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1.
Arterioscler Thromb Vasc Biol ; 40(9): 2212-2226, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32640908

RESUMO

OBJECTIVE: The ductus arteriosus (DA) is a fetal artery connecting the aorta and pulmonary arteries. Progressive matrix remodeling, that is, intimal thickening (IT), occurs in the subendothelial region of DA to bring anatomic DA closure. IT is comprised of multiple ECMs (extracellular matrices) and migrated smooth muscle cells (SMCs). Because glycoprotein fibulin-1 binds to multiple ECMs and regulates morphogenesis during development, we investigated the role of fibulin-1 in DA closure. Approach and Results: Fibulin-1-deficient (Fbln1-/-) mice exhibited patent DA with hypoplastic IT. An unbiased transcriptome analysis revealed that EP4 (prostaglandin E receptor 4) stimulation markedly increased fibulin-1 in DA-SMCs via phospholipase C-NFκB (nuclear factor κB) signaling pathways. Fluorescence-activated cell sorting (FACS) analysis demonstrated that fibulin-1 binding protein versican was derived from DA-endothelial cells (ECs). We examined the effect of fibulin-1 on directional migration toward ECs in association with versican by using cocultured DA-SMCs and ECs. EP4 stimulation promoted directional DA-SMC migration toward ECs, which was attenuated by either silencing fibulin-1 or versican. Immunofluorescence demonstrated that fibulin-1 and versican V0/V1 were coexpressed at the IT of wild-type DA, whereas 30% of versican-deleted mice lacking a hyaluronan binding site displayed patent DA. Fibulin-1 expression was attenuated in the EP4-deficient mouse (Ptger4-/-) DA, which exhibits patent DA with hypoplastic IT, and fibulin-1 protein administration restored IT formation. In human DA, fibulin-1 and versican were abundantly expressed in SMCs and ECs, respectively. CONCLUSIONS: Fibulin-1 contributes to DA closure by forming an environment favoring directional SMC migration toward the subendothelial region, at least, in part, in combination with EC-derived versican and its binding partner hyaluronan.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Permeabilidade do Canal Arterial/metabolismo , Canal Arterial/metabolismo , Células Endoteliais/metabolismo , Matriz Extracelular/metabolismo , Miócitos de Músculo Liso/metabolismo , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Movimento Celular , Células Cultivadas , Técnicas de Cocultura , Canal Arterial/anormalidades , Permeabilidade do Canal Arterial/genética , Permeabilidade do Canal Arterial/patologia , Células Endoteliais/patologia , Matriz Extracelular/genética , Matriz Extracelular/patologia , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos de Músculo Liso/patologia , NF-kappa B/metabolismo , Técnicas de Cultura de Órgãos , Proteína Quinase C/metabolismo , Ratos Wistar , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Transdução de Sinais , Fosfolipases Tipo C/metabolismo
2.
Nat Commun ; 11(1): 2717, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483112

RESUMO

Somatic inactivating mutations of ARID1A, a SWI/SNF chromatin remodeling gene, are prevalent in human endometrium-related malignancies. To elucidate the mechanisms underlying how ARID1A deleterious mutation contributes to tumorigenesis, we establish genetically engineered murine models with Arid1a and/or Pten conditional deletion in the endometrium. Transcriptomic analyses on endometrial cancers and precursors derived from these mouse models show a close resemblance to human uterine endometrioid carcinomas. We identify transcriptional networks that are controlled by Arid1a and have an impact on endometrial tumor development. To verify findings from the murine models, we analyze ARID1AWT and ARID1AKO human endometrial epithelial cells. Using a system biology approach and functional studies, we demonstrate that ARID1A-deficiency lead to loss of TGF-ß tumor suppressive function and that inactivation of ARID1A/TGF-ß axis promotes migration and invasion of PTEN-deleted endometrial tumor cells. These findings provide molecular insights into how ARID1A inactivation accelerates endometrial tumor progression and dissemination, the major causes of cancer mortality.


Assuntos
Carcinogênese/genética , Carcinoma Endometrioide/genética , Reprogramação Celular/genética , Proteínas de Ligação a DNA/genética , Neoplasias do Endométrio/genética , Fatores de Transcrição/genética , Animais , Carcinogênese/metabolismo , Carcinoma Endometrioide/metabolismo , Carcinoma Endometrioide/patologia , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Proteínas de Ligação a DNA/metabolismo , Neoplasias do Endométrio/metabolismo , Neoplasias do Endométrio/patologia , Endométrio/citologia , Endométrio/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Knockout , Camundongos Transgênicos , Mutação , Fatores de Transcrição/metabolismo
3.
Nat Commun ; 11(1): 2711, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483135

RESUMO

p16INK4a (CDKN2A) is a central tumor suppressor, which induces cell-cycle arrest and senescence. Cells expressing p16INK4a accumulate in aging tissues and appear in premalignant lesions, yet their physiologic effects are poorly understood. We found that prolonged expression of transgenic p16INK4a in the mouse epidermis induces hyperplasia and dysplasia, involving high proliferation rates of keratinocytes not expressing the transgene. Continuous p16INK4a expression increases the number of epidermal papillomas formed after carcinogen treatment. Wnt-pathway ligands and targets are activated upon prolonged p16INK4a expression, and Wnt inhibition suppresses p16INK4a-induced hyperplasia. Senolytic treatment reduces p16INK4a-expressing cell numbers, and inhibits Wnt activation and hyperplasia. In human actinic keratosis, a precursor of squamous cell carcinoma, p16INK4a-expressing cells are found adjacent to dividing cells, consistent with paracrine interaction. These findings reveal that chronic p16INK4a expression is sufficient to induce hyperplasia through Wnt-mediated paracrine stimulation, and suggest that this tumor suppressor can promote early premalignant epidermal lesion formation.


Assuntos
Transformação Celular Neoplásica/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Epiderme/metabolismo , Via de Sinalização Wnt/genética , Animais , Proliferação de Células/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Humanos , Hiperplasia/genética , Hiperplasia/metabolismo , Queratinócitos/metabolismo , Ceratose/genética , Ceratose/metabolismo , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Papiloma/genética , Papiloma/metabolismo , Papiloma/patologia
4.
Nat Commun ; 11(1): 2699, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483185

RESUMO

Nebulin is a giant protein that winds around the actin filaments in the skeletal muscle sarcomere. Compound-heterozygous mutations in the nebulin gene (NEB) cause typical nemaline myopathy (NM), a muscle disorder characterized by muscle weakness with limited treatment options. We created a mouse model with a missense mutation p.Ser6366Ile and a deletion of NEB exon 55, the Compound-Het model that resembles typical NM. We show that Compound-Het mice are growth-retarded and have muscle weakness. Muscles have a reduced myofibrillar fractional-area and sarcomeres are disorganized, contain rod bodies, and have longer thin filaments. In contrast to nebulin-based severe NM where haplo-insufficiency is the disease driver, Compound-Het mice express normal amounts of nebulin. X-ray diffraction revealed that the actin filament is twisted with a larger radius, that tropomyosin and troponin behavior is altered, and that the myofilament spacing is increased. The unique disease mechanism of nebulin-based typical NM reveals novel therapeutic targets.


Assuntos
Proteínas Musculares/genética , Mutação de Sentido Incorreto , Miofibrilas/metabolismo , Miopatias da Nemalina/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Animais , Heterozigoto , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/ultraestrutura , Miofibrilas/patologia , Miofibrilas/ultraestrutura , Miopatias da Nemalina/metabolismo , Sarcômeros/metabolismo , Sarcômeros/patologia , Sarcômeros/ultraestrutura , Tropomiosina/química , Tropomiosina/metabolismo , Troponina/química , Troponina/metabolismo , Difração de Raios X
5.
Nat Commun ; 11(1): 2491, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427842

RESUMO

Hox genes encode transcription factors (TFs) that establish morphological diversity in the developing embryo. The similar DNA-binding motifs of the various HOX TFs contrast with the wide-range of HOX-dependent genetic programs. The influence of the chromatin context on HOX binding specificity remains elusive. Here, we used the developing limb as a model system to compare the binding specificity of HOXA13 and HOXD13 (HOX13 hereafter), which are required for digit formation, and HOXA11, involved in forearm/leg development. We find that upon ectopic expression in distal limb buds, HOXA11 binds sites normally HOX13-specific. Importantly, these sites are loci whose chromatin accessibility relies on HOX13. Moreover, we show that chromatin accessibility specific to the distal limb requires HOX13 function. Based on these results, we propose that HOX13 TFs pioneer the distal limb-specific chromatin accessibility landscape for the proper implementation of the distal limb developmental program.


Assuntos
Cromatina/genética , Membro Anterior/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Botões de Extremidades/metabolismo , Animais , Sítios de Ligação/genética , Cromatina/metabolismo , Membro Anterior/embriologia , Perfilação da Expressão Gênica/métodos , Proteínas de Homeodomínio/metabolismo , Botões de Extremidades/embriologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Ligação Proteica
6.
PLoS Genet ; 16(5): e1008255, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32392211

RESUMO

mTOR, a serine/threonine protein kinase that is involved in a series of critical cellular processes, can be found in two functionally distinct complexes, mTORC1 and mTORC2. In contrast to mTORC1, little is known about the mechanisms that regulate mTORC2. Here we show that mTORC2 activity is reduced in mice with a hypomorphic mutation of the Ric-8B gene. Ric-8B is a highly conserved protein that acts as a non-canonical guanine nucleotide exchange factor (GEF) for heterotrimeric Gαs/olf type subunits. We found that Ric-8B hypomorph embryos are smaller than their wild type littermates, fail to close the neural tube in the cephalic region and die during mid-embryogenesis. Comparative transcriptome analysis revealed that signaling pathways involving GPCRs and G proteins are dysregulated in the Ric-8B mutant embryos. Interestingly, this analysis also revealed an unexpected impairment of the mTOR signaling pathway. Phosphorylation of Akt at Ser473 is downregulated in the Ric-8B mutant embryos, indicating a decreased activity of mTORC2. Knockdown of the endogenous Ric-8B gene in cultured cell lines leads to reduced phosphorylation levels of Akt (Ser473), further supporting the involvement of Ric-8B in mTORC2 activity. Our results reveal a crucial role for Ric-8B in development and provide novel insights into the signals that regulate mTORC2.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Animais , Células Cultivadas , Regulação para Baixo/genética , Embrião de Mamíferos , Desenvolvimento Embrionário/genética , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Transdução de Sinais/genética
7.
Am J Physiol Renal Physiol ; 318(5): F1199-F1209, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32249612

RESUMO

Lithium is widely used in psychiatry as the golden standard for more than 60 yr due to its effectiveness. However, its adverse effect has been limiting its long-term use in clinic. About 40% of patients taking lithium develop nephrogenic diabetes insipidus (NDI). Lithium can also induce proliferation of collecting duct cells, leading to microcyst formation in the kidney. Lithium was considered an autophagy inducer that might contribute to the therapeutic benefit of neuropsychiatric disorders. Thus, we hypothesized that autophagy may play a role in lithium-induced kidney nephrotoxicity. To address our hypothesis, we fed mice with a lithium-containing diet with chloroquine (CQ), an autophagy inhibitor, concurrently. Lithium-treated mice presented enhanced autophagy activity in the kidney cortex and medulla. CQ treatment significantly ameliorated lithium-induced polyuria, polydipsia, natriuresis, and kaliuresis accompanied with attenuated downregulation of aquaporin-2 and Na+-K+-2Cl- cotransporter protein. The protective effect of CQ on aquaporin-2 protein abundance was confirmed in cultured cortical collecting duct cells. In addition, we found that lithium-induced proliferation of collecting duct cells was also suppressed by CQ as detected by proliferating cell nuclear antigen staining. Moreover, both phosphorylated mammalian target of rapamycin and ß-catenin expression, which have been reported to be increased by lithium and associated with cell proliferation, were reduced by CQ. Taken together, our study demonstrated that CQ protected against lithium-induced NDI and collecting duct cell proliferation possibly through inhibiting autophagy.


Assuntos
Proliferação de Células/efeitos dos fármacos , Cloroquina/farmacologia , Diabetes Insípido Nefrogênico/prevenção & controle , Túbulos Renais Coletores/efeitos dos fármacos , Cloreto de Lítio , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Autofagia/efeitos dos fármacos , Linhagem Celular , Diabetes Insípido Nefrogênico/induzido quimicamente , Diabetes Insípido Nefrogênico/metabolismo , Diabetes Insípido Nefrogênico/patologia , Dinoprostona/urina , Modelos Animais de Doenças , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/patologia , Masculino , Camundongos da Linhagem 129 , Natriurese/efeitos dos fármacos , Fosforilação , Poliúria/induzido quimicamente , Poliúria/metabolismo , Poliúria/patologia , Poliúria/prevenção & controle , Membro 1 da Família 12 de Carreador de Soluto/genética , Membro 1 da Família 12 de Carreador de Soluto/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo , beta Catenina/metabolismo
8.
J Med Chem ; 63(8): 4205-4214, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32227946

RESUMO

Influenza and dengue viruses present a growing global threat to public health. Both viruses depend on the host endoplasmic reticulum (ER) glycoprotein folding pathway. In 2014, Sadat et al. reported two siblings with a rare genetic defect in ER α-glucosidase I (ER Glu I) who showed resistance to viral infections, identifying ER Glu I as a key antiviral target. Here, we show that a single dose of UV-4B (the hydrochloride salt form of N-(9'-methoxynonyl)-1-deoxynojirimycin; MON-DNJ) capable of inhibiting Glu I in vivo is sufficient to prevent death in mice infected with lethal viral doses, even when treatment is started as late as 48 h post infection. The first crystal structure of mammalian ER Glu I will constitute the basis for the development of potent and selective inhibitors. Targeting ER Glu I with UV-4B-derived compounds may alter treatment paradigms for acute viral disease through development of a single-dose therapeutic regime.


Assuntos
Dengue/prevenção & controle , Retículo Endoplasmático/efeitos dos fármacos , Inibidores de Glicosídeo Hidrolases/administração & dosagem , Influenza Humana/prevenção & controle , alfa-Glucosidases , Animais , Dengue/tratamento farmacológico , Dengue/enzimologia , Vírus da Dengue/efeitos dos fármacos , Vírus da Dengue/enzimologia , Relação Dose-Resposta a Droga , Retículo Endoplasmático/enzimologia , Humanos , Influenza Humana/tratamento farmacológico , Influenza Humana/enzimologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Estrutura Secundária de Proteína , alfa-Glucosidases/metabolismo
9.
Psychopharmacology (Berl) ; 237(7): 1973-1987, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32313981

RESUMO

RATIONALE: Obsessive-compulsive disorder (OCD) is characterized by repetitive behaviors exacerbated by stress. Many OCD patients do not respond to available pharmacotherapies, but neurosurgical ablation of the anterior cingulate cortex (ACC) can provide symptomatic relief. Although the ACC receives noradrenergic innervation and expresses adrenergic receptors (ARs), the involvement of norepinephrine (NE) in OCD has not been investigated. OBJECTIVE: To determine the effects of genetic or pharmacological disruption of NE neurotransmission on marble burying (MB) and nestlet shredding (NS), two animal models of OCD. METHODS: We assessed NE-deficient (Dbh -/-) mice and NE-competent (Dbh +/-) controls in MB and NS tasks. We also measured the effects of anti-adrenergic drugs on NS and MB in control mice and the effects of pharmacological restoration of central NE in Dbh -/- mice. Finally, we compared c-fos induction in the locus coeruleus (LC) and ACC of Dbh -/- and control mice following both tasks. RESULTS: Dbh -/- mice virtually lacked MB and NS behaviors seen in control mice but did not differ in the elevated zero maze (EZM) model of general anxiety-like behavior. Pharmacological restoration of central NE synthesis in Dbh -/- mice completely rescued NS behavior, while NS and MB were suppressed in control mice by anti-adrenergic drugs. Expression of c-fos in the ACC was attenuated in Dbh -/- mice after MB and NS. CONCLUSION: These findings support a role for NE transmission to the ACC in the expression of stress-induced compulsive behaviors and suggest further evaluation of anti-adrenergic drugs for OCD is warranted.


Assuntos
Comportamento Compulsivo/metabolismo , Modelos Animais de Doenças , Norepinefrina/metabolismo , Transtorno Obsessivo-Compulsivo/metabolismo , Estresse Psicológico/metabolismo , Antagonistas de Receptores Adrenérgicos alfa 1/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 1/uso terapêutico , Animais , Comportamento Compulsivo/tratamento farmacológico , Comportamento Compulsivo/psicologia , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Norepinefrina/antagonistas & inibidores , Transtorno Obsessivo-Compulsivo/tratamento farmacológico , Transtorno Obsessivo-Compulsivo/psicologia , Receptores Adrenérgicos/metabolismo , Roedores , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/psicologia
10.
J Pathol ; 251(2): 200-212, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32243583

RESUMO

Age-related macular degeneration (AMD) is a complex, multifactorial, progressive disease which represents a leading cause of irreversible visual impairment and blindness in older individuals. Human cytomegalovirus (HCMV), which infects 50-80% of humans, is usually acquired during early life and persists in a latent state for the life of the individual. In view of its previously described pro-angiogenic properties, we hypothesized that cytomegalovirus might be a novel risk factor for progression to an advanced form, neovascular AMD, which is characterized by choroidal neovascularization (CNV). The purpose of this study was to investigate if latent ocular murine cytomegalovirus (MCMV) infection exacerbated the development of CNV in vascular endothelial growth factor (VEGF)-overexpressing VEGF-Ahyper mice. Here we show that neonatal infection with MCMV resulted in dissemination of virus to various organs throughout the body including the eye, where it localized principally to the choroid in both VEGF-overexpressingVEGF-Ahyper and wild-type(WT) 129 mice. By 6 months post-infection, no replicating virus was detected in eyes and extraocular tissues, although virus DNA was still present in all eyes and extraocular tissues of both VEGF-Ahyper and WT mice. Expression of MCMV immediate early (IE) 1 mRNA was detected only in latently infected eyes of VEGF-Ahyper mice, but not in eyes of WT mice. Significantly increased CNV was observed in eyes of MCMV-infected VEGF-Ahyper mice compared to eyes of uninfected VEGF-Ahyper mice, while no CNV lesions were observed in eyes of either infected or uninfected WT mice. Protein levels of several inflammatory/angiogenic factors, particularly VEGF and IL-6, were significantly higher in eyes of MCMV-infected VEGF-Ahyper mice, compared to uninfected controls. Initial studies of ocular tissue from human cadavers revealed that HCMV DNA was present in four choroid/retinal pigment epithelium samples from 24 cadavers. Taken together, our data suggest that ocular HCMV latency could be a significant risk factor for the development of AMD. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Neovascularização de Coroide/virologia , Retinite por Citomegalovirus/virologia , Degeneração Macular/virologia , Muromegalovirus/patogenicidade , Retina/virologia , Latência Viral , Idoso , Idoso de 80 Anos ou mais , Animais , Neovascularização de Coroide/genética , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Retinite por Citomegalovirus/genética , Retinite por Citomegalovirus/metabolismo , Retinite por Citomegalovirus/patologia , Modelos Animais de Doenças , Progressão da Doença , Feminino , Humanos , Proteínas Imediatamente Precoces/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Camundongos da Linhagem 129 , Camundongos Transgênicos , Pessoa de Meia-Idade , Retina/metabolismo , Retina/ultraestrutura , Fatores de Risco , Transdução de Sinais , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
11.
Am J Physiol Renal Physiol ; 318(6): F1341-F1356, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32281415

RESUMO

We characterized mouse blood pressure and ion transport in the setting of commonly used rodent diets that drive K+ intake to the extremes of deficiency and excess. Male 129S2/Sv mice were fed either K+-deficient, control, high-K+ basic, or high-KCl diets for 10 days. Mice maintained on a K+-deficient diet exhibited no change in blood pressure, whereas K+-loaded mice developed an ~10-mmHg blood pressure increase. Following challenge with NaCl, K+-deficient mice developed a salt-sensitive 8 mmHg increase in blood pressure, whereas blood pressure was unchanged in mice fed high-K+ diets. Notably, 10 days of K+ depletion induced diabetes insipidus and upregulation of phosphorylated NaCl cotransporter, proximal Na+ transporters, and pendrin, likely contributing to the K+-deficient NaCl sensitivity. While the anionic content with high-K+ diets had distinct effects on transporter expression along the nephron, both K+ basic and KCl diets had a similar increase in blood pressure. The blood pressure elevation on high-K+ diets correlated with increased Na+-K+-2Cl- cotransporter and γ-epithelial Na+ channel expression and increased urinary response to furosemide and amiloride. We conclude that the dietary K+ maneuvers used here did not recapitulate the inverse effects of K+ on blood pressure observed in human epidemiological studies. This may be due to the extreme degree of K+ stress, the low-Na+-to-K+ ratio, the duration of treatment, and the development of other coinciding events, such as diabetes insipidus. These factors must be taken into consideration when studying the physiological effects of dietary K+ loading and depletion.


Assuntos
Pressão Arterial , Hipertensão/metabolismo , Túbulos Renais/metabolismo , Deficiência de Potássio/metabolismo , Potássio na Dieta/metabolismo , Cloreto de Sódio na Dieta/metabolismo , Ração Animal , Animais , Diabetes Insípido/etiologia , Diabetes Insípido/metabolismo , Diabetes Insípido/fisiopatologia , Canais Epiteliais de Sódio/metabolismo , Hipertensão/etiologia , Hipertensão/fisiopatologia , Transporte de Íons , Túbulos Renais/fisiopatologia , Masculino , Camundongos da Linhagem 129 , Natriurese , Fosforilação , Deficiência de Potássio/etiologia , Deficiência de Potássio/fisiopatologia , Potássio na Dieta/administração & dosagem , Potássio na Dieta/toxicidade , Simportadores de Cloreto de Sódio/metabolismo , Cloreto de Sódio na Dieta/toxicidade , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Transportadores de Sulfato/metabolismo
12.
Am J Physiol Renal Physiol ; 318(6): F1369-F1376, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32308018

RESUMO

Cytochrome P-450 (Cyp) epoxygenase-dependent metabolites of arachidonic acid (AA) have been shown to inhibit renal Na+ transport, and inhibition of Cyp-epoxygenase is associated with salt-sensitive hypertension. We used the patch-clamp technique to examine whether Cyp-epoxygenase-dependent AA metabolites inhibited the basolateral 40-pS K+ channel (Kir4.1/Kir5.1) in the distal convoluted tubule (DCT). Application of AA inhibited the basolateral 40-pS K+ channel in the DCT. The inhibitory effect of AA on the 40-pS K+ channel was specific because neither linoleic nor oleic acid was able to mimic the effect of AA on the K+ channel. Inhibition of Cyp-monooxygenase with N-methylsulfonyl-12,12-dibromododec-11-enamide or inhibition of cyclooxygenase with indomethacin failed to abolish the inhibitory effect of AA on the 40-pS K+ channel. However, the inhibition of Cyp-epoxygenase with N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide abolished the effect of AA on the 40-pS K+ channel in the DCT. Moreover, addition of either 11,12-epoxyeicosatrienoic acid (EET) or 14,15-EET also inhibited the 40-pS K+ channel in the DCT. Whole cell recording demonstrated that application of AA decreased, whereas N-methylsulfonyl-6-(propargyloxyphenyl)hexanamide treatment increased, Ba2+-sensitive K+ currents in the DCT. Finally, application of 14,15-EET but not AA was able to inhibit the basolateral 40-pS K+ channel in the DCT of Cyp2c44-/- mice. We conclude that Cyp-epoxygenase-dependent AA metabolites inhibit the basolateral Kir4.1/Kir5.1 in the DCT and that Cyp2c44-epoxygenase plays a role in the regulation of the basolateral K+ channel in the mouse DCT.


Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Ácido Araquidônico/farmacologia , Família 2 do Citocromo P450/metabolismo , Túbulos Renais Distais/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacologia , Amidas/farmacologia , Animais , Ácido Araquidônico/metabolismo , Família 2 do Citocromo P450/antagonistas & inibidores , Família 2 do Citocromo P450/genética , Inibidores Enzimáticos/farmacologia , Túbulos Renais Distais/metabolismo , Masculino , Potenciais da Membrana , Camundongos da Linhagem 129 , Camundongos Knockout , Bloqueadores dos Canais de Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo
13.
Am J Physiol Renal Physiol ; 318(5): F1067-F1073, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32200667

RESUMO

Protease-activated receptors (PARs) are coagulation protease targets, and they increase expression of inflammatory cytokines and chemokines in various diseases. Of all PARs, previous reports have shown that PAR1 or PAR2 inhibition is protective against diabetic glomerular injury. However, how PAR1 and PAR2 cooperatively contribute to diabetic kidney disease (DKD) pathogenesis and whether dual blockade of PARs is more effective in DKD remain elusive. To address this issue, male type I diabetic Akita mice heterozygous for endothelial nitric oxide synthase were used as a model of DKD. Mice (4 mo old) were divided into four treatment groups and administered vehicle, PAR1 antagonist (E5555, 60 mg·kg-1·day-1), PAR2 antagonist (FSLLRY, 3 mg·kg-1·day-1), or E5555 + FSLLRY for 4 wk. The results showed that the urinary albumin creatinine ratio was significantly reduced when both PAR1 and PAR2 were blocked with E5555 + FSLLRY compared with the vehicle-treated group. Dual blockade of PAR1 and PAR2 by E5555 + FSLLRY additively ameliorated histological injury, including mesangial expansion, glomerular macrophage infiltration, and collagen type IV deposition. Marked reduction of inflammation- and fibrosis-related gene expression in the kidney was also observed. In vitro, PAR1 and PAR2 agonists additively increased mRNA expression of macrophage chemoattractant protein 1 or plasminogen activator inhibitor-1 in human endothelial cells. Changes induced by the PAR1 agonist were blocked by a NF-κB inhibitor, whereas those of the PAR2 agonist were blocked by MAPK and/or NF-κB inhibitors. These findings suggest that PAR1 and PAR2 additively contribute to DKD pathogenesis and that dual blockade of both could be a novel therapeutic option for treatment of patients with DKD.


Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Iminas/farmacologia , Rim/efeitos dos fármacos , Oligopeptídeos/farmacologia , Piridinas/farmacologia , Receptor PAR-1/antagonistas & inibidores , Receptor PAR-2/antagonistas & inibidores , Albuminúria/genética , Albuminúria/metabolismo , Albuminúria/prevenção & controle , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo IV/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Modelos Animais de Doenças , Quimioterapia Combinada , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Fibrose , Humanos , Mediadores da Inflamação/metabolismo , Rim/metabolismo , Rim/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo III/deficiência , Óxido Nítrico Sintase Tipo III/genética , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Transdução de Sinais
14.
J Pharmacol Exp Ther ; 373(1): 81-91, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32024803

RESUMO

Oleoylethanolamide (OEA) is an endogenous peroxisome proliferator-activated receptor α (PPARα) agonist that acts on the peripheral control of energy metabolism. However, its therapeutic potential and related mechanisms in hepatic glucose metabolism under type 2 diabetes mellitus (T2DM) are not clear. Here, OEA treatment markedly improved glucose homeostasis in a PPARα-independent manner. OEA efficiently promoted glycogen synthesis and suppressed gluconeogenesis in mouse primary hepatocytes and liver tissue. OEA enhanced hepatic glycogen synthesis and inhibited gluconeogenesis via liver kinase B1 (LKB1)/5' AMP-activated protein kinase (AMPK) signaling pathways. PPARα was not involved in the roles of OEA in the LKB1/AMPK pathways. We found that OEA exerts its antidiabetic effect by increasing glycogenesis and decreasing gluconeogenesis via the LKB1/AMPK pathway. The ability of OEA to control hepatic LKB1/AMPK pathways may serve as a novel therapeutic approach for the treatment of T2DM. SIGNIFICANCE STATEMENT: Oleoylethanolamide (OEA) exerted a potent antihyperglycemic effect in a peroxisome proliferator-activated receptor α-independent manner. OEA played an antihyperglycemic role primarily via regulation of hepatic glycogen synthesis and gluconeogenesis. The main molecular mechanism of OEA in regulating liver glycometabolism is activating the liver kinase B1/5' AMP-activated protein kinase signaling pathways.


Assuntos
Diabetes Mellitus Tipo 2/sangue , Endocanabinoides/farmacologia , Gluconeogênese/fisiologia , Glicogênio/biossíntese , Fígado/metabolismo , Ácidos Oleicos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/induzido quimicamente , Diabetes Mellitus Tipo 2/tratamento farmacológico , Endocanabinoides/uso terapêutico , Gluconeogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Ácidos Oleicos/uso terapêutico , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
15.
mSphere ; 5(1)2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996415

RESUMO

Dissemination of antibiotic resistance (AR) genes, often on plasmids, leads to antibiotic-resistant bacterial infections, which is a major problem for animal and public health. Bacterial conjugation is the primary route of AR gene transfer in the mammalian gastrointestinal tract. Significant gaps in knowledge about which gastrointestinal communities and host factors promote plasmid transfer remain. Here, we used Salmonella enterica serovar Kentucky strain CVM29188 carrying plasmid pCVM29188_146 (harboring streptomycin and tetracycline resistance genes) to assess plasmid transfer to Escherichia coli under in vitro conditions and in various mouse strains with a conventional or defined microbiota. As an initial test, the transfer of pCVM29188_146 to the E. coli strains was confirmed in vitro Colonization resistance and, therefore, a lack of plasmid transfer were found in wild-type mice harboring a conventional microbiota. Thus, mice harboring the altered Schaedler flora (ASF), or ASF mice, were used to probe for host factors in the context of a defined microbiota. To assess the influence of inflammation on plasmid transfer, we compared interleukin-10 gene-deficient 129S6/SvEv ASF mice (proinflammatory environment) to wild-type 129S6/SvEv ASF mice and found no difference in transconjugant yields. In contrast, the mouse strain influenced plasmid transfer, as C3H/HeN ASF mice had significantly lower levels of transconjugants than 129S6/SvEv ASF mice. Although gastrointestinal members were identical between the ASF mouse strains, a few differences from C3H/HeN ASF mice were detected, with C3H/HeN ASF mice having significantly lower abundances of ASF members 356 (Clostridium sp.), 492 (Eubacterium plexicaudatum), and 502 (Clostridium sp.) than 129S6/SvEv ASF mice. Overall, we demonstrate that microbiota complexity and mouse genetic background influence in vivo plasmid transfer.IMPORTANCE Antibiotic resistance is a threat to public health. Many clinically relevant antibiotic resistance genes are carried on plasmids that can be transferred to other bacterial members in the gastrointestinal tract. The current study used a murine model to study the transfer of a large antibiotic resistance plasmid from a foodborne Salmonella strain to a gut commensal E. coli strain in the gastrointestinal tract. We found that different mouse genetic backgrounds and a different diversity of microbial communities influenced the level of Escherichia coli that acquired the plasmid in the gastrointestinal tract. This study suggests that the complexity of the microbial community and host genetics influence plasmid transfer from donor to recipient bacteria.


Assuntos
Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Microbioma Gastrointestinal , Plasmídeos/genética , Salmonella enterica/genética , Animais , Escherichia coli/efeitos dos fármacos , Feminino , Transferência Genética Horizontal , Intestinos/microbiologia , Masculino , Camundongos , Camundongos da Linhagem 129/genética , Camundongos Endogâmicos C3H/genética , Camundongos Knockout/genética , Salmonella enterica/efeitos dos fármacos
16.
Arterioscler Thromb Vasc Biol ; 40(3): 682-696, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31893950

RESUMO

OBJECTIVE: Activated perivascular mast cells (MCs) participate in different cardiovascular diseases. Many factors provoking MC degranulation have been described, while physiological counterregulators are barely known. Endothelial CNP (C-type natriuretic peptide) participates in the maintenance of vascular barrier integrity, but the target cells and mechanisms are unclear. Here, we studied whether MCs are regulated by CNP. Approach and Results: In cultured human and murine MCs, CNP activated its specific GC (guanylyl cyclase)-B receptor and cyclic GMP signaling. This enhanced cyclic GMP-dependent phosphorylation of the cytoskeleton-associated VASP (vasodilator-stimulated phosphoprotein) and inhibited ATP-evoked degranulation. To elucidate the relevance in vivo, mice with a floxed GC-B (Npr2) gene were interbred with a Mcpt5-CreTG line to generate mice lacking GC-B in connective tissue MCs (MC GC-B knockout). In anesthetized mice, acute ischemia-reperfusion of the cremaster muscle microcirculation provoked extensive MC degranulation and macromolecule extravasation. Superfusion of CNP markedly prevented MC activation and endothelial barrier disruption in control but not in MC GC-B knockout mice. Notably, already under resting conditions, such knockout mice had increased numbers of degranulated MCs in different tissues, together with elevated plasma chymase levels. After transient coronary occlusion, their myocardial areas at risk and with infarction were enlarged. Moreover, MC GC-B knockout mice showed augmented perivascular neutrophil infiltration and deep vein thrombosis in a model of inferior vena cava ligation. CONCLUSIONS: CNP, via GC-B/cyclic GMP signaling, stabilizes resident perivascular MCs at baseline and prevents their excessive activation under pathological conditions. Thereby CNP contributes to the maintenance of vascular integrity in physiology and disease.


Assuntos
Degranulação Celular , Células Endoteliais/metabolismo , Mastócitos/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Peptídeo Natriurético Tipo C/metabolismo , Comunicação Parácrina , Receptores do Fator Natriurético Atrial/metabolismo , Trombose/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Permeabilidade Capilar , Moléculas de Adesão Celular/metabolismo , Degranulação Celular/efeitos dos fármacos , Linhagem Celular , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Mastócitos/efeitos dos fármacos , Mastócitos/patologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos/metabolismo , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Peptídeo Natriurético Tipo C/farmacologia , Infiltração de Neutrófilos , Fosfoproteínas/metabolismo , Fosforilação , Receptores do Fator Natriurético Atrial/agonistas , Receptores do Fator Natriurético Atrial/genética , Transdução de Sinais , Trombose/genética , Trombose/patologia
17.
Neuron ; 105(3): 522-533.e4, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31806492

RESUMO

The timing and size of inhibition are crucial for dynamic excitation-inhibition balance and information processing in the neocortex. The underlying mechanism for temporal control of inhibition remains unclear. We performed dual whole-cell recordings from pyramidal cells (PCs) and nearby inhibitory interneurons in layer 5 of rodent neocortical slices. We found asynchronous release (AR) of glutamate occurs at PC output synapses onto Martinotti cells (MCs), causing desynchronized and prolonged firing in MCs and thus imprecise and long-lasting inhibition in neighboring PCs. AR is much stronger at PC-MC synapses as compared with those onto fast-spiking cells and other PCs, and it is also dependent on PC subtypes, with crossed-corticostriatal PCs producing the strongest AR. Moreover, knocking out synaptotagmin-7 substantially reduces AR strength and recurrent inhibition. Our results highlight the effect of glutamate AR on the operation of microcircuits mediating slow recurrent inhibition, an important mechanism for controlling the timing and size of cortical inhibition.


Assuntos
Ácido Glutâmico/metabolismo , Neocórtex/citologia , Neocórtex/metabolismo , Inibição Neural/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Knockout , Técnicas de Cultura de Órgãos , Ratos , Ratos Sprague-Dawley
18.
Scand J Med Sci Sports ; 30(2): 238-253, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31650583

RESUMO

Endurance training promotes exercise-induced adaptations in brain, like hippocampal adult neurogenesis and autophagy induction. However, resistance training effect on the autophagy response in the brain has not been much explored. Questions such as whether partial systemic autophagy or the length of training intervention affect this response deserve further attention. Therefore, 8-week-old male wild-type (Wt; n = 36) and systemic autophagy-deficient (atg4b-/- , KO; n = 36) mice were randomly distributed in three training groups, resistance (R), endurance (E), and control (non-trained), and in two training periods, 2 or 14 weeks. R and E maximal tests were evaluated before and after the training period. Forty-eight hours after the end of training program, cerebral cortex, striatum, hippocampus, and cerebellum were extracted for the analysis of autophagy proteins (LC3B-I, LC3B-II, and p62). Additionally, hippocampal adult neurogenesis was determined by doublecortin-positive cells count (DCX+) in brain sections. Our results show that, in contrast to Wt, KO were unable to improve R after both trainings. Autophagy levels in brain areas may be modified by E training only in cerebral cortex of Wt trained for 14 weeks, and in KO trained for 2 weeks. DCX + in Wt increased in R and E after both periods of training, with R for 14 weeks more effective than E. Interestingly, no changes in DCX + were observed in KO after 2 weeks, being even undetectable after 14 weeks of intervention. Thus, autophagy is crucial for R performance and for exercise-induced adult neurogenesis.


Assuntos
Autofagia , Córtex Cerebral/fisiologia , Neurogênese , Condicionamento Físico Animal , Adaptação Fisiológica , Animais , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Condicionamento Físico Animal/métodos , Proteína Sequestossoma-1/metabolismo
19.
Neuron ; 105(3): 464-474.e6, 2020 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-31812516

RESUMO

Many neuronal types occur as pairs that are similar in most respects but differ in a key feature. In some pairs of retinal neurons, called paramorphic, one member responds to increases and the other to decreases in luminance (ON and OFF responses). Here, we focused on one such pair, starburst amacrine cells (SACs), to explore how closely related neuronal types diversify. We find that ON and OFF SACs are transcriptionally distinct prior to their segregation, dendritic outgrowth, and synapse formation. The transcriptional repressor Fezf1 is selectively expressed by postmitotic ON SACs and promotes the ON fate and gene expression program while repressing the OFF fate and program. The atypical Rho GTPase Rnd3 is selectively expressed by OFF SACs and regulates their migration but is repressed by Fezf1 in ON SACs, enabling differential positioning of the two types. These results define a transcriptional program that controls diversification of a paramorphic pair.


Assuntos
Células Amácrinas/metabolismo , Interneurônios/metabolismo , Mitose/fisiologia , Proteínas Repressoras/biossíntese , Proteínas Repressoras/genética , Transcrição Genética/fisiologia , Células Amácrinas/química , Animais , Animais Recém-Nascidos , Feminino , Células HEK293 , Humanos , Interneurônios/química , Camundongos , Camundongos da Linhagem 129 , Camundongos Transgênicos , Gravidez , Proteínas Repressoras/análise
20.
Horm Behav ; 118: 104658, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31874139

RESUMO

The aim of the present study was to determine whether the TRPV1 channel is involved in the onset of sodium appetite. For this purpose, we used TRPV1-knockout mice to investigate sodium depletion-induced drinking at different times (2/24 h) after furosemide administration combined with a low sodium diet (FURO-LSD). In sodium depleted wild type and TRPV1 KO (SD-WT/SD-TPRV1-KO) mice, we also evaluated the participation of other sodium sensors, such as TPRV4, NaX and angiotensin AT1-receptors (by RT-PCR), as well as investigating the pattern of neural activation shown by Fos immunoreactivity, in different nuclei involved in hydromineral regulation. TPRV1 SD-KO mice revealed an increased sodium preference, ingesting a higher hypertonic cocktail in comparison with SD-WT mice. Our results also showed in SD-WT animals that SFO-Trpv4 expression increased 2 h after FURO-LSD, compared to other groups, thus supporting a role of SFO-Trpv4 channels during the hyponatremic state. However, the SD-TPRV1-KO animals did not show this early increase, and maybe as a consequence drank more hypertonic cocktail. Regarding the SFO-NaX channel expression, in both genotypes our findings revealed a reduction 24 h after FURO-LSD. In addition, there was an increase in the OVLT-NaX expression of SD-WT 24 h after FURO-LSD, suggesting the participation of OVLT-NaX channels in the appearance of sodium appetite, possibly as an anticipatory response in order to limit sodium intake and to induce thirst. Our work demonstrates changes in the expression of different osmo­sodium-sensitive channels at specific nuclei, related to the body sodium status in order to stimulate an adequate drinking.


Assuntos
Apetite/genética , Encéfalo/metabolismo , Dieta Hipossódica , Sódio na Dieta/administração & dosagem , Canais de Cátion TRPV/fisiologia , Animais , Apetite/efeitos dos fármacos , Dieta Hipossódica/efeitos adversos , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Líquidos/genética , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/genética , Furosemida/farmacologia , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sódio na Dieta/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Sede/efeitos dos fármacos , Sede/fisiologia
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