Chris Prior

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 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.

Nicotinic antagonist-produced frequency-dependent changes in acetylcholine release from rat motor nerve terminals

1. The frequency (0.5‐150 Hz) and calcium dependence (0.5‐2.0 mM) of the effects of the nicotinic antagonist tubocurarine (0.2 microM) on acetylcholine (ACh) liberation from motor nerve terminals has been examined using binomial analysis of quantal transmitter release. 2. At an extracellular calcium ion concentration ([Ca2+]o) of 2.0 mM, tubocurarine produced a decrease in the endplate current (EPC) quantal content of approximately 30% at high frequencies of motor nerve stimulation (50‐150 Hz). In contrast, at low frequencies of stimulation (0.5‐1.0 Hz), tubocurarine enhanced the EPC quantal content by approximately 20%. 3. The enhancement of EPC quantal content produced by tubocurarine at low frequencies of motor nerve stimulation was [Ca2+]o dependent, being abolished when [Ca2+]o was lowered from 2.0 to 0.5 mM. In contrast, the decrease in quantal content produced by tubocurarine at high frequencies of motor nerve stimulation was independent of [Ca2+]o, being approximately 30% at all calcium ion concentrations studied. 4. In direct contrast to tubocurarine, the nicotinic antagonist vecuronium (1.0 microM) produced no increase in EPC quantal content at low frequencies of nerve stimulation. However, at high frequencies of nerve stimulation it decreased EPC quantal content to a similar extent to 0.2 microM tubocurarine. The frequency‐dependent decrease in EPC quantal content produced by 1.0 microM vecuronium in 2.0 mM [Ca2+]o was very similar to that seen with 0.2 microM tubocurarine in 0.5 mM [Ca2+]o. 5. Binomial analysis revealed that all the changes in EPC quantal content associated with both nicotinic antagonists were due to changes in the size of the pool of quanta in the nerve terminal available for immediate release with no effect on the probability of release of an individual quantum. 6. The results are interpreted in terms of two separately identifiable prejunctional actions of the nicotinic antagonists, both involving an action at nicotinic ACh receptors situated on the motor nerve terminal. Thus, at high frequencies of motor nerve stimulation tubocurarine and vecuronium produce a [Ca2+]o‐independent decrease in ACh release, probably through an inhibitory action on a positive‐feedback prejunctional nicotinic autoreceptor closely related to the muscle‐type nicotinic ACh autoreceptor. However, at low frequencies of motor nerve stimulation we suggest that tubocurarine, but not vecuronium, produces a [Ca2+]o‐dependent increase in ACh release through an action at a negative‐feedback prejunctional neuronal‐type nicotinic ACh autoreceptor.

Acetylcholine recycling and release at rat motor nerve terminals studied using (‐)‐vesamicol and troxpyrrolium.

1. The presynaptic mechanisms governing the release and recycling of synaptic vesicles have been studied by examining the effects of nerve stimulation, (‐)‐vesamicol (an inhibitor of acetylcholine transport into synaptic vesicles) and troxypyrrolium (an inhibitor of the high‐affinity, sodium‐dependent, choline uptake system) on endplate currents (EPCs) and miniature endplate currents (MECPs) recorded from motor endplates in cut rat hemidiaphragm preparations. 2. In control experiments, 5 min of 10 Hz nerve stimulation had no effect on either the mean or the distribution of MEPC amplitudes. 3. Nerve stimulation in the presence of (‐)‐vesamicol (25 nM‐10 microM) revealed a population of MEPCs that was unaffected by the compound and a population of MEPCs whose mean amplitude was selectively reduced by the compound. 4. Nerve stimulation in the presence of troxypyrrolium (20 microM) produced a uniform reduction in the amplitude of all MEPCs with no change in the coefficient of variance of MEPC amplitudes. 5. The concentration‐dependent effects of (‐)‐vesamicol on the amplitude of the evoked EPCs paralleled the concentration‐dependent effects of the compound on MEPC amplitudes. 6. The results are consistent with the hypothesis that both recycled and performed synaptic vesicles are heterogeneously released from rat motor nerve terminals and that (‐)‐vesamicol acts selectively on recycling vesicles. In addition, a model of vascular loading that accounts for the different effects of nerve stimulation on MEPC amplitudes in the presence of (‐)‐vesamicol and troxypyrrolium is described.

Electrophysiological analysis of transmission at the skeletal neuromuscular junction

The review is divided into two sections. The first deals with methods and problems associated with performing electrophysiological experimentation on the skeletal muscle neuromuscular junction. The second section concentrates on the computer analysis of electrophysiological data. In the first section, the various techniques available for producing skeletal muscle paralysis are described. These include the use of pharmacological manipulations, such as an excess of magnesium ions or a competitive postjunctional nicotinic acetylcholine antagonist, physiological manipulations, such as cutting the muscle fibers, and the muscle fiber sodium channel toxin, mu-conotoxin. Also, in this section, a comparison is made of the use of voltage- and current-recording techniques, including descriptions of, and solutions to, the problems associated with membrane capacitance, nonlinear summation, membrane space constant, and electrical and mechanical interference. In the second section, details are given of the types of computer system commonly used for the analysis of electrophysiological data and also the requirements of the data analysis software. The use of computer algorithms for signal detection, signal evaluation, signal averaging, and curve fitting are qualitatively described, along with some of the problems and pitfalls often associated with these methods.

Presynaptic function is altered in snake K+-depolarized motor nerve terminals containing compromised mitochondria

1 Presynaptic function was investigated at K+‐stimulated motor nerve terminals in snake costocutaneous nerve muscle preparations exposed to carbonyl cyanide m‐chlorophenylhydrazone (CCCP, 2 μm), oligomycin (8 μg ml−1) or CCCP and oligomycin together. 2 Miniature endplate currents (MEPCs) were recorded at ‐150 mV with two‐electrode voltage clamp. With all three drug treatments, during stimulation by elevated K+ (35 mm), MEPC frequencies initially increased to values > 350 s−1, but then declined. The decline occurred more rapidly in preparations treated with CCCP or CCCP and oligomycin together than in those treated with oligomycin alone. 3 Staining with FM1‐43 indicated that synaptic vesicle membrane endocytosis occurred at some CCCP‐ or oligomycin‐treated nerve terminals after 120 or 180 min of K+ stimulation, respectively. 4 The addition of glucose to stimulate production of ATP by glycolysis during sustained K+ stimulation attenuated the decline in MEPC frequency and increased the percentage of terminals stained by FM1‐43 in preparations exposed to either CCCP or oligomycin. 5 We propose that the decline in K+‐stimulated quantal release in preparations treated with CCCP, oligomycin or CCCP and oligomycin together could result from a progressive elevation of intracellular calcium concentration ([Ca2+]i). For oligomycin‐treated nerve terminals, a progressive elevation of [Ca2+]i could occur as the cytoplasmic ATP/ADP ratio decreases, causing energy‐dependent Ca2+ buffering mechanisms to fail. The decline in MEPC frequency could occur more rapidly in preparations treated with CCCP or CCCP and oligomycin together because mitochondrial Ca2+ buffering and ATP production were both inhibited. Therefore, the proposed sustained elevation of [Ca2+]i could occur more rapidly.

A further study of the neuromuscular effects of vesamicol (AH5183) and of its enantiomer specificity

1 The effects of vesamicol (2‐(4‐phenylpiperidino) cyclohexanol), an inhibitor of acetylcholine storage, and its two optical isomers have been studied on neuromuscular transmission in rat and frog muscle, and on nerve conduction in frog nerve. 2 Racemic vesamicol produced a pre‐block augmentation of twitch tension that also occurred in directly‐stimulated muscle. This effect is thus at least partially due to an increase in muscle contractility. 3 (—)‐Vesamicol was approximately 20 times more potent than (+)‐vesamicol in blocking twitches elicited at 1 Hz. This degree of stereoselectivity is similar to that measured for inhibition of acetylcholine uptake by isolated synaptic vesicles. Both enantiomers were equally weak in reducing nerve action potential amplitude in frog nerve. 4 Further studies with the active isomer, (—)‐vesamicol, showed that, like that produced by racemic vesamicol, the neuromuscular block was highly frequency‐dependent. The block was not reversed by choline or neostigmine, but was partially reversed by 4‐ or 3,4‐aminopyridine. 5 Preliminary electrophysiological studies showed that vesamicol reduced miniature endplate potential amplitude in rapidly‐stimulated frog nerve‐muscle preparations. Addition of lanthanum ions increased the frequency of miniature endplate potentials and led to the appearance of apparently normal‐sized potentials amongst those of reduced amplitude. 6 The results show the close agreement between pharmacological and biochemical observations indicating the suitability of the rat diaphragm as a test model for substances of this nature. The degree of reversibility of the vesamicol‐induced neuromuscular block by aminopyridines was unexpected, and it is suggested that in the presence of a drug which greatly increases release, a pool of acetylcholine is capable of being released which is not normally releasable after block of storage by vesamicol. It is also considered possible that the results from the intracellular recording studies may be explained in these terms.

Prejunctional actions of muscle relaxants : synaptic vesicles and transmitter mobilization as sites of action

1. Nicotinic antagonists such as tubocurarine affect acetylcholine release from motor nerve terminals at the neuromuscular junction. 2. Electrophysiological studies comparing the prejunctional actions of tubocurarine to those of vesamicol and vecuronium have been used to provide an insight into the mechanisms involved in the prejunctional effects of tubocurarine-like compounds. 3. The observed prejunctional actions of tubocurarine can be accounted for by a model in which the compound has two separately identifiable effects on the nerve terminal. At low frequencies of nerve stimulation tubocurarine augments acetylcholine release while at high frequencies of nerve stimulation tubocurarine depresses acetylcholine release. 4. Both of the effects of tubocurarine on acetylcholine release are a consequence of a change in the number of quanta within the nerve terminal immediately available for release upon nerve stimulation. 5. On the basis of our experimental observations, we suggest that the two prejunctional effects of tubocurarine are mediated through two pharmacologically distinct prejunctional nAChRs.

α-Adrenoceptor blocking properties of vesamicol

Abstract The side-effects of vesamicol, an inhibitor of acetylcholine storage, on α 1 - and α 2 -adrenoceptors have been studied in the isolated rat vas deferens. Antagonism of α 1 -adrenoceptors was determined from the ability of vesamicol to reduce contractions elicited by exogenous noradrenaline. Antagonism of α 2 -adrenoceptors was determined from the ability of vesamicol to block the inhibitory effects of the α 2 -adrenoceptor agonist clonidine on electrically evoked twitches. In the absence of noradrenaline uptake block, (−) vesamicol, the isomer active at cholinergic synapses, produced a leftward shift of the noradrenaline concentration-effect curve. This effect was abolished by desipramine suggesting that it is due to an ability of (−)-vesamicol to block uptake 1 . In the presence of noradrenaline uptake blockers, (−)-vesamicol produced a competitive, non-selective block of both α 2 - and α 2 adrenoceptors with a K d of around 40 μM (pA 2 4.4). ( + )-Vesamicol, the isomer that has no activity at cholinergic synapses, was equipotent with the (−)-isomer for blocking α 2 adrenoceptors. In addition to its α-adrenoceptor antagonist activity, (−)-vesamicol augmented the maximum response of the tissue to exogenous and endogenous noradrenaline. This study was unable to determine the exact nature of this effect. We suggest that the α-adrenoceptor blocking activity of vesamicol is a function of the phenylpiperidino moiety of the molecule.

Hexamethonium‐ and methyllycaconitine‐induced changes in acetylcholine release from rat motor nerve terminals

1 The neuronal nicotinic receptor antagonists hexamethonium and methyllycaconitine (MLA) have been used to study the putative prejunctional nicotinic ACh receptors (AChRs) mediating a negative‐feedback control of ACh release from motor nerve terminals in voltage‐clamped rat phrenic nerve/hemidiaphragm preparations. 2 Hexamethonium (200 μM), but not MLA (0.4–2.0 μM), decreased the time constant of decay of both endplate currents (e.p.cs) and miniature endplate currents (m.e.p.cs), indicating endplate ion channel block with hexamethonium. However, driving function analysis and reconvolution of e.p.cs and m.e.p.cs indicated that this ion channel block did not compromise the analysis of e.p.c. quantal content. 3 At low frequencies of stimulation (0.5–2 Hz), hexamethonium (200 μM) and MLA (2.0 μM) increased e.p.c. quantal content by 30–40%. At high frequencies (50–150 Hz) neither compound affected e.p.c. quantal content. All effects on quantal content were paralleled by changes in the size of the pool of quanta available for release. 4 The low frequency augmentation of e.p.c. quantal content by hexamethonium was absent when extracellular [Ca2+] was lowered from 2.0 to 0.5 mM. 5 At the concentrations studied, MLA and hexamethonium produced a small (10–20%) decrease in the peak amplitude of m.e.p.cs. 6 Neither apamin (100 nM) nor charybdotoxin (80 nM) had effects on spontaneous or nerve evoked current amplitudes at any frequency of stimulation. Thus the ability of nicotinic antagonists to augment e.p.c. quantal content is not due to inhibition of Ca2+‐activated K+‐channels. 7 We suggest that hexamethonium and MLA increase evoked ACh release by blocking prejunctional nicotinic AChRs. These receptors exert a negative feedback control over evoked ACh release and are probably of the α‐bungarotoxin‐insensitive neuronal type.