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1.
Sci Rep ; 9(1): 8666, 2019 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-31209282

RESUMO

5-aminolevulinic acid (5-ALA) has recently been employed for photodynamic diagnosis (ALA-PDD) and photodynamic therapy (ALA-PDT) of various types of cancer because hyperproliferating tumor cells do not utilize oxidative phosphorylation and do not efficiently produce heme; instead, they accumulate protoporphyrin IX (PpIX), which is a precursor of heme that is activated by violet light irradiation that results in the production of red fluorescence and singlet oxygen. The efficiencies of ALA-PDD and ALA-PDT depend on the efficient cellular uptake of 5-ALA and the inefficient excretion of PpIX. We employed the JFCR39 cell panel to determine whether tumor cells originating from different tissues can produce and accumulate PpIX. We also investigated cellular factors/molecules involved in PpIX excretion by tumor cells with the JFCR39 cell panel. Unexpectedly, the expression levels of ABCG2, which has been considered to play a major role in PpIX extracellular transport, did not show a strong correlation with PpIX excretion levels in the JFCR39 cell panel, although an ABCG2 inhibitor significantly increased intracellular PpIX accumulation in several tumor cell lines. In contrast, the expression levels of dynamin 2, which is a cell membrane-associated molecule involved in exocytosis, were correlated with the PpIX excretion levels. Moreover, inhibitors of dynamin significantly suppressed PpIX excretion and increased the intracellular levels of PpIX. This is the first report demonstrating the causal relationship between dynamin 2 expression and PpIX excretion in tumor cells.


Assuntos
Ácido Aminolevulínico/farmacologia , Dinamina II/metabolismo , Exocitose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fármacos Fotossensibilizantes/metabolismo , Protoporfirinas/metabolismo , Linhagem Celular Tumoral , Dinamina II/antagonistas & inibidores , Dinamina II/genética , Exocitose/efeitos da radiação , Heme/antagonistas & inibidores , Heme/biossíntese , Humanos , Microscopia de Fluorescência , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Mitocôndrias/efeitos da radiação , Fotoquimioterapia , Compostos de Trimetil Amônio/farmacologia , Raios Ultravioleta
2.
Sci Rep ; 6: 27890, 2016 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-27293048

RESUMO

Ultraviolet (UV) irradiation induces skin pigmentation, which relies on the intercellular crosstalk of melanin between melanocytes to keratinocytes. However, studying the separate effects of UVA and UVB irradiation reveals differences in cellular response. Herein, we show an immediate shedding of extracellular vesicles (EVs) from the plasma membrane when exposing human melanocytes to UVA, but not UVB. The EV-shedding is preceded by UVA-induced plasma membrane damage, which is rapidly repaired by Ca(2+)-dependent lysosomal exocytosis. Using co-cultures of melanocytes and keratinocytes, we show that EVs are preferably endocytosed by keratinocytes. Importantly, EV-formation is prevented by the inhibition of exocytosis and increased lysosomal pH but is not affected by actin and microtubule inhibitors. Melanosome transfer from melanocytes to keratinocytes is equally stimulated by UVA and UVB and depends on a functional cytoskeleton. In conclusion, we show a novel cell response after UVA irradiation, resulting in transfer of lysosome-derived EVs from melanocytes to keratinocytes.


Assuntos
Vesículas Extracelulares/metabolismo , Raios Ultravioleta , Cálcio/metabolismo , Membrana Celular/metabolismo , Membrana Celular/efeitos da radiação , Células Cultivadas , Pré-Escolar , Técnicas de Cocultura , Exocitose/efeitos da radiação , Humanos , Concentração de Íons de Hidrogênio , Lactente , Recém-Nascido , Queratinócitos/citologia , Queratinócitos/metabolismo , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Microscopia Confocal , Microscopia Eletrônica de Transmissão
3.
Acta Derm Venereol ; 95(7): 792-7, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25669167

RESUMO

Ultraviolet (UV) irradiation is a risk factor for development of malignant melanoma. UVA-induced lysosomal exocytosis and subsequent cell growth enhancement was studied in malignant melanoma cell lines and human skin melanocytes. UVA irradiation caused plasma membrane damage that was rapidly repaired by calcium-dependent lysosomal exocytosis. Lysosomal content was released into the culture medium directly after irradiation and such conditioned media stimulated the growth of non-irradiated cell cultures. By comparing melanocytes and melanoma cells, it was found that only the melanoma cells spontaneously secreted cathepsins into the surrounding medium. Melanoma cells from a primary tumour showed pronounced invasion ability, which was prevented by addition of inhibitors of cathepsins B, D and L. Proliferation was reduced by cathepsin L inhibition in all melanoma cell lines, but did not affect melano-cyte growth. In conclusion, UVA-induced release of cathepsins outside cells may be an important factor that promotes melanoma growth and progression.


Assuntos
Catepsinas/metabolismo , Exocitose/efeitos da radiação , Lisossomos/enzimologia , Lisossomos/efeitos da radiação , Melanoma/enzimologia , Neoplasias Cutâneas/enzimologia , Raios Ultravioleta/efeitos adversos , Catepsinas/antagonistas & inibidores , Linhagem Celular Tumoral , Movimento Celular/efeitos da radiação , Proliferação de Células/efeitos da radiação , Progressão da Doença , Humanos , Melanócitos/enzimologia , Melanócitos/efeitos da radiação , Melanoma/secundário , Invasividade Neoplásica , Inibidores de Proteases/farmacologia , Neoplasias Cutâneas/patologia
4.
J Cell Biol ; 203(2): 283-98, 2013 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-24165939

RESUMO

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.


Assuntos
Exocitose , Neurônios/enzimologia , Vesículas Secretórias/enzimologia , Transmissão Sináptica , Vesículas Sinápticas/enzimologia , ATPases Vacuolares Próton-Translocadoras/metabolismo , Animais , Catecolaminas/metabolismo , Bovinos , Células Cromafins/enzimologia , Células Cromafins/metabolismo , Exocitose/efeitos dos fármacos , Exocitose/efeitos da radiação , Concentração de Íons de Hidrogênio , Cinética , Luz , Fusão de Membrana , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/efeitos da radiação , Células PC12 , Estrutura Terciária de Proteína , Interferência de RNA , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Vesículas Secretórias/efeitos dos fármacos , Vesículas Secretórias/metabolismo , Vesículas Secretórias/efeitos da radiação , Potenciais Sinápticos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/efeitos da radiação , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/efeitos da radiação , Transfecção , ATPases Vacuolares Próton-Translocadoras/genética
5.
Biochim Biophys Acta ; 1830(3): 2853-60, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23178861

RESUMO

BACKGROUND: Neurons signal to each other and to non-neuronal cells as those in muscle or glands, by means of the secretion of neurotransmitters at chemical synapses. In order to dissect the molecular mechanisms of neurotransmission, new methods for directly and reversibly triggering neurosecretion at the presynaptic terminal are necessary. Here we exploit the calcium permeability of the light-gated channel LiGluR in order to reversibly manipulate cytosolic calcium concentration, thus controlling calcium-regulated exocytosis. METHODS: Bovine chromaffin cells expressing LiGluR were stimulated with light. Exocytic events were detected by amperometry or by whole-cell patch-clamp to quantify membrane capacitance and calcium influx. RESULTS: Amperometry reveals that optical stimulation consistently triggers exocytosis in chromaffin cells. Secretion of catecholamines can be adjusted between zero and several Hz by changing the wavelength of illumination. Differences in secretion efficacy are found between the activation of LiGluR and native voltage-gated calcium channels (VGCCs). Our results show that the distance between sites of calcium influx and vesicles ready to be released is longer when calcium influx is triggered by LiGluR instead of native VGCCs. CONCLUSION: LiGluR activation directly and reversibly increases the intracellular calcium concentration. Light-gated calcium influx allows for the first time to control calcium-regulated exocytosis without the need of applying depolarizing solutions or voltage clamping in chromaffin cells. GENERAL SIGNIFICANCE: LiGluR is a useful tool to study the secretory mechanisms and their spatiotemporal patterns in neurotransmission, and opens a window to study other calcium-dependent processes such as muscular contraction or cell migration.


Assuntos
Potenciais de Ação/efeitos da radiação , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Exocitose/efeitos da radiação , Receptores de Glutamato/metabolismo , Transmissão Sináptica/efeitos da radiação , Potenciais de Ação/fisiologia , Adenoviridae/genética , Medula Suprarrenal/citologia , Medula Suprarrenal/metabolismo , Medula Suprarrenal/efeitos da radiação , Animais , Canais de Cálcio/genética , Catecolaminas/metabolismo , Bovinos , Células Cromafins/citologia , Células Cromafins/metabolismo , Células Cromafins/efeitos da radiação , Capacitância Elétrica , Exocitose/fisiologia , Expressão Gênica/efeitos da radiação , Vetores Genéticos , Luz , Técnicas de Patch-Clamp , Estimulação Luminosa , Cultura Primária de Células , Receptores de Glutamato/genética
6.
Artigo em Russo | MEDLINE | ID: mdl-23210366

RESUMO

Laserophoresis is a technique for the transcutaneous administration of biologically active compounds by means of low-intensity laser radiation (LFLR). It is currently regarded as a most promising method for the integrated application of a pharmaceutical substance and a physical factor. At present laserophoresis of various medicinal preparations is successfully used after preliminary experimental studies of their phoretic properties for the treatment of various inflammatory and dystrophic conditions as well as for the prevention of skin ageing. The most important route for the administration of the majority of drug preparations is through the shunts provided by perspiratory glands and hair follicles. Another essential factor determining the potential possibility of drug penetration through the skin is the characteristic of the substance chosen for the administration, such as its molecular weight, chemical structure, conformation, and hydrophilic properties. However, the most likely mechanism underlying the transport of the substance through the glandular cells of perspiratory glands and epithelial cells of hair follicles is pinocytosis, i.e. the process integrating exocytosis and endocytosis. To-day, the majority of the researchers lay emphasis on thermodynamic triggering of Ca2+-dependent processes as the primary mechanism behind the biological action of low-intensity laser radiation. Both exocytosis and endocytosis being the Ca2+-dependent processes, the liberation of Ca2+-ions under the influence of LFLR causes the activation of pinocytosis as a whole.


Assuntos
Células Epiteliais/metabolismo , Exocitose/efeitos da radiação , Folículo Piloso/metabolismo , Terapia com Luz de Baixa Intensidade/métodos , Pinocitose/efeitos da radiação , Glândulas Sudoríparas/metabolismo , Administração Tópica , Animais , Cálcio/metabolismo , Folículo Piloso/citologia , Humanos , Glândulas Sudoríparas/citologia
7.
Proc Natl Acad Sci U S A ; 109(13): 4904-9, 2012 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-22416118

RESUMO

The mitochondrial pathway of apoptosis is the major mechanism of physiological cell death in vertebrates. In this pathway, proapoptotic members of the Bcl-2 family cause mitochondrial outer membrane permeabilization (MOMP), allowing the release of cytochrome c, which interacts with Apaf-1 to trigger caspase activation and apoptosis. Despite conservation of Bcl-2, Apaf-1, and caspases in invertebrate phyla, the existence of the mitochondrial pathway in any invertebrate is, at best, controversial. Here we show that apoptosis in a lophotrochozoan, planaria (phylum Platyhelminthes), is associated with MOMP and that cytochrome c triggers caspase activation in cytosolic extracts from these animals. Further, planarian Bcl-2 family proteins can induce and/or regulate cell death in yeast and can replace Bcl-2 proteins in mammalian cells to regulate MOMP. These results suggest that the mitochondrial pathway of apoptosis in animals predates the emergence of the vertebrates but was lost in some lineages (e.g., nematodes). In further support of this hypothesis, we surveyed the ability of cytochrome c to trigger caspase activation in cytosolic extracts from a variety of organisms and found this effect in cytosolic extracts from invertebrate deuterostomes (phylum Echinodermata).


Assuntos
Apoptose , Mitocôndrias/metabolismo , Planárias/metabolismo , Planárias/efeitos da radiação , Transdução de Sinais , Animais , Apoptose/efeitos da radiação , Caspases/metabolismo , Extratos Celulares , Citocromos c/metabolismo , Ativação Enzimática/efeitos da radiação , Exocitose/efeitos da radiação , Raios gama , Mitocôndrias/efeitos da radiação , Membranas Mitocondriais/efeitos da radiação , Permeabilidade/efeitos da radiação , Fosfatidilserinas/metabolismo , Transporte Proteico/efeitos da radiação , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/efeitos da radiação , Ouriços-do-Mar/citologia , Ouriços-do-Mar/metabolismo , Ouriços-do-Mar/efeitos da radiação , Transdução de Sinais/efeitos da radiação , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo
8.
Apoptosis ; 16(1): 1-12, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20717727

RESUMO

Inhibitor of apoptosis (IAP) and Heat shock proteins (HSPs) provide assistance in protecting cells from stresses of hypoxia, imbalanced pH, and altered metabolic and redox states commonly found in the microenvironmental mixture of tumor and nontumor cells. HSPs are upregulated, cell-surface displayed and released extracellularly in some types of tumors, a finding that until now was not shared by members of the IAP family. The IAP Survivin has been implicated in apoptosis inhibition and the regulation of mitosis in cancer cells. Survivin exists in a number of subcellular locations such as the mitochondria, cytoplasm, nucleus, and most recently, the extracellular space. Our previous work showing that extracellular survivin was able to enhance cellular proliferation, survival and tumor cell invasion provides evidence that Survivin might be secreted via an unidentified exocytotic pathway. In the present study, we describe for the first time the exosome-release of Survivin to the extracellular space both basally and after proton irradiation-induced stress. To examine whether exosomes contributed to Survivin release from cancer cells, exosomes were purified from HeLa cervical carcinoma cells and exosome quantity and Survivin content were determined. We demonstrate that although proton irradiation does not influence the exosomal secretory rate, the Survivin content of exosomes isolated from HeLa cells treated with a sublethal dose of proton irradiation (3 Gy) is significantly higher than control. These data identify a novel secretory pathway by which Survivin can be actively released from cells in both the basal and stress-induced state.


Assuntos
Inibidores de Cisteína Proteinase/metabolismo , Exossomos/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Neoplasias do Colo do Útero/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Proliferação de Células , Inibidores de Cisteína Proteinase/genética , Citoesqueleto/metabolismo , Exocitose/efeitos da radiação , Exossomos/genética , Espaço Extracelular/metabolismo , Feminino , Expressão Gênica/efeitos da radiação , Células HeLa , Humanos , Proteínas Inibidoras de Apoptose/genética , Prótons , Radioisótopos , Via Secretória/efeitos da radiação , Survivina , Regulação para Cima , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/terapia
9.
PLoS One ; 5(7): e11773, 2010 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-20668710

RESUMO

Stratospheric ozone depletion, climate warming and acidification of aquatic ecosystems have resulted in elevated levels of solar radiation reaching many aquatic environments with an increased deleterious impact on a wide range of living organisms. While detrimental effects on living organisms are thought to occur primarily through DNA damage, solar UV can also damage cellular proteins, lipids and signalling pathways. Cryptosporidium, a member of the eukaryotic phylum Apicomplexa, contain numerous vesicular secretory organelles and their discharge via regulated exocytosis is essential for the successful establishment of infection. Using flow cytometric techniques we demonstrate that solar UV rapidly induces sporozoite exocytosis resulting in a significant reduction in the ability of sporozoites to attach and invade host cells. We found that solar UV induced sporozoite membrane depolarization, resulting in reduced cellular ATP and increased cytosolic calcium. These changes were accompanied by a reduction in the internal granularity of sporozoites, indicative of apical organelle discharge, which was confirmed by analysis of sporozoites with an exocytosis-sensitive dye. The precise timing of apical organelle discharge in the presence of a compatible host cell is critical for sporozoite attachment and invasion. Our results demonstrate for the first time how solar UV radiation can interfere with exocytosis, a fundamental cellular process in all eukaryotic cells. We contend that not only may the forecast increases in solar radiation in both aquatic and terrestrial environments significantly affect members of the Apicomplexa, solar UV-induced membrane depolarizations resulting in cytosolic calcium perturbation may affect a wider range of eukaryotic organisms through antagonistic effects on a myriad of calcium dependant cellular functions.


Assuntos
Cryptosporidium parvum/citologia , Cryptosporidium parvum/efeitos da radiação , Exocitose/efeitos da radiação , Luz Solar , Raios Ultravioleta , Animais , Citometria de Fluxo , Esporozoítos/citologia , Esporozoítos/efeitos dos fármacos
10.
Apoptosis ; 14(5): 655-64, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19259823

RESUMO

Sanazole has been tested clinically as a hypoxic cell radiosensitizer. In this study, we determined whether sanazole enhances the radiation-induced apoptosis of human lymphoma U937 cells. Our results revealed that, compared with 10 mM sanazole or radiation alone, the combination of both resulted in a significant enhancement of apoptosis after 6 h, which was evaluated on the basis of DNA fragmentation, morphological changes, and phosphatidylserine externalization. Sanazole alone enhanced intracellular superoxide and hydrogen peroxide formation, which further increased when the cells were irradiated. Significant enhancement of Fas externalization, loss of mitochondrial membrane potential (MMP), and activation of caspase-3 and caspase-8 were observed after the combined treatment. Moreover, this combination could also enhance Bid activation, reduction of Hsp70 expression level and release of cytochrome c from the mitochondria to the cytosol. An immediate increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) was observed after the combined treatment. These results suggest that the intracellular superoxide and peroxide generated by sanazole might be involved in the enhancement of radiation-induced apoptosis, and that these effects are associated with modulation of the Fas-mitochondria-caspase-dependent pathway, an increase in [Ca(2+)](i), and a decrease in the Hsp70 expression levels.


Assuntos
Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Radiossensibilizantes/farmacologia , Triazóis/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos da radiação , Caspases/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Fragmentação do DNA/efeitos da radiação , Ensaios de Seleção de Medicamentos Antitumorais , Exocitose/efeitos dos fármacos , Exocitose/efeitos da radiação , Humanos , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/efeitos da radiação , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/efeitos da radiação , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/efeitos da radiação , Estresse Oxidativo/efeitos dos fármacos , Radiação Ionizante , Espécies Reativas de Oxigênio/metabolismo , Células U937 , Receptor fas/metabolismo
11.
Chembiochem ; 10(2): 251-6, 2009 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-19132694

RESUMO

We have developed a method for the photomanipulation of lipid membrane morphology in which the shape of a vesicle can be switched by light through the use of a synthetic photosensitive amphiphile containing an azobenzene unit (KAON12). We prepared cell-sized liposomes from KAON12 and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and conducted real-time observations of vesicular transformation in the photosensitive liposome by phase-contrast microscopy. Budding transitions-either budding toward the centre of the liposome (endo-bud) or budding out of the liposome (exo-bud)-could be controlled by light. We discuss the mechanism of this transformation in terms of the change in the effective membrane surface area due to photoisomerization of the constituent molecules.


Assuntos
Endocitose , Exocitose , Luz , Lipossomos/química , Lipossomos/efeitos da radiação , Fosfolipídeos/química , Endocitose/efeitos da radiação , Exocitose/efeitos da radiação , Isomerismo , Microscopia de Contraste de Fase , Pressão Osmótica/efeitos da radiação
12.
J Neurosci ; 28(30): 7670-8, 2008 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-18650343

RESUMO

The mammalian cochlea is specialized to recognize and process complex auditory signals with remarkable acuity and temporal precision over a wide frequency range. The quality of the information relayed to the auditory afferent fibers mainly depends on the transfer characteristics of inner hair cell (IHC) ribbon synapses. To investigate the biophysical properties of the synaptic machinery, we measured changes in membrane capacitance (DeltaC(m)) in low-frequency (apical region, approximately 300 Hz) and high-frequency (basal, approximately 30 kHz) gerbil IHCs maintained in near physiological conditions (1.3 mm extracellular Ca(2+) and body temperature). With maturation, the Ca(2+) efficiency of exocytosis improved in both apical and basal IHCs and was more pronounced in the latter. Prehearing IHCs showed a similar Ca(2+) cooperativity of exocytosis despite the smaller DeltaC(m) in apical cells. After maturation, DeltaC(m) in high-frequency IHCs increased linearly with the Ca(2+) current, whereas, somewhat surprisingly, the relationship was significantly more nonlinear in low-frequency cells. This tonotopic difference seemed to be correlated with ribbon synapse morphology (spherical in apical and ellipsoid in basal IHCs) but not with the expression level of the proposed Ca(2+) sensor otoferlin or the spatial coupling between Ca(2+) channels and active zones. Repetitive stimulation of adult IHCs showed that vesicle pool refilling could become rate limiting for vesicle release, with high-frequency IHCs able to sustain greater release rates. Together, our findings provide the first evidence for a tonotopic difference in the properties of the synaptic machinery in mammalian IHCs, which could be essential for fine-tuning their receptor characteristics during sound stimulation.


Assuntos
Cálcio/metabolismo , Células Ciliadas Auditivas Internas/citologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Animais Recém-Nascidos , Cálcio/farmacologia , Cóclea/citologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Estimulação Elétrica/métodos , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Exocitose/efeitos da radiação , Proteínas do Olho/metabolismo , Gerbillinae , Células Ciliadas Auditivas Internas/efeitos da radiação , Indóis , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Proteínas de Membrana/metabolismo , Proteínas de Neurofilamentos/metabolismo , Técnicas de Patch-Clamp/métodos , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/efeitos da radiação , Fatores de Tempo
13.
Nat Neurosci ; 10(3): 331-9, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17310248

RESUMO

The release of transmitters from glia influences synaptic functions. The modalities and physiological functions of glial release are poorly understood. Here we show that glutamate exocytosis from astrocytes of the rat hippocampal dentate molecular layer enhances synaptic strength at excitatory synapses between perforant path afferents and granule cells. The effect is mediated by ifenprodil-sensitive NMDA ionotropic glutamate receptors and involves an increase of transmitter release at the synapse. Correspondingly, we identify NMDA receptor 2B subunits on the extrasynaptic portion of excitatory nerve terminals. The receptor distribution is spatially related to glutamate-containing synaptic-like microvesicles in the apposed astrocytic processes. This glial regulatory pathway is endogenously activated by neuronal activity-dependent stimulation of purinergic P2Y1 receptors on the astrocytes. Thus, we provide the first combined functional and ultrastructural evidence for a physiological control of synaptic activity via exocytosis of glutamate from astrocytes.


Assuntos
Astrócitos/metabolismo , Exocitose/fisiologia , Ácido Glutâmico/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Análise de Variância , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/efeitos da radiação , Astrócitos/ultraestrutura , Estimulação Elétrica/métodos , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Exocitose/efeitos dos fármacos , Exocitose/efeitos da radiação , Hipocampo/citologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Potenciais da Membrana/efeitos da radiação , Microscopia Imunoeletrônica/métodos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Neurônios/efeitos da radiação , Técnicas de Patch-Clamp/métodos , Via Perfurante/fisiologia , Via Perfurante/efeitos da radiação , Piperidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/ultraestrutura , Sinapses/ultraestrutura
14.
J Neurosci ; 26(45): 11606-14, 2006 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-17093082

RESUMO

The mechanism by which synaptic vesicles (SVs) are recruited to the release site is poorly understood. One candidate mechanism for trafficking of SVs is the myosin-actin motor system. Myosin activity is modulated by myosin light chain kinase (MLCK), which in turn is activated by calmodulin. Ca(2+) signaling in presynaptic terminals, therefore, may serve to regulate SV mobility along actin filaments via MLCK. Previous studies in different types of synapses have supported such a hypothesis. Here, we further investigated the role of MLCK in neurotransmitter release at glutamatergic synapses in cultured hippocampal neurons by examining the effects of two MLCK inhibitors, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine.HCl (ML-7) and wortmannin. Bath application of ML-7 enhanced short-term depression of EPSCs to repetitive stimulation, whereas it reduced presynaptic release probability. However, ML-7 also inhibited action potential amplitude and voltage-gated Ca(2+) channel currents. These effects were not mimicked by wortmannin, suggesting that ML-7 was not specific to MLCK in hippocampal neurons. When SV exocytosis was directly triggered by a Ca(2+) ionophore, calcimycin, to bypass voltage-gated Ca(2+) channels, ML-7 had no effect on neurotransmitter release. Furthermore, when SV exocytosis elicited by electrical field stimulation was monitored by styryl dye, FM1-43 [N-(3-triethylammoniumpropyl)-4-(4-(dibutylamino)styryl)pyridinium dibromide], the unloading kinetics of the dye was not altered in the presence of wortmannin. These data indicate that MLCK is not a major regulator of presynaptic SV trafficking during repetitive exocytosis at hippocampal synapses.


Assuntos
Exocitose/fisiologia , Hipocampo/citologia , Quinase de Cadeia Leve de Miosina/fisiologia , Neurônios/fisiologia , Vesículas Sinápticas/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Análise de Variância , Animais , Animais Recém-Nascidos , Axônios/metabolismo , Cálcio/metabolismo , Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Exocitose/efeitos dos fármacos , Exocitose/efeitos da radiação , Imuno-Histoquímica/métodos , Proteínas de Neurofilamentos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Compostos de Piridínio/metabolismo , Compostos de Amônio Quaternário/metabolismo , Ratos , Vesículas Sinápticas/efeitos dos fármacos
15.
J Neurosci ; 26(18): 4820-5, 2006 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-16672655

RESUMO

AMPA receptor (AMPAR) internalization provides a mechanism for long-term depression (LTD) in both hippocampal pyramidal neurons and cerebellar Purkinje cells (PCs). Cerebellar LTD at the parallel fiber (PF)-PC synapse is the underlying basis of motor learning and requires AMPAR activation, a large Ca2+ influx, and protein kinase C (PKC) activation. However, whether these requirements affect the constitutive AMPAR internalization in PF-PC synapses remains unclarified. Tetanus toxin (TeTx) infusion into PCs decreased PF-EPSC amplitude to 60% within 20-30 min (TeTx rundown), without change in paired-pulse facilitation ratio or receptor kinetics. Immunocytochemically measured glutamate receptor 2 (GluR2) internalization ratio decreased at the steady state of TeTx rundown. TeTx rundown did not require AMPAR activity nor an increase in intracellular Ca2+ concentration. TeTx rundown was suppressed partially by the inhibition of either conventional PKC or mitogen-activated protein kinase kinase (MEK) and completely by the inhibition of both kinases. The background PKC activity was shown to be sufficient, because a PKC activator did not facilitate TeTx rundown. The inhibition of protein phosphatase 1/2A (PP1/2A) enhanced TeTx rundown slightly, and both inhibition of PP1/2A and activation of PKC maximized it, but one-half of AMPARs at PF-PC synapses remained in the TeTx-resistant pool. The inhibition of actin depolymerization suppressed TeTx rundown and decreased the GluR2 internalization ratio. In contrast, the inhibition of actin polymerization enhanced TeTx rundown and increased the GluR2 internalization ratio. We suggest that the regulation of actin polymerization is involved in the surface expression of AMPARs and the surface expressing AMPARs are constitutively internalized through both basal PKC and MEK-ERK1/2 (extracellular signal-regulated kinase 1/2) activities at PF-PC synapses.


Assuntos
Cerebelo/citologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase C/metabolismo , Células de Purkinje/metabolismo , Receptores de AMPA/metabolismo , Acetamidas/farmacologia , Animais , Animais Recém-Nascidos , Proteínas de Bactérias/farmacologia , Células Cultivadas , Diagnóstico por Imagem/métodos , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Interações Medicamentosas , Estimulação Elétrica/métodos , Embrião de Mamíferos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Exocitose/efeitos da radiação , Técnicas In Vitro , Oxigenases de Função Mista/farmacologia , Técnicas de Patch-Clamp/métodos , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/fisiologia , Células de Purkinje/efeitos dos fármacos , Ratos , Estatísticas não Paramétricas
16.
FEBS Lett ; 580(9): 2201-6, 2006 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-16574108

RESUMO

Phaeocystis globosa, a leading agent in marine carbon cycling, releases its photosynthesized biopolymers via regulated exocytosis. Release is elicited by blue light and relayed by a characteristic cytosolic Ca(2+) signal. However, the source of Ca(2+) in these cells has not been established. The present studies indicate that Phaeocystis' secretory granules work as an intracellular Ca(2+) oscillator. Optical tomography reveals that photo-stimulation induces InsP(3)-triggered periodic lumenal [Ca(2+)] oscillations in the granule and corresponding out-of-phase cytosolic oscillations of [Ca(2+)] that trigger exocytosis. This Ca(2+) dynamics results from an interplay between the intragranular polyanionic matrix, and two Ca(2+)-sensitive ion channels located on the granule membrane: an InsP(3)-receptor-Ca(2+) channel, and an apamin-sensitive K(+) channel.


Assuntos
Sinalização do Cálcio/fisiologia , Exocitose/fisiologia , Phaeophyceae/fisiologia , Vesículas Secretórias/metabolismo , Sinalização do Cálcio/efeitos da radiação , Exocitose/efeitos da radiação , Canais Iônicos/metabolismo , Transporte de Íons/fisiologia , Transporte de Íons/efeitos da radiação , Luz , Phaeophyceae/citologia , Tomografia
17.
Electromagn Biol Med ; 25(1): 53-60, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16595334

RESUMO

Serial electron microscopic sections were prepared from half-ripened meristematic root cells of Vicia faba (Fabaceae) which had been exposed gradually to 700, 1000, 2500, 3500, and 5000 V/m static electric fields during seven days with and without Zn and Cd electrodes. At the end of five weeks, wall loosenings and very small nuclei were observed in those root cells which were exposed to static electric currents from the lower side of the medium without electrodes, while abnormalities in cell formation, e.g., two cells with one nucleus, and GER occurrence were present in an electrolytic (Cd upward and Zn downward) medium. The cells exposed to a static current from the upper side of the medium had small nuclei and abnormal cell divisions in the electrolyte, but in a non-electrolyte very large nuclei and thicker cell walls were observed, the cytoplasm was dense with GER, pinocytosis was seen filled with mitochondria, and protoplast formation with big nuclei was seen in exocytosis.


Assuntos
Divisão Celular/efeitos da radiação , Núcleo Celular/efeitos da radiação , Campos Eletromagnéticos , Raízes de Plantas/efeitos da radiação , Vicia faba , Cádmio/química , Núcleo Celular/ultraestrutura , Citoplasma/efeitos da radiação , Eletrodos , Eletrólitos , Exocitose/efeitos da radiação , Mitocôndrias/efeitos da radiação , Pinocitose/efeitos da radiação , Raízes de Plantas/citologia , Raízes de Plantas/ultraestrutura , Protoplastos/efeitos da radiação , Zinco/química
18.
Brain Res ; 1078(1): 1-8, 2006 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-16492381

RESUMO

Synaptic vesicle exocytosis in primary cultures of baroreceptor neurons is reduced during high-frequency stimulation. Calcium influx through voltage-gated calcium channels (VGCC) is a key step in neurotransmitter release. With the help of FM2-10, a marker of synaptic vesicle recycling, the present study investigates the differential contribution of several VGCC subtypes to exocytosis in neuronal processes and how this contribution is altered at high frequencies. In control experiments, field stimulation at 0.5 Hz evoked about 66 +/- 5% destaining. Combined blockade of N- and P/Q-subtypes with Ctx-MVIIC was far more effective in reducing exocytosis (11 +/- 8%) than blocking N-type (49 +/- 5%, Ctx-GVIA) or P-type (46 +/- 1%, Agatoxin) alone. The effectiveness of the blockers also varied with the duration of stimulation: Ctx-GVIA attenuating exocytosis significantly in the first 60 s and Agatoxin affecting exocytosis only towards the end of 180 s stimulation period. Field stimulation at 10 Hz evoked exocytosis (36 +/- 18%) comparable to that evoked by 0.5 Hz in the presence of Ctx-GVIA. While blockade with Agatoxin had no effects, Ctx-GVIA, Ctx-MVIIC and L-type blocker Nifedepine had small but similar inhibitory effects on exocytosis at 10 Hz. The data suggest that N-type is the major contributor to exocytosis at 0.5 Hz, and this contribution is reduced during prolonged stimulation periods and at high frequencies.


Assuntos
Canais de Cálcio Tipo N/fisiologia , Estimulação Elétrica/métodos , Exocitose/efeitos da radiação , Neurônios/efeitos da radiação , Análise de Variância , Animais , Cloreto de Cádmio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta à Radiação , Imuno-Histoquímica/métodos , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Ativação do Canal Iônico/efeitos da radiação , Neurônios/fisiologia , Gânglio Nodoso/citologia , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
19.
Plant Cell Physiol ; 45(5): 535-42, 2004 May.
Artigo em Inglês | MEDLINE | ID: mdl-15169935

RESUMO

Almost half of the global photosynthetic activity is carried out in the ocean. During blooms, Phaeocystis can fix CO(2) at rates up to 40 g C m(-2) month(-1). Most of this carbon is released as polysaccharides. However, the cellular mechanism whereby this huge amount of organic material is exported into the seawater remains unknown. A vaguely defined process of "exudation" is believed responsible for the release of these biopolymers. Here we report the first demonstration that Phaeocystis globosa does not "exude", but secretes microscopic gels. Secretion is stimulated by blue light (lambda = 470+/-20 nm), and it is transduced by a characteristic intracellular Ca(2+) signal that precedes degranulation. The polysaccharides that form the matrix of these gels remain in condensed phase while stored in secretory vesicles. Upon exocytosis, the exopolymer matrix undergoes a characteristic phase transition accompanied by extensive swelling resulting in the formation of microscopic hydrated gels. Owing to their tangled topology, once released into the seawater, the polymers that make these gels can reptate (axially diffuse), interpenetrate neighboring gels, and anneal them together forming massive mucilage accumulations that are characteristic of Phaeocystis blooms. These gel masses can supply a rich source of microbial substrates, disperse in the seawater, and/or eventually sediment to the ocean floor.


Assuntos
Secreções Corporais/fisiologia , Exocitose/fisiologia , Fitoplâncton/metabolismo , Polissacarídeos/metabolismo , Adesivos/metabolismo , Secreções Corporais/efeitos da radiação , Sinalização do Cálcio/fisiologia , Dióxido de Carbono/metabolismo , Exocitose/efeitos da radiação , Géis/metabolismo , Luz , Transdução de Sinal Luminoso/fisiologia , Estimulação Luminosa , Fotossíntese/fisiologia , Fitoplâncton/citologia , Fitoplâncton/efeitos da radiação , Polímeros/metabolismo , Vesículas Secretórias/metabolismo , Vesículas Secretórias/efeitos da radiação , Vesículas Secretórias/ultraestrutura
20.
Neurosci Lett ; 362(3): 249-52, 2004 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-15158025

RESUMO

For over 20 years, the bag cell neurons of the marine mollusk Aplysia have been used to investigate second-messenger pathways that mediate effects of synaptic stimulation on ion currents and membrane excitability, presumably leading to exocytotic release of the neuropeptide egg-laying hormone (ELH). It is widely cited that a train of action potentials, called an afterdischarge, is necessary for activating cellular events leading to ELH secretion. Using a combination of electrophysiology, optical imaging of calcium signaling, and radioimmunoassay of ELH secretion, we show that an afterdischarge is not required for ELH secretion. Electrical stimulation that failed to produce afterdischarges but did lead to prolonged membrane depolarization and a rise in intracellular calcium concentration was sufficient to stimulate significant ELH release.


Assuntos
Potenciais de Ação/efeitos da radiação , Hormônios de Invertebrado/metabolismo , Sinapses/efeitos da radiação , Potenciais de Ação/fisiologia , Animais , Aplysia , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Estimulação Elétrica/métodos , Eletrofisiologia/métodos , Exocitose/efeitos da radiação , Neurônios , Sinapses/fisiologia
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