William F. Dryden

Depolarization, desensitization and the effects of tubocurarine and neostigmine in cultured skeletal muscle

Abstract The actions of acetylcholine, carbachol, d-tubocurarine and neostigmine were studied in cultures of chick embryo skeletal muscle. Both agonists produced a rapid, concentration-dependent depolarization. The depolarization to a given concentration was greater in older cells with higher resting membrane potentials, but the response percent of resting potential was constant throughout development. Despite continued presence of the agonists, depolarization was not maintained, the membrane potential returning to control level in about 15 min. The rate of recovery increased with age of cells and concentration of agonist. Specific desensitization of acetylcholine receptors was demonstrated. Cross-desensitization between acetylcholine and carbachol was observed, but receptor desensitization was unaffected by potassium-induced depolarization. d-Tubocurarine competitively antagonised the cholinomimetic response. Neostigmine did not potentiate responses to low concentrations of acetylcholine, but did, itself, cause some desensitization. The results indicate that the acetylcholine receptors of cultured skeletal muscle are similar in many respects to adult nicotinic receptors.

Pharmacological studies on the bungarotoxins: separation of the fractions and their neuromuscular activity.

Abstract A commercial sample of Bungarus multicinctus venom was separated into its constituent fractions by column chromatography on CM-Sephadex C-50. The fractions were tested on the responses of the chick biventer cervicis preparation to nerve stimulation, and to added acetylcholine and carbachol. 13 fractions were obtained: 1 was inactive, 4 exhibited weak postjunctional neuromuscular blocking activity, 3 had potent postjunctional blocking activity, and the remaining 5 fractions exhibited predominately prejunctional blocking activity. Use of higher concentrations of the prejunctional toxins revelead that 2 of these fractions also possessed some postjunctional blocking activity. Because small differences in separation procedures may result in differences in the elution pattern of the venom, and because some fractions may possess nonspecific actions only obvious at high concentrations, it is suggested that, when using venom components as experimental tools, the separation method is detalied and the fractions thoroughly tested.

The effects of oral zinc supplementation in the mouse

Abstract C3H mice were given zinc sulphate in their drinking water for periods of up to one year. Histological confirmation of hypertrophy of the adrenal cortex and the pancreatic islets after three months was obtained and changes consistent with hyperactivity were noted in the pituitary. Further work is suggested to correlate structural changes with possible functional changes in the islets, the adrenal cortex and the pars distalis. Zinc content of the liver, spleen and skin was not altered. Plasma insulin and glucose concentrations in zinc-supplemented animals were not significantly different from control values.

The effects of putative neurotransmitters on the resting membrane potential of dissociated brain neurones in culture.

Cultures established from mechanically dissociated neonatal mouse brains were found to be suitable for electrophysiological investigation of drug action. During culture most cells were aggregated into either monolayer regions or thick cords joining monolayer regions. A few cells remained isolated. The neurones in the monolayer regions were distinguished from glial cells by differential staining, and were found to be the best subject for intracellular recording. Frequency of resting membrane potentials of these cells proved to be reproducible in cultures of the same age, and were a useful index of sensitivity to bath applied drugs. Acetylcholine, dopamine, histamine, serotonin and noradrenaline depolarized various neurones; GABA caused hyperpolarization, while glutamate and glycine had no significant effect. Antagonism of the responses to acetylcholine, dopamine, serotonin and GABA was seen using atropine, pimozide, methysergide and bicuculline respectively. It is concluded that dissociated brain neurones in culture show chemosensitivity and may be useful in further pharmacological studies.

Studies on the pharmacology of skeletal muscle in culture: specificity of receptors.

Abstract The specificity of the receptors of chick embryo skeletal muscle fibres in culture was examined. Noradrenaline, histamine, dopamine and γ -aminobutyric acid had no effect on resting membrane potential or on depolarization induced by carbachol. Adrenaline and 5-hydroxytryptamine did reduce the carbachol response but were without direct effect on membrane potential. Muscarinic agonists had no action but the muscarinic antagonists, atropine and hyoscine, reduced the response to carbachol. High concentration of atropine and hyoscine produced a well-maintained depolarization of the muscle fibres and this depolarization was not inhibited by tubocurarine, α -bungarotoxin or prior cholinoreceptor desensitization. The cultured muscle fibres were depolarized by a wide range of nicotinic agonists, and gallamine acted as a competitive antagonist. It is concluded that the receptors on cultured skeletal muscle are similar to normal nicotinic receptors and that atropine and hyoscine depolarize by an action at a site other than the cholinoreceptor, possibly at the ‘ion conductance modulator’.

Pharmacological studies on the bungarotoxins: an analysis of the site of action of nicotinic agonists

Abstract The possibility that nicotinin agonists may exert an indirect action by releasing endogenous acetylcholine from nerve terminals has been examined by the use of nerve-free cultured muscle, and by the use of the specifically prejunctionally active β-type bungarotoxins. In cultured muscle the depolarizations produced by acetylcholine and carbachol were not greatly affected by treatment of the cultures with β-type bungarotoxins for 60 min. In the chick biventer cervicis nerve-muscle preparations dose-response curves to acetylcholine, carbachol, dimethylphenylpiperazinium and nicotine were not affected even after abolition of responses to nerve stimulation by the β-types toxins. As the responses to nicotinic agonists were obtainable in nerve-free muscle and were unimpaired in the chick biventer after elimination of nerve function by β-type bungarotoxins, we conclude that such agonists exert a direct action on the postjunctional acetylcholine receptors.

The actions of some anticholinesterase drugs on skeletal muscle in culture

The actions of the anticholinesterase drugs, physostigmine, neostigmine and diisopropylfluorophosphate (DFP) on chick embryonic skeletal muscle in culture were studied. None of the anticholinesterases potentiated depolarization responses to acetylcholine. In high concentrations neostigmine and physostigmine produced depolarization. The neostigmine‐induced, but not the physostigmine‐induced, depolarization was antagonized by tubocurarine. DFP caused an increase in the rate of repolarization during the presence of a cholinomimetic. It is concluded that the cholinesterase present in cultured muscle fibres does not have a physiological role in hydrolysing acetylcholine and that physostigmine and DFP have an action at the ionic channels that are linked to the cholinoreceptor.

The toxicity of spermine and spermidine to cells in culture

Abstract Spermine tetrahydrochloride and spermidine trihydrochloride were examined for toxic effect on primary human embryo lung fibroblasts and on H.Ep.2 cells. Both substances inhibited the primary cells more than the H.Ep.2 cells and in each case a higher concentration of spermidine than of spermine was required to produce the same effect on cell morphology and replication. Neither substance influenced glucose uptake from the medium by either cell type during the first 24 hr of contact. Comparison of the toxicities towards some bacteria and towards these cultures of human cells leads to the conclusion that neither spermine nor spermidine is likely to be suitable for antibacterial chemotherapy.

Studies on the pharmacology of skeletal muscle in culture: Site of action of nicotinic agonists

Abstract The hypothesis that nicotinic agonists other than acetylcholine act by releasing endogenous acetylcholine was examined using nerve-free cultures of chick embryonic skeletal muscle. All nicotinic agonists tested (acetylcholine, carbachol, DMPP, tetramethylammonium and nicotine) depolarized the cultured skeletal muscle fibres. The depolarization was antagonised by triethylcholine, hemicholinium-3, tetraethylammonium and hexamethonium, substances that have been suggested to act by preventing agonists acting on nerve terminals. The results confirm that triethylcholine, hemicholinium-3, tetraethylammonium and hexamethonium have postjunctional blocking actions. We conclude that nicotinic agonists act directly on cholinoreceptors and do not require the presence of endogenous acetylcholine.

The effect of disulphide bond reduction on cholinoreceptors in cultured skeletal muscle

Abstract The effects of the disulphide bond reducing agent, dithiothreitol (DTT) were tested on cultured chick embryonic skeletal muscle. Thirty minutes treatment with 10−3 M DTT reduced the depolarization produced by acetylcholine and carbachol, but enhanced the depolarization produced by neostigmine. Hexamethonium was converted from an antagonist to an agonist by DTT treatment. Depolarization by high potassium concentrations was unaffected by DTT. The effects of DTT on carbachol and hexamethonium were completely reversed after exposure to an oxidizing agent. It was concluded that the receptors in cultured skeletal muscle are affected by disulphide bond reduction similarly to adult receptors and that the relationship between the disulphide bond and the normal function of the receptor is formed early in the differentiation of muscle.