Stanley M. Parsons

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.

Transport mechanisms in acetylcholine and monoamine storage

Sequence‐related vesicular acetylcholine transporter (VAChT) and vesicular monoamine transporter (VMAT) transport neurotransmitter substrates into secretory vesicles. This review seeks to identify shared and differentiated aspects of the transport mechanisms. VAChT and VMAT exchange two protons per substrate molecule with very similar initial velocity kinetics and pH dependencies. However, vesicular gradients of ACh in vivo are much smaller than the driving force for uptake and vesicular gradients of monoamines, suggesting the existence of a regulatory mechanism in ACh storage not found in monoamine storage. The importance of microscopic rather than macroscopic kinetics in structure‐function analysis is described. Transporter regions affecting binding or translocation of substrates, inhibitors, and protons have been found with photoaffinity labeling, chimeras, and single‐site mutations. VAChT and VMAT exhibit partial structural and mechanistic homology with lactose permease, which belongs to the same sequence‐defined super family, despite opposite directions of substrate transport. The vesicular transporters translocate the first proton using homologous aspartates in putative transmembrane domain X (ten), but they translocate the second proton using unknown residues that might not be conserved between them. Comparative analysis of the VAChT and VMAT transport mechanisms will aid understanding of regulation in neurotransmitter storage.—Parsons, S. M. Transport mechanisms in acetylcholine and monoamine storage. The FASEB J. 14, 2423–2434 (2000)

CHANGES IN THE ELASTIC PROPERTIES OF CHOLINERGIC SYNAPTIC VESICLES AS MEASURED BY ATOMIC FORCE MICROSCOPY

Cholinergic synaptic vesicles from Torpedo californica have been probed with the atomic force microscope in aqueous buffers to map and measure their elastic properties. Elastic properties were mapped with a new atomic force microscope technique known as force mapping. Force mapping of vesicles showed that the centers of the vesicles are harder or stiffer than the peripheral areas in the three buffers that were investigated. These were an isoosmotic buffer, a hypoosmotic buffer, and an isoosmotic buffer with 5 mM CaCl2 added. The hardness of the vesicular centers was quantified by calculation of the elastic modulus. Elastic moduli were in the range of 2-13 x 10(5) Pa. Vesicular centers were hardest in calcium-containing buffer and softest in isoosmotic buffer. Hypotheses are presented for the composition and function of the hard centers.

Acetylcholine Transport and Drug Inhibition Kinetics in Torpedo Synaptic Vesicles

Abstract: Steady‐state initial velocity uptake of [3H]acetylcholine ([3H]ACh) by purified Torpedo electric organ synaptic vesicles was studied. Transport specific activity decreased at higher vesicle concentration. Michaelis‐Menten type kinetics describe [3H]ACh active transport at constant vesicle concentration with no evidence of cooperativity or transporter heterogeneity. The ACh dissociation constant is about 0.3 mM, transport has a maximal velocity of about 1.6 nmol/min/mg protein, and both are dependent on the vesicle preparation. Nonradioactive ACh was a competitive inhibitor with respect to [3H]ACh. The potent transport inhibitor dl‐trans‐2‐(4‐phenylpiperidino)cyclohexanol (AH5183) is a non‐competitive inhibitor with respect to [3H]ACh, with an inhibition constant of 41 ± 7 nM. Inhibition by AH5183 is reversible. The results suggest that AH5183 does not bind to the ACh transporter recognition site on the outside of the vesicle membrane, and thus it might inhibit allosterically.

Vesamicol analogues as sigma ligands: Molecular determinants of selectivity at the vesamicol receptor

The present study compares the affinities of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1) and selected analogues of the latter at the vesamicol receptor (VR) with the corresponding affinities at sigma 1 and sigma 2 binding sites. For this study, the parent structure 1 was divided into three fragments: A (cyclohexyl), B (piperidyl) and C (phenyl). Vesamicol analogues were then selected to reflect structural modifications in these fragments. Consistent with earlier reports, vesamicol was found to exhibit nanomolar affinities at the VR and sigma 1 and sigma 2 sites, resulting in poor selectivity for the VR over the sigma sites. Vesamicol analogues characterized by an acyclic A-fragment showed moderate to low affinities at the VR and moderate to high affinities at sigma 1 and sigma 2 sites. As a result, many of these analogues showed poor selectivity for the VR. Replacement of the C4 carbon of 1 with a halobenzyl amine resulted in higher affinities at the VR coupled with moderate to low affinities at sigma 1 and sigma 2 sites. The introduction of a benzofused substituent at the C4 and C5 positions of 1 (compound 2) resulted in a 200-fold increase in affinity at the VR accompanied by a 5- to 6-fold decrease in affinity at sigma 1 and sigma 2 sites relative to the parent structure. Consequently, compound 2 showed 12,000-fold higher affinity at the VR than at sigma sites. Restricting the rotation of fragment C relative to B (by means of alkyl and alkenyl bridges) generally yielded analogues with subnanomolar affinities at the VR. The corresponding affinities of these spirofused conformationally restricted analogues were moderate to poor at sigma 1 and sigma 2 sites when fragment A was preserved. In contrast, the affinities at sigma 1 and sigma 2 sites were decreased 3- to 11-fold when fragment A was modified at position C4 and decreased up to 100-fold with benzofusion at the C4 and C5 positions of fragment A. Consequently, the spirofused analogues 15-19 were among the most selective VR ligands examined. Thus, the effect of conformational restriction in fragments A and B-C is to increase affinity at the VR while decreasing affinity at sigma 1 and sigma 2 sites, and thereby increasing selectivity for the VR over the sigma sites.

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.

Imaging of cholinergic terminals using the radiotracer [18F](+)-4-fluorobenzyltrozamicol: In vitro binding studies and positron emission tomography studies in nonhuman primates

The goal of the present set of studies was to characterize the in vitro binding properties and in vivo tissue kinetics for the vesicular acetylcholine transporter (VAcChT) radiotracer, [18F](+)‐4‐fluorobenzyltrozamicol ([18F](+)‐FBT). In vitro binding studies were conducted in order to determine the affinity of the (+)‐ and (−)‐ stereoisomers of FBT for the VAcChT as well as sigma (σ2 and σ2) receptors. (+)‐FBT was found to have a high affinity (Ki = 0.22 nM) for the VAcChT and lower affinities for σ1 (21.6 nM) and σ2 (35.9 nM) receptors, whereas (−)‐FBT had similar affinities for the VAcChT and σ1 receptors (∼20 nM) and a lower affinity for σ2 (110 nM) receptors. PET imaging studies were conducted in rhesus monkeys (n = 3) with [18F](+)‐FBT. [18F](+)‐FBT was found to have a high accumulation and slow rate of washout from the basal ganglia, which is consistent with the labeling of cholinergic interneurons in this brain region. [18F](+)‐FBT also displayed reversible binding kinetics during the 3 h time course of PET and produced radiolabeled metabolites that did not cross the blood‐brain barrier. The results from the current in vitro and in vivo studies indicate that [18F](+)‐FBT is a promising ligand for studying cholinergic terminal density, with PET, via the VAcChT. Synapse 25:368–380, 1997.

Synthesis and in Vitro and in Vivo Evaluation of 18F-Labeled Positron Emission Tomography (PET) Ligands for Imaging the Vesicular Acetylcholine Transporter

A new class of vesicular acetylcholine transporter inhibitor that incorporates a carbonyl group into the benzovesamicol structure was synthesized, and analogues were evaluated in vitro. (+/-)-trans-2-Hydroxy-3-(4-(4-[(18)F]fluorobenzoyl)piperidino)tetralin (9e) has K(i) values of 2.70 nM for VAChT, 191 nM for sigma(1), and 251 nM for sigma(2). The racemic precursor (9d) was resolved via chiral HPLC, and (+/-)-[(18)F]9e, (-)-[(18)F]9e, and (+)-[(18)F]9e were respectively radiolabeled via microwave irradiation of the appropriate precursors with [(18)F]/F(-) and Kryptofix/K(2)CO(3) in DMSO with radiochemical yields of approximately 50-60% and specific activities of >2000 mCi/micromol. (-)-[(18)F]9e uptake in rat brain was consistent with in vivo selectivity for the VAChT with an initial uptake of 0.911 %ID/g in rat striatum and a striatum/cerebellum ratio of 1.88 at 30 min postinjection (p.i.). MicroPET imaging of macaques demonstrated a 2.1 ratio of (-)-[(18)F]9e in putamen versus cerebellum at 2 h p.i. (-)-[(18)F]9e has potential to be a PET tracer for clinical imaging of the VAChT.

Cholinergic Synaptic Vesicles Contain a V-Type and a P-Type ATPase

Abstract: Fifty to eighty‐five percent of the ATPase activity in different preparations of cholinergic synaptic vesicles isolated from Torpedo electric organ was half‐inhibited by 7 μM vanadate. This activity is due to a recently purified phos‐phointermediate, or P‐type, ATPase. Acetylcholine (ACh) active transport by the vesicles was stimulated about 35% by vanadate, demonstrating that the P‐type enzyme is not the proton pump responsible for ACh active transport. Nearly all of the vesicle ATPase activity was inhibited by N‐ethyl‐maleimide. The P‐type ATPase could be protected from N‐ethylmaleimide inactivation by vanadate, and subsequently reactivated by complexation of vanadate with deferoxamine. The inactivation‐protection pattern suggests the presence of a vanadate‐insensitive, A‐ethylmaleimide‐sensitive ATPase consistent with a vacuolar, or V‐type, activity expected to drive ACh active transport. ACh active transport was half‐inhibited by 5 μM N‐ethylmaleimide, even in the presence of vanadate. The presence of a V‐type ATPase was confirmed by Western blots using antisera raised against three separate subunits of chromaffin granule vacuolar ATPase I. Both ATPase activities, the P‐type polypeptides, and the 38‐kilo‐dalton polypeptide of the V‐type ATPase precisely copurify with the synaptic vesicles. Solubilization of synaptic vesicles in octaethyleneglycol dodecyl ether detergent results in several‐fold stimulation of the P‐type activity and inactivation of the V‐type activity, thus explaining why the V‐type activity was not detected previously during purification of the P‐type ATPase. It is concluded that cholinergic vesicles contain a P‐type ATPase of unknown function and a V‐type ATPase which is the proton pump