Blockade Of Acetylcholine Receptors Research Articles

Role of dopamine receptor and muscarinic acetylcholine receptor blockade in the antiapomorphine action of neuroleptics

The authors analyze the role of dopamine and muscarinic acetylcholine receptor blocking components in the antistereotypic action of neuroleptics with different chemical structure. To determine dopamine-blocking activity in vitro, binding of /sup 3/H-spiperone with membranes of the rat striatum was measured. To study the blocking action of the substances on muscarinic acetylcholine receptors, binding of /sup 3/H-quinuclidinyl benzylate with brain membranes was chosen.

Blockade of cholinergic receptors by an irreversible antagonist, propylbenzilylcholine mustard (PrBCM), in the rat cerebral cortex causes deficits in passive avoidance learning

Studies were made on the effects of blockade of muscarinic acetylcholine (mACH) receptors in the rat cerebral cortex on learning and memory assessed by performance of a step-through passive avoidance task. Bilateral injection of propylbenzilylcholine mustard (PrBCM) into both the frontal and parietal cortex at doses of2.25 × 4 to 22.5 × 4μg decreased mACh receptors dose-dependently, as assessed by [ 3H]quinuclidinyl benzilate binding studies. When the training trial of a step-through passive avoidance task was performed 24 h after injection of7.5 × 4 to 22.5 × 4μg PrBCM into the frontal and parietal cortex, and then a retention test was made 24 h after the training trial, the treated rats showed shorter latencies than controls. In contrast, injection of PrBCM into the occipital cortex had no significant effect on performance in the rest. These results confirm the notion that cholinergic neurotransmission in the cerebral cortex, especially the frontoparietal cortex, is important in learning and memory. The effects of injection of PrBCM (22.5 × 4 μg) into the frontoparietal cortex on 3 postulated phases of the learning and memory process (i.e. registration, retention and recall) were also examined. (1) When PrBCM was injected 24 h before the training trial, no retention of the task was observed 14 days after the training trial. (2) However, when PrBCM was injected 24 h after the training trial, retention of the task 14 days after the training trial was not affected. (3) When PrBCM was injected 3–24 h after the initial training trial, the latencies in the retention test examined 24 h later were shorter than those of control rats. The above results suggest distinct effects of cholinergic neurotransmission on the postulated phases of the learning and memory process.

Functional blockade of neuronal acetylcholine receptors by antisera to a putative receptor from brain

Antisera to a putative acetylcholine receptor purified from chick brain specifically inhibit the acetylcholine response of chick ciliary ganglion neurons in cell culture. The putative brain receptor and a similar membrane component previously identified on ciliary ganglion neurons appear to be functional nicotinic acetylcholine receptors in the nervous system and are clearly distinct from membrane components in the tissues that bind alpha-bungarotoxin.

Drug Interactions at the Neuromuscular Junction

Summary Drugs may interact with muscle relaxants and/or general anaesthetics at the neuromuscular junction by producing a reduction in evoked acetylcholine release, by blockade of postjunctional acetylcholine receptors or acetylcholine-activated ion channels, by a depressant action on muscle contractility, or by a local anaesthetic action. The antibiotic agents are used as examples of drugs that act by some or all of the above mechanisms. The classes of clinically used antibiotics that possess neuromuscular blocking actions are the aminoglycosides, tetracyclines, polymyxins and lincosamides. Aminoglycoside-induced block may be overcome by calcium salts, but the block due to the other groups cannot be reliably reversed by pharmacological means.

Cholinergic learning deficits in the marmoset produced by scopolamine and ICV hemicholinium.

Common marmosets (Callithrix jacchus) were trained to perform daily position discrimination learning tasks in a Wisconsin General Test Apparatus. Acetylcholine receptor blockade with scopolamine was found to impair position learning. Testing on the day after scopolamine treatment suggested that a task learnt under scopolamine was not encoded into long term memory. Acetylcholine depletion achieved by the intraventricular injection of hemicholinium 4 h before testing resulted in a profound impairment of position discrimination learning. It is suggested that central acetylcholine depletion in primates may provide a useful model of senile dementia.

Evidence for acetylcholine receptor blockade by intracellular hydrogen ions in cultured chick myoballs.

Acetylcholine (ACh)-induced membrane currents were recorded in voltage-clamped myoballs in culture. The ACh sensitivity and the charge transfer per channel (Qc) were doubled in NH4+ at normal extracellular pH (pHo). Immediately after wash-out of NH4+, the ACh sensitivity and Qc undershot below the control. The recovery period of the ACh sensitivity and Qc was about 20 min. The ACh sensitivity and Qc greatly increased at alkaline pHo and decreased at acid pHo. We suggest that the intracellular H+ ions can block the ACh-induced channel. The pHo effects may be mediated by a similar (but smaller) change in the intracellular pH (pHi). Intracellular alkalinization can improve the channel function.

Anti-acetylcholine receptor antibodies directed against the alpha-bungarotoxin binding site induce a unique form of experimental myasthenia.

Of seven rat monoclonal antibodies directed against nicotinic acetylcholine receptor, the three whose binding was blocked by alpha-bungarotoxin produced acute paralysis in chicken hatchlings, whereas the four others had no effect. In the affected animals, weakness and decremental electromyographic responses appeared within 1 hr after intravenous injection and both abnormalities improved after anticholinesterase administration. No histological changes were seen in the muscle of injected animals. These data suggest that antibodies binding in relationship to the cholinergic binding site, and presumably producing pharmacologic blockade of acetylcholine receptor, may play an important role in the pathogenesis of the defective neuromuscular transmission in myasthenia gravis.

Functional activities of autoantibodies to acetylcholine receptors and the clinical severity of myasthenia gravis.

The pathogenesis of myasthenia gravis involves a humorally mediated autoimmune attack directed against acetylcholine receptors of skeletal muscles. Antibodies against acetylcholine receptors are detected in the serum of more than 80 per cent of patients, but the antibody titers correspond poorly with the severity of disease. To distinguish between antibody titers and antibody activity, we measured the ability of serum immunoglobulin from 49 patients to induce accelerated degradation or blockade of the binding sites of acetylcholine receptors, using a mammalian skeletal-muscle tissue-culture system. Immunoglobulin from 41 of 45 patients tested (91 per cent) increased the rate of degradation of acetylcholine receptors, and the relative increase in the degradation rate corresponded closely (P less than 0.001) with clinical status. Immunoglobulin from 42 of 48 patients tested (88 per cent) produced blockade of receptors, and the extent of the blockade also corresponded with clinical status (P less than 0.001). An index of the combined activities of the immunoglobulin in accelerating degradation and producing blockade of acetylcholine receptors was elevated in 43 of 44 patients (98 per cent) whose immunoglobulins were tested for both activities; this index predicted the patients' clinical status significantly better (P less than 0.001) than either measure alone. This finding suggests that the functional ability of antibodies to decrease the number of available acetylcholine receptors by these two mechanisms is clinically relevant in the pathogenesis of myasthenia gravis.

Dopamine depletion slows retinal transmission

In male hooded rats, depletion of norepinephrine and dopamine by α-methyl- para-tyrosine (AMT) significantly increased the latencies of early peaks in flash-evoked potentials recorded from the visual cortex, lateral geniculate nucleus, and optic tract. These effects were not produced by depletion of norepinephrine by FLA-63, blockade of muscarinic acetylcholine receptors by scopolamine, or blockade of opiate receptors by naloxone. AMT effects occurred only when flashes were used; optic tract stimulation failed to reveal the drug-induced changes. In a dose-response study, haloperidol produced effects similar to those of AMT. l-Dopa + RO4-4602 reversed some of the effects of AMT. It is concluded that depletion of retinal dopamine impairs the timing of retinal responses to light.

Acetylcholine in Intercostal Muscle from Myasthenia Gravis Patients and in Rat Diaphragm after Blockade of Acetylcholine Receptors

This chapter discusses whether the reduction in the number of AChRs in MG is accompanied by changes in the synthesis and release of ACh. It also investigates rat diaphragms, whose AChRs were blocked in vitro by a-bungarotoxin, or by immunization with AChR purified from Torpedo electroplax. ACh was extracted from the intercostal muscles from patients with acquired and congenital myasthenia gravis (MG) and from control patients with no clinical signs of muscle disease. ACh was estimated by mass fragmentography. It was found that the concentration of ACh was about two times higher in muscle from acquired MG than from congenital MG or control patients. Muscle from acquired MG and control patients was also incubated in Ringer with 50 mM KCl in order to stimulate the release of ACh. During the first 15 min of incubation with KCl more ACh was released from myasthenic than from control muscle, but this difference was not sustained on prolonged incubation. It was found that tetrodotoxin depressed the amount of ACh released into the medium in the presence of 50 mM KCI. Diaphragms from normal rats were treated with a-bungarotoxin, or taken from the animals that had been immunized against the ACh receptor protein from Torpedo marmorata. It was found that from these preparations KCl released about twice as much ACh as from control diaphragms.

Motor Nerve Sprouting and Acetylcholine Receptors

Sprouting of motor nerve terminals was evoked by functional denervation of skeletal muscles brought about by presynaptic blockade or disuse. The amount of sprouting, determined by morphometric measurement, was correlated with the level of extrajunctional acetylcholine receptors. Sprouting was inhibited by blockade of acetylcholine receptors with alpha-bungarotoxin. Extrajunctional acetylcholine receptors may play an important role in eliciting motor nerve terminal sprouting.

THE UPTAKE OF ATROPINE AND RELATED DRUGS BY INTESTINAL SMOOTH MUSCLE OF THE GUINEA-PIG IN RELATION TO ACETYLCHOLINE RECEPTORS.

In an attempt to study the properties of acetylcholine receptors in intestinal smooth muscle, measurements have been made of the uptake of tritium-labelled atropine and methylatropinium, and of 14 C-labelled methylfurmethide by the longitudinal muscle of guinea-pig small intestine in vitro . Substantial amounts of atropine were taken up from very dilute solutions, a clearance of 160 ml. per g tissue (wet weight) being achieved at the lowest concentration tested (1.5 × 10 -10 M). Analysis of the curve relating atropine uptake at equilibrium to the bath concentration, which was explored over a concentration range 1.5 × 10 -10 M to 2.5 × 10 -3 M, enabled three components to be distinguished: (1) A binding site with a capacity of 180 pmoles/g, and equilibrium constant 1.1 × 10 -9 M. (2) A binding site of capacity about 1000 pmoles/g and equilibrium constant about 5 × 10 -7 M. (3) A compartment with a clearance of 4.7 ml./g (nonsaturable). The equilibrium constant of the first binding site agreed exactly with that measured for acetylcholine antagonism in the same tissue. Methylatropinium was taken up in rather smaller amounts than atropine, and analysis of the uptake curve showed a binding site of capacity about 90 pmoles/g with an equilibrium constant 6.5 × 10 -10 M, an ill-defined series of binding sites with much higher equilibrium constants, and a constant clearance of about 0.4 ml. /g. Analysis of this curve was much less clear cut than that of atropine. The equilibrium constant for blockade of acetylcholine receptors by methylatropinium was 4.7 × 10 -10 M. Atropine was not taken up appreciably by striated muscle, nerve or tendon of the guineapig; hydrolysed atropine was not taken up by smooth muscle (and lacks atropinic activity); cocaine and d -tubocurarine in high concentrations did not affect atropine uptake; lachesine and benzhexol blocked atropine uptake competitively at low concentrations, and with lachesine the equilibrium constant for this interaction agreed with that measured for acetylcholine antagonism (1.4 × 10 -9 M). These findings suggested that the atropine taken up could be related to receptor-bound drug. The kinetics of atropine uptake and washout were studied over the concentration range 0.5-5 × 10 -9 M. Uptake and washout took place approximately exponentially between 2½ and 50 min, and the rate constant was 4.5-5 × 10 -4 s -1 for both uptake and washout. The uptake rate constant did not increase with concentration. This contrasted with the kinetics of receptor blockade, which took place much faster, with a rate constant which increased linearly with concentration, in accordance with the theoretical kinetic behaviour of a single binding site. This finding precluded a simple identification of atropine taken up with receptor-bound drug. Studies with various metabolic inhibitors suggested that no metabolic energy was required for the accumulation of atropine, and by dialysis experiments, the atropine taken up was shown to be bound in homogenized tissue. A theoretical study, using an analogue computer, was made of the kinetic properties of three passive binding systems, in order to see whether the observed kinetic behaviour could be simulated. It was found that a system of four binding sites in series, with only one communicating directly with the surrounding medium, could show these kinetic properties, and the outermost binding site could still show the kinetic behaviour of receptors. Experimental testing of this model demands more accurate kinetic measurements than can be made by the method used in this study. The acetylcholine-like stimulant, methylfurmethide, was taken up very slowly (taking more than 24 h to reach equilibrium), reaching a clearance of about 5 ml. /g after 6 h. This uptake was unaffected by atropine in a concentration sufficient to block 80% of acetylcholine receptors, but was blocked by depolarization in high potassium solution, suggesting that it was behaving passively as a slowly permeant cation. No uptake referable to acetylcholine receptors was detected. These findings are discussed in relation to the abundance and chemical behaviour of acetylcholine receptors in smooth muscle, and in relation to current theories of drug action.