RESUMEN
Coral snake venoms from the Micrurus genus are a natural library of components with multiple targets, yet are poorly explored. In Brazil, 34 Micrurus species are currently described, and just a few have been investigated for their venom activities. Micrurus venoms are composed mainly of phospholipases A2 and three-finger toxins, which are responsible for neuromuscular blockade-the main envenomation outcome in humans. Beyond these two major toxin families, minor components are also important for the global venom activity, including Kunitz-peptides, serine proteases, 5' nucleotidases, among others. In the present study, we used the two-microelectrode voltage clamp technique to explore the crude venom activities of five different Micrurus species from the south and southeast of Brazil: M. altirostris, M. corallinus, M. frontalis, M. carvalhoi and M. decoratus. All five venoms induced full inhibition of the muscle-type α1ß1δε nAChR with different levels of reversibility. We found M. altirostris and M. frontalis venoms acting as partial inhibitors of the neuronal-type α7 nAChR with an interesting subsequent potentiation after one washout. We discovered that M. altirostris and M. corallinus venoms modulate the α1ß2 GABAAR. Interestingly, the screening on KV1.3 showed that all five Micrurus venoms act as inhibitors, being totally reversible after the washout. Since this activity seems to be conserved among different species, we hypothesized that the Micrurus venoms may rely on potassium channel inhibitory activity as an important feature of their envenomation strategy. Finally, tests on NaV1.2 and NaV1.4 showed that these channels do not seem to be targeted by Micrurus venoms. In summary, the venoms tested are multifunctional, each of them acting on at least two different types of targets.
Asunto(s)
Serpientes de Coral , Venenos Elapídicos , Toxinas Biológicas , Animales , Brasil , Serpientes de Coral/fisiología , Venenos Elapídicos/química , Venenos Elapídicos/farmacología , Elapidae , Canales Iónicos , Fosfolipasas A2 , Toxinas Biológicas/química , Toxinas Biológicas/farmacología , Toxinas Biológicas/fisiologíaRESUMEN
Abstract The composition and pharmacological properties of Lippia alba (Mill.) (L. alba) (Verbenaceae) flower and leaf essential oils (EO) were determined in this study. The major constituents in the flower EO were geranial (49.83%) and neral (32.75%), and in the leaf EO were geranial (38.06%), neral (31.02%), and limonene (18.03%). Flower EO inhibited thrombolysis induced by Bothrops moojeni (B. moojeni) and Lachesis muta muta (L. muta muta) venoms (0.05-1.2 µL mL-1). When tested against L. muta muta venom, the protective effect was smaller in both EO. The EOs prolonged the clotting time induced by L. muta muta venom and a procoagulant effect was observed on B. moojeni. In the comet assay, the flower EO presented anti-genotoxic action (damage frequency of only 11.6 - 34.9%) against the L. muta muta venom. The positive control (Doxorubicin) and the venom alone presented a damage frequency of 80.3% and 70.7%, respectively. The flower EO protected DNA from damage induced by L. muta muta venom. L. alba leaf and flower EOs presented anti-genotoxic action
Asunto(s)
Productos Biológicos/análisis , Aceites Volátiles/análisis , Lippia/efectos adversos , Hojas de la Planta/clasificación , Ensayo Cometa/instrumentación , Flores/clasificación , Venenos Elapídicos/farmacología , Inhibidores Enzimáticos/administración & dosificación , HemostasisRESUMEN
A plasmin inhibitor, named tenerplasminin-1 (TP1), was isolated from Micrurus tener tener (Mtt) venom. It showed a molecular mass of 6542Da, similarly to Kunitz-type serine peptidase inhibitors. The amidolytic activity of plasmin (0.5nM) on synthetic substrate S-2251 was inhibited by 91% following the incubation with TP1 (1nM). Aprotinin (2nM) used as the positive control of inhibition, reduced the plasmin amidolytic activity by 71%. Plasmin fibrinolytic activity (0.05nM) was inhibited by 67% following incubation with TP1 (0.1nM). The degradation of fibrinogen chains induced by plasmin, trypsin or elastase was inhibited by TP1 at a 1:2, 1:4 and 1:20 enzyme:inhibitor ratio, respectively. On the other hand, the proteolytic activity of crude Mtt venom on fibrinogen chains, previously attributed to metallopeptidases, was not abolished by TP1. The tPA-clot lysis assay showed that TP1 (0.2nM) acts like aprotinin (0.4nM) inducing a delay in lysis time and lysis rate which may be associated with the inhibition of plasmin generated from the endogenous plasminogen activation. TP1 is the first serine protease plasmin-like inhibitor isolated from Mtt snake venom which has been characterized in relation to its mechanism of action, formation of a plasmin:TP1 complex and therapeutic potential as anti-fibrinolytic agent, a biological characteristic of great interest in the field of biomedical research. They could be used to regulate the fibrinolytic system in pathologies such as metastatic cancer, parasitic infections, hemophilia and other hemorrhagic syndromes, in which an intense fibrinolytic activity is observed.
Asunto(s)
Antifibrinolíticos/farmacología , Venenos Elapídicos/farmacología , Fibrinolisina/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/farmacología , Animales , Antifibrinolíticos/aislamiento & purificación , Venenos Elapídicos/aislamiento & purificación , Elapidae , Fibrinolisina/metabolismo , Humanos , Inhibidores de Serina Proteinasa/aislamiento & purificaciónRESUMEN
In this work, we have examined the neuromuscular activity of Micrurus laticollaris (Mexican coral snake) venom (MLV) in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations, the MLV induced an irreversible concentration- and time-dependent (1-30 µg/mL) neuromuscular blockade, with 50% blockade occurring between 8 and 30 min. Muscle contractures evoked by exogenous acetylcholine were completely abolished by MLV, whereas those of KCl were also significantly altered (86% ± 11%, 53% ± 11%, 89% ± 5% and 89% ± 7% for one, three, 10 and 30 µg of venom/mL, respectively; n = 4; p < 0.05). In mouse phrenic nerve-diaphragm preparations, MLV (1-10 µg/mL) promoted a slight increase in the amplitude of twitch-tension (3 µg/mL), followed by neuromuscular blockade (n = 4); the highest concentration caused complete inhibition of the twitches (time for 50% blockade = 26 ± 3 min), without exhibiting a previous neuromuscular facilitation. The venom (3 µg/mL) induced a biphasic modulation in the frequency of miniature end-plate potentials (MEPPs)/min, causing a significant increase after 15 min, followed by a decrease after 60 min (from 17 ± 1.4 (basal) to 28 ± 2.5 (t15) and 12 ± 2 (t60)). The membrane resting potential of mouse diaphragm preparations pre-exposed or not to d-tubocurarine (5 µg/mL) was also significantly less negative with MLV (10 µg/mL). Together, these results indicate that M. laticollaris venom induces neuromuscular blockade by a combination of pre- and post-synaptic activities.
Asunto(s)
Venenos Elapídicos/farmacología , Unión Neuromuscular/efectos de los fármacos , Animales , Pollos , Elapidae , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratones , Músculo Esquelético/efectos de los fármacos , Unión Neuromuscular/metabolismo , Nervio Frénico/efectos de los fármacos , Receptores Nicotínicos/efectos de los fármacos , Receptores Nicotínicos/metabolismo , Transmisión Sináptica/efectos de los fármacosRESUMEN
The intrinsic properties of spherical neurons play a fundamental role in the sensory processing of self-generated signals along a fast electrosensory pathway in electric fish. Previous results indicate that the spherical neuron's intrinsic properties depend mainly on the presence of two resonant currents that tend to clamp the voltage near the resting potential. Here we show that these are: a low-threshold potassium current blocked by 4-aminopyridine and a mixed cationic current blocked by cesium chloride. We also show that the low-threshold potassium current also causes the long refractory period, explaining the necessary properties that implement the dynamic filtering of the self-generated signals previously described. Comparative data from other fish and from the auditory system indicate that other single spiking onset neurons might differ in the channel repertoire observed in the spherical neurons of Gymnotus omarorum.
Asunto(s)
Gymnotiformes/fisiología , Potenciales de la Membrana/fisiología , Neuronas/fisiología , 4-Aminopiridina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Cesio/farmacología , Cloruros/farmacología , Venenos Elapídicos/farmacología , Técnicas In Vitro , Potenciales de la Membrana/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Mesencéfalo/efectos de los fármacos , Mesencéfalo/fisiología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Tetraetilamonio/farmacologíaRESUMEN
The therapeutic potential of snake venoms for pain control has been previously demonstrated. In the present study, the antinociceptive effects of Micrurus lemniscatus venom (MlV) were investigated in experimental models of pain. The antinociceptive activity of MIV was evaluated using the writhing, formalin, and tail flick tests. Mice motor performance was assessed in the rota rod and open field tests. In a screening test for new antinociceptive substances--the writhing test--oral administration of MlV (19.7-1600 µg/kg) produced significant antinociceptive effect. The venom (1600 µg/kg) also inhibited both phases of the formalin test, confirming the antinociceptive activity. The administration of MlV (1600 µg/kg) did not cause motor impairment in the rota rod and open field tests, which excluded possible non-specific muscle relaxant or sedative effects of the venom. The MIV (177-1600 µg/kg) also increases the tail flick latency response, indicating a central antinociceptive effect for the venom. In this test, the MlV-induced antinociceptive effect was long-lasting and higher than that of morphine, an analgesic considered the gold standard. In another set of experiments, the mechanisms involved in the venom-induced antinociception were investigated through the use of pharmacological antagonists. The MlV (1600 µg/kg) antinociceptive effect was prevented by naloxone (5 mg/kg), a non-selective opioid receptor antagonist, suggesting that this effect is mediated by activation of opioid receptors. In addition, the pre-treatment with the µ-opioid receptor antagonist CTOP (1 mg/kg) blocked the venom antinociceptive effect, while the k-opioid receptor antagonist nor-BNI (0.5 mg/kg) or the δ-opioid receptor antagonist naltrindole (3 mg/kg) only partially reduced the venom-induced antinociception. The present study demonstrates, for the first time, that oral administration of M. lemniscatus venom, at doses that did not induce any motor performance alteration, produced potent and long-lasting antinociceptive effect mediated by activation of opioid receptors.
Asunto(s)
Analgésicos/farmacología , Venenos Elapídicos/química , Venenos Elapídicos/farmacología , Administración Oral , Animales , Elapidae , Masculino , Ratones , Morfina/farmacología , Naltrexona/análogos & derivados , Naltrexona/farmacología , Dolor/tratamiento farmacológico , Dimensión del Dolor/efectos de los fármacos , Receptores Opioides/efectos de los fármacos , Receptores Opioides delta/antagonistas & inhibidores , Receptores Opioides mu/antagonistas & inhibidoresRESUMEN
BACKGROUND: The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. RESULTS: In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. CONCLUSION: Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process.
Asunto(s)
Anafilatoxinas/biosíntesis , Activación de Complemento/efectos de los fármacos , Venenos Elapídicos/farmacología , Elapidae/metabolismo , Animales , Proteína Inhibidora del Complemento C1/aislamiento & purificación , Proteína Inhibidora del Complemento C1/metabolismo , Complemento C3/metabolismo , Venenos Elapídicos/metabolismo , Humanos , Proteolisis/efectos de los fármacosRESUMEN
The venoms of coral snakes (genus Micrurus) are known to induce a broad spectrum of pharmacological activities. While some studies have investigated their potential human effects, little is known about their mechanism of action in terms of the ecological diversity and evolutionary relationships among the group. In the current study we investigated the neuromuscular blockade of the venom of two sister species Micrurus mipartitus and Micrurus dissoleucus, which exhibit divergent ecological characteristics in Colombia, by using the chick biventer cervicis nerve-muscle preparation. We also undertook a phylogenetic analysis of these species and their congeners, in order to provide an evolutionary framework for the American coral snakes. The venom of M. mipartitus caused a concentration-dependant inhibition (3-10 µg/ml) of nerve-mediated twitches and significantly inhibited contractile responses to exogenous ACh (1 mM), but not KCl (40 mM), indicating a postsynaptic mechanism of action. The inhibition of indirect twitches at the lower venom dose (3 µg/ml) showed to be triphasic and the effect was further attenuated when PLA2 was inhibited. M. dissoleucus venom (10-50 µg/ml) failed to produce a complete blockade of nerve-mediated twitches within a 3 h time period and significantly inhibited contractile responses to exogenous ACh (1 mM) and KCl (40 mM), indicating both postsynaptic and myotoxic mechanisms of action. Myotoxic activity was confirmed by morphological studies of the envenomed tissues. Our results demonstrate a hitherto unsuspected diversity of pharmacological actions in closely related species which exhibit divergent ecological characteristics; these results have important implications for both the clinical management of Coral snake envenomings and the design of Micrurus antivenom.
Asunto(s)
Venenos Elapídicos/farmacología , Elapidae , Unión Neuromuscular/efectos de los fármacos , Animales , Pollos , Venenos Elapídicos/química , Técnicas In Vitro , Contracción Muscular/efectos de los fármacos , Neostigmina/farmacología , Inhibidores de Fosfolipasa A2 , FilogeniaRESUMEN
Venoms of the redtail coral snake Micrurus mipartitus from Colombia and Costa Rica were analyzed by "venomics", a proteomic strategy to determine their composition. Proteins were separated by RP-HPLC, followed by SDS-PAGE, in-gel tryptic digestion, identification by MALDI or ESI tandem mass spectrometry, and assignment to known protein families by similarity. These analyses were complemented with a characterization of venom activities in vitro and in vivo. Proteins belonging to seven families were found in Colombian M. mipartitus venom, including abundant three-finger toxins (3FTx; ~60% of total proteins) and phospholipases A(2) (PLA(2); ~30%), with the remaining ~10% distributed among l-amino acid oxidase, P-III metalloproteinase, Kunitz-type inhibitor, serine proteinase, and C-type lectin-like families. The venoms of two M. mipartitus specimens from Costa Rica, also referred to as M. multifasciatus in some taxonomic classifications, were also analyzed. Both samples were highly similar to each other, and partially resembled the chromatographic and identity profiles of M. mipartitus from Colombia, although presenting a markedly higher proportion of 3FTxs (~83.0%) in relation to PLA(2)s (~8.2%), and a small amount of acetylcholinesterase, not detected in the venom from Colombia. An equine antivenom against the Central American coral snake, M. nigrocinctus, did not recognize venom components of M. mipartitus from Colombia or Costa Rica by enzyme-immunoassay. Four major components of Colombian M. mipartitus venom were isolated and partially characterized. Venomics of Micrurus species may provide a valuable platform for the rational design of immunizing cocktails to obtain polyspecific antivenoms for this highly diverse group of American elapids.
Asunto(s)
Venenos Elapídicos/química , Venenos Elapídicos/farmacología , Secuencia de Aminoácidos , Animales , Antivenenos/análisis , Cromatografía Líquida de Alta Presión , Venenos Elapídicos/metabolismo , Elapidae , L-Aminoácido Oxidasa/análisis , Ratones , Fosfolipasas A2/análisis , Proteoma/análisisRESUMEN
AIMS: We have isolated a new muscarinic protein (MT-Mlα) from the venom of the Brazilian coral snake Micrurus lemniscatus. MAIN METHODS: This small protein, which had a molecular mass of 7,048Da, shared high sequence homology with three-finger proteins that act on cholinergic receptors. The first 12 amino acid residues of the N-terminal sequence were determined to be: Leu-Ile-Cys-Phe-Ile-Cys-Phe-Ser-Pro-Thr-Ala-His. KEY FINDINGS: The MT-Mlα was able to displace the [(3)H]QNB binding in the hippocampus of rats. The binding curve in competition experiments with MT-Mlα was indicative of two types of [(3)H]QNB-binding site with pK(i) values of 9.08±0.67 and 6.17±0.19, n=4, suggesting that various muscarinic acetylcholine receptor (mAChR) subtypes may be the target proteins of MT-Mlα. The MT-Mlα and the M(1) antagonist pirenzepine caused a dose-dependent block on total [(3)H]inositol phosphate accumulation induced by carbachol. The IC(50) values for MT-Mlα and pirenzepine were, respectively, 33.1 and 2.26 nM. Taken together, these studies indicate that the MT-Mlα has antagonist effect on mAChRs in rat hippocampus. SIGNIFICANCE: The results of the present study show, for the first time, that mAChRs function is drastically affected by MT-Mlα since it not only has affinity for mAChRs but also has the ability to inhibit mAChRs.
Asunto(s)
Venenos Elapídicos/farmacología , Elapidae , Hipocampo/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Receptores Muscarínicos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Brasil , Venenos Elapídicos/administración & dosificación , Hipocampo/metabolismo , Concentración 50 Inhibidora , Fosfatos de Inositol/metabolismo , Masculino , Agonistas Muscarínicos/administración & dosificación , Agonistas Muscarínicos/aislamiento & purificación , Pirenzepina/administración & dosificación , Pirenzepina/farmacología , Ratas , Ratas Wistar , Receptores Muscarínicos/metabolismoRESUMEN
The coral snake Micrurus tener tener (Mtt) from the Elapidae family inhabits the southwestern United States and produces severe cases of envenomations. Although the majority of Mtt venom components are neurotoxins and phospholipase A2s, this study demonstrated, by SDS-PAGE and molecular exclusion chromatography (MEC), that these venoms also contain high-molecular-weight proteins between 50 and 150 kDa that target the hemostatic system. The biological aspects of other Micrurus venoms were also studied, such as the LD50s of Micrurus isozonus (from 0.52 to 0.61 mg/kg). A pool from these venoms presented a LD50 of 0.57 mg/kg, Micrurus f. fulvius (Mff) and Mtt had LD50s of 0.32 and 0.78 mg/kg, respectively. These venoms contained fibrino(geno)lytic activity, they inhibited platelet aggregation, as well as factor Xa and/or plasmin-like activities. M. isozonus venoms from different Venezuelan geographical regions inhibited ADP-induced platelet aggregation (from 50 to 68%). Micrurus tener tener venom from the United States was the most active with a 95.2% inhibitory effect. This venom showed thrombin-like activity on fibrinogen and human plasma. Fractions of Mtt showed fibrino(geno)lytic activity and inhibition on plasmin amidolytic activity. Several fractions degraded the fibrinogen Aα chains, and fractions F2 and F7 completely degraded both fibrinogen Aα and Bß chains. To our knowledge, this is the first report on thrombin-like and fibrino(geno)lytic activity and plasmin or factor Xa inhibitors described in Micrurus venoms. Further purification and characterization of these Micrurus venom components could be of therapeutic use in the treatment of hemostatic disorders.
Asunto(s)
Venenos Elapídicos/toxicidad , Fibrinolíticos/toxicidad , Hemostáticos/toxicidad , Animales , Coagulación Sanguínea/efectos de los fármacos , Venenos Elapídicos/química , Venenos Elapídicos/farmacología , Elapidae , Electroforesis en Gel de Poliacrilamida , Femenino , Fibrinolíticos/química , Fibrinolíticos/farmacología , Hemostáticos/química , Hemostáticos/farmacología , Humanos , Dosificación Letal Mediana , Ratones , Ratones Endogámicos BALB C , Plasma/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Especificidad de la EspecieRESUMEN
In adult neostriatal projection neurons, the intracellular Ca(2+) supplied by Ca(V)2.1 (P/Q) Ca(2+) channels is in charge of both the generation of the afterhyperpolarizing potential (AHP) and the release of GABA from their synaptic terminals, thus being a major target for firing pattern and transmitter release modulations. We have shown that activation of muscarinic M(1)-class receptors modulates Ca(V)2.1 channels in these neurons in rats. This modulation is reversible, is not membrane delimited, is blocked by the specific M(1)-class muscarinic antagonist muscarine toxin 7 (MT-7), and is neither mediated by protein kinase C (PKC) nor by protein phosphatase 2B (PP-2B). Hence, the signaling mechanism of muscarinic Ca(V)2.1 channel modulation has remained elusive. The present paper shows that inactivation of phospholipase C (PLC) abolishes this modulation while inhibition of phosphoinositide kinases, PI-3K and PI-4K, prevents its reversibility, suggesting that the reconstitution of muscarinic modulation depends on phosphoinositide rephosphorylation. In support of this hypothesis, the supply of intracellular phosphatidylinositol (4,5) bisphosphate [PI(4,5)P(2)] blocked all muscarinic modulation of this channel. The results indicate that muscarinic M(1) modulation of Ca(V)2.1 Ca(2+) channels in these neurons involves phosphoinositide hydrolysis.
Asunto(s)
1-Fosfatidilinositol 4-Quinasa/metabolismo , Canales de Calcio Tipo N/metabolismo , Neostriado/metabolismo , Neuronas/metabolismo , Receptor Muscarínico M1/metabolismo , Animales , Calcineurina/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Venenos Elapídicos/farmacología , Hidrólisis , Espacio Intracelular/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Neostriado/efectos de los fármacos , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Ratas , Receptor Muscarínico M1/antagonistas & inhibidores , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
The electrophysiological properties of voltage dependent potassium channels from freshly dissociated rat articular chondrocytes were studied. The resting membrane potential (-42.7+/-2.0 mV) was significantly depolarized by increasing concentrations of external potassium. No change was observed when external chloride concentration was varied. Addition of TEA, 4AP, alpha-Dendrotoxin and charybdotoxin depolarized resting membrane potential. Whole cell patch clamp studies revealed the presence of outwardly rectifying currents whose kinetic and pharmacological properties suggest the expression of voltage dependent potassium channels. Two kinds of currents were observed under the same experimental conditions. The first one, most frequently observed (80%), starts activating near -50 mV, with V(1/2)=-18 mV, G(max)=0.30 pS/pF. The second kind was observed in only 10% of cases; It activates near -40 mV, with(1/2)=+28.35 mV, G(max)=0.28 pS/pF pA/pF and does not inactivates. Inactivating currents were significantly inhibited by TEA (IC(50)=1.45 mM), 4AP (IC(50)=0.64 mM), CTX (IC(50) = 10 nM), alpha-Dendrotoxin (IC(50) < 100 nM) and Margatoxin (IC(50)=28.5 nM). These results show that rat chondrocytes express voltage dependent potassium currents and suggest a role of voltage-dependent potassium channels in regulating membrane potential of rat chondrocytes.
Asunto(s)
Condrocitos/fisiología , Canales de Potasio con Entrada de Voltaje/fisiología , Canales de Potasio/fisiología , Animales , Cartílago Articular/citología , Cloruros/farmacología , Condrocitos/citología , Condrocitos/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Venenos Elapídicos/farmacología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp/métodos , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Wistar , Venenos de Escorpión/farmacologíaRESUMEN
We have isolated a new phospholipase A2 (MiDCA1) from the venom of the coral snake Micrurus dumerilii carinicauda. This toxin, which had a molecular mass of 15,552Da, shared high sequence homology with the PLA2 toxins MICNI A and B from Micrurus nigrocinctus venom (77.7% and 73.1%, respectively). In chick biventer cervicis preparations, MiDCA1 produced concentration- and time-dependent neuromuscular blockade that reached 100% after 120 min (2.4 microM, n = 6); contractures to exogenously applied carbachol (8 microM) and KCl (13 mM) were still seen after complete blockade. In mouse phrenic-nerve diaphragm preparations, MiDCA1 (2.4 microM; n = 6) caused triphasic changes followed by partial neuromuscular blockade. Intracellular recordings of end-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) from mouse diaphragm preparations showed that MiDCA1 increased the quantal content by 386+/-12% after 10 min (n = 14; p<0.05) and caused a triphasic change in the frequency of MEPPs. MiDCA1 also decreased the resting membrane potential, an effect that was prevented by tetrodotoxin and/or low extracellular calcium, but not by d-tubocurarine. The toxin increased the amplitude of mouse sciatic-nerve compound action potentials by 30+/-9% (0.6 microM; p<0.05). Potassium currents elicited in freshly dissociated dorsal root ganglia neurones were blocked by 31+/-1% (n = 4; p<0.05) in the presence of 2.4 microM MiDCA1. These results show that MiDCA1 is a new presynaptic phospholipase A2 that produces neuromuscular blockade in vertebrate nerve-muscle preparations. The triphasic effects seen in mammalian preparations and the facilitatory response were probably caused mainly by the activation of sodium channels, complemented by the blockade of nerve terminal potassium channels. The inability of d-turocurarine to prevent the depolarization by MiDCA1 indicated that cholinergic nicotinic receptors were not involved in this phenomenon.
Asunto(s)
Venenos Elapídicos/enzimología , Elapidae , Fosfolipasas A/química , Fosfolipasas A/farmacología , Secuencia de Aminoácidos , Animales , Pollos , Diafragma/inervación , Venenos Elapídicos/farmacología , Masculino , Ratones , Datos de Secuencia Molecular , Fosfolipasas A/toxicidad , Fosfolipasas A2 , Nervio Frénico/efectos de los fármacos , Nervio Frénico/fisiología , Homología de Secuencia de AminoácidoRESUMEN
The venoms of Micrurus lemniscatus carvalhoi, Micrurus frontalis frontalis, Micrurus surinamensis surinamensis and Micrurus nigrocinctus nigrocinctus were assayed for biological activities. Although showing similar liposome disrupting and myotoxic activities, M. frontalis frontalis and M. nigrocinctus nigrocinctus displayed higher anticoagulant and phospholipase A2 (PLA2) activities. The latter induced a higher edema response within 30 min. Both venoms were the most toxic as well. In the isolated chick biventer cervicis preparation, M. lemniscatus carvalhoi venom blocked the indirectly elicited twitch-tension response (85+/-0.6% inhibition after a 15 min incubation at 5 microg of venom/mL) and the response to acetylcholine (ACh; 55 or 110 microM), without affecting the response to KCl (13.4 mM). In mouse phrenic nerve-diaphragm preparation, the venom (5 microg/mL) produced a complete inhibition of the indirectly elicited contractile response after 50 min incubation and did not affect the contractions elicited by direct stimulation. M. lemniscatus carvalhoi inhibited 3H-L-glutamate uptake in brain synaptosomes in a Ca2+-, but not time, dependent manner. The replacement of Ca2+ by Sr2+ and ethylene glycol-bis(beta-aminoethyl ether) (EGTA), or alkylation of the venom with p-bromophenacyl bromide (BPB), inhibited 3H-L-glutamate uptake. M. lemniscatus carvalhoi venom cross-reacted with postsynaptic alpha-neurotoxins short-chain (antineurotoxin-II) and long-chain (antibungarotoxin) antibodies. It also cross-reacted with antimyotoxic PLA2 antibodies from M. nigrocinctus nigrocinctus (antinigroxin). Our results point to the need of catalytic activity for these venoms to exert their neurotoxic activity efficiently and to their components as attractive tools for the study of molecular targets on cell membranes.
Asunto(s)
Venenos Elapídicos/enzimología , Venenos Elapídicos/farmacología , Elapidae/fisiología , Animales , Anticuerpos/inmunología , Bioensayo , Transporte Biológico/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Venenos Elapídicos/inmunología , Ácido Glutámico/metabolismo , Liposomas/metabolismo , Ratones , Músculos/efectos de los fármacos , Neurotoxinas/inmunología , Neurotoxinas/farmacología , Fosfolipasas A/metabolismo , Fosfolipasas A/farmacología , Fosfolipasas A2 , Nervio Frénico/efectos de los fármacosRESUMEN
Fasciculins are peptides isolated from mamba (Dendroaspis) venoms which exert their toxic action by inhibiting acetylcholinesterase (AChE). They contain a characteristic triple stranded antiparallel beta-sheet formed by residues 22-27, 34-39 and 48-53. A chimeric peptide named Fas-C, encompassing most of these sequences was synthesized using SPPS/Boc-chemistry and characterized chemically, structurally and functionally. Fas-C has two disulfide bridges, formed sequentially using dual cysteine protection. SDS-PAGE patterns, HPLC profiles and MS proved the peptide identity. Circular dichroism indicated the presence of 13.6% and 41.6% of beta-sheet and beta-turn, respectively, comparable to values observed in the native toxin. An inhibitory effect on eel AChE was displayed by the peptide (Ki71.6 +/- 18.3 microM), although not reaching the affinity level of the parent native toxin (Ki 0.3 nM). It is confirmed that the principal binding region of fasciculin to AChE resides within loop II.
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Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Venenos Elapídicos/química , Venenos Elapídicos/farmacología , Acetilcolinesterasa/química , Secuencia de Aminoácidos , Inhibidores de la Colinesterasa/síntesis química , Quimotripsina/química , Venenos Elapídicos/síntesis química , Datos de Secuencia Molecular , Péptidos/síntesis química , Péptidos/química , Prolil Oligopeptidasas , Estructura Secundaria de Proteína , Serina Endopeptidasas/químicaRESUMEN
We have examined the role of inflammatory cells, ischemia and serum complement on the development of acute experimental amoebic liver abscess in hamsters (AEALAH). In hamsters made leukopenic by whole body radiation (800 rad) and daily intraperitoneal glycogen injections, the absence of inflammatory cells and liver tissue damage surrounding the parasites resulted in their rapid (24 h) disappearance from the liver, which showed no lesions. Focal liver ischemia, always present in control AEALAH with inflammation and tissue destruction, was reproduced in radiated hamsters by injection of amoebae mixed with Superdex microspheres, but again in the absence of inflammation, amoebae caused no liver damage and disappeared in 24 h. In hamsters made hypocomplementemic by injection of purified cobra venom factor (CVF), amoebae caused AEALA indistinguishable from controls, but in leukopenic + hypocomplementemic hamsters, amoebae were unable to produce lesions and disappeared from the liver in 48 h. We conclude that inflammation and tissue damage are required for the survival of amoebae in AEALAH and for the progression of the experimental disease.
Asunto(s)
Proteínas del Sistema Complemento/inmunología , Entamoeba histolytica/fisiología , Hepatitis/patología , Isquemia/patología , Absceso Hepático Amebiano , Hígado/patología , Enfermedad Aguda , Animales , Proteínas Inactivadoras de Complemento/farmacología , Modelos Animales de Enfermedad , Venenos Elapídicos/farmacología , Entamoeba histolytica/efectos de la radiación , Femenino , Cobayas , Hepatitis/inmunología , Hepatitis/parasitología , Isquemia/inmunología , Isquemia/parasitología , Leucocitos/efectos de la radiación , Leucopenia/etiología , Hígado/inmunología , Hígado/parasitología , Absceso Hepático Amebiano/inmunología , Absceso Hepático Amebiano/parasitología , Absceso Hepático Amebiano/patología , Masculino , Traumatismos Experimentales por RadiaciónRESUMEN
Calciseptine is a natural peptide consisting of 60 amino acids with four disulfide bonds. The peptide is a natural L-type Ca2+-channel blocker in heart and other systems, but its actions in skeletal muscle have not been previously described. The aim of this study is to characterize the effects of calciseptine on L-type Ca2+ channels of skeletal muscle and on contraction. Whole-cell, patch-clamp experiments were performed to record Ca2+ currents (I(Ca)) from mouse myotubes, whereas Vaseline-gap voltage-clamp experiments were carried out to record I(Ca) from frog skeletal muscle fibers. We found that calciseptine acts as a channel agonist in skeletal muscle, increasing peak I(Ca) by 37% and 49% in these two preparations. Likewise, the peptide increased intramembrane charge movement, though it had little effect on contraction. The molecular analysis of the peptide indicated the presence of a local, electrostatic potential that resembles that of the 1,4-dihydropyridine agonist Bay K 8644. These observations suggest that calciseptine shares the properties of 1,4-dihydropyridine derivatives in modulating the permeation of divalent cations through L-type channels.
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Potenciales de Acción/efectos de los fármacos , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/efectos de los fármacos , Venenos Elapídicos/farmacología , Músculo Esquelético/efectos de los fármacos , Animales , Canales de Calcio Tipo L/fisiología , Células Cultivadas , Electrofisiología , Extremidades , Ratones , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/fisiología , Músculo Esquelético/fisiología , Ranidae , Ratas , Ratas WistarRESUMEN
Calcium signals participate in the differentiation of electrically excitable and nonexcitable cells; one example of this differentiation is the acquisition of mature neuronal phenotypes. For example, transient elevations of the intracellular calcium concentration have been recorded in the ectoderm of early embryos, and this elevation has been proposed to participate in neural induction. Here, we present molecular evidence indicating that voltage-sensitive calcium channels (VSCC) are involved in early developmental processes leading to the establishment of the dorsoventral (D-V) patterning of a vertebrate embryo. We report that alpha1S VSCC are expressed selectively in the dorsal marginal zone at the early gastrula stage. The expression of the VSCC correlates with elevated intracellular calcium levels, as evaluated by the fluorescence of the intracellular calcium indicator Fluo-3. Misexpression of VSCC leads to a strong dorsalization of the ventral marginal zone and induction of the secondary axis but no direct neuralization of the ectoderm. Moreover, specific inhibition of VSCC by the use of calcicludine results in ventralization of the dorsal mesoderm. Together, these results indicate that calcium channels regulate mesodermal patterning by specificating the D-V identity of the mesodermal cells. The D-V patterning of the mesoderm has been shown to depend on a gradient of BMPs activity. We discuss the possibility that VSCC affect or act downstream of BMPs activity.
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Canales de Calcio/genética , Canales de Calcio/metabolismo , Calcio/metabolismo , Gástrula/fisiología , Mesodermo/fisiología , Xenopus laevis/embriología , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/metabolismo , Canales de Calcio/efectos de los fármacos , Venenos Elapídicos/farmacología , Mesodermo/efectos de los fármacos , Subunidades de Proteína , Transducción de Señal/fisiología , Proteínas de XenopusRESUMEN
Intraperitoneal guanosine has been shown to prevent quinolinic acid-induced seizures in mice. In this study, we investigated the effect of orally administered guanosine on seizures induced by the glutamate agonists quinolinic acid and kainate, and the endogenous glutamate releaser alpha-dendrotoxin. Guanosine (7.5 mg/kg, per os), administered 75 min in advance, prevented 70% of seizures induced by i.c.v. quinolinic acid, being as efficient as the NMDA channel blocker MK-801 administered intraperitoneally. Guanosine was ineffective against kainate-induced seizures, but significantly reversed the potentiation of seizures and death caused by the concomitant injection of MK-801. Guanosine also significantly prevented seizures and death induced by i.c.v. alpha-dendrotoxin, whereas MK-801 and phenobarbital only prevented death. Altogether, our findings underscore the therapeutic potential of oral administration of guanosine for treating diseases involving glutamatergic excitotoxicity, including epilepsy.