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1.
Int J Mol Sci ; 22(7)2021 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-33810249

RESUMO

Transient receptor potential melastatin member 4 (TRPM4) encodes a Ca2+-activated, non-selective cation channel that is functionally expressed in several tissues, including the heart. Pathogenic mutants in TRPM4 have been reported in patients with inherited cardiac diseases, including conduction blockage and Brugada syndrome. Heterologous expression of mutant channels in cell lines indicates that these mutations can lead to an increase or decrease in TRPM4 expression and function at the cell surface. While the expression and clinical variant studies further stress the importance of TRPM4 in cardiac function, the cardiac electrophysiological phenotypes in Trpm4 knockdown mouse models remain incompletely characterized. To study the functional consequences of Trpm4 deletion on cardiac electrical activity in mice, we performed perforated-patch clamp and immunoblotting studies on isolated atrial and ventricular cardiac myocytes and surfaces, as well as on pseudo- and intracardiac ECGs, either in vivo or in Langendorff-perfused explanted mouse hearts. We observed that TRPM4 is expressed in atrial and ventricular cardiac myocytes and that deletion of Trpm4 unexpectedly reduces the peak Na+ currents in myocytes. Hearts from Trpm4-/- mice presented increased sensitivity towards mexiletine, a Na+ channel blocker, and slower intraventricular conduction, consistent with the reduction of the peak Na+ current observed in the isolated cardiac myocytes. This study suggests that TRPM4 expression impacts the Na+ current in murine cardiac myocytes and points towards a novel function of TRPM4 regulating the Nav1.5 function in murine cardiac myocytes.


Assuntos
Arritmias Cardíacas/genética , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Canais de Cátion TRPM/metabolismo , Potenciais de Ação , Animais , Células Cultivadas , Deleção de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/fisiologia , Canais de Cátion TRPM/genética , Função Ventricular
2.
Nat Commun ; 12(1): 1401, 2021 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-33658516

RESUMO

Effective treatments for patients suffering from heat hypersensitivity are lacking, mostly due to our limited understanding of the pathogenic mechanisms underlying this disorder. In the nervous system, activating transcription factor 4 (ATF4) is involved in the regulation of synaptic plasticity and memory formation. Here, we show that ATF4 plays an important role in heat nociception. Indeed, loss of ATF4 in mouse dorsal root ganglion (DRG) neurons selectively impairs heat sensitivity. Mechanistically, we show that ATF4 interacts with transient receptor potential cation channel subfamily M member-3 (TRPM3) and mediates the membrane trafficking of TRPM3 in DRG neurons in response to heat. Loss of ATF4 also significantly decreases the current and KIF17-mediated trafficking of TRPM3, suggesting that the KIF17/ATF4/TRPM3 complex is required for the neuronal response to heat stimuli. Our findings unveil the non-transcriptional role of ATF4 in the response to heat stimuli in DRG neurons.


Assuntos
Fator 4 Ativador da Transcrição/metabolismo , Nociceptividade/fisiologia , Células Receptoras Sensoriais/fisiologia , Canais de Cátion TRPM/metabolismo , Fator 4 Ativador da Transcrição/genética , Animais , Membrana Celular/metabolismo , Quimiocina CXCL12/metabolismo , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Células HEK293 , Temperatura Alta , Humanos , Injeções Espinhais , Cinesina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Técnicas de Patch-Clamp , Transporte Proteico , Receptores CXCR4/metabolismo , Canais de Cátion TRPM/genética
3.
J Laryngol Otol ; 135(2): 104-109, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33612130

RESUMO

BACKGROUND: The mechanism of nasal airflow sensation is poorly understood. This study aimed to examine the role of nasal mucosal temperature change in the subjective perception of nasal patency and the methods by which it can be quantified. METHOD: Medline and PubMed database searches were performed to retrieve literature relevant to the topic. RESULTS: The primary mechanism producing the sensation of nasal patency is thought to be the activation of transient receptor potential melastatin family member 8 ('TRPM8'), a thermoreceptor that is activated by nasal mucosal cooling. Computational fluid dynamics studies have demonstrated that increased airflow and heat flux are correlated with better patient-reported outcome measure scores. Similarly, physical measurements of the nasal cavity using temperature probes have shown a correlation between lower nasal mucosal temperatures and better patient-reported outcome measure scores. CONCLUSION: Nasal mucosal temperature change may be correlated with the perception of improved nasal patency. Future research should quantify the impact of mucosal cooling on the perception of nasal airway obstruction.


Assuntos
Temperatura Baixa/efeitos adversos , Mucosa Nasal/fisiologia , Obstrução Nasal/psicologia , Percepção/fisiologia , Resistência das Vias Respiratórias/fisiologia , Simulação por Computador , Humanos , Hidrodinâmica , Cavidade Nasal/anatomia & histologia , Cavidade Nasal/diagnóstico por imagem , Cavidade Nasal/fisiologia , Mucosa Nasal/metabolismo , Obstrução Nasal/diagnóstico , Obstrução Nasal/fisiopatologia , Obstrução Nasal/cirurgia , Medidas de Resultados Relatados pelo Paciente , Ventilação Pulmonar/fisiologia , Canais de Cátion TRPM/metabolismo , Temperatura , Termorreceptores/metabolismo
4.
Life Sci ; 268: 118967, 2021 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-33417951

RESUMO

AIMS: Sensory nerve activation modulates ureteral contractility by releasing neuropeptides including CGRP and neurokinin A (NKA). TRPM3 is a recently discovered thermosensitive channel expressed in nociceptive sensory neurons, and plays a key role in heat nociception and chronic pain. The aim of this study is to examine the role of TRPM3 activation in human ureter motility. MAIN METHOD: Human proximal ureters were obtained from fourteen patients undergoing nephrectomy. Spontaneous or NKA-evoked contractions of longitudinal ureter strips were recorded in an organ bath. Ureteral TRPM3 expression was examined by immunofluorescence. KEY FINDINGS: Spontaneous contractions were observed in 60% of examined strips. TRPM3 activation using pregnenolone sulphate (PS) or CIM0216 (specific TRPM3 agonists) dose-dependently reduced the frequency of spontaneous and NKA-evoked contractions, with IC50s of 241.7 µM and 4.4 µM, respectively. The inhibitory actions of TRPM3 agonists were mimicked by CGRP (10 to 100 nM) or a cAMP analogue (8-Br-cAMP; 1 mM). The inhibitory actions of TRPM3 agonists (300 µM PS or 30 µM CIM0216) were blocked by pretreatment with primidone (TRPM3 antagonist; 30 µM), tetrodotoxin (sodium channel blocker; 1 µM), olcegepant (CGRP receptor antagonist; 10 µM), or H89 (non-specific PKA inhibitor; 30 µM). TRPM3 was co-expressed with CGRP in nerves in the sub-urothelial and intermuscular regions of the ureter. SIGNIFICANCE: TRPM3 channels expressed on sensory terminals of the human ureter involve in inhibitory sensory neurotransmission and modulate ureter motility via the CGRP-cAMP-PKA signal pathway. Targeting TRPM3 may be a pharmacological strategy for promoting the ureter stone passage.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Células Receptoras Sensoriais/metabolismo , Canais de Cátion TRPM/metabolismo , Ureter/fisiologia , Adulto , Idoso , Capsaicina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Masculino , Pessoa de Meia-Idade , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Nefrectomia , Neurocinina A/metabolismo , Neurocinina A/farmacologia , Técnicas de Cultura de Órgãos , Pregnenolona/farmacologia , Primidona/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Canais de Cátion TRPM/agonistas , Canais de Cátion TRPM/antagonistas & inibidores , Ureter/efeitos dos fármacos
5.
Nat Commun ; 12(1): 617, 2021 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-33504784

RESUMO

The blood-brain barrier (BBB) is critical for neural function. We report here circadian regulation of the BBB in mammals. Efflux of xenobiotics by the BBB oscillates in mice, with highest levels during the active phase and lowest during the resting phase. This oscillation is abrogated in circadian clock mutants. To elucidate mechanisms of circadian regulation, we profiled the transcriptome of brain endothelial cells; interestingly, we detected limited circadian regulation of transcription, with no evident oscillations in efflux transporters. We recapitulated the cycling of xenobiotic efflux using a human microvascular endothelial cell line to find that the molecular clock drives cycling of intracellular magnesium through transcriptional regulation of TRPM7, which appears to contribute to the rhythm in efflux. Our findings suggest that considering circadian regulation may be important when therapeutically targeting efflux transporter substrates to the CNS.


Assuntos
Barreira Hematoencefálica/metabolismo , Relógios Circadianos , Xenobióticos/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Células Endoteliais/metabolismo , Regulação da Expressão Gênica , Humanos , Magnésio/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Permeabilidade , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Especificidade por Substrato , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo
6.
Life Sci ; 266: 118894, 2021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310045

RESUMO

As the most prevalent cancer for females, breast cancer is also the second most popular cancer type overall. More efforts are needed to research new drugs and combination therapies for this disease. A naturally derived transient receptor potential melastatin-like 7 channel (TRPM7) inhibitor, carvacrol, was found to have anti-cancer potentials. We hypothesized that carvacrol affects breast cancer cells through TRPM7 mediated cell cycle regulation. Cell viability and apoptosis of breast cancer cell lines BT-483, BT-474, MCF-7, MDA-MB-231, and MDA-MB-453 were determined using the CCK-8 assay and ELISA respectively. TRPM7 in MDA-MB-231, MCF-7 was knocked down. Functional TRPM7 in MDA-MB-231, MCF-7, and HEK293 cells were tested with western blotting, patch-clamp, and fura-2 quench assay. The cell cycle and the regulatory proteins were determined by flow cytometry and western blotting. Results showed that carvacrol inhibited the viability of breast cancer cells with different potency. At 200 µM, MDA-MB-231 was the most sensitive, and MCF-7 was the least sensitive. At >200 µM, the apoptosis was dramatically induced. Carvacrol inhibited TRPM7 functions in MDA-MB-231, MCF-7, and HEK293. Carvacrol at 200 µM increased cells in the G1/G0 phase and decreased cells in the S and G2/M phase by regulating some cyclin proteins in MDA-MB-231. These effects were blocked by the knockdown of TRPM7. This study demonstrated that carvacrol suppresses breast cancer cells by cell cycle regulation and the TRPM7 pathway is one of the pharmacological mechanisms.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Ciclo Celular , Cimenos/farmacologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Canais de Cátion TRPM/metabolismo , Apoptose , Biomarcadores Tumorais/genética , Neoplasias da Mama/patologia , Proliferação de Células , Feminino , Humanos , Proteínas Serina-Treonina Quinases/genética , Canais de Cátion TRPM/genética , Células Tumorais Cultivadas
7.
Chem Biol Interact ; 334: 109306, 2021 Jan 25.
Artigo em Inglês | MEDLINE | ID: mdl-33309544

RESUMO

Oxidative stress (OS)-induced glutathione (GSH) depletion plays an essential role in several kidney diseases such as chronic kidney disease and nephrotoxicity. The OS-dependent activation of TRPM2 cation channel in several neurons and cells were modulated by the concentration of intracellular GSH. However, the effects of GSH alteration on TRPM2 activation, OS, and apoptosis in the cortical collecting duct (mpkCCDc14) cells still remain elusive. We investigated the effects of GSH supplementation on OS-induced TRPM2 activation, mitochondrial oxidative stress, and apoptosis in the human embryonic kidney 293 (HEK293) and mpkCCDc14 cells treated with buthionine-sulfoximine (BSO), a GSH synthase inhibitor. The HEK293 and mpkCCDc14 cells were divided into five groups as control, GSH (10 mM for 2 h), BSO (0.5 mM for 6 h), BSO + GSH, and BSO + TRPM2 channel blockers. Apoptosis, cell death, mitochondrial OS, caspase -3, caspase -9, cytosolic free Zn2+, and Ca2+ concentrations were increased in the BSO group of the TRPM2 expressing mpkCCDc14 cells, although they were diminished by the treatments of GSH, PARP-1 inhibitors (PJ34 and DPQ), and TRPM2 blockers (ACA and 2-APB). The BSO-induced decreases in the levels of cell viability and cytosolic GSH were increased by the treatments of GSH, ACA, and 2-APB. However, the effects of BSO and GSH were not observed in the non-TRPM2 expressing HEK293 cells. Current results show that maintaining GSH homeostasis is not only important for quenching OS in the cortical collecting duct cells but equally critical to modulate TRPM2 activation. Thus, suppressing apoptosis and mitochondrial OS responses elicited by oxidant action of GSH depletion.


Assuntos
Apoptose/fisiologia , Glutationa/metabolismo , Córtex Renal/metabolismo , Estresse Oxidativo/fisiologia , Canais de Cátion TRPM/metabolismo , Animais , Apoptose/efeitos dos fármacos , Butionina Sulfoximina/farmacologia , Cálcio/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Citosol/efeitos dos fármacos , Citosol/metabolismo , Células HEK293 , Homeostase/efeitos dos fármacos , Homeostase/fisiologia , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Córtex Renal/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estresse Oxidativo/efeitos dos fármacos
8.
Science ; 370(6513)2020 10 09.
Artigo em Inglês | MEDLINE | ID: mdl-33033186

RESUMO

Excitotoxicity induced by NMDA receptors (NMDARs) is thought to be intimately linked to high intracellular calcium load. Unexpectedly, NMDAR-mediated toxicity can be eliminated without affecting NMDAR-induced calcium signals. Instead, excitotoxicity requires physical coupling of NMDARs to TRPM4. This interaction is mediated by intracellular domains located in the near-membrane portions of the receptors. Structure-based computational drug screening using the interaction interface of TRPM4 in complex with NMDARs identified small molecules that spare NMDAR-induced calcium signaling but disrupt the NMDAR/TRPM4 complex. These interaction interface inhibitors strongly reduce NMDA-triggered toxicity and mitochondrial dysfunction, abolish cyclic adenosine monophosphate-responsive element-binding protein (CREB) shutoff, boost gene induction, and reduce neuronal loss in mouse models of stroke and retinal degeneration. Recombinant or small-molecule NMDAR/TRPM4 interface inhibitors may mitigate currently untreatable human neurodegenerative diseases.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Descoberta de Drogas , Doenças Neurodegenerativas/metabolismo , Fármacos Neuroprotetores/química , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPM/metabolismo , Animais , Sinalização do Cálcio , AMP Cíclico/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Doenças Neurodegenerativas/tratamento farmacológico , Domínios Proteicos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/uso terapêutico , Proteínas Recombinantes/química , Proteínas Recombinantes/uso terapêutico , Canais de Cátion TRPM/genética , Ativação Transcricional
9.
PLoS One ; 15(8): e0237347, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785272

RESUMO

Here we examine a class of neurons that have been recently explored, the somatosensory neuronal subclass of cold thermosensors. We create a mathematical model of a cold sensing neuron that has been formulated to understand the variety of ionic channels involved. In particular this model showcases the role of TRPM8 and voltage gated potassium channels in setting the temperature dependent activation and inactivation threshold level. Bifurcation analysis of the model demonstrates that a Hodgkin-Huxley type model with additional TRPM8 channels is sufficient to replicate observable experimental features of when different threshold level cold thermosensors turn on. Additionally, our analysis gives insight into what is happening at the temperature levels at which these neurons shut off and the role sodium and leak currents may have in this. This type of model construction and analysis provides a framework moving forward that will help tackle less well understood neuronal classes and their important ionic channels.


Assuntos
Temperatura Baixa , Modelos Neurológicos , Canais de Cátion TRPM/metabolismo , Termorreceptores/fisiologia , Sensação Térmica/fisiologia , Animais , Potenciais da Membrana/fisiologia , Potássio/metabolismo , Sódio/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(33): 20298-20304, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32747539

RESUMO

In mammals, temperature-sensitive TRP channels make membrane conductance of cells extremely temperature dependent, allowing the detection of temperature ranging from noxious cold to noxious heat. We progressively deleted the distal carboxyl terminus domain (CTD) of the cold-activated melastatin receptor channel, TRPM8. We found that the enthalpy change associated with channel gating is proportional to the length of the CTD. Deletion of the last 36 amino acids of the CTD transforms TRPM8 into a reduced temperature-sensitivity channel (Q10 ∼4). Exposing the intracellular domain to a denaturing agent increases the energy required to open the channel indicating that cold drives channel gating by stabilizing the folded state of the CTD. Experiments in the presence of an osmoticant agent suggest that channel gating involves a change in solute-inaccessible volume in the CTD of ∼1,900 Å3 This volume matches the void space inside the coiled coil according to the cryogenic electron microscopy structure of TRPM8. The results indicate that a folding-unfolding reaction of a specialized temperature-sensitive structure is coupled to TRPM8 gating.


Assuntos
Domínios Proteicos , Dobramento de Proteína , Canais de Cátion TRPM/química , Animais , Temperatura Baixa , Microscopia Crioeletrônica , Humanos , Ativação do Canal Iônico , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Oócitos , Conformação Proteica , Canais de Cátion TRPM/metabolismo , Termodinâmica , Xenopus laevis
11.
Bratisl Lek Listy ; 1(5): 334-338, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32356430

RESUMO

AIM: Transient receptor potential (TRP) channels expression is enhanced significantly in colon cancer cells and  Low Lever Laser Treatment (LLLT), is known to have effects and is used clinically in the treatment ofmany diseases, including colon cancer. We aimed to reveal the effects of (LLLT) on apoptosis of colon cancer and on the efficacy of cyclophosphamide via Transient receptor potential melastatin 2 (TRPM2) channels. METHOD: Human colon cancer cells (Caco-2) were cultured and cells were divided into seven main groups. Cells were incubated with cyclophosphamide, TRPM2 channel inhibitor, stimulator and low level laser exposure separately and together. The effects of cyclophosphamide and low level laser were investigated on apoptosis. RESULTS: It was found that the levels of apoptosis in cyclophosphamide group were significantly increased in cancer cells compared to the control group. TRPM2 channel stimulator administration resulted in significantly increased apoptosis levels compared to the control group, in cyclophosphamide + low level laser group the apoptosis level was significantly increased compared to the cyclophosphamide-only group. CONCLUSIONS: It has been shown that apoptotic effects of cyclophosphamide on colon cancer cells were directly related to TRPM2 channels, low level laser increased apoptosis in colon cancer cells through TRPM2 channels and induced apoptotic effect of cyclophosphamide (Fig. 5, Ref. 26).


Assuntos
Apoptose , Neoplasias do Colo/metabolismo , Ciclofosfamida/farmacologia , Terapia com Luz de Baixa Intensidade , Canais de Cátion TRPM/metabolismo , Células CACO-2 , Humanos
12.
J Neurosci ; 40(25): 4813-4823, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32414784

RESUMO

During sleep, neurons in the thalamic reticular nucleus (TRN) participate in distinct types of oscillatory activity. While the reciprocal synaptic circuits between TRN and sensory relay nuclei are known to underlie the generation of sleep spindles, the mechanisms regulating slow (<1 Hz) forms of thalamic oscillations are not well understood. Under in vitro conditions, TRN neurons can generate slow oscillations in a cell-intrinsic manner, with postsynaptic Group 1 metabotropic glutamate receptor activation triggering long-lasting plateau potentials thought to be mediated by both T-type Ca2+ currents and Ca2+-activated nonselective cation currents (ICAN). However, the identity of ICAN and the possible contribution of thalamic circuits to slow rhythmic activity remain unclear. Using thalamic slices derived from adult mice of either sex, we recorded slow forms of rhythmic activity in TRN neurons, which were driven by fast glutamatergic thalamoreticular inputs but did not require postsynaptic Group 1 metabotropic glutamate receptor activation. For a significant fraction of TRN neurons, synaptic inputs or brief depolarizing current steps led to long-lasting plateau potentials and persistent firing (PF), and in turn, resulted in sustained synaptic inhibition in postsynaptic relay neurons of the ventrobasal thalamus (VB). Pharmacological approachesindicated that plateau potentials were triggered by Ca2+ influx through T-type Ca2+ channels and mediated by Ca2+- and voltage-dependent transient receptor potential melastatin 4 (TRPM4) channels. Together, our results suggest that thalamic circuits can generate slow oscillatory activity, mediated by an interplay of TRN-VB synaptic circuits that generate rhythmicity and TRN cell-intrinsic mechanisms that control PF and oscillation frequency.SIGNIFICANCE STATEMENT Slow forms of thalamocortical rhythmic activity are thought to be essential for memory consolidation during sleep and the efficient removal of potentially toxic metabolites. In vivo, thalamic slow oscillations are regulated by strong bidirectional synaptic pathways linking neocortex and thalamus. Therefore, in vitro studies in the isolated thalamus offer important insights about the ability of individual neurons and local circuits to generate different forms of rhythmic activity. We found that circuits formed by GABAergic neurons in the thalamic reticular nucleus and glutamatergic relay neurons in the ventrobasal thalamus generated slow oscillatory activity, which was accompanied by persistent firing in thalamic reticular nucleus neurons. Our results identify both cell-intrinsic and synaptic mechanisms that mediate slow forms of rhythmic activity in thalamic circuits.


Assuntos
Neurônios GABAérgicos/fisiologia , Núcleos Intralaminares do Tálamo/fisiologia , Canais de Cátion TRPM/metabolismo , Potenciais de Ação/fisiologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vias Neurais/fisiologia , Técnicas de Cultura de Órgãos , Sono/fisiologia
13.
Sci Rep ; 10(1): 5724, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32235895

RESUMO

Communication technologies based on radiofrequency (RF) propagation bring great benefits to our daily life. However, their rapid expansion raises concerns about possible impacts on public health. At intensity levels below the threshold to produce thermal effects, RF exposure has also recently been reported to elicit biological effects, resembling reactions to cold. The objective of the present study was to investigate the effects of non-thermal RF on body temperature in mice and the related mechanisms. 3-months-old C57BL/6 J mice were exposed to a continuous RF signal at 900 MHz, 20 ± 5 V.m-1 for 7 consecutive days, twice per day during the light phase, for one hour each time. The SAR was 0.16 ± 0.10 W.kg-1. We showed that body temperature patterns in mice change synchronously with the RF exposure periods. Average body temperature in the light phase in the exposed group was higher than in the control group. The expression of the TRPM8 gene was not affected by RF in trigeminal ganglia. Furthermore, the injection of a TRPM8 antagonist did not induce a temperature decrease in exposed mice, as this was the case for sham-controls. These findings indicate that 900 MHz RF exposure at non-thermal level produce a physiological effect on body temperature in mice. However, the involvement of TRPM8 receptors in the mechanism by which RF induced changes in body temperature of mice which remains to be further explored. It must then be assessed if this effect is extrapolable to man, and if this could lead to consequences on health.


Assuntos
Temperatura Corporal/efeitos da radiação , Peso Corporal/efeitos da radiação , Ondas de Rádio , Animais , Temperatura Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Camundongos , Naftiridinas/farmacologia , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , Gânglio Trigeminal/metabolismo , Gânglio Trigeminal/efeitos da radiação
14.
Int J Mol Sci ; 21(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218173

RESUMO

BACKGROUND: There is an increasing interest in TRPM8 ligands of medicinal interest, the rational design of which can be nowadays supported by structure-based in silico studies based on the recently resolved TRPM8 structures. Methods: The study involves the generation of a reliable hTRPM8 homology model, the reliability of which was assessed by a 1.0 µs MD simulation which was also used to generate multiple receptor conformations for the following structure-based virtual screening (VS) campaigns; docking simulations utilized different programs and involved all monomers of the selected frames; the so computed docking scores were combined by consensus approaches based on the EFO algorithm. Results: The obtained models revealed very satisfactory performances; LiGen™ provided the best results among the tested docking programs; the combination of docking results from the four monomers elicited a markedly beneficial effect on the computed consensus models. Conclusions: The generated hTRPM8 model appears to be amenable for successful structure-based VS studies; cross-talk modulating effects between interacting monomers on the binding sites can be accounted for by combining docking simulations as performed on all the monomers; this strategy can have general applicability for docking simulations involving quaternary protein structures with multiple identical binding pockets.


Assuntos
Canais de Cátion TRPM/metabolismo , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Canais de Cátion TRPM/genética
15.
Oxid Med Cell Longev ; 2020: 6724810, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32215176

RESUMO

Transient receptor potential melastatin subfamily member 7 (TRPM7) was essential in the growth and metastatic ability of prostate cancer cells. However, the effects and the relevant molecular mechanisms of TRPM7 on metastasis of prostate cancer under hypoxic atmosphere remain unclear. This study investigated the role of TRPM7 in the metastatic ability of androgen-independent prostate cancer cells under hypoxia. First, data mining was carried out to disclose the relationship between the TRPM7 gene level and the survival of prostate cancer patients. Specific siRNAs were used to knockdown target genes. Western blotting and qPCR were employed to determine protein and gene expression, respectively. The gene transcription activity was evaluated by luciferase activity assay of promoter gene. The protein interaction was determined by coimmunoprecipitation. Wound healing and transwell assays were employed to evaluated cell migration and invasion, respectively. Open access database results showed that high expression of TRPM7 was closely related to the poor survival of prostate cancer patients. Hypoxia simultaneously increased TRPM7 expression and induced HIF-1α accumulation in androgen-independent prostate cancer cells. Knockdown of TRPM7 significantly promoted HIF-1α degradation through the proteasome and inhibited EMT changes in androgen-independent prostate cancer cells under hypoxic condition. Moreover, TRPM7 knockdown increased the phosphorylation of RACK1 and strengthened the interaction between RACK1 and HIF-1α but attenuated the binding of HSP90 to HIF-1α. Whereas knockdown of RACK1 increased the binding of HSP90 to HIF-1α. Furthermore, both TRPM7 and HIF-1α knockdown significantly suppressed hypoxia-induced Annexin A1 protein expression, and suppression of HIF-1α/Annexin A1 signaling significantly inhibited hypoxia-induced cell migration and invasion of androgen-independent prostate cancer cells. Our findings demonstrate that TRPM7 knockdown promotes HIF-1α degradation via an oxygen-independent mechanism involving increased binding of RAKC1 to HIF-1α, and TRPM7-HIF-1α-Annexin A1 signaling axis plays a crucial role in the EMT, cell migration, and invasion of androgen-independent prostate cancer cells under hypoxic conditions.


Assuntos
Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias de Próstata Resistentes à Castração/patologia , Proteínas Serina-Treonina Quinases/genética , Receptores de Quinase C Ativada/metabolismo , Canais de Cátion TRPM/genética , Anexina A1/genética , Anexina A1/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Transição Epitelial-Mesenquimal , Regulação Neoplásica da Expressão Gênica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Masculino , Fosforilação , Prognóstico , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Canais de Cátion TRPM/metabolismo , Hipóxia Tumoral
16.
Proc Natl Acad Sci U S A ; 117(15): 8633-8638, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32220960

RESUMO

To adapt to habitat temperature, vertebrates have developed sophisticated physiological and ecological mechanisms through evolution. Transient receptor potential melastatin 8 (TRPM8) serves as the primary sensor for cold. However, how cold activates TRPM8 and how this sensor is tuned for thermal adaptation remain largely unknown. Here we established a molecular framework of how cold is sensed in TRPM8 with a combination of patch-clamp recording, unnatural amino acid imaging, and structural modeling. We first observed that the maximum cold activation of TRPM8 in eight different vertebrates (i.e., African elephant and emperor penguin) with distinct side-chain hydrophobicity (SCH) in the pore domain (PD) is tuned to match their habitat temperature. We further showed that altering SCH for residues in the PD with solvent-accessibility changes leads to specific tuning of the cold response in TRPM8. We also observed that knockin mice expressing the penguin's TRPM8 exhibited remarkable tolerance to cold. Together, our findings suggest a paradigm of thermal adaptation in vertebrates, where the evolutionary tuning of the cold activation in the TRPM8 ion channel through altering SCH and solvent accessibility in its PD largely contributes to the setting of the cold-sensitive/tolerant phenotype.


Assuntos
Adaptação Fisiológica , Temperatura Baixa , Elefantes/fisiologia , Ativação do Canal Iônico , Spheniscidae/fisiologia , Canais de Cátion TRPM/metabolismo , Sequência de Aminoácidos , Animais , Homologia de Sequência , Canais de Cátion TRPM/genética
17.
PLoS One ; 15(2): e0228938, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32074140

RESUMO

Calcifications can disrupt organ function in the cardiovascular system and the kidney, and are particularly common in patients with chronic kidney disease (CKD). Fetuin-A deficient mice maintained against the genetic background DBA/2 exhibit particularly severe soft tissue calcifications, while fetuin-A deficient C57BL/6 mice remain healthy. We employed molecular genetic analysis to identify risk factors of calcification in fetuin-A deficient mice. We sought to identify pharmaceutical therapeutic targets that could be influenced by dietary of parenteral supplementation. We studied the progeny of an intercross of fetuin-A deficient DBA/2 and C57BL/6 mice to identify candidate risk genes involved in calcification. We determined that a hypomorphic mutation of the Abcc6 gene, a liver ATP transporter supplying systemic pyrophosphate, and failure to regulate the Trpm6 magnesium transporter in kidney were associated with severity of calcification. Calcification prone fetuin-A deficient mice were alternatively treated with parenteral administration of fetuin-A dietary magnesium supplementation, phosphate restriction, or by or parenteral pyrophosphate. All treatments markedly reduced soft tissue calcification, demonstrated by computed tomography, histology and tissue calcium measurement. We show that pathological ectopic calcification in fetuin-A deficient DBA/2 mice is caused by a compound deficiency of three major extracellular and systemic inhibitors of calcification, namely fetuin-A, magnesium, and pyrophosphate. All three of these are individually known to contribute to stabilize protein-mineral complexes and thus inhibit mineral precipitation from extracellular fluid. We show for the first time a compound triple deficiency that can be treated by simple dietary or parenteral supplementation. This is of special importance in patients with advanced CKD, who commonly exhibit reduced serum fetuin-A, magnesium and pyrophosphate levels.


Assuntos
Calcinose/patologia , Microvasos/fisiologia , alfa-2-Glicoproteína-HS/metabolismo , Animais , Calcinose/genética , Difosfatos/metabolismo , Modelos Animais de Doenças , Feminino , Rim/patologia , Fígado/patologia , Magnésio/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Microvasos/metabolismo , Minerais , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Insuficiência Renal Crônica/complicações , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismo , alfa-2-Glicoproteína-HS/fisiologia , alfa-Fetoproteínas
18.
J Bone Miner Metab ; 38(4): 469-480, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32020290

RESUMO

INTRODUCTION: Cold intolerance is defined as abnormal pain resulting from exposure to cold stimulation after trauma. However, the pathophysiology remains unclear. We recently demonstrated that regional osteoporotic changes accompanied by high bone turnover were involved in causing pain-like behaviors in the unloaded hind limbs of tail-suspended mice. Bisphosphonate prevented pain-like behaviors and high bone turnover conditions in tail-suspended mice. The aims of this study were to examine the relationship between regional osteoporotic changes and the induction of hypersensitivity to cold stimulation. MATERIALS AND METHODS: The hind limbs of tail-suspended mice were unloaded for 2 weeks. The von Frey test and paw-flick test assessed pain-like behaviors and cold plate test evaluated cold escape behaviors. Furthermore, we examined whether cold hypersensitivity associated with regional osteoporotic changes could be improved by bisphosphonate, TRPV1 and TRPA1 antagonists. RESULTS: Hypersensitivity to cold stimulation was induced more noticeably in the tail-suspended mice, and this effect was related to the increased expression of bone metabolism markers. In addition, the cold hypersensitivity was improved by the resumption of weight bearing and prevented by bisphosphonate or a TRPV1 antagonist, and was accompanied with a decrease in the expression of bone metabolism markers. TRPA1 antagonist significantly improved the cold escape behavior, but had no significant effects on the expression of those markers. CONCLUSION: We demonstrated that the regional osteoporotic changes accompanying a high bone turnover state could be involved in the induction of hypersensitivity to cold stimulation in the tail-suspended mice.


Assuntos
Temperatura Baixa , Síndromes Periódicas Associadas à Criopirina/complicações , Elevação dos Membros Posteriores , Osteoporose/complicações , Animais , Comportamento Animal , Diferenciação Celular , Difosfonatos/uso terapêutico , Elevação dos Membros Posteriores/efeitos adversos , Masculino , Camundongos Endogâmicos C57BL , Osteoblastos/patologia , Osteoclastos/patologia , Osteoporose/tratamento farmacológico , Osteoporose/patologia , Dor/patologia , Pele/patologia , Medula Espinal/patologia , Canal de Cátion TRPA1/antagonistas & inibidores , Canal de Cátion TRPA1/metabolismo , Canais de Cátion TRPM/metabolismo , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/metabolismo
19.
Am J Physiol Regul Integr Comp Physiol ; 318(3): R579-R589, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31967850

RESUMO

In contrast to other species, humans are believed to lack hygroreceptors for sensing skin wetness. Yet, the molecular basis of human hygrosensation is currently unknown, and it remains unclear whether we possess a receptor-mediated sensing mechanism for skin wetness. The aim of this study was to assess the role of the cutaneous cold-sensitive transient receptor potential melastatin-8 (TRPM8) channel as a molecular mediator of human hygrosensation. To this end, we exploited both the thermal and chemical activation of TRPM8-expressing cutaneous Aδ cold thermoreceptors, and we assessed wetness sensing in healthy young men in response to 1) dry skin cooling in the TRPM8 range of thermosensitivity and 2) application of the TRPM8 agonist menthol. Our results indicate that 1) independently of contact with moisture, a cold-dry stimulus in the TRPM8 range of activation induced wetness perceptions across 12 different body regions and those wetness perceptions varied across the body following regional differences in cold sensitivity; and 2) independently of skin cooling, menthol-induced stimulation of TRPM8 triggered wetness perceptions that were greater than those induced by physical dry cooling and by contact with an aqueous cream containing actual moisture. For the first time, we show that the cutaneous cold-sensing TRPM8 channel plays the dual role of cold and wetness sensor in human skin and that this ion channel is a peripheral mediator of human skin wetness perception.


Assuntos
Temperatura Baixa , Temperatura Cutânea/fisiologia , Canais de Cátion TRPM/metabolismo , Termorreceptores/fisiologia , Temperatura Baixa/efeitos adversos , Gânglios Espinais/fisiologia , Humanos , Células Receptoras Sensoriais/fisiologia , Sensação Térmica/fisiologia , Canais de Receptores Transientes de Potencial/metabolismo
20.
Int J Mol Med ; 45(4): 1017-1026, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31985026

RESUMO

Clinical studies have proven that ultraviolet B (UVB) based phototherapy can induce perifollicular and marginal repigmentation patterns in the skin of vitiligo patients. It is, however, difficult to conceive how melanocytes can easily exit from their tightly interconnected epidermal microenvironment to re­enter a different location in the skin to establish a new network with neighboring keratinocytes. While it is known that matrix metalloprotease 9 (MMP9) is involved in the degradation of the extracellular matrix in physiological or pathological processes, little is known about whether MMP9 affects melanocyte migration in vitiligo repigmentation. To investigate the effects of the p53­ transient receptor potential cation channel subfamily M member 1 (TRPM1)/microRNA (miR/miRNA)­211­MMP9 axis to regulate melanocyte migration following exposure to UVB, the expression profile of MMP9 in cultured human melanocytes transfected with or without the miR­211­mimic and p53­GFP lentiviral vector, respectively were determined. Quantitative polymerase chain reaction and western blotting were used to examine p53, TRPM1 and MMP9 mRNA and protein levels in UVB­exposed and unexposed cells. The capacity of melanocytes to migrate on collagen IV substrate was estimated using a Transwell migration assay. Interestingly, the upregulation of p53 and MMP9 at the mRNA and protein levels was evident in melanocytes treated with single or repeat exposures to UVB, whereas levels of TRPM1 and miR­211 were significantly suppressed in UVB­exposed melanocytes compared with the UVB­unexposed control cells. These results indicate that the p53­TRPM1/miR­211­MMP9 axis is significantly activated in melanocytes exposed to UVB. Notably, the ability of melanocyte migration was altered by the overexpression of p53 using a lentiviral vector and by the upregulation of miR­211 using an miRNA mimic. That altered migration could be neutralized by co­treatment with GM6001 (a broad­spectrum MMP inhibitor). Overall, these results show that the MMP9­mediated migration of melanocytes is regulated by a novel mechanism driven by the p53­TRPM1/miR­211­MMP9 axis. Activation of the p53­TRPM1/miR­211­MMP9 axis potentially represents an attractive therapeutic target to improve repigmentation outcomes in vitiligo patients.


Assuntos
Metaloproteinase 9 da Matriz/metabolismo , Melanócitos/metabolismo , Melanócitos/efeitos da radiação , MicroRNAs/metabolismo , Canais de Cátion TRPM/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Raios Ultravioleta , Adolescente , Adulto , Western Blotting , Movimento Celular/efeitos da radiação , Células Cultivadas , Humanos , Adulto Jovem
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