Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.362
Filtrar
1.
Nat Commun ; 10(1): 4496, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31582750

RESUMO

Solitary chemosensory cells (SCCs) are epithelial sentinels that utilize bitter Tas2r receptors and coupled taste transduction elements to detect pathogenic bacterial metabolites, triggering host defenses to control the infection. Here we report that SCCs are present in mouse gingival junctional epithelium, where they express several Tas2rs and the taste signaling components α-gustducin (Gnat3), TrpM5, and Plcß2. Gnat3-/- mice have altered commensal oral microbiota and accelerated naturally occurring alveolar bone loss. In ligature-induced periodontitis, knockout of taste signaling molecules or genetic absence of gingival SCCs (gSCCs) increases the bacterial load, reduces bacterial diversity, and renders the microbiota more pathogenic, leading to greater alveolar bone loss. Topical treatment with bitter denatonium to activate gSCCs upregulates the expression of antimicrobial peptides and ameliorates ligature-induced periodontitis in wild-type but not in Gnat3-/- mice. We conclude that gSCCs may provide a promising target for treating periodontitis by harnessing innate immunity to regulate the oral microbiome.


Assuntos
Células Quimiorreceptoras/imunologia , Gengiva/imunologia , Imunidade Inata , Microbiota/imunologia , Periodontite/imunologia , Animais , Células Quimiorreceptoras/metabolismo , Modelos Animais de Doenças , Feminino , Gengiva/citologia , Gengiva/microbiologia , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/genética , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Mucosa Bucal/citologia , Mucosa Bucal/imunologia , Mucosa Bucal/metabolismo , Periodontite/microbiologia , Fosfolipase C beta/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/imunologia , Canais de Cátion TRPM/metabolismo
2.
Elife ; 82019 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-31513012

RESUMO

TRPM2 is critically involved in diverse physiological processes including core temperature sensing, apoptosis, and immune response. TRPM2's activation by Ca2+ and ADP ribose (ADPR), an NAD+-metabolite produced under oxidative stress and neurodegenerative conditions, suggests a role in neurological disorders. We provide a central concept between triple-site ligand binding and the channel gating of human TRPM2. We show consecutive structural rearrangements and channel activation of TRPM2 induced by binding of ADPR in two indispensable locations, and the binding of Ca2+ in the transmembrane domain. The 8-Br-cADPR-an antagonist of cADPR-binds only to the MHR1/2 domain and inhibits TRPM2 by stabilizing the channel in an apo-like conformation. We conclude that MHR1/2 acts as a orthostatic ligand-binding site for TRPM2. The NUDT9-H domain binds to a second ADPR to assist channel activation in vertebrates, but not necessary in invertebrates. Our work provides insights into the gating mechanism of human TRPM2 and its pharmacology.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Cálcio/metabolismo , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo , Sítios de Ligação , Cátions Bivalentes/metabolismo , Microscopia Crioeletrônica , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica
3.
Artif Cells Nanomed Biotechnol ; 47(1): 3448-3455, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31411068

RESUMO

Ventilator has been widely used for life support, but ventilator-induced lung injury (VILI) is still a major problem. Oxidative stress has been considered as a key contributor for VILI, but the specific mechanism remains unclear. The expression of NLRP3 inflammasome in cells and inflammatory factors in the supernatant were measured. Mitochondrial ROS and TRPM2 channel currents were investigated using flow cytometry and Patch-clamp technique, respectively. TRPM2-/- and NLRP3-/- mice were used for animal experiments. Lung tissues were stained by HE and the wet-dry ratio, bronchoalveolar lavage fluid (BALF) protein, MPO (marrow peroxidase), NLRP3 inflammasome were also investigated. Knockdown of NLRP3 or Caspase-1 or treatments with SS-31 or YVAD inhibited the expression of the NLRP3 inflammasome, and reduced IL-1ß and IL-18 levels in cell supernatant. These treatments suppressed the production of ROS and lowered the TRPM2 channel currents, but Rotenone exerted an opposite effect. High-tidal volume ventilation significantly increased the levels of IL-1ß, IL-18, NLRP3 inflammasome, wet-dry ratio of lung, MPO and BALF protein. However, these parameters were down-regulated in TRPM2-/- and NLRP3-/- mice. These parameters were suppressed in TRPM2-/- and NLRP3-/- mice indicate that oxidative stress might promote VILI through activating NLRP3 inflammasome and TRPM2 channel.


Assuntos
Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Estresse Oxidativo , Canais de Cátion TRPM/metabolismo , Lesão Pulmonar Induzida por Ventilação Mecânica/metabolismo , Animais , Técnicas de Silenciamento de Genes , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Espécies Reativas de Oxigênio/metabolismo , Canais de Cátion TRPM/deficiência , Canais de Cátion TRPM/genética , Lesão Pulmonar Induzida por Ventilação Mecânica/genética
4.
Nat Commun ; 10(1): 3740, 2019 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-31431622

RESUMO

The transient receptor potential melastatin 2 (TRPM2) channel plays a key role in redox sensation in many cell types. Channel activation requires binding of both ADP-ribose (ADPR) and Ca2+. The recently published TRPM2 structures from Danio rerio in the ligand-free and the ADPR/Ca2+-bound conditions represent the channel in closed and open states, which uncovered substantial tertiary and quaternary conformational rearrangements. However, it is unclear how these rearrangements are achieved within the tetrameric channel during channel gating. Here we report the cryo-electron microscopy structures of Danio rerio TRPM2 in the absence of ligands, in complex with Ca2+ alone, and with both ADPR and Ca2+, resolved to ~4.3 Å, ~3.8 Å, and ~4.2 Å, respectively. In contrast to the published results, our studies capture ligand-bound TRPM2 structures in two-fold symmetric intermediate states, offering a glimpse of the structural transitions that bridge the closed and open conformations.


Assuntos
Adenosina Difosfato Ribose/metabolismo , Cálcio/metabolismo , Estrutura Quaternária de Proteína , Canais de Cátion TRPM/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Linhagem Celular , Microscopia Crioeletrônica , Células HEK293 , Humanos , Ativação do Canal Iônico , Técnicas de Patch-Clamp , Células Sf9 , Spodoptera , Canais de Cátion TRPM/química , Peixe-Zebra , Proteínas de Peixe-Zebra/química
5.
Hypertension ; 74(4): 1021-1032, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31401881

RESUMO

Excessive salt consumption leads to cardiovascular diseases. Despite various measures designed to reduce salt intake, daily salt intake remains at a high level. Appropriate salt intake is balanced by salt taste preference triggered by epithelium sodium channel and salt taste aversion evoked by bitter taste sensor, transient receptor potential channel M5 (TRPM5). However, the behavioral mechanism of excessive salt intake remains largely elusive. In this study, wild type and TRPM5-/- mice were applied to study the influence of high-salt administration on epithelium sodium channel/TRPM5 and the associated behavior to salt consumption. We found that long-term high-salt intake impaired the aversive behavior to high-salt stimulation but did not alter the preference to low salt in mice. The mechanistic evidence demonstrated that high-salt intake blunted the TRPM5-mediated aversive behavior to noxious salt stimulation through inhibiting PKC (protein kinase C) activity and PKC-dependent threonine phosphorylation in the tongue epithelium but did not affect the epithelium sodium channel-dependent salt taste preference. Inhibition of TRPM5 also resulted in an impaired aversive response to high salt, with reduced taste perception in bitter cortical field of mice. TRPM5-/- mice showed a lowered aversion to high-salt diet and developed salt-induced hypertension. The impaired perception to bitter taste evoked by high-salt intake also existed in hypertensive patients with high-salt consumption. We demonstrate that long-term high-salt consumption impairs aversive response to concentrated salt by downregulating bitter taste sensor TRPM5. It suggests that enhancing TRPM5 function might antagonize excessive salt intake and high salt-induced hypertension.


Assuntos
Comportamento Alimentar/fisiologia , Hipertensão/metabolismo , Canais de Cátion TRPM/metabolismo , Percepção Gustatória/fisiologia , Paladar/fisiologia , Animais , Humanos , Hipertensão/genética , Hipertensão/fisiopatologia , Camundongos , Camundongos Knockout , Cloreto de Sódio na Dieta , Canais de Cátion TRPM/genética , Paladar/genética , Percepção Gustatória/genética , Língua/metabolismo
7.
Life Sci ; 232: 116658, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31310758

RESUMO

AIMS: To investigate the cardioprotective effects of hypothermic (25 °C) reperfusion on ischemia/reperfusion injury and the role of transient potential channel M8 (TRPM8) in this process. MAIN METHODS: Western blot and real-time PCR were used to monitor the expression of TRPM8 in myocardium. Myocardial ischemia/reperfusion injury was induced by 30 min of global ischemia followed by 120 min of reperfusion in Langendorff-perfused hearts from Sprague-Dawley rats. The reperfusion was either normothermic (37 °C) or hypothermic (25 °C). Infarct size and left ventricular function were assessed, and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) in the coronary effluent were measured spectrophotometrically, and cardiomyocyte apoptosis was detected by TUNEL assay. The expression of TRPM8, Bcl-2, Bax, cleaved capspase-3, RhoA, and ROCK2 was quantified. KEY FINDINGS: TRPM8 protein and mRNA were expressed in rat myocardium. Hypothermic reperfusion decreased the infarct size, LDH activity, MDA content, apoptosis, and expression of Bax, cleaved caspase-3, RhoA, and ROCK2 compared with normothermic reperfusion. These effects were associated with improved recovery of left ventricular contractility, and were reduced by BCTC, a TRPM8 antagonist. Ischemia/reperfusion injury and the increased expression of Bax, caspase-3, RhoA, and ROCK2 induced by normothermic reperfusion were reduced by Icilin, a TRPM8 agonist. SIGNIFICANCE: Hypothermic reperfusion at 25 °C has cardioprotective effects against ischemia/reperfusion injury via activation of TRPM8 to inhibit the oxidative stress-related RhoA/ROCK2 signal pathway.


Assuntos
Hipotermia/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Canais de Cátion TRPM/metabolismo , Animais , Apoptose , Hemodinâmica , L-Lactato Desidrogenase/metabolismo , Masculino , Proteínas Musculares/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/enzimologia , Miocárdio/metabolismo , Ratos , Ratos Sprague-Dawley
8.
PLoS One ; 14(6): e0217925, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31163064

RESUMO

There is an increasing amount of clinical evidence that hypomagnesemia (serum Mg2+ levels < 0.7 mmol/l) contributes to type 2 diabetes mellitus pathogenesis. Amongst other hypotheses, it has been suggested that Mg2+ deficiency affects insulin secretion. The aim of this study was, therefore, to investigate the acute effects of extracellular Mg2+ on glucose-stimulated insulin secretion in primary mouse islets of Langerhans and the rat insulinoma INS-1 cell line. Here we show that acute lowering of extracellular Mg2+ concentrations from 1.0 mM to 0.5 mM did not affect glucose-stimulated insulin secretion in islets or in insulin-secreting INS-1 cells. The expression of key genes in the insulin secretory pathway (e.g. Gck, Abcc8) was also unchanged in both experimental models. Knockdown of the most abundant Mg2+ channel Trpm7 by siRNAs in INS-1 cells resulted in a 3-fold increase in insulin secretion at stimulatory glucose conditions compared to mock-transfected cells. Our data suggest that insulin secretion is not affected by acute lowering of extracellular Mg2+ concentrations.


Assuntos
Espaço Extracelular/química , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Magnésio/farmacologia , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperglicemia/patologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Canais de Cátion TRPM/metabolismo
9.
Invest Ophthalmol Vis Sci ; 60(7): 2449-2460, 2019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31157834

RESUMO

Purpose: To investigate changes in corneal nerves positive to substance P (SP) and transient receptor potential melastatin 8 (TRPM8) and gene expression in the trigeminal ganglia (TG) following corneal surgery to unveil peripheral nerve mechanism of induced dry eye-like pain (DELP). Methods: Surgery was performed on mice by removing the central epithelial and anterior stromal nerves. Mice were euthanized at different times up to 15 weeks. Immunostaining was performed with TRPM8, SP, or protein gene product 9.5 (PGP9.5) antibodies, and epithelial nerve densities were calculated. The origin of TRPM8- and SP-TG neurons were analyzed by retrograde tracing. Gene expression in TG was studied by real-time PCR analysis. Results: SP-positive epithelial corneal nerves were more abundant than TRPM8 and were expressed in different TG neurons. After injury, epithelial nerve regeneration occurs in two distinct stages. An early regeneration of the remaining epithelial bundles reached the highest density on day 3 and then rapidly degraded. From day 5, the epithelial nerves originated from the underlying stromal nerves were still lower than normal levels by week 15. The SP- and TRPM8-positive nerve fibers followed the same pattern as the total nerves. TRPM8-positive terminals increased slowly and reached only half of normal values by 3 months. Corneal sensitivity gradually increased and reached normal values on day 12. Corneal injury also induced significant changes in TG gene expression, decreasing trpm8 and tac1 genes. Conclusions: Abnormal SP expression, low amounts of TRPM8 terminals, and hypersensitive nerve response occur long after the injury and changes in gene expression in the TG suggest a contribution to the pathogenesis of corneal surgery-induced DELP.


Assuntos
Lesões da Córnea/metabolismo , Epitélio Anterior/inervação , Regulação da Expressão Gênica/fisiologia , Plasticidade Neuronal/fisiologia , Nervo Oftálmico/fisiologia , Substância P/genética , Canais de Cátion TRPM/genética , Animais , Temperatura Baixa , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Camundongos , Modelos Animais , Regeneração Nervosa/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Substância P/metabolismo , Canais de Cátion TRPM/metabolismo , Lágrimas/fisiologia , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo
10.
Int J Mol Sci ; 20(9)2019 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-31035385

RESUMO

Several drugs including diuretics and proton-pump inhibitors can cause magnesium loss and hypomagnesemia. Magnesium and drugs use the same transport and metabolism pathways in the body for their intestinal absorption, metabolism, and elimination. This means that when one or more drug is taken, there is always a potential risk of interaction with the magnesium status. Consequently the action of a drug may be adversely affected by magnesium (e.g., magnesium, calcium, and zinc can interfere with the gastrointestinal absorption of tetracycline antibiotics) and simultaneously the physiological function of minerals such as magnesium may be impaired by a drug (e.g., diuretics induce renal magnesium loss). Given the ever-increasing number of drugs on the market and the frequency with which they are used, greater attention must be paid in daily medical and pharmaceutical practice focused in particular on the adverse effects of drug therapy on magnesium status in order to minimize the potential risk to the health of patients.


Assuntos
Magnésio/metabolismo , Animais , Humanos , Absorção Intestinal/efeitos dos fármacos , Deficiência de Magnésio/metabolismo , Inibidores da Bomba de Prótons/uso terapêutico , Canais de Cátion TRPM/metabolismo , Tetraciclina/metabolismo
11.
PLoS One ; 14(5): e0215952, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31042750

RESUMO

The sulfonylurea 1 transient receptor potential melastatin 4 (Sur1-Trpm4) receptor is selectively expressed after intracerebral hemorrhage (ICH). This upregulation contributes to increases in intracellular sodium. Water follows sodium through aquaporin channels, leading to cytotoxic edema. Even after edema is thought to have resolved, ionic dyshomeostasis persists, as does blood-brain barrier (BBB) damage. Glibenclamide, a hypoglycemic agent that inhibits Sur1-Trpm4, has been shown to reduce BBB damage and edema following infusion of autologous blood into the brain (ICH) as well as after other brain injuries. In order to further assess efficacy, we used the collagenase ICH model in rats to test whether glibenclamide reduces edema, attenuates ion dyshomeostasis, improves BBB damage, and reduces lesion volume. We tested a widely-used glibenclamide dose shown effective in other studies (10 µg/kg loading dose followed by 200 ng/hr for up to 7 days). Early initiation of glibenclamide did not significantly impact edema (72 hours), BBB permeability (72 hours), or lesion volume after ICH (28 days). Recovery from neurological impairments was also not improved by glibenclamide. These results suggest that glibenclamide will not improve outcome in ICH. However, the treatment appeared to be safe as there was no effect on bleeding or other physiological variables.


Assuntos
Hemorragia Cerebral/tratamento farmacológico , Colagenases/metabolismo , Glibureto/uso terapêutico , Hipoglicemiantes/uso terapêutico , Animais , Comportamento Animal/efeitos dos fármacos , Glicemia/análise , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Edema Encefálico/patologia , Hemorragia Cerebral/etiologia , Hemorragia Cerebral/patologia , Colagenases/toxicidade , Relação Dose-Resposta a Droga , Esquema de Medicação , Glibureto/farmacologia , Hipoglicemiantes/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Receptores Sulfonilureia/antagonistas & inibidores , Receptores Sulfonilureia/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Temperatura Ambiente
12.
Acta Neurobiol Exp (Wars) ; 79(1): 86-91, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31038487

RESUMO

Epilepsy is a life­threatening disorder that is marked by recurrent seizures. Febrile seizure is a common neurological disorder observed in neonates. In many cases, reducing body temperature can prevent febrile seizure. Transient receptor potential cation channel subfamily M member 8 (TRPM8) is a cation channel that is involved in body thermoregulation. It was reported that M8­B, a TRPM8 antagonist, can reduce body temperature. Thus, we aimed to investigate the effect of M8­B on different experimental seizure models. Eight­day­old male Wistar rat pups were used for induction of febrile seizure. M8­B and diazepam were injected intraperitoneally. The rat pups were then transferred to a heated plexiglas chamber and the latency to the first febrile seizure was measured. In addition, different groups of mice were pretreated with M8­B and received a convulsant dose of pentylenetetrazol (PTZ). Latencies to stages 2 and 4 and duration of stage 5 seizure episodes were measured. Furthermore, the effect of M8­B on electroshock­induced seizures was also investigated and hindlimb extension time was measured. The results showed that M8­B decreased the body temperature of rat pups and increased the latency to the first febrile seizure, implying a significant protective effect. It also induced a significant anticonvulsant effect in PTZ ­ but not electroshock­induced convulsions. M8­B showed anticonvulsant effects in both febrile­ and PTZ­induced seizures. M8­B had a hypothermic effect with significant protective effects on febrile­ and PTZ­induced seizures; however, it did not produce similarly protective effects on seizures induced by electroshock.


Assuntos
Anticonvulsivantes/uso terapêutico , Convulsivantes/toxicidade , Pentilenotetrazol/toxicidade , Convulsões Febris/tratamento farmacológico , Convulsões/induzido quimicamente , Convulsões/prevenção & controle , Tiofenos/uso terapêutico , Animais , Animais Recém-Nascidos , Temperatura Corporal/efeitos dos fármacos , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Masculino , Ratos , Ratos Wistar , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Fatores de Tempo
13.
Cell Mol Life Sci ; 76(17): 3301-3310, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31073743

RESUMO

The channel kinase (chanzyme) transient receptor potential melastatin-like 7 (TRPM7) has a unique dual protein structure composed of an ion channel with an α-kinase domain on its C-terminus. In the nervous system, under physiological conditions, TRPM7 contributes to critical neurobiological processes ranging from synaptic transmission to cognitive functions. Following certain pathological triggers, TRPM7 mediates neurotoxicity, neuro-injuries, and neuronal death. Here, we summarize the current knowledge of TRPM7 functions in neuronal systems in health and disease. The molecular mechanisms by which this chanzyme might regulate synaptic and cognitive functions are discussed. We also discuss the lack of knowledge regarding the molecular mechanisms responsible for turning TRPM7 into "a vicious tool" that mediates neuronal death following certain pathological triggers. Some synthetic and natural pharmacological modulators of the TRPM7 channel and its α-kinase are reviewed. We suggest that based on current knowledge, we should reshape our thinking regarding the implications of TRPM7 in neurological and neurodegenerative disorders. Moreover, we propose a paradigm shift concerning the targeting of TRPM7 as a therapeutic approach for treating certain neurological diseases. We agree that TRPM7 overexpression or overactivation may mediate neurodegenerative processes following certain triggers. However, TRPM7 dysfunction and/or downregulation might also be among the pathological changes leading to neurodegeneration. Consequently, further investigations are required before we decide whether blocking or activating the chanzyme is the correct therapeutic approach to treat certain neurological and/or neurodegenerative diseases.


Assuntos
Sistema Nervoso/metabolismo , Doenças Neurodegenerativas/patologia , Canais de Cátion TRPM/metabolismo , Humanos , Magnésio/metabolismo , Doenças Neurodegenerativas/metabolismo , Plasticidade Neuronal , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/genética , Zinco/metabolismo
14.
Toxicol Lett ; 312: 98-108, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31054354

RESUMO

BACKGROUND/AIMS: To investigate the effect of Arsenic Trioxide (ATO) on endothelial cells injury and explore the role of transient receptor potential melastatin 4 channel (TRPM4) in ATO-induced endothelial injury. METHODS: qRT-PCR was used to examine the mRNA expression of TRPM4 in human umbilical vein endothelial cells (HUVECs). The protein levels were measured by Western blot and immunostaining. The MTT, TUNEL, and transwell assays were used to evaluate the cell viability, apoptosis, and migration, respectively. The ultrastructural changes were observed by scanning electron microscopy. The membrane potential, cytosolic [Na+]i, cytosolic [Ca2+]i and reactive oxygen species (ROS) levels were detected by fluorescent probes. Isometric tension of mesenteric artery was recorded by using a multiwire myograph system. RESULTS: ATO induced HUVEC cells injury, the significant upregulation of TRPM4 in this process was inhibited by 9-phenanthrol or siRNA. ATO-induced apoptosis and decrease in the cell viability/ migration were all partially reversed upon the treatment with 9-phenanthrol. Whereas, ATO-mediated increase in membrane potential, cytosolic [Na+]i, cytosolic [Ca2+]i and the ROS levels were also abolished by 9-phenanthrol or siRNA, suggesting that oxidative stress may be the potential mechanisms underlying ATO-induced endothelial injury. Additionally, 9-phenanthrol treatment prevented ATO-mediated impairment of acetylcholine-induced endothelium-dependent relaxations. CONCLUSION: TRPM4 is involved in endothelial injury induced by ATO and may be a promising therapeutic target for endothelial injury.


Assuntos
Antineoplásicos/toxicidade , Trióxido de Arsênio/toxicidade , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Canais de Cátion TRPM/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fenantrenos/farmacologia , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Canais de Cátion TRPM/genética , Regulação para Cima/efeitos dos fármacos
15.
Mol Med Rep ; 19(6): 5015-5022, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31059088

RESUMO

Sweet taste receptors (STRs) expressed on ß­cells stimulate insulin secretion in response to an increase in the circulating level of glucose, maintaining glucose homeostasis. 3­Deoxyglucosone (3DG), a highly reactive α­dicarbonyl compound, has been previously described as an independent factor associate with the development of prediabetes. In our previous study, pathological plasma levels of 3DG were induced in normal rats with a single intravenous injection of 50 mg/kg 3DG, and an acute rise in circulating 3DG induced glucose intolerance by impairing the function of pancreatic ß­cells. The present study aimed to investigate whether the deleterious effects of pathological plasma levels of 3DG on ß­cell function and insulin secretion were associated with STRs. INS­1 cells, an in vitro model to study rat ß­cells, were treated with various concentrations of 3DG (1.85, 30.84 and 61.68 mM) or lactisole (5 mM). Pancreatic islets were collected from rats 2 h after a single intravenous injection of 50 mg/kg 3DG + 0.5 g/kg glucose. The insulin concentration was measured by ELISA. The protein expression levels of components of the STR signaling pathways were determined by western blot analysis. Treatment with 3DG and 25.5 mM glucose for 1 h significantly reduced insulin secretion by INS­1 cells, which was consistent with the phenotype observed in INS­1 cells treated with the STR inhibitor lactisole. Accordingly, islets isolated from rats treated with 3DG exhibited a significant reduction in insulin secretion following treatment with 25.5 mM glucose. Furthermore, acute exposure of INS­1 cells to 3DG following treatment with 25.5 mM glucose for 1 h significantly reduced the protein expression level of the STR subunit taste 1 receptor member 3 and its downstream factors, transient receptor potential cation channel subfamily M member 5 and glucose transporter 2. Notably, islet tissues collected from rats treated with 3DG exhibited a similar downregulation of these factors. The present results suggested that acute exposure to pathologically relevant levels of 3DG in presence of high physiological levels of glucose decreased insulin secretion from ß­cells by, at least in part, downregulating the STR signaling pathway.


Assuntos
Desoxiglucose/análogos & derivados , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Derivados de Benzeno/farmacologia , Células Cultivadas , Desoxiglucose/farmacologia , Regulação para Baixo/efeitos dos fármacos , Transportador de Glucose Tipo 2/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Masculino , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas-G/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo
16.
BMC Immunol ; 20(1): 14, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-31077146

RESUMO

BACKGROUND: Natural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/- and CD56DimCD16+ NK cells. Intracellular calcium (Ca2+) is fundamental to regulate a number of intracellular signalling pathways and functions in NK cells, which are essential in mediating their natural cytotoxic function. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable non-selective cation channel that possesses a critical role in calcium-dependent cell signalling to maintain cellular homeostasis. TRPM2 and CD38 protein surface expression has yet to be determined on NK cells using flow cytometry. Characterisation of TRPM2 has been previously identified by in vivo models, primarily using methods such as genetic remodification, immunohistochemistry and whole cell electrophysiology. The aim of this study was to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously applied using flow cytometry. RESULTS: At 2 h/1 h, TRPM2 (Fig. 2 A, B, p < 0.05) and TRPM2/CD38 (Fig. 3A, B, p < 0.05) surface expression significantly increased between 1:300 and 1:50 at 2 h/1 h. TRPM2/CD38 surface expression furthermore increased between 1:100 and 1:50 at 2 h/1 h (Fig. 3A, p < 0.05). Interestingly, TRPM2/CD38 surface expression significantly decreased from 1:50 to 1:5 on CD56BrightCD16Dim/- NK cells. These consistent findings highlight that 1:50 is the optimal antibody dilution and threshold to measure TRPM2 and TRPM2/CD38 surface expression on NK subsets. 2 h/1 h was determined as the optimal incubation period to ensure a sufficient timeframe for maximal antibody binding and surface expression. CONCLUSION: For the first time, we describe an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cells in healthy participants. Finally, using an antibody that has not been previously applied in flow cytometry, we determined an antibody concentration and incubation time that is robust, rapid and sensitive for the application of flow cytometry.


Assuntos
ADP-Ribosil Ciclase 1/metabolismo , Citometria de Fluxo/métodos , Células Matadoras Naturais/metabolismo , Canais de Cátion TRPM/metabolismo , ADP-Ribosil Ciclase 1/genética , Antígeno CD56/metabolismo , Sinalização do Cálcio , Separação Celular , Células Cultivadas , Citotoxicidade Imunológica , Regulação da Expressão Gênica , Voluntários Saudáveis , Humanos , Canais de Cátion TRPM/genética
17.
Int J Mol Sci ; 20(11)2019 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-31141957

RESUMO

The transient receptor potential melastatin subtype 8 (TRPM8) is a nonselective, multimodal ion channel, activated by low temperatures (<28 °C), pressure, and cooling compounds (menthol, icilin). Experimental evidences indicated a role of TRPM8 in cold thermal transduction, different life-threatening tumors, and other pathologies, including migraine, urinary tract dysfunction, dry eye disease, and obesity. Hence, the modulation of the TRPM8 channel could be essential in order to understand its implications in these pathologies and for therapeutic intervention. This short review will cover recent progress on the TRPM8 agonists and antagonists, describing newly reported chemotypes, and their application in the pharmacological characterization of TRPM8 in health and disease. The recently described structures of the TRPM8 channel alone or complexed with known agonists and PIP2 are also discussed.


Assuntos
Moduladores de Transporte de Membrana/química , Canais de Cátion TRPM/agonistas , Animais , Sítios de Ligação , Humanos , Moduladores de Transporte de Membrana/farmacologia , Ligação Proteica , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/química , Canais de Cátion TRPM/metabolismo
18.
Curr Protein Pept Sci ; 20(8): 777-788, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31060485

RESUMO

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) that is a global health concern associated with high morbidity and mortality. So far, no specific interventions limit injury or improve recovery and survival. Transient receptor potential melastatin 7 (TRPM7), a bifunctional membrane protein, plays key roles in inflammation and cell death. However, the precise role and underlying mechanism of TRPM7 in IR-induced AKI have not been well defined. Herein, we reviewed the structure and function of TRPM7 as a non-selective ion channel, but Ca2+ and Mg2+-conducting, that mediated the elevation of cytosolic Ca2+ and Mg2+. We then comprehensively reviewed the mechanism of TRPM7 involved in the pathophysiology of renal IRI, including inflammatory response, apoptosis and necroptosis, renal microvasculature, as well as maladaptive fibrogenesis leading to chronic kidney disease (CKD). Our previous study has shown that the dynamic change and underlying mechanism of TRPM7 involving in inflammation and apoptosis in in vitro hypoxia/reoxygenation and in vivo renal IRI models. The association between TRPM7, inflammatory response and apoptosis, as well as related caspase-3, HMGB1 and Bax/Bcl-2 ratio, was also discussed. Disclosing the involvement of TRPM7 in renal IRI might provide new mechanistic insights for a potential biomarker as diagnostic and therapeutic target of AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Traumatismo por Reperfusão/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Apoptose , Movimento Celular , Proliferação de Células , Humanos , Inflamação/metabolismo , Traumatismo por Reperfusão/patologia , Transdução de Sinais
19.
Oxid Med Cell Longev ; 2019: 4619865, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30984336

RESUMO

Numerous studies have reported a strong association between increased production of reactive oxygen species (ROS) and the pathobiology of several diseases, and cancer in particular. Therefore, manipulation of cellular oxidative stress levels represents an important therapeutic target. Recently, resveratrol (RESV), a naturally occurring phytochemical, has been shown to sensitize several cell lines to the anticancer effects of other chemotherapeutic agents, including paclitaxel (PAX). However, the molecular mechanisms of action of RESV through oxidative sensitive TRPM2 channel activation remain unclear. The aim of this study was to evaluate the effect of combination therapy of RESV and PAX on activation of TRPM2 in DBTRG glioblastoma cells. DBTRG cells were divided into four treatment groups: control, RESV (50 µM), PAX (50 µM), and PAX + RESV for 24 hours. Our data shows that markers for apoptosis, mitochondrial membrane depolarization and mitochondrial function, intracellular steady-state ROS levels, caspase 3 activity, TRPM2 current density, and Ca2+ florescence intensity were significantly increased in DBTRG cells following treatment with PAX and RESV, respectively, although cell viability was also decreased by these treatments. These biochemical markers were further increased to favor the anticancer effects of PAX in DBTRG cells in combination with RESV. The PAX and RESV-mediated increase in current density and Ca2+ florescence intensity was decreased with a TRPM2 blocker. This suggests that for this combination therapy to have a substantial effect on apoptosis and cell viability, the TRPM2 channel must be stimulated.


Assuntos
Anti-Inflamatórios não Esteroides/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Glioblastoma/tratamento farmacológico , Oxidantes/metabolismo , Paclitaxel/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Resveratrol/uso terapêutico , Canais de Cátion TRPM/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Humanos , Paclitaxel/farmacologia , Resveratrol/farmacologia
20.
Int J Mol Sci ; 20(8)2019 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-31022885

RESUMO

Transient receptor potential melastatin member 4 (TRPM4) and 5 (TRPM5) channels are Ca2+-activated nonselective cation channels. Intracellular Ca2+ is the most important regulator for them to open, though PI(4,5)P2, a membrane phosphoinositide, has been reported to regulate their Ca2+-sensitivities. We previously reported that negatively-charged amino acid residues near and in the TRP domain are necessary for the normal Ca2+ sensitivity of TRPM4. More recently, a cryo-electron microscopy structure of Ca2+-bound (but closed) TRPM4 was reported, proposing a Ca2+-binding site within an intracellular cavity formed by S2 and S3. Here, we examined the functional effects of mutations of the amino acid residues related to the proposed Ca2+-binding site on TRPM4 and also TRPM5 using mutagenesis and patch clamp techniques. The mutations of the amino acid residues of TRPM4 and TRPM5 reduced their Ca2+-sensitivities in a similar way. On the other hand, intracellular applications of PI(4,5)P2 recovered Ca2+-sensitivity of desensitized TRPM4, but its effect on TRPM5 was negligible. From these results, the Ca2+-binding sites of TRPM4 and TRPM5 were shown to be formed by the same amino acid residues by functional analyses, but the impact of PI(4,5)P2 on the regulation of TRPM5 seemed to be smaller than that on the regulation of TRPM4.


Assuntos
Cálcio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Masculino , Modelos Moleculares , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Ratos , Canais de Cátion TRPM/química , Canais de Cátion TRPM/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA