The action of cholinomimetic and cholinolytic agents, hemicholinium-3 and alpha- and beta-bungarotoxin on the body wall muscle of the earthworm, Lumbricus terrestris

Strips of muscle, approximately 12 segments in length, were prepared from the body wall of the earthworm, Lumbricus terrestris, from which the nerve cord and viscera had been removed. Contractions to electrical stimulation and acetylcholine agonists were recorded using an isometric transducer. A range of nicotinic and muscarinic agonists and antagonists were tested on this preparation and the results indicate that the acetylcholine receptor on this muscle cannot be classified as either nicotinic or muscarinic. Hemicholinium-3 abolished electrically induced muscle twitches at concentrations which had no effect on the acetylcholine response. Alpha-Bungarotoxin blocked the responses to both electrical stimulation and acetylcholine while beta-bungarotoxin blocked the contractions induced by electrical stimulation but potentiated the acetylcholine contraction.     

Presynaptic effect of the aziridinium ion of acetylcholine mustard (methyl-2-acetoxyethyl-2'-chloroethylamine) on the phrenic nerve--rat diaphragm preparation.

The rat diaphragm has been used to investigate the neuromuscular blocking action of acetylcholine mustard which yields a potent nicotinic agonist, an aziridinium ion, in aqueous medium. Evidence was obtained that the acetylcholine mustard aziridinium ion impaired neuromuscular activity when the phrenic nerve was stimulated and that the ion did not directly inhibit muscle contraction. Impairment of neuromuscular activity was characterized by a latent period and depended both on the concentration of aziridinium ion and the frequency of stimulation of the phrenic nerve. Elevated concentrations of Ca-2+ and choline changed the response of the rat diaphragm to the aziridinium ion, the former increasing the rate of development of neuromuscular block and the latter protecting against neuromuscular block. These results indicated that the aziridinium ion may act either at the site of choline uptake or have an effect on acetylcholine synthesis in the nerve ending and that impairment of neuromuscular transmission in the rat diaphragm involved the availability of acetylcholine. Similar results were obtained with acetylcholine mustard aziridinium ion subjected to alkaline hydrolysis. This substance is thought to be choline mustard aziridinium ion. Although difficult to prove with the rat diaphragm it is possible that acetylcholinesterase of this preparation could hydrolyze acetylcholine mustard aziridinium ion at the neurotransmitter site and the resultant choline mustard aziridinium ion would interfere with the uptake of choline and eventually prevent neuromuscular transmission. This hemicholinium-like hypothesis for the mechanism of action of choline mustard aziridinium ion is compatible with reported date for toxicity of acetylcholine mustard aziridinium ion in the mouse.     

Nerve-Muscle Interaction In Vitro : Role of acetylcholine

Nerve and muscle cells from clonal lines interact in vitro, resulting in the association on the muscle surface of an area of increased acetylcholine sensitivity with a site of nerve-muscle contact. This localization of acetylcholine sensitivity on the muscle cell to a site of contact between nerve and muscle was found to occur when acetylcholine receptors on the muscle had been blocked with α-neurotoxin. Localization was also found to occur when the nerve cell had been prevented from releasing acetylcholine. It is concluded that neither the presence of active acetylcholine receptors on the muscle, nor the release of acetylcholine from the nerve, was required for the events leading to the localization of acetylcholine sensitivity in vitro.     

The Kinetics of Acetylcholine Turnover in a Resting Cholinergic Nerve Terminal and the Magnitude of the Cytoplasmic Compartment

Abstract: The magnitude of the cytosolic pool of acetylcholine in the cholinergic electromotor nerve terminals of Torpedo marmorata has been calculated to be 22 ± 3% of the total by comparing the isotopic ratio of acetylcholine with that of choline when slices of electric organ were incubated with 10 μM deuterated choline. The calculation is based on a two-compartment model that assumes the presence, in unstimulated tissue, of a vesicular pool of acetylcholine that does not exchange, under resting conditions, with a second cytosolic pool; the latter, by contrast, is subject to 'futile recycling' and comes into isotopic equilibrium with the tissue choline pool.     

Crystallization of kappa-bungarotoxin: preliminary X-ray data obtained from the venom-derived protein.

kappa-Bungarotoxin is a 66 residue polypeptide found in the venom of the Taiwanese banded krait, Bungarus multicinctus. It binds tightly to neuronal nicotinic acetylcholine receptors and inhibits nerve transmission mediated by these postsynaptic receptors. It is related, by similarity in amino acid sequence, to alpha-bungarotoxin and other alpha-neurotoxins, but differs sharply in physiologic action. The alpha-neurotoxins inhibit nerve transmission in nicotinic acetylcholine receptors associated with vertebrate skeletal muscle and fish electric organs. The kappa-neurotoxins inhibit nerve transmission in neuronal nicotinic acetylcholine receptors such as those found in chick ciliary ganglia. The kappa-neurotoxins display a low level of interaction with receptors that are strongly affected by alpha-neurotoxins, but alpha-neurotoxins are completely without effect on receptors that are affected by kappa-bungarotoxin. The structural basis for this physiologic differentiation is not known. Crystals of kappa-bungarotoxin have now been obtained that diffract to at least 2.3 A. These crystals are hexagonal, space group P6, and have dimensions of a = b = 80.2 A, c = 39.6 A, and angles of alpha = beta = 90 degrees and gamma = 120 degrees. Each unit cell contains 12 molecules of the 66 residue protein or two molecules per asymmetric unit. Comparison of the structure of kappa-bungarotoxin, which will result from further diffraction analysis of these crystals, with the structures of the alpha-neurotoxins that have been determined may provide information on the structural basis of physiologic action in these acetylcholine receptor antagonists.     

Phencyclidine inhibition of the acetylcholine receptor: measurement of cation flux in a sympathetic neuronal cell line using 22Na+ and spectroscopic detection of Cs+

The site of action of phencyclidine, a powerful and increasingly abused drug, in sympathetic nerve cells has not previously been identified. Here it is demonstrated that phencyclidine is a powerful, noncompetitive inhibitor of the nicotinic acetylcholine receptor in a sympathetic nerve cell line, PC-12. In the presence of 1 mM carbamoylcholine the rate of the receptor-controlled influx of 22Na+ is reduced by a factor of 2 by 0.7 microM phencyclidine. Increasing concentrations of carbamoylcholine cannot reverse the inhibitory effect of the drug. Both the transmission of electrical signals between nerve cells and the secretion of catecholamines in the PC-12 cell line depend on the receptor-controlled ion flux. Thus phencyclidine interferes with at least two specific, physiologically important functions of these nerve cells. A new spectroscopic method has been developed to measure cation flux in cells. It is shown that this method can replace measurements of tracer ion flux.     

ON THE TURNOVER OF ACETYLCHOLINE IN NERVE ENDINGS OF MOUSE BRAIN IN VIVO

Abstract— Acetylcholine is synthesized and stored in the nerve endings from which the liberation of the nerve transmittor is regulated by the nerve activity. The aim of the present investigation was to measure the in vivo turnover of acetylcholine in this subcellular acetylcholine pool. This has been carried out by injecting labelled choline intravenously and then by measuring at different time intervals the ratio between labelled choline and acetylcholine in the fractions obtained after subcellular fractionation. It was found that the ratio radioactive choline to radioactive acetylcholine was the same (2:1) in whole brain and in the nerve ending fraction 2 to 20 min after injection. Since it was assumed that the same ratio is true also for the endogenous compounds the choline pool in the nerve terminals was considered to make up 13 nmoles/g brain. The results also indicate that plasma choline is rapidly equilibrated with the nerve terminals and transformed to acetylcholine at a rate of about 5 nmoles/g brain/min.     

The effect of phenylephrine on excretion of fluid and electrolytes by the parotid and mandibular glands of the rat

The effect has been investigated of the alpha-adrenergic agonist, phenylephrine, on excretion of water and electrolytes (Na, K, and HCO3) by the parotid and mandibular glands of the rat. In the mandibular glands the agonist was as effective as acetylcholine (or parasympathetic nerve stimulation) in stimulating secretion, and the electrolyte excretory patterns seen in the two modes of stimulation were similar. In the parotid gland, phenylephrine was only one-fifth as potent as acetylcholine (or parasympathetic nerve stimulation) in evoking a secretory response but, when due allowance for flow rate differences is made, the electrolyte excretion patterns were similar. In both glands the secretory response to phenylephrine was totally different, in magnitude and in electrolyte excretion pattern, to that evoked by the beta-adrenergic agonist, isoprenaline. It is concluded, as has already been established for secretion of exportable protein, that alpha-adrenergic agonists have very similar effects to muscarinic agonists both on endpiece and on duct cells and that these actions are completely different from those evoked by activation of beta-adrenergic receptors.     

Cholinesterases in the Carotid Body of the Cat as seen with the Electron Microscope

THE response of the carotid body of the cat to the stimuli of hypoxia and hypercapnia is indicated by a large increase in signal traffic in the afferent nerve, but the transducer mechanism is unknown. There are two principal schools of thought, one proposing the involvement of an adrenergic mechanism, the other a cholinergic mechanism. Among the evidence for the cholinergic mechanism is the response of the carotid body to local injections of acetylcholine, which cause an increase in the afferent nerve impulses¹. Recently acetylcholine, or a very similar substance, has been found in the carotid body in considerable amounts².     

Analysis of the reactivity of the sensorimotor neurons of the rat cerebral cortex to acetylcholine

Several types of cholinoceptive neurons have been identified in rat's sensomotor cortex according to response character to ionophoretical application of acetylcholine. The neurons investigated form continuous range according to duration of the excitatory component of reaction to transmitter application. It has been suggested that the duration of this component reflects the important functional properties of the nerve cell.     

The effect of indomethacin and substance P on the guinea pig urinary bladder

The effects of indomethacin on the responses of the guinea pig urinary bladder to nerve stimulation, acetylcholine, adenosine 5′ triphosphate and Substance P have been investigated. Indomethacin alone had no significant effect on responses of the bladder to nerve stimulation but did significantly reduce the atropine-resistant contractions. Responses of the tissue to acetylcholine were unaffected by indomethacin but responses to Substance P were significantly reduced. Only the highest dose of ATP (10^?3 M) was significantly reduced by indomethacin. The possibility that Substance P is the transmitter responsible for the atropine-resistant contractions of the urinary bladder to nerve stimulation is discussed.     

Synaptic vesicles in electromotoneurones. II. Heterogeneity of populations is expressed in uptake properties; exocytosis and insertion of a core proteoglycan into the extracellular matrix.

The three populations of synaptic vesicles in electromotor nerve terminals were analysed quantitatively. Empty vesicles (VP0), fully charged vesicles (VP1) and charged but smaller VP2-type vesicles are present in approximately equal amounts in the nerve terminal. The populations show differences in the kinetics of in vitro uptake of acetylcholine, ATP and Ca2+. VP0 and VP2 accumulate acetylcholine and ATP but no Ca2+, whereas VP1 shows negligible acetylcholine and ATP but high Ca2+ uptake. Thus the expression of uptake properties of this secretory organelle depend on the stage it has reached in its life cycle and might constitute a signal for processing. VP2 was found to contain much less core proteoglycan than VP0 and VP1 indicating that part of it has been lost by exocytosis. In synaptic extracellular matrix containing fractions an antigen is detectable that cross-reacts with an antiserum against the vesicle proteoglycan. This material elutes upon gel filtration in a position similar to a smaller form of proteoglycan found in vesicles. We conclude that the electromotor nerve terminal releases a proteoglycan by the regulated secretory pathway that is deposited in the extracellular matrix. It might have a function in keeping pre- and postsynaptic structures in alignment constituting a transsynaptic signal. Based on the findings described, a model of the vesicles' life-cycle is discussed, whereby the VP2 population is the major source of quantal release of acetylcholine.     

Chromatographic Evidence for the Presence of Multiple Tachykinins in the Bovine Adrenal Medulla

Among the mammalian tachykinins, substance P (SP) has been shown to be the most potent at modulating the response due to nicotinic acetylcholine receptor stimulation of bovine adrenal chromaffin cells. SP-like immunoreactivity has been detected in nerve terminals innervating the adrenal medulla; however, little is known of the presence of other tachykinins in this tissue. In this study, reverse-phase HPLC was used to fractionate peptides in bovine adrenal medullary extracts, and the fractions were analyzed by radioimmunoassay using antisera to SP or neurokinin A (NKA). The results show that both NKA- and SP-like immunoreactivities are present in the adrenal medulla. The presence of neurokinin B is also indicated. The presence of multiple tachykinins in this tissue raises questions as to their functions in the adrenal medulla.     

Both A1 and A2a Purine Receptors Regulate Striatal Acetylcholine Release

The receptors responsible for the adenosine-mediated control of acetylcholine release from immunoaffinity-purified rat striatal cholinergic nerve terminals have been characterized. The relative affinities of three analogues for the inhibitory receptor were (R)-phenylisopropyladenosine greater than cyclohexyladenosine greater than N-ethylcarboxamidoadenosine (NECA), with binding being dependent of the presence of Mg2+ and inhibited by 5'-guanylylimidodiphosphate [Gpp(NH)p] and adenosine receptor antagonists. Adenosine A1 receptor agonists inhibited forskolin-stimulated cholinergic adenylate cyclase activity, with an IC50 of 0.5 nM for (R)-phenylisopropyladenosine and 500 nM for (S)-phenylisopropyladenosine. A1 agonists inhibited acetylcholine release at concentrations approximately 10% of those required to inhibit the cholinergic adenylate cyclase. High concentrations (1 microM) of adenosine A1 agonists were less effective in inhibiting both adenylate cyclase and acetylcholine release, due to the presence of a lower affinity stimulatory A2 receptor. Blockade of the A1 receptor with 8-cyclopentyl-1,3-dipropylxanthine revealed a half-maximal stimulation by NECA of the adenylate cyclase at 10 nM, and of acetylcholine release at approximately 100 nM. NECA-stimulated adenylate cyclase activity copurified with choline acetyltransferase in the preparation of the cholinergic nerve terminals, suggesting that the striatal A2 receptor is localized to cholinergic neurones. The possible role of feedback inhibitory and stimulatory receptors on cholinergic nerve terminals is discussed.     

Prolonged effects of a post-synaptic blocking fraction of Naja siamensis venom of skeletal muscle of the mouse.

A sublethal dose of a post-synaptic blocking fraction of Naja siamensis venom was injected into the soleus muscle of the mouse inhibiting neuromuscular transmission for 2-3 days. The paralysed soleus muscle behaved as if denervated, developing extra-junctional sensitivity to acetylcholine and accepting innervation by an implanted foreign nerve. Since the only known action of the post-synaptic blocking fraction of this venom is due to its affinity to acetylcholine receptors, the results suggest that the spread in the sensitivity of muscle fibres to acetylcholine and their ability to accept a foreign nerve is a consequence of neuromuscular blockade.     

Conformational Requirements of Acetylcholine Analogues and Local Anaesthetics

ATTEMPTS have been made to view the roles of acetylcholine and of cholinergic agonists as triggering permeability changes in excitable membranes through attachment to acetylcholine receptor biopolymers^1,2. Similarly, it has been proposed that local anaesthetics might block nerve conduction through attachment to axonal acetylcholine receptors^3,4. These considerations raise a number of questions. Are certain specific conformations essential for molecules related to acetylcholine either to “trigger” or to “block” depolarization of excitable membranes ? Are the conformations of such molecules identical in the crystal and in solution ? What are the rotational barriers to conformational alteration of such molecules ?     

Effects of Enucleation on High-Affinity Binding Sites in Chick Optic Tecta

Abstract: The effect of unilateral eye extirpation on the development of the chick optic tectum has been studied in both the embryo and the newly hatched chick. Although the prevention of normal afferentation of the embryonic tectum retarded its growth, there appeared to be a significant increase of muscarinic acetylcholine binding site in the noninnervated tectum. This phenomenon was repeated also in the posthatch denervated system wherein the functioning optic nerve is severed. A significant increase in the number of binding sites as well as reduced dissociation constant of the interactions of this receptor with [³H]quinuclindinyl benzilate was found in the deafferented optic tectum. This may suggest the presence of a denervation-supersensitivity-like modulation. Similar increases were not detected with other binding sites studied in either the noninnervated embryonic or deafferented posthatch optic lobes. The possibility that acetylcholine is a primary neurotransmitter of the optic system is discussed.     

Effect of ethanol on young nerve endings

The ethanol changes the quantal spontaneous release of acetylcholine and it affects the reinnervation time course. The effects of ethanol on regenerated nerve endings have been tested. 20 days after crushing sciatic nerve, the m.e.p.p. frequency at the end plate of rat extensor digitorum longus muscle keep in Ringer solution without and with ethanol has been estimated by intracellular recordings. The increase of the m.e.p.p. frequency produced by ethanol is greater in immature, than in normal nerve endings.     

Simultaneous monitoring of acetylcholine and catecholamine release in the in vivo rat adrenal medulla

To simultaneously monitor acetylcholine release from pre-ganglionic adrenal sympathetic nerve endings and catecholamine release from post-ganglionic adrenal chromaffin cells in the in vivo state, we applied microdialysis technique to anesthetized rats. Dialysis probe was implanted in the left adrenal medulla and perfused with Ringer's solution containing neostigmine (a cholinesterase inhibitor). After transection of splanchnic nerves, we electrically stimulated splanchnic nerves or locally administered acetylcholine through dialysis probes for 2 min and investigated dialysate acetylcholine, choline, norepinephrine and epinephrine responses. Acetylcholine was not detected in dialysate before nerve stimulation, but substantial acetylcholine was detected by nerve stimulation. In contrast, choline was detected in dialysate before stimulation, and dialysate choline concentration did not change with repetitive nerve stimulation. The estimated interstitial acetylcholine levels and dialysate catecholamine responses were almost identical between exogenous acetylcholine (10 microM) and nerve stimulation (2 Hz). Dialysate acetylcholine, norepinephrine and epinephrine responses were correlated with the frequencies of electrical nerve stimulation, and dialysate norepinephrine and epinephrine responses were quantitatively correlated with dialysate acetylcholine responses. Neither hexamethonium (a nicotinic receptor antagonist) nor atropine (a muscarinic receptor antagonist) affected the dialysate acetylcholine response to nerve stimulation. Microdialysis technique made it possible to simultaneously assess activities of pre-ganglionic adrenal sympathetic nerves and post-ganglionic adrenal chromaffin cells in the in vivo state and provided quantitative information about input-output relationship in the adrenal medulla.     

Evaluation of acetate uptake and its relative conversion to acetylcholine in Torpedo electric organ synaptosomes under different ionic and metabolic conditions

The uptake of acetate and its incorporation into acetylcholine were measured under various conditions in nerve terminals isolated from the electric organ in order to characterize acetate uptake and to study the relationship between acetate uptake and acetylcholine synthesis in a pure cholinergic preparation. It was found that increasing extracellular choline up to 10^?4 M had no effect on either acetate uptake or the conversion of acetate to ACh, while the addition of hemicholinium-3 to the incubation medium led to decreases in both parameters. Hence, it appears that endogenous levels of choline are sufficient to support ongoing acetylcholine synthesis in this preparation and that this synthesis depends to some extent on the uptake of extracellular choline. Nonetheless, in the absence of choline uptake, both the uptake of acetate and the conversion of acetate to acetylcholine remained substantial, indicating that internal sources of choline as well can be used for acetylcholine synthesis.Acetate uptake displayed a marked requirement for external Na⁺ and was decreased following depolarization of the synaptosomes by an elevated K⁺ concentration. The conversion of acetate to acetylcholine followed a similar pattern, except that a small reduction in acetylcholine synthesis was observed in the absence of external Ca²⁺, while acetate uptake was unaffected. The addition of ATP, AMP-PNP or phosphate to the incubation medium caused an increase in both the uptake and incorporation of acetate, but adenosine had no effect on either of these functions. Choline uptake, meanwhile, was unchanged in the presence of ATP, phosphate or adenosine. Acetate uptake appears to be more closely linked to its intracellular metabolism than to the transmembrane movement of choline itself.The mechanism by which acetate crosses the nerve terminal membrane has not been established, but the possibility that acetate is a substrate for a monocarboxylate transport system such as has been described in other systems can be ruled out as inhibitors of anion permeability do not block acetate uptake in this preparation.