RESUMO
The "plasticity hypothesis" proposes that major depression is caused by morphological and biochemical modifications in neurons and astrocytes and those beneficial pharmacological effects of selective-serotonin-reuptake-inhibitors (SSRI) are at least partially associated with modifications of cellular communications between these cells. In this study we examined effects of the antidepressant fluoxetine on cultured astrocytes that were, in some cases, pretreated with dexamethasone, a cortisol analog known to trigger depressive disorder. Primary rat astrocytes were purified and treated with dexamethasone and the SSRI fluoxetine in physiological concentrations so that both drugs did not affect cell viability. Expression of interleukin-2 (IL-2) and glia-derived-neurotrophic-factor (GDNF) were analyzed and monitored and cell viability, apoptosis, cluster formation, particle-removing capacity and cell mobility were also monitored. Pre-studies without any drugs on mixed neuron-astrocyte co-cultures suggested that astrocytes interacted with neurons and other brain cells in vitro by actively assembling them into clusters. Treatment of purified astrocytes with dexamethasone significantly decreased their mobility compared to controls but had no effect on cluster formation. Dexamethasone-treated cells removed fewer extracellular particles derived from dead cells and cell debris. Both effects were abolished by simultaneous application of fluoxetine. Intracellular IL-2 increased, while GDNF amount expression was diminished following dexamethasone treatment. Simultaneous administration of fluoxetine reversed dexamethasone-triggered IL-2 elevation but had no effect on decreased GDNF concentration. These results suggest that mobility and growth factor equilibrium of astrocytes are affected by dexamethasone and by fluoxetine and that fluoxetine could reverse some changes induced by dexamethasone.
Assuntos
Astrócitos/efeitos dos fármacos , Citocinas/metabolismo , Dexametasona/farmacologia , Fluoxetina/farmacologia , Glucocorticoides/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Astrócitos/fisiologia , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Feminino , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Interleucina-2/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Ratos Sprague-DawleyRESUMO
Metabolic and functional studies of the amyloid precursor protein (APP) in platelets have advanced our understanding of Alzheimer's disease (AD). Here we report that human platelets contain Abeta peptides, process and secrete them constitutively. Platelets generate formerly unkown Abeta-species by differential processing of APP. Release of Abeta peptides were also increased by platelet activation with thrombin, indicating the existence of a regulated exocytotic pathway. We showed that Abeta-levels, Abeta-processing patterns and Abeta-release kinetics were regulated by thrombin. In controls, release of Abeta peptide species (Abeta 1-40/42 and 1-37/38/39/) continued for more than 4 h, while thrombin activated cells ceased secretion after 1 h at large. Treatment of platelets with prostaglandine 2 slowed this process down. Intracellular Abeta peptide concentrations decreased steadily until no peptides could be detected after 20 h (control) or after 4 h (thrombin) in cultured platelets.
Assuntos
Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Plaquetas/fisiologia , Dinoprostona/metabolismo , Trombina/metabolismo , Plaquetas/metabolismo , Células Cultivadas , Colágeno/metabolismo , Eletroforese em Gel de Poliacrilamida , Exocitose/fisiologia , Espaço Extracelular/metabolismo , Humanos , Imunoprecipitação , Espaço Intracelular/metabolismo , Cinética , Fragmentos de Peptídeos/metabolismo , Fatores de TempoRESUMO
The diagnostic potential of large A beta-peptide binding particles (LAPs) in the cerebrospinal fluid (CSF) of Alzheimer's dementia (AD) patients and non-AD controls (nAD) was evaluated. LAPs were detected by confocal spectroscopy in both groups with high inter-individual variation in number. Molecular imaging by confocal microscopy revealed that LAPs are heterogeneous superaggregates that could be subdivided morphologically into four main types (LAP 1-4). LAP-4 type, resembling a 'large chain of pearls', was detected in 42.1% of all nAD controls but it was virtually absent in AD patients. LAP-4 type could be selectively removed by protein A beads, a clear indication that it contained immunoglobulins in addition to beta-amyloid peptides (A beta 1-42). We observed a close correlation between LAPs and immunoglobulin G (IgG) concentration in CSF in controls but not in AD patients. Double labeling of LAPs with anti-A beta and anti-IgG antibodies confirmed that LAP-4 type consisted of A beta and IgG aggregates. Our results assign a central role to the immune system in regulating A beta1-42 homeostasis by clustering this peptide in immunocomplexes.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/imunologia , Complexo Antígeno-Anticorpo/líquido cefalorraquidiano , Autoanticorpos/líquido cefalorraquidiano , Demência/líquido cefalorraquidiano , Imunoglobulina G/líquido cefalorraquidiano , Fragmentos de Peptídeos/imunologia , Doença de Alzheimer/imunologia , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Autoanticorpos/classificação , Biomarcadores/líquido cefalorraquidiano , Estudos de Casos e Controles , Sistema Nervoso Central/imunologia , Demência/classificação , Demência/imunologia , Humanos , Imunoglobulina G/imunologia , Fragmentos de Peptídeos/líquido cefalorraquidiano , Valores de ReferênciaRESUMO
A newly discovered system of photoreceptors for circadian rhythms works non-visual and responds to blue light (460 nm). We report a longitudinal study in 44 adults, showing that a significant increase in alertness and speed of information processing could be achieved by blue light as compared to normal light.
Assuntos
Cognição/fisiologia , Luz , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Projetos PilotoRESUMO
Acid sphingomyelinase (A-SMase) and its reaction product ceramide may play a role in the pathophysiology of depressive disorders and in the therapeutic action of antidepressive drugs. In a prospective case-control study, A-SMase activity was measured in peripheral blood mononuclear cells of 17 patients with a major depressive episode who were free of antidepressant drug therapy for at least 10 days and 8 healthy volunteers. In the patient group, A-SMase activity was correlated to the score (n=17, r=0.64, P=0.005). The patient group exhibited higher A-SMase activity compared to healthy volunteers (T=2.09, df=21.33, P<0.05). In addition, we demonstrate that the antidepressants imipramine and amitriptyline induce a long-term reduction of the activity of A-SMase in cultured cells.
Assuntos
Química Encefálica/fisiologia , Encéfalo/enzimologia , Transtorno Depressivo Maior/enzimologia , Esfingomielina Fosfodiesterase/sangue , Regulação para Cima/fisiologia , Adulto , Antidepressivos Tricíclicos/farmacologia , Encéfalo/fisiopatologia , Química Encefálica/efeitos dos fármacos , Estudos de Casos e Controles , Células Cultivadas , Ceramidas/metabolismo , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/diagnóstico , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Feminino , Humanos , Masculino , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Pessoa de Meia-Idade , Bainha de Mielina/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Estudos Prospectivos , Agregação de Receptores/efeitos dos fármacos , Agregação de Receptores/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Esfingomielina Fosfodiesterase/análise , Esfingomielinas/metabolismoRESUMO
Aging of population, and increasing life expectancy result in an increasing number of patients with dementia. This symptom can be a part of a completely curable disease of the central nervous system (e.g, neuroinflammation), or a disease currently considered irreversible (e.g, Alzheimer's disease, AD). In the latter case, several potentially successful treatment approaches are being tested now, demanding reasonable standards of pre-mortem diagnosis. Cerebrospinal fluid and serum analysis (CSF/serum analysis), whereas routinely performed in neuroinflammatory diseases, still requires standardization to be used as an aid to the clinically based diagnosis of AD. Several AD-related CSF parameters (total tau, phosphorylated forms of tau, Abeta peptides, ApoE genotype, p97, etc.) tested separately or in a combination provide sensitivity and specificity in the range of 85%, the figure commonly expected from a good diagnostic tool. In this review, recently published reports regarding progress in neurochemical pre-mortem diagnosis of dementias are discussed with a focus on an early and differential diagnosis of AD. Novel perspectives offered by recently introduced technologies, e.g, fluorescence correlation spectroscopy (FCS) and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) are briefly discussed.
Assuntos
Doença de Alzheimer , Consenso , Idoso , Doença de Alzheimer/sangue , Doença de Alzheimer/líquido cefalorraquidiano , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/líquido cefalorraquidiano , Apolipoproteínas E/genética , Biomarcadores/sangue , Biomarcadores/líquido cefalorraquidiano , Diagnóstico Diferencial , Genótipo , Humanos , Immunoblotting , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios/metabolismo , Fosforilação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Proteínas tau/líquido cefalorraquidianoRESUMO
This study was focussed on the identification of the endocytic organelles in chromaffin cells which retrieve large, dense core vesicle (LDCV)-membrane components from the plasma membrane. For this purpose, 'on-cell' capacitance measurements and electron microscopy were employed. We found capacitance steps and capacitance flickers, corresponding to single exo- and endocytic events. The analysis revealed that the total membrane surface of completely fused LDCVs is recycled by large endocytic vesicles and smaller, most likely clathrin-coated vesicles, at approximately the same ratio. These results were confirmed by rapid-freeze immuno-electron microscopy, where an extracellular marker was rapidly internalized into endocytic vesicles that morphologically resembled LDCVs.
Assuntos
Células Cromafins/ultraestrutura , Animais , Bovinos , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Células Cultivadas , Células Cromafins/fisiologia , Endocitose , Potenciais da Membrana , Microscopia EletrônicaRESUMO
Exocytosis and 'kiss-and-run' secretion coexist in chromaffin cells. Our findings suggest that these mechanisms are closely related, based on their common molecular machinery. Here we present a model that describes how chromaffin cells regulate catecholamine release by switching the mode of secretion between the two pathways, a process controlled by phosphorylation. Stimulation-dependent vesicle-plasma membrane interactions in chromaffin cells were analysed by simultaneous 'on-cell' capacitance and conductance measurements, a technique that allows the monitoring of single vesicles. Capacitance steps represent fusions of large dense-core vesicles with the plasma membrane, whereas capacitance flickers correspond to transient connections of the vesicle lumen with the extracellular space. All these events require the presence of extracellular calcium in millimolar concentrations. 'Kiss-and-run' type of release is enhanced by the kinase inhibitor staurosporine, which suggests that this secretion mode is regulated by protein phosphorylation. We also observed capacitance bursts, which most probably represent 'hot spots' of secretion and we found that 'kiss-and-run' is the prevalent mechanism during these episodes. The significance of 'kiss-and run' for neurohormone release is even higher at physiological temperature, because up to half of all secretion events are mediated by this mechanism.
Assuntos
Células Cromafins/fisiologia , Exocitose/fisiologia , Animais , Cálcio/metabolismo , Carbacol/farmacologia , Bovinos , Células Cultivadas , Células Cromafins/enzimologia , Células Cromafins/metabolismo , Meios de Cultura/farmacologia , Capacitância Elétrica , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Modelos Biológicos , Técnicas de Patch-Clamp , Fosforilação , Inibidores de Proteínas Quinases , Estaurosporina/farmacologiaRESUMO
Dictyostelium discoideum myosin Ik (MyoK) is a novel type of myosin distinguished by a remarkable architecture. MyoK is related to class I myosins but lacks a cargo-binding tail domain and carries an insertion in a surface loop suggested to modulate motor velocity. This insertion shows similarity to a secondary actin-binding site present in the tail of some class I myosins, and indeed a GST-loop construct binds actin. Probably as a consequence, binding of MyoK to actin was not only ATP- but also salt-dependent. Moreover, as both binding sites reside within its motor domain and carry potential sites of regulation, MyoK might represent a new form of actin crosslinker. MyoK was distributed in the cytoplasm with a significant enrichment in dynamic regions of the cortex. Absence of MyoK resulted in a drop of cortical tension whereas overexpression led to significantly increased tension. Absence and overexpression of MyoK dramatically affected the cortical actin cytoskeleton and resulted in reduced initial rates of phagocytosis. Cells lacking MyoK showed excessive ruffling, mostly in the form of large lamellipodia, accompanied by a thicker basal actin cortex. At early stages of development, aggregation of myoK null cells was slowed due to reduced motility. Altogether, the data indicate a distinctive role for MyoK in the maintenance and dynamics of the cell cortex.
Assuntos
Movimento Celular/fisiologia , Dictyostelium/citologia , Miosina Tipo I , Miosinas/genética , Fagocitose/fisiologia , Actinas/metabolismo , Animais , Agregação Celular/fisiologia , Reagentes de Ligações Cruzadas/metabolismo , Citoesqueleto/química , Citoesqueleto/fisiologia , Dictyostelium/química , Dictyostelium/genética , Imunofluorescência , Regulação da Expressão Gênica no Desenvolvimento , Mutagênese/fisiologia , Miosinas/análise , Miosinas/fisiologia , Fenótipo , Proteínas de Protozoários , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/fisiologia , Estresse MecânicoRESUMO
Constitutive exo- and endocytic events are expected to increase and diminish the cell surface area in small spontaneous steps. Indeed, cell-attached patch-clamp measurements in resting chromaffin cells revealed spontaneous upward and downward steps in the electrical capacitance of the plasma membrane. The most frequent step size indicated cell surface changes of <0.04 microm(2), corresponding to vesicles of <110 nm diameter. Often downward steps followed upward steps within seconds, and vice versa, as if vesicles transiently opened and closed their lumen to the external space. Transient openings and closings sometimes alternated rhythmically for tens of seconds. The kinase inhibitor staurosporine dramatically increased the occurrence of such rhythmic episodes by making vesicle closure incomplete and by inhibiting fission. Staurosporine also promoted transient closures of large endocytic vesicles possibly representing remnants of secretory granules. We suggest that staurosporine blocks a late step in the endocytosis of both small and large vesicles, and that endocytosis involves a reaction cascade that can act as a chemical oscillator.
Assuntos
Células Cromafins/fisiologia , Endocitose/fisiologia , Exocitose/fisiologia , Organelas/fisiologia , Animais , Bovinos , Células Cultivadas , Condutividade Elétrica , Eletrofisiologia , Endocitose/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Exocitose/efeitos dos fármacos , Estaurosporina/farmacologia , Propriedades de SuperfícieRESUMO
Kex2 in the yeast Saccharomyces cerevisiae is a transmembrane, Ca2+-dependent serine protease of the subtilisin-like pro-protein convertase (SPC) family with specificity for cleavage after paired basic amino acids. At steady state, Kex2 is predominantly localized in late Golgi compartments and initiates the proteolytic maturation of pro-protein precursors that transit the distal secretory pathway. However, Kex2 localization is not static, and its itinerary apparently involves transiting out of the late Golgi and cycling back from post-Golgi endosomal compartments during its lifetime. We tested whether the endocytic pathway could deliver small molecules to Kex2 from the extracellular medium. Here we report that intramolecularly quenched fluorogenic substrates taken up into intact yeast revealed fluorescence due to specific cleavage by Kex2 protease in endosomal compartments. Furthermore, the endocytic delivery of protease inhibitors interfered with Kex2 activity for precursor protein processing. These observations reveal that the endocytic pathway does intersect with the cycling itinerary of active Kex2 protease. This strategy of endocytic drug delivery has implications for modulating SPC protease activity needed for hormone, toxin and viral glycoprotein precursor processing in human cells.
Assuntos
Endocitose , Endossomos/metabolismo , Pró-Proteína Convertases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Subtilisinas/metabolismo , Transporte Biológico , Humanos , Saccharomyces cerevisiae/ultraestruturaRESUMO
The major component of black widow spider venom, alpha-latrotoxin, triggers massive exocytosis in a variety of neurosecretory cells. An important trigger for exocytosis is the calcium influx via alpha-latrotoxin-induced channels in biological membranes. However, this mechanism fails to explain exocytosis which occurred in the complete absence of extracellular calcium. Recently, sophisticated biochemical and molecular techniques have led to the discovery of novel alpha latrotoxin-binding membrane receptors: neurexins and latrophilin/CIRL (calcium-independent receptor for alpha-latrotoxin). Neurexins are single transmembrane proteins which bind to alpha-latrotoxin in a calcium-dependent manner and also interact with the synaptic vesicle protein, synaptotagmin. On the other hand, latrophilin is a seven-transmembrane protein and belongs to the family of G-protein-coupled receptors. The multitude of effects of alpha-latrotoxin on exocytosis in different cell systems and the nature of its membrane targets are discussed in this article. The molecular details of how alpha-latrotoxin binding is transduced eventually to exocytosis remain to be elucidated.
Assuntos
Canais de Cálcio/fisiologia , Cálcio/metabolismo , Venenos de Aranha/farmacocinética , Venenos de Aranha/toxicidade , Animais , Viúva Negra , Exocitose/efeitos dos fármacos , Modelos Biológicos , Proteínas do Tecido Nervoso/metabolismo , Receptores de Peptídeos/metabolismoRESUMO
The past year has witnessed progress in identifying late steps in exocytosis that are so short-lived as to be difficult to study biochemically. Recent studies have also revealed a novel and surprisingly fast mechanism of endocytosis that may be triggered by a rise in the intracellular concentration of Ca2+ and that retrieves exocytosed membrane in seconds.
Assuntos
Glândulas Endócrinas/fisiologia , Endocitose , Exocitose , Animais , Cálcio/metabolismo , Clatrina/fisiologia , Invaginações Revestidas da Membrana Celular/fisiologia , Dinaminas , Condutividade Elétrica , Glândulas Endócrinas/citologia , GTP Fosfo-Hidrolases/fisiologia , Humanos , Membranas Intracelulares/metabolismo , Fatores de TempoRESUMO
We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals. We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.
Assuntos
Terminações Pré-Sinápticas/química , Vesículas Sinápticas/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Toxinas Botulínicas/farmacologia , Estimulação Elétrica , Inibidores Enzimáticos/farmacologia , Éteres Cíclicos/farmacologia , Corantes Fluorescentes , Lasers , Neurônios Motores/química , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Fibras Nervosas/química , Fibras Nervosas/fisiologia , Fibras Nervosas/ultraestrutura , Ácido Okadáico , Fotoquímica , Terminações Pré-Sinápticas/metabolismo , Compostos de Piridínio , Compostos de Amônio Quaternário , Rana pipiens , Coloração e Rotulagem , Vesículas Sinápticas/efeitos dos fármacosRESUMO
Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion. This dye appears to label the membranes of recycled synaptic vesicles by being trapped during endocytosis. Stained terminals can subsequently be destained by repeating nerve stimulation in the absence of dye; the destaining evidently reflects escape of dye into the bathing medium from membranes of exocytosing synaptic vesicles. In the present study we tested two key aspects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually released into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobenzidine. This created an electron-dense reaction product that could be visualized in the electron microscope. Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated into the medium from tubocurarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle-forming detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude.
Assuntos
Corantes Fluorescentes , Compostos de Piridínio , Compostos de Amônio Quaternário , Vesículas Sinápticas/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Exocitose , Microscopia Eletrônica , Microscopia de Fluorescência , Mitocôndrias/ultraestrutura , Dados de Sequência Molecular , Placa Motora , Junção Neuromuscular/fisiologia , Fotoquímica , Rana pipiensAssuntos
Clatrina/metabolismo , Músculo Esquelético/inervação , Terminações Nervosas/fisiologia , Junção Neuromuscular/fisiologia , Sinaptofisina/fisiologia , Animais , Clatrina/análise , Estimulação Elétrica , Imuno-Histoquímica , Terminações Nervosas/ultraestrutura , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/ultraestrutura , Rana pipiens , Venenos de Aranha/farmacologia , Vesículas Sinápticas/fisiologia , Vesículas Sinápticas/ultraestrutura , Sinaptofisina/análiseRESUMO
The protein kinase inhibitor staurosporine inhibited, and often abolished, activity-dependent destaining of frog motor nerve terminals that had been preloaded with the fluorescent dye FM1-43. Staurosporine did not, however, block synaptic transmission; staurosporine treated muscles twitched in response to nerve stimulation, and the amplitudes of evoked end plate potentials were reduced only slightly, and in some cases not at all. The blockade of FM1-43 destaining was not reversed by washing, although treatment with black widow spider venom caused complete destaining. Nerve terminal pretreated with staurosporine could subsequently be stained with FM1-43 (and then destained by black widow spider venom). Thus, staurosporine blocked destaining but not staining of nerve terminals. Staurosporine treatment had little effect on the ultra-structure of resting terminals, the main difference we noted being a somewhat closer packing of synaptic vesicles after exposure to staurosporine. However, staurosporine blocked completely the ultrastructural changes produced by prolonged nerve stimulation, such as depletion of synaptic vesicles, appearance of intraterminal cisternae, and the uptake of horseradish peroxidase. The effects of staurosporine were not mimicked by KN-62, H7, calmidozolium, or trifluoroperazine. These and other observations are consistent with, but do not prove the hypothesis that, after exposure to staurosporine, the exocytotic fusion pore behaved like a valve, letting FM1-43 in, but not out, as if staurosporine interfered with the postexocytotic collapse of synaptic vesicles into the surface membrane.
Assuntos
Acetilcolina/metabolismo , Alcaloides/farmacologia , Neurônios Motores/metabolismo , Terminações Nervosas/metabolismo , Compostos de Piridínio/antagonistas & inibidores , Compostos de Amônio Quaternário/antagonistas & inibidores , Animais , Anuros , Estimulação Elétrica , Eletrofisiologia , Corantes Fluorescentes , Lasers , Placa Motora/efeitos dos fármacos , Placa Motora/fisiologia , Neurônios Motores/ultraestrutura , Músculos/efeitos dos fármacos , Músculos/inervação , Músculos/metabolismo , Terminações Nervosas/efeitos da radiação , Proteína Quinase C/antagonistas & inibidores , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Venenos de Aranha/farmacologia , EstaurosporinaRESUMO
The neurotoxin Black Widow Spider Venom (BWSV) triggers massive release of neurotransmitter at synapses. Here we demonstrate that the action of BWSV on the frog neuromuscular junction can be visualized in vivo by the use of the fluorescent styryl dye FM1-43. This vital dye stains recycled synaptic vesicles upon nerve stimulation. Motor nerve terminals were stained with FM1-43 via electrical stimulation, washed and then exposed to BWSV or alpha-Latrotoxin. All terminals destained completely, independent of external calcium. Exposure of frog nerve terminals to BWSV in the presence of FM1-43 and calcium led to staining of terminals. The staining pattern appeared to be exactly the same as in control preparations, stimulated electrically via the nerve. When the same experiment was performed in the absence of calcium, only a minute quantity of dye was taken up into the nerve terminals, and the synapses looked swollen and puffed. Addition of external calcium to these preparations elicited an immediate shrinking of the nerve terminals, indicating endocytosis. These observations support electron-microscopic data that suggest an important role for extracellular calcium in endocytosis of BWSV poisoned nerve terminals.
Assuntos
Viúva Negra , Corantes Fluorescentes , Junção Neuromuscular/efeitos dos fármacos , Compostos de Piridínio , Compostos de Amônio Quaternário , Venenos de Aranha/farmacologia , Animais , Cálcio/metabolismo , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Técnicas In Vitro , Junção Neuromuscular/ultraestrutura , Rana pipiens , Vesículas Sinápticas/ultraestruturaRESUMO
We have investigated basic fibroblast growth factor (FGF-2) localization in and release from isolated bovine adrenal chromaffin cells. In contrast to previous reports, we found no evidence of fibroblast growth factor (FGF) storage in catecholamine-containing chromaffin granules. Subcellular fractionation studies did not show enrichment of FGF-2 immunoreactivity in granules, and cholinergic stimulation failed to release FGF-2 into the medium. Our results suggest that adrenal chromaffin cells resemble other FGF-2-synthesizing cell types with respect to FGF storage and secretion.