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1.
In Vivo ; 33(5): 1431-1437, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31471389

RESUMO

BACKGROUND/AIM: Transient receptor potential vanilloid 1 (TRPV1)-expressing sensory nerves innervate the pancreatic islets. Sensory neuropeptides, including calcitonin gene-related peptide (CGRP) and substance P (SP), participate in insulin secretion. This study aimed to investigate the role of TRPV1 in glucose-induced insulin secretion. MATERIALS AND METHODS: TRPV1-/- and wild-type (WT) mice were fed a normal diet for 24 weeks. Glucose tolerance and insulin secretion were measured at the end of the experiments. RESULTS: TRPV1-/- mice had greater impairments in glucose tolerance and higher decrease in glucose-induced insulin secretion than WT mice. Capsaicin (a TRPV1 agonist) increased insulin secretion in WT, but not in TRPV1-/- mice. Glucose-induced insulin secretion was blunted in TRPV1-/- mice, and was attenuated by AMG9810 (a TRPV1 inhibitor), CGRP8-37 (a CGRP receptor antagonist), or RP67580 (a NK-1 receptor antagonist) in WT mice. Glucose-induced SP and CGRP release from WT pancreas was higher than that from TRPV1-/- pancreas. CONCLUSION: TRPV1 mediates glucose-induced insulin secretion likely through CGRP and SP release.


Assuntos
Glucose/metabolismo , Secreção de Insulina , Neuropeptídeos/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Intolerância à Glucose , Masculino , Camundongos , Camundongos Knockout , Canais de Cátion TRPV/genética
2.
Int J Mol Sci ; 20(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394715

RESUMO

Muscular dystrophy and dilated cardiomyopathy are intractable diseases and their treatment options are very limited. Transient receptor potential cation channel subfamily V, member 2 (TRPV2), is a stretch-sensitive Ca2+-permeable channel that causes sustained intracellular Ca2+ increase in muscular cells, which is a pathophysiological feature of degenerative muscular disease. Recent reports have clarified that TRPV2 is concentrated and activated in the sarcolemma of cardiomyocytes/myocytes during cardiomyopathy/heart failure and muscular dystrophy. Furthermore, these reports showed that inactivation of TRPV2 ameliorates muscle dysgenesis to improve cardiac function and survival prognosis. Although TRPV2 is a potential therapeutic target for cardiomyopathy, there were no TRPV2 inhibitors available until recently. In this review, we introduce our recent findings and discuss the current progress in the development of TRPV2 inhibitors and their therapeutic applications for cardiomyopathy associated with muscular dystrophy.


Assuntos
Cardiomiopatia Dilatada/tratamento farmacológico , Cardiomiopatia Dilatada/etiologia , Terapia de Alvo Molecular , Distrofias Musculares/complicações , Distrofias Musculares/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Animais , Ensaios Clínicos como Assunto , Descoberta de Drogas , Expressão Gênica , Humanos , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofias Musculares/etiologia , Domínios e Motivos de Interação entre Proteínas , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
3.
Int J Mol Sci ; 20(16)2019 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426314

RESUMO

The vanilloid transient receptor potential channel TRPV3 is a putative molecular thermosensor widely considered to be involved in cutaneous sensation, skin homeostasis, nociception, and pruritus. Repeated stimulation of TRPV3 by high temperatures above 50 °C progressively increases its responses and shifts the activation threshold to physiological temperatures. This use-dependence does not occur in the related heat-sensitive TRPV1 channel in which responses decrease, and the activation threshold is retained above 40 °C during activations. By combining structure-based mutagenesis, electrophysiology, and molecular modeling, we showed that chimeric replacement of the residues from the TRPV3 cytoplasmic inter-subunit interface (N251-E257) with the homologous residues of TRPV1 resulted in channels that, similarly to TRPV1, exhibited a lowered thermal threshold, were sensitized, and failed to close completely after intense stimulation. Crosslinking of this interface by the engineered disulfide bridge between substituted cysteines F259C and V385C (or, to a lesser extent, Y382C) locked the channel in an open state. On the other hand, mutation of a single residue within this region (E736) resulted in heat resistant channels. We propose that alterations in the cytoplasmic inter-subunit interface produce shifts in the channel gating equilibrium and that this domain is critical for the use-dependence of the heat sensitivity of TRPV3.


Assuntos
Citoplasma/metabolismo , Canais de Cátion TRPV/metabolismo , Células HEK293 , Temperatura Alta , Humanos , Simulação de Dinâmica Molecular , Mutação , Domínios Proteicos , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Canais de Cátion TRPV/química , Canais de Cátion TRPV/genética
4.
Int J Mol Sci ; 20(14)2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31295806

RESUMO

The increase in cytosolic Ca2+ is essential in key effector functions of dendritic cells (DCs), including differentiation, maturation, cytokine expression, and phagocytosis. Although several Ca2+-permeable ion channels have been described in DCs, the contribution of transient receptor potential (TRP) channels remains poorly understood. Here, we investigated whether TRPV4 plays a role in the differentiation, maturation, and phagocytosis of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced mouse bone marrow-derived cells (BMDCs). Using intracellular Ca2+ imaging experiments, we found that TRPV4 was functionally expressed in the plasma membrane of immature CD11c+ BMDCs and that its activity and expression were downregulated in CD11c+ BMDCs matured with lipopolysaccharide (LPS). Comparative analysis of the GM-CSF-stimulated cells showed that Trpv4 knockout and wild-type bone marrow cultures had a similar distribution of differentiated cells, generating a heterogenous culture population rich in CD11c+, CD11b+ cells, and low levels of F4/80+ cells. The lack of TRPV4 did not prevent the LPS-induced nuclear translocation of NF-κB, the upregulation of the proinflammatory cytokines IL-6 and IL-12, or the upregulation of the maturation markers CD40, CD80, and CD86. In contrast, TRPV4-deficient CD11c+ BMDCs exhibited a significantly reduced endocytic capacity of IgG-coated beads, but the internalization of uncoated beads in the absence of TRPV4 was not affected. Taken together, our results demonstrate that TRPV4 was dispensable in the differentiation and maturation of mouse CD11c+ BMDCs but contributed to the mechanism underlying Fc receptor-mediated phagocytosis. Overall, our results further strengthen the role of TRPV4 in immune-related processes.


Assuntos
Células da Medula Óssea/metabolismo , Antígeno CD11c/metabolismo , Expressão Gênica , Canais de Cátion TRPV/genética , Animais , Biomarcadores , Células da Medula Óssea/citologia , Cálcio/metabolismo , Sinalização do Cálcio , Diferenciação Celular , Células Cultivadas , Humanos , Imuno-Histoquímica , Imunofenotipagem , Camundongos , Camundongos Knockout , Imagem Molecular , Fagocitose , Fenótipo , Transporte Proteico , Canais de Cátion TRPV/metabolismo
5.
PLoS Pathog ; 15(6): e1007746, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31194856

RESUMO

Toxoplasma gondii is a prevalent protozoan parasite that can infect any nucleated cell but cannot replicate outside of its host cell. Toxoplasma is auxotrophic for several nutrients including arginine, tryptophan, and purines, which it must acquire from its host cell. The demands of parasite replication rapidly deplete the host cell of these essential nutrients, yet Toxoplasma successfully manages to proliferate until it lyses the host cell. In eukaryotic cells, nutrient starvation can induce the integrated stress response (ISR) through phosphorylation of an essential translation factor eIF2. Phosphorylation of eIF2 lowers global protein synthesis coincident with preferential translation of gene transcripts involved in stress adaptation, such as that encoding the transcription factor ATF4 (CREB2), which activates genes that modulate amino acid metabolism and uptake. Here, we discovered that the ISR is induced in host cells infected with Toxoplasma. Our results show that as Toxoplasma depletes host cell arginine, the host cell phosphorylates eIF2 via protein kinase GCN2 (EIF2AK4), leading to induced ATF4. Increased ATF4 then enhances expression of the cationic amino acid transporter CAT1 (SLC7A1), resulting in increased uptake of arginine in Toxoplasma-infected cells. Deletion of host GCN2, or its downstream effectors ATF4 and CAT1, lowers arginine levels in the host, impairing proliferation of the parasite. Our findings establish that Toxoplasma usurps the host cell ISR to help secure nutrients that it needs for parasite replication.


Assuntos
Arginina/metabolismo , Fator de Iniciação 2 em Eucariotos/metabolismo , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/metabolismo , Toxoplasma/crescimento & desenvolvimento , Toxoplasmose/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Animais , Arginina/genética , Transporte Biológico Ativo/genética , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Fator de Iniciação 2 em Eucariotos/genética , Camundongos , Camundongos Knockout , Proteínas Serina-Treonina Quinases/genética , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Toxoplasmose/genética , Toxoplasmose/patologia
6.
J Physiol Pharmacol ; 70(1)2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31172971

RESUMO

Dexamethasone inhibits mucin secretion considering the primary option for treating acute asthma exacerbation. However, the mechanism underlying dexamethasone-induced decreased in mucosecretion is unclear. Recent studies have reported that dexamethasone exerts an inhibitory effect on mucosecretion in the lung by modulating the expression of calcium processing genes. However, the expression of the calcium processing genes in the trachea is not examined yet. Thus, the present study is the first to report the localization of calcium processing proteins such as transient receptor potential vanilloid-4 (Trpv4), transient receptor potential vanilloid-6 (Trpv6), calbindin-D9k (CaBP-9k) and plasma membrane Ca2+-ATPase 1 (Pmca1) in the mouse trachea and their glucocorticoid-induced response. In this study, mice were subcutaneously injected with dexamethasone for 5 days, and their tracheal samples were collected by dividing the trachea into the cervical, and thoracic sections based on its anatomical structure. The localization of TRPV4, TRPV6, CaBP-9k, and PMCA1 proteins was detected in the tracheal epithelium, submucosal glands, cartilages and muscles. Dexamethasone treatment downregulated the mRNA expression of the four calcium processing genes and mucin producing genes. The dexamethasone-induced decrease in the secretion of mucosubstances in the trachea was determined by performing Alcian blue-periodic acid-Schiff staining. Thus, the findings of the present study suggest that glucocorticoids simultaneously can regulate the expression of calcium processing genes and tracheal mucosecretion.


Assuntos
Dexametasona/farmacologia , Glucocorticoides/farmacologia , Mucosa Respiratória/efeitos dos fármacos , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Mucosa Respiratória/metabolismo , Proteína G de Ligação ao Cálcio S100/genética , Proteína G de Ligação ao Cálcio S100/metabolismo , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Traqueia/efeitos dos fármacos , Traqueia/metabolismo
7.
Biomed Res Int ; 2019: 6712536, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31183372

RESUMO

Background/Aims: TRPV1 is a nonselective Ca2+ channel which has recently been observed in many cancers, while its effect on cell proliferation, apoptosis, metabolism, and cancer development in colorectal cancer (CRC) is still unclear. In this study, we hypothesized that TRPV1 is a tumor suppressor in CRC development as well as the underlying mechanism. Methods: Immunohistochemistry assay was applied to detect the expression of TRPV1 protein in CRC tissues. HCT116 cell proliferation and apoptosis were measured by CCK-8 and flow cytometry, respectively. Cellular Ca2+ concentration was measured by Fluo-4/AM-based flow cytometer. Apoptosis-related proteins were measured by Western blotting. Results: In this study, we found that TRPV1 expression was significantly decreased in CRC tissues, compared with CRC-adjacent tissues and normal tissues, respectively. Then, we found that the TRVP1 agonist capsaicin treatment inhibited CRC growth and induced apoptosis by activating P53. Subsequent mechanistic study revealed that the TRPV1 induced cytosolic Ca2+ influx to regulate cell apoptosis and p53 activation through calcineurin. Conclusions: This study suggests that TRPV1 served as a tumor suppressor in CRC and contributed to the development of novel therapy of CRC.


Assuntos
Apoptose , Calcineurina/metabolismo , Sinalização do Cálcio , Neoplasias Colorretais/metabolismo , Fatores de Transcrição NFATC/metabolismo , Canais de Cátion TRPV/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Calcineurina/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Células HCT116 , Humanos , Fatores de Transcrição NFATC/genética , Canais de Cátion TRPV/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética
8.
Life Sci ; 228: 158-166, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31055086

RESUMO

AIMS: Chondrocyte apoptosis is the most common pathological feature of cartilage in osteoarthritis (OA). Excessive mechanical stress can induce chondrocyte apoptosis and destroy cartilage tissue. Transient receptor potential channel vanilloid 4 (TRPV4) is a mechanosensitive ion channel that mediates chondrocyte response to mechanical stress. Here, we investigated the potential role of TRPV4 in chondrocyte apoptosis induced by excessive mechanical stress. MAIN METHODS: Using a rat OA anterior cruciate-ligament transection (ALCT) model, we detected immunolocalization of calmodulin protein and mRNA and protein levels of TRPV4, calmodulin, and cleaved caspase-8 in articular cartilage. Primary chondrocytes were isolated and cultured in vitro, and Fluo-4AM staining was used to assess intracellular Ca2+ levels in order to evaluate TRPV4-mediated Ca2+ influx. Flow cytometry and western blot were performed to detect apoptosis and apoptosis-related protein levels in chondrocytes, respectively. KEY FINDINGS: TRPV4 was upregulated in ALCT-induced OA articular cartilage, and we found that administration of a TRPV4 inhibitor attenuated cartilage degeneration. Additionally, TRPV4 specifically mediated extracellular Ca2+ influx, leading to chondrocyte apoptosis in vitro, which was inhibited by transfection of TRPV4 small-interfering RNA or administration of a TRPV4 inhibitor. Moreover, increased Ca2+ influx triggered apoptosis by upregulating FAS-associated protein with death domain and cleaved caspase-3, -6, -7, and -8 levels, with these effects abolished by TRPV4 knockdown or TRPV4 inhibition. SIGNIFICANCE: These results indicated that TRPV4 was upregulated in OA articular cartilage, and that excessive mechanical stress might induce chondrocyte apoptosis via TRPV4-mediated Ca2+ influx, suggesting TRPV4 as a potential drug target in OA.


Assuntos
Ligamento Cruzado Anterior/patologia , Apoptose , Condrócitos/patologia , Osteoartrite/patologia , Estresse Mecânico , Canais de Cátion TRPV/metabolismo , Animais , Ligamento Cruzado Anterior/metabolismo , Células Cultivadas , Condrócitos/metabolismo , Modelos Animais de Doenças , Masculino , Osteoartrite/genética , Osteoartrite/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Canais de Cátion TRPV/análise , Canais de Cátion TRPV/genética , Regulação para Cima
9.
Mol Med Rep ; 19(6): 5386-5396, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059059

RESUMO

Stress is a pivotal factor for inflammation, reactive oxygen species (ROS) production and formation of visceral hypersensitivity (VH) in the process of gastroesophageal reflux disease (GERD). In the present study, the effects of stress on esophageal inflammation, oxidative stress and VH were investigated in a chronic restraint stress mouse model. C57BL/6J male mice were subjected to 2 weeks of intermittent restraint stress, and histopathological analysis revealed that stress induced esophageal inflammation and fibrosis, while no distinct changes were detected in non­stressed control mice. In addition, increased NADPH oxidase 4 expression was observed in the plasma and esophagus of stressed mice, indicating accumulation of ROS. The expression levels of antioxidants, including Mn­superoxide dismutase (MnSOD), Cu/Zn­SOD, catalase and glutathione peroxidase, were also analyzed using reverse transcription­quantitative polymerase chain reaction (RT­qPCR). In addition, transient receptor potential vanilloid 1 (TRPV­1) and protease­activated receptor 2 (PAR­2), which are crucial receptors for VH, were measured by immunohistochemistry and RT­qPCR. The results demonstrated that stress markedly reduced antioxidant expression, while it significantly upregulated TRPV­1 and PAR­2 expression levels in the mouse esophagus. Finally, 2 weeks of restraint stress significantly increased the esophageal and plasma levels of inflammatory cytokines, including interleukin (IL)­6, IL­8, interferon­Î³ and tumor necrosis factor­α. Taken together, the present study results indicated that stress­induced esophageal inflammation and ROS generation involves VH.


Assuntos
Esôfago/patologia , Inflamação , Receptor PAR-2/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Catalase/genética , Catalase/metabolismo , Citocinas/sangue , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Esôfago/citologia , Esôfago/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 4/genética , NADPH Oxidase 4/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptor PAR-2/genética , Estresse Fisiológico , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Regulação para Cima
10.
Oncol Rep ; 42(1): 243-252, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31059092

RESUMO

Iodine­125 (125I) seed radiation applied around the celiac ganglion can relieve the refractory pain in pancreatic cancer. In an in vitro cell radiation model of human neuroblastoma cell lines, the impact of 125I radiation on the expression of transient receptor potential vanilloid­1 (TRPV1) was investigated. The results indicated that the radiation delivering doses <2.13 Gy did not significantly affect cell growth, whereas the doses >3.12 Gy significantly reduced cell viability. The reduced TRPV1 mRNA level was dependent on the doses, while the reduced protein level occurred at lower doses (2.63 and 4.27 Gy), then returned to normal at an intermediate dose of 5.09 Gy, and decreased again at higher doses (5.91 and 6.73 Gy). The miRNA profiling at the dose of 2.63 Gy revealed 32 and 22 miRNAs that were significantly upregulated and downregulated, respectively. In addition, the upregulated miR­1246 target, regulated the expression of TRPV1, indicating that miR­1246 may be a new therapeutic target for pancreatic pain.


Assuntos
Regulação para Baixo , Radioisótopos do Iodo/farmacologia , MicroRNAs/genética , Neuroblastoma/genética , Canais de Cátion TRPV/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta à Radiação , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Neuroblastoma/metabolismo , Neuroblastoma/radioterapia , Análise de Sequência com Séries de Oligonucleotídeos , Canais de Cátion TRPV/metabolismo
11.
Gastroenterology ; 157(2): 522-536.e2, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31075226

RESUMO

BACKGROUND & AIMS: Proper colon function requires signals from extrinsic primary afferent neurons (ExPANs) located in spinal ganglia. Most ExPANs express the vanilloid receptor TRPV1, and a dense plexus of TRPV1-positive fibers is found around myenteric neurons. Capsaicin, a TRPV1 agonist, can initiate activity in myenteric neurons and produce muscle contraction. ExPANs might therefore form motility-regulating synapses onto myenteric neurons. ExPANs mediate visceral pain, and myenteric neurons mediate colon motility, so we investigated communication between ExPANs and myenteric neurons and the circuits by which ExPANs modulate colon function. METHODS: In live mice and colon tissues that express a transgene encoding the calcium indicator GCaMP, we visualized levels of activity in myenteric neurons during smooth muscle contractions induced by application of capsaicin, direct colon stimulation, stimulation of ExPANs, or stimulation of preganglionic parasympathetic neuron (PPN) axons. To localize central targets of ExPANs, we optogenetically activated TRPV1-expressing ExPANs in live mice and then quantified Fos immunoreactivity to identify activated spinal neurons. RESULTS: Focal electrical stimulation of mouse colon produced phased-locked calcium signals in myenteric neurons and produced colon contractions. Stimulation of the L6 ventral root, which contains PPN axons, also produced myenteric activation and contractions that were comparable to those of direct colon stimulation. Surprisingly, capsaicin application to the isolated L6 dorsal root ganglia, which produced robust calcium signals in neurons throughout the ganglion, did not activate myenteric neurons. Electrical activation of the ganglia, which activated even more neurons than capsaicin, did not produce myenteric activation or contractions unless the spinal cord was intact, indicating that a complete afferent-to-efferent (PPN) circuit was necessary for ExPANs to regulate myenteric neurons. In TRPV1-channel rhodopsin-2 mice, light activation of ExPANs induced a pain-like visceromotor response and expression of Fos in spinal PPN neurons. CONCLUSIONS: In mice, ExPANs regulate myenteric neuron activity and smooth muscle contraction via a parasympathetic spinal circuit, linking sensation and pain to motility.


Assuntos
Colo/fisiopatologia , Neurônios Aferentes/fisiologia , Peristaltismo/fisiologia , Dor Visceral/fisiopatologia , Animais , Técnicas Biossensoriais/métodos , Capsaicina/administração & dosagem , Colo/efeitos dos fármacos , Colo/inervação , Modelos Animais de Doenças , Feminino , Gânglios Espinais/citologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Músculo Liso/inervação , Músculo Liso/fisiopatologia , Plexo Mientérico/citologia , Plexo Mientérico/efeitos dos fármacos , Neurônios Aferentes/efeitos dos fármacos , Optogenética , Peristaltismo/efeitos dos fármacos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Dor Visceral/induzido quimicamente
12.
Nat Commun ; 10(1): 2134, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086183

RESUMO

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a prototypical molecular sensor for noxious heat in mammals. Its role in sustained heat response remains poorly understood, because rapid heat-induced desensitization (Dh) follows tightly heat-induced activation (Ah). To understand the physiological role and structural basis of Dh, we carried out a comparative study of TRPV1 channels in mouse (mV1) and those in platypus (pV1), which naturally lacks Dh. Here we show that a temperature-sensitive interaction between the N- and C-terminal domains of mV1 but not pV1 drives a conformational rearrangement in the pore leading to Dh. We further show that knock-in mice expressing pV1 sensed heat normally but suffered scald damages in a hot environment. Our findings suggest that Dh evolved late during evolution as a protective mechanism and a delicate balance between Ah and Dh is crucial for mammals to sense and respond to noxious heat.


Assuntos
Aclimatação/fisiologia , Temperatura Alta/efeitos adversos , Domínios Proteicos/fisiologia , Canais de Cátion TRPV/metabolismo , Animais , Evolução Biológica , Temperatura Corporal/fisiologia , Dermatite , Feminino , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Filogenia , Ornitorrinco/fisiologia , Canais de Cátion TRPV/genética
13.
J Neuroinflammation ; 16(1): 114, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31142341

RESUMO

BACKGROUND: Hypoxic-ischemic encephalopathy (HIE) is a serious birth complication with high incidence in both advanced and developing countries. Children surviving from HIE often have severe long-term sequela including cerebral palsy, epilepsy, and cognitive disabilities. The severity of HIE in infants is tightly associated with increased IL-1ß expression and astrocyte activation which was regulated by transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel in the TRP family. METHODS: Neonatal hypoxic ischemia (HI) and oxygen-glucose deprivation (OGD) were used to simulate HIE in vivo and in vitro. Primarily cultured astrocytes were used for investigating the expression of glial fibrillary acidic protein (GFAP), IL-1ß, Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), and activation of the nucleotide-binding, oligomerization domain (NOD)-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome by using Western blot, q-PCR, and immunofluorescence. Brain atrophy, infarct size, and neurobehavioral disorders were evaluated by Nissl staining, 2,3,5-triphenyltetrazolium chloride monohydrate (TTC) staining and neurobehavioral tests (geotaxis reflex, cliff aversion reaction, and grip test) individually. RESULTS: Astrocytes were overactivated after neonatal HI and OGD challenge. The number of activated astrocytes, the expression level of IL-1ß, brain atrophy, and shrinking infarct size were all downregulated in TRPV1 KO mice. TRPV1 deficiency in astrocytes attenuated the expression of GFAP and IL-1ß by reducing phosphorylation of JAK2 and STAT3. Meanwhile, IL-1ß release was significantly reduced in TRPV1 deficiency astrocytes by inhibiting activation of NLRP3 inflammasome. Additionally, neonatal HI-induced neurobehavioral disorders were significantly improved in the TRPV1 KO mice. CONCLUSIONS: TRPV1 promotes activation of astrocytes and release of astrocyte-derived IL-1ß mainly via JAK2-STAT3 signaling and activation of the NLRP3 inflammasome. Our findings provide mechanistic insights into TRPV1-mediated brain damage and neurobehavioral disorders caused by neonatal HI and potentially identify astrocytic TRPV1 as a novel therapeutic target for treating HIE in the subacute stages (24 h).


Assuntos
Astrócitos/metabolismo , Encéfalo/metabolismo , Hipóxia-Isquemia Encefálica/metabolismo , Interleucina-1beta/metabolismo , Canais de Cátion TRPV/deficiência , Animais , Astrócitos/patologia , Encéfalo/patologia , Células Cultivadas , Feminino , Hipóxia-Isquemia Encefálica/genética , Hipóxia-Isquemia Encefálica/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canais de Cátion TRPV/genética
14.
Cutan Ocul Toxicol ; 38(3): 212-220, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30935251

RESUMO

Background: Poor ocular tolerance of sunscreens is partially responsible for poor compliance in use of sunscreens. A three-tiered approach for the testing of ocular tolerance for such products is described that includes an in vitro test for ocular irritation, an in vitro test for the activation of pain receptors, and finally a clinical study involving ocular instillation of the product under controlled conditions followed by ophthalmologic and subjective self-evaluation on a graded scale. We report the results for a new water-based facial sunscreen (SCFW) with very good ocular tolerance. Methods: The ocular irritation potential of SCFW was determined using the EpiOcular™ human cell construct which constituted the first-tier testing. Briefly, the tissues were exposed to SCFW and appropriate positive and negative controls for 15 minutes to 24 hours. After treatment, the tissues were rinsed and cytotoxicity determined. The calculated ET50 value (time at which relative viability decreased 50%) was then used to determine the ocular irritation potential. In the second-tier testing, the sting potential of SCFW was determined by employing the NociOcular assay that measures the activation of TRPV1 (transient receptor potential cation channel subfamily V member 1) specific receptors linked to pain sensation in a neuronal model with over-expression of functional TRPV1 channels. Finally, as the third-tier testing, SCFW was tested in a clinical study with instillation of product into the ocular cul-de-sac and ocular irritation was evaluated after 30 seconds, 15 minutes, and 60 minutes by an ophthalmologist. Participating subjects were also asked to score sensation on a scale of 0 to 3 from slight prickliness to severe stinging. Assay control reference product with known good ocular tolerability (10% baby shampoo) was concurrently tested. Results: In the in vitro topical application assay using the EpiOcular™ construct, no significant cytotoxicity was observed in the tissues exposed to SCFW, indicating minimal ocular irritation potential. In the in vitro NociOcular assay, the cells exposed to the prepared dilutions of SCFW showed minimal TRPV1 specific activity, indicating minimal ocular sting potential. In the in vivo study, no statistically significant differences were found in terms of subjective or objective eye irritation assessment between SCFW and 10% baby shampoo. Conclusion: SCFW showed negligible ocular irritation potential in tier 1, minimal potential to activate pain receptors in tier 2, and good ocular tolerability that was comparable to 10% baby shampoo in tier 3 testing. The results suggest that SCFW has good eye tolerance and that the tiered approach can be used to evaluate facial sunscreens for ocular tolerability.


Assuntos
Olho/efeitos dos fármacos , Protetores Solares/toxicidade , Idoso , Linhagem Celular Tumoral , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Testes de Toxicidade/métodos
15.
Int J Mol Sci ; 20(8)2019 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-31003519

RESUMO

Bone is a dynamic tissue, whose homeostasis is maintained by a fine balance between osteoclast (OC) and osteoblast (OB) activity. The endocannabinoid/endovanilloid (EC/EV) system's receptors are the cannabinoid receptor type 1 (CB1), the cannabinoid receptor type 2 (CB2), and the transient receptor potential cation channel subfamily V member 1 (TRPV1). Their stimulation modulates bone formation and bone resorption. Bone diseases are very common worldwide. Osteoporosis is the principal cause of bone loss and it can be caused by several factors such as postmenopausal estrogen decrease, glucocorticoid (GC) treatments, iron overload, and chemotherapies. Studies have demonstrated that CB1 and TRPV1 stimulation exerts osteoclastogenic effects, whereas CB2 stimulation has an anti-osteoclastogenic role. Moreover, the EC/EV system has been demonstrated to have a role in cancer, favoring apoptosis and inhibiting cell proliferation. In particular, in bone cancer, the modulation of the EC/EV system not only reduces cell growth and enhances apoptosis but it also reduces cell invasion and bone pain in mouse models. Therefore, EC/EV receptors may be a useful pharmacological target in the prevention and treatment of bone diseases. More studies to better investigate the biochemical mechanisms underlining the EC/EV system effects in bone are needed, but the synthesis of hybrid molecules, targeting these receptors and capable of oppositely regulating bone homeostasis, seems to be a promising and encouraging prospective in bone disease management.


Assuntos
Endocanabinoides/genética , Osteogênese/genética , Osteoporose/genética , Osteossarcoma/genética , Apoptose/genética , Reabsorção Óssea/genética , Reabsorção Óssea/patologia , Proliferação de Células/genética , Endocanabinoides/metabolismo , Glucocorticoides/genética , Humanos , Osteoporose/patologia , Osteossarcoma/patologia , Receptor CB1 de Canabinoide/genética , Receptor CB2 de Canabinoide/genética , Canais de Cátion TRPV/genética
16.
J Headache Pain ; 20(1): 39, 2019 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-31014225

RESUMO

BACKGROUND: Some variables have been proposed as predictors of efficacy of OnabotulinumtoxinA in chronic migraine patients, but data available are inconclusive. We aimed to analyse the influence of single nucleotide polymorphisms in the response to OnabotulinumtoxinA. METHODS: We included 156 female patients treated with OnabotulinumtoxinA accordingly to PREEMPT paradigm in three headache units. OnabotulinumtoxinA was offered to patients that had not responded to topiramate and at least one other preventative. Age at first procedure was 43.7 ± 11.8 years (16-74). Patients with a reduction of at least 50% in the number of migraine days after two OnabotulinumtoxinA procedures were considered as responders. We analysed 25 polymorphisms selected for their relevance regarding migraine pathophysiology and their association with migraine according to previously published genome-wide association studies. Genotyping was performed using KASP probes and a LightCycler-480 (Roche-Diagnostics). Allelic, genotypic frequencies and dominance/recesivity hypothesis of the allelic variants were compared between responders and non-responders by Fisher's exact test. RESULTS: Response to treatment with OnabotulinumtoxinA was achieved in 120 patients (76,9%). Two polymorphisms showed differences: CALCA rs3781719, where allele C represents 26.9% in responders and 40.9% in non-responders (p = 0.007, OR = 3.11 (1.33-7.26)); and TRPV1 rs222749, where allele A represents 4.17% in responders and 12.5% in non-responders (p = 0.013, OR = 3.29 (1.28-8.43)). No significant differences in rest of polymorphisms or clinical or demographic variables were found. CONCLUSIONS: Polymorphic variations of CALCA and TRPV1 genes might play a role as prognostic markers of efficacy of OnabotulinumtoxinA in chronic migraine female patients in our population.


Assuntos
Toxinas Botulínicas Tipo A/uso terapêutico , Peptídeo Relacionado com Gene de Calcitonina/genética , Transtornos de Enxaqueca/tratamento farmacológico , Transtornos de Enxaqueca/genética , Polimorfismo de Nucleotídeo Único/genética , Canais de Cátion TRPV/genética , Adolescente , Adulto , Idoso , Doença Crônica , Feminino , Frequência do Gene , Estudo de Associação Genômica Ampla/métodos , Humanos , Pessoa de Meia-Idade , Fármacos Neuromusculares/uso terapêutico , Estudos Prospectivos , Topiramato/uso terapêutico , Resultado do Tratamento , Adulto Jovem
17.
Methods Mol Biol ; 1987: 23-37, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028671

RESUMO

Transient receptor potential (TRP) channels are polymodal sensory transducers that respond to chemicals, temperature, mechanical stress, and membrane voltage and are involved in vision, taste, olfaction, hearing, touch, thermal perception, and nociception. TRP channels are implicated in numerous devastating diseases, including various forms of cancer, and represent important drug targets. The large sizes, low expression levels, and conformational dynamics of TRP channels make them challenging targets for structural biology. Here, we present the methodology used in structural studies of TRPV6, a TRP channel that is highly selective for calcium and mediates Ca2+ uptake in epithelial tissues. We provide a protocol for the expression, purification, and crystallization of TRPV6. Similar approaches can be used to determine crystal structures of other membrane proteins, including different members of the TRP channel family.


Assuntos
Cristalografia por Raios X/métodos , Canais de Cátion TRPV/isolamento & purificação , Animais , Baculoviridae , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células HEK293 , Humanos , Células Sf9 , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo
18.
Methods Mol Biol ; 1987: 223-231, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028683

RESUMO

Ion channels participate in several biological processes. Among these channels, the ionotropic TRP family is the most prominent group being TRPV1 the most studied. The activation of these channels can elicit pain sensation; thus, the development of blockers for these channels is receiving increasing attention. TRP channels are the responsible for thermonociception but also, they are involved in osmolarity, taste, and chemical substances perception such as capsaicin or menthol which can evoke pain. The needed of testing new compounds implies the use of animal models of pain and nociceptive tests in order to evaluate their potential efficacy for the treatment of painful symptoms. Several methods have been developed. Here, I describe the standard, current, and available tests to explore nociception in rodents, especially when thermal or mechanical stimuli are applied.


Assuntos
Nociceptividade , Canais de Cátion TRPV/fisiologia , Animais , Hiperalgesia/etiologia , Hiperalgesia/genética , Camundongos , Camundongos Knockout , Dor/etiologia , Dor/genética , Ratos , Canais de Cátion TRPV/genética
19.
Molecules ; 24(7)2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30979007

RESUMO

Endocannabinoid (eCB)-binding receptors can be modulated by several ligands and membrane environment, yet the effect of glycosylation remains to be assessed. In this study, we used human neuroblastoma SH-SY5Y cells to interrogate whether expression, cellular localization, and activity of eCB-binding receptors may depend on N-linked glycosylation. Following treatment with tunicamycin (a specific inhibitor of N-linked glycosylation) at the non-cytotoxic dose of 1 µg/mL, mRNA, protein levels and localization of eCB-binding receptors, as well as N-acetylglucosamine (GlcNAc) residues, were evaluated in SH-SY5Y cells by means of quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR), fluorescence-activated cell sorting (FACS), and confocal microscopy, respectively. In addition, the activity of type-1 and type-2 cannabinoid receptors (CB1 and CB2) was assessed by means of rapid binding assays. Significant changes in gene and protein expression were found upon tunicamycin treatment for CB1 and CB2, as well as for GPR55 receptors, but not for transient receptor potential vanilloid 1 (TRPV1). Deglycosylation experiments with N-glycosidase-F and immunoblot of cell membranes derived from SH-SY5Y cells confirmed the presence of one glycosylated form in CB1 (70 kDa), that was reduced by tunicamycin. Morphological studies demonstrated the co-localization of CB1 with GlcNAc residues, and showed that tunicamycin reduced CB1 membrane expression with a marked nuclear localization, as confirmed by immunoblotting. Cleavage of the carbohydrate side chain did not modify CB receptor binding affinity. Overall, these results support N-linked glycosylation as an unprecedented post-translational modification that may modulate eCB-binding receptors' expression and localization, in particular for CB1.


Assuntos
Endocanabinoides/genética , Neuroblastoma/tratamento farmacológico , Receptores de Canabinoides/química , Tunicamicina/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Endocanabinoides/química , Endocanabinoides/farmacologia , Citometria de Fluxo , Glicosilação/efeitos dos fármacos , Humanos , Ligantes , Microscopia Confocal , Neuroblastoma/genética , Neuroblastoma/patologia , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Receptores de Canabinoides/genética , Canais de Cátion TRPV/genética , Tunicamicina/química
20.
Biotech Histochem ; 94(4): 244-251, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30916584

RESUMO

We investigated the dynamic expression of calcium transporters, TRPV5 and TRPV6, in placenta and bone to determine their role in maternal and fetal calcium balance during gestation. In placenta, TRPV5 was expressed predominantly in syncytiotrophoblasts of the labyrinthine zone, whereas TRPV6 was expressed in spongiotrophoblasts of the junction zone. In bone, the two transporters were found in osteoblasts, osteoclasts, cartilage and bone matrices. During the first half of gestation, TRPV5 and TRPV6 levels in bone were increased on pregnancy day (P) 0.5, then decreased on P3.5 followed by a slight increase on P6.5. During the second half of pregnancy, both the proteins and their mRNAs gradually increased from P9.5 to P15.5-P17.5 in both bone and placenta, followed at parturition by relatively high amounts in placenta, but markedly decreased amounts in bone. The expression pattern is likely related to the fetal and maternal calcium requirement during gestation, which may be regulated by estrogen and other hormones, because the fetal demand for calcium is greatest during the last few days of gestation for rats; maternal calcium metabolism is designed to meet the calcium needs of the fetus during this period. We found that TRPV5 and TRPV6 are involved in calcium transport in the placenta and bone, and therefore play a role in calcium homeostasis during embryonic and fetal development.


Assuntos
Osso e Ossos/metabolismo , Canais de Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Placenta/metabolismo , Prenhez , Canais de Cátion TRPV/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/genética , Feminino , Camundongos , Gravidez , Prenhez/fisiologia , Canais de Cátion TRPV/genética
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