RESUMO
Transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a cation channel covalently linked to a protein kinase domain. TRPM7 is ubiquitously expressed and regulates key cellular processes such as Mg(2+) homeostasis, motility, and proliferation. TRPM7 is involved in anoxic neuronal death, cardiac fibrosis, and tumor growth. The goal of this work was to identify small molecule activators of the TRPM7 channel and investigate their mechanism of action. We used an aequorin bioluminescence-based assay to screen for activators of the TRPM7 channel. Valid candidates were further characterized using patch clamp electrophysiology. We identified 20 drug-like compounds with various structural backbones that can activate the TRPM7 channel. Among them, the δ opioid antagonist naltriben was studied in greater detail. Naltriben's action was selective among the TRP channels tested. Naltriben activates TRPM7 currents without prior depletion of intracellular Mg(2+) even under conditions of low PIP2. Moreover, naltriben interfered with the effect of the TRPM7 inhibitor NS8593. Finally, our experiments with TRPM7 variants carrying mutations in the pore, TRP, and kinase domains indicate that the site of TRPM7 activation by this small-molecule ligand is most likely located in or near the TRP domain. In conclusion, we identified the first organic small-molecule activators of TRPM7 channels, thus providing new experimental tools to study TRPM7 function in native cellular environments.
Assuntos
Bibliotecas de Moléculas Pequenas/farmacologia , Canais de Cátion TRPM/agonistas , Potenciais de Ação , Animais , Células HEK293 , Humanos , Magnésio/metabolismo , Camundongos , Naltrexona/análogos & derivados , Naltrexona/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Bibliotecas de Moléculas Pequenas/química , Canais de Cátion TRPM/metabolismoRESUMO
The luminal composition of the auditory tube influences its function. The mechanisms involved in the monitoring are currently not known. For the lower respiratory epithelium, such a sentinel role is carried out by cholinergic brush cells. Here, using two different mouse strains expressing eGFP under the control of the promoter of choline acetyltransferase (ChAT), we show the presence of solitary cholinergic villin-positive brush cells also in the mouse auditory tube epithelium. They express the vesicular acetylcholine (ACh) transporter and proteins of the taste transduction pathway such as α-gustducin, phospholipase C beta 2 (PLC(ß2)) and transient receptor potential cation channel subfamily M member 5 (TRPM5). Immunoreactivity for TRPM5 and PLCß2 was found regularly, whereas α-gustducin was absent in approximately 15% of the brush cells. Messenger RNA for the umami taste receptors (TasR), Tas1R1 and 3, and for the bitter receptors, Tas2R105 and Tas2R108, involved in perception of cycloheximide and denatonium were detected in the auditory tube. Using a transgenic mouse that expresses eGFP under the promotor of the nicotinic ACh receptor α3-subunit, we identified cholinoceptive nerve fibers that establish direct contacts to brush cells in the auditory tube. A subpopulation of these fibers displayed also CGRP immunoreactivity. Collectively, we show for the first time the presence of brush cells in the auditory tube. These cells are equipped with all proteins essential for sensing the composition of the luminal microenvironment and for communication of the changes to the CNS via attached sensory nerve fibers.
Assuntos
Células Quimiorreceptoras/citologia , Neurônios Colinérgicos/citologia , Tuba Auditiva/citologia , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Língua/citologiaRESUMO
Investigations into Drosophila mutants with impaired vision due to mutations in the transient receptor potential gene (trp) initiated a systematic search for TRP homologs in other species, finally leading to the discovery of a whole new family of plasma membrane cation channels involved in multiple physiological processes. Among the recently discovered TRP cation channels two homologous proteins, TRPM6 and TRPM7, display unique domain compositions and biophysical properties. These remarkable genes are vital for Mg(2+) homeostasis in vertebrates and, if disrupted, lead to cell death or human disease.
Assuntos
Magnésio/metabolismo , Fosfotransferases/fisiologia , Transdução de Sinais/fisiologia , Canais de Cátion TRPM/fisiologia , Animais , Sequência de Bases , Humanos , Camundongos , Dados de Sequência Molecular , Fosfotransferases/genética , Fosfotransferases/metabolismo , Proteínas Serina-Treonina Quinases , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
Specialized epithelial cells in the respiratory tract such as solitary chemosensory cells and brush cells sense the luminal content and initiate protective reflexes in response to the detection of potentially harmful substances. The majority of these cells are cholinergic and utilize the canonical taste signal transduction cascade to detect "bitter" substances such as bacterial quorum sensing molecules. Utilizing two different mouse strains reporting expression of choline acetyltransferase (ChAT), the synthesizing enzyme of acetylcholine (ACh), we detected cholinergic cells in the submucosal glands of the murine larynx and trachea. These cells were localized in the ciliated glandular ducts and were neither found in the collecting ducts nor in alveolar or tubular segments of the glands. ChAT expression in tracheal gland ducts was confirmed by in situ hybridization. The cholinergic duct cells expressed the brush cell marker proteins, villin and cytokeratin-18, and were immunoreactive for components of the taste signal transduction cascade (Gα-gustducin, transient receptor potential melastatin-like subtype 5 channel = TRPM5, phospholipase C(ß2)), but not for carbonic anhydrase IV. Furthermore, these cells expressed the bitter taste receptor Tas2r131, as demonstrated utilizing an appropriate reporter mouse strain. Our study identified a previously unrecognized presumptive chemosensory cell type in the duct of the airway submucosal glands that likely utilizes ACh for paracrine signaling. We propose that these cells participate in infection-sensing mechanisms and initiate responses assisting bacterial clearance from the lower airways.
Assuntos
Acetilcolina/metabolismo , Células Quimiorreceptoras/metabolismo , Células Epiteliais/metabolismo , Laringe/citologia , Traqueia/citologia , Animais , Colina O-Acetiltransferase/genética , Colina O-Acetiltransferase/metabolismo , Regulação Enzimológica da Expressão Gênica , Proteínas de Fluorescência Verde , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
In organized tissues, the precise geometry and the overall shape are critical for the specialized functions that the cells carry out. Odontoblasts are major matrix-producing cells of the tooth and have also been suggested to participate in sensory transmission. However, refined morphologic data on these important cells are limited, which hampers the analysis and understanding of their cellular functions. We took advantage of fluorescent color-coding genetic tracing to visualize and reconstruct in 3 dimensions single odontoblasts, pulp cells, and their assemblages. Our results show distinct structural features and compartments of odontoblasts at different stages of maturation, with regard to overall cellular shape, formation of the main process, orientation, and matrix deposition. We demonstrate previously unanticipated contacts between the processes of pulp cells and odontoblasts. All reported data are related to mouse incisor tooth. We also show that odontoblasts express TRPM5 and Piezo2 ion channels. Piezo2 is expressed ubiquitously, while TRPM5 is asymmetrically distributed with distinct localization to regions proximal to and within odontoblast processes.
Assuntos
Imageamento Tridimensional/métodos , Odontoblastos/citologia , Ameloblastos/citologia , Ameloblastos/ultraestrutura , Animais , Compartimento Celular , Núcleo Celular/ultraestrutura , Forma Celular , Extensões da Superfície Celular/ultraestrutura , Polpa Dentária/citologia , Polpa Dentária/ultraestrutura , Dentina/ultraestrutura , Matriz Extracelular/ultraestrutura , Imunofluorescência , Incisivo/citologia , Incisivo/ultraestrutura , Canais Iônicos/ultraestrutura , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/ultraestrutura , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica de Varredura/métodos , Odontoblastos/ultraestrutura , Canais de Cátion TRPM/ultraestruturaRESUMO
We studied the influence of plasma membrane depolarization on cAMP content in presynaptic nerve endings (synaptosomes) isolated from brain hemispheres (HS) and cerebellum (CS). Depolarization by elevated [K+]o decreased basal cAMP level in both types of synaptosomes; reduced cAMP content in HS and increased cAMP in CS in the presence of IBMX; and lowered forskolin-stimulated cAMP accumulation in both the HS and the CS. Similar results were obtained when depolarization was induced by veratrine or when [Ca2+]i was elevated by treatment of the synaptosomes with the ionophore A23187. In Ca2+-free media, depolarization was not able to affect the synaptosomal cAMP levels. These data suggest that in brain synaptosomes intracellular cAMP pathway is modulated by alterations in [Ca2+]i.
Assuntos
Encéfalo/metabolismo , AMP Cíclico/metabolismo , Sinaptossomos/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Adenilil Ciclases/metabolismo , Animais , Encéfalo/ultraestrutura , Calcimicina/farmacologia , Cálcio/metabolismo , Membrana Celular/fisiologia , Cerebelo/enzimologia , Cerebelo/fisiologia , Eletrofisiologia , Técnicas In Vitro , Ionóforos/farmacologia , Masculino , Potássio/metabolismo , Ratos , Ratos Wistar , Veratrina/farmacologiaRESUMO
The effects of long-term prenatal gamma-radiation (0.5 Gy) on the glucagon signalling through adenylyl cyclase in adult rat liver have been investigated. The coupling of receptor/Gs-protein/adenylyl cyclase was tested using kinetic constants for stimulation of adenylyl cyclase by GTP, Gp(NH)pp, AlF4- and glucagon. The estimated data suggests that prenatal chronic gamma-radiation prompted (i) a decrease in rate of GTP hydrolysis on Gs-protein; (ii) a reduction of glucagon potency to accelerate the exchange GDP for GTP on Gs-protein.
Assuntos
Adenilil Ciclases/efeitos da radiação , Fígado/metabolismo , Fígado/efeitos da radiação , Exposição Materna/efeitos adversos , Adenilil Ciclases/metabolismo , Animais , Radioisótopos de Césio , AMP Cíclico/metabolismo , Feminino , Proteínas de Ligação ao GTP/metabolismo , Raios gama , Glucagon/metabolismo , Hidrólise , Gravidez , Doses de Radiação , Radioimunoensaio , Ratos , Receptores de Glucagon/metabolismoRESUMO
In the present work the effects of long-term prenatal gamma-irradiation (0.5 Gy) on the glucagon signalling through adenylyl cyclase have been investigated. In utero gamma-irradiation resulted in the increase of basal and GTP-stimulated adenylyl cyclase activity, whereas, the adenylyl cyclase response to glucagon was essentially reduced. The comparison of kinetic constants estimated from dose-response data for GTP and glucagon suggests that prenatal chronic irradiation prompted (i) the decrease in rate of GTP hydrolysis on Gs-protein; (ii) the reduction of glucagon potency to accelerate the exchange GDP for GTP on Gs-protein.
Assuntos
Adenilil Ciclases/metabolismo , Fígado/efeitos da radiação , Efeitos Tardios da Exposição Pré-Natal , Animais , AMP Cíclico/biossíntese , Ativação Enzimática , Feminino , Raios gama , Glucagon/biossíntese , Glucagon/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Fígado/enzimologia , Fígado/metabolismo , Gravidez , Ratos , Transdução de Sinais/efeitos da radiaçãoRESUMO
BACKGROUND AND PURPOSE: Transient receptor potential cation channel subfamily M member 7 (TRPM7) is a bifunctional protein comprising a TRP ion channel segment linked to an α-type protein kinase domain. TRPM7 is essential for proliferation and cell growth. Up-regulation of TRPM7 function is involved in anoxic neuronal death, cardiac fibrosis and tumour cell proliferation. The goal of this work was to identify non-toxic inhibitors of the TRPM7 channel and to assess the effect of blocking endogenous TRPM7 currents on the phenotype of living cells. EXPERIMENTAL APPROACH: We developed an aequorin bioluminescence-based assay of TRPM7 channel activity and performed a hypothesis-driven screen for inhibitors of the channel. The candidates identified were further assessed electrophysiologically and in cell biological experiments. KEY RESULTS: TRPM7 currents were inhibited by modulators of small conductance Ca²âº -activated K⺠channels (K(Ca)2.1-2.3; SK) channels, including the antimalarial plant alkaloid quinine, CyPPA, dequalinium, NS8593, SKA31 and UCL 1684. The most potent compound NS8593 (IC50 1.6 µM) specifically targeted TRPM7 as compared with other TRP channels, interfered with Mg²âº -dependent regulation of TRPM7 channel and inhibited the motility of cultured cells. NS8593 exhibited full and reversible block of native TRPM7-like currents in HEK 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. CONCLUSIONS AND IMPLICATIONS: This study reveals a tight overlap in the pharmacological profiles of TRPM7 and K(Ca)2.1-2.3 channels. NS8593 acts as a negative gating modulator of TRPM7 and is well-suited to study functional features and cellular roles of endogenous TRPM7.
Assuntos
Descoberta de Drogas , Magnésio/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Baixa/antagonistas & inibidores , Canais de Cátion TRPM/antagonistas & inibidores , 1-Naftilamina/efeitos adversos , 1-Naftilamina/análogos & derivados , 1-Naftilamina/farmacologia , Animais , Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Potenciais da Membrana/efeitos dos fármacos , Moduladores de Transporte de Membrana/efeitos adversos , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Miócitos de Músculo Liso/metabolismo , Podócitos/citologia , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Bloqueadores dos Canais de Potássio/efeitos adversos , Isoformas de Proteínas/antagonistas & inibidores , Proteínas Serina-Treonina Quinases , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Canais de Cátion TRPM/genética , Canais de Cátion TRPM/metabolismoRESUMO
Neuropeptide hormones like bombesin/gastrin-releasing peptide, galanin or bradykinin, acting via auto and paracrine growth loops, represent the principal mitogens of small cell lung cancer (SCLC). These mitogenic neuropeptides activate G(q/11)-coupled receptors which stimulate phospholipase Cbeta activity, followed by rises of the intracellular calcium concentration ([Ca2+](i)) and activation of protein kinase C (PKC). We report here that proline-rich tyrosine kinase Pyk2 is highly expressed in SCLC cells and provides a functional link between neuropeptide-induced increases in [Ca2+](i) and tumor cell proliferation. Activation of Pyk2 and its association with Src kinases critically depends on the elevation of [Ca2+](i), but is independent of PKC. Src kinase activities are crucial for neuropeptide-mediated GTP-loading of Ras and activation of extracellular signal-regulated kinases in SCLC cells. Pyk2 and Src kinases essentially contribute to anchorage-independent proliferation of SCLC cells. Inhibition of either Pyk2 or Src kinases by lentiviral RNAi or pharmacological inhibition with PP2, respectively, attenuated basal and neuropeptide-elicited survival and proliferation of SCLC cells in liquid culture and in soft agar. Thus, neuropeptides stimulate anchorage-independent survival and proliferation of SCLC cells via pathways involving Pyk2 and Src kinases. Therefore, Ca2+-induced Pyk2/Src complex formation may be a rewarding molecular target for novel therapeutic strategies in SCLC cells.