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The effects of a water-soluble carbodiimide were examined at the frog neuromuscular junction. Acetylcholine sensitivity was measured using a fluid electrode technique and intracellular recording of miniature end-plate potentials. The carbodiimide blocked synaptic sensitivity by a reversible, curare-like action. Irreversible blockade was also observed, probably due to covalent binding. The conditions of reaction and irreversibility suggest that several different residues may be attacked. The inability of cholinergic antagonists to protect the receptor from attack indicates that nonspecific sites, and not the acetylcholine binding site, are involved.

Patients with myasthenia gravis (MG) have increased tolerance to the neuromuscular blocking properties of suxamethonium (SCh) and decamethonium (C10) and exhibit a reversal of the C10-induced block by neostigmine. The effects of these drugs were compared in forelimb flexor digitorum longus muscle from normal rats and from rats with experimental autoimmune myasthenia gravis (EAMG) to investigate the similarity of EAMG to MG. The depolarization induced by 1, 5, 10 and 25 microM SCh or C10 at the motor end-plates was significantly higher in normal than in EAMG muscle. However, both normal and EAMG end-plates responded in a similar qualitative manner to each drug. The depolarization produced by SCh was typically maintained until the drug was washed from the bath. The depolarization produced by C10 tended to decrease after reaching its peak despite continued application of the drug. With both drugs, miniature end-plate potential (MEPP) amplitude reduction is maintained until a saline wash. Neostigmine interaction with SCh and C10 in normal and EAMG muscle was compared by measuring isometric twitch tension in vitro. Neostigmine potentiated the neuromuscular block produced by either SCh or C10 in both normal and EAMG muscle. Thus muscle from rats with EAMG shares with MG an increased tolerance to SCh and C10 when compared to normal muscle but does not exhibit the qualitatively different interaction of C10 affected muscle with neostigmine that is found in MG patients. This and other studies comparing EAMG and MG indicate that EAMG is an appropriate model of MG but differences such as we have noted should be considered when extrapolating data from EAMG to the human disease.

Equilibrium measurements of the interaction of a reversible ligand with a specific membrane site which constitutes only a small component of the membrane are complicated by several factors, which are usually not considered sufficiently. These are the volume occupied by the membrane from which the ligand is excluded, and unspecific binding of the ligand. A simple method for correction of the observed measurements in a single experiment is described. The importance of this correction is illustrated in experiments conducted with the acetylcholine receptor.

1. (Methyl-3H)decamethonium dichloride was injected intravenously into the tail vein of dystrophic and normal mice of the Bar Harbour strain 129 ReJ/dy in paralysing doses. 2. Scintillation counts were made of 1 mm sections of diaphragm and tibialis anterior which showed a normal distribution of tritiated decamethonium in the dystrophic animals. 3. Intraperitoneal injections of L-(gamma-3H)leucine were made into dystrophic and normal mice. Examination of diaphragm and tibialis anterior by scintillation counting showed the abnormal uptake typical of dystrophic involvement of murine muscle. 4. The normal distribution of (methyl-3H)decamethonium dichloride uptake by the dystrophic muscle does not support the concept of active denervation in this disease.

The 24-hr, ip LD50 of triethylene glycol dinitrate (TEGDN) in rats is 995 (1063-932) mg/kg. The ip LD50 of TEGDN in rats anesthetized with pentobarbital is only 321 (605-170) mg/kg. Significant increases in pentobarbital sleep times were observed in rats pretreated with 50 mg of TEGDN/kg ip. Higher doses of TEGDN ameliorated Metrazol toxicity and increased zoxazolamine paralysis times. The peripheral nervous system toxicity of TEGDN was compared to that of decamethonium (C10). Both compounds caused severe tremors and clonic convulsions in vivo, and in vitro both selectively blocked nerve stimulated contractions of the cholinergic rat phrenic nerve diaphragm (PND) preparation, with C10 being 60 times more potent in this respect than was TEGDN. When TEGDN and C10 were applied to PND preparations in combination, the inhibition of the force of contraction occurred more rapidly and was greater than that expected from either compound applied alone. It appears that TEGDN and C10 act at the same site in vivo in the peripheral nervous system to block cholinergic transmission.

A cobra neurotoxin (Naja naja siamensis 3) acts by blocking acetylcholine receptors of mouse fast and slow muscles in an irreversible way.

Low-frequency stimulation (1–10 Hz) of the cervical sympathetic nerve is shown to increase twitch tension in the non-fatigued orbicularis oculi and oris muscles by up to 15 % and to increase contraction and half-relaxation times. The effect sets in 15–45 s following start of sympathetic stimulation, reaches a peak in 50–100 s and outlasts the stimulation by up to 90 s. The potentiating effect is not accompanied by any signs of changes in neuromuscular transmission or conduction. It is blocked by the /ff-blocker propranolol. The mechanism of sympathetic action is discussed as well as its possible physiological significance. The possible presence of non-twitch fibers of the slow tonic type in facial muscles is tested by i.v. injections of suxamethonium and decamethonium. Even relatively small doses not affecting neuromuscular transmission may produce long-lasting contractions in the orbicularis oculi and oris. It is concluded that these contractions suggest specific properties of the twitch fibers rather than pointing to the presence of slow tonic muscle fibers.

Mechanoreceptor channels were localized by using the ligands, tubocurarine (TC), decamethonium (Deca), and gallamine (Gall), which have been shown to bind specifically to these channels. The binding of radioactively labeled TC (TC*) was found to be directly proportional to the cell surface area suggesting that the channels are uniformly distributed over the cell surface. Intracellular TC and Gall injections did not depress mechanical stimulus sensitivity though these drugs did depress sensitivity when applied extracellularly at the same concentrations; therefore, the ligand binding sites are on or near the external surface of the cell. Autoradiographs revealed that radioactively labeled Deca (Deca*) bound to the pigmented stripes but not to the ciliary stripes or membranellar band. Further, Stentor induced to shed their membranellar band through exposure to 8% urea were more sensitive to mechanical stimuli than were controls; therefore, the membranellar cilia do not appear to contain mechanoreceptor channels. Collectively, these data indicate that the mechanoreceptor channels are located in the somatic surface covering the pigmented stripes. The density of mechanoreceptor channels in the plasma membrane covering the somatic surface is tentatively estimated to be between 5500 and 14,500 microns-2 based on the density of TC* binding, the apparent number of TC molecules binding per mechanoreceptor channel, and data suggesting that only one fifth to one fourth of the bound TC* is bound to structures in the plasma membrane.

1. Tritium-labelled decamethonium accumulated in diaphragm muscles of the rat in vitro with a peak at the end-plate region and the distributions were fitted by Gaussian curves. 2. Prolonged wash in physiological saline (10 hr) produced some loss in radioactivity but no detectable spread of the labelled compound along the fibres, which indicated that the decamethonium was not in a mobile form. 3. Rats injected with labelled decamethonium showed radioactivity in the diaphragm muscles after 21 days. 4. A slow spread of the labelled compound along the fibres was detected, and from the widening of the Gaussian curves the apparent diffusion coefficient was 1.2 X 10(-8) cm2sec-1, which is less than 1/500 of that in free solution.

Cations caused a decrease in the apparent Km and an increase in the Vmax. for the oxidation of exogenous NADH by both Jerusalem-artichoke (Helianthus tuberosus) and Arum maculatum (cuckoo-pint) mitochondria prepared and suspended in a low-cation medium (approximately or equal to 1 mM-K+). In Arum mitochondria the addition of cations caused a much greater stimulation of the oxidation of NAD(P)H via the cytochrome oxidase pathway than via the alternative, antimycin-insensitive, pathway. This shows that cations affected a rate-limiting step in the electron-transport chain at or beyond ubiquinone, the branch-point of electron transport in plant mitochondria. The effects were only dependent on the valency of the cation (efficiency C3+ greater than C2+ greater than C+) and not on its chemical nature, which is consistent with the theory of the diffuse layer. The results are interpreted to show that the screening of fixed negative membrane changes on lipids and protein complexes causes a conformational change in the mitochondrial inner membrane, leading to a change in a rate-limiting step of NAD(P)H oxidation. More specifically, it is proposed that screening removes electrostatic restrictions on lateral diffusion and thus accelerates diffusion-limited steps in electron transport.

The properties of the choline transport system are fundamentally altered in saline solution containing 5 mM imidazole buffer instead of 5 mM phosphate: (i) The system no longer exhibits accelerated exchange. (ii) Choline in the external compartment fails to increase the rate of inactivation of the carrier by N-ethylmaleimide. (iii) Depending on the relative concentrations of choline and imidazole, transport may be activated or inhibited. The maximum rates are increased more than fivefold by imidazole, but at moderate substrate concentrations activation is observed with low concentrations of imidazole and inhibition with high concentrations. (iv) The imidazole effect is asymmetric, there being a greater tendency to activate exit than entry. All this behavior is predicted by the carrier model if imidazole is a substrate of the choline carrier having a high maximum transport rate but a relatively low affinity, and if imidazole rapidly enters the cell by simple diffusion, so that it can add to carrier sites on both sides of the membrane. Addition at the cis side inhibits, and at the trans side activates. According to the carrier model, asymmetry is a necessary consequence of the potassium ion gradient in red cells, potassium ion being another substrate of the choline system.

There have been some articles in regard to the pharmacological study on the extraocular muscles. Katz and Eakins (1) reported the in situ effect of drugs on electrically stimulated extraocular muscle of cats, and Kern (2, 3) studied the effect of sympathomimetic amines on the isolated extraocular muscles. However, there has been no publication, to our knowledge, in regard to the electrical stimulation of the isolated extraocular muscle-nerve preparations. The investigations on the neuromuscular junction in the extraocular muscle were generally carried out in the in situ experiments. In the in situ experiments a part of responses of the extraocular muscles to drugs may be due to secondary effects accompanied with responses of the intraocular muscles to the drugs. In the present study, the effects of drugs on the electrically stimulated extraocular muscle-nerve preparations isolated from rabbits were investigated and were compared with actions of the drugs in the in situ experiments. Furthermore, actions of drugs on the isolated extraocular muscles were examined.

An attempt has been made to evaluate the antibacterial activity which, in some publications, has been suggested or attributed to halothane and methoxy-flurane Such activity would indeed offer definite advantages in the prevention of postoperative pulmonary complications and cros-infection through anaesthetic apparatus

Exposure of frog sartorius muscle to the disulfide bond reducing agent, dithiothreitol, caused a decrease in the apparent affinity of their acetylcholine receptors for some agonists. Exposure to the oxidizing agent K3Fe(CN)6 reversed this effect. After reduction, the response to agonists was irreversibly blocked by exposure of the muscles to N-ethyl maleimide or 4-(N′-maleimido)phenyltrimethylammonium. However, contrary to what had been observed with electroplax, the blocking reaction did not occur at the acetylcholine binding site of the receptor.

The effects of edrophonium and choline have been compared with those of the depolarizing substances acetylcholine, decamethonium, and suxamethonium, in both innervated and chronically denervated tibialis anterior muscles of cats under chloralose anaesthesia. Both edrophonium and choline were more potent antagonists to paralysis by tubocurarine than could be accounted for by their ability to stimulate the motor end-plates directly. It appeared likely that direct depolarization of the end-plate played no part in the anti-curare action of edrophonium and only some part in the anti-curare action of choline. A paralysis produced by the neuromuscular blocking agent, benzoquinonium, was more readily antagonized by a tetanus or by acetylcholine, suxamethonium, and decamethonium than a similar paralysis produced by tubocurarine. The tetraethyl ammonium ion was also slightly more effective against a paralysis by benzoquinonium. On the other hand, edrophonium was about 300 times and choline about five times less potent as an antagonist to benzoquinonium than to tubocurarine. Furthermore, the previous administration of benzoquinonium abolished the antagonistic action to tubocurarine of normally effective doses of edrophonium and reduced that of choline. These results were similar to those previously obtained with neostigmine, physostigmine and ethyl pyrophosphate and suggested that there was some similarity in the mechanism of action of all of these substances. Benzoquinonium, therefore, showed promise as a useful pharmacological tool for distinguishing compounds with this particular type of action. These anti-curare compounds did not appear to act by cholinesterase inhibition, not by an increase in the sensitivity of the motor end-plates. In common with other workers, we suggest that there is a pre-synaptic mechanism of action.

Decamethonium is concentrated as the unchanged compound in rat kidney cortex slices incubated (oxygen-carbon dioxide atmosphere 95:5 v/v %) in Krebs-Ringer bicarbonate medium (37°, pH 7.4) containing [ 14 C]-decamethonium. Decamethonium uptake was a saturable and metabolically dependent process. When added to medium at relatively low concentrations carbamoylcholine, choline, neostigmine and tetraethylammonium stimulated 1 hr uptake of decamethonium(2 × 10 −6 M), whereas these monoquaternary ammonium compounds at higher concentrations inhibited uptake. Initial decamethonium uptake (5 min) was increased when slices were preincubated (90 min) with a monoquaternary ammonium compound (without decamethonium) before transfer to a medium containing only decamethonium (2 × 10 −6 M). This suggests that stimulation is related to the presence of monoquaternary ammonium compounds in the slices. No decamethonium efflux (30 min) occurred from slices preincubated (1 hr) with decamethonium (2 × 10 −6 M), which means that stimulation cannot be attributed to inhibition of decamethonium efflux by monoquaternary ammonium compounds. The stimulation phenomenon can be interpreted as an example of substrate-facilitated carrier transport, which implies that efflux of monoquaternary ammonium compounds accelerates decamethonium influx. This interpretation is consistent with the conclusion from the experiments on mutual inhibition that the polymethylene-bisquaternary ammonium compound decamethonium and the above monoquaternary ammonium compounds share a common carrier mechanism.

1. 1. The question whether the protozoan Tetrahymena pyriformis possesses a phosphagen was investigated. An approach to the problem was made by characterization of the guanidine compounds of this organism and by hydrolysis studies of the phosphate-containing compounds. 2. 2. The predominant guanidine present was arginine. Two other unidentified guanidine compounds were found in trace amounts. Apparently neither of these three compounds served as a phosphagen . 3. 3. Several bases which could possibly serve as phosphagens were tested for ability to stimulate ATPase activity. Neither creatine, arginine, taurocyamine, histidine nor arcaine functioned in this respect. The addition of boiled supernatant also had no effect on ATPase activity. 4. 4. Production of inorganic phosphate by extracts of Tetrahymena under mild acid conditions was also determined. The amount produced could be accounted for by the hydrolysis of ATP. 5. 5. When decamethionium bromide was used to inhibit motility of the cells, there was no increase in acid-labile phosphate which would correspond to an increase in the amount of phosphagen present. 6. 6. No evidence for the presence of inorganic polyphosphate was found.

This report deals with 18 months' experience with electronarcosis. The establishment of a technique by the use of thiopentone and C.10 is described. It is dependent upon an efficient team and correct doses of thiopentone and C.10, as well as upon the anaesthetist's ability in caring for the apnoeic patient. Electronarcosis is a relatively safe form of physical therapy, and complications should not arise if the technique as described above is correctly carried out. In this series the few experienced could all be traced to errors in technique. Patients are surprisingly undisturbed throughout the whole course of treatment, and are able to carry out their usual activities within 3 hours. In this hospital there has been a gradual reduction in the use of chemical narcosis through the introduction of E.L.N. It is particularly noticeable that patients who are familiar with the nature of epileptiform convulsions prefer “shock” treatment with anaesthesia and curarization rather than without it, and have no hesitation in accepting E.L.N. and E.C.T. under the above conditions. In contrast to d-tubocurarine, C.10 has the following advantages : it has a quicker action, and produces as good, if not better, relaxation of the vocal cords. There is less involvement of the respiratory muscles. Its effects wear off more quickly, and the period under observation in the recovery room is much reduced. With increasing experience and confidence in the technique an increasingly larger number of senile patients are being treated. At this stage no claim is made that the method described is superior to other forms of shock therapy, but, in certain patients, it does produce striking results where other methods have failed. There is some evidence to suggest that the paranoid reaction in, older patients responds well to electronarcosis.

Hydrophobic lipoproteins (i.e. proteolipids) from rat diaphragm were separated by column chromatography with Sephadex LH20. A protein peak 2-R, eluted with chloroform, was found to bind 14 C-acetylcholine and other cholinergic ligands. Following denervation there was an increase in this protein peak which was accompanied by an increase in the binding for 14 C-acetylcholine. This effect was more marked in the non-innervated portion of the muscle. The large amount of protein and of molecules of ligand bound to peak 2-R favors the view that in normal skeletal muscle most receptors are in a non-reactive form and that they may be reactivated by denervation or by extraction with organic solvents.

A. ABDEL-AZIZ and N. BAKRY. The action and interaction of diphenhydramine (Bendryl) hydrochloride at the neuromuscular function, European J. Pharmacol. 22 (1973) 169–174. Diphenhydramine(Benadryl) · HCl, in concentrations of 5–30 μg/ml augmented indirect muscle twitches of the rat phrenic nerve-diaphragm preparation, but in higher concentrations (40–100 μg/ml) resulted in failure of neuromuseular transmission which was usually preceded by transient augmentation of twitches. This blockade was not antagonized by neostigmine. The drug had an anti-tetanic action, reduced post-tetanic potentiation and blocked augmentation of indirect muscle twitches caused by neostigmine on the rat phrenic nerve-diaphragm preparation and on the cat tibialis. Diphenhydramine reduced the action of acetylcholine on the frog rectus muscle. In the rat phrenic nerve-diaphragm preparation, depending on the concentrations of the drugs used, interaction of diphen-hydramine with thiopentone, pentobarbital and phenobaibitone, resulted in either augmentation of muscle twitches or neuromuseular blockade. In the cat tibialis, diphenhydramine antagonized the biphasic actions of decamethonium and succinylcholine, while in the case of the rat and human phrenic nerve-diaphragm preparations, it potentiated the blockade caused by decamcthonium.

1. End-plate depolarization and associated ;membrane noise' due to decamethonium, acetylthiocholine and suberyldicholine have been examined and compared with the effects of acetylcholine and carbachol.2. The transient ionic channels resulting from the action of decamethonium and acetylthiocholine are of much shorter duration than those produced by acetylcholine. They are, therefore, similarly to carbachol channels, less effective in causing membrane depolarization.3. Suberyldicholine, on the other hand, produces ion channels whose duration exceeds that of acetylcholine.4. Lowering the temperature causes an increase in amplitude of the elementary potential change produced by decamethonium and acetylthiocholine.5. The relation between the characteristic features of drug-induced membrane noise and the underlying molecular reaction rates is discussed.

The conductances in water of hexamethonium bromide and iodide, decamethonium bromide and iodide, hexadecamethonium iodide, and decaethonium iodide have been measured. The values of the limiting cationic conductances have been interpreted in terms of molecular shape, and the internitrogen distance for the above compounds calculated, using hydrodynamic relationships. The internitrogen distances found were 6·3 Å for hexamethonium, 9·5 Å for decamethonium, 13·5 Å for hexadecamethonium, and 10·2–13·5 Å for decaethonium. The effect of the hydrocarbon/water interfacial energy on the extended lengths of these molecules is discussed.

The binding properties of the purified acetylcholine receptor from Torpedo californica were investigated. One type of binding was observed for acetylcholine (KD = 2.3 μM), dimethyl tubocurarine (KD = 6.2 μM), and decamethonium (KD = 55 μM). No cooperativity was observed in ligand binding. By virtue of its ligand binding properties, the purified receptor is nicotinic in nature.

Compression of the transected spinal cord below C7 excites sympathetic neurones to elicit a vasomotor response (SCVR) and compression of the sacral cord excites parasympathetic neurones to elicit an increase in intravesical pressure (UBR). These responses were reproducible enabling a study of the spinal autonomic effects of the peripheral neuromuscular blocking agents to be made. Localization of drugs in the segments of the spinal cord was achieved by intrathecal or closed intra-arterial injection of drugs. The sciatic gastrocnemius preparation served as control to detect any possible peripheral leakage. Chloralose anaesthesia was found more suitable than pentobarbital for the study of drug effect on the autonomic neurones. d -Tubocurarine and other non-depolarizing agents facilitated the SCVR and UBR, whereas decamethonium and other depolarizing agents inhibited these responses. There was, however, transient facilitation of the SCVR following the administration of the depolarizing agents. These effects were independent of their peripheral actions. Intrathecal acetylcholine initially facilitated then depressed the spinal autonomic responses and depressed the facilitation of these responses by d -tubocurarine. Atropinization enhanced the spinal autonomic effect of d -tubocurarine, whereas GABA (intrathecal) antagonized the effect of d -tubocurarine. It is concluded that the peripheral neuromuscular blocking agents have spinal autonomic effects similar to their effects on somatic motoneurones.

The elimination from CSF of the quaternary ammonium compounds choline, methylatropine and decamethonium and the tertiary amine atropine has been investigated by ventriculo-cisternal perfusion in anesthetized dogs and rabbits. The elimination of the amines from the perfusate was partly concentration dependent. Further, in the rabbit experiments they decreased the elimination of each other. In the dog 63 per cent of the infused choline was eliminated between the lateral ventricle and cisterna magna at an infusate concentration of 5·10--6M and a rate of 150 μl/min. The corresponding figure for the rabbit was 78 per cent at 30 μl/min. Brain drug concentrations after the experiments revealed that diffusion into the brain contributed to the elimination only to a minor extent. Results were compared to in vitro studies on choroid plexa. A correlation was found between the rate of removal from perfusate for the quaternary drugs and their uptake characteristics in vitro. The conclusion was drawn that the investigated amines are eliminated from CSF partly by a carrier mediated process. One important site of this process is probably the choroid plexus.

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 μ. 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.

The effects of suxamethonium on the electroencephalogram (e.e.g.) heart rate and blood pressure were studied in patients anaesthetized with halothane. Intravenous injection of suxamethonium led to an arousal response in the e.e.g. which was accompanied by increases in heart rate and blood pressure. Similar responses were seen with decameth-onium and carbolonium. Whilst gallamine or alcuronium produced no arousal pattern in the e.e.g. they did prevent the changes produced by a subsequent injection of suxamethonium. It is postulated that the depolarizing drugs produce an increased activity in the afferent discharge of the muscle spindles which leads to the e.e.g. arousal pattern. This arousing effect may have some role in the tachycardia and hypertension produced by these drugs.

Tris-(choline chloride) phosphate (TCCP) has been observed to slowly and progressively inhibit eel acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7). The kinetics of the inhibition reaction are consistent with a mechanism involving binding of the inhibitor to the enzyme followed by a rate-limiting irreversible reaction producing inactive enzyme. The dissociation constant of the enzyme-inhibitor complex and the rate constant of the inactivation reaction have been determined to be 0.036 M and 0.2 min−1, respectively. The quaternary reversible inhibitor tetraethylammonium bromide competes with TCCP for the reaction site. TCCP does not noticeably reduce the inhibition of enzyme by decamethonium bromide as does the structurally analogous compound flaxedil. The results obtained with TCCP are compared with available data on the relative enzymic reactivity of alkoxy and thiolo esters of phosphoric acid.

Dantrolene causes skeletal muscle relaxation in animals without prominent CNS actions, and it has little or no measurable effect on smooth or cardiac muscle. Dantrolene reduces rigidity in decerebrate cats. Unlike centrally acting muscle relaxants, it has no preferential effect on polysynaptic flexor reflexes and still produces its maximum effect on the muscle twitch in an isolated, neurally intact, perfused hindlimb. Dantrolene blocks the twitch response when stimulated through the motor nerve in a way different from the action of tubocurarine or decamethonium. It inhibits direct twitch responses in denervated muscle and is equally effective in attenuating the direct and indirect twitch responses. It is hypothesized that dantrolene causes skeletal muscle relaxation by a direct action on muscle at a site beyond the neuromuscular junction.

The effects of cholinergic drugs on transmission at the cercal nervegiant fiber synapse in the desheathed sixth abdominal ganglion of the American cockroach, Periplaneta americana (L.) were evaluated. When due consideration was given to slow diffusion rates of drugs into the ganglion, and to the enzymic hydrolysis of exogenously applied choline esters by endogenous cholinesterase, the synapse was found to respond in ways consistent with cholinergic transmission. Botulinus toxin, magnesium, and hemicholinium-3, all working presynaptically blocked transmission. Atropine, D-tubocurarine, and hexamethonium produced non-depolarizing blockade at concentrations of 10−7M or 10−6M. Atropine and d-tubocurarine also blocked the action of exogenously applied acetylcholine. Postsynaptic responses were obtained from acetylcholine (10−7M), acetyl s-methyl choline (10−8M), and nicotine (10−8M). The results were discussed with respect to criteria which have been proposed for the indentification of transmitters. It was concluded that considering all of the information available on the cercal nerve-giant fiber synapse, the transmitter is almost certainly Ach.

Binding of radioactive cholinergic ligands was assayed by equilibrium dialysis to mitochondrial and synaptosomal preparations from mouse brain. Whereas 3H-atropine bound to two sites. 3H-nicotine. 3H-pilocarpine and 3H-acetylcholine (ACh) (after inhibition of cholinesterases in the tissue preparation with pyridostigmine) bound reversibly to single sites with high affinities (7·2, 8·1 and 23 nm. respectively). The 3H-nicotine binding sites (3·1 pmol/g brain) were inhibited by nicotinic but not muscarinic drugs, and the reverse effect was on the 3H-pilocarpine binding sites (3·7 pmol/g brain). The 3H-ACh binding sites were inhibited by both nicotinic and muscarinic drugs and α-bungarotoxin. and equalled approximately the total concentration of the nicotinic and muscarinic sites. It is suggested that the ACh binding sites are on the ACh receptor (AChR), which is found at concentrations similar to acetylcholinesterase in the mouse brain. These AChR sites are divided almost equally between muscarinic and nicotinic types. The data also suggest that most of the binding sites for 3H-atropine or 125I-α-bungarotoxin in the mouse brain are not on AChR.

In his response to drugs, the newborn infant is qualitatively arul quantitatively different from the mother arul even the same infant several months hence. The newborn shows marked susceptibility to the depressant effects of drugs used in labor. This heightened sensitivity is related in part to increased permeability of the blood-brain barrier, inefficient renal function, absence of microsomal enzymes necessary for drug metabolism, arul asphyxia and physical trauma associated with the delivery process. Drugs used during labor arul delivery are considered in terms of their transmission across the placenta arul pharmacologic effects on the fetus and newborn.

1 Purified acetylcholinesterase from toluene-treated organs of the electric eel (form B) was inhibited in a noncompetitive manner by the acridine derivatives proflavine and acriflavine. With proflavine secondary plots of slopes and intercepts revealed hyperbolic inhibition patterns. The secondary plots of acriflavine showed a biphasic inhibition pattern with two different slopes (slope1 > slope2). A similar biphasic inhibition pattern was observed in the Dixon plot. From the two slopes two apparent Ki-values were obtained. Between 0 and 1 μM acriflavine (low concentrations of acriflavine) the apparent inhibition constant was 0.6 μM, at acriflavine concentrations greater than 1 μM (high concentrations of acriflavine) the apparent inhibition constant was 2.2 μM. The apparent Km values for acetylthiocholine were 52 μM and 99 μM respectively. The relative V were 0.37 IU and 0.26 IU respectively. At high concentrations of acriflavine the double-reciprocal plot of substrate concentration versus enzyme activity became hyperbolic and the Hill coefficient for acetylthiocholine was 0.6. However, in presence of 2 mM hexamethonium it increased to 0.9. 2 Double inhibitor studies were carried out with acriflavine and atropine, d-(+)tubocurarine, hexamethonium, decamethonium, nicotine or tensilone. At a constant concentration of acriflavine, atropine, d-(+)tubocurarine, hexamethonium and to a lesser extent decamethonium decreased the inhibition by acriflavine. Nicotine and tensilone showed no such effect. Yonetani-Theorell plots with atropine, d-(+)tubocurarine, hexamethonium or decamethonium as constant inhibitors and acriflavine as variable inhibitor revealed a decrease in slopes2 at acriflavine concentrations above 1 μM. At acriflavine concentrations below 1 μM slopes1 did not significantly differ from each other for the pairs of inhibitors investigated. From the Yonetani-Theorell plots α-values for acriflavine (fixed inhibitor) and nicotine and tensilone (variable inhibitors) were determined. For slopes1 the values ranged between 10 and 100 depending on substrate concentration, for slope2 the values were 1–10 at 0.1 mM substrate and reached infinity at 5 mM substrate. 3 Addition of proflavine and acriflavine to acetylcholinesterase produced a bathochromic and hypochromic shift in the visible absorption spectrum. A maximum difference in the spectra was obtained at 465 nm for proflavine and at 472 nm for acriflavine. Spectral titration of acetylcholinesterase with proflavine revealed normal binding with proflavine concentrations up to 22 μM. At higher concentrations the change in absorbance was significantly greater than expected for a simple stoichiometry of one mole of dye bound per mole of active site. The Hill coefficients were 1.03 and 2.25 respectively. The binding of acriflavine followed a theoretical binding curve up to a concentration of 10 μM. At higher concentrations the change in absorbance was again greater. For the acriflavine binding the Hill coefficients were 1.05 and 1.75 respectively. Addition of a second ligand decreased the minimum and maximum of the difference spectrum, indicating a displacement of acriflavine by the second ligand. Atropine and hexamethonium were more effective in displacing acriflavine than nicotine and tensilone. 4 From these data it nas concluded that acetylcholinesterase exists in two catalytically different states. At high concentrations of acriflavine an apparent activation of the enzyme was observed in presence of atropine, d-(+)tubocurarine, hexamethonium and substrate. At low concentrations of acriflavine the enzyme was insensitive to these compounds.

The intra-arterial administration of vasodilator drugs (acetylcholine, histamine, isoproterenol, adenosine triphosphate, bradykinin, and isoxsuprine hydrochloride) induced reversible depression in the maximal contractile force of the intermittently stimulated gastrocnemius-soleus muscles in the cat. In general, blood flow to the muscles was held constant and perfusion pressure was observed. The agents were effective in depressing the muscle at doses the same as those required to produce dilatation in the resting muscle. Increase in blood flow during infusion could reverse the depression. Atropine, in amounts sufficient to block the hypotensive action of intravenously administered acetylcholine, prevented the muscle depressant action of the latter, while leaving unaffected the action of histamine. Further experiments, utilizing mainly acetylcholine as the prototype, were performed to test the hypothesis that the depressant effect on the gastrocnemius was closely related to a vascular action of the drugs. Evidence serving both to exclude neuromuscular blockade and to support a vascular effect was adduced from three sets of observations. 1) In vitro tests of blocking action of acetylcholine and histamine on nerve-muscle preparations indicated that any such action would require doses several hundred times greater than those effective in the cat gastrocnemius. 2) Under conditions where dependency of contractile force on blood flow varied, comparison of the effects of acetylcholine to d -tubocurarine and decamethonium on the cat gastrocnemius showed that when dependency on flow was reduced the effect of acetylcholine was also, while neuromuscular blockade was affected little. 3) A decrease occurred in the I 131 clearance rate from the gastrocnemius parallel to the reduction in muscle force from acetylcholine, while a much smaller, or delayed, reduction in rate occurred when force was reduced equally by d -tuboeurarine. A redistribution of blood flow within the muscle and associated connective tissue may account for the results.

Papaverine had a biphasic action on indirectly submaximally-stimulated red (m. soleus) and white (m. tibialus anterior) muscle of the cat, but no apparent effect on directly stimulated muscles. In the indirectly stimulated muscles, the initial augmentation of twitches was followed by a later decrease of contraction amplitude. The initial phase of the action of papaverine on muscle twitches was temporarily related to respiration increase and blood pressure decrease. This phase of papaverine action was observed on both supramaximally-and submaximally-stimulated muscles. There was not a large difference between the effects on the red and white muscles. Inhibition of muscle contraction evoked by d -tubocurarine was temporarily antagonized by the first phase of papaverine action. After pretreatment with adrenaline, papaverine failed to cause further increase in muscle contraction. The possible mechanism in this phase of the action of papaverine is discussed. The phase of decrease of contraction-amplitude could be seen only when the nerve was stimulated at submaximal intensity. Soleus muscle was more sensitive than anterior tibialis muscle to the inhibitory effect of papaverine. Adrenaline partially and neostigmine completely abolished the inhibition or blockade evoked by papaverine. The quantitative differences in the curare-like potency of papaverine in in vitro and in vivo experiments are discussed.

There have been various theories about the way in which drugs exert their effects. If recent progress in the isolation of receptors continues, it should soon be possible to look directly at the interaction between drugs and tissues.

The effect of temperature on the direct twitch response of the normal and denervated rat diaphragm, and the sensitivity of the contractural response of the denervated muscle to acetylcholine, suxamethonium, decamethonium, and potassium salts, was studied. Cooling the normal diaphragm from 39 to 20 degrees increased the size of the direct twitch, but below this temperature the twitch response of the muscle was progressively depressed. The denervated muscle twitch was, by contrast, maximal at 39 degrees and decreases as the temperature was lowered. This property of the denervated muscle first appeared about the third day after denervation. The contractural response of the denervated diaphragm to applied acetylcholine was enhanced by cooling, while the response to potassium salts was depressed. The depression of the contracture induced by potassium followed closely the depression of the direct muscle twitch of the denervated muscle over the same temperature range.

1. The transfer of choline was studied with sacs of everted intestine of rat and hamster. 2. The choline transfer can be divided into two components, a diffusion process and a saturable process. The latter plays a relatively greater part at low concentrations of choline, which include the physiological concentration in the plasma. The saturable process is better seen in the hamster than in the rat. 3. Intestinal transfer of choline is influenced by substances altering the availability of energy in the cell, and by some substances chemically or pharmacologically related to choline. These findings are consistent with some kind of specific mechanism for choline transfer. 4. Part of the choline taken up by the cell appears as a metabolite not yet identified. The formation of the metabolite is a saturable process and is abolished by anaerobic conditions and by homogenization. 5. The results are also discussed in relation to parameters of transfer.