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1.
J Neurochem ; 147(1): 40-57, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29920676

RESUMO

Neuritin is a neurotrophic factor that is activated by neural activity and neurotrophins. Its major function is to promote neurite growth and branching; however, the underlying mechanisms are not fully understood. To address this issue, this study investigated the effects of neuritin on neurite and spine growth and intracellular Ca2+ concentration in rat cerebellar granule neurons (CGNs). Incubation of CGNs for 24 h with neuritin increased neurite length and spine density; this effect was mimicked by insulin and abolished by inhibiting insulin receptor (IR) or mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (ERK) activity. Calcium imaging and western blot analysis revealed that neuritin enhanced the increase in intracellular Ca2+ level induced by high K+ , and stimulated the cell surface expression of CaV 1.2 and CaV 1.3 α subunits of the L-type calcium channel, which was suppressed by inhibition of IR or mitogen-activated protein kinase kinase/ERK. Treatment with inhibitors of L-type calcium channels, calmodulin, and calcineurin (CaN) abrogated the effects of neuritin on neurite length and spine density. A similar result was obtained by silencing nuclear factor of activated T cells c4, which is known to be activated by neuritin in CGNs. These results indicate that IR and ERK signaling as well as the Ca2+ /CaN/nuclear factor of activated T cells c4 axis mediate the effects of neuritin on neurite and spine growth in CGNs. OPEN PRACTICES: Open Science: This manuscript was awarded with the Open Materials Badge. For more information see: https://cos.io/our-services/open-science-badges/ Cover Image for this issue: doi: 10.1111/jnc.14195.


Assuntos
Canais de Cálcio Tipo L/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Cerebelo/citologia , Espinhas Dendríticas/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Neuropeptídeos/farmacologia , Animais , Canais de Cálcio/metabolismo , Canais de Cálcio Tipo L/metabolismo , Cerebelo/efeitos dos fármacos , Cerebelo/crescimento & desenvolvimento , Grânulos Citoplasmáticos/efeitos dos fármacos , Feminino , Proteínas Ligadas por GPI/farmacologia , Inativação Gênica , Humanos , Insulina/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fatores de Transcrição NFATC/antagonistas & inibidores , Fatores de Transcrição NFATC/genética , Ratos , Ratos Sprague-Dawley , Receptor de Insulina/antagonistas & inibidores
2.
Neurosci Bull ; 34(5): 759-768, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29671217

RESUMO

Cyproheptadine (CPH), a first-generation antihistamine, enhances the delayed rectifier outward K+ current (IK) in mouse cortical neurons through a sigma-1 receptor-mediated protein kinase A pathway. In this study, we aimed to determine the effects of CPH on neuronal excitability in current-clamped pyramidal neurons in mouse medial prefrontal cortex slices. CPH (10 µmol/L) significantly reduced the current density required to generate action potentials (APs) and increased the instantaneous frequency evoked by a depolarizing current. CPH also depolarized the resting membrane potential (RMP), decreased the delay time to elicit an AP, and reduced the spike threshold potential. This effect of CPH was mimicked by a sigma-1 receptor agonist and eliminated by an antagonist. Application of tetraethylammonium (TEA) to block IK channels hyperpolarized the RMP and reduced the instantaneous frequency of APs. TEA eliminated the effects of CPH on AP frequency and delay time, but had no effect on spike threshold or RMP. The current-voltage relationship showed that CPH increased the membrane depolarization in response to positive current pulses and hyperpolarization in response to negative current pulses, suggesting that other types of membrane ion channels might also be affected by CPH. These results suggest that CPH increases the excitability of medial prefrontal cortex neurons by regulating TEA-sensitive IK channels as well as other TEA-insensitive K+ channels, probably ID and inward-rectifier Kir channels. This effect of CPH may explain its apparent clinical efficacy as an antidepressant and antipsychotic.


Assuntos
Ciproeptadina/farmacologia , Antagonistas dos Receptores Histamínicos H1/farmacologia , Córtex Pré-Frontal/efeitos dos fármacos , Células Piramidais/efeitos dos fármacos , Animais , Feminino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Córtex Pré-Frontal/fisiologia , Células Piramidais/fisiologia , Receptores sigma/agonistas , Receptores sigma/metabolismo , Tetraetilamônio/farmacologia , Técnicas de Cultura de Tecidos
3.
Acta Pharmacol Sin ; 39(9): 1414-1420, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29595190

RESUMO

Neuritin is a member of the neurotrophic factor family, which is activated by neural activity and neurotrophins, and promotes neurite growth and branching. It has shown to play an important role in neuronal plasticity and regeneration. It is also involved in other biological processes such as angiogenesis, tumorigenesis and immunomodulation. Thus far, however, the primary mechanisms of neuritin, including whether or not it acts through a receptor or which downstream signals might be activated following binding, are not fully understood. Recent evidence suggests that neuritin may be a potential therapeutic target in several neurodegenerative diseases. This review focuses on the recent advances in studies regarding the newly identified functions of neuritin and the signaling pathways related to these functions. We also discuss current hot topics and difficulties in neuritin research.


Assuntos
Neuropeptídeos/fisiologia , Transdução de Sinais/fisiologia , Animais , Proteínas Ligadas por GPI/fisiologia , Humanos , Transtornos Mentais/etiologia , Transtornos Mentais/fisiopatologia , Neurogênese/fisiologia , Plasticidade Neuronal/fisiologia , Sinapses/fisiologia
4.
Cereb Cortex ; 27(7): 3842-3855, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28475719

RESUMO

Neuritin is a neurotrophic factor involved in neural development and synaptic plasticity. However, its role in modulating synaptic transmission remains unclear. Here, we investigated the effects of neuritin on miniature excitatory postsynaptic currents (mEPSCs) and glutamate release in the medial prefrontal cortex (mPFC) in mice. Incubation of mPFC slices with neuritin for 45 min significantly increased mEPSC frequency and glutamate release as measured by high-performance liquid chromatography, which was mimicked by insulin and abrogated by an insulin receptor (IR) inhibitor. Neuritin-induced upregulation of synaptic transmission was correlated with activation of ERK, and inhibition of mitogen-activated protein kinases/extracellular signal-regulated kinases (MEK/ERK) activity attenuated the neuritin-induced increase in mEPSC frequency and glutamate release. T-type calcium channel inhibitors but not the L-type inhibitor abolished the inward calcium current and the effects of neuritin on mEPSC frequency and glutamate release. Western blotting of membrane proteins showed that neuritin promoted surface expression of CaV3.3 α-subunit, which was also eliminated by inhibition of IR or MEK/ERK activity. The effects of neuritin on mEPSC frequency, glutamate release, and CaV3.3 α-subunit expression were inhibited by an intracellular protein-transport inhibitor. These results confirm involvement of the IR and ERK signaling pathway, and provide novel insights into the mechanisms of neuritin function in synaptic transmission.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neuropeptídeos/farmacologia , Córtex Pré-Frontal/citologia , Transmissão Sináptica/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Relação Dose-Resposta a Droga , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Feminino , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/farmacologia , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Neuropeptídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Coloração pela Prata , Transmissão Sináptica/fisiologia , Fatores de Tempo , Transdução Genética
5.
Sci Rep ; 7: 44521, 2017 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-28303965

RESUMO

Mounting evidence suggests that exposure to radiofrequency electromagnetic radiation (RF-EMR) can influence learning and memory in rodents. In this study, we examined the effects of single exposure to 1.8 GHz RF-EMR for 30 min on subsequent recognition memory in mice, using the novel object recognition task (NORT). RF-EMR exposure at an intensity of >2.2 W/kg specific absorption rate (SAR) power density induced a significant density-dependent increase in NORT index with no corresponding changes in spontaneous locomotor activity. RF-EMR exposure increased dendritic-spine density and length in hippocampal and prefrontal cortical neurons, as shown by Golgi staining. Whole-cell recordings in acute hippocampal and medial prefrontal cortical slices showed that RF-EMR exposure significantly altered the resting membrane potential and action potential frequency, and reduced the action potential half-width, threshold, and onset delay in pyramidal neurons. These results demonstrate that exposure to 1.8 GHz RF-EMR for 30 min can significantly increase recognition memory in mice, and can change dendritic-spine morphology and neuronal excitability in the hippocampus and prefrontal cortex. The SAR in this study (3.3 W/kg) was outside the range encountered in normal daily life, and its relevance as a potential therapeutic approach for disorders associated with recognition memory deficits remains to be clarified.


Assuntos
Campos Eletromagnéticos/efeitos adversos , Radiação Eletromagnética , Reconhecimento Visual de Modelos/efeitos da radiação , Células Piramidais/efeitos da radiação , Potenciais de Ação/efeitos da radiação , Animais , Espinhas Dendríticas/patologia , Espinhas Dendríticas/efeitos da radiação , Hipocampo/fisiopatologia , Hipocampo/efeitos da radiação , Memória , Transtornos da Memória/etiologia , Transtornos da Memória/fisiopatologia , Camundongos , Células Piramidais/patologia , Ondas de Rádio/efeitos adversos
6.
Sheng Li Xue Bao ; 69(1): 109-121, 2017 Feb 25.
Artigo em Chinês | MEDLINE | ID: mdl-28217814

RESUMO

Growth differentiation factor-15 (GDF-15) is a member of the transforming growth factor beta superfamily. GDF-15 expression is dramatically upregulated during acute brain injury, cancer, cardiovascular disease, and inflammation, suggesting its potential value as a disease biomarker. It has been suggested that GDF-15 has neurotropic effects in the nervous system. Our studies showed that GDF-15 modulated the expression of neuronal K+ and Ca2+ ion channels and increased the release of excitatory transmitter in the medial prefrontal cortex of mice. GDF-15 is also involved in the complex modulation of cancer and cardiovascular disease. Here, we reviewed studies involving the modulation of GDF-15 expression and its mechanisms, the primary pathological and physiological functions of GDF-15 in neurological and cardiovascular systems, and its role in cancer progression. The biological effects and the values of GDF-15 in basic research and clinical applications were also addressed.


Assuntos
Doenças Cardiovasculares/fisiopatologia , Fator 15 de Diferenciação de Crescimento/metabolismo , Neoplasias/fisiopatologia , Sistema Nervoso/metabolismo , Animais , Lesões Encefálicas/fisiopatologia , Canais de Cálcio/metabolismo , Progressão da Doença , Humanos , Inflamação , Camundongos , Canais de Potássio/metabolismo , Córtex Pré-Frontal/metabolismo , Fator de Crescimento Transformador beta , Regulação para Cima
7.
Hypertension ; 68(3): 785-95, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27432863

RESUMO

Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism.


Assuntos
Córtex Suprarrenal/citologia , Aldosterona/metabolismo , Agonistas dos Canais de Cálcio/farmacologia , Hiperaldosteronismo/fisiopatologia , Canais de Potássio/metabolismo , Córtex Suprarrenal/metabolismo , Adulto , Idoso , Análise de Variância , Angiotensina II/administração & dosagem , Apamina/administração & dosagem , Células Cultivadas/efeitos dos fármacos , Feminino , Humanos , Hiperaldosteronismo/metabolismo , Masculino , Pessoa de Meia-Idade , Canais de Potássio/efeitos dos fármacos , RNA Mensageiro/metabolismo , Amostragem
8.
J Biol Chem ; 291(33): 17369-81, 2016 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-27307045

RESUMO

Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current (IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca(2+)/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca(2+) and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4(-/-) mice but not in Nfatc2(-/-) mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4(-/-) mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions.


Assuntos
Calcineurina/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Espinhas Dendríticas/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores de Transcrição NFATC/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/metabolismo , Canais de Potássio Shal/biossíntese , Animais , Calcineurina/genética , Cerebelo/metabolismo , Espinhas Dendríticas/genética , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células HeLa , Humanos , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Proteínas do Tecido Nervoso/genética , Neuropeptídeos/genética , Canais de Potássio Shal/genética
9.
Sci Rep ; 6: 28653, 2016 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-27353765

RESUMO

Growth differentiation factor-15 (GDF-15) has been implicated in ischemic brain injury and synapse development, but its involvement in modulating neuronal excitability and synaptic transmission remain poorly understood. In this study, we investigated the effects of GDF-15 on non-evoked miniature excitatory post-synaptic currents (mEPSCs) and neurotransmitter release in the medial prefrontal cortex (mPFC) in mice. Incubation of mPFC slices with GDF-15 for 60 min significantly increased the frequency of mEPSCs without effect on their amplitude. GDF-15 also significantly elevated presynaptic glutamate release, as shown by HPLC. These effects were blocked by dual TGF-ß type I receptor (TßRI) and TGF-ß type II receptor (TßRII) antagonists, but not by a TßRI antagonist alone. Meanwhile, GDF-15 enhanced pERK level, and inhibition of MAPK/ERK activity attenuated the GDF-15-induced increases in mEPSC and glutamate release. Blocking T-type calcium channels reduced the GDF-15 induced up-regulation of synaptic transmission. Membrane-protein extraction and use of an intracellular protein-transport inhibitor showed that GDF-15 promoted CaV3.1 and CaV3.3 α-subunit expression by trafficking to the membrane. These results confirm previous findings in cerebellar granule neurons, in which GDF-15 induces its neurobiological effects via TßRII and activation of the ERK pathway, providing novel insights into the mechanism of GDF-15 function in cortical neurons.


Assuntos
Canais de Cálcio Tipo T/biossíntese , Ácido Glutâmico/metabolismo , Fator 15 de Diferenciação de Crescimento/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Córtex Pré-Frontal/metabolismo , Transmissão Sináptica/fisiologia , Animais , Feminino , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/metabolismo
11.
Biochem J ; 473(13): 1895-904, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27114559

RESUMO

GDF-15 (growth/differentiation factor 15) is a novel member of the TGF (transforming growth factor)-ß superfamily that has critical roles in the central and peripheral nervous systems. We reported previously that GDF-15 increased delayed rectifier outward K(+) currents and Kv2.1 α subunit expression through TßRII (TGF-ß receptor II) to activate Src kinase and Akt/mTOR (mammalian target of rapamycin) signalling in rat CGNs (cerebellar granule neurons). In the present study, we found that treatment of CGNs with GDF-15 for 24 h increased the intracellular Ca(2+) concentration ([Ca(2+)]i) in response to membrane depolarization, as determined by Ca(2+) imaging. Whole-cell current recordings indicated that GDF-15 increased the inward Ca(2+) current (ICa) without altering steady-state activation of Ca(2+) channels. Treatment with nifedipine, an inhibitor of L-type Ca(2+) channels, abrogated GDF-15-induced increases in [Ca(2+)]i and ICa The GDF-15-induced increase in ICa was mediated via up-regulation of the Cav1.3 α subunit, which was attenuated by inhibiting Akt/mTOR and ERK (extracellular-signal-regulated kinase) pathways and by pharmacological inhibition of Src-mediated TßRII phosphorylation. Given that Cav1.3 is not only a channel for Ca(2+) influx, but also a transcriptional regulator, our data confirm that GDF-15 induces protein expression via TßRII and activation of a non-Smad pathway, and provide novel insight into the mechanism of GDF-15 function in neurons.


Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Cerebelo/citologia , Fator 15 de Diferenciação de Crescimento/farmacologia , Neurônios/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neurônios/efeitos dos fármacos , Nifedipino/farmacologia , Proteína Oncogênica v-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
12.
Sci Rep ; 6: 21774, 2016 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-26887777

RESUMO

Accumulating evidence suggests significant biological effects caused by extremely low frequency electromagnetic fields (ELF-EMF). Although exo-endocytosis plays crucial physical and biological roles in neuronal communication, studies on how ELF-EMF regulates this process are scarce. By directly measuring calcium currents and membrane capacitance at a large mammalian central nervous synapse, the calyx of Held, we report for the first time that ELF-EMF critically affects synaptic transmission and plasticity. Exposure to ELF-EMF for 8 to 10 days dramatically increases the calcium influx upon stimulation and facilitates all forms of vesicle endocytosis, including slow and rapid endocytosis, endocytosis overshoot and bulk endocytosis, but does not affect the RRP size and exocytosis. Exposure to ELF-EMF also potentiates PTP, a form of short-term plasticity, increasing its peak amplitude without impacting its time course. We further investigated the underlying mechanisms and found that calcium channel expression, including the P/Q, N, and R subtypes, at the presynaptic nerve terminal was enhanced, accounting for the increased calcium influx upon stimulation. Thus, we conclude that exposure to ELF-EMF facilitates vesicle endocytosis and synaptic plasticity in a calcium-dependent manner by increasing calcium channel expression at the nerve terminal.


Assuntos
Canais de Cálcio/fisiologia , Cálcio/química , Campos Eletromagnéticos , Endocitose , Terminações Pré-Sinápticas/fisiologia , Sinapses/fisiologia , Animais , Comunicação Celular , Exocitose , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal , Neurônios/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Proteínas SNARE/fisiologia
13.
Cell Physiol Biochem ; 37(5): 1903-13, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26584289

RESUMO

BACKGROUND/AIMS: Arachidonic acid (AA) and its metabolites are important endogenous lipid messengers. In this study, we test the effect of Leukotriene B4 (LTB4), a 5-lipoxygenase metabolite of AA, on L-type calcium channels in A7r5 rat aortic vascular smooth muscle cells. METHODS: L-type calcium channel currents were recorded by a patch-clamp technique. The mRNA expression of CaV1.2 was determined by Real-time RT-PCR. The protein expression of CaV1.2 and p38 activity was determined by Western blot analysis. RESULTS: LTB4 inhibits L-type channel currents in A7r5 cells in a dose-and time- dependent manner. LTB4 reduced the mRNA/protein expression of CaV1.2 channels in A7r5 cells. BLT1 receptor antagonist LY29311 abrogated the inhibitory effect of LTB4, while BLT2 receptor antagonist LY255283 had no effect. 5Z-7-oxozeaenol and SB203580, which block TAK1 and p38 kinase respectively, abrogated the LTB4 inhibitory effect on L-type calcium channels. LTB4 increased p38 activity in A7r5 cells. Blockage of Src, PI3K, JNK and NF-x03BA;B kinase had no effects on LTB4 inhibition of L-type calcium channel currents in A7r5 cells. CONCLUSION: We conclude that LTB4 inhibits L-type calcium channels through BLT1-TAk1-p38 signaling pathway. The LTB4 inhibitory effect on L-type calcium channels may be involved in its pathological processes such as atherosclerosis.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Leucotrieno B4/farmacologia , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Benzoatos/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Imidazóis/farmacologia , MAP Quinase Quinase Quinases/antagonistas & inibidores , MAP Quinase Quinase Quinases/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Técnicas de Patch-Clamp , Piridinas/farmacologia , RNA Mensageiro/metabolismo , Ratos , Receptores do Leucotrieno B4/antagonistas & inibidores , Receptores do Leucotrieno B4/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
14.
PLoS One ; 10(10): e0140715, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26485043

RESUMO

Arachidonic acid (AA) and its metabolites are important second messengers for ion channel modulation. The effects of extracellular application of AA and its non-metabolized analogue on muscle rNaV1.4 Na+ current has been studied, but little is known about the effects of intracellular application of AA on this channel isoform. Here, we report that intracellular application of AA significantly augmented the rNaV1.4 current peak without modulating the steady-state activation and inactivation properties of the rNaV1.4 channel. These results differed from the effects of extracellular application of AA on rNaV1.4 current. The effects of intracellular AA were mimicked by prostaglandin E2 but not eicosatetraynoic acid (ETYA), the non-metabolized analogue of AA, and were eliminated by treatment with cyclooxygenase inhibitors, flufenamic acid, or indomethacin. AA/PGE2-induced activation of rNaV1.4 channels was mimicked by a cAMP analogue (db-cAMP) and eliminated by a PKA inhibitor, PKAi. Furthermore, inhibition of EP2 and EP4 (PGE2 receptors) with AH6809 and AH23848 reduced the intracellular AA/PGE2-induced increase of rNaV1.4 current. Two mutated channels, rNaV1.4S56A and rNaV1.4T21A, were designed to investigate the role of predicted phosphorylation sites in the AA/PGE2-mediated regulation of rNaV1.4 currents. In rNaV1.4S56A, the effects of intracellular db-cAMP, AA, and PGE2 were significantly reduced. The results of the present study suggest that intracellular AA augments rNaV1.4 current by PGE2/EP receptor-mediated activation of the cAMP/PKA pathway, and that the S56 residue on the channel protein is important for this process.


Assuntos
Ácido Araquidônico/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Dinoprostona/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.4/metabolismo , Transdução de Sinais/efeitos dos fármacos , Inibidores de Ciclo-Oxigenase/farmacologia , Ácido Flufenâmico/farmacologia , Células HEK293 , Humanos , Indometacina/farmacologia , Fosforilação/efeitos dos fármacos , Transdução de Sinais/fisiologia
15.
J Cell Mol Med ; 19(10): 2413-22, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26176998

RESUMO

Previous work from both our lab and others have indicated that exposure to 50 Hz magnetic fields (ELF-MF) was able to modify ion channel functions. However, very few studies have investigated the effects of MF on γ-aminobutyric acid (GABA) type A receptors (GABA(A) Rs) channel functioning, which are fundamental to overall neuronal excitability. Here, our major goal is to reveal the potential effects of ELF-MF on GABA(A) Rs activity in rat cerebellar granule neurons (CGNs). Our results indicated that exposing CGNs to 1 mT ELF-MF for 60 min. significantly increased GABA(A) R currents without modifying sensitivity to GABA. However, activation of PKA by db-cAMP failed to do so, but led to a slight decrease instead. On the other hand, PKC activation or inhibition by PMA or Bis and Docosahexaenoic acid (DHA) mimicked or eliminated the field-induced-increase of GABA(A) R currents. Western blot analysis indicated that the intracellular levels of phosphorylated PKC (pPKC) were significantly elevated after 60 min. of ELF-MF exposure, which was subsequently blocked by application of DHA or EP1 receptor-specific (prostaglandin E receptor 1) antagonist (SC19220), but not by EP2-EP4 receptor-specific antagonists. SC19220 also significantly inhibited the ELF-MF-induced elevation on GABA(A) R currents. Together, these data obviously demonstrated for the first time that neuronal GABA(A) currents are significantly increased by ELF-MF exposure, and also suggest that these effects are mediated via an EP1 receptor-mediated PKC pathway. Future work will focus on a more comprehensive analysis of the physiological and/or pathological consequences of these effects.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Ativação do Canal Iônico , Campos Magnéticos , Neurônios/metabolismo , Proteína Quinase C/metabolismo , Receptores de GABA-A/metabolismo , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação do Canal Iônico/efeitos dos fármacos , Modelos Biológicos , Neurônios/efeitos dos fármacos , Ratos Sprague-Dawley , Receptores de Prostaglandina E Subtipo EP1/antagonistas & inibidores , Análise de Regressão , Transdução de Sinais/efeitos dos fármacos , Ácido gama-Aminobutírico/farmacologia
16.
Cell Physiol Biochem ; 36(5): 1699-711, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26183394

RESUMO

AIMS: PGE2 is one of the most abundant prostanoids in mammalian tissues, but its effect on neuronal receptors has not been well investigated. This study examines the effect of PGE2 on GABAA receptor currents in rat cerebellar granule neurons. METHODS: GABAA currents were recorded using a patch-clamp technique. Cell surface and total protein of GABAA ß1/2/3 subunits was carried out by Western blot analysis. RESULTS: Upon incubation of neurons with PGE2 (1 µM) for 60 minutes, GABAA currents were significantly potentiated. This PGE2-driven effect could be blocked by PKC or CaMKII inhibitors as well as EP1 receptor antagonist, and mimicked by PMA or EP1 receptor agonist. Furthermore, Western blot data showed that PGE2 did not increase the total expression level of GABAA receptors, but significantly increased surface levels of GABAA ß1/2/3 subunits after 1 h of treatment. Consistently, both PKC and CaMKII inhibitors were able to reduce PGE2-induced increases in cell surface expression of GABAA receptors. CONCLUSION: Activation of either the PKC or CaMKII pathways by EP1 receptors mediates the PGE2-induced increase in GABAA currents. This suggests that upregulation of postsynaptic GABAA receptors by PGE2 may have profound effects on cerebellar functioning under physiological and pathological conditions.


Assuntos
Dinoprostona/fisiologia , Receptores de GABA-A/fisiologia , Receptores de Prostaglandina E Subtipo EP1/metabolismo , Transdução de Sinais , Animais , Células Cultivadas , Cerebelo/metabolismo , Grânulos Citoplasmáticos/metabolismo , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley
17.
Sci Rep ; 5: 11768, 2015 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-26138388

RESUMO

Animal studies have shown that electromagnetic field exposure may interfere with the activity of brain cells, thereby generating behavioral and cognitive disturbances. However, the underlying mechanisms and possible preventions are still unknown. In this study, we used a mouse model to examine the effects of exposure to extremely low-frequency (50 Hz) electromagnetic fields (ELF MFs) on a recognition memory task and morphological changes of hippocampal neurons. The data showed that ELF MFs exposure (1 mT, 12 h/day) induced a time-dependent deficit in novel object associative recognition memory and also decreased hippocampal dendritic spine density. This effect was observed without corresponding changes in spontaneous locomotor activity and was transient, which has only been seen after exposing mice to ELF MFs for 7-10 days. The over-expression of hippocampal neuritin, an activity-dependent neurotrophic factor, using an adeno-associated virus (AAV) vector significantly increased the neuritin level and dendritic spine density. This increase was paralleled with ELF MFs exposure-induced deficits in recognition memory and reductions of dendritic spine density. Collectively, our study provides evidence for the association between ELF MFs exposure, impairment of recognition memory, and resulting changes in hippocampal dendritic spine density. Neuritin prevented this ELF MFs-exposure-induced effect by increasing the hippocampal spine density.


Assuntos
Campos Eletromagnéticos/efeitos adversos , Hipocampo/fisiopatologia , Transtornos da Memória/prevenção & controle , Proteínas do Tecido Nervoso/fisiologia , Animais , Espinhas Dendríticas/patologia , Dependovirus/genética , Feminino , Proteínas Ligadas por GPI/fisiologia , Vetores Genéticos , Hipocampo/metabolismo , Hipocampo/patologia , Transtornos da Memória/etiologia , Transtornos da Memória/fisiopatologia , Camundongos Endogâmicos ICR , Reconhecimento Visual de Modelos , Fatores de Proteção , Reconhecimento Psicológico
18.
Biochem J ; 460(1): 35-47, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24597762

RESUMO

GDF15 (growth/differentiation factor 15), a novel member of the TGFß (transforming growth factor ß) superfamily, plays critical roles in the central and peripheral nervous systems, but the signal transduction pathways and receptor subtypes involved are not well understood. In the present paper, we report that GDF15 specifically increases the IK (delayed-rectifier outward K+ current) in rat CGNs (cerebellar granule neurons) in time- and concentration-dependent manners. The GDF15-induced amplification of the IK is mediated by the increased expression and reduced lysosome-dependent degradation of the Kv2.1 protein, the main α-subunit of the IK channel. Exposure of CGNs to GDF15 markedly induced the phosphorylation of ERK (extracellular-signal-regulated kinase), Akt and mTOR (mammalian target of rapamycin), but the GDF15-induced IK densities and increased expression of Kv2.1 were attenuated only by Akt and mTOR, and not ERK, inhibitors. Pharmacological inhibition of the Src-mediated phosphorylation of TGFßR2 (TGFß receptor 2), not TGFßR1, abrogated the effect of GDF15 on IK amplification and Kv2.1 induction. Immunoprecipitation assays showed that GDF15 increased the tyrosine phosphorylation of TGFßRII in the CGN lysate. The results of the present study reveal a novel regulation of Kv2.1 by GDF15 mediated through the TGFßRII-activated Akt/mTOR pathway, which is a previously uncharacterized Smad-independent mechanism of GDF15 signalling.


Assuntos
Cerebelo/fisiologia , Fator 15 de Diferenciação de Crescimento/fisiologia , Proteína Oncogênica v-akt/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Fatores de Crescimento Transformadores beta/fisiologia , Canais de Potássio Shab/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Cerebelo/citologia , Humanos , Ratos , Ratos Sprague-Dawley , Receptor do Fator de Crescimento Transformador beta Tipo II , Transdução de Sinais/genética
19.
J Cell Mol Med ; 18(6): 1060-70, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24548607

RESUMO

Although melatonin (MT) has been reported to protect cells against oxidative damage induced by electromagnetic radiation, few reports have addressed whether there are other protective mechanisms. Here, we investigated the effects of MT on extremely low-frequency electromagnetic field (ELF-EMF)-induced Nav activity in rat cerebellar granule cells (GCs). Exposing cerebellar GCs to ELF-EMF for 60 min. significantly increased the Nav current (INa ) densities by 62.5%. MT (5 µM) inhibited the ELF-EMF-induced INa increase. This inhibitory effect of MT is mimicked by an MT2 receptor agonist and was eliminated by an MT2 receptor antagonist. The Nav channel steady-state activation curve was significantly shifted towards hyperpolarization by ELF-EMF stimulation but remained unchanged by MT in cerebellar GC that were either exposed or not exposed to ELF-EMF. ELF-EMF exposure significantly increased the intracellular levels of phosphorylated PKA in cerebellar GCs, and both MT and IIK-7 did not reduce the ELF-EMF-induced increase in phosphorylated PKA. The inhibitory effects of MT on ELF-EMF-induced Nav activity was greatly reduced by the calmodulin inhibitor KN93. Calcium imaging showed that MT did not increase the basal intracellular Ca(2+) level, but it significantly elevated the intracellular Ca(2+) level evoked by the high K(+) stimulation in cerebellar GC that were either exposed or not exposed to ELF-EMF. In the presence of ruthenium red, a ryanodine-sensitive receptor blocker, the MT-induced increase in intracellular calcium levels was reduced. Our data show for the first time that MT protects against neuronal INa that result from ELF-EMF exposure through Ca(2+) influx-induced Ca(2+) release.


Assuntos
Cálcio/metabolismo , Cerebelo/citologia , Grânulos Citoplasmáticos/metabolismo , Campos Eletromagnéticos/efeitos adversos , Melatonina/farmacologia , Substâncias Protetoras/farmacologia , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Antioxidantes/farmacologia , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cerebelo/efeitos da radiação , Grânulos Citoplasmáticos/efeitos dos fármacos , Grânulos Citoplasmáticos/efeitos da radiação , Masculino , Camundongos , Oxirredução , Técnicas de Patch-Clamp , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/efeitos da radiação
20.
J Neurochem ; 128(3): 350-62, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24118019

RESUMO

In addition to their neurotoxic role in Alzheimer's disease (AD), ß-amyloid peptides (Aßs) are also known to play physiological roles. Here, we show that recombinant Aß40 significantly increased the outward current of the GABA(A) receptor containing (GABA(A)α6) in rat cerebellar granule neurons (CGNs). The Aß40-mediated increase in GABA(A)α6 current was mediated by an increase in GABA(A)α6 protein expression at the translational rather than the transcriptional level. The exposure of CGNs to Aß40 markedly induced the phosphorylation of ERK (pERK) and mammalian target of rapamycin (pmTOR). The increase in GABA(A)α6 current and expression was attenuated by specific inhibitors of ERK or mTOR, suggesting that the ERK and mTOR signaling pathways are required for the effect of Aß40 on GABA(A)α6 current and expression in CGNs. A pharmacological blockade of the p75 neurotrophin receptor (p75(NTR)), but not the insulin or α7-nAChR receptors, abrogated the effect of Aß40 on GABA(A)α6 protein expression and current. Furthermore, the expression of GABA(A)α6 was lower in CGNs from APP(-/-) mice than in CGNs from wild-type mice. Moreover, the internal granule layer (IGL) in APP(-/-) mice was thinner than the IGL in wild-type mice. The injection of Aß40 into the cerebellum reversed this effect, and the application of p75(NTR) blocking antibody abolished the effects of Aß40 on cerebellum morphology in APP(-/-) mice. Our results suggest that low concentrations of Aß40 play a role in regulating CGN maturation through p75(NTR).


Assuntos
Peptídeos beta-Amiloides/farmacologia , Cerebelo/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neurônios/metabolismo , Fragmentos de Peptídeos/farmacologia , Receptores de GABA-A/biossíntese , Serina-Treonina Quinases TOR/efeitos dos fármacos , Precursor de Proteína beta-Amiloide/genética , Animais , Biotinilação , Western Blotting , Cerebelo/citologia , Cerebelo/efeitos dos fármacos , Feminino , Imunoprecipitação , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase em Tempo Real , Receptor de Fator de Crescimento Neural/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácido gama-Aminobutírico/fisiologia
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