RESUMO
Evidence suggests that individual differences in emotion control are associated with frontoparietal-limbic networks and linked to emotional traits and executive functions. In a first attempt to directly target the link between emotional traits and executive functions using resting-state fMRI analysis, 43 healthy adults completed a test battery including executive tasks and emotional trait self-assessments that were subjected to a principal component analysis. Of the three factors detected, two explained 40.4% of the variance and were further investigated. Both factors suggest a relation between emotional traits and executive functions. Specifically, the first factor consisted of measures related to inhibitory control and negative affect, and the second factor was related to reward and positive affect. To investigate whether this interplay between emotional traits and executive functions is reflected in neural connectivity, we used resting-state fMRI to explore the functional connectivity of the amygdala as a starting point, and progressed to other seed-based analyses based on the initial findings. We found that the first factor predicted the strength of connectivity between brain regions known to be involved in the cognitive control of emotion, including the amygdala and the dorsolateral prefrontal cortex, whereas the second factor predicted the strength of connectivity between brain regions known to be involved in reward and attention, including the amygdala, the caudate and the thalamus. These findings suggest that individual differences in the ability to inhibit negative affect are mediated by prefrontal-limbic pathways, while the ability to be positive and use rewarding information is mediated by a network that includes the amygdala and thalamostriatal regions.
Assuntos
Tonsila do Cerebelo/fisiologia , Conectoma/métodos , Emoções/fisiologia , Função Executiva/fisiologia , Individualidade , Córtex Pré-Frontal/fisiologia , Adulto , Feminino , Humanos , Inibição Psicológica , Imageamento por Ressonância Magnética , Masculino , Análise de Componente Principal , Autoavaliação (Psicologia) , Adulto JovemRESUMO
Membrane currents of src-transformed NIH3T3 mouse fibroblasts were analyzed in comparison with their non-transformed counterparts using the patch-clamp technique. Normal NIH3T3 cells exhibit two types of Ca2+ currents and a membrane current of ohmic behaviour (current amplitude 135 pA at +30 mV) that can partially be blocked by Cd2+. Src-transformed NIH3T3 cells show an additional membrane current that becomes activated after the establishment of the whole-cell configuration with a maximum amplitude of 1040 pA at +30 mV within 30-60 s. This current then inactivates irreversibly within 5-10 min. The additional current is highly K(+)-selective and Ca(2+)-dependent but voltage-independent. It can be blocked by charybdotoxin (IC50 = 20 nM) and by internal tetraethylammonium (TEA; IC50 = 2.9 mM), but it is not sensitive to external TEA (up to 30 mM). Single-channel analysis revealed only one K+ channel type with a conductance of 37 pS at negative potentials and 18 pS at positive potentials (in symmetrical 145 mM K+ solutions), a voltage-independent open-state probability of 0.6 and the same pharmacological properties as the macroscopic KCa current. The properties of the KCa current and the underlying channels of src-transformed NIH3T3 cells are identical to those observed in ras-transformed NIH3T3 cells. In contrast, src- or ras-transformation affects differently the voltage-dependent, transient (T-type) Ca2+ current. While ras-transformation of NIH3T3 cells suppresses their T-type Ca2+ current, this current remains unchanged in src-transformed NIH3T3 cells.
Assuntos
Cálcio/química , Genes src , Potássio/química , Células 3T3 , Animais , Linhagem Celular Transformada , Membrana Celular/química , Eletroquímica , Genes ras , Canais Iônicos/química , Camundongos , Técnicas de Patch-ClampRESUMO
Effects of the lysophospholipids sphingosine-1-phosphate and lysophosphatidic acid were studied in cultured murine microglia using the patch-clamp and video imaging techniques. Both lysophospholipids induced transient membrane hyperpolarization and K(+) current activation. The lysophospholipid-induced K(+) current was blocked by charybdotoxin or iberiotoxin, but was unaffected by apamin. In recordings with 1 microM intracellular free Ca(2+), Ca(2+)-dependent K(+) currents of microglia showed a similar pharmacological profile to lysophospholipid-induced currents. The Ca(2+)-dependent K(+) channels activated in microglia by lysophospholipids are most likely encoded by the IKCa1 channel gene. The presence of IKCa1 mRNA in microglia was demonstrated by reverse transcriptase-polymerase chain reaction studies. Ca(2+) imaging experiments revealed increases in the intracellular free Ca(2+) concentration of microglia to a mean value of about 400 nM after application of 1 microM sphingosine-1-phosphate or 1 microM lysophosphatidic acid. We suggest that the transient membrane hyperpolarization seen in microglia following exposure to sphingosine-1-phosphate or lysophosphatidic acid is caused by activation of IKCa1 Ca(2+)-dependent K(+) channels. Increases in the concentration of intracellular free Ca(2+) evoked by the lysophospholipids are sufficient to activate microglial Ca(2+)-dependent K(+) channels.
Assuntos
Encéfalo/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Lisofosfolipídeos/metabolismo , Microglia/metabolismo , Canais de Potássio/metabolismo , Esfingosina/análogos & derivados , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/efeitos dos fármacos , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Charibdotoxina/farmacologia , Gliose/metabolismo , Gliose/fisiopatologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Lisofosfolipídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos , Microglia/efeitos dos fármacos , Peptídeos/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Esfingosina/metabolismo , Esfingosina/farmacologiaRESUMO
Sugar beet ( Beta vulgaris L.) is a biennial species. Shoot elongation (bolting) starts after a period of low temperature. The dominant allele of locus B causes early bolting without cold treatment. This allele is abundant in wild beets whereas cultivated beets carry the recessive allele. Fifteen AFLP markers, tightly linked to the bolting locus, have been identified using bulked segregant analysis. The F(2)-population consisted of 2,134 individuals derived after selfing a single F(1)-plant ( Bb). In a first step, a linkage map was established with 249 markers based on 775 F(2)-individuals with a coverage of 822.3 cM. The loci are dispersed over nine linkage groups corresponding to the haploid chromosome number of Beta species. Seventeen marker loci were placed at a distance less than 3.2 cM around the bolting gene. In a second step, four of those markers most closely linked to B were mapped with the entire F(2)-population. Two of the markers were mapped flanking the B gene at distances of 0.14 and 0.23 cM. The other two markers were mapped at a distance of 0.5 cM from the gene. The tight linkage could be verified by testing 88 unrelated plants from a breeding program. The closely linked markers will enable breeders to select for the non-bolting character without laborious test crossings. Moreover, these markers are being used for map-based cloning of the bolting gene.
RESUMO
The effects of botulinum A neurotoxin and tetanus toxin were studied in cultured mouse spinal cord neurons. In approximately 60% of the neurons (n = 150), botulinum A neurotoxin caused paroxysmal depolarizing events. In two cells hyperpolarizing shifts were observed. The pattern of the burst-like activity varied in shape and frequency in individual cells. Between the paroxysmal events, ongoing synaptic activity could be recorded. The other 40% of the treated neurons did not develop a characteristic pattern of bursts, but there was a decrease in frequency of synaptically generated events. In contrast to botulinum A neurotoxin, tetanus toxin invariably produced well organized paroxysmal events without any synaptic activity between them. At later stages botulinum A neurotoxin and tetanus toxin blocked inhibitory and excitatory postsynaptic potentials in all neurons studied. These results have demonstrated, for the first time using electrophysiological techniques, that botulinum A neurotoxin blocks both excitatory and inhibitory synaptic transmission in the mammalian central nervous system. There are however differences between these effects of botulinum A neurotoxin and the actions of tetanus toxin on these cells. It is suggested that at the femtomolar range tetanus toxin blocks selectively central inhibitory systems and botulinum A neurotoxin the motor endplate. At the picomolar range both toxins affect many if not all, transmitter systems.
Assuntos
Toxinas Botulínicas/farmacologia , Medula Espinal/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacos , Animais , Células Cultivadas , Depressão Química , Potenciais Evocados/efeitos dos fármacos , Feto , Camundongos , Inibição Neural/efeitos dos fármacos , Sinapses/efeitos dos fármacosRESUMO
The hypothesis according to which BoTx and TeTx block neuromuscular transmission by impairing Ca2+ influx was tested by recording presynaptic membrane currents at motor endplates of the mouse by means of external electrodes. The use of K-channel blockers allowed us to observe inward Ca current which was unaffected by bath application of BoTx or TeTx at doses which block neuromuscular transmission.
Assuntos
Toxinas Botulínicas/farmacologia , Cálcio/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Toxina Tetânica/farmacologia , Animais , Canais Iônicos/efeitos dos fármacos , Camundongos , Neurônios Motores/efeitos dos fármacos , Potássio/metabolismo , Sódio/metabolismo , Transmissão Sináptica/efeitos dos fármacosRESUMO
The quantal transmitter release of tetanus (TeTx) and botulinum A (BoTx) toxin paralyzed mouse diaphragms was studied. The very low release probability could be enhanced by increasing the frequency of nerve stimulation to 50 Hz or by the application of 4-aminopyridine. In the BoTx-muscles the endplate potentials were strongly coupled to the stimuli with synaptic delays similar to unpoisoned terminals. In contrast, in the TeTx-muscles large variations in the delay of release of quanta in response to stimulation were observed. From these findings it is suggested that TeTx and BoTx act at different sites of the depolarization-transmitter release process.
Assuntos
Toxinas Botulínicas/farmacologia , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Toxina Tetânica/farmacologia , Animais , Diafragma/metabolismo , Estimulação Elétrica , Potenciais Evocados , Técnicas In Vitro , Camundongos , Junção Neuromuscular/fisiologia , Nervo Frênico/fisiologia , Tempo de Reação , Transmissão Sináptica , Fatores de TempoRESUMO
beta-Bungarotoxin (beta-BuTx), at concentrations of 0.45-45 nmol/l, selectively reduced a portion of the noninactivating potassium current (IsK) in dorsal root ganglion neurones of the guinea pig, measured by voltage clamp of internally perfused cells. The average reduction of IsK obtainable with beta-BuTx was 34% and usually not completed within 20 min, but irreversible upon washing for 20 min. The I/V-characteristic of the current blocked by beta-BuTx was almost linear. It is suggested that beta-BuTx selectively blocks a noninactivating subtype of potassium channel.
Assuntos
Bungarotoxinas/farmacologia , Gânglios Espinais/metabolismo , Canais Iônicos/efeitos dos fármacos , Neurônios/metabolismo , Potássio/metabolismo , Animais , Venenos Elapídicos/farmacologia , Eletrofisiologia , Gânglios Espinais/citologia , Cobaias , Canais Iônicos/fisiologia , Concentração OsmolarRESUMO
Purified heavy chain of botulinum toxin type A and light chain of tetanus toxin were combined to form a chimeric toxin. It was active on the mouse phrenic nerve-hemidiaphragm with a potency 6 times higher than that of native tetanus toxin. Electrophysiological data from poisoned neuromuscular junctions revealed that the pattern of nerve-evoked and spontaneous transmitter release was equivalent to that seen with tetanus toxin i.e. asynchronous release, and did not resemble that after botulinum toxin type A poisoning. We conclude that the light chain of tetanus toxin alone is responsible for the characteristic effects on spontaneous and nerve-evoked transmitter release of the native toxin and that these properties can be introduced into a new, more potent complex with the heavy chain of botulinum toxin A.
Assuntos
Toxinas Botulínicas/farmacologia , Placa Motora/metabolismo , Neurotransmissores/metabolismo , Toxina Tetânica/farmacologia , Animais , Química Encefálica/efeitos dos fármacos , Estimulação Elétrica , Eletrofisiologia , Técnicas In Vitro , Camundongos , Placa Motora/efeitos dos fármacos , Nervo Frênico/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
Tetanus toxin causes a block of the neuromuscular transmission. The kinetic aspects of the block were studied in vitro on the mouse phrenic nerve-hemidiaphragm exposed to toxin (1 microgram/ml). 1. The toxin action on the nerve ending involves three sequential steps: binding, "translocation" and paralysis. 2. Diffusion and binding of tetanus toxin molecules to the presynaptic membrane is complete in about 60 min. The binding step is irreversible, independent of transmitter release and of the temperature. Tetanus antitoxin, however, inactivates the bound toxin molecules. 3. After a second step which is probably due to a "translocation" of the toxin molecules into or through the presynaptic membrane the antitoxin molecules are now ineffective to prevent the toxin-induced inhibition of transmitter release. This so called "translocation" step requires transmitter release and therefore depends strongly on the frequency of nerve stimulation. 4. The paralytic step does not depend on the transmitter release. It, however, depends strongly on temperature with a break in the Arrhenius-plot around 33 degrees C which suggests the involvement of a phase transition rather than of an enzymatic activity of the toxin.
Assuntos
Junção Neuromuscular/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Toxina Tetânica/toxicidade , Animais , Estimulação Elétrica , Técnicas In Vitro , Camundongos , Contração Muscular/efeitos dos fármacos , Neurotransmissores/metabolismo , Paralisia/fisiopatologia , Membranas Sinápticas/metabolismo , Temperatura , Antitoxina Tetânica/farmacologiaRESUMO
Sphingosine-1-phosphate (S1P) is a phospholipid that acts through G-protein-coupled plasma membrane receptors and induces a broad spectrum of cellular responses, including proliferation, migration, differentiation and apoptosis. Here we report that in NIH3T3 and C3H10T1/2 mouse fibroblasts S1P activates a Ca2+-dependent, voltage-independent K+ current (EC50-value 113 nM) that is blocked by the K+ channel blockers charybdotoxin, margatoxin, and iberiotoxin. The K+ current activation by S1P is transient and leads to a large membrane hyperpolarization. Recently, we showed that lysophosphatidic acid (LPA), a serum lipid with similar biological effects compared to those of S1P, can activate a Ca2+-dependent K+ current in NIH3T3 cells that has identical properties compared to the one that is activated by S1P. When applied consecutively, both S1P and LPA induced a K+ current response in NIH3T3 cells, which indicates that the K+ current activation is not subjected to cross-desensitization between S1P and LPA. In C3H10T1/2 mouse fibroblasts that overexpress the nonreceptor protein tyrosine kinase c-Src, the amplitude of the S1P-induced K+ current was almost doubled compared to the one that we found in control cells. Expression of a non-myristylated c-Src mutant led to a further increase in the K+ current response to S1P, whereas expression of a kinase-defective c-Src mutant reduced it to about 40% compared to the control value. Our data show that S1P activates Ca2+-dependent K+ channels in mouse fibroblasts via an intracellular signalling pathway that involves the non-receptor protein tyrosine kinase c-Src.
Assuntos
Cálcio/fisiologia , Canais de Potássio/efeitos dos fármacos , Proteínas Tirosina Quinases/fisiologia , Esfingosina/farmacologia , Células 3T3/efeitos dos fármacos , Células 3T3/enzimologia , Células 3T3/fisiologia , Animais , Proteína Tirosina Quinase CSK , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Fibroblastos/fisiologia , Lisofosfolipídeos/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Endogâmicos C3H , Mutação , Bloqueadores dos Canais de Potássio , Canais de Potássio/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Esfingosina/análogos & derivados , Quinases da Família srcRESUMO
Lysophosphatidic acid (LPA) is a bioactive lipid that acts through G protein-coupled plasma membrane receptors and mediates a wide range of cellular responses. Here we report that LPA activates a K+ current in NIH3T3 mouse fibroblasts that leads to membrane hyperpolarization. The activation occurs with an EC50 value of 1.7 nM LPA. The K+ current is Ca2+-dependent, voltage-independent, and completely blocked by the K+ channel blockers charybdotoxin, margatoxin, and iberiotoxin with IC50 values of 1.7, 16, and 62 nM, respectively. The underlying K+ channels possess a single channel conductance of 33 pS in symmetrical K+ solution. Pretreatment of cells with pertussis toxin (PTX), Clostridium sordellii lethal toxin, or a farnesyl protein transferase inhibitor reduced the K+ current amplitude in response to LPA to about 25% of the control value. Incubation of cells with the protein tyrosine kinase inhibitor genistein or microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 reduced it by more than 50%. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase A activator 8-bromo-cAMP had no effect. These results indicate that the K+ channel activation by LPA is mediated by a signal transduction pathway involving a PTX-sensitive G protein, a protein tyrosine kinase, and Ras. LPA is already known to activate Cl- channels in various cell types, thereby leading to membrane depolarization. In conjunction with our results that demonstrate LPA-induced membrane hyperpolarization by activation of K+ channels, LPA appears to be significantly involved in the regulation of the cellular membrane potential.
Assuntos
Cálcio/fisiologia , Proteínas de Ligação ao GTP/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Toxina Pertussis , Canais de Potássio/efeitos dos fármacos , Fatores de Virulência de Bordetella/farmacologia , Proteínas ras/efeitos dos fármacos , Células 3T3/citologia , Células 3T3/efeitos dos fármacos , Células 3T3/fisiologia , Animais , Proteínas de Ligação ao GTP/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Canais de Potássio/fisiologia , Reprodutibilidade dos Testes , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas ras/metabolismoRESUMO
The pore-forming activity of tetanus toxin, its chains and fragments was studied on membrane patches from spinal cord neurons of fetal mice using the outside-out patch-clamp configuration. 1. The dichain tetanus toxin forms pores at pH 5, but not at pH 7.4. The elementary pore conductance is 38.4 +/- 1.1 pS and nonselective for small cations. The open probability of the pores is voltage-dependent and increases with membrane depolarisation. The pores activate at +80 mV with a time constant of about 20 ms and deactivate at -80 mV with two time constants of about 2 ms and 10 ms. Besides the elementary pore conductance, larger pore conductances which are multiples of the elementary conductance were observed. With increasing conductances, their frequency of occurrence decreases exponentially. 2. The light chain of tetanus toxin alone does not form pores in neuronal membranes at pH 5 or at pH 7.4. 3. The heavy chain of tetanus toxin forms pores at pH 5 as well as at pH 7.4. The single pore conductance increases from 35.0 +/- 1.2 pS at pH 5 to 43.2 +/- 1.8 pS at pH 7.4. The pores allow mono- and divalent cations and chloride ions to pass. Only at pH 5 do they have a voltage dependence with time constants identical to those obtained with tetanus toxin. 4. Secondary structure predictions show a high density of presumably helically organized elements in fragment beta 2 (45 kDa) of the heavy chain between residues 700-850.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Estrutura Secundária de Proteína , Toxina Tetânica/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Células Cultivadas , Fenômenos Químicos , Físico-Química , Estudos de Avaliação como Assunto , Concentração de Íons de Hidrogênio , Camundongos , Peso Molecular , Neurônios/ultraestrutura , Fragmentos de Peptídeos/química , Medula Espinal/citologia , Medula Espinal/efeitos dos fármacos , Relação Estrutura-Atividade , Toxina Tetânica/químicaRESUMO
Electrophysiological and electron microscopic techniques were used to investigate the actions of potassium depolarization, black widow venom (BWSV), Ca2+-ionophore A 23187 and hyperosmotic solution on mouse hemidiaphragms poisoned in vitro with botulinum A toxin (BoTx) and tetanus toxin (TeTx). These neurotoxins reduced the frequency of miniature endplate potentials (m.e.p.ps) from 5/s of the control to 2/min and 21/min, respectively. High potassium (25 mmol/l) increased the m.e.p.p.-frequency at BoTx- and TeTx-poisoned endplates to 30/min and 50/s, respectively. The ultrastructure of endplates showed no obvious changes. BWSV (0.04 glands/ml) was just as effective in promoting transmitter release from BoTx-treated endplates as in control preparations. Electron micrographs revealed depletion of vesicles as well as swollen and disrupted mitochondria. When preparations were pretreated with TeTx, BWSV only moderately increased transmitter release and no alterations of the ultrastructure could be observed. At TeTx- or BoTx-poisoned endplates Ca2+-ionophore A 23187 usually produced an extreme reduction of m.e.p.p.-frequency (0.005/s), sometimes preceded by a short burst-like release. The ultrastructure of these endplates was not obviously affected. Application of hyperosmotic solution to BoTx- or TeTx-poisoned preparations further reduced the already low m.e.p.p.-frequency. These results further demonstrate that TeTx and BoTx act at different sites in the transmitter releasing process.
Assuntos
Toxinas Botulínicas/farmacologia , Neurônios Motores/metabolismo , Terminações Nervosas/metabolismo , Neurotransmissores/metabolismo , Toxina Tetânica/farmacologia , Animais , Calcimicina/farmacologia , Soluções Hipertônicas/farmacologia , Técnicas In Vitro , Camundongos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/ultraestrutura , Terminações Nervosas/efeitos dos fármacos , Terminações Nervosas/ultraestrutura , Fármacos Neuromusculares Despolarizantes , Potássio/farmacologia , Venenos de Aranha/farmacologiaRESUMO
The effects of dendrotoxin (DTX), a toxic peptide from Dendroaspis angusticeps venom, were studied electrophysiologically on peripheral frog nerve fibres, and biochemically on large synaptosomes from rat brain. On nerve fibres, DTX reduced the amplitude and prolonged the duration of the action potential; even at 0.1 nmol/l DTX produced significant effects. Maximum block of potassium currents occurred at about 30 nmol/l. Turning on of the remaining current was slowed. Reversibility was incomplete. The reduction of potassium currents was between 31% and 85% at 85 nmol/l DTX (n = 8). The remainder appeared to be resistant to DTX. Sodium channels were not affected. On large synaptosomes DTX (above 1 nmol/l) produced a slight depolarization, indicated by an outward shift of the lipophilic cation tetraphenylphosphonium, and promoted the release of radioactivity after preloading with [3H] GABA. DTX had similar potency but lower efficacy in this respect than sea anemone toxin II (ATX II). In contrast to the effects of ATX II, those due to DTX were only partially inhibited by tetrodotoxin. The actions of 4-aminopyridine resembled those of DTX, but the latter was about 500 times more potent. The electrophysiological data provide direct evidence for blockade of a potassium channel by DTX. This action is sufficient to explain the biochemical observations, although additional effects on synaptosomes cannot be excluded.
Assuntos
Venenos Elapídicos/farmacologia , Canais Iônicos/efeitos dos fármacos , Neurotoxinas/farmacologia , Potássio/metabolismo , Potenciais de Ação/efeitos dos fármacos , Animais , Cálcio/fisiologia , Técnicas In Vitro , Potenciais da Membrana/efeitos dos fármacos , Fibras Nervosas/efeitos dos fármacos , Oniocompostos , Compostos Organofosforados , Rana esculenta , Ratos , Sinaptossomos/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K+ current that is absent in control cells. The activation of the K+ current was Ca2+-dependent, voltage-independent, and was completely blocked by the K+ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC50 values of 3 nM, 18 nM and 76 nM, respectively. To identify signalling components that mediate the activation of this K+ current, NIH3T3 cells that express different mutants of the wild-type v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K+ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K+ current. Treatment of wild-type v-Fms cells with Clostiridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K+ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K+ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K+ channel activation by the oncogenic RTK v-Fms.
Assuntos
Cálcio/fisiologia , Proteína Oncogênica gp140(v-fms)/fisiologia , Canais de Potássio/fisiologia , Células 3T3 , Animais , Toxinas Bacterianas/farmacologia , Camundongos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio , Proteínas ras/fisiologiaRESUMO
Dracotoxin, a protein possessing toxic, membrane depolarizing and hemolytic activities, was isolated from the crude venom of the greater weever fish Trachinus draco. The purification involved ammonium sulfate precipitation of crude venom followed by gel filtration on a high performance liquid chromatograph column. About 300 micrograms of dracotoxin was obtained from 18 mg of crude venom proteins extracted from one average size fish. Dracotoxin consists of a single polypeptide of about 105,000 mol. wt. It hemolyzed rabbit erythrocytes with an EC50 of 3 ng/ml. Rabbit erythrocytes possessed binding sites for dracotoxin on their surface. Preincubation of dracotoxin with rabbit ghosts increased its EC50 value for rabbit erthrocytes from 3 to 25 ng/ml. Incubation of dracotoxin with enriched glycophorin fraction from rabbit erythrocytes also led to an increase in the EC50 to 70 ng/ml. The high specificity of dracotoxin for rabbit erythrocytes resembles that of staphylococcal alpha-toxin. Dracotoxin, however, caused hemolysis even at 4 degrees C and did not interact with cholesterol indicating substantial differences between the two hemolysins. Dracotoxin represents a major toxic component of T. draco venom.
Assuntos
Venenos de Peixe/química , Animais , Toxinas Bacterianas/toxicidade , Precipitação Química , Cromatografia Líquida de Alta Pressão , Venenos de Peixe/isolamento & purificação , Venenos de Peixe/toxicidade , Peixes , Proteínas Hemolisinas/toxicidade , Hemólise/efeitos dos fármacos , Dose Letal Mediana , Camundongos , Peso Molecular , StaphylococcusRESUMO
Crude venom of the greater weever fish, Trachinus draco was analyzed to assess its toxicity, stability and biological properties. The best yield of venom was obtained by extraction in physiological saline of the whole venom apparatus of the fish which were shock-frozen and stored at -70 degrees C. This extract had a mouse i.v. minimum lethal dose of 1.8 micrograms protein per gram mouse and a total of 61,000 minimum lethal doses were obtained from venom apparatus of one fish. The lethal activity was unstable at room temperature especially at lower protein concentrations. Stability was achieved either by storing the extract at -70 degrees C or by precipitation with ammonium sulfate at 50% saturation. Toxicity of the crude venom was abolished by trypsin treatment. The crude venom did not possess any proteolytic or histamine-releasing activities. The venom caused an outflow of tetraphenylphosphonium from preloaded rat brain particles in a concentration-dependent manner. Like toxicity, this effect was also abolished by trypsin treatment or by keeping the venom at higher temperatures. The crude venom also possessed hemolytic activity with an EC50 for rabbit erythrocytes of 75 ng/ml venom protein. The hemolytic activity was also sensitive to heat and proteolytic treatment. Rabbit erythrocytes were most sensitive to venom followed by rat erythrocytes. Mouse and cattle erythrocytes were only slightly sensitive, whereas human, chicken and guinea pig erythrocytes were totally resistant.
Assuntos
Venenos de Peixe/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Venenos de Peixe/química , Venenos de Peixe/isolamento & purificação , Peixes , Hemólise/efeitos dos fármacos , Liberação de Histamina/efeitos dos fármacos , Humanos , Indicadores e Reagentes , Dose Letal Mediana , Mastócitos/efeitos dos fármacos , Camundongos , Oniocompostos/metabolismo , Compostos Organofosforados/metabolismo , Ratos , Temperatura , Tripsina/farmacologiaRESUMO
The binding and effects of tetanus and botulinum A neurotoxins were studied on mouse spinal cord cultures treated with neuraminidase. In untreated cultures both neurotoxins blocked synaptic transmission. Treatment of the cell cultures with neuraminidase, 25 mU/ml for 24 hr, decreased the potency of botulinum A neurotoxin. At 7 X 10(-11) M no toxin effect on inhibitory or excitatory synapses was observed, whereas at higher concentrations of the toxin the concentration-response curve was shifted to the right by a factor of about 30. Surprisingly, the action of tetanus toxin over a large concentration range was unaffected by pretreatment of the neurones with the enzyme. Accordingly, neurones treated with neuraminidase failed to bind 125I-botulinum A neurotoxin, whereas labelled tetanus toxin was still fixed by cell bodies, as well as by neurites, as shown by histoautoradiography. Chromatographic extraction of gangliosides from cultures prelabelled with 14C-glucosamine showed a dramatic loss in the contents of polysialogangliosides following treatment with neuraminidase. Our results indicate that neuraminidase-sensitive structures might be important for the action of botulinum A neurotoxin. The effect of tetanus toxin appears to be mediated by a different site which is insensitive to neuraminidase.
Assuntos
Toxinas Botulínicas/farmacologia , Proteínas de Membrana , Neuraminidase , Neurônios/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Animais , Sítios de Ligação , Toxinas Botulínicas/antagonistas & inibidores , Toxinas Botulínicas/metabolismo , Técnicas de Cultura , Gangliosídeos/metabolismo , Camundongos , Receptores Colinérgicos/metabolismo , Sinapses/efeitos dos fármacos , Toxina Tetânica/metabolismo , Toxina Tetânica/farmacologiaRESUMO
The antiproliferative action of the guanine-specific ribonuclease secreted by Bacillus intermedius (binase) was studied in different chicken and mouse cell lines. The proliferation rate of chicken embryo fibroblasts, either normal or Rous sarcoma virus-transformed, was significantly reduced by binase treatment. Among mouse fibroblasts, v-ras-transformed NIH3T3 cells were sensitive to binase, whereas the growth of non-transformed, v-src-transformed or v-fms-transformed NIH3T3 cells was not affected. A 48 h treatment with binase inhibited the Ca2+-dependent K+ current of v-ras-transformed NIH3T3 cells but had no effect on this membrane current in non-transformed and in v-src- or v-fms-transformed NIH3T3 cells. Our results suggest that mammalian cells expressing the ras-oncogene are a potential target for the antiproliferative action of binase.