RESUMO
Although most examples of modulation of synaptic transmission have been obtained from excitatory rather than from inhibitory connections, serotonin (5HT) is now shown to cause a presynaptic facilitation of release of the inhibitory neurotransmitter glycine. Brief local injections of this amine, or application of a 5HT uptake blocker, produce a long-lasting enhancement of both spontaneous and evoked inhibitory currents in the teleost Mauthner cell. Quantal analysis showed that the probability of release is increased. Focal recording indicated that 5HT acts directly on the inhibitory terminals, possibly reducing potassium conductances. Double staining with specific antibodies demonstrated a morphological substrate for this effect. Nerve endings that contain 5HT contact inhibitory terminals directly apposed to postsynaptic glycine receptors.
Assuntos
Glicina/metabolismo , Serotonina/fisiologia , Sinapses/fisiologia , Vias Aferentes , Animais , Carpa Dourada , Potenciais da Membrana , Microeletrodos , Canais de Potássio/metabolismoRESUMO
Klotho mutant mice, defective in the klotho gene, develop multiple age-related disorders with very short lifespans. Introduction of the exogenous klotho gene into these mutant mice leads to an improvement in their phenotypes, while overexpression of this gene in wild-type mice significantly extends their lifespan. These observations suggest that the klotho gene/protein has an anti-aging function. Since there have been only a few reports with some disagreement about results on the CNS of the mutant mice, we tried to clarify whether the CNS neurons generate aging-like features, even in premature stages, using biochemical and morphological approaches. Results obtained from the mutant mice, when compared with wild-type mice, were as follows. Neurofilaments (NFs) were increased significantly in axons, with the subunit proteins showing a significant enhancement in phosphorylation or expression of NF-H or NF-L, respectively. Microtubules in Purkinje cell dendrites were closer to each other, and in the CNS tissue tubulin was unaltered, but microtubule-associated protein (MAP) 2 was significantly reduced in expression. Neuronal cellular organelles were morphologically disordered. Lysosomes, cathepsin D and light chain 3 of MAP1A/B (LC3) were augmented with the appearance of putative autophagy-related structures. Antiapoptotic Bcl-xL and proapoptotic Bax were reduced and enhanced, respectively, and mitogen-activated protein kinase was reduced. Synapse-related proteins and structures were decreased. Neuronal degeneration was evident in hippocampal pyramidal cells, and possibly in Purkinje cells. Astrocytic glial filaments and glial fibrillary acidic protein were increased in density and expression, respectively. Together, the CNS neuronal alterations in klotho mutant mice were quite similar to those found in aged animals, including even premature death, so this mouse should be a more appropriate animal model for CNS aging than those previously reported.
Assuntos
Envelhecimento/fisiologia , Sistema Nervoso Central , Regulação da Expressão Gênica/genética , Glucuronidase/deficiência , Doenças Neurodegenerativas , Neurônios/patologia , Animais , Axônios/metabolismo , Axônios/patologia , Axônios/ultraestrutura , Catepsina D/metabolismo , Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/patologia , Sistema Nervoso Central/ultraestrutura , Proteínas Klotho , Masculino , Camundongos , Camundongos Knockout , Microscopia Eletrônica de Transmissão/métodos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/metabolismo , Neurônios/ultraestrutura , Proteína X Associada a bcl-2/metabolismoRESUMO
Bcl-2, an anti-apoptotic protein, is believed to be localized in the outer mitochondrial membrane, endoplasmic reticulum, and nuclear envelope. However, Bcl-2 has also been suggested as playing a role in the maintenance of mitochondrial membrane potential, indicating its possible association with the inner mitochondrial membrane. We therefore further examined the exact localization of Bcl-2 in mitochondria purified from wild-type and bcl-2-transfected PC12 cells and pre- and postnatal rat brains. Double immunostaining demonstrated that Bcl-2 was co-localized with subunit beta of F1F0ATPase in the inner mitochondrial membrane. Biochemical analysis of isolated mitochondria using digitonin and trypsin suggests an association of Bcl-2 with the inner mitochondrial membrane. More interestingly, the majority of Bcl-2 disappeared from the inner membrane of mitochondria when cultured under serum deprivation. These results suggest that Bcl-2 acts as an anti-apoptotic regulator by localizing mainly to the inner mitochondrial and smooth ER membranes.
Assuntos
Retículo Endoplasmático Liso/química , Membranas Intracelulares/química , Membranas Intracelulares/ultraestrutura , Mitocôndrias/química , Neurônios/química , Proteínas Proto-Oncogênicas c-bcl-2/análise , Animais , Meios de Cultura Livres de Soro , Digitonina/farmacologia , Retículo Endoplasmático Liso/ultraestrutura , Humanos , Imuno-Histoquímica , Camundongos , Microscopia Confocal , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Neurônios/ultraestrutura , Células PC12 , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , ATPases Translocadoras de Prótons/análise , ATPases Translocadoras de Prótons/imunologia , Ratos , Proteínas Recombinantes , Frações Subcelulares , TransfecçãoRESUMO
Mcm2-7 proteins that play an essential role in eukaryotic DNA replication contain DNA-dependent ATPase motifs in a central domain that, from yeast to mammals, is highly conserved. Our group has reported that a DNA helicase activity is associated with a 600 kDa human Mcm4, 6 and 7 complex. The structure of the Mcm4,6,7 complex was visualized by electron microscopy after negative staining with uranyl acetate. The complex contained toroidal forms with a central channel and also contained structures with a slit. Gel-shift analysis indicated that the level of affinity of the Mcm4,6,7 complex for single-stranded DNA was comparable to that of SV40 T antigen, although the Mcm4,6,7 complex required longer single-stranded DNA for the binding than did SV40 T antigen. The nucleoprotein complexes of Mcm4,6,7 and single-stranded DNA were visualized as beads in a queue or beads on string-like structures. The formation of these nucleoprotein complexes was erased by Mcm2 that is a potential inhibitor of the Mcm4,6,7 helicase. We also found that the DNA helicase activity of Mcm4,6,7 complex was inhibited by the binding of Mcm3,5 complex. These results support the notion that the Mcm4,6,7 complex functions as a DNA helicase and the formation of 600 kDa complex is essential for the activity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/ultraestrutura , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Proteínas de Saccharomyces cerevisiae , Animais , Antígenos Transformantes de Poliomavirus/metabolismo , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona , DNA Helicases/antagonistas & inibidores , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/química , Dimerização , Células HeLa , Humanos , Camundongos , Microscopia Eletrônica , Componente 2 do Complexo de Manutenção de Minicromossomo , Componente 3 do Complexo de Manutenção de Minicromossomo , Componente 4 do Complexo de Manutenção de Minicromossomo , Componente 6 do Complexo de Manutenção de Minicromossomo , Componente 7 do Complexo de Manutenção de Minicromossomo , Peso Molecular , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/química , Compostos Organometálicos , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Recombinantes/metabolismo , Proteínas de Schizosaccharomyces pombe , Técnica Histológica de Sombreamento , Especificidade por SubstratoRESUMO
Neurofilament (NF) structure and ability to form cross-bridges were examined by quick-freeze deep-etch mica and low-angle rotary-shadow electron microscopy in NFs purified from bovine spinal cord and reassembled in various combinations of NF subunits. When NFs were reassembled from triplet proteins, NF-L, NF-M and NF-H, they were oriented randomly and often fragmented, but their elongated filaments (12-15 nm wide) and the cross-bridges (4-5 nm wide) connecting them were similar in appearance to those of isolated bovine NFs or in vivo rat NFs. Projections extended from the wall of the core filament in almost the same pattern as the cross-bridges and were the same in width and interval (minimum interval, 20-25 nm) as the cross-bridges. Projections were more conspicuous when core filaments were separated by 60 to 80 nm or more, while cross-bridges were more conspicuous when core filaments were close to each other. Projections or cross-bridges extended bilaterally at intervals of 20 to 25 nm where core filaments expanded and formed a network between filaments which were far from one another. When NFs were reconstructed from NF-L alone, only core filaments appeared, the same width as the filaments of triplet NFs. The core filaments were occasionally in almost direct contact with each other, with no projection or cross-bridge. When NFs were reassembled from NF-M alone or NF-L + NF-M, although NF-M core filaments were shorter and slightly thinner than NF-L + NF-M core filaments, both had projections, and both had cross-bridges, but cross-bridges were less evident. Cross-bridges were almost the same in width as those of triplet NFs, but significantly shorter and much less frequent although the minimum interval was the same, and core filaments were not attached to each other. In contrast, when NFs were reconstituted from NF-H alone or NF-L + NF-H, both had conspicuous projections and cross-bridges, similar to those of triplet NFs. Thus, when NFs contained NF-H, they formed frequent cross-bridges and long projections with extensive peripheral branching. When NFs contained NF-M but no NF-H, they tended to form cross-bridges, and to form projections that were shorter and straighter and without peripheral branching. That is, there appears to be a significant difference between NF-M and NF-H in ability to form cross-bridges and thus in interaction with adjacent NFs.
Assuntos
Filamentos Intermediários/ultraestrutura , Proteínas de Neurofilamentos/ultraestrutura , Animais , Química Encefálica , Bovinos , Técnica de Congelamento e Réplica , Filamentos Intermediários/química , Proteínas de Neurofilamentos/química , Ratos , Ratos Wistar , Medula Espinal/químicaRESUMO
The postnatal maturation of the GABAergic innervation of the rat inferior olive was studied with an antiserum to glutamic acid decarboxylase (GAD), the GABA-synthesizing enzyme. GAD-positive axons were present at a very low density in the periolivary and interlamellar regions of newborn rats, as well as in certain precise areas of the lamellae, at the mediodorsal limit. The immature distribution indicates that the GABAergic projections reach the inferior olive shortly before birth and that the greater part of synaptogenesis and the establishment of the adult organization occurs postnatally. Light and electron microscopic analyses disclosed that the maturation of this system of olivary afferents passes through three well-defined stages: (1) During the first, or immature stage (from PO to P5), GAD immunoreactivity is not confined to axon terminals, as in adult rats. The labeled fibers penetrate progressively into the periphery of the lamellae and reach their centers in an irregular manner by the end of the immature stage. This staggered invasion of the lamellae accentuates intraregional olivary differences and begins to take the adult configuration. As fiber penetration advances, the density of labeled axons establishing synaptic contacts increases, while the number of completely immunostained fibers decreases. This distribution prevails until the end of the immature stage and suggests that the GABAergic afferent projections remain in a "waiting compartment" from their prenatal arrival until the moment they invade the olivary parenchyma. (2). The second stage is designated as an intermediate stage of maturation and lasts from P7 to P10. During this period, GAD axoplasmic compartmentation occurs, and henceforth only axon terminals exhibit GAD immunoreactivity. Concomitantly, intraregional differences in the pattern of innervation become more marked, because of the continuing irregular distribution of the growing labeled axons. This intermediate maturational stage is also characterized by a rapid increase in labeled axon terminals bearing synaptic complexes and by the formation of complex synaptic arrangements, the protoglomeruli. From the beginning of protoglomeruli formation, GAD-positive axon terminals are one of their constituents, and they are systematically localized at the periphery of the incipient dendritic protrusions. (3) The final stage of maturation takes place from P10 to P15. During this stage, the adultlike pattern of GABAergic innervation of the inferior olive is attained. Toward P15, intraregional differences in GAD immunoreactivity are similar to those of the adult rat.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Animais Recém-Nascidos/fisiologia , Neurônios Aferentes/fisiologia , Núcleo Olivar/crescimento & desenvolvimento , Ratos/crescimento & desenvolvimento , Ratos/fisiologia , Sinapses/fisiologia , Ácido gama-Aminobutírico/fisiologia , Animais , Glutamato Descarboxilase/imunologia , Imuno-Histoquímica , Microscopia Eletrônica , Neurônios Aferentes/ultraestrutura , Núcleo Olivar/imunologia , Núcleo Olivar/ultraestrutura , Ratos EndogâmicosRESUMO
Immunocytochemical and electron microscopic methods were used to examine the GABAergic innervation of the inferior olivary nucleus in adult rats. This neuronal system was visualized with an antibody against glutamic acid decarboxylase (GAD, EC 4.1.1.15), the GABA-synthesizing enzyme. A GAD-positive reaction product was encountered only in short segments of preterminal axons and in axon terminals. Their relative number per unit area of neuropil was very similar in all olivary subnuclei. Despite this homogeneity in density, obvious intraregional differences existed. Some regions were strongly immunoreactive (the "c" subgroup, the beta nucleus, and the mediolateral outgrowth of the medial accessory olive), whereas others were weakly labeled (the dorsomedial cell column and the central zones of the medial accessory and principal olives). The strongly immunoreactive areas contained the largest and most intensively labeled axon terminals. Areas of weak labeling were filled with small, weakly immunoreactive nerve terminals. Thus, variations in size and in intensity of labeling create a specific pattern of GABA innervation, revealed by an almost continuous gradient between the above-mentioned extremes. The GAD-positive axon terminals established conventional synapses with dendrites (94% of the samples) or with cell bodies (6%). The vast majority of these synapses were type II (84%) and only a small proportion formed type I synaptic contacts (16%), regardless of the nature of the postsynaptic element. Immunoreactive terminals were also involved in the complex synaptic arrangements--the glomeruli, which characterize the olivary neuropil. Within these formations, olivary neurons were electrotonically coupled through dendrodendritic gap junctions. There was a constant association between GAD-positive axon terminals and small dendritic appendages linked by gap junctions. This association was revealed not only by the systematic presence of immunolabeled terminals directly apposed to the dendritic appendages but, more importantly, by the frequent presence of type II synapses straddling both elements. These synapses were in close proximity to the low-resistance pathways represented by the gap junctions. The strategic location of these GABA synapses is discussed in relation to recent findings indicating the possibility of a synaptic modulation of the electrical coupling: the release of GABA, by increasing nonjunctional membrane conductance, could shunt the coupling between olivary neurons. The functional decoupling of selected gap junctions would be responsible for the spatial organization of the olivary electrotonic coupling.
Assuntos
Glutamato Descarboxilase/metabolismo , Núcleo Olivar/enzimologia , Ácido gama-Aminobutírico/fisiologia , Animais , Feminino , Técnicas Imunoenzimáticas , Masculino , Núcleo Olivar/fisiologia , Ratos , Ratos Endogâmicos , Sinapses/ultraestrutura , Transmissão SinápticaRESUMO
The morphology and distribution of the serotoninergic (5-HT) input to the Mauthner cell (M cell) of a teleost, Carassius auratus, were analyzed at the light microscopic level. Immunohistochemical methods revealed that 1) most fibers innervating the M cell originate from the ventral and lateroventral regions of the rhombencephalon; 2) two groups of fibers contribute to this innervation, thick ones (type I, 0.4-0.7 microns in diameter) with terminal endings and thin ones (type II, less than 0.2 microns) that issue numerous beaded varicosities 4-10 microns from the target cell and only occasional side endings contacting it; 3) the density of immunoreactive profiles is uneven over the whole cell and predominates on the ventral dendrite; and 4) the two sets of axons, although overlapping, do not have the same distribution. Specifically, both classes are present on the ventral dendrite, whereas type II fibers are the only ones observed on the soma, in the region of the initial segment of the axon, and in the vicinity of the lateral dendrite. Functionally identified inputs on the M cell also have a regionalized distribution, depending, for example, on whether they belong to excitatory or inhibitory networks. Thus we propose that 5-HT inputs have specific influences that are a function of their respective localization.
Assuntos
Tronco Encefálico/metabolismo , Cyprinidae/metabolismo , Carpa Dourada/metabolismo , Rombencéfalo/metabolismo , Serotonina/metabolismo , Animais , Tronco Encefálico/citologia , Carpa Dourada/anatomia & histologia , Imuno-Histoquímica , Rombencéfalo/citologiaRESUMO
Neurofilaments accumulated in perikarya and dendrites of anterior horn cells and Purkinje cells of rabbit treated by aluminum chloride were analysed with a variety of techniques. Four different monoclonal antibodies against phosphorylated and nonphosphorylated epitopes on neurofilament H subunit were used to compare phosphorylation state of these accumulated neurofilaments with that of axonal neurofilaments. Although immunoblotting revealed no significant difference in phosphorylation between control and aluminum-treated brains, accumulated neurofilaments were immunocytochemically more phosphorylated than control perikaryal or dendritic neurofilaments. With detailed analysis of cryothin-section immunogold labeling, accumulated neurofilaments were, however, significantly less phosphorylated than axonal neurofilaments. With quick-freeze deep etching, core filaments of accumulated neurofilaments are as dense as axonal neurofilaments but much less regularly aligned. Cross-bridges of accumulated neurofilaments were less frequent and more branched than those of axonal neurofilaments, and when examined with combined immunocytochemistry and deep etching, were less phosphorylated. These results suggest that there is a relationship between the phosphorylation and the structural organization of neurofilaments. The phosphorylation of neurofilament H subunit may be necessary for formation of frequent and straight cross-bridges and resulting regular alignment of core filaments.
Assuntos
Alumínio/farmacologia , Filamentos Intermediários/ultraestrutura , Proteínas de Neurofilamentos/metabolismo , Células de Purkinje/ultraestrutura , Animais , Tronco Encefálico/imunologia , Tronco Encefálico/ultraestrutura , Córtex Cerebelar/imunologia , Córtex Cerebelar/ultraestrutura , Imunoquímica , Injeções Intraventriculares , Filamentos Intermediários/efeitos dos fármacos , Masculino , Microscopia Eletrônica , Fosforilação , Células de Purkinje/metabolismo , Coelhos , Nervo Isquiático/imunologia , Nervo Isquiático/ultraestrutura , Medula Espinal/imunologia , Medula Espinal/ultraestruturaRESUMO
Cytoplasmic architecture of axon terminals in rat central nervous tissue was examined by quick-freeze deep-etch method to determine how synaptic vesicles and their associated cytoplasmic environment are organized in the terminal and to know how these structures participate in the mechanism for neurotransmitter release. The axoplasm is divisible into two domains: one occupied by mitochondria in the middle of the terminal, called the mitochondrial domain, the other situated in the periphery and exclusively filled with spherical synaptic vesicles, 50-60 nm in diameter, the synaptic vesicle domain. The most characteristic feature of the mitochondrial domain was the appearance of many microtubules connected with mitochondria by filamentous strands. Large vesicles, 80-100 nm in diameter, were preferentially associated with the mitochondrial domain, and linked with microtubules wherever they appeared. The cytoplasmic matrix of the synaptic vesicle domain showed a more fibrillar texture than that of the mitochondrial domain because of the distribution of filamentous strands associated with synaptic vesicles. These strands were significantly thicker and longer (mean 11.7 nm thick and 42.7 nm long) than those linking membrane-bound organelles to microtubules (mean 8.3 nm thick and 23.0 nm long), and connected vesicles to one another or to the plasma membrane, making a complicated network around the vesicles. Further, both strands were significantly different in dimension from actin filaments (mean 9.9 nm thick and 73.5 nm long) showing 5-nm axial periodicity. These strands, especially synaptic vesicle-associated ones including their network, were readily broken down in the most part by detergent treatment or chemical fixation, indicating that they are very delicate in nature. Granular materials, which are spherical and vary in size (6-20 nm in diameter), are also more conspicuous in the synaptic vesicle domain than in the mitochondrial domain. More fibrillar and granular cytoplasmic structure of the synaptic vesicle domain may be crucial for synaptic vesicles to perform an essential role in releasing the transmitter.
Assuntos
Axônios/ultraestrutura , Citoplasma/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Animais , Detergentes/farmacologia , Fixadores/farmacologia , Organelas/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
To examine the participation of annexin V, a member of Ca(2+)-dependent phospholipid-binding proteins, in the process of synaptic vesicle exocytosis, rat central nervous tissue was analysed using biochemical and morphological techniques. By both fluorescence and confocal laser scanning microscopy, immunoreactivity for annexin V was predominantly localized around neuronal somata and dendrites, and the reactivity was mostly co-labeled with that for synaptophysin. The annexin V immunoreactivity was also detectable, but less intensely, in neuronal perikarya, glial cells and endothelial cells. Both immunoblot and immunoelectron microscopic analyses with intact tissues, synaptosomes and purified synaptic vesicles showed that annexin V was expressed in neurons, preferentially concentrated in axon terminals and associated with synaptic vesicles. Purified synaptic vesicles were relatively homogeneously distributed in the medium where Ca2+ was removed and thus the amount of annexin V was reduced drastically. The vesicles tended to be clustered in the fraction where endogenous annexin V is maintained, and the clusters were more conspicuous when purified human annexin V was added. Synaptic vesicles forming the clusters were not directly fused with each other but separated by a 10-15 nm gap that corresponded well with the size of single annexin V molecules. In axon terminals, globular structures 12-13 nm in diameter, similar in dimension to annexin V molecules, were distinctly found to be attached to the cytoplasmic surface of both vesicle membranes when the two vesicles were close to each other. These results suggest that annexin V belongs to the group of synaptic vesicle-associated proteins. Although its localization and significance in non-neuronal cells were not analysed here, at least in the axon terminal annexin V may participate in the cluster formation of synaptic vesicles by linking with the cytoplasmic surface of the vesicles in a Ca(2+)-dependent manner.
Assuntos
Anexina A5/metabolismo , Terminações Pré-Sinápticas/metabolismo , Terminações Pré-Sinápticas/ultraestrutura , Animais , Anexina A5/fisiologia , Encéfalo/citologia , Encéfalo/ultraestrutura , Química Encefálica/fisiologia , Feminino , Humanos , Immunoblotting , Imuno-Histoquímica , Técnicas In Vitro , Microscopia Confocal , Microscopia Eletrônica , Microscopia de Fluorescência , Gravidez , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Wistar , Vesículas Sinápticas/metabolismo , Vesículas Sinápticas/ultraestrutura , Sinaptossomos/metabolismo , Sinaptossomos/ultraestruturaRESUMO
PC12 cells undergo apoptosis when cultured under conditions of serum deprivation. In this situation, the activity of caspase-3-like proteinases was elevated, and the survival rate could be maintained by treatment with acetyl-DEVD-cho, a specific inhibitor of caspase-3. In a culture of PC12 cells treated with acetyl-DEVD-cho, where caspase-3-like proteinases are not activated, CA074, a specific inhibitor of cathepsin B induced active death of the cells. Cathepsin B antisense oligonucleotides showed a similar effect to CA074 on the induction of active cell death. By double staining of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling and activated caspase-3, the dying cells treated with CA074 were positive for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling staining but negative for activated caspase-3. Ultrastructurally, the cells were relatively large and had nuclei with chromatin condensation. The initiation of cell death by CA074 or the cathepsin B antisense were inhibited by the addition of pepstatin A, a lysosomal aspartic proteinase inhibitor, or by cathepsin D antisense. To examine whether this cell death pathway was present in cell types other than PC12 cells, we analysed dorsal root ganglion neurons obtained from rat embryos on the 15th gestational day, a time when they require nerve growth factor for survival and differentiation in culture. When cultured in the absence of nerve growth factor, the neurons survived in the presence of acetyl-DEVD-cho or acetyl-YVAD-cho. Under these conditions, CA074 reduced the survival rate of the neurons, which was subsequently restored by the further addition of pepstain A. These results suggest that a novel pathway for initiating cell death exists which is regulated by lysosomal cathepsins, and in which cathepsin D acts as a death factor. We speculate that this death-inducing activity is normally suppressed by cathepsin B.
Assuntos
Ácido Aspártico Endopeptidases/fisiologia , Cisteína Endopeptidases/fisiologia , Lisossomos/enzimologia , Animais , Caspase 3 , Caspases/metabolismo , Catepsina B/biossíntese , Catepsina B/genética , Catepsina D/biossíntese , Catepsina D/genética , Morte Celular/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Inibidores de Cisteína Proteinase/farmacologia , Precursores Enzimáticos/metabolismo , Marcação In Situ das Extremidades Cortadas , Microscopia Eletrônica , Fatores de Crescimento Neural/farmacologia , Oligonucleotídeos Antissenso/farmacologia , Células PC12 , Inibidores de Proteases/farmacologia , RatosRESUMO
A single BPSP (excitatory-inhibitory postsynaptic potential) was monosynaptically produced in an identified Onchidium neuron, Be-1, with a beating rhythm upon stimulation of the cardiac nerve. The BPSPs summated to produce an inhibition of long duration (ILD) upon blockage of the beating rhythm after repeated stimulation, so that the BPSPs seemed to be functionally inhibitory. Ten stimuli (1-2 Hz) applied to the cardiac nerve usually evoked an ILD (0.5-1 min) of about 10 mV. The early and middle phases of this ILD reversed near -80 to -85 mV, but the late phase did not reverse at more negative potentials. None of the phases was significantly affected by low Cl or Na solutions or by high Ca solutions. However, by changing the external K, the shift of the reversal potentials for the early and middle phases reached about 65% of that predicted for the K electrode, although the late phase was insensitive to the external K. Intracellular tetraethylammonium (TEA) attenuated the amplitude of the ILD but did not shorten the duration. These suggest that the ILD has another conductance-independent mechanism simultaneously with the increase in K conductance. Several lines of evidence suggested that a ouabain-sensitive Na pump does not contribute to the ILD. Inhibitors of energy supply, 2,4-dinitrophenol sodium salt (DNP) and cyanide, selectively and reversibly reduced the ILD. Simultaneous applications of intracellular TEA and DNP completely abolished the ILD. As for the ionic basis, the histamine-induced inhibitory response in Be-1 was closely related to the ILD. Cimetidine specifically blocked the ILD and histamine-induced inhibitory response, which were mimicked by 2-methylhistamine, but not by dimaprit. It is concluded that the ILD, mediated by some histamine receptor other than the H1 or H2 type, results from an increase in K conductance and a hyperpolarizing ion pump insensitive to ouabain.
Assuntos
Gânglios/fisiologia , Histamina/fisiologia , Animais , Antimetabólitos/farmacologia , Cálcio/farmacologia , Cloretos/farmacologia , Técnicas In Vitro , Moluscos , Inibição Neural , Periodicidade , Potássio/farmacologia , Sódio/farmacologia , Transmissão Sináptica , Tetraetilamônio , Compostos de Tetraetilamônio/farmacologiaRESUMO
The photoresponse of an extraocular photoreceptor, the photoresponsive neuron (A-P-1) in the abdominal ganglion of Onchidium verruculatum, was studied by using a voltage-clamp with two micropipettes and a monochromatic light. When the A-P-1 was voltage-clamped at resting membrane potential levels, light induced a slowly developing inward current which peaked at about 20 s. A decrease in membrane conductance accompanied this light-induced current which corresponded to the depolarizing photoreceptor potential in the unclamped A-P-1. The relationship between the peak of the current response and light intensity could be predicted by using the modified Michaelis-Menten equation. The spectral sensitivity for the photoresponse had a peak at 490 nm. The steady-state light-induced current was a non-linear function of the membrane potential. The current-voltage relationship for the instantaneous light-induced current was almost linear. In normal (10 mM K+) saline, the polarity of the instantaneous light current reversed from inward to outward at about -67 mV, and doubling the external K+ from 10 to 20 mM shifted the reversal potential to about-50 mV, similar to that predicted by a K+-electrode. These results suggest that the light-induced current or the depolarizing receptor potential of A-P-1 is due to the light suppression of a voltage- and time-dependent K+ current.
Assuntos
Moluscos/fisiologia , Células Fotorreceptoras/fisiologia , Animais , Potenciais da Membrana , Estimulação LuminosaRESUMO
The internal messengers mediating the photocurrent of the molluscan extraocular photoreceptor, A-P-1, were examined. In the dark, pressure-injection of cGMP into the A-P-1, voltage-clamped at resting levels, produced a rapid outward current, associated with an increase in conductance. However, the cGMP-induced current and increase in conductance were suppressed by subsequent photostimulation, suggesting hydrolysis of cGMP by light. The steady-state I/V relation for the cGMP-induced current was non-linear. The I/V relation for the instantaneous cGMP-induced current, measured 50 ms after the beginning of a voltage step, was linear, and reversed at the membrane potential, -67 mV, which corresponded to the K+ equilibrium potential of A-P-1 in 10 mM K+ normal saline. These findings indicate that the internal cGMP induces a voltage- and time-dependent K+ current. Since the photocurrent results from the suppression of a voltage- and time-dependent K+ current similar to above, the photocurrent is considered to be equivalent to the suppression of the cGMP-induced current. Short pressure-injection of GDP-beta-S into A-P-1 reduced the subsequent photocurrent. The photocurrent was also suppressed after an external application of Pertussis toxin. On the other hand, the photocurrent was amplified by prior pressure-injection of inositol 1,4,5-trisphosphate (IP3). However, a short pressure-injection of neomycin into A-P-1 depressed the subsequent photocurrent. These results suggested that the cGMP-induced (dark) current is mediated by cGMP, and that hydrolysis of cGMP by light leads to the photocurrent, then being modified by another messenger, IP3, to be amplified.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
GMP Cíclico/fisiologia , Inositol 1,4,5-Trifosfato/fisiologia , Células Fotorreceptoras/fisiologia , Transdução de Sinais/efeitos dos fármacos , Caramujos/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , GMP Cíclico/farmacologia , Dibutiril GMP Cíclico/farmacologia , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Inositol 1,4,5-Trifosfato/farmacologia , Iontoforese , Potenciais da Membrana/efeitos dos fármacos , Microeletrodos , Neomicina/farmacologia , Neurônios/efeitos dos fármacos , Toxina Pertussis , Células Fotorreceptoras/efeitos dos fármacos , Canais de Potássio/efeitos dos fármacos , Cloreto de Potássio/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Tionucleotídeos/farmacologia , Fatores de Virulência de Bordetella/farmacologiaRESUMO
The depolarizing photoreceptor potential in the molluscan extraocular photoreceptor, A-P-1, results from the light-induced suppression of specific K+ currents. An application of cGMP or IBMX (inhibitor of phosphodiesterase) to A-P-1-evoked light-suppressed currents, similar to the above specific K+ currents. Thus, the light-induced decrease of cGMP levels in A-P-1 may be responsible for the photoreceptor potential, like vertebrate phototransductions.
Assuntos
GMP Cíclico/fisiologia , Iluminação , Moluscos/fisiologia , Células Fotorreceptoras/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , Animais , GMP Cíclico/metabolismo , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Moluscos/metabolismo , Estimulação Luminosa , Células Fotorreceptoras/efeitos dos fármacos , Células Fotorreceptoras/metabolismoRESUMO
Intrinsic beating activity in the identified molluscan neuron Be-1 can be inhibited for a relatively long time by a short application of histamine. Cyclic AMP enhanced both the amplitude and duration of this inhibitory histamine response (H2-response), while cyclic GMP depressed the H2-response. However, when only these nucleotides were applied, they produced no significant, change in the beating spike activity.
Assuntos
AMP Cíclico/farmacologia , GMP Cíclico/farmacologia , Histamina/farmacologia , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Bucladesina/farmacologia , Dibutiril GMP Cíclico/farmacologia , Potenciais Evocados/efeitos dos fármacos , Gânglios/citologia , Moluscos , Transmissão Sináptica/efeitos dos fármacosRESUMO
The phototransduction mechanism of the extra-ocular photoreceptor cells Ip-2 and Ip-1 in the mollusc Onchidium ganglion was examined. Previous work showed that the depolarizing receptor potential of another extra-ocular photoreceptor cell, A-P-1 is produced by a decrease of the light-sensitive K+ conductance activated by a second messenger, cGMP and is inactivated by the hydrolysis of cGMP. Here, a hyperpolarizing receptor potential of Ip-2 or Ip-1 was associated with an increase in membrane conductance. When Ip-2 or Ip-1 was voltage-clamped near the resting membrane potential, light induced an outward photocurrent corresponding to the above hyperpolarization. The spectral sensitivity had a peak at 510 nm. The shift of reversal potentials of the photocurrent depended on the Nernst equation of K(+)-selective conductance. The photocurrent was blocked by 4-AP and L-DIL, which are effective blockers of the A-P-1 light-sensitive K+ conductance. These results suggested that the hyperpolarization is mediated by increasing a similar light-sensitive K+ conductance to that of A-P-1. The injection of cGMP or Ca2+ into a cell produced a K+ current that mimicked the photocurrent. 4-AP and L-DIL both abolished the cGMP-activated K+ current, while TEA suppressed only the Ca(2+)-activated K+ current. These results indicated that cGMP is also a second messenger that regulates the light-sensitive K+ conductance. The photocurrent was blocked by LY-83583, a guanylate cyclase (GC) inhibitor, but was unaltered by zaprinast, a phosphodiesterase (PDE) inhibitor. Together, the present results suggest that increasing the internal cGMP in Ip-2 or Ip-1 cells light-activates GC rather than inhibits PDE, thereby leading to an increase of the light-sensitive K+ conductance and the hyperpolarization.
Assuntos
GMP Cíclico/metabolismo , Células Fotorreceptoras de Invertebrados/química , Células Fotorreceptoras de Invertebrados/metabolismo , Potássio/metabolismo , Visão Ocular/fisiologia , 1-Metil-3-Isobutilxantina/farmacologia , 4-Aminopiridina/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Abdome/inervação , Aminoquinolinas/farmacologia , Animais , GMP Cíclico/antagonistas & inibidores , Condutividade Elétrica , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Moluscos , Inibidores de Fosfodiesterase/farmacologia , Estimulação Luminosa , Purinonas/farmacologia , Sistemas do Segundo Mensageiro/fisiologia , Tetraetilamônio/farmacologiaRESUMO
We report the first recordings of the light-sensitive channel which is active during dark and is closed by light in the Onchidium extra-ocular photoreceptor cells. This light-sensitive channel was K-selective and was not blocked by extracellular Ca2+ and Mg2+. Application of cyclic GMP to excised inside-out patches activated (opened) a channel that appeared to be the same as the light-sensitive channel recorded from the same membrane in the intact cell.
Assuntos
GMP Cíclico/farmacologia , Moluscos/fisiologia , Células Fotorreceptoras de Invertebrados/efeitos dos fármacos , Células Fotorreceptoras de Invertebrados/efeitos da radiação , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/efeitos da radiação , Animais , Cálcio/farmacologia , Eletrofisiologia , Gânglios dos Invertebrados/citologia , Gânglios dos Invertebrados/efeitos dos fármacos , Gânglios dos Invertebrados/metabolismo , Luz , Magnésio/farmacologia , Células Fotorreceptoras de Invertebrados/metabolismoRESUMO
The cGMP-activated K+ channels closed by light lead to the depolarizing photocurrent of photoreceptors in the Onchidium ganglion. Whole-cell current records showed that external application of 100-200 microM 4-aminopyridine or 200-400 microM l-cis-diltiazem completely blocked the macroscopic photocurrent at any depolarizing and hyperpolarizing potentials. Single-channel current recordings suggested that both 4-aminopyridine and l-cis-diltiazem act to block the cGMP-activated K+ channels in their open state from inside the cell.