Insights of the effects of polyethylene glycol 400 on mammalian and avian nerve terminals.

Polyethylene glycol (PEG) has been widely used as a solvent among other applications. An ideal solvent is one that does not interfere with an in vitro biological system, unless it is a bioactive agent. Herein, a facilitatory neurotransmission effect was exhibited by PEG (20 microM) in mammalian (67 +/- 12.5%, n = 4) and avian (74 +/- 6.8%, n = 6) neuromuscular preparations. In curarized preparations, PEG did not reverse the neurotransmission blockade induced by D-tubocurarine (D-Tc, 5.8 microM, n = 6) as promoted by neostigmine (12 microM, n = 4). A possible presynaptic action of PEG was ruled out, because quantal acetylcholine (ACh) content was similar to the control Tyrode-incubated mammalian preparation. PEG showed improved sarcolemmal sensitivity, both under direct (sarcolemma) and indirect stimulation (motor axon), because it was able to release calcium from the sarcoplasmic reticulum, even when 30 microM dantrolene (n = 5) was previously applied. Neurotransmission decreased at a higher PEG concentration (100 microM, n = -6) in the depolarized membrane, but it did not alter normal muscle fiber morphology. In addition, it partially recovered twitch tension amplitude (55 +/- 5.7%) after washing the preparations. More than a simple solvent, we suggest that PEG 400 is able to act on the sarcolemmal membrane, probably at the triad level, which is in line with its well-known ability as drug carrier.

Pharmacological and structural characterization of a novel phospholipase A2 from Micrurus dumerilii carinicauda venom.

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.

Neuromuscular action of Bothrops lanceolatus (Fer de lance) venom and a caseinolytic fraction.

A protein capable of inducing neuromuscular blockade in avian preparations and of depolarizing mouse diaphragm muscle was isolated from Bothrops lanceolatus venom using gel filtration and ion-exchange chromatography. The purified protein was a single chain polypeptide with an estimated molecular mass of 27.5 kDa by SDS-PAGE and had caseinolytic activity (13.3 units/mg), but no phospholipase A(2). B.lanceolatus venom (50 micro g/ml) and the caseinolytic protein (20 micro g/ml) produced contracture and progressive irreversible blockade (50% in 25+/-5 min (SEM) and 45+/-15 min, respectively), in indirectly stimulated chick biventer cervicis preparations. The contractile responses to acetylcholine (ACh; 37 and 74 micro M, n=6) were inhibited by venom and the caseinolytic protein, whereas those to potassium (13.4mM, n=6) were not. Membrane resting potential measurements in mouse hemidiaphragm preparations showed that B.lanceolatus venom and the purified protein caused depolarization which was prevented by D-tubocurarine (14.6mM). The venom produced a slight increase in the amplitude and frequency of miniature end-plate potentials, but this effect was not seen with the purified fraction. These results suggest that the purified protein acts exclusively post-synaptically.

The presynaptic activity of bothropstoxin-I, a myotoxin from Bothrops jararacussu snake venom.

Bothropstoxin-I from Bothrops jararacussu snake venom is a lysine-49 phospholipase A(2) with myotoxic and neurotoxic activities. In this study, we used mouse phrenic nerve-diaphragm preparations in the absence and presence of manganese (Mn(2+)), a presynaptic blocker, to investigate a possible presynaptic action of bothropstoxin-I. At concentrations of 0.9 mM and 1.8 mM, Mn(2+) produced 50% neuromuscular blockade in less than 4 min., which was spontaneously reversible at the lower concentration. Bothropstoxin-I (1.4 microM) irreversibly inhibited neuromuscular blockade by 50% in 31+/-4 min. (mean+/-S.E.M., n = 9). Pretreating preparations with 0.9 mM Mn(2+) prevented the blockade by bothropstoxin-I. When added after bothropstoxin-I, Mn(2+) produced its characteristic blockade and, after washing, the twitch tension returned to pre-Mn(2+) levels, indicating that bothropstoxin-I caused irreversible damage before the addition of Mn(2+). Electrophysiological measurements showed that a concentration of bothropstoxin-I (0.35 microM), which did not produce neuromuscular blockade, caused the appearance of giant miniature end-plate potentials with no change in the membrane resting potential but increased the quantal content. Preparations preincubated with Mn(2+) (0.9 mM, 30 min.) were protected against the depolarizing action of bothropstoxin-I (0.7 microM). These results show that, in addition to its well-known myotoxic effect, bothropstoxin-I also has a presynaptic action.

Heparin at low concentration acts as antivenom against Bothrops jararacussu venom and bothropstoxin-I neurotoxic and myotoxic actions.

Heparin has been shown to antagonize myotoxic effects of crotaline venoms. Here a very low heparin concentration (LHC) was examined in its ability to antagonize the neurotoxic/myotoxic effects of Bothrops jararacussu venom and its phospholipase A(2) myotoxin, bothropstoxin-I (BthTX-I), in an in vitroz nerve-muscle preparation and in mice gastrocnemius. Normalization of results was done by assays with commercial antibothropic antivenom (CBA). LHC (1IU/ml) added to the incubation bath reduced by 4- and 4.5-fold (vs 2.8- and 2.5-fold by CBA) the neuromuscular paralysis, by 5.4 and 4.4-fold (vs 2.5- and 13.3-fold by CBA) the percentage of fibers damaged and by 6- and 1.7-fold (vs 30- and 1.6-fold by CBA) the CK activity induced by B. jararacussu and BthTX-I, respectively. Protamine sulphate added 15min after the incubation of the preparation with LHC+venom, avoided the LHC neutralizing effect against venom neurotoxicity. This strongly attests that given the polycationic nature of protamine, it probably complexed with the polyanionic heparin making it unattainable for binding to basic components of venom, reducing toxicity. Since heparin antagonism is generally stronger against venom effects than is myotoxin we discuss that other venom components than the BthTX-I are likely target for the antagonism promoted by the polyanionic heparin.

Neurotoxicity of Micrurus altirostris (Uruguayan coral snake) venom and its neutralization by commercial coral snake antivenom and specific antiserum raised in rabbits.

In this work, we studied the neuromuscular blockade caused by Micrurus altirostris venom (0.1-10 microg/mL) in indirect stimulated chick biventer cervicis and mouse phrenic nerve-diaphragm preparations and the ability of commercial antivenom (Instituto Butantan) and antiserum raised in rabbits to neutralize neurotoxicity and lethality in chicks and mice (LD(50) 0.042 and 0.255 mg/kg), injected i.m. and i.p., respectively, with venom (5 LD(50)):antivenom or antiserum mixtures (n = 6) of 1:1-1:2.5-1:5-1:10-1:20. The venom caused a complete and irreversible neuromuscular blockade in both preparations, inhibited the acetylcholine and carbachol contractures, without interfering on KCl response. The neuromuscular blockade was not Ca(2+) or temperature-dependent and did not affect the response to direct stimulation. Only a venom:antivenom or antiserum ratio of 1:20 neutralized the neuromuscular blockade in vitro and protected chicks and mice against 5 LD(50) of venom. Our results indicated that Micrurus altirostris venom interferes with postsynaptic neurotransmission and that commercial antivenom and rabbit antiserum have low efficacy in neutralizing the neurotoxicity and lethality of this venom.

Actions of Androctonus australis and Leiurus quinquestriatus venoms in the rat isolated atria and anesthetized rats; effect of magnesium and lidocaine.

In rat isolated atria, Androctonus australis (Aa), Leiurus quinquestriatus quinquestriatus (Lqq), and L. q. hebraceus (Lqh) venoms produced intense contracture, alterations in the force and frequency of the spontaneous atrial contractions, and delayed afterdepolarizations (DAD). It was shown by means of tetrodotoxin-induced blockade of neurotransmitter release that the contracture and DAD were produced by the action of the venoms on the atrial cell membrane (direct action) while alterations in the force and frequency of the spontaneous atrial contractions were caused by acetylcholine and norepinephrine released by the venoms (indirect action). The irregularities in the spontaneous contractions and DAD were suppressed by magnesium and lidocaine which, however, caused only a small reduction in the intensity of the atrial contracture. The venom-induced DAD was also abolished by ryanodine and intensified by an increase in [Ca2+]0. In anesthetized rats, Aa, Lqq, and Lqh venoms induced hypertension, arrhythmias, and T wave inversion. The arrhythmias included bradycardia, ventricular and supraventricular extrasystoles, unsustained and sustained ventricular tachycardia with torsade de pointes episodes. Magnesium and lidocaine abolished them, and magnesium also counteracted the hypertension. These results suggest that magnesium and lidocaine, particularly magnesium, may be useful in the treatment of the arrhythmias, hypertension, and other disorders associated with Buthinae scorpion envenomation.

New evidence for a presynaptic action of prednisolone at neuromuscular junctions.

The action of prednisolone at the neuromuscular junction was studied in mouse isolated phrenic nerve-diaphragm and rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. Prednisolone (0.03 mM and 0.3 mM) did not alter the twitch-tension in phrenic nerve-diaphragm preparations after 120 min, but increased the frequency (170 +/- 4%) and amplitude (200 +/- 13%) of miniature end-plate potentials. Quantal content was not influenced by the glucocorticoid treatment. Prednisolone (400 microg/kg) did not change the twitch-tension in rat external popliteal/sciatic nerve-tibialis anterior muscle preparations. However, this steroid (0.3 mM) prevented the neuromuscular blockade by d-tubocurarine (1.45 microM) in mouse preparations by 70 +/- 10% (P < 0.05). A similar effect (82 +/- 6% protection, P < 0.05) occurred in rats treated with prednisolone (400 microg/kg) before d-tubocurarine (225 microg/kg). In phrenic nerve-diaphragm preparations, prednisolone (0.3 mM) increased (13 +/- 4%, p < 0.05) the twitch-tension in the presence of beta-bungarotoxin (1 microM), and prevented the blockade produced by this toxin (0.15 microM) in its third phase of action. This presynaptic facilitatory effect may contribute to the usefulness of prednisolone in myasthenia gravis.

A study of the myotoxicity of bothropstoxin-i using manganese in mouse phrenic nerve-diaohragm and extensor digitorum longus preparations

Bothropstoxin-I (BthTX-I) from Bothrops jararacussu snake venom has a predominantly postsynaptic action that is responsible for this toxin´s myotoxicity. However, BthTX-I also has a presynaptic action that is counteracted by Mn2+, a reversible neuromuscular blocker that acts predominantly presynaptically. In this work, we used two nerve-muscle preparations (mouse phrenic nerve-diaphragm - PND and extensor digitorum longus - EDL) to investigate the ability of Mn2+ to protect against the myotoxicity of BthTX-I. The preparations were incubated with Tyrode solution (control), BthTX-I, or Mn2+ alone. BthTX-I (1.4 µM) produced irreversible blockade in both preparations, whereas the blockade by Mn2+ (0.9 mM) was total and reversible in PND but just partially reversible in EDL. Pretreating the preparations with Mn2+ resulted in 100% and 80% protection against BthTX-I-induced blockade, respectively. However, when Mn2+ (0.9 or 1.8 mM) and BthTX-I (1.4 µM) were co-incubated for 30 min before testing, the blockade was faster and sustained. Washing the preparations resulted in complete, sustained recovery in those exposed to 1.8 mM Mn2+ but not to 0.9 mM Mn2+. Morphological analysis showed that the extent of fiber damage by BthTXI (1.4 µM) was 82% (PND) and 68.5% (EDL), and that Mn2+ (0.9 mM) afforded 40% protection in both preparations and reduced the increase in muscle fiber cross-sectional area by 20% and 15%, respectively, compared to BthTX-I alone. Mn2+ (0.9 mM) significantly attenuated the release of creatine kinase by BthTXI. The low creatine kinase activity resulted from a protective action of Mn2+ on the sarcolemma and from direct inactivation of the released enzyme. These results show that Mn2+ prevents membrane disruption by BthTX-I and can protect against the myotoxicity and neurotoxicity caused by this toxin.

Modo de açäo da peçonha da aranha armadeira, Phoneutreia nigriventer (Keyserling, 1891) nas aurículas isoladas de cobaia / Mode of action of phoneutria nigriventer (Keyserling, 1891), on isolated guinea-pig auricles

Nos acidentes causados no homem pela aranha armadeira, observam-se, a par de outros efeitos, pertubaçöes cardíacas, caracterizadas por taquicardia e arritmias. Este fato sugeriu o estudo das açöes cardíacas da peçonha. Na presente pesquisa, investigaram-se os seus efeitos e mecanismo de açäo nas aurículas isoladas de cobaia. A peçonha nas concentraçöes de 1 microng/ml e de 5 microng/ml produziu inicialmente efeitos cronotrópico negativos, seguidos de aumento da freqüência e tensäo das contraçöes auriculares espontâneas. Os efeitos conotrópico e inotrópico negativos foram potencializados pela neostigmina, abolidos pela atropina e grandemente reduzidos pelo hemicolínio. O propranolol e o bretílio bloquearam o aumento da freqüência e da tensäo das contraçöes de aurículas atropinizadas, induzido pela peçonha. Em aurículas atropinizadas provenientes de cobaias reserpinizadas, a peçonha näo produziu efeitos cronotrópicos e inotrópico positivo. A retrodotoxina, em concentraçäo que reduz apenas ligeiramente (23,6 ñ 6,2%) a tensäo das contraçöes auriculares, aboliu os efeitos da peçonha sobre o cronotropismo e inotropismo. Estes resultados mostram que os efeitos da peçonha de P. nigriventer nas aurículas de cobaia säo produzidos através de liberaçäo dos neuro-transmissores acetilcolina e noradrenalina pelas terminaçöes nervosas autonômicas e que essa liberaçäo decorre de açäo da peçonha nos canais do sódio da membrana das terminaçöes nervosas

O íon magnésio no tratamento de acidentes causados pelos escorpiöes Tityus serrulatus, T. bahiensis e Centruroides sculpturatus: estudo experimental / Magnesium in the treatment of accidents caused by the scorpions Tityus serrulatus, T. bahiensis and Centruroides sculpturatus: an experimental study

Os efeitos produzidos pelas peçonhas escorpiônicas säo consequentes, em sua maioria, à liberaçäo de acetilcolina (ACh) e catecolaminas. A verificaçäo de que o magnésio (Mg2+) inibe a liberaçäo de ACh em razäo de bloquear o influxo de cálcio nas terminaçöes nervosas, levou-nos a investigar a açäo deste cátion sobre os distúrbios produzidos pelas peçonhas escorpiônicas. Relatamos na presente comunicaçäo a açäo do Mg2+ sobre os efeitos induzidos pelas peçonhas dos escorpiöes Tityus serrulatus, T. bahiensis e Centruroides sculpturatus nas preparaçöes isoladas nervo frênico-diafragma, íleo, canal deferente e átrios de rato e in vivo, em ratos anestesiados com registro da pressäo arterial e do eletrocardiograma. Os efeitos da peçonha dos escorpiöes nas preparaçöes isoladas foram abolidos ou muito atenuados pelo Mg2+. O Mg2+, no entanto, somente antagonizou os efeitos da peçonha de C. sculpturatus no íleo de rato. Em ratos anestesiados, a hipertensäo e arritmias provocadas pela peçonha de T. serrulatus foram revertidas com exclusäo de bradicardia pela injeçäo do Mg2+. A peçonha de C. sculpturatus na maioria das experiências causou hipotensäo e arritmias de pequena gravidade. O Mg2+ reverteu as arritmias, mas causou quedas acentuadas da pressäo arterial. Os resultados da pesquisa sugerem o emprego do Mg2+ em acidentes graves na ausência de hipotensäo e bradicardia, produzidos por T. serrulatus e T. bahiensis. Parece contra-indicado nos acidentes causados por C. sculpturatus em vista de seu efeito acima referido na pressäo arterial.