restored the endothelial permeability to baseline levels. Additionally, 8-pCPT-2'-O-Me-cAMP rescued eNOS activity and NO production. In mouse models of retinopathy, i.e., oxygen-induced retinopathy and the spontaneous diabetic heterozygous Ins2Akita mice, EPAC-1 levels are decreased which is associated with an increase in microRNA-7-5p expression and reduced eNOS activity. CONCLUSION/INTERPRETATION: In retinopathy, EPAC-1 expression is decreased in a microRNA-7-mediated manner, contributing to endothelial dysfunction. Pharmacological activation of remnant EPAC-1 rescues endothelial function. Collectively, these data indicate that EPAC-1 resembles an efficacious and druggable target molecule for the amelioration of (diabetic) retinopathy.


Assuntos
Permeabilidade Capilar/genética , Diabetes Mellitus Experimental/genética , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Endotélio Vascular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , MicroRNAs/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Regulação para Baixo/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
12.
Life Sci ; 155: 102-9, 2016 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-27142830

RESUMO

AIMS: We investigated the implication of PKA and Epac proteins in the endothelium-independent vasorelaxant effects of cyclic AMP (cAMP). MAIN METHODS: Cytosolic Ca(2+) concentration ([Ca(2+)]c) was measured by fura-2 imaging in rat aortic smooth muscle cells (RASMC). Contraction-relaxation experiments were performed in rat aortic rings deprived of endothelium. KEY FINDINGS: In extracellular Ca(2+)-free solution, cAMP-elevating agents induced an increase in [Ca(2+)]c in RASMC that was reproduced by PKA and Epac activation and reduced after depletion of intracellular Ca(2+) reservoirs. Arginine-vasopressin (AVP)-evoked increase of [Ca(2+)]c and store-operated Ca(2+) entry (SOCE) were inhibited by cAMP-elevating agents, PKA or Epac activation in these cells. In aortic rings, the contractions induced by phenylephrine in absence of extracellular Ca(2+) were inhibited by cAMP-elevating agents, PKA or Epac activation. In these conditions, reintroduction of Ca(2+) induced a contraction that was inhibited by cAMP-elevating agents, an effect reduced by PKA inhibition and reproduced by PKA or Epac activators. SIGNIFICANCE: Our results suggest that increased cAMP depletes intracellular, thapsigargin-sensitive Ca(2+) stores through activation of PKA and Epac in RASMC, thus reducing the amount of Ca(2+) released by IP3-generating agonists during the contraction of rat aorta. cAMP rise also inhibits the contraction induced by depletion of intracellular Ca(2+), an effect mediated by reduction of SOCE after PKA or Epac activation. Both effects participate in the cAMP-induced endothelium-independent vasorelaxation.


Assuntos
Cálcio/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Vasoconstrição , Animais , Aorta/citologia , Aorta/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Ativação Enzimática , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Ratos , Ratos Endogâmicos WKY
13.
Eur J Med Chem ; 82: 407-17, 2014 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-24929291

RESUMO

Five series of 1,4-phthalazinedione derivatives were synthesized in good yields. Vasorelaxant activity of these new derivatives was measured on either intact or endothelium-denuded isolated rat thoracic aortic rings pre-contracted with phenylephrine. Most of studied compounds, substituted in both nitrogen atoms, attained practically the total relaxation of the organ at low micromolar concentrations. The presence of functional endothelium significantly reduced the EC50 values for most of studied compounds. Some structure-activity relationships were established and compounds 2d and 5d can be considered as new leads for further modifications.


Assuntos
Aorta Torácica/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Ftalazinas/farmacologia , Vasodilatadores/farmacologia , Animais , Aorta Torácica/citologia , Linhagem Celular , Relação Dose-Resposta a Droga , Masculino , Estrutura Molecular , Músculo Liso/citologia , Ftalazinas/síntese química , Ftalazinas/química , Ratos , Ratos Endogâmicos WKY , Ratos Wistar , Relação Estrutura-Atividade , Vasodilatadores/síntese química , Vasodilatadores/química
14.
Vascul Pharmacol ; 60(3): 95-101, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24469067

RESUMO

Vascular relaxation induced by 3',5'-cyclic adenosine monophosphate (cAMP) is both endothelium-dependent and endothelium-independent, although the underlying signaling pathways are not fully understood. Aiming to uncover potential mechanisms, we performed contraction-relaxation experiments on endothelium-denuded and intact rat aorta rings and measured NO levels in isolated human endothelial cells using single cell fluorescence imaging. The vasorelaxant effect of forskolin, an adenylyl cyclase activator, was decreased after selective inhibitor of protein kinase A (PKA), a cAMP-activated kinase, or L-NAME, an endothelial nitric oxide synthase (eNOS) inhibitor, only in intact aortic rings. Both selective activation of PKA with 6-Bnz-cAMP and exchange protein directly activated by cAMP (Epac) with 8-pCPT-2'-O-Me-cAMP significantly relaxed phenylephrine-induced contractions. The vasorelaxant effect of the Epac activator, but not that of the PKA activator, was reduced by endothelium removal. Forskolin, dibutyryl cAMP (a cAMP analogue), 6-Bnz-cAMP and 8-pCPT-2'-O-Me-cAMP increased NO levels in endothelial cells and the forskolin effect was significantly inhibited by inactivation of both Epac and PKA, and eNOS inhibition. Our results indicate that the endothelium-dependent component of forskolin/cAMP-induced vasorelaxation is partially mediated by an increase in endothelial NO release due to an enhanced eNOS activity through PKA and Epac activation in endothelial cells.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/farmacologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Óxido Nítrico/biossíntese , Vasodilatação/fisiologia , Animais , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Masculino , Técnicas de Cultura de Órgãos , Ratos , Ratos Endogâmicos WKY , Vasodilatação/efeitos dos fármacos
15.
Vascul Pharmacol ; 58(1-2): 98-104, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22960580

RESUMO

Despite a large number of studies, the mechanism by which 3',5'-cyclic monophosphate (cAMP) induces vasorelaxation is not fully understood. The comparison between results obtained in different vessels or species has often been the source of conflicting reports. In order to shed more light onto this mechanism, we studied the effects of forskolin in phenylephrine-pre-contracted endothelium-denuded rat aorta and measured cAMP levels in rat aortic myocytes by enzyme-immunoassay. Nanomolar forskolin relaxed phenylephrine-induced contractions. This effect was mimicked by dibutyryl-cAMP and was potentiated by rolipram or a p38-mitogen-activated protein kinase (p38-MAPK) inhibitor (SB-203580). Nifedipine and verapamil partially relaxed phenylephrine-induced contractions, while further application of cAMP-elevating agents fully relaxed these contractions. In Ca(2+)-free extracellular solution, forskolin reduced phenylephrine-induced transient contractions and reduced the Ca(2+)-induced contraction after depletion of intracellular stores. Nanomolar concentrations of forskolin increased basal cAMP levels only in the presence of rolipram or phenylephrine, which did not modify intracellular levels of cAMP by themselves. In conclusion, relaxation by cAMP is mediated in part by decrease of depletion of intracellular Ca(2+) stores and inhibition of capacitative calcium entry. This study provides the first evidence that inhibition of PDE4 or p38-MAPK potentiates the vasodilator effect of cAMP-elevating agents in rat aortic myocytes.


Assuntos
Aorta Torácica/metabolismo , Cálcio/metabolismo , AMP Cíclico/metabolismo , Músculo Liso Vascular/metabolismo , Animais , Aorta Torácica/citologia , Aorta Torácica/efeitos dos fármacos , Colforsina/farmacologia , Imidazóis/farmacologia , Masculino , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Nifedipino/farmacologia , Fenilefrina/farmacologia , Inibidores da Fosfodiesterase 4/farmacologia , Piridinas/farmacologia , Ratos , Ratos Endogâmicos WKY , Rolipram/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Verapamil/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
16.
Adv Exp Med Biol ; 740: 461-82, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22453954

RESUMO

The role of Ca(2+) as a key and pivotal second messenger in cells depends largely on a wide number of heterogeneous so-called calcium binding proteins (CBP), which have the ability to bind this ion in specific domains. CBP contribute to the control of Ca(2+) concentration in the cytosol and participate in numerous cellular functions by acting as Ca(2+) transporters across cell membranes or as Ca(2+)-modulated sensors, i.e., decoding Ca(2+) signals. In this chapter we review the main Ca(2+)-modulated CBP, starting with those intracellular CBP that contain the structural EF-hand domain: parvalbumin, calmodulin, S100 proteins and calcineurin. Then, we address intracellular CBP lacking the EF-hand domain: CBP within intracellular Ca(2+) stores (paying special attention to calreticulin and calsequestrin), annexins and proteins that contain a C2 domain, such as protein kinase C (PKC) or sinaptotagmin. Finally, extracellular CBP have been classified in six groups, according to their Ca(2+) binding structures: (i) EF-hand domains; (ii) EGF-like domains; (iii) γ-carboxyl glutamic acid (GLA)-rich domains; (iv) cadherin domains; (v) Ca(2+)-dependent (C)-type lectin-like domains; (vi) Ca(2+)-binding pockets of family C G-protein-coupled receptors. For all proteins, we briefly review their structure, location and function and additionally their potential as pharmacological targets in several human diseases.


Assuntos
Proteínas de Ligação ao Cálcio/fisiologia , Animais , Anexinas/fisiologia , Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/química , Humanos , Estrutura Terciária de Proteína , Receptores de Detecção de Cálcio/fisiologia
17.
Mol Nutr Food Res ; 55(8): 1237-48, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21710562

RESUMO

SCOPE: The aim of this study was to investigate whether the dietary polyphenol trans-resveratrol (t-Resv) increases [Ca(2+)](c) in endothelial cells, leading to a simultaneous augmentation of nitric oxide (NO) biosynthesis. METHODS AND RESULTS: We have separately and simultaneously measured [Ca(2+)](c) and NO in human endothelial cells using the Ca(2+) indicator fura-2 and the NO-sensitive fluorescent probe 4,5-diaminofluorescein. In ∼30% of cells, t-Resv (30 µM) induced an increase in [Ca(2+)](c) with a transient as well as sustained component and a simultaneous increase in NO biosynthesis. This effect was reduced by non-selective Ca(2+) channel blockers, inhibition of intracellular Ca(2+) release, inhibition of endothelial nitric oxide synthase (eNOS) and, to a lesser extent, inhibition of extracellular signal-regulated kinase 1/2 (ERK 1/2) or 5' adenosine monophosphate-activated protein kinase (AMPK). t-Resv did not modify in vitro eNOS activity, suggesting that the observed stimulation of NO generation proceeds via mobilisation of Ca(2+) and not through direct effects on eNOS. CONCLUSION: We therefore show, for the first time, that t-Resv induces a concentration-dependent, simultaneous increase in [Ca(2+)](c) and NO biosynthesis that could be linked to its endothelium-dependent vasorelaxant effect. Under the assumption that t-Resv exhibits similar behaviour in human blood vessels in vivo, the pharmacological properties described here may contribute to the beneficial cardiovascular effects of this polyphenol by improving endothelial function.


Assuntos
Cálcio/metabolismo , Citoplasma/metabolismo , Células Endoteliais/efeitos dos fármacos , Óxido Nítrico/metabolismo , Estilbenos/farmacologia , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/fisiologia , Células Cultivadas , Células Endoteliais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Humanos , Óxido Nítrico Sintase Tipo III/metabolismo , Resveratrol
18.
Eur J Pharmacol ; 577(1-3): 91-9, 2007 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-17822692

RESUMO

Although the natural polyphenol resveratrol posses a direct vasorelaxant effect, its effects on cytoplasmic Ca(2+) concentration ([Ca(2+)](i)) in vascular cells remain still unclear. Here, we have investigated the effects of the isomers trans- and cis-resveratrol on agonist- and high-K(+)-induced [Ca(2+)](i) increases and on voltage-activated transmembrane Ca(2+) fluxes using imaging and patch-clamp techniques in vascular A7r5 myocytes. Arginine vasopressin (AVP) or angiotensin II caused a biphasic increase in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol and cis-resveratrol. Both isomers also reduced the agonist-induced increase in [Ca(2+)](i) in absence of extracellular Ca(2+). In high-K(+) Ca(2+)-free solution, reintroduction of Ca(2+) caused a sustained rise in [Ca(2+)](i) that was reduced by preincubation with trans-resveratrol or cis-resveratrol. When the isomers were applied during the plateau phase of the agonist- or the high-K(+)-induced response, a biphasic change in [Ca(2+)](i) was observed: a transient reduction of the plateau (<5 min) followed by an increase (>10 min). Finally, trans-resveratrol and cis-resveratrol inhibited voltage-dependent L-type Ca(2+) currents (I(Ca(L))). In conclusion, resveratrol isomers exert a dual effect on [Ca(2+)](i) handling in A7r5 myocytes: 1) a blockade of I(Ca(L)) and 2) an increase in [Ca(2+)](i) by depletion of intracellular Ca(2+) stores (which interferes with the agonist-induced release of intracellular Ca(2+)) and influx of Ca(2+), mainly due to activation of capacitative Ca(2+) entry, although other Ca(2+)-permeable channels are also involved. Taken together, these effects may explain, in part, the endothelium-independent vasorelaxant effects of resveratrol in rat aorta.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Cálcio/metabolismo , Miócitos de Músculo Liso/metabolismo , Estilbenos/farmacologia , Angiotensina II/farmacologia , Animais , Arginina Vasopressina/farmacologia , Vasos Sanguíneos/citologia , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Corantes Fluorescentes , Fura-2 , Potenciais da Membrana/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Resveratrol , Rianodina/farmacologia , Estereoisomerismo
19.
Biochem Biophys Res Commun ; 359(3): 599-603, 2007 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-17544372

RESUMO

A secretagogue-stimulated pancreatic acinar cell releases digestive enzymes from its apical pole. We attempted to identify the SNAREs involved in zymogen granule exocytosis. Antibodies against syntaxins 2 and 3, SNAP-23 and VAMP 8, and the corresponding recombinant SNAREs, inhibited amylase secretion from streptolysin O-permeabilised acini; other anti-SNARE antibodies and SNAREs had no effect. Botulinum neurotoxin C, which cleaved syntaxin 2 and (to a lesser extent) syntaxin 3, but not syntaxins 4, 7 or 8, also inhibited exocytosis. We propose that syntaxin 2, SNAP-23 and VAMP 8 mediate primary granule-plasma membrane fusion. Syntaxin 3 may be involved in secondary granule-granule fusion.


Assuntos
Precursores Enzimáticos/metabolismo , Exocitose , Proteínas SNARE/metabolismo , Animais , Toxinas Botulínicas/farmacologia , Células Cultivadas , Exocitose/efeitos dos fármacos , Ratos , Proteínas SNARE/genética
20.
Life Sci ; 80(23): 2147-53, 2007 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-17499810

RESUMO

The effects of (-)-epigallocatechin-3-gallate (EGCG), the most abundant catechin of tea, on Ca(2+)-permeable non-selective cation currents (NSCC) and voltage-operated Ca(2+) channels (VOCC) have been investigated in cultured rat aortic smooth muscle cells using the whole-cell voltage-clamp technique. Under the Cs(+)/tetraethylammonium (TEA)-containing internal solution, and in the presence of nifedipine (1 microM), EGCG (30 microM) activated a long-lasting inward current, with a reversal potential (E(rev)) of approximately 0 mV. This current was not significantly altered by the replacement of [Cl(-)](i) or [Cl(-)](o), implying that the inward current was not a chloride channel, but a NSCC. SKF 96365 (30 microM) and Cd(2+) (500 microM) almost completely abolished the EGCG-induced NSCC. A higher dose of EGCG (100 microM) additionally activated a nifedipine-sensitive inward current in the absence of depolarization protocol. EGCG (100 microM) also potentiated a nifedipine-sensitive voltage-dependent Ba(2+)-current during the first 5 min of incubation. However, after > 10 min of incubation with EGCG, this current was significantly inhibited. Our results suggest that EGCG caused a Ca(2+) influx into smooth muscle cells via VOCC (probably L-type) and other SKF-96365- and Cd(2+)-sensitive Ca(2+)-permeable channels. The action described here may be responsible for the contraction induced by EGCG in rat aortic rings and for the rise of the intracellular concentration of Ca(2+) in rat aortic smooth muscle cells evoked by this catechin. On the other hand, the inhibition of VOCC after > 10 min of incubation may be, in part, responsible for the relaxation of rat aorta induced by EGCG.


Assuntos
Canais de Cálcio/química , Catequina/análogos & derivados , Cátions/química , Músculo Liso Vascular/citologia , Animais , Antioxidantes/farmacologia , Aorta/citologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Catequina/química , Catequina/farmacologia , Imidazóis/farmacologia , Masculino , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar
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