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1.
Redox Biol ; 38: 101769, 2020 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-33126056

RESUMO

Nitrate and nitrite supplement deficient endogenous nitric oxide (NO) formation. While these anions may generate NO, recent studies have shown that circulating nitrite levels do not necessarily correlate with the antihypertensive effect of oral nitrite administration and that formation of nitrosylated species (RXNO) in the stomach is critically involved in this effect. This study examined the possibility that RXNO formed in the stomach after oral nitrite administration promotes target protein nitrosylation in the vasculature, inhibits vasoconstriction and the hypertensive responses to angiotensin II. Our results show that oral nitrite treatment enhances circulating RXNO concentrations (measured by ozone-based chemiluminescence methods), increases aortic protein kinase C (PKC) nitrosylation (measured by resin-assisted capture SNO-RAC method), and reduces both angiotensin II-induced vasoconstriction (isolated aortic ring preparation) and hypertensive (in vivo invasive blood pressure measurements) effects implicating PKC nitrosylation as a key mechanism for the responses to oral nitrite. Treatment of rats with the nitrosylating compound S-nitrosoglutathione (GSNO) resulted in the same effects described for oral nitrite. Moreover, partial depletion of thiols with buthionine sulfoximine prevented PKC nitrosylation and the blood pressure effects of oral nitrite. Further confirming a role for PKC nitrosylation, preincubation of aortas with GSNO attenuated the responses to both angiotensin II and to a direct PKC activator, and this effect was attenuated by ascorbate (reverses GSNO-induced nitrosylation). GSNO-induced nitrosylation also inhibited the increases in Ca2+ mobilization in angiotensin II-stimulated HEK293T cells expressing angiotensin type 1 receptor. Together, these results are consistent with the idea that PKC nitrosylation in the vasculature may underlie oral nitrite treatment-induced reduction in the vascular and hypertensive responses to angiotensin II.

2.
World Neurosurg ; 141: e86-e96, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32371077

RESUMO

BACKGROUND: Transorbital and subtemporal keyhole approaches have recently been proposed to approach lesions in the lateral wall of the cavernous sinus (CS) and Meckel's cave (MC). Our goal was to compare these approaches and suggest indications for each of them. METHODS: Five cadaver heads (10 sides, 40 procedures) were used. The lateral transorbital approaches were carried out without and with the removal of the lateral orbital rim, herein referred to as the lateral transorbital approach (LTOA) and the lateral orbital wall approach (LOWA). The subtemporal approaches were performed without and with the removal of the zygomatic arch, referred to as the subtemporal approach (STA) and the subtemporozygomatic approach (STZA). Five targets were chosen and 2 triangles were created representing the lateral wall of the CS and MC. Stereotactic measurements were quantified to calculate angles of attack, surgical freedom, and temporal lobe retraction for each approach. RESULTS: LTOA presented the smaller horizontal angles of attack. LOWA increased the angles to the same level of STA and STZA. STA and STZA presented larger vertical angles of attack. The surgical freedom presented gradual increase from LTOA to LOWA, STA, and STZA. STA and STZA needed greater temporal lobe retraction for most targets. CONCLUSIONS: LTOA is a good option to biopsy a lesion in the lateral wall of the CS and LOWA increased the surgical corridor to work with microsurgical techniques. STA and STZA could be better options when wide exposure is necessary, but temporal lobe retraction should be taken into consideration.

3.
Invest Ophthalmol Vis Sci ; 60(12): 3842-3853, 2019 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-31529081

RESUMO

Purpose: Outer blood retinal barrier breakdown is a neglected feature of diabetic retinopathy (DR). We demonstrated that the agonism of the δ opioid receptor (DOR) by epicatechin preserves the tight junction proteins in ARPE-19 cells under diabetic conditions. Presently, we aimed to evaluate the possible role of the DOR on the maintenance of tight junction of RPE layer and on the early markers of experimental DR. Methods: DR markers and external retinal tight junction proteins were evaluated in CL57B diabetic mice submitted to intravitreous injection of short hairpin RNA (shRNA)-DOR (108 transducing units [TU]/mL) treated or not with DOR agonist (0.05 g/animal/d of epicatechin in drinking water) for 16 weeks. The presence of DOR in human retina from postmortem eyes from diabetic and nondiabetic donors were also performed. Results: DOR is present in RPE layer and in neuro retina. The treatment with DOR agonist prevented the upregulation of the early markers of retinopathy (glial fibrillary acidic protein, VEGF) and the downregulation of pigment epithelium-derived factor, occludin, claudin-1, and zonula occludens-1 tight junction expressions. The silencing of DOR in retina of diabetic mice partially abolished the protective effects of epicatechin. In human retina specimens, DOR is present throughout the retina, similarly in nondiabetic and diabetic donors. Conclusions: This set of experiments strongly indicates that the DOR agonism preserves RPE tight junctions and reduces the early markers of retinopathy in model of diabetes. These novel findings designate DOR as a potential therapeutic tool to treat DR with preservation of the RPE tight junction proteins.


Assuntos
Catequina/farmacologia , Diabetes Mellitus Experimental/prevenção & controle , Retinopatia Diabética/prevenção & controle , Receptores Opioides delta/agonistas , Epitélio Pigmentado da Retina/metabolismo , Junções Íntimas/metabolismo , Idoso , Animais , Glicemia/metabolismo , Western Blotting , Claudina-1/metabolismo , Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética/metabolismo , Retinopatia Diabética/patologia , Impedância Elétrica , Proteínas do Olho/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Fatores de Crescimento Neural/metabolismo , Ocludina/metabolismo , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Receptores Opioides delta/metabolismo , Serpinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo
4.
J. bras. nefrol ; 41(3): 384-392, July-Sept. 2019. tab, graf
Artigo em Inglês | LILACS | ID: biblio-1040258

RESUMO

Abstract Introduction: Chronic kidney disease (CKD) has a high prevalence and is a worldwide public health problem. Reuse of dialyzers is a cost reduction strategy used in many countries. There is controversy over its effects on clinical parameters and microbiological safety. Methods: In this clinical crossover study, 10 patients performed consecutive hemodialysis (HD) sessions divided in two phases: "single use" sessions (N = 10 HD sessions) followed by "dialyzer reuse" sessions (N = 30 HD sessions). Clinical, laboratory, and microbiological parameters were collected in the following time points: "single use", 1st, 6th, and 12th sessions with reuse of dialyzers, including bacterial cultures, endotoxins quantification in serum and dialyzer blood chamber, and detection of hemoglobin and protein residues in dialyzers. Results: Mean age of the sample was 37 ± 16 years, 6 (60%) were men, and 5 (50%) were white. CKD and HD vintage were 169 ± 108 and 47 (23-111) months, respectively. Serum C-reactive protein (CRP) [4.9 (2.1) mg/mL], ferritin (454 ± 223 ng/mL), and endotoxin levels [0.76 (0.61-0.91) EU/mL] were high at baseline. Comparison of pre- and post-HD variations of serum levels of CRP and endotoxins in the "single use" versus "reuse" phases did not result in differences (p = 0.8 and 0.4, respectively). Samples of liquid in the dialyzer inner chamber were negative for the growth of bacteria or endotoxins. There was no significant clinical manifestation within and between the phases. Conclusion: Dialyzers reuse was safe from a clinical, microbiological, and inflammatory point of view. The dialyzer performance remained adequate until the 12th reuse.


Resumo Introdução: A doença renal crônica (DRC) é um problema de saúde pública mundial de alta prevalência. O reúso de dialisadores é uma estratégia de redução de custos empregada em muitos países. Seus efeitos sobre parâmetros clínicos e de segurança microbiológica são alvo de controvérsia. Métodos: No presente estudo clínico cruzado, 10 pacientes realizaram sessões consecutivas de hemodiálise (HD) divididas em duas fases: a primeira com sessões de "uso único" (N = 10 sessões de HD) e a segunda com sessões com "reúso de dialisadores" (N = 30 sessões de HD). Parâmetros clínicos, laboratoriais e microbiológicos foram registrados nos seguintes momentos: "uso único", 1a, 6a e 12a sessões com reúso de dialisadores, incluindo culturas bacterianas, quantificação de endotoxinas no soro e na câmara interna do dialisador e detecção de hemoglobina e resíduos de proteína nos dialisadores. Resultados: A idade média da amostra foi de 37 ± 16 anos seis (60%) eram homens e cinco (50%) eram brancos. Os tempos com DRC e em HD foram de 169 ± 108 e 47 (23-111) meses, respectivamente. Os níveis séricos de proteína C-reativa (PCR) [4,9 (2,1) mg/mL], ferritina (454 ± 223 ng/mL) e endotoxinas [0,76 (0,61-0,91) UE/mL] estavam elevados no início do estudo. A diferença dos níveis séricos de PCR e endotoxinas pré e pós-HD nas fases de "uso único" e "reúso" não foi significativa (p = 0,8 e 0,4, respectivamente). As amostras de líquido retiradas da câmara interna do dialisador foram negativas para crescimento de bactérias e endotoxinas. Não houve registro de manifestações clínicas significativas nas fases do estudo. Conclusão: O reúso de dialisadores foi seguro dos pontos de vista clínico, microbiológico e inflamatório. O desempenho do dialisador permaneceu adequado até o 12º reuso.


Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Adulto Jovem , Diálise Renal/instrumentação , Reutilização de Equipamento , Rins Artificiais/efeitos adversos , Rins Artificiais/microbiologia , Proteína C-Reativa/análise , Projetos Piloto , Seguimentos , Estudos Cross-Over , Endotoxinas/sangue , Insuficiência Renal Crônica/terapia , Ferritinas/sangue , Inflamação/sangue
5.
Front Pharmacol ; 10: 628, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31214037

RESUMO

LASSBio-579, an N-phenylpiperazine antipsychotic lead compound, has been previously reported as a D2 receptor (D2R) ligand with antipsychotic-like activities in rodent models of schizophrenia. In order to better understand the molecular mechanism of action of LASSBio-579 and of its main metabolite, LQFM 037, we decided to address the hypothesis of functional selectivity at the D2R. HEK-293T cells transiently coexpressing the human long isoform of D2 receptor (D2LR) and bioluminescence resonance energy transfer (BRET)-based biosensors were used. The antagonist activity was evaluated using different concentrations of the compounds in the presence of a submaximal concentration of dopamine (DA), after 5 and 20 min. For both signaling pathways, haloperidol, clozapine, and our compounds act as DA antagonists in a concentration-dependent manner, with haloperidol being by far the most potent, consistent with its nanomolar D2R affinity measured in binding assays. In our experimental conditions, only haloperidol presented a robust functional selectivity, being four- to fivefold more efficient for inhibiting translocation of ß-arrestin-2 (ß-arr2) than for antagonizing Gi activation. Present data are the first report on the effects of LASSBio-579 and LQFM 037 on the ß-arr2 signaling pathway and further illustrate that the functional activity could vary depending on the assay conditions and approaches used.

6.
J Bras Nefrol ; 41(3): 384-392, 2019.
Artigo em Inglês, Português | MEDLINE | ID: mdl-30720850

RESUMO

INTRODUCTION: Chronic kidney disease (CKD) has a high prevalence and is a worldwide public health problem. Reuse of dialyzers is a cost reduction strategy used in many countries. There is controversy over its effects on clinical parameters and microbiological safety. METHODS: In this clinical crossover study, 10 patients performed consecutive hemodialysis (HD) sessions divided in two phases: "single use" sessions (N = 10 HD sessions) followed by "dialyzer reuse" sessions (N = 30 HD sessions). Clinical, laboratory, and microbiological parameters were collected in the following time points: "single use", 1st, 6th, and 12th sessions with reuse of dialyzers, including bacterial cultures, endotoxins quantification in serum and dialyzer blood chamber, and detection of hemoglobin and protein residues in dialyzers. RESULTS: Mean age of the sample was 37 ± 16 years, 6 (60%) were men, and 5 (50%) were white. CKD and HD vintage were 169 ± 108 and 47 (23-111) months, respectively. Serum C-reactive protein (CRP) [4.9 (2.1) mg/mL], ferritin (454 ± 223 ng/mL), and endotoxin levels [0.76 (0.61-0.91) EU/mL] were high at baseline. Comparison of pre- and post-HD variations of serum levels of CRP and endotoxins in the "single use" versus "reuse" phases did not result in differences (p = 0.8 and 0.4, respectively). Samples of liquid in the dialyzer inner chamber were negative for the growth of bacteria or endotoxins. There was no significant clinical manifestation within and between the phases. CONCLUSION: Dialyzers reuse was safe from a clinical, microbiological, and inflammatory point of view. The dialyzer performance remained adequate until the 12th reuse.


Assuntos
Reutilização de Equipamento , Rins Artificiais/efeitos adversos , Rins Artificiais/microbiologia , Diálise Renal/instrumentação , Adulto , Proteína C-Reativa/análise , Estudos Cross-Over , Endotoxinas/sangue , Feminino , Ferritinas/sangue , Seguimentos , Humanos , Inflamação/sangue , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Insuficiência Renal Crônica/terapia , Adulto Jovem
7.
Front Pharmacol ; 8: 825, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29184503

RESUMO

The histamine receptors (HRs) are traditional G protein-coupled receptors of extensive therapeutic interest. Recently, H3R and H4R subtypes have been targeted in drug discovery projects for inflammation, asthma, pain, cancer, Parkinson's, and Alzheimer's diseases, which includes searches for dual acting H3R/H4R ligands. In the present work, nine 1-[(2,3-dihydro-1-benzofuran-2-yl)methyl]piperazine (LINS01 series) molecules were synthesized and evaluated as H3R and H4R ligands. Our data show that the N-allyl-substituted compound LINS01004 bears the highest affinity for H3R (pKi 6.40), while the chlorinated compound LINS01007 has moderate affinity for H4R (pKi 6.06). In addition, BRET assays to assess the functional activity of Gi1 coupling indicate that all compounds have no intrinsic activity and act as antagonists of these receptors. Drug-likeness assessment indicated these molecules are promising leads for further improvements. In vivo evaluation of compounds LINS01005 and LINS01007 in a mouse model of asthma showed a better anti-inflammatory activity of LINS01007 (3 g/kg) than the previously tested compound LINS01005. This is the first report with functional data of these compounds in HRs, and our results also show the potential of their applications as anti-inflammatory.

8.
Sci Rep ; 7(1): 11903, 2017 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-28928410

RESUMO

The renin-angiotensin system (RAS) plays a key role in the control of vasoconstriction as well as sodium and fluid retention mediated mainly by angiotensin (Ang) II acting at the AT1 receptor (AT1R). Ang-(1-7) is another RAS peptide, identified as the endogenous ligand of the Mas receptor and known to counterbalance many of the deleterious effects of AngII. AT1R signaling triggered by ß-arrestin-biased agonists has been associated to cardioprotection. Because position 8 in AngII is important for G protein activation, we hypothesized that Ang-(1-7) could be an endogenous ß-arrestin-biased agonist of the AT1R. Here we show that Ang-(1-7) binds to the AT1R without activating Gq, but triggering ß-arrestins 1 and 2 recruitment and activation. Using an in vivo model of cardiac hypertrophy, we show that Ang-(1-7) significantly attenuates heart hypertrophy by reducing both heart weight and ventricular wall thickness and the increased end-diastolic pressure. Whereas neither the single blockade of AT1 or Mas receptors with their respective antagonists prevented the cardioprotective action of Ang1-7, combination of the two antagonists partially impaired the effect of Ang-(1-7). Taken together, these data indicate that Ang-(1-7) mediates at least part of its cardioprotective effects by acting as an endogenous ß-arrestin-biased agonist at the AT1R.


Assuntos
Angiotensina I/uso terapêutico , Cardiomegalia/tratamento farmacológico , Cardiotônicos/uso terapêutico , Fragmentos de Peptídeos/uso terapêutico , Receptor Tipo 1 de Angiotensina/metabolismo , beta-Arrestinas/agonistas , Angiotensina I/metabolismo , Animais , Cardiomegalia/metabolismo , Cardiomegalia/fisiopatologia , Cardiotônicos/metabolismo , Diástole/efeitos dos fármacos , Células HEK293 , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ratos , Ratos Endogâmicos WF , Transdução de Sinais/efeitos dos fármacos , beta-Arrestinas/metabolismo
9.
Proc Natl Acad Sci U S A ; 114(34): 9044-9049, 2017 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-28784762

RESUMO

The neurohormone oxytocin is a key player in the modulation of reproductive and social behavioral traits, such as parental care. Recently, a correlation between different forms of oxytocin and behavioral phenotypes has been described in the New World Monkeys (NWMs). Here, we demonstrate that, compared with the Leu8OXT found in most placental mammals, the Cebidae Pro8OXT and Saguinus Val3Pro8OXT taxon-specific variants act as equi-efficacious agonists for the Gq-dependent pathway but are weaker agonists for the ß-arrestin engagement and subsequent endocytosis toward the oxytocin receptor (OXTR). Upon interaction with the AVPR1a, Pro8OXT and the common Leu8OXT yielded similar signaling profiles, being equally efficacious on Gq and ß-arrestin, while Val3Pro8OXT showed reduced relative efficacy toward ß-arrestin. Intranasal treatment with either of the variants increased maternal behavior and also promoted unusual paternal care in rats, as measured by pup-retrieval tests. We therefore suggest that Val3Pro8OXT and Pro8OXT are functional variants, which might have been evolutionarily co-opted as an essential part of the adaptive genetic repertoire that allowed the emergence of taxon-specific complex social behaviors, such as intense parental care in the Cebidae and the genus Saguinus.


Assuntos
Comportamento Animal/efeitos dos fármacos , Comportamento Materno/efeitos dos fármacos , Ocitocina/farmacologia , Comportamento Paterno/efeitos dos fármacos , Administração Intranasal , Animais , Animais Recém-Nascidos , Feminino , Variação Genética , Células HEK293 , Humanos , Masculino , Ocitocina/administração & dosagem , Ocitocina/genética , Platirrinos , Ratos , Receptores de Ocitocina/agonistas , Receptores de Ocitocina/genética , Receptores de Ocitocina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética
10.
Eur J Pharmacol ; 800: 70-80, 2017 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-28216049

RESUMO

Fluoxetine, a serotonin reuptake inhibitor (SSRI), has other effects in addition to blocking serotonin reuptake, including changes in the vasomotor tone. Whereas many studies focused on the acute effects of fluoxetine in the vasculature, its chronic effects are still limited. In the present study, we tested the hypothesis that chronic fluoxetine treatment modulates adrenergic vascular responses by interfering with post- and pre-synaptic mechanisms. Wistar rats were treated with vehicle (water) or chronic fluoxetine (10mg/kg/day) for 21 days. Blood pressure (BP) and heart rate were measured. Vascular reactivity was evaluated in perfused mesenteric arterial beds (MAB) and in mesenteric resistance arteries. Protein expression by western blot analysis or immunohistochemistry, ß-arrestin recruitment by BRET and calcium influx by FLIPR assay. Fluoxetine treatment decreased phenylephrine (PE)-induced, but not electrical-field stimulation (EFS)-induced vasoconstriction. Fluoxetine-treated rats exhibited increased KCl-induced vasoconstriction, which was abolished by prazosin. Desipramine, an inhibitor of norepinephrine (NA) reuptake, increased EFS-induced vasoconstrictor response in vehicle-treated, but not in fluoxetine-treated rats. Chronic treatment did not alter vascular expression of α1 adrenoceptor, phosphorylation of PKCα or ERK 1/2 and RhoA. On the other hand, vascular contractions to calcium (Ca2+) as well as Ca2+ influx in mesenteric arteries were increased, while intracellular Ca2+ storage was decreased by the chronic treatment with fluoxetine. In vitro, fluoxetine decreased vascular contractions to PE, EFS and Ca2+, but did not change ß-arrestin activity. In conclusion, chronic treatment with fluoxetine decreases sympathetic-mediated vascular responses by mechanisms that involve inhibition of NA release/reuptake and decreased Ca2+ stores.


Assuntos
Fluoxetina/farmacologia , Receptores Adrenérgicos alfa 1/metabolismo , Inibidores de Captação de Serotonina/farmacologia , Sinapses/efeitos dos fármacos , Animais , Pressão Arterial/efeitos dos fármacos , Cálcio/metabolismo , Estimulação Elétrica , Frequência Cardíaca/efeitos dos fármacos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Masculino , Proteínas da Membrana Plasmática de Transporte de Norepinefrina/metabolismo , Cloreto de Potássio/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiologia , Fatores de Tempo , Vasoconstrição/efeitos dos fármacos , beta-Arrestinas/metabolismo
11.
Invest Ophthalmol Vis Sci ; 57(10): 4356-66, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27564518

RESUMO

PURPOSE: Müller cells (MCs) are a major source of VEGF in diabetic retinopathy (DR). Vascular endothelial growth factor is the main therapeutic target for treating DR. This study aimed to investigate whether autophagy is involved in MC response under high glucose (HG). METHODS: Rat retinal Müller cells (rMCs) were exposed to normal or high glucose in and out of presence of pharmacologic inhibitors and activators and small interfering RNA (siRNA) for p62/SQTSM1 for 24 hours. RESULTS: High glucose induces increase of early and late autophagic markers, accumulation of p62/SQTSM1 and endoplasmic reticulum (ER) stress response associated with apoptosis augmentation (P < 0.01). The inhibition of autophagy in HG leads to higher rMC apoptotic rate (P < 0.001). By silencing the p62/SQTSM1, ER stress is ameliorated (p<0.0001), preventing apoptosis. Retinal MCs in HG treated with rapamycin (mTOR inhibitor) show autophagy machinery activation and reestablishment of cargo degradation, protecting cells from apoptosis (P < 0.0001). Rapamycin improves lysosomal proteolytic activity by improving cathepsin L activity restoring autophagic cargo degradation, and preventing increased VEGF release (P < 0.0001). In experimental model of diabetes, Beclin-1 and p62/SQTSM-1 were found to be marked increased in retinas from diabetic Wystar Kyoto rats compared with control group (P < 0.003) with reduction of cathepsin L activity. CONCLUSIONS: High glucose upregulates autophagy but accumulates p62/SQTSM1 cargo due to lysosomal dysfunction, leading to massive VEGF release and cell death of rMCs. Lysosomal impairment and autophagic dysfunction are early events present in the pathogenesis of diabetic retinopathy (DR). This might be valuable for developing a novel therapeutic strategy to treat DR.


Assuntos
Autofagia/fisiologia , Diabetes Mellitus Experimental , Retinopatia Diabética/patologia , Regulação da Expressão Gênica , RNA Interferente Pequeno/genética , Retina/metabolismo , Proteína Sequestossoma-1/genética , Animais , Apoptose , Autofagia/efeitos dos fármacos , Western Blotting , Células Cultivadas , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Células Ependimogliais/metabolismo , Células Ependimogliais/ultraestrutura , Glucose/farmacologia , Microscopia Eletrônica de Transmissão , Estresse Oxidativo , RNA Interferente Pequeno/metabolismo , Ratos , Retina/patologia , Proteína Sequestossoma-1/biossíntese , Edulcorantes/farmacologia
12.
Sci Rep ; 6: 28282, 2016 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-27320846

RESUMO

Prior research has shown that in experimental diabetes mellitus, green tea reduces albuminuria by decreasing podocyte apoptosis through activation of the WNT pathway. We investigated the effect of green tea polyphenols (GTP) on residual albuminuria of diabetic subjects with nephropathy. We conducted a randomised, double-blind study in 42 diabetic subjects with a urinary albumin-creatinine ratio (UACR) >30 mg/g, despite administration of the maximum recommended dose of renin-angiotensin (RAS) inhibition. Patients were randomly assigned to two equal groups to receive either GTP (containing 800 mg of epigallocatechin gallate, 17 with type 2 diabetes and 4 with type 1 diabetes) or placebo (21 with type 2 diabetes) for 12 weeks. Treatment with GTP reduced UACR by 41%, while the placebo group saw a 2% increase in UACR (p = 0.019). Podocyte apoptosis (p = 0.001) and in vitro albumin permeability (p < 0.001) were higher in immortalized human podocytes exposed to plasma from diabetic subjects compared to podocytes treated with plasma from normal individuals. In conclusion, GTP administration reduces albuminuria in diabetic patients receiving the maximum recommended dose of RAS. Reduction in podocyte apoptosis by activation of the WNT pathway may have contributed to this effect.


Assuntos
Albuminúria/tratamento farmacológico , Catequina/análogos & derivados , Nefropatias Diabéticas/tratamento farmacológico , Ervilhas/química , Polifenóis/administração & dosagem , Idoso , Albuminúria/metabolismo , Albuminúria/patologia , Apoptose/efeitos dos fármacos , Catequina/administração & dosagem , Catequina/química , Células Cultivadas , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Podócitos/metabolismo , Podócitos/patologia , Polifenóis/química
13.
PLoS One ; 11(2): e0147978, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26836609

RESUMO

Bone marrow-derived cells were demonstrated to improve organ function, but the lack of cell retention within injured organs suggests that the protective effects are due to factors released by the cells. Herein, we tested cell therapy using early outgrowth cells (EOCs) or their conditioned media (CM) to protect the retina of diabetic animal models (type 1 and type 2) and assessed the mechanisms by in vitro study. Control and diabetic (db/db) mice (8 weeks of age) were randomized to receive a unique intravenous injection of 5×105EOCs or 0.25 ml thrice weekly tail-vein injections of 10x concentrated CM and Wystar Kyoto rats rendered diabetic were randomized to receive 0.50 ml thrice weekly tail-vein injections of 10x concentrated CM. Four weeks later, the animals were euthanized and the eyes were enucleated. Rat retinal Müller cells (rMCs) were exposed for 24 h to high glucose (HG), combined or not with EOC-conditioned medium (EOC-CM) from db/m EOC cultures. Diabetic animals showed increase in diabetic retinopathy (DR) and oxidative damage markers; the treatment with EOCs or CM infusions significantly reduced this damage and re-established the retinal function. In rMCs exposed to diabetic milieu conditions (HG), the presence of EOC-CM reduced reactive oxygen species production by modulating the NADPH-oxidase 4 system, thus upregulating SIRT1 activity and deacetylating Lys-310-p65-NFκB, decreasing GFAP and VEGF expressions. The antioxidant capacity of EOC-CM led to the prevention of carbonylation and nitrosylation posttranslational modifications on the SIRT1 molecule, preserving its activity. The pivotal role of SIRT1 on the mode of action of EOCs or their CM was also demonstrated on diabetic retina. These findings suggest that EOCs are effective as a form of systemic delivery for preventing the early molecular markers of DR and its conditioned medium is equally protective revealing a novel possibility for cell-free therapy for the treatment of DR.


Assuntos
Células da Medula Óssea/metabolismo , Meios de Cultivo Condicionados/farmacologia , Substâncias Protetoras/farmacologia , Retina/efeitos dos fármacos , Retina/metabolismo , Animais , Biomarcadores , Glicemia , Linhagem Celular Transformada , Retinopatia Diabética/diagnóstico , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Eletrorretinografia , Células Ependimogliais/metabolismo , Masculino , Camundongos , Estresse Oxidativo , Substâncias Protetoras/administração & dosagem , Ratos , Espécies Reativas de Oxigênio , Retina/patologia , Transdução de Sinais , Sirtuína 1/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
14.
Pharmacol Res ; 112: 49-57, 2016 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26836887

RESUMO

G protein-coupled receptors (GPCRs) are the most important targets for drug discovery and not surprisingly ∼40% of all drugs currently in the market act on these receptors. Currently, one of the most active areas in GPCRs signaling is biased agonism, a phenomenon that occurs when a given ligand is able to preferentially activate one (or some) of the possible signaling pathways. In this review, we highlight the most recent findings about biased agonism, including an extension of this concept to intracellular signaling, allosterism, strategies for assessment and interpretation, and perspectives of therapeutic applications for biased agonists.


Assuntos
Descoberta de Drogas , Receptores Acoplados a Proteínas-G/agonistas , Sítio Alostérico , Animais , Sítios de Ligação , Humanos , Ligantes , Terapia de Alvo Molecular , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais
15.
Front Pharmacol ; 6: 131, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26191004

RESUMO

G protein-coupled receptors (GPCRs) are involved in essentially all physiological processes in mammals. The classical GPCR signal transduction mechanism occurs by coupling to G protein, but it has recently been demonstrated that interaction with ß-arrestins leads to activation of pathways that are independent of the G protein pathway. Also, it has been reported that some ligands can preferentially activate one of these signaling pathways; being therefore called biased agonists for G protein or ß-arrestin pathways. The angiotensin II (AngII) AT1 receptor is a prototype GPCR in the study of biased agonism due to the existence of well-known ß-arrestin-biased agonists, such as [Sar(1), Ile(4), Ile(8)]-AngII (SII), and [Sar(1), D-Ala(8)]-AngII (TRV027). The aim of this study was to comparatively analyze the two above mentioned ß-arrestin-biased agonists on downstream phosphorylation events and gene expression profiles. Our data reveal that activation of AT1 receptor by each ligand led to a diversity of activation profiles that is far broader than that expected from a simple dichotomy between "G protein-dependent" and "ß-arrestin-dependent" signaling. We observed clusters of activation profiles common to AngII, SII, and TRV027, as well as downstream effector activation that are unique to AngII, SII, or TRV027. Analyses of ß-arrestin conformational changes after AT1 receptor stimulation with SII or TRV027 suggests that the observed differences could account, at least partially, for the diversity of modulated targets observed. Our data reveal that, although the categorization "G protein-dependent" vs. "ß-arrestin-dependent" signaling can be of pharmacological relevance, broader analyses of signaling pathways and downstream targets are necessary to generate an accurate activation profile for a given ligand. This may bring relevant information for drug development, as it may allow more refined comparison of drugs with similar mechanism of action and effects, but with distinct side effects.

16.
J Nutr Biochem ; 26(1): 64-74, 2015 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-25448608

RESUMO

Cocoa is rich in flavonoids, which are potent antioxidants with established benefits for cardiovascular health but unproven effects on neurodegeneration. Sirtuins (SIRTs), which make up a family of deacetylases, are thought to be sensitive to oxidation. In this study, the possible protective effects of cocoa in the diabetic retina were assessed. Rat Müller cells (rMCs) exposed to normal or high glucose (HG) or H2O2 were submitted to cocoa treatment in the presence or absence of SIRT-1 inhibitor and small interfering RNA The experimental animal study was conducted in streptozotocin-induced diabetic rats randomized to receive low-, intermediate-, or high-polyphenol cocoa treatments via daily gavage for 16 weeks (i.e., 0.12, 2.9 or 22.9 mg/kg/day of polyphenols). The rMCs exposed to HG or H2O2 exhibited increased glial fibrillary acidic protein (GFAP) and acetyl-RelA/p65 and decreased SIRT1 activity/expression. These effects were cancelled out by cocoa, which decreased reactive oxygen species production and PARP-1 activity, augmented the intracellular pool of NAD(+), and improved SIRT1 activity. The rat diabetic retinas displayed the early markers of retinopathy accompanied by markedly impaired electroretinogram. The presence of diabetes activated PARP-1 and lowered NAD(+) levels, resulting in SIRT1 impairment. This augmented acetyl RelA/p65 had the effect of up-regulated GFAP. Oral administration of polyphenol cocoa restored the above alterations in a dose-dependent manner. This study reveals that cocoa enriched with polyphenol improves the retinal SIRT-1 pathway, thereby protecting the retina from diabetic milieu insult.


Assuntos
Cacau/química , Retinopatia Diabética/prevenção & controle , Proteína Glial Fibrilar Ácida/metabolismo , Polifenóis/farmacologia , Substâncias Protetoras/farmacologia , Animais , Antioxidantes/farmacologia , Catequina/sangue , Cromatografia Líquida , Diabetes Mellitus Experimental/etiologia , Diabetes Mellitus Experimental/prevenção & controle , Relação Dose-Resposta a Droga , Proteína Glial Fibrilar Ácida/genética , Glucose/metabolismo , Peróxido de Hidrogênio/metabolismo , Masculino , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Endogâmicos SHR , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Estreptozocina/efeitos adversos , Espectrometria de Massas em Tandem , Fator de Transcrição RelA/genética , Fator de Transcrição RelA/metabolismo
17.
Invest Ophthalmol Vis Sci ; 55(9): 6090-100, 2014 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-25190662

RESUMO

PURPOSE: Retinal pigment epithelium cells, along with tight junction (TJ) proteins, constitute the outer blood retinal barrier (BRB). Contradictory findings suggest a role for the outer BRB in the pathogenesis of diabetic retinopathy (DR). The aim of this study was to investigate whether the mechanisms involved in these alterations are sensitive to nitrosative stress, and if cocoa or epicatechin (EC) protects from this damage under diabetic (DM) milieu conditions. METHODS: Cells of a human RPE line (ARPE-19) were exposed to high-glucose (HG) conditions for 24 hours in the presence or absence of cocoa powder containing 0.5% or 60.5% polyphenol (low-polyphenol cocoa [LPC] and high-polyphenol cocoa [HPC], respectively). RESULTS: Exposure to HG decreased claudin-1 and occludin TJ expressions and increased extracellular matrix accumulation (ECM), whereas levels of TNF-α and inducible nitric oxide synthase (iNOS) were upregulated, accompanied by increased nitric oxide levels. This nitrosative stress resulted in S-nitrosylation of caveolin-1 (CAV-1), which in turn increased CAV-1 traffic and its interactions with claudin-1 and occludin. This cascade was inhibited by treatment with HPC or EC through δ-opioid receptor (DOR) binding and stimulation, thereby decreasing TNF-α-induced iNOS upregulation and CAV-1 endocytosis. The TJ functions were restored, leading to prevention of paracellular permeability, restoration of resistance of the ARPE-19 monolayer, and decreased ECM accumulation. CONCLUSIONS: The detrimental effects on TJs in ARPE-19 cells exposed to DM milieu occur through a CAV-1 S-nitrosylation-dependent endocytosis mechanism. High-polyphenol cocoa or EC exerts protective effects through DOR stimulation.


Assuntos
Cacau/química , Caveolina 1/metabolismo , Endocitose/fisiologia , Polifenóis/farmacologia , Receptores Opioides/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Junções Íntimas/metabolismo , Animais , Barreira Hematorretiniana , Western Blotting , Linhagem Celular , Claudina-1/metabolismo , Dextranos/metabolismo , Impedância Elétrica , Técnica Indireta de Fluorescência para Anticorpo , Glucose/farmacologia , Humanos , Óxido Nítrico Sintase Tipo II/metabolismo , Nitrosação , Ocludina/metabolismo , Permeabilidade , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Suínos , Fator de Necrose Tumoral alfa/metabolismo
18.
Invest Ophthalmol Vis Sci ; 55(5): 2921-32, 2014 May 02.
Artigo em Inglês | MEDLINE | ID: mdl-24699383

RESUMO

PURPOSE: Diabetic retinopathy (DR) is associated with nitrosative stress. The purpose of this study was to evaluate the beneficial effects of S-nitrosoglutathione (GSNO) eye drop treatment on an experimental model of DR. METHODS: Diabetes (DM) was induced in spontaneously hypertensive rats (SHR). Treated animals received GSNO eye drop (900 nM or 10 µM) twice daily in both eyes for 20 days. The mechanisms of GSNO effects were evaluated in human RPE cell line (ARPE-19). RESULTS: In animals with DM, GSNO decreased inducible nitric oxide synthase (iNOS) expression and prevented tyrosine nitration formation, ameliorating glial dysfunction measured with glial fibrillary acidic protein, resulting in improved retinal function. In contrast, in nondiabetic animals, GSNO induced oxidative/nitrosative stress in tissue resulting in impaired retinal function. Nitrosative stress was present markedly in the RPE layer accompanied by c-wave dysfunction. In vitro study showed that treatment with GSNO under high glucose condition counteracted nitrosative stress due to iNOS downregulation by S-glutathionylation, and not by prevention of decreased GSNO and reduced glutathione levels. This posttranslational modification probably was promoted by the release of oxidized glutathione through GSNO denitrosylation via GSNO-R. In contrast, in the normal glucose condition, GSNO treatment promoted nitrosative stress by NO formation. CONCLUSIONS: In this study, a new therapeutic modality (GSNO eye drop) targeting nitrosative stress by redox posttranslational modification of iNOS was efficient against early damage in the retina due to experimental DR. The present work showed the potential clinical implications of balancing the S-nitrosoglutathione/glutathione system in treating DR.


Assuntos
Diabetes Mellitus Experimental/metabolismo , Retinopatia Diabética , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , S-Nitrosoglutationa/farmacologia , Análise de Variância , Animais , Biomarcadores/metabolismo , Linhagem Celular , Retinopatia Diabética/tratamento farmacológico , Retinopatia Diabética/metabolismo , Modelos Animais de Doenças , Eletrorretinografia/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Glutationa/metabolismo , Humanos , Doadores de Óxido Nítrico/uso terapêutico , Soluções Oftálmicas/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Retina/efeitos dos fármacos , Retina/metabolismo , S-Nitrosoglutationa/uso terapêutico , Tirosina/análogos & derivados , Tirosina/metabolismo , Regulação para Cima
19.
Clin Sci (Lond) ; 126(11): 753-74, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24559183

RESUMO

GPCRs (G-protein-coupled receptors) are among the most important targets for drug discovery due to their ubiquitous expression and participation in cellular events under both healthy and disease conditions. These receptors can be activated by a plethora of ligands, such as ions, odorants, small ligands and peptides, including angiotensins and kinins, which are vasoactive peptides that are classically involved in the pathophysiology of cardiovascular events. These peptides and their corresponding GPCRs have been reported to play roles in other systems and under pathophysiological conditions, such as cancer, central nervous system disorders, metabolic dysfunction and bone resorption. More recently, new mechanisms have been described for the functional regulation of GPCRs, including the transactivation of other signal transduction receptors and the activation of G-protein-independent pathways. The existence of such alternative mechanisms for signal transduction and the discovery of agonists that can preferentially trigger one signalling pathway over other pathways (called biased agonists) have opened new perspectives for the discovery and development of drugs with a higher specificity of action and, therefore, fewer side effects. The present review summarizes the current knowledge on the non-canonical signalling and roles of angiotensins and kinins.


Assuntos
Angiotensinas/metabolismo , Regulação da Expressão Gênica , Cininas/metabolismo , Transdução de Sinais , Angiotensina II/metabolismo , Animais , Arrestinas/metabolismo , Reabsorção Óssea , Bradicinina/metabolismo , Doenças do Sistema Nervoso Central/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Ligantes , Neoplasias/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , beta-Arrestinas
20.
Br J Ophthalmol ; 98(6): 726-9, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24511082

RESUMO

BACKGROUND: Assessment of the perifoveal capillary network (PCN) might indicate macular function and could reflect the systemic microcirculation. The quantification and reliability of this measurement is currently unknown. The aim of this study was to validate quantification of the PCN by a non-invasive technique from high-resolution retinal images. METHODS: Ten healthy volunteers were included in this validation study. At least 320 high-resolution retinal images were used for assessment of inter- and intra-observer reliability. Non-invasive capillary perfusion mapping was performed using a retinal function imager. After the images were enhanced and segmented, the reproducibility was verified by comparing the values of two independent examiners and of a single examiner at two different time points. RESULTS: The inter-observer concordance coefficients were highly significant for PCN (intraclass correlation coefficient (ICC)=0.901, 95% CI 0.655 to 0.975, p<0.001) and normalised PCN (ICC=0.727, 95% CI 0.262 to 0.923, p=0.004). The intra-observer measurements at two different time points were also highly concordant for PCN (ICC=0.879, 95% CI 0.598 to 0.968, p<0.001) and for normalised PCN (ICC=0.960, 95% CI 0.851 to 0.990, p<0.001). CONCLUSIONS: The reliability of PCN measurement is reproducible and could be used as a new tool to quantify the capillary perfusion network of the macular area.


Assuntos
Diagnóstico por Imagem/métodos , Técnicas de Diagnóstico Oftalmológico/instrumentação , Fóvea Central/irrigação sanguínea , Vasos Retinianos/anatomia & histologia , Adulto , Capilares/anatomia & histologia , Diagnóstico por Imagem/instrumentação , Voluntários Saudáveis , Humanos , Microcirculação , Variações Dependentes do Observador , Reprodutibilidade dos Testes
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