RESUMO
ABA is important for plant growth and development; however, it also inhibits photosynthesis by regulating the stomatal aperture and ribulose-1,5-bisphosphate carboxylase/oxygenase activity. Noteworthy, this negative effect can be alleviated by antioxidants including ascorbic acid (AsA) and catalase (CAT), but the underlying mechanism remains unclear. Two rice cultivars, Zhefu802 (recurrent parent) and its near-isogenic line, fgl were selected and planted in a greenhouse with 30/24⯰C (day/night) under natural sunlight conditions. Compared to fgl, Zhefu802â¯had significantly lower net photosynthetic rate (PN) and stomatal conductance (Cond) as well as significantly higher ABA and H2O2 contents. However, AsA and CAT increased PN, Cond, and stomatal aperture, which decreased H2O2 and malondialdehyde (MDA) levels. In this process, AsA and CAT significantly increased the ribulose-1,5-bisphosphate carboxylase activity, while they strongly decreased the ribulose-1,5-bisphosphate oxygenase activity, and finally caused an obvious decrease in the ratio of photorespiration (Pr) to PN. Additionally, AsA and CAT significantly increased the expression levels of RbcS and RbcL genes of leaves, while H2O2 significantly decreased them, especially the RbcS gene. In summary, the removal of H2O2 by AsA and CAT can improve the leaf photosynthesis by alleviating the inhibition on the stomatal conductance and ribulose-1,5-bisphosphate carboxylase capacity caused by ABA.
Assuntos
Antioxidantes/metabolismo , Oryza/fisiologia , Fotossíntese , Estômatos de Plantas/fisiologia , Ribulose-Bifosfato Carboxilase/metabolismoRESUMO
Essentials ARHGEF10 single-nucleotide polymorphism provides risk of ischemic and atherothrombotic stroke. The role of ARHGEF10 in platelet function was examined using ARHGEF10 knockout mice. ARHGEF10 deficiency inhibits platelet function and arterial thrombus formation. ARHGEF10 knockout protects mice from stroke-induced infarction. SUMMARY: Background ARHGEF10, a member of the Rho guanine nucleotide exchange factor (GEF) family, stimulates Rho GTPases. Rho GTPases have been reported to regulate a variety of cellular behaviors, such as cell polarity, cytoskeletal organization, and gene transcription. ARHGEF10 single-nucleotide polymorphisms are linked to the risk of ischemic stroke. However, the role of ARHGEF10 in platelet function remains unknown. Objective To examine the role of ARHGEF10 in platelet function. Methods ARHGEF10-/- were generated. We examined the in vitro and in vivo effects of ARHGEF10 knockout on platelet function and arterial thrombosis formation. Results ARHGEF10-/- mice had normal platelet counts, but showed altered aggregation in response to thrombin, collagen, ADP, protease-activated receptor-4 peptide, and U46619 stimulation. ARHGEF10 knockout influenced platelet spreading on fibrinogen-coated surfaces, and caused the platelets to show less lamellipodia-like extension than wild-type platelets. ARHGEF10 knockout also inhibited platelet clot retraction induced by thrombin stimulation. ARHGEF10 knockout resulted in prolonged tail bleeding time and inhibited the stable thrombus formation induced by FeCl3 in the carotid artery. Conclusions ARHGEF10 serves as an important regulator in platelet shape change, spreading, and aggregation. Moreover, ARHGEF10 also plays an important role in arterial thrombosis formation.
Assuntos
Arteriopatias Oclusivas/prevenção & controle , Plaquetas/metabolismo , Doenças das Artérias Carótidas/prevenção & controle , Hemostasia , Agregação Plaquetária , Fatores de Troca de Nucleotídeo Guanina Rho/deficiência , Trombose/prevenção & controle , Animais , Arteriopatias Oclusivas/sangue , Arteriopatias Oclusivas/genética , Doenças das Artérias Carótidas/sangue , Doenças das Artérias Carótidas/genética , Forma Celular , Cloretos , Modelos Animais de Doenças , Compostos Férricos , Técnicas de Inativação de Genes , Genótipo , Masculino , Camundongos da Linhagem 129 , Camundongos Knockout , Cadeias Leves de Miosina/metabolismo , Fenótipo , Fosforilação , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/sangue , Fatores de Troca de Nucleotídeo Guanina Rho/genética , Selenoproteína P/sangue , Trombose/sangue , Trombose/genética , Fatores de TempoRESUMO
OBJECTIVE: Bone marrow mesenchymal stem cells (BMMSCs) have been widely applied in osteonecrosis. However, lack of biomechanical support limited application of BMMSCs. And porous tantalum (PTA) has been identified as a cell-friendly scaffold for bone regeneration. Herein, we aimed to investigate the efficacy of PTA seeded with BMMSCs in the treatment of osteonecrosis. MATERIALS AND METHODS: After the production of PTA seeded with BMMSCs, MTT and GFP were performed to identify the proliferation and adhesion of BMMSCs respectively, which was further examined by scanning electron microscopy (SEM). And real-time PCR was also used to determine mRNA level of osteogenic markers, including Alp, OCN, OPN, Col I and Runx-2 in BMMSCs. Nineteen adult rabbits were applied for building steroid-associated osteonecrosis (SAON) models. Bone formation rate (BFR) and mineral apposition rate (MAR) were determined. And Goldner Trichrome Staining was used in these SAON models, which further confirmed the efficacy of PTA seeded with BMMSCs in SAON. RESULTS: PTA seeded with BMMSCs showed excellent biocompatibility. Additionally, SEM assay showed that BMMSCs adhered tightly and spread fully in the pores of PTA. Next, the expression of ALP and OPN mRNA in BMMSCs were significantly (p < 0.05) higher in the PTA-treated group compared to those in the PTA-untreated group. Furthermore, compared to those treated by only PTA, the dynamic bone formation in rabbits treated by PTA seeded with BMMSCs was significantly increased (p < 0.001) at both week 3rd and week 6th. CONCLUSIONS: The product, PTA seeded with BMMSCs, was successfully produced, and was determined as high efficacy for treatment of steroid-associated osteonecrosis. PTA seeded with BMMSCs may afford a promising option for treating osteonecrosis.
Assuntos
Células da Medula Óssea/citologia , Tantálio , Animais , Regeneração Óssea , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Osteonecrose/terapiaRESUMO
OBJECTIVE: To investigate some important issues for diagnosis and treatment of idiopathic normal-pressure hydrocephalus (iNPH), such as standardized pre-operative assessment, initial pressure value of diverter pump, and pressure regulation during follow-up. METHODS: Twenty six iNPH patients (21 males) who treated in Department of Neurosurgery of 2nd Affiliated Hospital of Zhejiang University School of Medicine from 2011 to 2015 were analyzed retrospectively. The average age was 60.5 year. The analysis focused on the treatment process of iNPH, initial pressure value of diverter pump, choice of diverter pump, and pressure regulation during follow-up. RESULTS: As a result, 24 cases (92.3%) had a good prognosis based on their imaging and clinical manifestations. Based on the literature and their clinical experiences, this department established a diagnosis and treatment procedure of iNPH and a pressure regulation procedure for the follow-up of iNPH. Moreover, it is proposed that choosing an anti-gravity diverter pump and making an initial pressure value 20 mmH2O less than pre-surgical cerebrospinal pressure may be beneficial for the prognosis. CONCLUSION: This standardized diagnosis and treatment procedure for iNPH is practical and effective.
Assuntos
Derivações do Líquido Cefalorraquidiano/métodos , Hidrocefalia de Pressão Normal/diagnóstico , Hidrocefalia de Pressão Normal/cirurgia , Procedimentos Neurocirúrgicos/métodos , Adulto , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: The efficacy of opioids typically decreases after long-term use owing to the development of tolerance. Glial activation and the upregulation of proinflammatory cytokines are related to the induction of tolerance. We investigated the effect of leukemia inhibitory factor (LIF) on morphine analgesia and tolerance. METHODS: LIF concentrations in rat spinal cords were measured by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) after morphine administration. LIF distribution was examined using confocal microscopy. To evaluate the effects of LIF on morphine analgesia and tolerance, LIF was intrathecally administered 30 min before morphine injection. The analgesic effect of morphine was evaluated by measuring tail-flick latency. Human LIF concentrations from the cerebrospinal fluid (CSF) of opioid tolerant patients were also determined by specific ELISA. RESULTS: Chronic morphine administration upregulated LIF concentrations in rat spinal cords. Intrathecal injection of LIF potentiated the analgesic action of morphine. Patch clamp recording of spinal cord slices showed that LIF enhanced DAMGO ([D-Ala2, N-MePhe4, Gly-ol]-enkephalin)-induced outward potassium current. The development of tolerance was markedly suppressed by exogenous LIF, whereas neutralizing the endogenously released LIF with anti-LIF antibodies accelerated the tolerance induction. Moreover, LIF concentrations in the CSF of opioid-tolerant patients were higher than those in the opioid-naive controls. CONCLUSIONS: Intrathecal administration of LIF potentiated morphine antinociceptive activity and attenuated the development of morphine tolerance. Upregulation of endogenously released LIF by long-term use of opioids might counterbalance the tolerance induction effects of other proinflammatory cytokines. LIF might be a novel drug candidate for inhibiting opioid tolerance induction.
Assuntos
Analgésicos Opioides/farmacologia , Fator Inibidor de Leucemia/fisiologia , Morfina/farmacologia , Animais , Citocinas/análise , Tolerância a Medicamentos , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Recent studies found that hepatitis C virus (HCV) may invade the central nervous system, and both HCV and Parkinson's disease (PD) have in common the overexpression of inflammatory biomarkers. We analysed data from a community-based integrated screening programme based on a total of 62,276 subjects. We used logistic regression models to investigate association between HCV infection and PD. The neurotoxicity of HCV was evaluated in the midbrain neuron-glia coculture system in rats. The cytokine/chemokine array was performed to measure the differences of amounts of cytokines released from midbrain in the presence and absence of HCV. The crude odds ratios (ORs) for having PD were 0.62 [95% confidence interval (CI), 0.48-0.81] and 1.91 (95% CI, 1.48-2.47) for hepatitis B virus (HBV) and HCV. After controlling for potential confounders, the association between HCV and PD remained statistically significant (adjusted OR = 1.39; 95% CI, 1.07-1.80), but not significantly different between HBV and PD. The HCV induced 60% dopaminergic neuron death in the midbrain neuron-glia coculture system in rats, similar to that of 1-methyl-4-phenylpyridinium (MPP(+) ) but not caused by HBV. This link was further supported by the finding that HCV infection may release the inflammatory cytokines, which may play a role in the pathogenesis of PD. In conclusion, our study demonstrated a significantly positive epidemiological association between HCV infection and PD and corroborated the dopaminergic toxicity of HCV similar to that of MPP(+) .
Assuntos
Hepatite C Crônica/complicações , Doença de Parkinson/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células Cultivadas , Técnicas de Cocultura , Feminino , Hepatite C Crônica/patologia , Humanos , Masculino , Mesencéfalo/patologia , Pessoa de Meia-Idade , Neuroglia/virologia , Neurônios/virologia , Doença de Parkinson/patologia , Ratos Wistar , Medição de RiscoRESUMO
UNLABELLED: Onion powder has been reported to decrease the ovariectomy-induced bone resorption of rats. However, the molecular mechanism of onion powder on the bone cells has not been reported. Here, we report that water solution of onion crude powder decreases the osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells. Additionally, water solution of onion crude powder inhibits the RANKL-induced ERK, p38 and NF-kappaB activation in macrophages. In summary, our data showed that onion powder may benefit bone through an anti-resorption effect on the osteoclasts. INTRODUCTION: A nutritional approach is important for both prevention and treatment of osteoporosis. Onion has been reported to decrease the ovariectomy-induced bone resorption. However, the functional effects of onion on the cultured osteoclasts and osteoblasts remain largely unknown. Here, we found that water solution of onion crude powder markedly inhibited the receptor activator of nuclear factor kappa B ligand (RANKL)-induced osteoclastogenesis through ERK, p38 and NF-kappaB pathways. Other studies were also designed to investigate the potential signaling pathways involved in onion-induced decrease in osteoclastogenesis. METHODS: The osteoclastogenesis was examined using the TRAP staining method. The MAPKs and NF-kappaB pathways were measured using Western blot analysis. A transfection protocol was used to examine NF-kappaB activity. RESULTS: Water solution of onion crude powder inhibited the RANKL plus M-CSF-induced osteoclastic differentiation from either bone marrow stromal cells or from RAW264.7 macrophage cells. Treatment of RAW264.7 macrophages with RANKL could induce the activation of ERK, p38 and NF-kappaB that was inhibited by water solution of onion crude powder. On the other hand, it did not affect the cell proliferation and differentiation of human cultured osteoblasts. CONCLUSIONS: Our data suggest that water solution of onion crude powder inhibits osteoclastogenesis from co-cultures of bone marrow stromal cells and macrophage cells via attenuation of RANKL-induced ERK, p38 and NF-kappaB activation.
Assuntos
Reabsorção Óssea , Dieta , Cebolas , Osteoclastos/fisiologia , Transdução de Sinais/fisiologia , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Feminino , Fator Estimulador de Colônias de Macrófagos/farmacologia , Macrófagos/citologia , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Ligante RANK/metabolismo , Ligante RANK/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
BACKGROUND: Large cranial defects combined with hydrocephalus after decompressive craniectomy are a common, harsh reality among patients with head trauma. Typically, a shunt is first used to relieve the hydrocephalus. However, subsequently the patients may develop a severe sinking scalp flap over the skull defect before cranioplasty, which would make the procedure difficult. METHODS: This problem was overcome by temporarily adjusting the shunt pressure using a programmable ventriculoperitoneal shunt tube, which allowed expansion of the depressed scalp flap and facilitated the subsequent cranioplasty. This study describes two patients who were treated for this problem after severe head trauma. RESULTS: When performing a titanium mesh cranioplasty after a shunt, this new method facilitated the separation of the scalp from the underlying muscle or dura and obliterated the dead space between the titanium mesh and the underlying tissue. Both patients had satisfactory outcomes without complications. CONCLUSIONS: This method is easy and safe and it facilitates the cranioplasty, reducing the potential complications, including intracranial haematoma, effusions and infection, and thereby improving the patient outcome.
Assuntos
Traumatismos Craniocerebrais/cirurgia , Hidrocefalia/cirurgia , Fratura do Crânio com Afundamento/cirurgia , Crânio/cirurgia , Adulto , Descompressão Cirúrgica/métodos , Feminino , Humanos , Masculino , Procedimentos de Cirurgia Plástica , Retalhos Cirúrgicos , Derivação VentriculoperitonealRESUMO
BACKGROUND AND PURPOSE: An inflammatory response in the central nervous system mediated by the activation of microglia is a key event in the early stages of the development of neurodegenerative diseases. LPS has been reported to cause marked microglia activation. It is very important to develop drugs that can inhibit microglia activation and neuroinflammation. Here, we investigated the inhibitory effect of YC-1, a known activator of soluble guanylyl cyclase, against LPS-induced inflammatory responses in microglia. EXPERIMENTAL APPROACH: To understand the inhibitory effects of YC-1 on LPS-induced neuroinflammation, primary cultures of rat microglia and the microglia cell line BV-2 were used. To examine the mechanism of action of YC-1, LPS-induced nitric oxide (NO) and prostaglandin E2 (PGE2) production, iNOS, COX-2 and cytokine expression were analyzed by Griess reaction, ELISA, Western blotting and RT-PCR, respectively. The effect of YC-1 on LPS-induced activation of nuclear factor kappa B (NF-kappaB) was studied by NF-kappaB reporter assay and immunofluorocytochemistry. KEY RESULTS: YC-1 inhibited LPS-induced production of NO and PGE2 in a concentration-dependent manner. The protein and mRNA expression of iNOS and COX-2 in response to LPS application were also decreased by YC-1. In addition, YC-1 effectively reduced LPS-induced expression of the mRNA for the proinflammatory cytokines, TNF-alpha and IL-1beta. Furthermore, YC-1 inhibited LPS-induced NF-kappaB activation in microglia. CONCLUSIONS AND IMPLICATIONS: YC-1 was able to inhibit LPS-induced iNOS and COX-2 expression and NF-kappaB activation, indicating that YC-1 may be developed as an anti-inflammatory neuroprotective agent.
Assuntos
Indazóis/farmacologia , Lipopolissacarídeos/farmacologia , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , GMP Cíclico/análogos & derivados , GMP Cíclico/farmacologia , Proteínas Quinases Dependentes de GMP Cíclico/antagonistas & inibidores , Ciclo-Oxigenase 2/genética , Ciclo-Oxigenase 2/metabolismo , Dinoprostona/biossíntese , Relação Dose-Resposta a Droga , Ativadores de Enzimas/farmacologia , Expressão Gênica/efeitos dos fármacos , Guanilato Ciclase/antagonistas & inibidores , Luciferases/genética , Luciferases/metabolismo , Microglia/citologia , Microglia/metabolismo , NF-kappa B/genética , Óxido Nítrico/biossíntese , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Oxidiazóis/farmacologia , Prolina/análogos & derivados , Prolina/farmacologia , Quinoxalinas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tiocarbamatos/farmacologia , Tionucleotídeos/farmacologiaRESUMO
Neonatal peripheral inflammation has been shown to alter the neural circuitry of the spinal cord in adult rats. However, the temporal and spatial changes in the distribution of primary afferent terminals immediately following a neonatal inflammatory stimulus remains unclear. In the present study we found that intraplantar injection of complete Freund's adjuvant (CFA) or saline alone on postnatal day 1 (P1) causes CGRP immunoreactivity (CGRP-Ir) to gradually increase from P6 to P15 in laminae I and II, and return to baseline at P22. In laminae III and IV, CGRP-Ir markedly increased beginning at P6, and remained elevated thereafter. CGRP-Ir in lamina V remained unchanged throughout the observation period. These findings show that intraplantar CFA induces CGRP-fiber sprouting in laminae III and IV, but not in laminae I, II or V. We suggest that neonatal inflammation causes changes in the neural circuitry pattern in various regions of the dorsal horn during the critical neonatal development period in rats.
Assuntos
Animais Recém-Nascidos/fisiologia , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Inflamação/metabolismo , Terminações Nervosas/metabolismo , Células do Corno Posterior/metabolismo , Animais , Adjuvante de Freund , Imuno-Histoquímica , Inflamação/induzido quimicamente , Masculino , Fibras Nervosas/metabolismo , Neurônios Aferentes/metabolismo , RatosRESUMO
Extracellular matrix proteins, such as fibronectin, laminin, and collagen, have been implicated in a wide variety of cellular properties, which include cell adhesion, migration, differentiation, and proliferation. In this study, we investigated the modulation of protein kinase A (PKA) activity by matrix proteins at developing motoneurons. The cultures of spinal neurons and myotomal cells were prepared from 1-day-old Xenopus laevis embryos. Spontaneous synaptic currents (SSC) were recorded from innervated myocytes of natural synapses by whole-cell voltage-clamped recordings (V(h) = -60 to approximately -65 mV). Bath application of agents, which directly or indirectly activate PKA, such as forskolin (20 microM), dibutyryl cAMP (DBcAMP) (1 mM), isoproterenol (10 microM), or albuterol (10 microM), significantly increased SSC frequency in cultures grown on fibronectin (FN)-coated substratum, but not on laminin- or collagen-coated glasses. The evoked synaptic currents increased in response to forskolin in neurons grown on FN substratum. Triflavin, an Arg-Gly-Asp-dependent disintegrin, inhibited potentiating action of isoproterenol in neurons grown on FN substratum, suggesting that integrin is involved in the potentiation of the PKA pathway in the regulation of acetylcholine (ACh) release. There is collaboration of neurotrophic factors and the FN matrix in regulating synaptic transmission in response to DBcAMP. Chronic treatment with neurotrophic factors, such as ciliary neurotrophic factor (150 ng/ml), glial cell line-derived neurotrophic factor (30 ng/ml), or neurotrophin-3 (50 ng/ml), enhanced the SSC-increasing action of DBcAMP in neurons grown on FN-coated glasses. These results suggest that the FN matrix potentiates synaptic transmission in response to PKA activation. Neurotrophic factors may collaborate with FN to regulate spontaneous ACh secretion at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular synapses.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fibronectinas/metabolismo , Sinapses/fisiologia , Acetilcolina/metabolismo , Animais , Bucladesina/farmacologia , Colforsina/farmacologia , Eletrofisiologia , Ativação Enzimática , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Isoproterenol/farmacologia , Fatores de Crescimento Neural/metabolismo , Simpatomiméticos/farmacologia , Sinapses/efeitos dos fármacos , Sinapses/enzimologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Xenopus laevisRESUMO
Integrins mediate cell-extracellular matrix connections and are particularly important during neuronal development. We here investigated the regulatory role of extracellular matrix (ECM) proteins on the synaptic transmission at developing motoneurons. Synaptic currents were recorded from innervated myocytes of 1-day-old Xenopus cultures by whole-cell recordings. Soluble fibronectin and laminin had no significant effect on the frequency of spontaneous synaptic currents (SSCs) by themselves and markedly increased SSC frequency in the presence of low concentration of protein kinase C (PKC) activators. Pretreatment with Gly-Arg-Gly-Asp-Ser peptide inhibited the SSC increasing action of 12-o-tetradecanoyl-phorbol-13-acetate (TPA, 0.5 microM) plus fibronectin, but not that of TPA plus laminin. Genistein but not cytochalasin D inhibited the SSC increasing action of TPA plus fibronectin or laminin. High concentration of TPA (5 microM) markedly increased the SSC frequency by itself and occluded the SSC increasing action of fibronectin. Very low concentration of TPA (0.05 microM) markedly enhanced the SSC frequency when the cells were plated onto fibronectin- or laminin-coated substratum for 1 day. The SSC frequency increased markedly right after a train stimulation, which was defined as post-train potentiation (PTrP), when the cultures were plated onto fibronectin substratum and chronically treated with brain-derived neurotrophic factor (BDNF). The PTrP phenomenon is not observed upon chronic treatment with neurotrophin-3, glial cell line-derived neurotrophic factor, or ciliary neurotrophic factor. Our results suggest that the activation of PKC and tyrosine kinase but not actin reorganization plays a role in the SSC potentiating action of fibronectin. BDNF exerts synergistic effects in increasing synaptic transmission in neurons grown on fibronectin substratum. ECMs in concert with neurotrophic factor may play a role in regulating synaptic function at developing motoneurons.
Assuntos
Acetilcolina/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Neurônios Motores/enzimologia , Fatores de Crescimento Neural , Proteína Quinase C/metabolismo , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Carcinógenos/farmacologia , Células Cultivadas , Fator Neurotrófico Ciliar/farmacologia , Fibronectinas/farmacologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Integrinas/metabolismo , Laminina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Proteínas do Tecido Nervoso/farmacologia , Fármacos Neuroprotetores/farmacologia , Técnicas de Patch-Clamp , Solubilidade , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , XenopusRESUMO
Integrins mediate cell-extracellular matrix connection and are particularly important during neuronal development. We here investigated the regulation of fibronectin (FN) matrix and neurotrophins on the embryonic synaptic transmission. Spontaneous synaptic currents (SSCs) were recorded from innervated myocytes of 1-day-old Xenopus cultures by whole-cell recordings. The SSC increasing action of alpha,beta-methylene adenosine triphosphate was enhanced in neurons grown on FN substratum, which was further potentiated by chronic treatment with brain-derived neurotrophic factor (BDNF). The SSC increasing action of thapsigargin, carbonyl cyanide m-chlorophenylhydrazone and N-methyl-D-aspartate was also markedly potentiated in neurons grown on FN-coated glass coverslips and chronically treated with BDNF. FN matrix or BDNF alone only exerts slight potentiation on the SSC increasing action of these three drugs. Our results suggest that FN matrix can collaborate with neurotrophin in regulating synaptic transmission at developing motoneurons, which may play an important role in the maturation of embryonic neuromuscular junction.
Assuntos
Matriz Extracelular/metabolismo , Fibronectinas/metabolismo , Fatores de Crescimento Neural/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Transmissão Sináptica/fisiologia , Acetilcolina/metabolismo , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Cálcio/metabolismo , Carbonil Cianeto m-Clorofenil Hidrazona/farmacologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Inibidores Enzimáticos/farmacologia , Matriz Extracelular/efeitos dos fármacos , Fibronectinas/farmacologia , Integrinas/efeitos dos fármacos , Integrinas/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , N-Metilaspartato/farmacologia , Fatores de Crescimento Neural/farmacologia , Junção Neuromuscular/embriologia , Neurotrofina 3/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Tapsigargina/farmacologia , Desacopladores/farmacologia , Xenopus laevisRESUMO
Mechanisms underlying the acute effects of amphetamine (AMP) were examined by monitoring the expression of metabotropic glutamate receptor 5 (mGluR5) and specific 3H-glutamate binding in the developing rat brain. Each of the postnatal day (P) 4, P21 and P60 rats received one intraperitoneal injection of AMP, 5 mg/kg or saline and were sacrificed one hour later. In situ hybridization analysis revealed that the AMP treatment raised the levels of the mGluR5 mRNA by 9-28% in the neurons of the layer 5 of motor and somatosensory cortices, whereas reduced the levels by 12-28% in the layer 5 of perirhinal cortex and the ventromedial part of caudate-putamen of the 3 ages. In the layer 2/3 neurons of cingular cortex, an 18% higher and 14% and 22% lower than control levels of the mRNA were detected in the P4 and in the P21 and P60 rats injected with AMP. Moreover, the levels of mGluR5 mRNA in the hippocampi and dentate gyri were elevated by AMP to 110-151% of controls in the rats of 3 ages. Reversible 3H-glutamate binding assay showed an increase of 25% and a 12% decrease in the binding levels in the cortices of AMP-treated P4 and P21 rats. The AMP administration also produced a 27% reduction and 62% elevation in the binding of the hippocampi of P4 and P60 rats. The results reveal age- and region-dependent changes in the expression of the glutamate receptors induced by AMP and may indicate differential plastic capability of the neurons to the drug perturbation.
Assuntos
Adrenérgicos/farmacologia , Anfetamina/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/crescimento & desenvolvimento , Neurônios/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , Receptores de Glutamato Metabotrópico/genética , Fatores Etários , Transtornos Relacionados ao Uso de Anfetaminas/genética , Transtornos Relacionados ao Uso de Anfetaminas/metabolismo , Transtornos Relacionados ao Uso de Anfetaminas/fisiopatologia , Animais , Comportamento Animal/efeitos dos fármacos , Comportamento Animal/fisiologia , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Encéfalo/metabolismo , Diencéfalo/efeitos dos fármacos , Diencéfalo/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Ácido Glutâmico/metabolismo , Giro do Cíngulo/efeitos dos fármacos , Giro do Cíngulo/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Mesencéfalo/efeitos dos fármacos , Mesencéfalo/metabolismo , Córtex Motor/efeitos dos fármacos , Córtex Motor/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Ensaio Radioligante , Ratos , Ratos Wistar , Receptor de Glutamato Metabotrópico 5 , Córtex Somatossensorial/efeitos dos fármacos , Córtex Somatossensorial/metabolismo , TrítioRESUMO
Our previous study has shown that tunicamycin irreversibly downregulates the expression of GABA(A)R and causes cell death in cultured brain neurons by biochemical and light microscopic methods. In this study, we examined mechanisms underlying the degeneration of the neurons mainly employing electron microscopic analysis. Cultured neurons derived from embryonic chicken brains were incubated with 5 microg/ml of tunicamycin (TM) for 24 h, followed by continual incubation or removal of TM for additional 3 h or 24 h. Neurons treated with TM for 24 h showed dilated rough endoplasmic reticulum (rER), nuclear envelope and components of Golgi apparatus, in addition to the degranulation of rER and disaggregation of ribosomal rosettes. In neurons subjected to the prolonged incubation, some ribosomes reattached to the membranes of rER; the polyribosomes reappeared, and the swelling of Golgi apparatus subsided. However, the distention of rER persisted, and an uncommon spindle-like structure appeared in the perikarya. This structure is implicated to involve the neuronal degeneration. Moreover, extracellular cell debris was increased with time of incubation. The ratio of the light neurons, defined as containing lower cytoplasmic matrix density than the untreated control, decreased from 28% at 3 h to 3% at 24 h after the removal of TM, and 45% at further 3 h to 6% at further 24 h incubation of TM, whereas dense neurons only appeared in the two 24 h groups, as 44% and 34%. The light neurons resemble necrotic cells, but the dense neurons exhibit distinct morphological features from necrosis and apoptosis. The gel electrophoresis assay revealed the absence of DNA fragmentation in all cultures. In addition, whole cell recordings exhibited a 40% decrease of the GABA-elicited current in the neurons exposed to TM for 24 h. The results indicate irreversible toxicity of chronic TM treatment to the neurons and suggest differential mechanisms for the neuronal death among various populations of cells. It is evident that the N-glycosylation plays a critical role for neuronal survival.
Assuntos
Antibacterianos/toxicidade , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Tunicamicina/toxicidade , Animais , Células Cultivadas , Embrião de Galinha , Potenciais Evocados/efeitos dos fármacos , Glicosilação , Microscopia Eletrônica , Degeneração Neural/fisiopatologia , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Receptores de GABA-A/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
1. Myocyte-dependent regulation of acetylcholine (ACh) quantal secretion from developing motoneurons was studied in day-3 Xenopus nerve-muscle co-cultures. Spontaneous synaptic currents (SSCs) were measured in manipulated synapses by using whole-cell voltage-clamped myocytes. Changes in SSC amplitude were assumed to reflect changes in the ACh content of secreted quantal packets. Compared with natural synapses, motoneurons without any contact with a myocyte (naive neurons) released ACh in smaller quantal packets. 2. Bipolar cultured motoneurons, which were in contact with a myocyte with one axon branch (contact-end) but remained free at another axon branch (free-end), were further used to examine quantal ACh secretion. The ACh quantal size recorded at free-end terminals was similar to that of naive neurons and was smaller than that at the contact-end, indicating that myocyte contact exerts differential regulation on quantal secretion in the same neuron. 3. Some of the neurons that formed a natural synapse with a myocyte continued to grow forward and ACh quantal secretion from the free growth cone was examined. The ACh quantal size recorded at free growth cones was inversely proportional to the distance to the natural synapse, implying localized regulation of quantal secretion by the myocyte. 4. Chronic treatment of day-1 cultures with veratridine and d-tubocurarine, respectively, increased and decreased the neurotrophic action of myocytes when assayed on day 3. 5. Taken together, these findings suggest that the myocyte is an important postsynaptic target in the regulation of quantal secretion and that the trophic action is spatially restricted to the neighbourhood of the neuromuscular junction.
Assuntos
Acetilcolina/metabolismo , Neurônios Motores/fisiologia , Músculos/fisiologia , Junção Neuromuscular/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Axônios/fisiologia , Células Cultivadas , Fator Neurotrófico Ciliar , Técnicas de Cocultura , Embrião não Mamífero , Cinética , Neurônios Motores/citologia , Neurônios Motores/efeitos dos fármacos , Músculos/citologia , Fatores de Crescimento Neural/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neurotrofina 3 , Técnicas de Patch-Clamp , Teoria Quântica , Sinapses/efeitos dos fármacos , Fatores de Tempo , Tubocurarina/farmacologia , Veratridina/farmacologia , XenopusRESUMO
The activation of autoreceptors is known to be important in the modulation of presynaptic transmitter secretion in peripheral and central neurons. Using whole-cell recordings made from the free growth cone of myocyte-contact motoneurons of Xenopus cell cultures, we have observed spontaneous nerve terminal currents (NTCs). These spontaneous NTCs are blocked by d-tubocurarine (d-TC) and alpha-bungarotoxin (alpha-BuTx), indicating that endogenously released acetylcholine (ACh) can produce substantial membrane depolarization in the nerve terminals. Local application of NMDA to the growth cone increased the frequency of spontaneous NTCs. When the electrical stimulations were applied at the soma to initiate evoked-release of ACh, evoked ACh-induced potentials were recorded in the nerve terminals, which were inhibited by d-TC and hexamethonium but not by atropine. Replacement of normal Ringer's solution with high-Mg2+, low-Ca2+ solution also reversibly inhibited evoked ACh-induced potentials. The possible regulatory role of presynaptic nicotinic autoreceptors on the synaptic transmission was also examined. When the innervated myocyte was whole-cell voltage-clamped to record synaptic currents, application of hexamethonium inhibited the amplitude of evoked synaptic currents at a higher degree than that of iontophoretic ACh-induced currents. Furthermore, hexamethonium markedly reduced the frequency of spontaneous synaptic currents at high-activity synapses. Pretreatment of neurons with alpha-BuTx also inhibited the evoked synaptic currents in manipulated synapses. These results suggest that ACh released spontaneously or by electrical stimulation may act on the presynaptic nicotinic autoreceptors of the same nerve terminals to produce membrane potential change and to regulate synaptic transmission.
Assuntos
Acetilcolina/metabolismo , Autorreceptores/fisiologia , Terminações Pré-Sinápticas/metabolismo , Receptores Nicotínicos/fisiologia , Transmissão Sináptica/fisiologia , Animais , Atropina/farmacologia , Cálcio/farmacologia , Agonistas de Aminoácidos Excitatórios/farmacologia , Hexametônio/farmacologia , Antagonistas Muscarínicos/farmacologia , N-Metilaspartato/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Técnicas de Patch-Clamp , Terminações Pré-Sinápticas/química , Transmissão Sináptica/efeitos dos fármacos , XenopusRESUMO
Prostaglandin E (PGE)-induced morphological changes of osteoblasts and its possible mechanisms were investigated in cultured calvaria and isolated osteoblasts from long bone fragments of neonatal rats. The control osteoblasts, either on the calvaria or isolated from the long bone fragments, were flat, polygonal in shape, and arranged in a monolayer under scanning electron microscopy (SEM) or phase contrast microscopy. Treatment with 1 mumol/L of prostaglandin E2 (PGE2, 2 h) caused these bone cells to contract a soma, whereas 10 and 100 mumol/L PGE2 (2 h) caused 18%-30% of the bone cells to elongate and expose the undersurface. Incubation of the cultured osteoblasts with PGE2 at different time periods showed a bell-shaped pattern with the optimal response at 2 h of incubation. A similar reaction can be induced by treatment with prostaglandin E1 (PGE1) or dibutyryl cyclic adenosine monophosphate (DBcAMP) in combination with 3-isobutyl-1-methylxanthine (IBMX). Furthermore, we assessed the percentage of responsive isolated bone cells to investigate interactions with other agents. The morphological changes induced by PGEs were inhibited by H-8, a protein kinase inhibitor. On the other hand, elevated intracellular calcium enhanced the PGE-induced morphological changes. Fluorescence labeling showed that PGEs caused the breakdown of the actin microfilaments, but spared the microtubules and vimentin filaments in the isolated osteoblast-like cells. These results suggest that the morphological changes of osteoblasts induced by PGEs may be related to the intracellular cAMP and calcium levels.
Assuntos
Cálcio/metabolismo , Osteoblastos/efeitos dos fármacos , Ocitócicos/farmacologia , Prostaglandinas E/farmacologia , 1-Metil-3-Isobutilxantina/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Bucladesina/farmacologia , Tamanho Celular/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Isoquinolinas/farmacologia , Microscopia Eletrônica de Varredura , Osteoblastos/ultraestrutura , Inibidores de Fosfodiesterase/farmacologia , Inibidores de Proteínas Quinases , Ratos , Ratos Wistar , Crânio , Vimentina/metabolismoRESUMO
1. Acetylcholine (ACh) is important as the transmitter responsible for neuromuscular transmission. Here we report the non-quantal release of ACh from embryonic myocytes. 2. Co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos. Single channel currents were recorded in the non-innervated myocytes. When the patch pipette was filled with Ringer solution alone, spontaneous single channel currents occurred, which were inhibited by d-tubocurarine (d-Tc). 3. The channel conductance appearing in Ringer solution (37.3 pS) was similar to that of an embryonic-type ACh channel (36.9 pS), indicating that ACh is probably released from myocytes in normal Ringer solution. 4. When the patch pipette was filled with anticholinesterase alone to prevent hydrolysis of ACh released from myocytes, both physostigmine and neostigmine in a concentration-dependent manner increased channel open probability; it was reduced by d-Tc or alpha-bungarotoxin. 5. Vesamicol and quinacrine, vesicular transporter inhibitors, reduced the channel open probability caused by ACh released from myocytes in the presence of neostigmine or physostigmine. 6. Intracellular alkalinization with NH4Cl inhibited the ACh release from myocytes, whereas, extracellular alkalinization, brought about by replacing normal Ringer solution, with pH 8.6 Ringer solution enhanced ACh release. 7. The immunocytochemistry of choline acetyltransferase (ChAT) showed that ChAT exists in both myocytes and neuronal cells but not in fibroblasts. 8. These results suggest that embryonic myocytes are capable of synthesizing and releasing ACh in a non-quantal manner. Extracellular alkalinization enhanced and intracellular alkalinization inhibited ACh release from myocytes.