Phrenic Nerve-diaphragm Research Articles

Presynaptic neuromuscular action of a methanolic extract from the venom of Rhinella schneideri toad

BackgroundRhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity.MethodsThe neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording – in response to indirect stimulation – and for electrophysiological measurements.ResultsVenom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively).ConclusionThese results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.

Effect of experimental hyperthyroidism on the maximum acute exercise tolerance and neuromuscular performance in rats

Background Thyroid hormones regulate many physiologic functions such as energy and heat production, synthesis of proteins, and muscle contraction. Objective This study aims to demonstrate the mechanism by which hyperthyroidism affects neuromuscular performance and acute exercise tolerance and whether this effect is reversible or not after recovery in rats. Materials and methods Rats used in this study were divided into three groups, each containing 10 rats: (a) in the euthyroid group, rats were selected by measurement of serum FT3 (triiodothyronin), FT4 (tetraiodothyronine), and thyroid-stimulating hormone levels. (b) In the hyperthyroid group, hyperthyroidism was induced by Eltroxin in increasing doses, beginning from 50 μg to reach 200 μg/kg body weight daily by intragastric administration for 3 weeks. (c) In the recovery group, hyperthyroid rats are allowed to recover by exogenous thyroxine withdrawal for 25 days. After induction, body weight and the maximal swimming time were estimated, and then retro-orbital blood samples were collected for estimation of malondialdehyde and the total antioxidant capacity. Rats were then killed by cervical decapitation. Phrenic nerve diaphragm preparations were excised, connected to a four-channel oscillograph for measuring the strength of muscle contraction at the start of the experiment and after 30 min activity using both direct and indirect stimulation. Glucose uptake by the muscle during 30 min of activity and 30 min of recovery was estimated. Results The data show that hyperthyroidism induced by Eltroxin elevates serum malondialdehyde significantly when compared with the euthyroid group, whereas reduces total antioxidant capacity significantly. Hyperthyroidism was also associated with reduction of body weight, the maximal swimming time, the strength of skeletal muscle contraction induced by direct and indirect repetitive stimulation, and its glucose uptake. Conclusion Oxidative stress plays a critical role in the pathogenesis of hyperthyroidism, especially on skeletal muscle performance and exercise tolerance. This effect is reversible after restoration of the euthyroid state.

Paradoxical neostigmine-induced TOFfade: On the role of presynaptic cholinergic and adenosine receptors

Neuromuscular transmission is clinically monitored using the train-of-four ratio (TOFratio), which is the quotient between twitch tension produced by the fourth (T4) and by the first (T1) stimulus within a train-of-four stimulation at 2Hz. Neostigmine causes a paradoxical depression of the TOFratio (TOFfade) that is amplified by intra-arterial atropine in cats. This led us to question the usefulness of the TOFratio as a sole testing element to control neostigmine-induced reversal of neuromuscular transmission block caused by non-depolarizing agents. We hypothesized that the inhibition of cholinesterase activity might increase acetylcholine bioavailability and consequently cholinoceptor activation, leading to concomitant adenosine release from nerve endings and skeletal muscle fibers. The involvement of presynaptic muscarinic and adenosine receptors in neostigmine-induced TOFfade in the rat phrenic nerve diaphragm was investigated. Blockade of adenosine A2A receptors with ZM241385 and of muscarinic M2 receptors with methoctramine or atropine amplified neostigmine-induced TOFfade. Notwithstanding TOFfade amplification, the blockade of M2 or A2A receptors increased both T1 and T4 twitch tensions above control during the first 3min of neostigmine application. Beyond that period, the T1 twitch tension returned to baseline, whereas T4 decreased further until the control value with neostigmine alone. Blockade of M1 receptors by pirenzepine did not change neostigmine-induced TOFfade, unless A2A receptors were concurrently blocked with ZM241385. Data indicate that the paradoxical neostigmine-induced fade involves presynaptic mechanisms that regulate transmitter release and synaptic adenosine accumulation, including the activation of adenosine A2A and muscarinic M2 receptors.

Inosine induces presynaptic inhibition of acetylcholine release by activation of A3 adenosine receptors at the mouse neuromuscular junction

The role of inosine at the mammalian neuromuscular junction (NMJ) has not been clearly defined. Moreover, inosine was classically considered to be the inactive metabolite of adenosine. Hence, we investigated the effect of inosine on spontaneous and evoked ACh release, the mechanism underlying its modulatory action and the receptor type and signal transduction pathway involved. End-plate potentials (EPPs) and miniature end-plate potentials (MEPPs) were recorded from the mouse phrenic-nerve diaphragm preparations using conventional intracellular electrophysiological techniques. Inosine (100 μM) reduced MEPP frequency and the amplitude and quantal content of EPPs; effects inhibited by the selective A3 receptor antagonist MRS-1191. Immunohistochemical assays confirmed the presence of A3 receptors at mammalian NMJ. The voltage-gated calcium channel (VGCC) blocker Cd(2+) , the removal of extracellular Ca(2+) and the L-type and P/Q-type VGCC antagonists, nitrendipine and ω-agatoxin IVA, respectively, all prevented inosine-induced inhibition. In the absence of endogenous adenosine, inosine decreased the hypertonic response. The effects of inosine on ACh release were prevented by the Gi/o protein inhibitor N-ethylmaleimide, PKC antagonist chelerytrine and calmodulin antagonist W-7, but not by PKA antagonists, H-89 and KT-5720, or the inhibitor of CaMKII KN-62. Our results suggest that, at motor nerve terminals, inosine induces presynaptic inhibition of spontaneous and evoked ACh release by activating A3 receptors through a mechanism that involves L-type and P/Q-type VGCCs and the secretory machinery downstream of calcium influx. A3 receptors appear to be coupled to Gi/o protein. PKC and calmodulin may be involved in these effects of inosine.

Population Divergence in Venom Bioactivities of Elapid Snake Pseudonaja textilis: Role of Procoagulant Proteins in Rapid Rodent Prey Incapacitation

This study looked at how toxic proteins in venoms of adult Australian eastern Brown snakes Pseudonaja textilis from South Australian and Queensland populations interact with physiological functions of the lab SD rat Rattus norvegicus. Circulatory collapse and incoagulable blood occurred instantly after injection of venom under the dorsal skin of anaesthetised and mechanically ventilated rats in an imitation of a P. textilis bite. Intravenous injection of purified P. textilis (Mackay, QLD) venom prothrombin activator proteins caused instant failure of circulation, testifying of high toxicity of these proteins and suggesting their role in rapid incapacitation of rodent prey. The hypothesis is further supported by circulatory collapse occurring instantly despite artificial respiration in envenomed rats and the finding of extremely high venom procoagulant potency in rat plasma. LC-MS and physiology assays revealed divergent venom composition and biological activity of South Australian (Barossa locality) and Queensland (Mackay locality) populations, which may be driven by selection for different prey. The Queensland venom of P. textilis was found to be more procoagulant and to exhibit predominately presynaptic neurotoxicity, while the South Australian venom contained diverse postsynaptic type II and III α-neurotoxins in addition to the presynaptic neurotoxins and caused significantly faster onset of neuromuscular blockade in the rat phrenic nerve-diaphragm preparation. LC-MS analysis found evidence of multiple coagulation factor X-like proteins in P. textilis venoms, including a match to P. textilis coagulation factor X isoform 2, previously known to be expressed only in the liver.

Effects of rocuronium and vecuronium on initial rundown of endplate potentials in the isolated phrenic nerve diaphragm preparation of rats.

Rocuronium and vecuronium, two non-depolarizing neuromuscular blockers, have been widely used in surgery procedures. However, their electrophysiological properties need to be more widely explored. We examined the effects of rocuronium and vecuronium on initial rundown of endplate potential amplitudes in the non-uniform stretched muscle preparation of the rat isolated phrenic nerve diaphragm. More specifically, the endplate potentials were recorded with one microelectrode from a single endplate. The effects of rocuronium or vecuronium each at 4 concentrations (0.5 ×, l ×, 2 ×, 4 × EC95; EC95 = concentration of the drug required to produce the inhibitory effect by 95%) on the amplitude of endplate potentials and its rundown were observed. Treatment of the isolated rat phrenic nerve-diaphragm preparation with rocuronium (2.5–20 μg/ml) or vecuronium (0.5–4 μg/ml) decreased the amplitude of endplate potentials and inhibited its rundown in a concentration-dependent manner. At the concentration (2.5 μg/ml for rocuronium and 0.5 μg/ml for vecuronium) that did not alter the endplate potential amplitude, the onset of reduced endplate potential rundown was 3 and 5 min after administration of rocuronium or vecuronium, respectively. The results suggest that rocuronium and vecuronium block the neuromuscular junction presynaptically and that rocuronium does it faster than vecuronium.

Species differences in the neuromuscular activity of post-synaptic neurotoxins from two Australian black snakes (Pseudechis porphyriacus and Pseudechis colletti)

Bites by Australian black snakes (Pseudechis spp.) do not cause neurotoxicity in human envenoming. This is unusual as in vitro neurotoxicity has been reported for all Pseudechis spp. venoms. The present study aimed to identify, isolate and characterise neurotoxins from the venoms of Pseudechis porphyriacus and Pseudechis colletti to elucidate the reason for the lack of neurotoxicity in humans. α-Elapitoxin-Ppr1 and α-elapitoxin-Pc1 were isolated from P. porphyriacus and P. colletti, respectively, using reverse-phase high performance liquid chromatography. Each toxin consisted of 62 amino acids with molecular weights of 6746.5Da and 6759.6Da, respectively. α-Elapitoxin-Ppr1 and α-elapitoxin-Pc1 caused concentration-dependent (0.1–0.3μM) inhibition of indirect twitches in the chick biventer cervicis nerve-muscle preparation. Both toxins inhibited contractile responses to exogenous ACh and CCh, but not KCl, suggesting a post-synaptic mode of action at the nicotinic acetylcholine receptor (nAChR). CCh concentration–response curves obtained in the presence or absence of α-elapitoxin-Ppr1 or α-elapitoxin-Pc1 indicated pA2 values of 6.97±0.03 and 7.04±0.07, respectively. Neither α-elapitoxin-Ppr1 (0.1μM) nor α-elapitoxin-Pc1 (0.1μM) had a significant effect on the electrically-induced twitches of the rat isolated phrenic nerve-diaphragm preparation. When the venom with the toxin removed (10μg/ml) was added to both the rat and chick preparations, the inhibition was significantly less than that caused by the intact whole venoms (10μg/ml). The current study shows that α-elapitoxin-Ppr1 and α-elapitoxin-Pc1 act as pseudo-irreversible antagonists at the nAChR of the skeletal neuromuscular junction and that the avian preparation is more sensitive to the neurotoxic effects of these toxins than the mammalian preparation.

Pharmacological study of a new Asp49 phospholipase A2 (Bbil-TX) isolated from Bothriopsis bilineata smargadina (forest viper) venom in vertebrate neuromuscular preparations

The neuromuscular activity of Bbil-TX, a PLA2 with catalytic activity isolated from Bothriopsis bilineata smargadina venom, was examined in chick biventer cervicis (BC) and mouse phrenic nerve–diaphragm (PND) preparations. In BC preparations, Bbil-TX (0.5–10 μg/ml) caused time- and concentration-dependent blockade that was not reversed by washing; the times for 50% blockade were 87 ± 7, 41 ± 7 and 19 ± 2 min (mean ± SEM; n = 4–6) for 1, 5 and 10 μg/ml, respectively. Muscle contractures to exogenous ACh and KCl were unaffected. The toxin (10 μg/ml) also did not affect the twitch-tension of directly-stimulated, curarized (10 μg/ml) BC preparations. However, Bbil-TX (10 μg/ml) produced mild morphological alterations (edematous and/or hyperchromic fibers) in BC; there was also a progressive release of CK (from 116 ± 17 IU/ml (basal) to 710 ± 91 IU/ml after 45 min). Bbil-TX (5 μg/ml)-induced blockade was markedly inhibited at 22–24 °C and pretreatment with p-bromophenacyl bromide (p-BPB) abolished the neuromuscular blockade. Bbil-TX (3–30 μg/ml, n = 4–6) caused partial time- and concentration-dependent blockade in PND preparations (52 ± 2% at the highest concentration). Bbil-TX (30 μg/ml) also markedly reduced the MEPPs frequency [from 26 ± 2.5 (basal) to 10 ± 1 after 60 min; n = 5; p < 0.05] and the quantal content [from 94 ± 14 (basal) to 24 ± 3 after 60 min; n = 5; p < 0.05] of PND preparations, but caused only minor depolarization of the membrane resting potential [from −80 ± 1 mV (basal) to −66 ± 2 mV after 120 min; n = 5; p < 0.05], with no significant change in the depolarizing response to exogenous carbachol. These results show that Bbil-TX is a presynaptic PLA2 that contributes to the neuromuscular blockade caused by B. b. smargadina venom.

Biotin labeling and receptor analysis of huwentoxin-I

Huwentoxin-I (HWTX-I) is the highest content peptidic neurotoxin in the crude venom of spider Selenocosmia huwena. This toxin blocks the nerve–muscle conduction of isolated phrenic nerve–hemidiaphragm preparation reversibly, with the action site in the presynaptic membrane. In order to further investigate the action mechanism of HWTX-I, the present work optimized the experimental conditions for the biotin labeling of HWTX-I and used the site-selectively biotinylated toxin to affinity purify and identify its receptor proteins from the plasma membrane in rat phrenic nerve–diaphragm synapses. The experiments demonstrated that when the biotinylation reagent reacted with HWTX-I at a molar ratio of 2 : 1 on ice for 2 h, most HWTX-I molecules were labeled by a single biotin group, with a yield of 57%. Tandem mass spectrometry and electrophysiological experiments indicated that the single biotin labeling occurred at the Lys13 residue of HWTX-I and this biotinylation did not significantly affect the bioactivity of HWTX-I. After the affinity purified protein fraction was ultrafiltered, digested with trypsin and analyzed by CapLC-MS/MS, several membrane proteins including voltage-gated calcium channel proteins that HWTX-I potentially interacted with were identified and their roles in the action mechanism of HWTX-I were discussed.

In VitroAntiophidian Mechanisms ofHypericum brasilienseChoisy Standardized Extract: Quercetin-Dependent Neuroprotection

The neuroprotection induced by Hypericum brasiliense Choisy extract (HBE) and its main active polyphenol compound quercetin, against Crotalus durissus terrificus (Cdt) venom and crotoxin and crotamine, was enquired at both central and peripheral mammal nervous system. Cdt venom (10 μg/mL) or crotoxin (1 μg/mL) incubated at mouse phrenic nerve-diaphragm preparation (PND) induced an irreversible and complete neuromuscular blockade, respectively. Crotamine (1 μg/mL) only induced an increase of muscle strength at PND preparations. At mouse brain slices, Cdt venom (1, 5, and 10 μg/mL) decreased cell viability. HBE (100 μg/mL) inhibited significantly the facilitatory action of crotamine (1 μg/mL) and was partially active against the neuromuscular blockade of crotoxin (1 μg/mL) (data not shown). Quercetin (10 μg/mL) mimicked the neuromuscular protection of HBE (100 μg/mL), by inhibiting almost completely the neurotoxic effect induced by crotoxin (1 μg/mL) and crotamine (1 μg/mL). HBE (100 μg/mL) and quercetin (10 μg/mL) also increased cell viability in mice brain slices. Quercetin (10 μg/mL) was more effective than HBE (100 μg/mL) in counteracting the cell lysis induced by Cdt venom (1 and 10 μg/mL, resp.). These results and a further phytochemical and toxicological investigations could open new perspectives towards therapeutic use of Hypericum brasiliense standardized extract and quercetin, especially to counteract the neurotoxic effect induced by snake neurotoxic venoms.

In vitro neurotoxic effects of Pseudechis spp. venoms: A comparison of avian and murine skeletal muscle preparations

Two common in vitro skeletal muscle preparations used for the study of venom neurotoxicity are the indirectly stimulated chick isolated biventer cervicis nerve-muscle preparation and the rat isolated phrenic nerve-diaphragm preparation. The aim of the current study was to compare the in vitro neurotoxicity of six Pseudechis spp. (Black snakes) venoms in both avian (chicken) and mammalian (rat) skeletal muscle preparations to determine differences in sensitivity. All Pseudechis spp. venoms significantly inhibited indirect twitches, in both preparations, indicating the presence of post synaptic neurotoxins. The inhibitory effects of all venoms were more rapid in the avian preparation, except for Pseudechis colletti venom where no significant difference was seen between the murine and avian muscles. Time taken to produce 50% reduction in stimulated twitches (i.e. t50) was markedly shorter in the avian preparation. We have shown that the avian in vitro preparation is more sensitive to the neurotoxic activity of Pseudechis spp. than the murine preparation. This difference is likely to be due to species differences in the interaction between the neurotoxins and the nicotinic receptor binding sites as well as differences in the ‘safety factor’ between the preparations.

Vecuronium suppresses transmission at the rat phrenic neuromuscular junction by inhibiting presynaptic L-type calcium channels

Background and objectivesThe non-depolarizing muscle relaxant vecuronium inhibits contraction by competitive inhibition of postsynaptic acetylcholine receptors (AchRs), which decreases the number of quanta released per impulse in response to 50Hz stimulation. The specific role of calcium influx through L-type calcium channels is the promotion of endocytosis and vesicle recycling during high-frequency stimulation. Vecuronium also induces four pulse tetanic fade, a proxy measure of decreased quanta release. We examined whether vecuronium suppresses neuromuscular transmission during high-frequency stimulation by inhibiting presynaptic L-type calcium channels. MethodsFifty male Sprague-Dawley rats were divided into five treatment groups: unstimulated control group, α-bungarotoxin (BTX) group, nifedipine group, vecuronium group, and nifedipine plus vecuronium group. Rat phrenic nerve–diaphragm neuromuscular juctions were stimulated at 50Hz and field excitatory post-synaptic potentials (fEPSPs) were recorded. Expression levels of the presynaptic Ca2+-binding, protein synaptotagmin 1, and the presynaptic plasma membrane protein, syntaxin 1, were measured by Western blots. ResultsThe fEPSPs evoked by 50Hz stimulus trains were decreased by vecuronium, nifedipine, and by vecuronium plus nifedipine. Nifedipine, an L-type calcium channel blocker, reduced the expression of synaptogamin and syntaxin and blocked the suppressive effect of vecuronium, suggesting that both agents inhibit presynaptic L-type calcium channels. ConclusionsVecuronium which blocked L-type calcium channels may suppress activity of the α3β2 nAChR subunit, which exists in the presynaptic membrane and enhances quantal release. This α3β2 nAChR-mediated positive feedback effect may be facilitated by L-type Ca2+ channel activity under high-frequency stimulation. Vecuronium may disrupt this positive feedback cycle, leading to suppression of fEPSPs. Vercuronium may reduce neuromuscular transmission through presynaptic and postsynaptic mechanisms.

Guanidine affects differentially the twitch response of diaphragm, extensor digitorum longus and soleus nerve-muscle preparations of mice.

Guanidine has been used with some success to treat myasthenia gravis and myasthenic syndrome because it increases acetylcholine release at nerve terminals through K+, Na+ and Ca2+ channels-involving mechanisms. Currently, guanidine derivatives have been proposed for treatment of several diseases. Studies aimed at providing new insights to the drug are relevant. Experimentally, guanidine (10 mM) induces on mouse phrenic nerve-diaphragm (PND) preparations neurotransmission facilitation followed by blockade and a greatest secondary facilitation after its removal from bath. Herein, we hypothesized that this peculiar triphasic response may differ in muscles with distinct twitch/metabolic characteristics. Morphological alterations and contractile response of PND, extensor digitorum longus (EDL) and soleus (SOL) preparations incubated with guanidine (10 mM) for 15, 30, 60 min were analyzed. Guanidine concentrations of 5 mM (for PND and EDL) and 1 mM (for EDL) were also tested. Guanidine triphasic effect was only observed on PND regardless the concentration. The morphological alterations in muscle tissue varied along time but did not impede the PND post-wash facilitation. Higher doses (20–25 mM) did not increase EDL or SOL neurotransmission. The data suggest a complex mechanism likely dependent on the metabolic/contractile muscle phenotype; muscle fiber types and density/type of ion channels, sarcoplasmic reticulum and mitochondria organization may have profound impact on the levels and isoform expression pattern of Ca2+ regulatory membrane proteins so reflecting regulation of calcium handling and contractile response in different types of muscle.

Protection by Mikania laevigata (guaco) extract against the toxicity of Philodryas olfersii snake venom

Philodryas olfersii is responsible for most colubrid snakebites in Brazil. In this work, we examined the ability of an ethanolic extract from Mikania laevigata (guaco) leaves to protect against the in vitro neuromuscular activity of P. olfersii venom in mouse phrenic nerve-diaphragm (PND) and chick biventer cervicis (BC) preparations. M. laevigata extract caused moderate twitch-tension facilitation at low concentrations (107.4 ± 6.2% with 20 μl/ml and 118.9 ± 9.3% with 40 μl/ml in PND, and 120.7 ± 7.7% with 40 μl/ml and 114.5 ± 4.4% with 50 μl/ml in BC after 120 min; n = 4–6, mean ± SEM). In PND, the ethanol alone (40 μl/ml, n = 4) did not change the twitch-tension when compared with control. However, in BC, the ethanol produced a higher facilitation when compared to control. At higher concentrations (>50 μl/ml) the extract caused total and reversible blockade in both preparations. Venom (50 μg/ml) caused partial blockade in PND (58.5 ± 12%, n = 4) and almost total blockade in BC (93.5 ± 2.2%, n = 4). Pretreatment of the preparations with extract (40 μl/ml) for 30 min before incubation with venom (50 μg/ml) completely protected PND from neuromuscular blockade and delayed the blockade in BC. The extract alone caused only mild morphological alterations (12.5 ± 0.5% and 10.9 ± 2.3% fiber damage in PND and BC, respectively, compared to 2.3 ± 0.3% and 3 ± 0 in controls; n = 3), with no increase in expression of the inflammatory cytokines TNFα and IFNγ. The ethanol alone also caused slight muscle damage: 4.3 ± 2.4% in PND and 6.7 ± 3.3% in BC (both n = 3) and little or no TNFα and IFNγ expression in both preparations as observed in control. Venom (50 μg/ml) caused 53.5 ± 8.5% and 55.8 ± 4.3% fiber damage in PND and BC, respectively; (n = 3, p < 0.05 vs. controls) and enhanced expression of TNFα and IFNγ. Pretreatment of the preparations with extract protected against venom-induced muscle damage by 80.3 and 60.4 in PND and BC, respectively, and prevented TNFα and IFNγ expression. These results indicate that the M. laevigata extract protected nerve-muscle preparations against the myotoxic, neurotoxic and inflammatory effects of P. olfersii venom.

Peripheral nerve inflammation in ALS mice: cause or consequence

Neuroinflammation is a prominent pathologic feature in the spinal cord of patients with amyotrophic lateral sclerosis (ALS), and is characterized by glial activation and infiltrating T cells.1 A similar inflammatory response is present in spinal cords of ALS mice1 and is preceded by evidence of a “dying back phenomenon” which includes motor axon degeneration and alterations of the neuromuscular junction.2 The presence of monocytes/macrophages surrounding the degenerating peripheral nerve fibers is an early event that occurs prior to the onset of clinical signs of motor weakness,3 and thus raises the question whether the peripheral nerve inflammatory response initiates, or is in response to, the neurodegenerative process. To address this question, we evaluated the time course of denervation and accompanying inflammatory responses in the lumbar spinal cord–sciatic nerve–gastrocnemius and the cervical spinal cord–phrenic nerve–diaphragm motor units of ALS mice, and found that denervation occurred prior to inflammation; also, both denervation and inflammation occurred earlier in the sciatic nerve motor unit than in the phrenic nerve motor unit. Therefore, peripheral nerve inflammation is probably not the cause of denervation, but rather a response to the neurodegenerative process.

Augmentation of catecholamine release elicited by an Eugenia punicifolia extract in chromaffin cells

Plant extracts of Eugenia punicifolia (Kunth) DC., Myrtaceae, are used in Amazon region of Brazil to treat diarrhea and stomach disturbances, and as hypoglycemic medicine. We have recently shown that an aqueous extract of E. punicifolia augmented cholinergic neurotransmission in a rat phrenic nerve-diaphragm preparation. In this study, we investigated the effects of an E. punicifolia dichloromethane extract (EPEX) in a neuronal model of cholinergic neurotransmission, the bovine adrenal chromaffin cell. EPEX augmented the release of catecholamine triggered by acetylcholine (ACh) pulses but did not enhance ACh-evoked inward currents, which were inhibited by 30%. Since EPEX did not cause a blockade of acetylcholinesterase or butyrylcholinesterase, it seems that EPEX is not directly activating the cholinergic system. EPEX also augmented K+-elicited secretion without enhancing the whole-cell inward calcium current. This novel and potent effect of EPEX in enhancing exocytosis might help to identify the active component responsible for augmenting exocytosis. When elucidated, the molecular structure of this active principle could serve as a template to synthesise novel compounds to regulate the exocytotic release of neurotransmitters.

Presynaptic action of Bothriopsis bilineata smargadina (forest viper) venom in vitro

In this work, we examined the neuromuscular activity of Bothriopsis bilineata smargadina (forest viper) venom in vertebrate isolated nerve-muscle preparations. In chick biventer cervicis preparations the venom caused concentration-dependent (0.1–30μg/ml) neuromuscular blockade that was not reversed by washing, with 50% blockade occurring in 15–90min. Muscle contractures to exogenous acetylcholine and KCl were unaffected by venom, but there was a slight increase in creatine kinase release after 120min (from 80±15 to 206±25U/ml, n=6, p<0.05). In mouse phrenic nerve-diaphragm preparations, the venom (1, 10 and 30μg/ml) produced marked facilitation (∼120% increase above basal) at the highest concentration followed by neuromuscular blockade; the effects at lower concentrations were considerably less marked. Venom increased the quantal content values after 15 and 30min followed by significant inhibition at ≥90min. However, venom did not alter the muscle membrane resting potential or the response to exogenous carbachol. In both preparations, incubation at 22°C instead of 37°C delayed the onset of blockade, as did inhibition of venom PLA2 activity. In curarized mouse preparations, the venom produced only muscle facilitation. These results indicate that B. b. smargadina venom causes neuromuscular blockade in vitro by a presynaptic mechanism involving PLA2.

Phrenic nerve-hemidiaphragm as a highly sensitive replacement assay for determination of functional botulinum toxin antibodies

Botulinum neurotoxins induce a prolonged muscle paralysis by specifically blocking the release of neuronal transmitters from peripheral nerve junctions. Potency testing of toxin and antitoxin therapies is entirely dependent on mouse lethality bioassay which is associated with extreme suffering of large numbers of animals to ensure high precision. The mouse phrenic nerve-diaphragm assay is an ex vivo assay that closely mimics in vivo respiratory paralysis offering substantial refinement and reduction in the number of animals used. A range of botulinum antitoxin standards, one licenced product and experimental antitoxins were tested for neutralising potency using ex vivo hemidiaphragm assay and compared with in vivo determined activities. Overall, there was an excellent agreement between neutralising activity detected by the two assay systems and for each toxin serotype using only 4–7 replicates for each product (almost perfect concordance for type A antitoxins: ρ = 0.997, and substantial concordance for type B antitoxins: ρ = 0.991 and type E antitoxins: ρ = 0.964, respectively). These findings confirm that the mouse nerve-diaphragm preparation can provide a functional ex vivo replacement assay for specific, sensitive and precise assessment of toxin and antitoxin activity.

A quantitative comparison of the antagonism of tubocurarine and diallylnortoxiferine by four anticurare agents.

Abstract The antagonism of tubocurarine and diallylnortoxiferine by neostigmine, physostigmine, edrophonium and ambenonium, has been examined on the rat phrenic nerve diaphragm preparation. Ambenonium showed the greatest activity and physostigmine and edrophonium were the least potent and equally active. This order applied to both neuromuscular blocking agents, but all four antagonists show significantly greater activity against diallylnortoxiferine. The possibility that neostigmine and physostigmine have some qualitative differences in anticurare mechanism compared with edrophonium and ambenonium, on this preparation, is discussed.

The Action of Procaine At the Neuromuscular Junction

AbstractThe mode of action of procaine at the neuromuscular junction has been studied in the rat isolated phrenic nerve diaphragm preparation. Procaine had no effect on the response to indirect stimulation in concentrations below 8 × 10−5. But 2 × 10−5 procaine reduced the neostigmine potentiated twitch to its normal size, and caused a significant reduction in the amount of acetylcholine released by a period of tetanus. This is thought to be due to a selective local anaesthetic action on the fine motor nerve terminals.