A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action or alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX stowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation. (C) 2000 Elsevier B.V. Ireland Ltd. All rights reserved.

Comparative structural analysis of neuromuscular junctions in mice at different ages

The authors studied the histochemical and ultrastructural modifications that occur in the neuromuscular junctions (NMJ) of fibularis longus muscles of mice with an age range of 3 to 21 months. Twenty-four male and female animals were killed at 3, 5, 14 and 21 months of age: 7 of them at 3 months, 4 of them at 5 month, 9 at 14 months and 4 at 21 months. The fibularis longus muscles were processed and their NMJ examined with the transmission electron microscope. The most relevant changes were associated with the degeneration and retraction of terminal axons, i.e., axons poor in synaptic vesicles with degenerated mitochondria, and exhibiting multivesicular bodies and vacuoles; exposed and widened junctional folds and cytoplasmic processes of Schwann cells located in the synaptic gutter. The presence of lysosomes or lipofuchsin in the juxtajunctional sarcoplasm was also noted. These observations suggest that the phenomena of retraction and budding occur in the NMJ with advancing age, with a predominance of events associated with degeneration, leading to profound changes in NMJ shape.

Novel wasp toxin discriminates between neuronal and cardiac sodium channels

Pompilidotoxins (PMTXs), derived from the venom of solitary wasp has been known to facilitate synaptic transmission in the lobster neuromuscular junction, and a recent further study from rat trigeminal neurons revealed that the toxin slows Na+ channel inactivation without modifying activation process. Here we report that beta -PMTX modifies rat brain type II Na+ channel alpha -subunit (rBII) expressed in human embryonic kidney cells but fails to act on the rat heart alpha -subunit (rH1) at similar concentrations. We constructed a series of chimeric mutants of rBII and rH1 Na+ channels and compared modification of the steady-state Na+ currents by beta -PMTX. We found that a difference in a single amino acid between Glu-1616 in rBII and Gln-1615 in rH1 at the extracellular loop of D4S3-S4 is crucial for the action of beta -PMTX. PMTXs, which are small peptides with 13 amino acids, would be a potential tool for exploring a new functional moiety of Na+ channels.

Features of myoneural junctions in the extraocular muscles of the opossum (Didelphis albiventris)

The myoneural junctions present in the opossum (Didelphis albiventris) extraocular muscles were studied through histochemical and ultrastructural techniques. Three different types of junction were shown: two single types, 'en grappe' and 'en plaque', present in the middle third of the muscle fibers; and one multiple type, more rare and observed in the distal third of the muscle.

Morphology and histochemistry of the hyolingual apparatus in chameleons

We reexamined the morphological and functional properties of the hyoid, the tongue pad, and hyolingual musculature in chameleons. Dissections and histological sections indicated the presence of five distinctly individualized pairs of intrinsic tongue muscles. An analysis of the histochemical properties of the system revealed only two fiber types in the hyolingual muscles: fast glycolytic and fast oxidative glycolytic fibers. In accordance with this observation, motor-endplate staining showed that all endplates are of the en-plaque type. All muscles show relatively short fibers and large numbers of motor endplates, indicating a large potential for fine muscular control. The connective tissue sheet surrounding the entoglossal process contains elastin fibers at its periphery, allowing for elastic recoil of the hyolingual system after prey capture. The connective tissue sheets surrounding the m. accelerator and m. hyoglossus were examined under polarized light. The collagen fibers in the accelerator epimysium are configured in a crossed helical array that will facilitate limited muscle elongation. The microstructure of the tongue pad as revealed by SEM showed decreased adhesive properties, indicating a change in the prey prehension mechanics in chameleons compared to agamid or iguanid lizards. These findings provide the basis for further experimental analysis of the hyolingual system. © 2001 Wiley-Liss, Inc.

Histochemical and SEM evaluation of the neuromuscular junctions from alcoholic rats

In the present study morphological changes occurring in the neuromuscular junctions (NMJ) of the extensor digitorum longus (EDL) and soleus muscles from albino rats (Rattus norvegicus) submitted to experimental chronic alcoholism were evaluated. Seventy two male animals aged 4 months and weighing on average 400 g were divided into three groups: control, alcoholic and isocaloric. Six rats from each group were anesthetized and sacrificed after 5, 10, 15 and 18 months. The NMJ did not show detectable morphological changes in either muscle after treatment when examined by light microscopy. With respect to the dimensions, statistical analysis demonstrated a tendency to a statistically significant treatment x time interaction for the length of soleus muscle NMJ. The ultrastructural study, however, revealed that the NMJ of the soleus muscle of animals submitted to 18 months of experimental alcoholism presented important morphological alterations. Characteristically, the NMJ of these muscles is located on an elevation on the surface of the muscle fiber, presenting a regular round, oval or elliptical shape and continuous and not very deep synaptic grooves. Approximately 30% of the NMJ of alcoholic rats are irregular in shape, with the sarcolemmal elevations typical of the synapse region being flattened on at least one side, with discontinuous synaptic grooves, and deep and punctiform contacts of the synaptic buds. These data suggest that, although skeletal muscle has a greater natural resistance against the direct or indirect effects of alcohol, some submicroscopic morphological alterations are detectable in the NMJ, especially in muscles with oxidative metabolism (soleus) following long periods of ingestion of alcohol. (C) 2002 Elsevier B.V. Ltd. All rights reserved.

The tof-guard neuromuscular transmission monitor and its use in horses

Quando da utilização de bloqueadores neuromusculares, já foi enfatizado na literatura médica humana, que é de vital imporância a monitoração do bloqueio neuromuscular e que estes agentes nunca devem ser utilizados sem a mesma. O objetivo deste estudo foi o de avaliar o uso do monitor da transmissão neuromuscular TOF-Guard em eqüinos. Para tanto, doze eqüinos foram separados aleatoriamente para receberem como bloqueadores neuromusculares o pancurônio ou o atracúrio. Todos os eqüinos foram pré-medicados com romifidina, induzidos com diazepam e quetamina e mantidos com halotano. Foi administrado o atracúrio ou o pancurônio, seguindo-se a apnéia e início da ventilação mecânica controlada. O tempo entre a administração do relaxante muscular e a obtenção de um bloqueio máximo (T1=0), o retorno do T1 para 25% e da razão do TOF para 0,7 e o tempo do retorno do T1 de 25 para 75% foram mensurados. Concluiu-se que é de grande importância a monitoração do bloqueio neuromuscular quando da utilização de um bloqueador neuromuscular não-despolarizante, uma vez que ela torna o ato anestésico e cirúrgico mais seguro, com a utilização de doses adequadas que inviabilizam a superficialização do bloqueio durante o procedimento cirúrgico. O monitor TOF-Guard mostrou ser uma boa opção para a monitoração do bloqueio neuromuscular em eqüinos.

Ultrastructural Study of the Neuromuscular Junctions of Extensor Digitorum Longus Muscle (M. extensor digitorum longus) of the Mice

The neuromuscular junction of the extensor digitorum longus muscle of fingers was analyzed in 21 young (three months) and old (from six to 25 months) mice, from both genders. Morphologic changes were found throughout the mouse life, being more frequent and visible with aging. According with the data described in the literature consulted and the observations taken in this research, it becomes clear that a continuous process of morphological remodeling occurs in all neuromuscular ultrastructural junctions of the extensor digitorum longus muscle of fingers, during the life of the animal. Theses changes are characterized by figures of myelin in the cytoplasm of Schwann cells, pleomorphic and multivesiclar bodies, mitochondrias with morphologically altered crests in the axon terminal and degenerated junction folders. Coated vesicles are common in older animals and rare in young animals.

Effects of two proprioceptive neuromuscular facilitation techniques in different planes on hamstrings muscles of healthy subjects

Introduction: The proprioceptive neuromuscular facilitation technique (PNF) has been proven to be efficient, since it was found higher gain of joint range-of-motion compared to the classic stretching. This study aimed to perform a comparison between the muscular stretching techniques and the PNF hold-relax on the internal and external sagittal/diagonal plane.Method: Randomly divided in 3 groups by a drawing, 30 healthy male individuals have undergone the test. In group I the hold-relax technique was utilized on the sagittal plane, grupo II receveid hold-relax on the internal and external diagonals, and group III, on which an evaluation was performed, worked as control. All the groups went through tests on the first, fifth and fifteenth day after the application of the different approaches. In this evaluation it was used a Flexis (R) Fleximeter.Result: Group II (diagonal) obtained statistically significant gain of 13.99% in the immediate post-test and post test later obtained a loss of 4.81%, group I (sagittal) showed no statistical difference as the group III (control).Conclusion: We conclude that the technique of PNF in the diagonal plane is effective in the flexibility of the hamstring muscles.

Anestesia peribulbar com ropivacaína como alternativa ao bloqueio neuromuscular para facectomia em cães

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Morphometric and morphological analysis of neuromuscular junction alterations in the denervated rat diaphragm

The morphological and structural alterations that occur in the neuromuscular junctions of the denervated rat diaphragm were studied. Fifteen adult male albino rats (Rattus norvegicus) aged about 60 days and with a mean weight of 200 g were used. Chronically denervated diaphragms were obtained and the animals were sacrificed after 4, 8 and 12 weeks of denervation. The left antimere of the diaphragm was denervated by section of the phrenic nerve and the right antimere was used as control. Each antimere was divided into three fragments: one was used for histochemical (nonspecific esterase) and morphometric study of neuromuscular junctions, and the other two were used for transmission and scanning electron microscopy (SEM) analysis. Histochemical analysis of the diaphragm neuromuscular junctions after denervation showed only small changes in junction morphology. However, these junctions became smaller and elongated and presented less visible contours with increasing time of denervation. Ultrastructural analysis of neuromuscular junctions after 12 weeks showed more or less organized junctional folds on the muscle fiber surface. The junctional cytoplasm exhibited important alterations such as mitochondrial degeneration and the presence of numerous filaments. SEM revealed the presence of deep primary synaptic grooves with peripheral excavations which housed the nerve terminal boutons and exhibited internally the secondary synaptic clefts present among the junctional folds of the sarcolemma. This study showed that some of the morphological changes demonstrated in other denervated striated skeletal muscles are not repeated at the same intensity or in the same temporal pattern in the rat diaphragm.

Paralyzing and myotoxic effects of a recombinant bothropstoxin-1 (BthTX-I) on mouse neuromuscular preparations

As a first step to investigate the structure-function relationship of bothropstoxin-1 (BthTX-1), a myotoxin from Bothrops jararacussu snake venom, Our group previously cloned a recombinant toxin (rBthTX-1) in Escherichia coli. The aim or this work was to characterize the biological activities of this rBthTX-1 (1.0 mu M) in both phrenic-diaphragm and extensor digitorum longus preparations in vitro, by means of myographic and morphologic techniques. Native BthTX-1 (1.0 mu M) was used as a standard. The influence of heparin (27.5 mu g/ml) upon the biological activities of both toxins was also investigated. rBthTX-1 had similar effects to the native toxin inducing blockage of both directly and indirectly evoked contractions in phrenic-diaphragm preparations, and muscle damage characterized by edema, round fibers, and cell areas devoid of myofibrils. Interestingly the paralyzing activity of rBthTX-1 was slightly more potent than the native toxin. Heparin prevented paralyzing and myotoxic effects of both the native and recombinant toxins. This work shows that rBthTX-1 was expressed in a fully active form, and presents a biological profile similar to the native toxin. (c) 2005 Elsevier GmbH All rights reserved.

Reduction of crotoxin-induced neuromuscular blockade by gamma radiation

A comparative study between crotoxin and gamma irradiated crotoxin was performed on the indirectly evoked twitches and tetani of sciatic nerve-extensor digitorum longus muscle of rats. Crotoxin (3 to 14 mu g/ml) decreased the amplitude of twitches and induced a slight tetanic fade, and irradiated crotoxin did not significantly affect either twitch amplitude or tetanic tension. Since gamma radiation reduced the neurotoxicity of crotoxin it may be useful for the production of anticrotalic serum. (C) 1998 Elsevier B.V. Ltd. All rights reserved.

Neutralization of snake venom phospholipase A(2) toxins by aqueous extract of Casearia sylvestris (Flacourtiaceae) in mouse neuromuscular preparation

Aqueous extract of Casearia sylvestris (Flacourtiaceae) has been shown to inhibit enzymatic and biological properties of some Bothrops and Crotalus venoms and their purified phospholipase A(2) (PLA(2)) toxins. In this work we evaluated the influence of C sylvestris aqueous extract upon neuromuscular blocking and muscle damaging activities of some PLA(2)S (crotoxin from C. durissus terrificus, bothropstoxin-I from B.jararacussu, piratoxin-I from B. pirajai and myotoxin-II from B. moojeni) in mouse phrenic-diaphragm preparations. Crotoxin (0.5 mu M) and all other PLA2 toxins (1.0 mu M) induced irreversible and time-dependent blockade of twitches. Except for crotoxin, all PLA2 toxins induced significant muscle damage indices, assessed by microscopic analysis. Preincubation of bothropstoxin-I, piratoxin-I or myotoxin-II with C. sylvestris extract (1:5 (w/w), 30 min, 37 degrees C significantly prevented the neuromuscular blockade of preparations exposed to the mixtures for 90 min; the extent of protection ranged from 93% to 97%. The vegetal extract also neutralized the muscle damage (protection of 80-95%). Higher concentration of the C. sylvestris extract (1: 10, w/w) was necessary to neutralize by 90% the neuromuscular blockade induced by crotoxin. These findings expanded the spectrum of C. sylvestris antivenom activities, evidencing that it may be a good source of potentially useful PLA2 inhibitors. (c) 2007 Elsevier B.V.. All rights reserved.