Biventer Cervicis Nerve-muscle Preparation Research Articles

In Vitro Toxicity of Chinese Russell's Viper (Daboia siamensis) Venom and Neutralisation by Antivenoms.

Daboia siamensis (Russell’s viper) is a highly venomous and medically important snake in China, as well as much of Asia. There is minimal information on the pharmacological activity of the venom of the Chinese species, and currently no commercially available specific antivenom in China. This has led to the use of non-specific antivenoms to treat D. siamensis envenomation. In this study, the in vitro neurotoxicity and myotoxicity of D. siamensis venom was examined and the efficacy of four antivenoms was investigated, including the recently developed Chinese D. siamensis monovalent antivenom (C-DsMAV) and three commercially available antivenoms (Thai D. siamensis (Thai-DsMAV) monovalent antivenom, Deinagkistrodon acutus monovalent antivenom (DaAV), and Gloydius brevicaudus monovalent antivenom (GbAV). D. siamensis venom (10–30 µg/mL) caused the concentration-dependent inhibition of indirect twitches in the chick biventer cervicis nerve muscle preparation, without abolishing contractile responses to exogenous agonists ACh or CCh, indicating pre-synaptic neurotoxicity. Myotoxicity was also evident at these concentrations with inhibition of direct twitches, an increase in baseline tension, and the partial inhibition of ACh, CCh, and KCl responses. The prior addition of C-DsMAV or Thai-DsMAV prevented the neurotoxic and myotoxic activity of D. siamensis venom (10 µg/mL). The addition of non-specific antivenoms (GbAV and DaAV) partially prevented the neurotoxic activity of venom (10 µg/mL) but failed to neutralize the myotoxic effects. We have shown that D. siamensis venom exhibits in vitro weak presynaptic neurotoxicity and myotoxicity, which can be prevented by the pre-addition of the Chinese and Thai Russell’s viper antivenoms. Non-specific antivenoms were poorly efficacious. There should be further development of a monospecific antivenom against D. siamensis envenomation in China.

The Dragon's Paralysing Spell: Evidence of Sodium and Calcium Ion Channel Binding Neurotoxins in Helodermatid and Varanid Lizard Venoms.

Bites from helodermatid lizards can cause pain, paresthesia, paralysis, and tachycardia, as well as other symptoms consistent with neurotoxicity. Furthermore, in vitro studies have shown that Heloderma horridum venom inhibits ion flux and blocks the electrical stimulation of skeletal muscles. Helodermatids have long been considered the only venomous lizards, but a large body of robust evidence has demonstrated venom to be a basal trait of Anguimorpha. This clade includes varanid lizards, whose bites have been reported to cause anticoagulation, pain, and occasionally paralysis and tachycardia. Despite the evolutionary novelty of these lizard venoms, their neuromuscular targets have yet to be identified, even for the iconic helodermatid lizards. Therefore, to fill this knowledge gap, the venoms of three Heloderma species (H. exasperatum, H. horridum and H. suspectum) and two Varanus species (V. salvadorii and V. varius) were investigated using Gallus gallus chick biventer cervicis nerve–muscle preparations and biolayer interferometry assays for binding to mammalian ion channels. Incubation with Heloderma venoms caused the reduction in nerve-mediated muscle twitches post initial response of avian skeletal muscle tissue preparation assays suggesting voltage-gated sodium (NaV) channel binding. Congruent with the flaccid paralysis inducing blockage of electrical stimulation in the skeletal muscle preparations, the biolayer interferometry tests with Heloderma suspectum venom revealed binding to the S3–S4 loop within voltage-sensing domain IV of the skeletal muscle channel subtype, NaV1.4. Consistent with tachycardia reported in clinical cases, the venom also bound to voltage-sensing domain IV of the cardiac smooth muscle calcium channel, CaV1.2. While Varanus varius venom did not have discernable effects in the avian tissue preparation assay at the concentration tested, in the biointerferometry assay both V. varius and V. salvadorii bound to voltage-sensing domain IV of both NaV1.4 and CaV1.2, similar to H. suspectum venom. The ability of varanid venoms to bind to mammalian ion channels but not to the avian tissue preparation suggests prey-selective actions, as did the differential potency within the Heloderma venoms for avian versus mammalian pathophysiological targets. This study thus presents the detailed characterization of Heloderma venom ion channel neurotoxicity and offers the first evidence of varanid lizard venom neurotoxicity. In addition, the data not only provide information useful to understanding the clinical effects produced by envenomations, but also reveal their utility as physiological probes, and underscore the potential utility of neglected venomous lineages in the drug design and development pipeline.

Widespread and Differential Neurotoxicity in Venoms from the Bitis Genus of Viperid Snakes.

Research into the neurotoxic activity of venoms from species within the snake family Viperidae is relatively neglected compared with snakes in the Elapidae family. Previous studies into venoms from the Bitis genus of vipers have identified the presence of presynaptic phospholipase A2 neurotoxins in B. atropos and B. caudalis, as well as a postsynaptic phospholipase A2 in B. arietans. Yet, no studies have investigated how widespread neurotoxicity is across the Bitis genus or if they exhibit prey selectivity of their neurotoxins. Utilising a biolayer interferometry assay, we were able to assess the binding of crude venom from 14 species of Bitis to the neuromuscular α-1 nAChR orthosteric site across a wide range of vertebrate taxa mimotopes. Postsynaptic binding was seen for venoms from B. arietans, B. armata, B. atropos, B. caudalis, B. cornuta, B. peringueyi and B. rubida. To further explore the types of neurotoxins present, venoms from the representatives B. armata, B. caudalis, B. cornuta and B. rubida were additionally tested in the chick biventer cervicis nerve muscle preparation, which showed presynaptic and postsynaptic activity for B. caudalis and only presynaptic neurotoxicity for B. cornuta and B. rubida, with myotoxicity also evident for some species. These results, combined with the biolayer interferometry results, indicate complex neurotoxicity exerted by Bitis species, which varies dramatically by lineage tested upon. Our data also further support the importance of sampling across geographical localities, as significant intraspecific variation of postsynaptic neurotoxicity was reported across the different localities.

Novel long-chain neurotoxins from Bungarus candidus distinguish the two binding sites in muscle-type nicotinic acetylcholine receptors

αδ-Bungarotoxins, a novel group of long-chain α-neurotoxins, manifest different affinity to two agonist/competitive antagonist binding sites of muscle-type nicotinic acetylcholine receptors (nAChRs), being more active at the interface of α-δ subunits. Three isoforms (αδ-BgTx-1-3) were identified in Malayan Krait (Bungarus candidus) from Thailand by genomic DNA analysis; two of them (αδ-BgTx-1 and 2) were isolated from its venom. The toxins comprise 73 amino acid residues and 5 disulfide bridges, being homologous to α-bungarotoxin (α-BgTx), a classical blocker of muscle-type and neuronal α7, α8, and α9α10 nAChRs. The toxicity of αδ-BgTx-1 (LD50 = 0.17-0.28 µg/g mouse, i.p. injection) is essentially as high as that of α-BgTx. In the chick biventer cervicis nerve-muscle preparation, αδ-BgTx-1 completely abolished acetylcholine response, but in contrast with the block by α-BgTx, acetylcholine response was fully reversible by washing. αδ-BgTxs, similar to α-BgTx, bind with high affinity to α7 and muscle-type nAChRs. However, the major difference of αδ-BgTxs from α-BgTx and other naturally occurring α-neurotoxins is that αδ-BgTxs discriminate the two binding sites in the Torpedo californica and mouse muscle nAChRs showing up to two orders of magnitude higher affinity for the α-δ site as compared with α-ε or α-γ binding site interfaces. Molecular modeling and analysis of the literature provided possible explanations for these differences in binding mode; one of the probable reasons being the lower content of positively charged residues in αδ-BgTxs. Thus, αδ-BgTxs are new tools for studies on nAChRs.

Pharmacological characterization of α-elapitoxin-Al2a from the venom of the Australian pygmy copperhead (Austrelaps labialis): An atypical long-chain α-neurotoxin with only weak affinity for α7 nicotinic receptors

Despite the in vivo lethality of venom, neurotoxicity has not previously been considered a significant complication of envenoming by the Australian pygmy copperhead (Austrelaps labialis). However, recent evidence has emerged demonstrating that this venom contains potent presynaptic and postsynaptic neurotoxicity. The present study describes the isolation and pharmacological characterization of the first postsynaptic neurotoxin, α-EPTX-Al2a, from the venom of A. labialis. α-EPTX-Al2a (8072.77Da) caused a concentration-dependent block of twitch contractions and a complete block of responses to cholinergic agonists in the chick biventer cervicis nerve–muscle preparation. This action is consistent with postjunctional neurotoxicity. Monovalent tiger snake antivenom prevented the onset of neurotoxicity if applied prior to toxin administration, but was only able to partially reverse neurotoxicity once muscle paralysis had developed. α-EPTX-Al2a produced a potent pseudo-irreversible antagonism of chick muscle nicotinic acetylcholine receptors (nAChRs), with an estimated pA2 value of 7.902 (KB=12.5nM). Interestingly, the toxin only produced a modest block of neuronal α7 nAChRs, with an IC50 of 1.2μM, and failed to inhibit ganglionic α3β2/α3β4 nAChRs in a fluorescence-based FLIPR assay using SH-SY5Y cells. α-EPTX-Al2a contained 75 amino acid residues with five disulfide bonds that had significant homology to classical long-chain α-neurotoxins. While α-EPTX-Al2a retains most pharmacophore residues critical for binding to muscle-type (α1)2βγδ nAChRs it lacks the key Ala28 and Arg36 residues important for α7 nAChR affinity. Given that A. labialis venom contains both irreversible presynaptic and postsynaptic neurotoxins, clinicians need to be aware of potential neurotoxic complications associated with pygmy copperhead envenomation.

Intersexual variations in the pharmacological properties of Coremiocnemis tropix (Araneae, Theraphosidae) spider venom

This study aimed to determine the biochemical, insecticidal and neurotoxic properties of venom from both sexes of the Australian spider Coremiocnemis tropix (Araneae, Theraphosidae). Insecticidal properties were tested in crickets, while in vitro neurotoxicity was determined in an avian skeletal muscle preparation. Some intersexual differences in venom composition were identified by rp-HPLC and by LC–MS, but the majority of components were found in venoms of both sexes. Injecting the venom into crickets revealed that venom from male specimens was slightly more potent, while female venom induced more prominent effects in the chick biventer cervicis nerve–muscle preparation. The results from the chick assay suggest the presence of at least two vertebrate-active neurotoxins. A pre-synaptic neurotoxin may explain the reversible inhibition of muscle twitches and the unaffected response to nicotinic agonists at medium concentrations of female and medium to high concentrations of male venom. In addition, the presence of a neurotoxin that blocks post-synaptic nicotinic receptors might explain the irreversible inhibition of muscle twitches and the reduced response to nicotinic agonists at high concentrations (5–10 μg/ml) of venom from female specimens only.

Neurotoxic and insecticidal properties of venom from the Australian theraphosid spider Selenotholus foelschei

The present study is the first report on the bioactivity of venom from the Australian theraphosid spider Selenotholus foelschei. Venom from female specimens was used in all experiments. Adult spiders yielded an average of 2.2 mg dried venom per milking with a maximum yield of 7.2 mg. To evaluate the activity of pooled S. foelschei venom in invertebrates, a toxicity test in crickets ( Acheta domesticus) was used. The results suggest the presence of several insecticidal toxins with different but synergistic modes of action, leading to a fast onset of paralysis as well as persistent paralysis and lethal effects (starting at 4 h after injection) in crickets. Vertebrate activity of S. foelschei venom was tested by using the isolated chick biventer cervicis nerve–muscle preparation. The venom produced a significant reduction in baseline tension (at a concentration of 10 μg/mL) and twitch height (at 0.2–10 μg/mL). Twitches were not restored after repeated washing. The response of the muscle to exogenous acetylcholine (1 mM) and carbachol (0.02 mM) was not reduced by the venom. These results indicate the presence of a vertebrate-active neurotoxin in S. foelschei that irreversibly blocks muscle twitches by acting either on voltage-activated Na +-channels or on other pre-synaptically located receptors.

Intersexual variations in Northern ( Missulena pruinosa) and Eastern ( M. bradleyi) mouse spider venom

Venoms of both sexes of Australian Northern ( Missulena pruinosa) and Eastern ( Missulena bradleyi) mouse spiders were studied in order to determine intersexual variations in venom yield, composition and bioactivity. Females of both species yielded more venom than males. High-performance liquid chromatography (HPLC) and mass spectrometry data further indicate a substantial degree of intersexual variation in the venom composition of both species. In a cricket ( Acheta domestica) acute toxicity assay, only small intersexual differences were observed, but M. bradleyi venom was found to be considerably more potent than M. pruinosa venom. In the chick biventer cervicis nerve–muscle preparation, male but not female M. bradleyi venom induced large and sustained muscle contractions with fasciculation and decreased twitch height that could be reversed by CSL funnel-web spider antivenom. In contrast, venoms of both sexes of M. pruinosa did not induce significant effects in the chick biventer cervicis nerve–muscle preparation. We therefore conclude that female M. bradleyi venom and venoms from male and female M. pruinosa appear to contain few, if any, orthologs of δ-missulenatoxin-Mb1a, the toxin responsible for the effects of male M. bradleyi venom in vertebrates. These findings are consistent with clinical reports that mouse spiders, particularly species other than male M. bradleyi, do not appear to be a major medical problem in humans.

Isolation and characterization of rufoxin, a novel protein exhibiting neurotoxicity from venom of the psammophiine, Rhamphiophis oxyrhynchus (Rufous beaked snake)

Colubrid snake venoms potentially represent a vast source of novel biological actives and structural motifs owing to their diverse phylogeny. The present study describes the identification of rufoxin, a neurotoxin from the venom of Rhamphiophis oxyrhynchus (Rufous beaked snake) which is a member of the African colubrid lineage, the psammophiines. Rufoxin (1 μM) displayed reversible post-synaptic neurotoxic activity as evidenced by significant inhibition of indirect twitches and responses to exogenous nicotinic agonists in the chick biventer cervicis nerve–muscle preparation. Rufoxin (0.1–1.0 μM) also caused a rightward parallel shift of cumulative concentration–response curves to carbachol (CCh; 0.6–80 μM) without a significant depression of the maximum response, suggestive of classical competitive antagonism at the skeletal muscle nicotinic receptor. Rufoxin lacks NH 2-terminal sequence homology to previously identified snake venom toxins. This work indicates a wider distribution of neurotoxins across the advanced snake superfamily than previously described.

Isolation and pharmacological characterisation of hostoxin-1, a postsynaptic neurotoxin from the venom of the Stephen's banded snake ( Hoplocephalus stephensi)

Envenoming by the Stephen's banded snake ( Hoplocephalus stephensi) is not usually characterised by neurotoxicity. The present study describes the pharmacological characterisation of hostoxin-1 (MW 6660 Da), the first neurotoxin to be isolated from the venom of the Stephen's banded snake. Hostoxin-1 (0.3–1.0 μM) caused concentration-dependent inhibition of indirect twitches of the chick biventer cervicis nerve–muscle preparation. The neurotoxic activity of hostoxin-1 (0.3 μM) was irreversible by washing, but significantly reversed by the addition of CSL tiger snake antivenom (5 units/ml) added at t 90 (i.e. time at which twitches were inhibited by 90%). In addition, hostoxin-1 (0.3 μM) inhibited responses to exogenous acetylcholine and carbachol, but not KCl, indicating a postsynaptic mode of action. Hostoxin-1 (5–30 nM) displayed pseudo-irreversible antagonism at the skeletal muscle nicotinic receptor with a pA 2 value of 8.45 ± 0.32 (i.e. approximately 100-fold more potent than tubocurarine). H. stephensi venom displayed a high level of PLA 2 activity (specific activity 100.1 ± 4.4 μmol/min/mg). However, the activity of hostoxin-1 was negligible. Partial N-terminal sequencing of hostoxin-1 indicates that it has high sequence homology with other elapid short-chain neurotoxins.

A biochemical and pharmacological examination of Rhamphiophis oxyrhynchus (Rufous beaked snake) venom

The venom of R. oxyrhynchus, a member of the psammophiine subfamily of the colubrid assemblage, was examined for biological activity using biochemical and pharmacological techniques. Venom displayed a high protein content, a complex electrophorectic profile and PLA 2 activity but no detectable proteolytic or haematological activities. In the chick biventer cervicis nerve muscle preparation, venom (1–10 μg/ml) displayed postsynaptic neurotoxic activity as evidenced by inhibition of indirect (0.1 Hz, 0.2 ms, supramaximal V) twitches and responses to exogenous acetylcholine (1 mM) and carbachol (20 μM). This inhibitory effect was poorly reversible by washing. Venom (30–50 μg/ml) caused a rapid and readily reversible inhibition of direct (0.1 Hz, 2 ms, supramaximal V) twitches of the chick biventer cervicis nerve muscle preparation without morphological changes to the muscle fibers. Venom (30–100 μg/ml) inhibited electrically-evoked (0.2 Hz, 0.3 ms, 70–100 V) twitches of the prostatic segment of the rat vas deferens. This inhibitory effect was not significantly attenuated by 8-phenyltheophylline (8-PT; 20 μM), idazoxan (1 μM), a combination of ranitidine (0.2 μM) and thioperamide (10 μM) or capsazepine (10 μM). Venom (5 mg/kg) induced hypotension with subsequent cardiovascular collapse in the anaesthetised rat. The cardiovascular collapse was prevented by artificial respiration of the animals prior to venom administration. The biological activities demonstrated by R. oxyrhynchus venom may aid in prey envenomation strategies such as prey immobilisation. This study provides further evidence that colubrid venoms are comprised of multiple components which can display a variety of actions, some of which may be novel, therefore reinforcing the largely untapped potential of colubrid venoms.

Clinical and in vitro evidence for the efficacy of Australian red-back spider ( Latrodectus hasselti) antivenom in the treatment of envenomation by a Cupboard spider ( Steatoda grossa)

We report the case of a 22-year-old female who was bitten on the shoulder by a spider subsequently identified as a female Cupboard spider ( Steatoda grossa). She developed nausea, vomiting, and severe local and regional pain, similar to that seen in latrodectism. Symptoms were treated successfully with red-back spider antivenom (RBSAV). We also present in vitro data, which supports this clinical observation, and suggests that S. grossa venom is immunogenically reactive with both RBSAV and latrotoxin (LTx)-specific antibodies by Western blotting. Moreover, the effects of S. grossa venom on the isolated chick biventer cervicis nerve–muscle preparation are dose-dependent and similar to those seen with Latrodectus spp. venoms. S. grossa venom produced a sustained muscle contracture which could be prevented by pre-incubation of venom with RBSAV. Venom effects could also be reversed by the addition of antivenom after application of venom to the preparation. Although severe envenomation is uncommon following the bite of Steatoda spp. it may resemble latrodectism. These results indicate that RBSAV is likely to be effective in reversing symptoms of envenomation and should be considered in the treatment of patients with distressing or persisting symptoms.

Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web ( Atrax and Hadronyche) spider venoms

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve–muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5/10) produced muscle contracture and fasciculation when applied to the nerve–muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders.

Use of the biventer cervicis nerve-muscle preparation to detect early changes following exposure to organophosphates inducing delayed neuropathy

Indices of organophosphorus (OP)-induced delayed neuropathy (OPIDN) in the hen model have traditionally been restricted to the early inhibition of neuropathy target esterase (NTE) and ataxia with associated pathological changes in hind limb peripheral nerve which occur more than 7 days after OP exposure. The biventer cervicis nerve-muscle preparation was used to evaluate OPIDN in adult hens at various time periods after treatment with either the protoxicant tri- o-tolyl phosphate (TOTP), 360 mg/kg po, or the active congener phenyl saligenin phosphate (PSP), 2.5 mg/kg im. NTE activity was 21 and 48% of control for TOTP and PSP, respectively, 4 days after administration. Clinical signs were notable by 10 days and progressed in severity to paralysis by 21 days. Partial clinical recovery was evident at 37 days. Denervation hypersensitivity of biventer cervicis muscle to acetylcholine (ACh) was evident as early as 4 days following TOTP or PSP treatment. The sensitivity to ACh was greatest 21 days after OP administration, with partial recovery at 37 days. Strength-duration curves (SDC) of preparations from OP-treated hens showed an increase in excitability thresholds and elevated rheobase with shorter chronaxie than did preparations from controls as early as 4 days following treatment with either compound. SDC at 37 days indicated partial reinnervation. Peripheral nerve myelinated fiber degeneration and regeneration consistent with these physiological changes was seen on histopathological examination. This study suggests that the biventer cervicis nerve-muscle preparation may prove useful for detection of functional and morphological changes that occur during the interval between NTE inhibition and appearance of clinical deficits.

Effect of verapamil on organophosphorus-induced delayed neuropathy in hens

Verapamil, a calcium channel blocker, was administered to adult white leghorn hens to determine if inhibition of calcium entry could alter delayed neuropathy induced by administration of phenyl saligenin phosphate (PSP). Verapamil was given im in doses of 7 mg/kg/day for 4 days beginning 24 hr before administration of PSP (2.5 mg/kg im). Ataxia was less pronounced in hens given PSP plus verapamil than in hens given PSP alone during observations made 8–28 days after PSP administration. Myelinated fiber lesions were less extensive and regeneration more notable in the biventer cervicis nerve in chickens given PSP plus verapamil, with samples obtained both 17 and 28 days after PSP. In the absence of verapamil, rheobase and chronaxie values of strength-duration curves were higher and shorter, respectively, and sensitivity to acetylcholine was increased in biventer cervicis nerve-muscle preparations from hens given PSP. Verapamil did not alter PSP-induced inhibition of neurotoxic esterase, indicating that the mechanism involved in amelioration of these indices of delayed neuropathy was not associated with initial enzyme inhibition caused by this organophosphorus ester.

The interaction of diazepam with myoneural blocking agents.

The interaction of diazepam with neuromuscular blocking agents was studied in patients and in several animal nerve-muscle preparations. This drug did not alter the recovery slope of blockade produced by d-tubocurarine, decamethonium, or gallamine. In higher doses diazepam produced a decrease in muscle contractions and blocked the actions of exogenously applied nicotine and acetylcholine in the superfused chick biventer cervicis nerve—muscle preparation. In low doses the commercially available drug given intra-arterially reversed the myoneural blockades produced by both depolarizing and nondepolarizing blockers. However, this property was due to the solvent system of the drug.

Effects of halogenoacetylcholines on the neuromuscular junction

The effect of halogenoacetylcholines (HACh's) has been studied on the chick biventer cervicis nerve muscle preparation. All of HACH's studied were shown to be neuromuscular blocking agents of a depolarizing type. Neither the nicotinic effect of HACH's or of ACh was affected by triethylcholine (TEC) and hexamethonium (C 6) whereas that of nicotine, tetramethylammonium and dimethylphenylpiperazinium was abolished by TEC and C 6. These results suggest that HACh's act directly on ACh receptor to produce nicotinic effects similar to ACh. The brief contact of HACh's with the biventer cervicis muscle using superfusion technique caused slow contraction of the muscle followed by an immediate relaxation. This transient reversible contracture can be blocked by d-tubocurarine, indicating that it is induced through depolarization of the muscle at the cholinergic receptor site. On the other hand, if the muscle was treated by bromoacetylcholine and iodoacetylcholine for 10 min or longer, it produced a persistent contracture which was not reversible after repeated washing. It was questioned whether the inhibition of 3-phosphoglyceraldehyde dehydrogenase in the Embden-Meyerhof pathway and consequential reduction in the availability of adenosine triphosphate to the muscle is the reason for the persistent muscle contracture in this case.

Effects of ganglionic blocking agents on the neuromuscular junction

Effects of ganglionic blocking agents on neuromuscular transmission and on responses of nicotinic agents have been studied using the baby chick biventer cervicis nerve muscle preparation. Responses of nicotinic agents such as nicotine, tetramethylammonium, dimethylphenylpiperazinium and carbachol were markedly inhibited by 10 mg % of hexamethonium (C 6) and 100 mg % of tetraethylammonium (TEA) and almost completely abolished by 100 mg % of C 6 and 1 g% of TEA. However, responses induced by ACh were not significantly affected by these ganglionic blocking agents, indicating that nicotinic agents were blocked by these ganglionic blocking agents at a site other than the ACh receptor, probably at the motor nerve endings. Neuromuscular transmission was also blocked by C 6 and TEA without affecting ACh responses suggesting that C 6 and TEA might block the uptake of choline bynerve across the membrane and thus inhibit ACh synthesis and thereby block neuromuscular transmission. Initial facilitation and concomitant contraction of the biventer cervicis muscle preceded neuromuscular blockade induced by TEA indicating that part of the TEA effect might be due to the release of endogenous ACh.

Acetylcholine-releasing effects of some nicotinic agents on chick biventer cervicis nerve muscle preparation.

SummaryThe mechanism of action of some nicotinic agents has been studied on the baby chick biventer cervicis nerve muscle preparation. The effects of various nicotinic agents on this preparation were blocked by 1.2 × 10—2M triethylcholine (TEC) or by neuromuscular blockade achieved by TEC treatment with interrupted tetanic nerve stimulation, whereas the dose-responsive curve of acetylcholine (ACh) was not altered. These results suggest that the nicotinic agents tested are presumably acting primarily at the nerve terminals rather than at the receptor sites on the postjunctional membrane. After the effects of the nicotinic agents were completely blocked by TEC alone, the muscle was still responsive to nerve stimulation. This suggests that the TEC blockade of the nicotinic effects of the drugs tested may be at the membrane site of the nerve terminal since ACh is still available in the storage site for release after TEC blockade. In the same preparation, the responses to most of the nicotinic agents were mark...