RESUMO
Serotonin (5-hydroxytryptamine; 5-HT) is abundantly present throughout the gastrointestinal tract and stored mostly in enterochromaffin (EC) cells, which are located on the mucosal surface. 5-HT released from EC cells stimulate both intrinsic and extrinsic nerves, which results in various physiological and pathophysiological responses, such as gastrointestinal contractions. EC cells are believed to have the ability to respond to the chemical composition of the luminal contents of the gut; however, the underlying molecular and cellular mechanisms have not been identified. Here, we demonstrate that the transient receptor potential (TRP) cation channel TRPA1, which is activated by pungent compounds or cold temperature, is highly expressed in EC cells. We also found that TRPA1 agonists, including allyl isothiocyanate and cinnamaldehyde, stimulate EC cell functions, such as increasing intracellular Ca(2+) levels and 5-HT release, by using highly concentrated EC cell fractions and a model of EC cell function, the RIN14B cell line. Furthermore, we showed that allyl isothiocyanate promotes the contraction of isolated guinea pig ileum via the 5-HT(3) receptor. Taken together, our results indicate that TRPA1 acts as a sensor molecule for EC cells and may regulate gastrointestinal function.
Assuntos
Canais de Cálcio/metabolismo , Células Enterocromafins/metabolismo , Motilidade Gastrointestinal , Proteínas do Tecido Nervoso/metabolismo , Serotonina/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Anquirinas , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular , Regulação da Expressão Gênica/genética , Cobaias , Humanos , Masculino , Camundongos , Proteínas do Tecido Nervoso/genética , Ratos , Canal de Cátion TRPA1 , Canais de Cátion TRPC , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Hepatocellular carcinoma is an aggressive cancer with poor prognosis. Fibroblast growth factor 19, a member of the fibroblast growth factor family, is a ligand for fibroblast growth factor receptor 4. Moreover, it plays a crucial role in the progression of hepatocellular carcinoma. ASP5878 is a novel inhibitor of fibroblast growth factor receptors 1, 2, 3, and 4 that is under development. It inhibits fibroblast growth factor receptor 4 kinase activity with an IC50 of 3.5 nmol/L. ASP5878 potently suppressed the growth of the fibroblast growth factor 19-expressing hepatocellular carcinoma cell lines Hep3B2.1-7, HuH-7, and JHH-7. In the Hep3B2.1-7 cell line, ASP5878 inhibited the phosphorylation of fibroblast growth factor receptor 4 and its downstream signaling molecules as well as induced apoptosis. Oral administration of ASP5878 at 3 mg/kg induced sustained tumor regression in a subcutaneous xenograft mouse model using Hep3B2.1-7. In HuH-7, an orthotopic xenograft mouse model, ASP5878 induced complete tumor regression and dramatically extended the survival of the mice. These results suggest that ASP5878 is a potentially effective therapeutic agent for hepatocellular carcinoma patients with tumors expressing fibroblast growth factor 19. Mol Cancer Ther; 16(1); 68-75. ©2016 AACR.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/genética , Fatores de Crescimento de Fibroblastos/genética , Expressão Gênica , Neoplasias Hepáticas/genética , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/química , Pirimidinas/química , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The NMDA receptor (NMDAR) is a Ca (2+)-permeable cation channel that plays a critical role in neural network formation during brain development. Since it is blocked in a voltage-dependent manner by extracellular Mg(2+), in order for the NMDA to be activated, the membrane must be strongly depolarized. Immature neurons in the developing neocortex can be depolarized by ligand-gated Cl(-) channels, such as the glycine receptor (GlyR) or GABA(A) receptor (GABA(A) R). We here assess the contribution of GlyRs to Ca(2+) influx via NMDARs in neonatal mouse cortical neurons. The GlyR antagonist, strychnine, was more effective in suppressing postsynaptic Ca(2+) influx than the GABA(A) R antagonist, picrotoxin, suggesting greater potentiation of NMDARs by GlyRs than by GABA(A) Rs. The GlyR, known to be endogenously activated at this stage, may play a critical role in neocortical development.
Assuntos
Glicinérgicos/farmacologia , Ácido Cinurênico/análogos & derivados , Neocórtex/crescimento & desenvolvimento , Neocórtex/metabolismo , Neurônios/metabolismo , Receptores de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Estricnina/farmacologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Interações Medicamentosas/fisiologia , Estimulação Elétrica , Antagonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas GABAérgicos/farmacologia , Antagonistas de Receptores de GABA-A , Ácido Cinurênico/farmacologia , Magnésio/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Knockout , N-Metilaspartato/farmacologia , Neocórtex/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Picrotoxina/farmacologia , Receptores de GABA-A/metabolismo , Receptores de Glicina/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/deficiência , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/genética , Transmissão Sináptica/efeitos dos fármacosRESUMO
Spontaneous correlated activity regulates the precision of developing neural circuits. A synchronized elevation of intracellular calcium ion concentration, [Ca(2+)](i), occurred in 5-50 adjacent neurons--known as a "neuronal domain"--in developing neocortex. This coordinated response of neuronal cells is mediated by the diffusion of inositol trisphosphate (IP(3)) via gap-junction channels. In this study, we utilized the N-methyl-D-aspartate (NMDA)-type glutamate receptor epsilon 2 (GluR epsilon 2/NR2B)(-/-) mouse, which does not possess any functional NMDA receptors in the developing neocortex, and showed that NMDA receptors are essential for the generation of "neuronal domains". First, the frequency of spontaneously occurring neuronal domains in brain slices from GluR epsilon 2(-/-) mice was significantly reduced compared to that seen in brain slices from wild-type mice. Secondly, IP(3) injection into a single neuron in a cortical slice from a GluR epsilon 2(-/-) brain resulted in very few neuronal domains being observed, but an injection similarly made into a neuron in a wild-type slice promptly resulted in neuronal domains. Even in the GluR epsilon 2(-/-) brain, the elevation of intracellular [Ca2+](i) was observed frequently in single neurons and microinjection of IP(3) produced an elevation of [Ca2+](i) in the injected cells. These results suggest that the diffusion of IP(3) into the surrounding neurons via gap junctions is almost completely absent in the GluR epsilon 2(-/-) brain. Our results may reflect the critical role of NMDA receptors in the formation of cortical circuitry, probably via the regulation of gap-junction channels between immature cortical neurons.
Assuntos
Cálcio/metabolismo , Neocórtex/citologia , Neocórtex/metabolismo , Neurônios/metabolismo , Receptores de N-Metil-D-Aspartato/deficiência , Animais , Animais Recém-Nascidos , Canais de Cálcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato , Camundongos , Camundongos Mutantes , Morfogênese , Neocórtex/crescimento & desenvolvimento , Rede Nervosa , Neurônios/citologia , Receptores Citoplasmáticos e Nucleares/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologiaRESUMO
To find a novel human ion channel gene we have executed an extensive search by using a human genome draft sequencing data base. Here we report a novel two-pore domain K+ channel, TRESK (TWIK-related spinal cord K+ channel). TRESK is coded by 385 amino acids and shows low homology (19%) with previously characterized two-pore domain K+ channels. However, the most similar channel is TREK-2 (two-pore domain K+ channel), and TRESK also has two pore-forming domains and four transmembrane domains that are evolutionarily conserved in the two-pore domain K+ channel family. Moreover, we confirmed that TRESK is expressed in the spinal cord. Electrophysiological analysis demonstrated that TRESK induced outward rectification and functioned as a background K+ channel. Pharmacological analysis showed TRESK to be inhibited by previously reported K+ channel inhibitors Ba2+, propafenone, glyburide, lidocaine, quinine, quinidine, and triethanolamine. Functional analysis demonstrated TRESK to be inhibited by unsaturated free fatty acids such as arachidonic acid and docosahexaenoic acid. TRESK is also sensitive to extreme changes in extracellular and intracellular pH. These results indicate that TRESK is a novel two-pore domain K+ channel that may set the resting membrane potential of cells in the spinal cord.