Apparent cooperative effects in acetylcholine receptor-mediated ion flux in electroplax membrane preparations
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Decamethonium
Cooperativity
Efflux
The accumulation of decamethonium by mouse kidney slices was investigated with particular reference to the possibility that this agent uses a choline transport system. Slices of mice kidneys were incubated (1 hour) in Krebs‐Ringer bicarbonate medium (37°, pH 7.4) containing 14 C‐decamethonium (2 × 10 ‐6 M) with or without the addition of other drugs. Choline and neostigmine stimulated decamethonium uptake at relatively low concentrations (10 ‐3 M and 3 × 10 ‐3 M choline, 5 × 10 ‐5 M and 10 ‐4 M neostigmine), whereas both agents at higher concentrations (3 × 10 ‐2 M choline, 10 ‐3 M and 10 ‐2 M neostigmine) depressed the uptake. Hemicholinium‐3 (10 ‐3 M), atropine (2 × 10 ‐5 M) and physostigmine (2 × 10 ‐4 M) inhibited decamethonium uptake, indicating that these agents in addition to choline and neostigmine share a common transport mechanism with decamethonium. The initial decamethonium influx (3 minutes incubation) could be stimulated by pre‐incubating the slices (1 hour) with 10 ‐3 M choline or 3 × 10 ‐4 M neostigmine (in the absence of decamethonium) before transfer to a final medium containing only decamethonium. Stimulation can thus be interpreted as an example of accelerative exchange diffusion, which should mean that efflux of choline or neostigmine accumulated by the slices accelerates decamethonium influx. It is concluded that decamethonium uses a specialized transport system, which seems to involve a choline carrier and to be in part at least identical with the system responsible for organic cation secretion by the intact kidney.
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Choline
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Decamethonium
Affinity label
Cys-loop receptors
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Efflux
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1. The recovery of contractile responses and appearance of new alpha-bungarotoxin-binding sites were studied in the baby chick biventer cervicis and the rat diaphragm muscles after saturating the existing acetylcholine (ACh) receptors (AChR) with alpha-bungarotoxin in vitro.2. Washout of alpha-bungarotoxin restored gradually the response to exogenous ACh attaining about 30% recovery in 3 hr either in the chick muscle or in the denervated rat diaphragm. No recovery was obtained, however, for the response to nerve stimulation.3. The recovery of ACh-response was abolished by decreasing the bath temperature to 9 degrees C during the washout of the toxin whereas the recovery was not reduced in the presence of cycloheximide.4. The half-life of [(3)H]acetyl alpha-bungarotoxin bound specifically on the existing AChRs, junctional and extrajunctional receptors combined, was 16 hr in the chick muscle. That on the extrajunctional AChR was estimated to be 8 hr.5. New toxin-binding sites were found to be incorporated on the membrane of extrajunctional site rapidly after treatment with alpha-bungarotoxin in the chick and the denervated rat muscles along the muscle fibres but not in the innervated rat diaphragm. Treatment with (+)-tubocurarine, ACh or decamethonium did not cause an appreciable increase of the toxin-binding sites.6. The appearance of new binding sites was progressive during 5 hr at a rate of 24 sites/mum(2).hr in the chick muscle and 42 sites/mum(2).hr in the rat diaphragm denervated for 7 days. The existing extrajunctional AChR were about 50/mum(2) and 192/mum(2), respectively.7. ACh effectively antagonized the binding of alpha-bungarotoxin with the new sites whereas (+)-tubocurarine was less effective than its effect on the existing AChR.8. The new toxin-binding sites appeared to have a reduced capacity to evoke ACh response.9. The incorporation of new binding sites was reduced by lowering of the temperature, treatment with dinitrophenol, high K(+), high Ca(2+) and by the stimulation of either nerve or muscle. Cycloheximide, ACh, decrease of [Na(+)](o) and increase of [Mg(2+)](o) were without effect.10. It is suggested that binding of the extrajunctional AChRs with alpha-bungarotoxin cause a change of membrane architecture and trigger the incorporation of cytoplasmic AChR-precursor or hidden AChR into the membrane.
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Bungarotoxin
Neuromuscular transmission
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1. In rat diaphragms immersed in solution containing 5 m M potassium the maximum uptake of labelled decamethonium was found at the end‐plate region. In muscles depolarized in solution containing potassium methyl sulphate the uptake was reduced but a peak concentration at the end‐plate region was still demonstrated. 2. The uptake of labelled decamethonium increased steadily with time and was interpreted in terms of the entry of decamethonium into the fibres. The permeability at 10–100 μ M was similar to that of sodium. 3. The uptake of decamethonium at the end‐plate region was dependent on the concentration. At low values the uptake in depolarized muscle was uniform along the fibres. Increase in concentration produced a peak at the end‐plate region. This was interpreted as a change in permeability such that half the maximum effect was present at a concentration of approximately 5 μ M . 4. At high concentrations the influx showed saturation and carrier‐like kinetics with a half‐saturation concentration of 400 μ M . 5. Tubocurarine inhibited the peak uptake in depolarized diaphragm. The results were consistent with competitive inhibition with an inhibitory constant of 0·07 μ M . 6. Acetylcholine in high concentration also inhibited the uptake of decamethonium in the end‐plate region of depolarized muscle.
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1. When a steady plasma level of decamethonium was maintained by infusion in rats, the labelled compound became concentrated in the region of the end-plate of skeletal muscle, as shown by scintillation counting.2. The distribution of tritium-labelled decamethonium in single muscle fibres was studied by autoradiography of frozen sections, with resolution less than 1 mu.3. After intravenous injection of a dose of decamethonium which produced partial paralysis it was shown that the labelled compound had entered muscle fibres in the region of the end-plate, and for several hundred microns on either side of the end-plate.4. Entry of decamethonium could be demonstrated as early as 30 sec after intra-arterial injection. There was no evidence of any redistribution of labelled drug for a period of 2 hr after the initial entry.5. Previous administration of tubocurarine markedly reduced the entry of labelled decamethonium.
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Interaction of cholinergic effectors with eel acetylcholinesterase was investigated in physiological eel Ringer9s solution, pH 7. The acceleration of methanesulfonation of the soluble enzyme by the cholinergic agonists decamethonium and trimethylbutylammonium ions was indistinguishable from that observed with the membrane-bound enzyme. Maximum acceleration of the methanesulfonation reaction by decamethonium and succinylcholine with the particulate acetylcholinesterase was essentially the same. A limited acceleration caused by trimethylbutylammonium ion is attributed to its partial overlap with the esteratic site, in contrast to the postulated exo mode of binding for the bisquaternary accelerator, decamethonium. Kinetic studies with trimethylbutylammonium and decamethonium ions using acetylcholine and phenyl acetate as substrates, as well as equilibrium binding studies to the enzyme, support the exo mode of binding for decamethonium, the endo mode of binding for 3-hydroxyphenyltrimethylammonium ion, and an intermediate mode of binding for trimethylbutylammonium ion. The effects produced by the quaternary nitrogen agonists on acetylcholinesterase in physiological eel Ringer9s solution can be explained on the basis of binding of the effectors to the anionic subsite of the active center.
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Interaction between different choline derivatives has been studied by applying them simultaneously to a motor end-plate and recording the resulting changes in the membrane potential of the muscle fibre. Choline potentiates the depolarizing effect of acetylcholine ( Ach ) when applied in normal Ringer. Decamethonium has a ‘diphasic’ action, initial depression of the Ach effect being followed by more prolonged potentiation. When these experiments are made after treating the muscle with an esterase inhibitor (prostigmine 10 -6 w/v), the potentiation of the Ach effect, by decamethonium or choline, is absent and replaced by simple ‘curare-like’ inhibition. When decamethonium is allowed to interact with a rapidly acting stable ester (carbaminoylcholine or succinylcholine), it produces simple 'curare-like’ inhibition. The triple effects of choline and decamethonium, i. e. (i) weak depolarization, (ii) potentiation of Ach in normal Ringer solution, (iii) inhibition of Ach in the presence of prostigmine, can be explained by competitive reactions between the drugs and receptor as well as Ach -esterase molecules. It is suggested that the first step in a depolarizing end-plate reaction is the formation of an intermediate, inactive, compound between drug and receptor.
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