Chick Biventer Cervicis Muscle Research Articles

Chromatographic separation of the venom of Egyptian black snake ( Walterinnesia aegyptia) and pharmacological characterization of its components

Three neurotoxic components (W-III, W-IV and W-V) were isolated from the venom of Walterinnesia aegyptia by CM-cellulose column chromatography, followed by gel filtration on a Sephadex G-50 column. The ld 50 in mice (i.p.) was 0.95 μg per g body wt for W-III, 0.14 μg per g for W-IV and 0.2 μg per g for W-V, while in baby chicks (i.m.) it was over 0.5 μg per g for W-III, 0.12 μg per g for W-IV and 0.23 μg per g for W-V. All of the three toxins blocked the neuromuscular transmission of the isolated rat phrenic nerve-diaphragm and the chick biventer cervicis muscle and inhibited the response of the latter to acetylcholine, just like other postsynaptically acting snake neurotoxins. The neuromuscular block produced by W-III was reversible in both muscle preparations, whereas that by W-IV as well as W-V was irreversible in the chick biventer cervicis but partially reversible in the rat diaphragm.

Neurotoxic and myotoxic effects of crotalus phospholipase A and its complex with crotapotin.

1. The acidic peptide crotapotin potentiated the toxicity of the basic crotalus phospholipase A in all species tested. The order of sensitivity to the lethal action of the phospholipase was: chick greater than mouse larger than or equal to rabbit greater than rat. After a latency period of a least 20 min the animals died of respiratory paralysis. Rabbits survived for more than 10 h, if they were artificially respired. The animals recovered very slowly from respiratory depression. 2. In rabbits even high doses of the basic crotalus phospholipase A or its complex with crotapotin did not affect the respiratory center nor its reactivity to asphyxia. Conduction of action potentials in the phrenic nerve was not changed. 3. Phospholipase-crotapotin complexes decreased the contractile response of isolated phrenic-hemidiaphragms of rats to direct and indirect stimulation in an irreversible manner. A latency period 20-100 min preceded the paralysis. 4. A similar block of neuromuscular transmission developed in vivo. After i. v. injection of PC-complexes the contractile response of isolated rat phrenic diaphragms to nerve stimulation was considerably lower than to direct stimulation. 5. Crotalus phospholipase A alone as well as its complex with crotapotin reduced the contractions of the isolated chick biventer cervicis muscle but did not cause a contracture, thus indicating that the phospholiphase PC-complexes act not as depolarizing blockers. 6. Blood pressure, heart rate and electrocardiogram were not substantially altered, when phospholipase A or PC-complex were injected slowly i.v. into rabbits or rats. No cardiotoxic effect was observed in the Langendorff preparation of rat hearts perfused with phospholipase A alone (6 x 10(-6) M) or together with crotapotin (10(-5) M). Rapid i.v. injection of the venom produced hypotension. The degree of haemolysis correlated with the enzymatic activity, and was low for the highly toxic PC-complexes.

Further evidence that extrinsic acetylcholine acts preferentially on extrajunctional receptors in the chick biventer cervicis muscle

The specificity of action of extrinsic acetylcholine on extrajunctional and junctional receptors in the chick biventer cervicis muscle was studies by determining its ability to protect the responses evoked by acetylcholine and by tetanic nerve stimulation from the blockade by α-bungarotoxin, an irreversible binding agent of acetylcholine receptors. At concentrations of 50–100 μg/ml, acetylcholine caused a desensitization to extrinsic acetylcholine but not to nerve stimulation and protected only the contractile response to extrinsic acetylcholine from the toxin blockade whereas neither the response to tetanic nerve stimulation nor the endplate potentials were protected. For the protection of the latter, higher concentrations of acetylcholine were needed. In the presence of physostigmine, a concentration of acetylcholine as low as 10 μg/ml protected the endplate potentials from the toxin blockade. By contrast, d-tubocurarine protected the tetanic contraction and the endplate potentials induced by nerve stimulation at a concentration which produced the same protection of acetylcholine-induced contraction as that produced by 50–100 μg/ml acetylcholine. These results indicate that in contrast to d-tubocurarine, extrinsic acetylcholine at low concentrations acts preferentially on the extrajunctional receptors in the absence of an anticholinesterase.

A quantification of acetylcholine receptors of the chick biventer cervicis muscle

A quantification of acetylcholine receptors of the chick biventer cervicis muscle

Pharmacology of scorpion toxin II in the skeletal muscle.

1. Scorpion toxin II is potent in inducing contracture and spontaneous contractions of the chick biventer cervicis muscle. In addition, this toxin induces membrane depolarization and blockade of neuromuscular transmission in this muscle preparation. The purpose of the present study is to explore the possible mechanism of actions of toxin II. 2. The muscle contracture induced by toxin II is moderately accelerated by Ca2+-free Krebs solution, delayed by high Ca2+ (10 mM), high Mg2+ (10 mM) and low Na+ (60mM) Krebs solution. Moreover, this action is inhibited slightly by d-tubocurarine and completely by either procaine or tetrodotoxin, but unaffected by beta-bungarotoxin. All these findings suggest that toxin II induces contracture mainly by increasing the Na+ permeability of the muscle membrane. 3. Spontaneous contractions induced by toxin II are abolished by Ca2+-free Krebs solution, inhibited partially by either d-tubocurarine or beta-bungarotoxin and completely by tetrodotoxin or procaine. These results suggest that toxin II induces spontaneous contractions partially by releasing acetylcholine from nerve endings and partially by increasing the Na+ permeability of the muscle membrane.

Cholinergic receptor labelling specific irreversible binding of bromoacetylcholine to cholinergic receptors at neuromuscular junctions

This study shows that (a) the slopes of dose-response curves of bromoacetylcholine (BrACh) and acetylcoline (ACh) were the same; (b) the dose-response curves of BrACh to contract superfused chick biventer cervicis and frog rectus abdominis muscles were shifted to the right by d-tubocurarine in a parallel fashio; and (c) the responses of BrACh were not affected by triethylcholine, which paralyzes cholinergic neurons through inhibition of ACh synthesis. These results indicate that BrACh interacts with cholinergic receptors specifically and directly. When the incubation time was prolonged to 15 min or longer in the organ bath, BrACh (2×10 −3 M) produced irreversible blockade of the neuromuscular transmission in chick biventer cervicis and rat diaphragm preparations. The latter blockade was prevented partially by ACh (2×10 −2 M) and d-tubocurarine (3.4×10 −6 M) (25–38%) and markedly by carbachol (1×10 −4 M) and decamethonium (6.2×10 −4 M)_(76–79%), indicating agani that BrACh binds specifically and irreversibly to the cholinergic receptor. Therefore, 14C-BrACh is recommended for use in mapping, estimation, and isolation of cholinergic receptors. In order to do such experiments, the biological preparations would have to be pretreated with cold organophosphorus compounds in order to prevent irreversible binding of BrACh to cholinesterases.

Differentiation between intrinsic and extrinsic acetylcholine receptors of the chick biventer cervicis muscle.

The intrinsic and extrinsic acetylcholine receptors were studied by analysing the effects of α-bungarotoxin and desipramine on the contractile response elicited by nerve stimulation (single shocks) and that by application of acetylcholine. In the absence of anticholinesterases, α-bungarotoxin as well as desipramine blocked the response to exogenous acetylcholine with a time-course much faster than that of the inhibition of the response to nerve stimulation. After complete blockade of neuromuscular transmission by α-bungarotoxin, washout of the toxin gradually resulted in recovery of the response to acetylcholine, whereas the response to nerve stimulation was not appreciably restored. (+)-Tubocurarine, on the other hand, blocked both types of responses to a parallel extent. (+)-Tubocurarine as well as acetylcholine at high concentration protected both responses from blockade by α-bungarotoxin. In the presence of anticholinesterases, the inhibition by α-bungarotoxin of responses to nerve stimulation and to acetylcholine was parallel. The recovery of acetylcholine-induced responses after washout of the toxin, however, was smaller than in the absence of anticholinesterases.

Further studies on the site of action of nicotinic blocking drugs

The effects of combinations of tubocarine and physostigmine, and of physostigmine and edrophonium alone, have been studied on the responses of the chick biventer cervicis muscle to nerve stimulation and to exogenous acetylcholine. The effects of hexamethonium, tetraethylammonium and triethylcholine have also been studied on the dose-response curves for acetylcholine, determined in the presence of low concentrations of physostigmine. Suitable combinations of tubocurarine and physostigmine reduced responses of the muscle to nerve stimulation, whilst leaving the responses to acetylcholine unaffected, whereas physostigmine and edrophonium augmented responses to acetylcholine, whilst leaving responses to nerve stimulation unaffected. These results indicate that the multiply innervated fibres of the muscle are particularly sensitive to inhibition of cholinesterase. Hexamethonium, tetraethylammonium and triethylcholine, drugs that are known to exhibit actions similar to that of the combination of tubocurarine and physostigmine, and also to possess anticholinesterase activity, depressed responses to acetylcholine determined in the presence of concentrations of physostigmine sufficiently low to possess a specific inhibitory action on cholinesterase. The results indicate that the neuromuscular block produced by large concentrations of these drugs is probably post-junctional in origin.

Some actions of 4,17a-dimethyl-4,17a-diaza-D-homo-5alpha-androstane dimethiodide (HS-342), a new neuromuscular blocking drug.

Abstract The effects of the newly-synthesized bisquaternary steroid 4,17a-dimethyl-4,17a-diaza-D-homo-5α-androstane dimethiodide (HS-342) have been investigated on the rat isolated phrenic nerve-hemidiaphragm, chick biventer cervicis and intact cat tibialis anterior muscle preparations, on the guinea-pig isolated ileum and on the rat isolated vas deferens preparations. The anticholinesterase activity of HS-342 was also measured. In the skeletal muscle preparations HS-342 exhibited properties of a post-junctionally active neuromuscular blocking agent of the non-depolarizing class. In the anaesthetized cat HS-342 exhibited approximately equal activity to that of tubocurarine, but its duration of action was about one-third of that of tubocurarine. HS-342 exhibited low anticholinesterase activity, low muscarinic blocking activity and apparently had no effect on the adrenergic neuroeffector junction studied.

Studies on the action of some cholinoceptor agonists on the chick biventer cervicis muscle preparation.

Dose-response curves constructed for the nicotinic agonists: acetylcholine, carbachol, choline, tetramethyl-ammonium, nicotine, decamethonium and dimethylpiperazinium, after treatment of chick biventer cervicis muscle preparations with a combination of rapid nerve stimulation, and the drug 2-(4-phenylpiperidino)-cyclohexanol (AH5183), were indistinguishable from control dose-response curves for these agonists. As AH5183 reduced the acetylcholine content of, and output from, chick biventer cervicis muscles after nerve stimulation, these results provide evidence that nicotinic agonists do not release acetylcholine from the same source as that released by nerve stimulation.

The effects of 2-(4-phenylpiperidino)-cyclohexanol (AH5183) on the acetylcholine content of, and output from, the chick biventer cervicis muscle preparation.

The effects of the tertiary base 2-(4-phenylpiperidino)-cyclohexanol (AH5183) were studied on the acetylcholine content of, and output from, the chick biventer cervicis nerve-muscle preparation. Neither treatment with AH5183 (4 μg/ml) alone, nor continued rapid nerve stimulation alone, produced significant changes in the acetylcholine content of the preparations. However, after ninety minutes rapid nerve stimulation in the presence of AH5183 the acetylcholine content of the preparations was significantly reduced. The output of acetylcholine from the nerve-muscle preparations was markedly reduced after two and three hours nerve stimulation in the presence of AH5183. The results provide further evidence that AH5183 possesses a potent prejunctional action at the neuromuscular junction. The potential usefulness of AH5183 in producing acute chemical denervation is indicated.

Mode of action of purified basic proteins from three rattlesnake venoms on neuromuscular junctions of the chick biventer cervicis muscle: C. Y. Lee, M. C. Huang and C. A. Bonilla.∗ (Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, China and ∗Department of Physiology and Biophysics, Colorado State University, Fort Collins, Colorado, U.S.A.)

Mode of action of purified basic proteins from three rattlesnake venoms on neuromuscular junctions of the chick biventer cervicis muscle: C. Y. Lee, M. C. Huang and C. A. Bonilla.∗ (Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan, China and ∗Department of Physiology and Biophysics, Colorado State University, Fort Collins, Colorado, U.S.A.)

Some effects of hexamethonium and tetraethylammonium at a neuromuscular junction of the chicken

The effects of the ganglion-blocking drugs hexamethonium and tetraethylammonium were studied on the responses of the chick biventer cervicis muscle to acetylcholine and carbachol, and their anticholinesterase activities were measured using the same muscle as the source of enzyme. Large concentrations of hexamethonium and tetraethylammonium had little effect on responses to acetylcholine but blocked responses to carbachol. However, responses to acetylcholine were blocked by these compounds in the presence of physostigmine. Essentially similar effects were produced in chronically denervated muscles. Large concentrations of hexamethonium and tetraethylammonium possessed anticholinesterase activity in chick muscle. It was concluded that the main action of hexamethonium and tetraethylammonium in the chick biventer cervicis muscle is a post-junctional antagonistic action which, in the presence of acetylcholine, is masked by a weak anticholinesterase activity, and that these drugs owe little of their effects to an action nervous tissue.

Actions of lidoflazine on neuromascular transmission

Lidoflazine, 2.0 × 10 −5−1.2 × 10 −4M, was tested on the twitch responses elicited by direct and indirect stimulation of the isolated rat diaphragm. The drug produced a marked depression of the responses to indirect stimulation. Recovery from lidoflazine-induced depression was prolonged and sometimes incomplete. Neostigmine failed to reverse lidoflazine-induced neuromascular blockade except when blockade was slight. In indirectly stimulated chick biventer cervicis muscle, lidoflazine induced a depression of the twitch response followed by a contracture of the muscle. The characteristics of the neuromascular action of lidoflazine differ from those of the classical neuromascular blocking agents.

Effects of Nicotine and Acetylcholine On Denervated Chick Biventer Cervicis Muscles

Chick biventer cervicis muscle was chronically denervated for various periods of time. The muscle lost its responses to indirect electrical stimulation on the nerve as expected. It remained responsive to ACh although the contraction force was somewhat reduced, depending on the length of time after denervation. The denervated muscle was approximately 10-fold more sensitive to ACh than the innervated one was. This is presumably due to the reduction of cholinesterase activity in the denervated muscle. The responses of nicotine were reduced much more than those of ACh in denervated muscles. The result supports the hypothesis that ACh is released by nicotine to produce nicotine responses. The residual responses produced by nicotine in denervated muscle could be due to the release of ACh from the fine diffuse nerve fibres which can not be denervated completely with surgical procedures, and to the reduction of cholinesterase activity which potentiates the responses of minute quantities of ACh released by nicotine.

Actions of acetylcholine and carbachol on the chick biventer cervicis muscle.

1. The possibility that carbachol may act on the chick biventer cervicis muscle by releasing acetylcholine was investigated.2. Log dose-response curves for acetylcholine and carbachol were constructed in the presence of triethylcholine, tubocurarine and physostigmine, in both innervated and denervated chick biventer cervicis muscles.3. The log dose-response curve for acetylcholine was shallower than that for carbachol and was moved to the left by triethylcholine, indicating potentiation. In the presence of physostigmine, the curve was as steep as that for carbachol, and like that for carbachol was moved to the right by triethylcholine, indicating antagonism. In the presence of tubocurrine, the log dose-response curves for both acetylcholine and carbachol were moved to the right.4. The log dose-response curves for both acetylcholine and carbachol were moved to the left by chronic denervation, indicating increased sensitivity to these drugs. Both curves were moved similar distances to the right by excess magnesium ion concentration. No tachyphylaxis developed to repeated administration of carbachol in the presence of triethylcholine or tubocurarine, either in innervated or denervated muscles.5. It was concluded that the main action of triethylcholine in the chick biventer cervicis muscle is a tubocurarine-like action, which, in the presence of acetylcholine, is masked by a weak anticholinesterase action. It was also concluded that the diminished response to carbachol in the presence of triethylcholine is due to the postjunctional blocking action of triethylcholine, and that carbachol owes none of its activity to an ability to release acetylcholine from the nerve terminals.

The effects of some hemicholinium-like substances on the chick biventer cervicis muscle preparation

The effects of tubocurarine, hemicholinium-3, triethylcholine, troxypyrrolium and bretylium were examined on the chick biventer cervicis muscle. Relative pre- and post-junctional activities were assessed by the ability of the compounds to depress carbachol contractures and twitches elicited at 1/sec and 1/10 sec. Tubocurarine, hemicholinium, bretylium and triethylcholine showed post-junctional blocking action. Troxypyrrolium was the most specific pre-junctional blocking drug, and along with triethylcholine was reversible by choline. In addition to their blocking actions bretylium and triethylcholine produced an initial twitch augmentation and an anti-tubocurarine action. This was shown to be due to anticholinesterase and acetylcholine-releasing propertie respectively. The chick muscle was more sensitive than mammalian muscle to the post-junctional blocking and pre-junctional facilitatory components of action, and these effects tended to obscure the pre-junctional blocking action of the drugs. It is suggested that these different sensitivites to drugs are a reflection of a small safety margin in transmitter release at fowl motor nerve-endings.

Species defferences in susceptibility to elapid venoms.

Birds (pigeon, chick and Formosan sharp-tailed munia) are extremely susceptible to the lethal action of the venom of Bungarus multicinctus as compared with mammals. Comparison of the neuromuscular blocking activity of α- and β-bungarotoxin in chick's biventer cervicis muscle reveals that avian muscle is particularly susceptible to β- but not to α-bungarotoxin. It is concluded that the transmitter-releasing mechanism of avian motor nerve endings is particularly vulnerable to the action of β-bungarotoxin. On the other hand, cats are highly resistant to cobra venom, if given s.c. It was found that the skeletal muscle of the cat is highly resistant to the neuromuscular blocking action of cobra venom.

Pharmacological properties of cardiotoxin isolated from Formosan cobra venom

Lyophilized venom of Naja naja atra was fractionated on column of CM-Sephadex C-50 into 12 fractions by gradient elution with ammonium acetate buffer at pH 5–7. Among them 5 fractions (V–IX) were found to be neurotoxic and 3 (X–XII) to be cardiotoxic. Intraperitoneal LD50 in mice was 0.074 µg/g for the major neurotoxic component (Fr. VIII) and 1.48 µg/g for the major cardiotoxic one (Fr. XII). The latter, named cardiotoxin, caused contracture followed by paralysis of the chick biventer cervicis muscle, the frog sartorius muscle and the rat diaphragm, in consequence of irreversible depolarization of the cell membrane. In the absence of calcium no contracture was produced, although the depolarizing effect remained unchanged. Cardiotoxin caused systolic arrest of the isolated frog heart and the rat atrium, probably by the same mechanism. It also caused a contraction of the guinea-pig ileum, which was largely antagonized by atropine but not by hexamethonium or pyribenzamine. In the presence of cardiotoxin the responses of the ileum to nicotine and to submaximal transmural stimulation were first enhanced and then depressed. Cardiotoxin caused vasoconstriction of the rabbit ear vessels, which was largely antagonized by phenoxybenzamine. It also produced local irritation of the rabbit conjunctiva and the rat hind paw. In cats, cardiotoxin caused a fall in the systolic pressure much more than the diastolic pressure, accompanied by various ECG changes, such as P–R interval prolongation, decreased amplitude of QRS, S–T and T changes, ventricular premature beats, complete A–V block, idioventricular rhythm etc. It is concluded that cardiotoxin acts on various kinds of cells in the animal body, causing irreversible depolarization of the cell membrane and consequently impairing its functions.