I.G. Marshall

Acetylcholine Transport, Storage, And Release

ACh is released from cholinergic nerve terminals under both resting and stimulated conditions. Stimulated release is mediated by exocytosis of synaptic vesicle contents. The structure and function of cholinergic vesicles are becoming known. The concentration of ACh in vesicles is about 100-fold greater than the concentration in the cytoplasm. The AChT exhibits the lowest binding specificity among known ACh-binding proteins. It is driven by efflux of protons pumped into the vesicle by the V-type ATPase. A potent pharmacology of the AChT based on the allosteric VR has been developed. It has promise for clinical applications that include in vivo evaluation of the density of cholinergic innervation in organs based on PET and SPECT. The microscopic kinetics model that has been developed and the very low transport specificity of the vesicular AChT-VR suggest that the transporter has a channel-like or multidrug resistance protein-like structure. The AChT-VR has been shown to be tightly associated with proteoglycan, which is an unexpected macromolecular relationship. Vesamicol and its analogs block evoked release of ACh from cholinergic nerve terminals after a lag period that depends on the rate of release. Recycling quanta of ACh that are sensitive to vesamicol have been identified electrophysiologically, and they constitute a functional correlate of the biochemically identified VP2 synaptic vesicles. The concept of transmitter mobilization, including the observation that the most recently synthesized ACh is the first to be released, has been greatly clarified because of the availability of vesamicol. Differences among different cholinergic nerve terminal types in the sensitivity to vesamicol, the relative amounts of readily and less releasable ACh, and other aspects of the intracellular metabolism of ACh probably are more apparent than real. They easily could arise from differences in the relative rates of competing or sequential steps in the complicated intraterminal metabolism of ACh rather than from fundamental differences among the terminals. Nonquantal release of ACh from motor nerve terminals arises at least in part from the movement of cytoplasmic ACh through the AChT located in the cytoplasmic membrane, and it is blocked by vesamicol. Possibly, the proteoglycan component of the AChT-VR produces long-term residence of the macromolecular complex in the cytoplasmic membrane through interaction with the synaptic matrix. The preponderance of evidence suggests that a significant fraction of what previously, heretofore, had been considered to be nonquantal release from the motor neuron actually is quantal release from the neuron at sites not detected electrophysiologically.(ABSTRACT TRUNCATED AT 400 WORDS)

The vesicular acetylcholine transport system

Abstract In recent years an acetylcholine uptake mechanism has been described in isolated synaptic vesicles. The drug 2-(4-phenylpiperidino) cyclohexanol (AH5183; vesamicol) has been shown to be a potent inhibitor of vesicular acetylcholine storage. Vesamicol acts in a stereoselective non-competitive manner at a site distinct from the transport ATPase and the acetylcholine transporter active site. The drug represents the prototype of a new series of pharmacological agents used to investigate prejunctional cholinergic transmission.

Feedback control of transmitter release at the neuromuscular junction

Abstract There is evidence that acetylcholine, in addition to acting on postjunctional receptors to fulfil its transmitter role, also acts on prejunctional nicotinic autoreceptors to facilitate mobilization of the transmitter. Hence, the availability of the transmitter at the sites of release keeps pace with the demand for it . Bill Bowman and colleagues demonstrate that block of the prejunctional receptors by tubocurarine and related drugs leads to a reduction in acetylcholine output and consequently to the characteristic fade of tetanic tension and rundown in trains of endplate currents .

Presynaptic Receptors in the Neuromuscular Junction

The suggestion that motor nerve endings in skeletal muscle possess cholinoceptors, though still controversial, is not new. Early evidence in support of their presence at this site notably includes that of Masland and Wigton,’ and Riker, Hubbard, and Blaber, and their coworkers. Much of their early work has been described in reviews.’“ The question of whether such prejunctional cholinoceptors, assuming they exist, play a part in the normal transmission process or merely reflect some general, perhaps vestigeal, tendency for nonmyelinated neuronal membranes to respond pharmacologically to acetylcholine is also controversial. However, several ~ o r k e r s , ’ ~ , ~ . ’ ~ ~ including ourselves, have ascribed autoreceptor functions, a t least to some of them, and have incorporated them into postulated physiological mechanisms for controlling evoked transmitter release.

The neuromuscular and autonomic blocking activities of pancuronium, Org NC 45, and other pancuronium analogues, in the cat

Twenty‐six mono‐or bis‐quaternary salts of 3,17‐dioxy‐2β,16β‐dipiperidino‐5α‐androstanes (including pancuronium) and one 17‐desoxy congener were tested for neuromuscular blocking and autonomic blocking activities in the chloralose‐anaesthetized cat. The 17β‐acetoxy series, all the members of which contain an acetylcholine‐like fragment in the steroidal D‐ring, was most selective for effecting neuromuscular blockade. The salient member of this series is 3α,17β‐diacetoxy‐2β,16β‐dipiperidino‐5α‐androstane 16β‐N‐monomethobromide (Org NC 45) which is highly selective in blocking neuromuscular transmission in that a dose approximately sixty times greater than the neuromuscular blocking dose was required to block responses to vagal stimulation. In contrast, in doses sufficient to produce neuromuscular block, pancuronium simultaneously blocked responses to vagal stimulation. Moreover, pancuronium and Org NC 45 exhibited the same order of neuromuscular blocking activity and therefore the latter potentially represents a useful addition to the armamentarium of neuromuscular blocking agents currently in clinical use.

Studies on the blocking action of 2‐(4‐phenyl piperidino) cyclohexanol (AH5183)

1 . AH5183 (2‐(4‐phenyl piperidino) cyclohexanol) produced neuromuscular block of slow onset in rapidly stimulated nerve‐skeletal muscle preparations of the rat, chicken and cat. 2 . The neuromuscular block was not antagonized by neostigmine, tetraethylammonium (TEA) or choline. The rate of onset of transmission failure was enhanced by factors which increase the release of acetylcholine. 3 . It was concluded that the neuromuscular blocking activity was primarily pre‐junctional in origin, being due either to a non‐competitive action on the choline transport mechanism, or to an intracellular action on acetylcholine metabolism. 4 . In high doses AH5183 possessed local anaesthetic activity, but this was considered insufficient to bring about the failure of neuromuscular transmission. 5 . AH5183 also produced a block of sympathetically innervated preparations that was indistinguishable from that produced by an α‐adrenoceptor blocking drug.

The actions of three diaminopyridines on the chick biventer cervicis muscle

The effects of 2,3-, 2,6- and 3,4-diaminopyridine were investigated on the isolated chick biventer cervicis muscle preparation. All three compounds reversed tubocurarine blockade and augmented twitch height in indirectly stimulated preparations. Less twitch augmentation was observed in directly stimulated preparations. 3,4-Diaminopyridine was the most effective of the compounds in facilitating neuromuscular transmission, but exhibited less convulsant activity than 4-aminopyridine. 3,4- and 2,3-diaminopyridine in high concentrations caused contractures that were inhibited by erabutoxin b or by beta-bungarotoxin. It is suggested that the diaminopyridines increase both evoked and spontaneous acetylcholine release.

Nicotinic antagonists produce differing amounts of tetanic fade in the isolated diaphragm of the rat

1 The effects of nicotine antagonists on single twitches, trains of four twitches and tetanic contractions of the isolated diaphragm of the rat were examined. 2 Different drugs were found to produce different amounts of tetanic fade relative to depression of twitch tension. 3 The order of activity from most able, to least able to produce fade was: hexamethonium> trimetaphan = atracurium = tubocurarine>pancuronium>erabutoxin b. 4 The effect of erabutoxin b was distinctive for its almost complete lack of tetanic fade. 5 3,4‐Diaminopyridine increased tetanic fade produced by tubocurarine, atracurium and hexamethonium, but not that produced by erabutoxin b. 6 It is concluded that nicotinic antagonists act at more than one site at the neuromuscular junction. Assuming block of the postjunctional acetylcholine receptor produces tension depression, a second or third site must be involved in producing tetanic fade. 7 The possibility that tetanic fade results from block of the ion channel associated with the postjunctional acetycholine receptor or from the block of a prejunctional nicotinic receptor is discussed.

The pharmacology of vesamicol: An inhibitor of the vesicular acetylcholine transporter

1. Vesamicol (2-[4-phenylpiperidino] cyclohexanol) inhibits the transport of acetylcholine into synaptic vesicles in cholinergic nerve terminals. 2. Recent pharmacological studies of the effects of vesamicol on skeletal neuromuscular transmission have revealed a pattern of activity for the compound consistent with the neurochemical observation of the mechanism of action of the compound. 3. Pharmacological manipulation of vesicular acetylcholine transport has been used to investigate the recycling and mobilization of synaptic vesicles within cholinergic nerve terminals. 4. In addition to its effects on vesicular acetylcholine transport, vesamicol also possesses some sodium channel and alpha-adrenoceptor blocking activity. 5. Vesamicol clearly represents a unique tool for investigating presynaptic mechanisms in cholinergic nerve terminals.

The actions of aminopyridines on avian muscle

SummaryThe effects of 2-, 3-, and 4-aminopyridines were investigated on the isolated chick biventer cervicis nerve-muscle preparation and on nerve-free cell cultures of embryonic chick skeletal muscle. All 3 compounds reversed tubocurarine blockade and augmented twitch height in indirectly stimulated biventer cervicis preparations. 4-Aminopyridine was approximately 10 times more potent than 2-, or 3-aminopyridine. Twitch augmentation was also seen in directly stimulated preparations but to a much lesser extent. The compounds did not have significant anticholinesterase activity, nor did they have any depolarizing activity when tested on nerve-free cultured muscle fibres. At high concentrations the aminopyridines produced a maintained contracture in the biventer preparations which was enhanced by neostigmine and inhibited by tubocurarine. It is suggested that the aminopyridines facilitate neuromuscular transmission by increasing acetylcholine release in response to nerve stimulation, and that the compounds can also increase spontaneous transmitter release.