piperidines and vesamicol

The vesicular acetylcholine transporter (VAChT), a presynaptic cholinergic neuron marker, is a potential internal molecular target for the development of an imaging agent for early diagnosis of neurodegenerative disorders with cognitive decline such as Alzheimer's disease (AD). Since vesamicol has been reported to bind to VAChT with high affinity, many vesamicol analogs have been studied as VAChT imaging agents for the diagnosis of cholinergic neurodeficit disorder. However, because many vesamicol analogs, as well as vesamicol, bound to sigma receptors (

Topics: Alzheimer Disease; Animals; Humans; Neurodegenerative Diseases; Piperidines; Radiopharmaceuticals; Structure-Activity Relationship; Vesicular Acetylcholine Transport Proteins

The human cardiac nervous system consists of a sympathetic and a parasympathetic branch with (-)-norepinephrine and acetylcholine as the respective endogenous neurotransmitters. Dysfunction of the cardiac nervous system is implicated in various types of cardiac disease, such as heart failure, myocardial infarction and diabetic autonomic neuropathy. In vivo assessment of the distribution and function of cardiac sympathetic and parasympathetic neurones with positron emission tomography (PET) and single-photon emission tomography (SPET) can be achieved by means of a number of carbon-11-, fluorine-18-, bromine-76- and iodine-123-labelled tracer molecules. Available tracers for mapping sympathetic neurones can be divided into radiolabelled catecholamines, such as 6-[18F]fluorodopamine, (-)-6-[18F]fluoronorepinephrine and (-)-[11C]epinephrine, and radiolabelled catecholamine analogues, such as [123I]meta-iodobenzylguanidine, [11C]meta-hydroxyephedrine, [18F]fluorometaraminol, [11C]phenylephrine and meta-[76Br]bromobenzylguanidine. Resistance to metabolism by monoamine oxidase and catechol-O-methyl transferase simplifies the myocardial kinetics of the second group. Both groups of compounds are excellent agents for an overall assessment of sympathetic innervation. Biomathematical modelling of tracer kinetics is complicated by the complexity of the steps governing neuronal uptake, retention and release of these agents as well as by their high neuronal affinity, which leads to partial flow dependence of uptake. Mapping of cardiac parasympathetic neurones is limited by a low density and focal distribution pattern of these neurones in myocardium. Available tracers are derivatives of vesamicol, a molecule that binds to a receptor associated with the vesicular acetylcholine transporter. Compounds like (-)-[18F]fluoroethoxybenzovesamicol display a high degree of non-specific binding in myocardium which restricts their utility for cardiac neuronal imaging.

Topics: Catecholamines; Heart; Heart Conduction System; Humans; Parasympathetic Nervous System; Piperidines; Radiopharmaceuticals; Sympathetic Nervous System; Tomography, Emission-Computed; Tomography, Emission-Computed, Single-Photon

Topics: Acetylcholine; Animals; Carrier Proteins; Kinetics; Membrane Transport Proteins; Models, Biological; Piperidines; Proteoglycans; Receptors, Cholinergic; Substrate Specificity; Synaptic Vesicles; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Topics: Acetylcholine; Adenosine; Animals; Biological Transport; Calcium; Cats; Cell Compartmentation; Electric Stimulation; Ganglia, Sympathetic; Neurotoxins; Parasympatholytics; Piperidines; Synaptic Vesicles

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.

Topics: Acetylcholine; Animals; Carrier Proteins; Humans; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Piperidines; Synaptic Vesicles; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Sigma-1 receptor imaging probes for determining the expression levels are desirable for diagnoses of various diseases and companion diagnoses of therapeutic agents targeting the sigma-1 receptor. In this study, we aimed to develop probes with higher affinity for the sigma-1 receptor. For this purpose, we synthesized and evaluated compounds, namely, vesamicol derivatives, in which alkyl chains of varying chain length were introduced between a piperazine ring and a benzene ring. The binding affinity of the vesamicol derivatives for the sigma-1 receptor tended to increase depending on the length of the alkyl chain between the benzene ring and the piperazine ring. The sigma-1 receptor of 2-(4-(3-phenylpropyl)piperazin-1-yl)cyclohexan-1-ol (5) (K

Topics: Animals; Aza Compounds; Cell Line, Tumor; Humans; Iodine Radioisotopes; Isotope Labeling; Male; Mice; Mice, Nude; Neoplasms; Piperidines; Protein Binding; Radiopharmaceuticals; Receptors, sigma; Sigma-1 Receptor; Tissue Distribution; Tomography, Emission-Computed, Single-Photon; Transplantation, Heterologous

Sigma-1 receptor is a target for tumor imaging. In a previous study, we synthesized a vesamicol analog, (+)-2-[4-(4-bromophenyl)piperidino]cyclohexanol [(+)-pBrV], with a high affinity for sigma-1 receptor, and synthesized radiobrominated (+)-pBrV. This radiobrominated (+)-pBrV showed high tumor uptake in tumor-bearing mice; however, radioactivity accumulation in normal tissues, such as the liver, was high. We assumed that the accumulation of (+)-pBrV in the non-target tissues was partially derived from its high lipophilicity; therefore, we synthesized and evaluated (+)-4-[1-(2-hydroxycyclohexyl)piperidine-4-yl]-2-bromophenol [(+)-BrV-OH], which is a more hydrophilic compound. Although we aimed to develop a PET tracer using. (+)-[. The lipophilicity and affinity for sigma-1 receptor of (+)-[. These results indicate that (+)-[

Topics: Animals; Biological Transport; Bromine Radioisotopes; Half-Life; Hydrophobic and Hydrophilic Interactions; Isotope Labeling; Male; Mice; Piperidines; Positron-Emission Tomography; Receptors, sigma; Sigma-1 Receptor; Tissue Distribution

The vesicular acetylcholine transporter (VAChT) is an important target for in vivo imaging of neurodegenerative processes using positron emission tomography (PET). So far the development of VAChT PET radioligands is based on the single known lead compound vesamicol. In this study we investigated a recently published spiroindoline based compound class (Sluder et al., 2012), which was suggested to have potential in the development of VAChT ligands. Therefore, we synthesized a small series of N,N-substituted spiro[indoline-3,4'-piperidine] derivatives and determined their in vitro binding affinities toward the VAChT. In order to investigate the selectivity, the off-target binding toward σ

Topics: Animals; Brain Chemistry; Female; Indoles; Ligands; Liver; PC12 Cells; Piperidines; Positron-Emission Tomography; Radioisotopes; Rats; Rats, Sprague-Dawley; Spiro Compounds; Vesicular Acetylcholine Transport Proteins

First highly enantioselective synthesis of biologically important vesamicol, benzovesamicol, and their derivatives was achieved via the desymmetrization of meso-epoxides with secondary aliphatic amines (4-phenylpiperidine derivatives) using a chiral [salenCo(iii)-BF

Topics: Amines; Catalysis; Cobalt; Epoxy Compounds; Molecular Structure; Optical Phenomena; Organometallic Compounds; Piperidines; Stereoisomerism

To verify vesamicol as lead structure in the development of radioligands for imaging of VAChT in the brain by PET, we systematically modified this molecule and investigated four different groups of derivatives. Structural changes were conducted in all three ring systems A, B, and C resulting in a library of different vesamicol analogs. Based on their in vitro binding affinity toward VAChT as well as σ1 and σ2 receptors, we performed a structure-affinity relationship (SAR) study regarding both affinity and selectivity. The compounds possessed VAChT affinities in the range of 1.32 nM (benzovesamicol) to >10 μM and selectivity factors from 0.1 to 73 regarding σ1 and σ2 receptors, respectively. We could confirm the exceptional position of benzovesamicols as most affine VAChT ligands. However, we also observed that most of the compounds with high VAChT affinity demonstrated considerable affinity in particular to the σ1 receptor. Finally, none of the various vesamicol analogs in all four groups showed an in vitro binding profile suitable for specific VAChT imaging in the brain.

Topics: Animals; Brain; Dose-Response Relationship, Drug; Female; Molecular Imaging; Molecular Structure; PC12 Cells; Piperidines; Positron-Emission Tomography; Radioligand Assay; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Vesicular Acetylcholine Transport Proteins

In this study, the regional rat brain distribution of radioiodinated o-iodo-trans-decalinvesamicol ([(125) I]OIDV) was determined in vivo to evaluate its potential as a single-photon emission computed tomography (SPECT) imaging probe for vesicular acetylcholine transporter (VAChT). Following intravenous injection, [(125) I]OIDV passed freely across the blood-brain barrier and accumulated in rat brain. The accumulation of [(125) I]OIDV in rat brain was significantly reduced by coadministration of (+/-)-vesamicol (0.125 µmol). In contrast, the coadministration of σ-receptor ligands, such as (+)-pentazocine (0.125 µmol) as a σ-1 receptor ligand and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine (0.125 µmol) as a σ-1 and σ-2 receptor ligands, barely affected the accumulation of [(125) I]OIDV in rat brain. These findings in vivo were corroborated by autoradiographic analysis ex vivo. The authors found that the tracer binds with pharmacological selectivity to VAChT in rat brain and predicted that it may likewise serve in translational SPECT imaging studies of this marker in the integrity of cholinergic innervations.

Topics: Animals; Autoradiography; Blood-Brain Barrier; Brain; Iodine Radioisotopes; Ligands; Male; Narcotic Antagonists; Pentazocine; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, sigma; Sigma-1 Receptor; Tomography, Emission-Computed, Single-Photon; Vesicular Acetylcholine Transport Proteins

We investigated the characteristics of the regional rat brain distribution of radio-brominated o-bromo-decalinvesamicol (OBDV) in vivo to evaluate its potential as a PET ligand for vesicular acetylcholine transporter (VAChT). In in vivo biodistribution study, the specific brain regional accumulation of [(77) Br]OBDV was revealed 30 min after intravenous injection. The specific brain regional accumulation of [(77) Br]OBDV was significantly inhibited by co-injection of (+/-)-vesamicol. In contrast, no significant inhibition of the uptake of [(77) Br]OBDV in all brain regions was observed with co-injection of (+)-pentazocine (selective σ-1 receptor agonist) and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine, [(+)-3-PPP] (σ-1 and σ-2 receptor agonist) with [(77) Br]OBDV. [(77) Br]OBDV accumulation in VAChT-rich brain regions was observed in ex vivo autoradiography. These results showed that [(77) Br]OBDV selectively bound to VAChT with high affinity in rat brain in vivo. Hence, OVBDV radiolabelled with more suitable (76) Br was suggested to be a potent VAChT ligand for PET.

Topics: Animals; Autoradiography; Brain; Bromine Radioisotopes; Central Nervous System Agents; Male; Pentazocine; Piperidines; Positron-Emission Tomography; Radiopharmaceuticals; Rats, Sprague-Dawley; Receptors, sigma; Vesicular Acetylcholine Transport Proteins

Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by α7-, α3β2-, and β3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II-induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy.

Topics: Acetylcholine; Adenocarcinoma, Bronchiolo-Alveolar; Animals; Apoptosis; Blotting, Western; Cell Line, Tumor; Cells, Cultured; Humans; Immunohistochemistry; Lung Neoplasms; Male; Mice; Mice, Nude; Neuromuscular Depolarizing Agents; Phosphorylation; Piperidines; Proto-Oncogene Proteins c-akt; RNA Interference; Signal Transduction; Vesicular Acetylcholine Transport Proteins; Xenograft Model Antitumor Assays

Effects of organophosphorous acetylcholinesterase inhibitor paraoxon were studied in the isolated atrial and ventricular myocardium preparations of a fish (cod), an amphibian (frog) and a mammal (rat) using the microelectrode technique. Incubation of isolated atrium with paraoxon (5 × 10(-6)-5 × 10(-5) M) caused significant reduction of action potential duration and marked slowing of sinus rhythm. These effects were abolished by muscarinic blocker atropine and therefore are caused by acetylcholine, which accumulates in the myocardium due to acetylcholinesterase inhibition even in the absence of vagal input. Hemicholinium III is a blocker of high affinity choline-uptake transporters, which are believed to mediate non-quantal release of acetylcholine from cholinergic terminals in different tissues. In the atrial myocardium of all the three studied species, hemicholinium III (10(-5) M) significantly suppressed all the effects of paraoxon. Blocker of parasympathetic ganglionic transmission hexamethonium bromide (10(-4) M) and inhibitor of vesicular acetylcholine transporters vesamicol (10(-5) M) failed to attenuate paraoxon effects. Among ventricular myocardium preparations of three species paraoxon provoked marked cholinergic effects only in frog, hemicholinium III abolished these effects effectively. We conclude that paraoxon stops degradation of acetylcholine in the myocardium and helps to reveal the effects of acetylcholine, which is continuously secreted from the cholinergic nerves in non-quantal manner. Thus, non-quantal release of acetylcholine in the heart is not specific only for mammals, but is also present in the hearts of different vertebrates.

Topics: Acetylcholine; Animals; Atropine; Cholinesterase Inhibitors; Gadus morhua; Heart; Heart Atria; Heart Ventricles; Hemicholinium 3; In Vitro Techniques; Male; Myocardium; Paraoxon; Piperidines; Rana temporaria; Rats; Rats, Wistar; Vertebrates

This Letter describes the synthesis of two regioisomers of a new class of vesamicol analogs as possible ligands for imaging the vesicular acetylcholine transporter in future PET studies. The two pyrrolovesamicols (±)-6a and (±)-6b were synthesized by nucleophilic ring opening reaction of a tetrahydroindole epoxide precursor with 4-phenylpiperidine. The reaction mechanism of the synthesis was studied by HPLC and the molecular structures were determined by X-ray structure analysis. Unexpected low binding affinities to VAChT (K(i)=312±73 nM for (±)-6a and K(i)=7320±1840 nM for (±)-6b) were determined by competitive binding analysis using a cell line stably transfected with ratVAChT and (-)-[(3)H]vesamicol.

Topics: Animals; Binding Sites; Binding, Competitive; Cell Line; Chromatography, High Pressure Liquid; Contrast Media; Crystallography, X-Ray; Epoxy Compounds; Fluorine Radioisotopes; Humans; Ligands; Molecular Structure; Piperidines; Positron-Emission Tomography; Pyrroles; Rats; Stereoisomerism; Tritium; Vesicular Acetylcholine Transport Proteins

Release of stored calcium ions during activation of ryanodine receptors with ryanodine or caffeine elevates the mean amplitude of spontaneous miniature end-plate potentials. Blockade of these receptors with selective antagonists abolishes this effect. Preliminary loading of the motor nerve terminals with intracellular calcium buffer EGTA-AM, but not with BAPTA-AM, also completely prevented the ryanodine-induced increment of miniature end-plate potential amplitude probably induced by the release of stored calcium. Vesamicol, a selective blocker of acetylcholine transport into vesicles, prevented the ryanodine-induced increment of the mean amplitude of miniature end-plate potentials. This increment was 2-fold more pronounced after preliminary blockade of protein kinase C with chelerythrine and was completely abolished by blockade of protein kinase A with H-89.

Topics: Acetylcholine; Animals; Benzophenanthridines; Caffeine; Calcium; Cations, Divalent; Cholinergic Antagonists; Cyclic AMP-Dependent Protein Kinases; Egtazic Acid; Isoquinolines; Mice; Miniature Postsynaptic Potentials; Neuromuscular Junction; Piperidines; Presynaptic Terminals; Protein Kinase C; Ryanodine; Ryanodine Receptor Calcium Release Channel; Sulfonamides; Tissue Culture Techniques

In the resting state, motor neurons continuously release ACh through quantal and non-quantal mechanisms, the latter through vesicular ACh transporter (VAChT) and choline transporter (ChT). Although in skeletal muscle these mechanisms have been extensively studied, the non-quantal release (NQR) from parasympathetic neurons of airway smooth muscle has not been described. Here we corroborated that the organophosphate paraoxon (acetylcholinesterase inhibitor) induced a contraction blocked by atropine (muscarinic antagonist) in guinea-pig tracheal rings. This contraction was not modified by two blockers of evoked quantal release, tetrodotoxin (voltage-dependent Na(+) channel blocker) and ω-conotoxin GVIA (N-type Ca(2+) channel blocker), nor by the nicotinic blocker hexamethonium, suggesting that acetylcholine NQR could be responsible of the paraoxon-induced contraction. We confirmed that tetrodotoxin, and to some extent -conotoxin, abolished the evoked quantal ACh release induced by electrical field stimulation. Hemicholinium-3 (ChT inhibitor), but not vesamicol (VAChT inhibitor), caused a concentration-dependent inhibition of the response to paraoxon. The highest concentration of hemicholinium-3 left ∼75% of the response to electrical field stimulation, implying that inhibition of paraoxon-induced contraction was not due to depletion of neuronal vesicles. Non-neuronal sources of ACh released through organic cation transporters were discarded because their inhibition by quinine or corticosterone did not modify the response to paraoxon. Calcium-free medium abolished the effect of paraoxon, and NiCl(2), 2-aminoethyl diphenyl-borate and SKF 96365 partly inhibited it, suggesting that non-specific cation channels were involved in the acetylcholine NQR. We concluded that a Ca(2+)-dependent NQR of ACh is present in cholinergic nerves from guinea-pig airways, and that ChT is involved in this phenomenon.

Topics: Acetylcholine; Animals; Atropine; Calcium; Cation Transport Proteins; Cholinesterase Inhibitors; Electric Stimulation; Guinea Pigs; Hemicholinium 3; Hexamethonium; Male; Membrane Transport Proteins; Muscle Contraction; Muscle, Smooth; Neurons; omega-Conotoxin GVIA; Organophosphates; Paraoxon; Piperidines; Tetrodotoxin; Trachea; Vesicular Acetylcholine Transport Proteins

Acetylcholine is a neurotransmitter that has a major role in the function of the insulin-secreting pancreatic beta cell. Parasympathetic innervation of the endocrine pancreas, the islets of Langerhans, has been shown to provide cholinergic input to the beta cell in several species, but the role of autonomic innervation in human beta cell function is at present unclear. Here we show that, in contrast to the case in mouse islets, cholinergic innervation of human islets is sparse. Instead, we find that the alpha cells of human islets provide paracrine cholinergic input to surrounding endocrine cells. Human alpha cells express the vesicular acetylcholine transporter and release acetylcholine when stimulated with kainate or a lowering in glucose concentration. Acetylcholine secretion by alpha cells in turn sensitizes the beta cell response to increases in glucose concentration. Our results demonstrate that in human islets acetylcholine is a paracrine signal that primes the beta cell to respond optimally to subsequent increases in glucose concentration. Cholinergic signaling within islets represents a potential therapeutic target in diabetes, highlighting the relevance of this advance to future drug development.

Topics: Acetylcholine; Alkenes; Animals; Dose-Response Relationship, Drug; Glucagon-Secreting Cells; Glucose; Humans; Insulin; Insulin Secretion; Insulin-Secreting Cells; Mice; Physostigmine; Piperidines; Receptors, Cholinergic; Secretory Vesicles; Signal Transduction; Vesicular Acetylcholine Transport Proteins

To identify selective high-affinity inhibitors of the vesicular acetylcholine transporter (VAChT), we have interposed a carbonyl group between the phenyl and piperidyl groups of the prototypical VAChT ligand vesamicol and its more potent analogues benzovesamicol and 5-aminobenzovesamicol. Of 33 compounds synthesized and tested, 6 display very high affinity for VAChT (K(i), 0.25-0.66 nM) and greater than 500-fold selectivity for VAChT over sigma(1) and sigma(2) receptors. Twelve compounds have high affinity (K(i), 1.0-10 nM) and good selectivity for VAChT. Furthermore, 3 halogenated compounds, namely, trans-3-[4-(4-fluorobenzoyl)piperidinyl]-2-hydroxy-1,2,3,4-tetrahydronaphthalene (28b) (K(i) = 2.7 nM, VAChT/sigma selectivity index = 70), trans-3-[4-(5-iodothienylcarbonyl)piperidinyl]-2-hydroxy-1,2,3,4-tetrahydronaphthalene (28h) (K(i) = 0.66 nM, VAChT/sigma selectivity index = 294), and 5-amino-3-[4-(p-fluorobenzoyl)piperidinyl]-2-hydroxy-1,2,3,4,-tetrahydronaphthalene (30b) (K(i) = 2.40 nM, VAChT/sigma selectivity index = 410) display moderate to high selectivity for VAChT. These three compounds can be synthesized with the corresponding radioisotopes so as to serve as PET/SPECT probes for imaging the VAChT in vivo.

Topics: Animals; Drug Discovery; Guinea Pigs; Inhibitory Concentration 50; Piperidines; Rats; Structure-Activity Relationship; Substrate Specificity; Torpedo; Vesicular Acetylcholine Transport Proteins

Invariant E309 is in contact with critical and invariant D398 in a three-dimensional homology model of vesicular acetylcholine transporter (VAChT, TC 2.A.1.2.13) [Vardy, E., et al. (2004) Protein Sci. 13, 1832-1840]. In the work reported here, E309 and D398 in human VAChT were mutated singly and together to test their functions, assign pK values to them, and determine whether the residues are close to each other in three-dimensional space. Mutants were stably expressed in the PC12(A123.7) cell line, and transport and binding properties were characterized at different pH values using radiolabeled ligands and filtration assays. Contrary to a prior conclusion, the results demonstrate that most D398 mutants do not bind the allosteric inhibitor vesamicol even weakly. Earlier work showed that most D398 mutants do not transport ACh. D398 therefore probably is the residue that must deprotonate with a pK of 6.5 for binding of vesamicol and with a pK of approximately 5.9 for transport of ACh. Because E309Q has no effect on VAChT functions at physiological pH, E309 has no apparent critical role. However, radical mutations in E309 cause decreases in ACh and vesamicol affinities and total loss of ACh transport. Unlike wild-type VAChT, which exhibits a peak of [(3)H]vesamicol binding centered at pH 7.4, mutants E309Q, E309D, E309A, and E309K all exhibit peaks of binding centered at pH >or=9. The combination of high pH and mutated E309 apparently produces a relaxed (in contrast to tense) conformation of VAChT that binds vesamicol exceptionally tightly. No compensatory interactions between E309 and D398 in double mutants were discovered. Proof of a close spatial relationship between E309 and D398 was not found. Nevertheless, the data are more consistent with the homology model than an alternative hydropathy model of VAChT that likely locates E309 far from D398 and the ACh binding site in three-dimensional space. Also, a probable network of interactions involving E309 and an unknown residue having a pK of 10 has been revealed.

Topics: Acetylcholine; Animals; Biological Transport; Humans; Hydrogen-Ion Concentration; Models, Molecular; Mutation; PC12 Cells; Piperidines; Protein Conformation; Radioligand Assay; Rats; Vesicular Acetylcholine Transport Proteins

The method describes production and the selection of neurosecretory PC12A123.7 cells stably transfected with human vesicular acetylcholine transporter (hVAChT). Transfected cells provide postnuclear supernatant used to characterize equilibrium binding of the neurotransmitter acetylcholine (ACh), the pH dependence for transport of ACh, and the rate behavior for dissociation of the allosteric, high-affinity inhibitor vesamicol. Retention of radiolabeled ACh or vesamicol, mediated by hVAChT in synaptic-like microvesicles of postnuclear supernatant, is measured using filter assays. The procedure for regression analysis of data also is described.

Topics: Acetylcholine; Animals; Biological Transport; Blotting, Western; Hydrogen-Ion Concentration; PC12 Cells; Piperidines; Protein Binding; Rats; Vesicular Acetylcholine Transport Proteins

High-performance liquid chromatography enantioseparation of vesamicol and six novel azaspirovesamicols (amino alcohols) was accomplished on different chiral stationary phases (CSPs) by using an optical rotation based chiral detector for identification of the resolved enantiomers. The Pirkle-type column Reprosil Chiral-NR was found to be most suitable for chiral resolution in normal phase (NP) mode; all compounds could be enantioseparated successfully. Also the cellulose-based column Reprosil Chiral-OM showed appropriate separation properties by using NP conditions. The amylose-type column Reprosil Chiral-AM-RP was most suitable for enantioseparation in reversed phase (RP) mode; five out of seven compounds were resolved. This CSP showed a considerably higher capability for chiral recognition of vesamicol derivatives in RP mode than the corresponding cellulose-based column Reprosil Chiral-OM-RP. Enantioseparation with the teicoplanin aglycone-based column Reprosil Chiral-AA was successful under polar ionic mobile phase conditions.

Topics: Amylose; Cellulose; Chromatography, High Pressure Liquid; Hydrophobic and Hydrophilic Interactions; Piperidines; Polyvinyls; Siloxanes; Stereoisomerism; Teicoplanin

The amount of neurotransmitter released from a presynaptic terminal is the product of the quantal content (number of vesicles) and the presynaptic quantal size (QSpre, amount of transmitter per vesicle). QSpre varies with synaptic use, but its regulation is poorly understood. The motor nerve terminals at the neuromuscular junction (NMJ) contain TGF-beta receptors. We present evidence that TGF-beta2 regulates QSpre at the NMJ. Application of TGF-beta2 to the rat diaphragm NMJ increased the postsynaptic response to both spontaneous and evoked release of acetylcholine, whereas antibodies to TGF-beta2 or its receptor had the converse effect. L-vesamicol and bafilomycin blocked the actions of TGF-beta2, indicating that TGF-beta2 acts by altering the extent of vesicular filling. Recordings of the postsynaptic currents from the diaphragm were consistent with TGF-beta2 having this presynaptic action and a lesser postsynaptic effect on input resistance. TGF-beta2 also decreased quantal content by an atropine-sensitive pathway, indicating that this change is secondary to cholinergic feedback on vesicular release. Consequently, the net actions of TGF-beta2 at the NMJ were to amplify the postsynaptic effects of spontaneous transmission and to diminish the number of vesicles used per evoked stimulus, without diminishing the amount of acetylcholine released.

Topics: Acetylcholine; Animals; Atropine; Dose-Response Relationship, Drug; Evoked Potentials; Macrolides; Male; Mice; Mice, Inbred C57BL; Miniature Postsynaptic Potentials; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Neurotransmitter Agents; Parasympatholytics; Piperidines; Presynaptic Terminals; Rats; Receptors, Transforming Growth Factor beta; Synaptic Transmission; Transforming Growth Factor beta2

Vesicular acetylcholine transporter (VAChT; TC 2.A.1.2.13) mediates storage of acetylcholine (ACh) by synaptic vesicles. A three-dimensional homology model of VAChT is available, but the binding sites for ACh and the allosteric inhibitor (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) are unknown. In previous work, mutations of invariant W331 in the lumenal beginning of transmembrane helix VIII (TM VIII) of rat VAChT led to as much as ninefold loss in equilibrium affinity for ACh and no loss in affinity for vesamicol. The current work investigates the effects of additional mutations in and around W331 and the nearby lumenal end of the substrate transport channel. Mutants of human VAChT were expressed in the PC12(A123.7) cell line and characterized using radiolabeled ligands and filtration assays for binding and transport. Properties of a new and a repeat mutation in W331 are consistent with the original observations. Of 16 additional mutations in 13 other residues (Y60 in the beginning of lumenal Loop I/II, F231 in the lumenal end of TM V, W315, M316, K317, in the lumenal end of TM VII, M320, A321, W325, A330 in lumenal Loop VII/VIII, A334 in the lumenal beginning of TM VIII, and C388, C391, F392 in the lumenal beginning of TM X), only A334F impairs binding. This mutation decreases ACh and vesamicol equilibrium binding affinities by 14- and 4-fold, respectively. The current results, combined with previous results, demonstrate existence of a spatial cluster of residues close to vesicular lumen that decreases affinity for ACh and/or vesamicol when the cluster is mutated. The cluster is composed of invariant W331, highly conserved A334, and invariant F335 in TM VIII and invariant C391 in TM X. Different models for the locations of the ACh and vesamicol binding sites relative to this cluster are discussed.

Topics: Acetylcholine; Allosteric Regulation; Amino Acid Substitution; Animals; Binding Sites; Binding, Competitive; Conserved Sequence; Genetic Variation; Humans; Membrane Proteins; Mutagenesis, Site-Directed; PC12 Cells; Piperidines; Protein Structure, Tertiary; Protein Transport; Rats; Vesicular Acetylcholine Transport Proteins

Located in presynaptic cholinergic nerve terminals, the vesicular acetylcholine transporter (VAChT) represents a potential target for quantitative visualization of early degeneration of cholinergic neurons in Alzheimer's disease using PET. Benzovesamicol derivatives are proposed as radioligands for this purpose. We report QSAR studies of vesamicol and benzovesamicol derivatives taking into account the stereoselectivity of the VAChT binding site. Use of different data sets and different models in this study revealed that both enantiomers of 5-fluoro-3-(4-phenyl-piperidin-1-yl)-1,2,3,4-tetrahydro-naphthalen-2-ol (5-FBVM) are promising candidates, with predicted VAChT affinities between 6.1 and 0.05 nM. The synthesis of enantiopure (R,R)- and (S,S)-5-FBVM and their corresponding triazene precursors for future radiofluorination is reported. Both enantiomers exhibited high in vitro affinity for VAChT [(+)-5-FBVM: K(i)=6.95 nM and (-)-5-FBVM: K(i)=3.68 nM] and were selective for σ(2) receptors (∼70-fold), only (+)-5-FBVM is selective for σ(1) receptors (∼fivefold). These initial results suggest that (+)-(S,S)-5-FBVM warrants further investigation as a potential radioligand for in vivo PET imaging of cholinergic nerve terminals.

Topics: Binding Sites; Ligands; Naphthols; Piperidines; Positron-Emission Tomography; Quantitative Structure-Activity Relationship; Radiopharmaceuticals; Stereoisomerism; Vesicular Acetylcholine Transport Proteins

Different RP-HPLC columns (phenyl, conventional ODS, cross-linked C(18) and special end-capped C(8) and C(18) phases) were used to investigate the separation of four basic ionizable isomers. Using ACN/20mM NH(4)OAc aq., a separation was observed exclusively on RP columns with higher silanol activity at unusual high ACN concentration, indicating cation-exchange as main retention mechanism. Using MeOH/20mM NH(4)OAc aq., another separation at low MeOH concentrations was observed on both, RP columns with higher as well as RP columns with lower silanol activity, which is mainly based on hydrophobic interactions. The isomers were also separated on a bare silica column at higher MeOH content using NH(4)OAc. Since cation-exchange governs this retention, the elution order was different compared to the RP phases. A strong retention on the silica column was observed in ACN, which could be attributed to partition processes as additional retention mechanism.

Topics: Acetonitriles; Aza Compounds; Binding Sites; Chromatography, High Pressure Liquid; Chromatography, Reverse-Phase; Isomerism; Piperidines; Silanes

(-)-o-Methylvesamicol ((-)-OMV) exhibited in vitro a high affinity for a vesicular acetylcholine transporter (VAChT) (Ki, 6.7 nM), and (-)-o-[(11)C]methylvesamicol [(-)-[(11)C]OMV] exhibited appropriate kinetics and bound mainly to VAChTs in the rat brain. In this study, the in vivo distribution and kinetics of (-)-[(11)C]OMV were evaluated in comparison with [(11)C]SA4503 in disability model monkeys produced by selectively destroying the p75NTR-positive cells in the right hemisphere of the brain using positron emission tomography. Time-activity curves of (-)-[(11)C]OMV showed peaks within 20 min in regions rich in acetylcholine transporters (AchT). (-)-[(11)C]OMV binding in the ipsilateral cortex to the lesion was significantly reduced by 22.0% +/- 6.7% when compared with that in the contralateral region. The decrease (19.3% +/- 2.2%) in (-)-[(11)C]OMV binding in the ipsilateral temporal cortex to the lesion was greater than that (7.4% +/- 4.6%) of [(11)C]SA4503. These results suggested that (-)-[(11)C]OMV may be useful in the study of dementia characterized by degeneration of the cholinergic neurotransmitter system.

Topics: Acetylcholine; Animals; Brain; Carbon Radioisotopes; Dementia; Macaca mulatta; Male; Piperidines; Positron-Emission Tomography; Radiopharmaceuticals; Vesicular Acetylcholine Transport Proteins

It has been reported that sigma receptors are highly expressed in a variety of human tumors. In this study, we selected (+)-2-[4-(4-iodophenyl)piperidino] cyclohexanol [(+)-pIV] as a sigma receptor ligand and evaluated the potential of radioiodinated (+)-pIV for tumor imaging and therapy. (+)-[(125/131)I]pIV was prepared by an iododestannylation reaction under no-carrier-added conditions with radiochemical purity over 99% after HPLC purification. Biodistribution experiments were performed by the intravenous injection of (+)-[(125)I]pIV into mice bearing human prostate tumors (DU-145). Blocking studies were performed by intravenous injection of (+)-[(125)I]pIV mixed with an excess amount of unlabeled sigma ligand into DU-145 tumor-bearing mice. For therapeutic study, (+)-[(131)I]pIV was injected at a dose of 7.4 MBq followed by measurement of the tumor size. In biodistribution experiments, (+)-[(125)I]pIV showed high uptake and long residence in the tumor. High tumor to blood and muscle ratios were achieved because the radioactivity levels of blood and muscle were low. However, the accumulations of radioactivity in non-target tissues, such as liver and kidney, were high. The radioactivity in the non-target tissues slowly decreased over time. Co-injection of (+)-[(125)I]pIV with an excess amount of unlabeled sigma ligand resulted in a significant decrease in the tumor/blood ratio, indicating sigma receptor-mediated tumor uptake. In therapeutic study, tumor growth in mice treated with (+)-[(131)I]pIV was significantly inhibited compared to that of an untreated group. These results indicate that radioiodinated (+)-pIV has a high potential for sigma receptor imaging in tumor and radionuclide receptor therapy.

Topics: Animals; Iodine Radioisotopes; Male; Mice; Mice, Inbred BALB C; Neoplasms, Experimental; Piperidines; Radionuclide Imaging; Radiopharmaceuticals; Receptors, sigma; Tissue Distribution

Vesicular acetylcholine transporter (VAChT) is inhibited by (-)-vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol], which binds tightly to an allosteric site. The tertiary alkylamine center in (-)-vesamicol is protonated and positively charged at acidic and neutral pH and unprotonated and uncharged at alkaline pH. Deprotonation of the amine has been taken to explain loss of (-)-vesamicol binding at alkaline pH. However, binding data deviate from a stereotypical bell shape, and more binding occurs than expected at alkaline pH. The current study characterizes the binding of (-)-vesamicol from pH 5 to pH 10 using filter assays, (-)-[3H]vesamicol (hereafter called [3H]vesamicol), and human VAChT expressed in PC12(A123.7) cells. At acidic pH, protons and [3H]vesamicol compete for binding to VAChT. Preexposure or long-term exposure of VAChT to high pH does not affect binding, thus eliminating potential denaturation of VAChT and failure of the filter assay. The dissociation constant for the complex between protonated [3H]vesamicol and VAChT decreases from 12 nM at neutral pH to 2.1 nM at pH 10. The simplest model of VAChT that explains the behavior requires a proton at site 1 to dissociate with pK1 = 6.5 +/- 0.1, a proton at site A to dissociate with pKA = 7.6 +/- 0.2, and a proton at site B to dissociate with pKB = 10.0 +/- 0.1. Deprotonation of the site 1 proton is obligatory for [3H]vesamicol binding. Deprotonation of site A decreases affinity (2.2 +/- 0.5)-fold, and deprotonation of site B increases affinity (18 +/- 4)-fold. Time-dependent dissociation of bound [3H]vesamicol is biphasic, but equilibrium saturation curves are not. The contrasting phasicity suggests that the pathway to and from the [3H]vesamicol binding site exists in open and at least partially closed states. The potential significance of the findings to development of PET and SPECT ligands based on (-)-vesamicol for human diagnostics also is discussed.

Topics: Allosteric Site; Animals; Binding, Competitive; Enzyme Stability; Humans; Hydrogen-Ion Concentration; In Vitro Techniques; Kinetics; Models, Chemical; PC12 Cells; Piperidines; Protons; Rats; Recombinant Proteins; Tritium; Vesicular Acetylcholine Transport Proteins

We assessed the integrity of cholinergic neurotransmission in parietal cortex of young adult (7 months) and aged (17 months) transgenic APPswe/PS1dE9 female mice compared to littermate controls. Choline acetyltransferase and acetylcholinesterase activity declined age-dependently in both genotypes, whereas both age- and genotype-dependent decline was found in butyrylcholinesterase activity, vesicular acetylcholine transporter density, muscarinic receptors and carbachol stimulated binding of GTP gamma S in membranes as a functional indicator of muscarinic receptor coupling to G-proteins. Notably, vesicular acetylcholine transporter levels and muscarinic receptor-G-protein coupling were impaired in transgenic mice already at the age of 7 months compared to wild type littermates. Thus, brain amyloid accumulation in this mouse model is accompanied by a serious deterioration of muscarinic transmission already before the mice manifest significant cognitive deficits.

Topics: Acetylcholinesterase; Age Factors; Amyloid beta-Protein Precursor; Analysis of Variance; Animals; Animals, Genetically Modified; Dose-Response Relationship, Drug; Female; Guanosine 5'-O-(3-Thiotriphosphate); Humans; Mice; Mutation; N-Methylscopolamine; Piperidines; Presenilin-1; Protein Binding; Receptors, Muscarinic; Synaptic Transmission; Vesicular Acetylcholine Transport Proteins

With the aim of producing selective radiotracers for in vivo imaging of the vesicular acetylcholine transporter (VAChT) using positron mission tomography (PET), here, we report synthesis and analysis of a new class of conformationally constrained vesamicol analogues with moderate lipophilicity. The sequential ring opening on trans-1,4-cyclohexadiene dioxide enabled an approach to synthesize 6-arylpiperidino-octahydrobenzo[1,4]oxazine-7-ols [morpholino vesamicols]. The radiosynthesis of the [18F]fluoroacetyl-substituted derivative ([18F]FAMV) was achieved starting from a corresponding bromo precursor [2-Bromo-1-[7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazin-4-yl]-ethanone] and using a modified commercial computer-controlled module system with a radiochemical yield of 27+/-4%, a high radiochemical purity (99%) and a specific activity of 35 GBq/micromol. In competitive binding assays using a PC12 cell line overexpressing VAChT and [3H]-(-) vesamicol, 2-fluoro-1-[7-hydroxy-6-(4-phenyl-piperidin-1-yl)-octahydro-benzo[1,4]oxazin-4-yl]-ethanone (FAMV) demonstrated a high selectivity for binding to VAChT (K(i): 39.9+/-5.9 nM) when compared to its binding to sigma 1/2 receptors (Ki>1500 nM). The compound showed a moderate lipophilicity (logD (pH 7)=1.9) and a plasma protein binding of 49%. The brain uptake of [18F]FAMV was about 0.1% injected dose per gram at 5 min after injection and decreased continuously with time. Notably, an increasing accumulation of radioactivity in the lateral brain ventricles was observed. After 1 h, the accumulation of [18F]FAMV, expressed as ratio to the cerebellum, was 4.5 for the striatum, 2.0 for the cortical and 1.5 for the hippocampal regions, measured on brain slices using ex vivo autoradiography. At the present time, 75% of [18F]FAMV in the plasma was shown to be metabolized to various hydrophilic compounds, as detected by high-performance liquid chromatography. The degradation of [18F]FAMV was also detected in brain extracts as early as 15 min post injection (p.i.) and increased to 50% at 1 h postinjection. In conclusion, although the chemical properties of [18F]FAMV and the selectivity of binding to VAChT appear to be promising indicators of a useful PET tracer for imaging VAChT, a low brain extraction, in combination with only moderate specific accumulation in cholinergic brain regions and an insufficient in vivo stability prevents the application of this compound for neuroimaging in humans.

Topics: Animals; Autoradiography; Binding, Competitive; Brain; Female; Fluorine Radioisotopes; Isotope Labeling; Metabolic Clearance Rate; Morpholines; PC12 Cells; Piperidines; Positron-Emission Tomography; Protein Binding; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Receptors, sigma; Sigma-1 Receptor; Vesicular Acetylcholine Transport Proteins

Vesamicol derivatives are promising candidates as ligands for the vesicular acetylcholine transporter (VAChT) to enable in vivo imaging of cholinergic deficiencies if applied as positron emission tomography radiotracers. So far, optimization of the binding affinity of vesamicol-type ligands was hampered by the lack of respective quantitative structure-activity relationships. We developed the first quantitative model to predict, from molecular structure, the binding affinity of vesamicol-type ligands toward VAChT employing comparative molecular field analysis (CoMFA) for a set of 37 ligands, covering three different structural types (4-phenylpiperidine, spiro, and tropan derivatives of vesamicol). The prediction capability was assessed by leave-one-out cross-validation (LOO) and through leaving out and predicting 50% of the compounds selected such that both the training and the prediction sets cover almost the whole range of experimental data. The statistics indicate a significant prediction power of the models ( q (2) (LOO) = 0.66, q (2) (50% out) = 0.59-0.74). The discussion includes detailed analyses of CoMFA regions critical for ligand-VAChT binding, identifying structural implications for high binding affinity.

Topics: Binding Sites; Fluorobenzenes; Iodobenzenes; Ligands; Models, Molecular; Molecular Structure; Piperidines; Quantitative Structure-Activity Relationship; Reproducibility of Results; Static Electricity; Stereoisomerism; Vesicular Acetylcholine Transport Proteins

We synthesized methylvesamicol analogs 13-16 and investigated the binding characteristics of 2-[4-phenylpiperidino]cyclohexanol (vesamicol) and methylvesamicol analogs 13-16, with a methyl group introduced into the 4-phenylpiperidine moiety, to sigma receptors (sigma-1, sigma-2) and to vesicular acetylcholine transporters (VAChT) in membranes of the rat brain and liver. In competitive inhibition studies, (-)-o-methylvesamicol [(-)-OMV] (13) (Ki=6.7 nM), as well as (-)-vesamicol (Ki=4.4 nM), had a high affinity for VAChT. (+)-p-Methylvesamicol [(+)-PMV] (16) (Ki=3.0 nM), as well as SA4503 (Ki=4.4 nM), reported as a sigma-1 mapping agent for positron emission tomography (PET), had a high affinity for the sigma-1 receptor. The binding affinity of (+)-PMV (16) for the sigma-1 receptor (Ki=3.0 nM) was about 13 times higher than that for the sigma-2 (sigma-2) receptor (Ki=40.7 nM). (+)-PMV (16) (Ki=199 nM) had a much lower affinity for VAChT than SA4503 (Ki=50.2 nM) and haloperidol (Ki=41.4 nM). These results showed that the binding characteristics of (-)-OMV (13) to VAChT were similar to those of (-)-vesamicol and that (+)-PMV (16) bound to the sigma-1 receptor with high affinity. In conclusion, (-)-OMV (13) and (+)-PMV (16), which had a suitable structure, with a methyl group for labeling with 11C, may become not only a new VAChT ligand and a new type of sigma receptor ligand, respectively, but may also become a new target compound of VAChT and the sigma-1 receptor radioligand for PET, respectively.

Topics: Animals; Brain; Liver; Magnetic Resonance Spectroscopy; Male; Piperidines; Protein Binding; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, sigma; Vesicular Acetylcholine Transport Proteins

Vesamicol is a leading compound for positron emission tomography (PET) and single photon emission computed tomography (SPECT) tracers for mapping the vesicular acetylcholine transporter (VAChT). Recently, we found that (+)-p-methylvesamicol ((+)-PMV) has low affinity for VAChT (K(i)=199 nM), but has moderate to high affinity for sigma receptors: K(i)=3.0 nM for sigma1 and K(i)=40.7 nM for sigma2, and that sigma1-selective SA4503 (K(i)=4.4 nM for sigma1 and K(i)=242 nM for sigma2) has moderate affinity for VAChT (K(i)=50.2 nM). In the present study, we examined the potential of (+)-[11C]PMV as a PET radioligand for mapping sigma1 receptors as compared with [11C]SA4503. In rat brain, similar regional distribution patterns of (+)-[11C]PMV and [11C]SA4503 were shown by tissue dissection and by ex vivo autoradiography. Blocking experiments using (+/-)-PMV, (-)-vesamicol, SA4503, haloperidol and (+/-)-pentazocine showed that the two tracers specifically bound to sigma1 receptors, and that [11C]SA4503 exhibited greater specific binding than (+)-[11C]PMV. No sign of VAChT-specific binding by [11C]SA4503 was observed in the striatum, which is rich in VAChT sites. In conclusion, (+)-[11C]PMV specifically bound to sigma1 receptors in the brain, but to a lesser extent than [11C]SA4503, suggesting that (+)-[11C]PMV is a less preferable PET ligand than [11C]SA4503. On the other hand, the moderate affinity of [11C]SA4503 for VAChT is negligible in vivo.

Topics: Brain; Carbon Radioisotopes; Metabolic Clearance Rate; Piperazines; Piperidines; Positron-Emission Tomography; Radiopharmaceuticals; Receptors, sigma; Sigma-1 Receptor; Tissue Distribution

The mesolimbic dopamine (DA) system, projecting from the ventral tegmental area (VTA) to the nucleus accumbens (nAcc), is involved in reward-related behaviours and addictive processes, such as alcoholism and drug addiction. It was recently suggested that strychnine-sensitive glycine receptors (GlyR) in the nAcc regulate both basal and ethanol-induced mesolimbic DA activity via a neuronal loop involving endogenous activation of nicotinic acetylcholine receptors (nAChR) in the VTA. However, as the nAcc appears to contain few glycine-immunoreactive cell bodies or fibres, the question as to what may be the endogenous ligand for GlyRs in this brain region remains open. Here we have investigated whether the amino acid taurine could serve this purpose using in vivo microdialysis in awake, freely moving male Wistar rats. Local perfusion of taurine (1, 10 or 100 mm in the perfusate) increased DA levels in the nAcc. The taurine (10 mm)-induced DA increase was, similarly to that previously observed after ethanol, completely blocked by (i) perfusion of the competitive GlyR antagonist strychnine in the nAcc, (ii) perfusion of the nAChR antagonist mecamylamine (100 microm) in the VTA, and (iii) systemic administration of the acetylcholine-depleting drug vesamicol (0.4 mg/kg, i.p). The present results suggest that taurine may be an endogenous ligand for GlyRs in the nAcc and that the taurine-induced elevation of DA levels in this area, similarly to that observed after local ethanol, is mediated via a neuronal loop involving endogenous activation of nAChRs in the VTA.

Topics: Animals; Dopamine; Ganglionic Blockers; Glycine Agents; Male; Mecamylamine; Microdialysis; Neuromuscular Depolarizing Agents; Nucleus Accumbens; Piperidines; Rats; Rats, Wistar; Receptors, Glycine; Strychnine; Taurine

(-)-O-Methylvesamicol ((-)-OMV) exhibited in vitro a high affinity for vesicular acetylcholine transporter (VAChT) (Ki, 6.7 nM) and a relatively low affinity for sigmal receptor (Ki, 33.7 nM). We prepared (-)-[11C]OMV by a palladium-promoted cross-coupling reaction using [11C]methyl iodide, in a radiochemical yield of 38 +/- 6.9% (n=3), a radiochemical purity of 98 +/- 2.3% (n = 5), and a specific activity of 11 +/- 7.0 TBq/mmol at 30 minutes after EOB (n=5). Then, we evaluated in vivo whether (-)-[11C]OMV has properties as a PET radioligand for mapping VAChT. In rats, the brain uptake of (-)-[11C]OMV was 1.1%ID/g at 5 minutes postinjection, and was retained of a high level for 60 minutes. The brain uptake was significantly inhibited by the co-injection (500 nmol/kg) of cold (-)-OMV (58-66%), (-)-vesamicol (57-65%), and two sigma receptor ligands with modulate affinities for VAChTs: SA4503 (56-71%) and haloperidol (39-64%) in all of the brain regions, including the cerebellum with a low density of VAChTs, but not of sigmal-selective ligand (+)-pentazocine. However, the pretreatment with a large excess amount of (+/-)-pentazocine (50 micromol/kg) reduced the uptake in a different manner in the brain regions: 25% reduction in the striatum with a high density of VAChTs, and a 50-55% reduction in the other regions with a lower density of VAChTs. Ex vivo autoradiography using (-)-[11C]OMV showed a similar regional brain distribution of [3H](-)-vesamicol. In the PET study of the monkey brain, the regional brain distribution pattern of (-)-[11C]OMV was different from that of [11C]SA4503. The uptake of (-)-[11C]OMV was relatively higher in the striatum, was reversible, and an apparent equilibrium state was found at 20-40 minutes. In conclusion, (-)-[11C]OMV exhibited appropriate brain kinetics during the time frame of 11C-labeled tracers and bound mainly to VAChTs; however, the binding to sigmal receptors was not disregarded. Therefore, (-)-[11C]OMV-PET together with help of [11C]SA4503-PET may evaluate VAChTs.

Topics: Animals; Brain; Carbon Radioisotopes; Haplorhini; Isotope Labeling; Ligands; Metabolic Clearance Rate; Organ Specificity; Piperidines; Positron-Emission Tomography; Radiopharmaceuticals; Rats; Rats, Wistar; Tissue Distribution; Vesicular Acetylcholine Transport Proteins

The vesicular acetylcholine transporter (VAChT) contains six conserved sequence motifs that are rich in proline and glycine. Because these residues can have special roles in the conformation of polypeptide backbone, the motifs might have special roles in conformational changes during transport. Using published bioinformatics insights, the amino acid sequences of the 12 putative, helical, transmembrane segments of wild-type and mutant VAChTs were analyzed for propensity to form non-alpha-helical conformations and molecular notches. Many instances were found. In particular, high propensity for kinks and notches are robustly predicted for motifs D2, C and C'. Mutations in these motifs either increase or decrease Vmax for transport, but they rarely affect the equilibrium dissociation constants for ACh and the allosteric inhibitor, vesamicol. The near absence of equilibrium effects implies that the mutations do not alter the backbone conformation. In contrast, the Vmax effects demonstrate that the mutations alter the difficulty of a major conformational change in transport. Interestingly, mutation of an alanine to a glycine residue in motif C significantly increases the rates for reorientation across the membrane. These latter rates are deduced from the kinetics model of the transport cycle. This mutation is also predicted to produce a more flexible kink and tighter tandem notches than are present in wild-type. For the full set of mutations, faster reorientation rates correlate with greater predicted propensity for kinks and notches. The results of the study argue that conserved motifs mediate conformational changes in the VAChT backbone during transport.

Topics: Acetylcholine; Amino Acid Motifs; Animals; Binding Sites; Biological Transport; Computational Biology; Dose-Response Relationship, Drug; Drug Interactions; Glycine; Mutation; PC12 Cells; Piperidines; Proline; Protein Binding; Rats; Sequence Alignment; Transfection; Tritium; Vesicular Acetylcholine Transport Proteins

This research investigated the roles of 7 conserved ionic residues in the 12 putative transmembrane domains (TMDs) of vesicular acetylcholine transporter (VAChT). Rat VAChT in wild-type and mutant forms was expressed in PC12(A123.7) cells. Transport and ligand binding were characterized at different pH values using filter assays. The ACh binding site is shown to exhibit high or low affinity (K(d) values are approximately 10 and 200 mM, respectively). Mutation of the lysine and aspartate residues in TMDs II and IV, respectively, can decrease the fraction of sites having high affinity. In three-dimensional structures of related transporters, these TMDs lie next to each other and distantly from TMDs VIII and X, which probably contain the binding sites for ACh and the allosteric inhibitor vesamicol. Importantly, mutation of the aspartate in TMD XI can create extra-high affinities for ACh (K(d) approximately 4 mM) and vesamicol (K(d) approximately 2 nM compared to approximately 20 nM). Effects of different external pH values on transport indicate a site that must be protonated (apparent pK(a) approximately 7.6) likely is the aspartate in TMD XI. The observations suggest a model in which the known ion pair between lysine in TMD II and aspartate in TMD XI controls the conformation or relative position of TMD XI, which in turn controls additional TMDs in the C-terminal half of VAChT. The pH effects also indicate that sites that must be unprotonated for transport (apparent pK(a) approximately 6.4) and vesamicol binding (apparent pK(a) approximately 6.3) remain unidentified.

Topics: Acetylcholine; Animals; Aspartic Acid; Binding Sites; Biological Transport; Blotting, Western; Cell Line; Cell Membrane; Fluorescent Antibody Technique, Direct; Histidine; Hydrogen-Ion Concentration; Lysine; Membrane Transport Proteins; Microscopy, Confocal; Mutagenesis, Site-Directed; Piperidines; Protein Structure, Tertiary; Rats; Static Electricity; Vesicular Acetylcholine Transport Proteins

The acetylcholine-binding site in vesicular acetylcholine transporter faces predominantly toward the outside of the vesicle when resting but predominantly toward the inside when transporting. Transport-related reorientation is detected by an ATP-induced decrease in the ability of saturating substrate to displace allosterically bound [(3)H]vesamicol. The assay was used here to determine whether structurally diverse compounds are transported by rat VAChT expressed in PC12(A123.7) cells. Competition by ethidium, tetraphenylphosphonium and other monovalent organic cations with [(3)H]vesamicol is decreased when ATP is added, and the effect depends on proton-motive force. The results indicate that many organic molecules carrying +1 charge are transported, even though the compounds do not resemble acetylcholine in structural details.

Topics: Acetylcholine; Adenosine Triphosphate; Allosteric Regulation; Animals; Binding Sites; Binding, Competitive; Biological Transport, Active; Cations; Ethidium; Membrane Transport Proteins; Molecular Structure; Neuromuscular Depolarizing Agents; Neurons; Onium Compounds; Organophosphorus Compounds; PC12 Cells; Piperidines; Rats; Synaptic Transmission; Synaptic Vesicles; Vesicular Acetylcholine Transport Proteins

18F labelled vesamicol analogues, which bind to the vesicular acetylcholine transporter (VAChT) in central cholinergic nerve terminals, are expected to be potential radioligands for the visualisation of cholinergic transmission deficits via positron emission tomography (PET). In this report the regioselective synthesis of five novel vesamicol analogues as well as their in vitro binding properties to the VAChT are described. Beside having the 4-fluorobenzylether-substitution at the cyclohexyl ring as an unique structural feature, the new compounds are additionally modified at the phenyl and piperidine moiety of the vesamicol skeleton. The affinity and selectivity to the VAChT were analysed by competitive binding studies using tritium labelled radioligands. The VAChT affinities (Ki-values) of the novel compounds were estimated ranging between 7.8+/-3.5 nM and 161.6+/-17.3 nM, thus some of them are binding with higher affinity to the transporter than vesamicol. However, the compounds tested demonstrated also affinities to the sigma receptors sigma1 and sigma2 ranging between 4.1+/-1.5 nM and 327.5+/-75.9 nM. Nevertheless, these data provide the basis for future structure-binding-studies and further underline the potential and usefulness of vesamicol analogues for imaging of the VAChT.

Topics: Animals; Binding Sites; Female; In Vitro Techniques; Liver; Molecular Conformation; Piperidines; Protein Binding; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Vesicular Acetylcholine Transport Proteins

At a variety of fast chemical synapses, spent synaptic vesicles are recycled via a large 'reserve' vesicle pool at high stimulus frequencies, and via fast 'local cycling' near release sites (e.g. 'kiss and run' transmitter release) at low stimulus frequencies. We have investigated recycling at the snake neuromuscular junction (NMJ), specifically seeking evidence for local cycling. Activity-dependent staining and destaining of the endocytic probe FM1-43 were directly compared to transmitter release over a range of stimulus frequencies. We found a fixed proportionality between staining/destaining and summed endplate potentials (EPPs) representing total transmitter release. There was no direct dependence of staining or destaining on stimulus frequency, as would be expected if local cycling (and consequent altered FM1-43 retention) were more prevalent at one frequency than another. In other experiments the drug vesamicol was used to abolish refilling of vesicles with transmitter, thereby blocking EPPs contributed by recycled vesicles. Control and vesamicol-treated NMJs had identical quantal content for the first 10 min of 1 Hz stimulation. Afterwards EPP amplitudes at vesamicol-treated NMJs declined at a rate consistent with use of a large pool containing approximately 130,000 vesicles. Finally, calibrated paired stimulations show that regenerated vesicles have poorer than random probability of re-release. Our findings are inconsistent with local cycling and suggest that the snake motor terminal utilizes exclusively a single large vesicle pool.

Topics: Animals; Colubridae; Electric Stimulation; Fluorescent Dyes; In Vitro Techniques; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Piperidines; Pyridinium Compounds; Quaternary Ammonium Compounds; Synaptic Transmission; Synaptic Vesicles; Time Factors

Both the membrane-bound choline acetyltransferase (MChAT) and soluble ChAT (SChAT) were found to be activated by ATP-mediated protein phosphorylation. ATP activation of MChAT but not SChAT was found to depend on the integrity of proton gradient of synaptic vesicles because conditions disrupting the proton gradient also abolished the activation of MChAT by ATP. Among the kinases studied, Ca2+/calmodulin kinase II is most effective in activation of MChAT. Transport of ACh into synaptic vesicles by vesicular acetylcholine transporter (VAChT) is also proton gradient-dependent; therefore we proposed that there is a functional coupling between ACh synthesis and its packaging into synaptic vesicles. This notion is supported by the following findings: first, the newly synthesized [3H]-ACh from [3H]-choline was taken up much more efficiently than the pre-existing ACh; second, ATP-activation of MChAT was abolished when VAChT was inhibited by the specific inhibitor vesamicol; third, the activity of ChAT was found to be markedly increased when neurons are under depolarizing conditions.

Topics: Adenosine Triphosphate; Animals; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Choline; Choline O-Acetyltransferase; Enzyme Activation; Enzyme Inhibitors; Gramicidin; Membrane Transport Proteins; Piperidines; Protein Kinase Inhibitors; Protein Kinases; Swine; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Detection of the central cholinergic deficits, a consistent feature of Alzheimer's disease, is essential to allow preventive measures and/or symptomatic treatment already at a very early stage of the disease. The vesicular acetylcholine transporter (VAChT) represents an appropriate target to establish PET radiotracer that are adequate for brain imaging the loss of cholinergic terminals. Here we describe the synthesis and binding characteristics of novel derivatives of vesamicol, known to represent a specific antagonist of VAChT sites. Novel benzyl ether derivatives of vesamicol either 4- or 5-substituted at the cyclohexylring have been synthesized by different regioselective ring opening reactions of a same epoxide precursor. The affinity and selectivity of the novel compounds to VAChT sites were analyzed by competitive radioligand binding studies in rat brain and liver membrane preparations using tritium labeled radioligands. The 4-substituted fluorobenzylether of vesamicol 10b was shown to exhibit a high affinity to VAChT sites (K(i)-value(10b)=10.7+/-1.7 nM), but demonstrated also binding capacities to sigma receptors (K(i-)value(10b)=18.5+/-6.9 nM, [(3)H]DTG; K(i)-value(10b)=30.6+/-9.6 nM, [(3)H]haloperidol). The data suggest the potential of vesamicol derivatives to design appropriate radiotracer for PET imaging of central cholinergic deficits.

Topics: Animals; Binding Sites; Binding, Competitive; Brain; Ether; Female; In Vitro Techniques; Liver; Membrane Transport Proteins; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, sigma; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

An enantioseparation high performance liquid chromatographic (HPLC) method was developed and validated to determine D-(+)- and L-(-)-vesamicol in human plasma. The assay involved the use of a solid phase extraction for plasma sample clean up prior to HPLC analysis utilizing a C18 Bond-Elute column. Chromatographic resolution of the vesamicol enantiomers was performed on a vancomycin macrocyclic antibiotic chiral stationary phase (CSP) known as Chirobiotic V with a polar ionic mobile phase (PIM) consisting of methanol:glacial acetic acid:triethylamine (100:0.1:0.05 (v/v/v)) at a flow rate of 1.0 ml/min and UV detection set at 262 nm. All analyses were conducted at ambient temperature. The method was validated over the range of 1-20 microg/ml for each enantiomer concentration (R2>0.999). Recoveries for D-(+)- and L-(-)-vesamicol enantiomers were in the ranges of 96-105% at 3-16 microg/ml level. The method proved to be precise (within-run precision ranged from 1.3 to 2.7% and between-run precision ranged from 1.5 to 3.4%) and accurate (within-run accuracies ranged from 0.8 to 3.4% and between-run accuracies ranged from 1.7 to 5.0%). The limit of quantitation (LOQ) and limit of detection (LOD) for each enantiomer in human plasma were 1.0 and 0.5 microg/ml (S/N=3), respectively.

Topics: Chromatography, Liquid; Humans; Piperidines; Stereoisomerism; Technology, Pharmaceutical; Vancomycin

Active transport of acetylcholine (ACh) by vesicular ACh transporter (VAChT) is driven by a proton-motive force established by V-ATPase. A published microscopic kinetics model predicts the ACh-binding site is primarily oriented toward the outside for nontransporting VAChT and toward the inside for transporting VAChT. The allosteric ligand [(3)H]vesamicol cannot bind when the ACh-binding site is outwardly oriented and occupied by ACh, but it can bind when the ACh site is inwardly oriented. The kinetics model was tested in the paper reported here using rat VAChT expressed in PC12(A1237) cells. Equilibrium titrations of [(3)H]vesamicol binding and ACh competition show that ATP blocks competition between vesamicol and ACh in over one-half of the VAChT. NaCl did not mimic ACh chloride, and bafilomycin A(1) and FCCP completely blocked the ATP effect, which shows that it is mediated by a proton-motive force. The data are consistent with reorientation of over one-half of the ACh-binding sites from the outside to the inside of vesicles upon activation of transport. The observations support the proposed microscopic kinetics model, and they should be useful in characterizing effects of mutations on the VAChT transport cycle.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Binding Sites; Binding, Competitive; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; Cell Membrane; Gene Expression Regulation, Neoplastic; Kinetics; Ligands; Macrolides; Membrane Transport Proteins; PC12 Cells; Piperidines; Proton-Motive Force; Rats; Sodium Chloride; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

This study sought primarily to locate the acetylcholine (ACh) binding site in the vesicular acetylcholine transporter (VAChT). The design of the study also allowed us to locate residues linked to (a) the binding site for the allosteric inhibitor vesamicol and (b) the rates of the two transmembrane reorientation steps of a transport cycle. In more characterized proteins, ACh is known to be bound in part through cation-pi solvation by tryptophan, tyrosine, and phenylalanine residues. Each of 11 highly conserved W, Y, and F residues in putative transmembrane domains (TMDs) of rat VAChT was mutated to A and a different aromatic residue to test for loss of cation-pi solvation. Mutated VAChTs were expressed in PC12(A123.7) cells and characterized with the goal of determining whether mutations widely perturbed structure. The thermodynamic affinity for ACh was determined by displacement of trace [(3)H]-(-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol) with ACh, and Michaelis-Menten parameters were determined for [(3)H]ACh transport. Expression levels were determined with [(3)H]vesamicol saturation curves and Western blots, and they were used to normalize V(max) values. "Microscopic" parameters for individual binding and rate steps in the transport cycle were calculated on the basis of a published kinetics model. All mutants were expressed adequately, were properly glycosylated, and bound ACh and vesamicol. Subcellular mistargeting was shown not to be responsible for poor transport by some mutants. Mutation of residue W331, which lies in the beginning of TMD VIII proximal to the vesicular lumen, produced 5- and 9-fold decreased ACh affinities and no change in other parameters. This residue is a good candidate for cation-pi solvation of bound ACh. Mutation of four other residues decreased the ACh affinity up to 6-fold and also affected microscopic rate constants. The roles of these residues in ACh binding and transport thus are complex. Nine mutations allowed us to resolve the ACh and vesamicol binding sites from each other. Other mutations affected only the rates of the transmembrane reorientation steps, and four mutations increased the rate of one or the other. Two mutations increased the value of K(M) up to 5-fold as a result of rate effects with no ACh affinity effect. The results demonstrate that analysis of microscopic kinetics is required for the correct interpretation of mutational effects in VAChT. Results also are discussed in terms of recently det

Topics: Acetylcholine; Alanine; Allosteric Site; Animals; Binding Sites; Membrane Transport Proteins; Mutagenesis, Site-Directed; PC12 Cells; Phenylalanine; Piperidines; Protein Transport; Rats; Subcellular Fractions; Synaptic Vesicles; Transfection; Tritium; Tyrosine; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

The use of acetylcholinesterase (AChE) inhibitors is the primary therapeutic strategy in the treatment of Alzheimer's disease. However, these drugs have been reported to have effects beyond the simple stimulation of neuronal acetylcholine receptors (AChRs) by elevated acetylcholine (ACh), interfering directly with the nAChR. Therefore, a pure pharmacological blockade of AChE is not usually obtained. In this study, the patch-clamp technique was utilized to determine the effects of methamidophos, a pesticide that is considered a selective AChE inhibitor, on nAChRs of substantia nigra dopaminergic neurons. In spite of the fact that methamidophos has been reported to be devoid of direct nicotinic actions, our main observation was that it selectively and reversibly blocked nAChR responses, without directly affecting the holding current. Methamidophos produced a downward shift in the dose response curve for nicotine; the mechanism accounting for this non-competitive antagonism was open channel block, in view of its voltage dependence. Pre-treatment with vesamicol did not prevent the reduction of nicotine-induced currents, indicating that the effect on nAChRs was independent from the activity of methamidophos as a cholinesterase inhibitor. Our results conclude that methamidophos has a complex blocking action on neuronal nAChRs that is unlinked to the inhibition of AChE. Therefore, it should not be considered a selective AChE inhibitor and part of its toxic effects could reside in an interference with the nicotinic neurotransmission.

Topics: alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Excitatory Amino Acid Agonists; Excitatory Postsynaptic Potentials; In Vitro Techniques; Ion Channel Gating; Membrane Potentials; Neural Inhibition; Neuromuscular Depolarizing Agents; Neurons; Nicotine; Organothiophosphorus Compounds; Patch-Clamp Techniques; Piperidines; Rats; Rats, Wistar; Receptors, Nicotinic; Substantia Nigra

Previously published results appeared to show that vesicular acetylcholine transporter (VAChT) does not transport choline (Ch). Because it is uniquely suited to detect transport of weakly bound substrates, a recently developed assay that detects transmembrane reorientation of the substrate binding site was used to re-examine transport selectivity. Rat VAChT was expressed in PC12(A1237) cells, postnuclear supernatant-containing microvesicles was prepared, and the reorientation assay was conducted with unlabeled Ch and tetramethylammonium (TMA). Also, [(14)C]Ch and [(3)H]acetylcholine (ACh) were used in an optimized accumulation assay. The results demonstrate that Ch is transported at least as well as ACh is, but with sevenfold lower affinity. Even TMA is transported, but with 26-fold lower affinity. Ch transport by VAChT is of interest in view of the possibilities that Ch (i) occurs at higher concentration than ACh does in terminal cytoplasm under some conditions, and (ii) is an agonist for alpha 7 nicotinic receptors.

Topics: Acetylcholine; Animals; Binding, Competitive; Biological Transport; Carbon Radioisotopes; Choline; Intracellular Membranes; Membrane Transport Proteins; PC12 Cells; Piperidines; Quaternary Ammonium Compounds; Radioligand Assay; Rats; Subcellular Fractions; Tritium; Vesicular Acetylcholine Transport Proteins

We studied the effect of the venom of the Brazilian spider, Lasiodora sp. (Mygalomorphae, Theraphosidae), on force generation and electrical activity in the isolated rat heart. Previous work showed that this venom is excitotoxic to excitable cells due to Na(+) channel gating modifier activity [Toxicon 39 (2001) 991]. In the isolated heart, the venom (10-100 microg bolus administration) caused a dose-dependent bradycardia, with transient cardiac arrest and rhythm disturbances. The electrocardiogram showed that the reduction of heart rate was due to sinus bradycardia, sinus arrest and partial or complete A-V block. All of the effects were reversible upon washout of the venom. The effect of the venom was potentiated by the anticholinesterase neostigmine (3.3 microM), suppressed by the muscarinic acetylcholine receptor antagonist atropine (1.4 microM), and inhibited by the vesicular acetylcholine transporter inhibitor (-)-vesamicol (10 microM). Tetrodotoxin (200 nM) did not inhibit the effect of the venom. Together, these data suggest that this Lasiodora venom evokes vesicular release of acetylcholine from parasympathetic nerve terminals by activating tetrodotoxin-resistant Na(+) channels.

Topics: Animals; Atropine; Dose-Response Relationship, Drug; Heart Conduction System; Heart Rate; Male; Neostigmine; Piperidines; Rats; Rats, Wistar; Spider Venoms; Spiders

The protein kinase A-deficient PC12 cell line PC12A123.7 lacks both choline acetyltransferase and the vesicular acetylcholine transporter. This cell line has been used to establish a stably transfected cell line expressing recombinant rat vesicular acetylcholine transporter that is appropriately trafficked to small synaptic vesicles. Acetylcholine is transported by the rat vesicular acetylcholine transporter at a maximal rate of 1.45 nmol acetylcholine/min/mg protein and exhibits a Km for transport of 2.5 mM. The transporter binds vesamicol with a Kd of 7.5 nM. The ability of structural analogs of acetylcholine to inhibit both acetylcholine uptake and vesamicol binding was measured. The results demonstrate that like Torpedo vesicular acetylcholine transporter, the mammalian transporter can bind a diverse group of acetylcholine analogs.

Topics: Acetylcholine; Animals; Biological Transport; Carrier Proteins; Cyclic AMP-Dependent Protein Kinases; Membrane Transport Proteins; PC12 Cells; Piperidines; Rats; Synaptic Vesicles; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Topics: Acetylcholine; Animals; Biochemistry; Biological Transport; Cell Nucleus; Dose-Response Relationship, Drug; Kinetics; Membrane Transport Proteins; PC12 Cells; Piperidines; Protein Binding; Protein Transport; Rats

We evaluated the potencies of radioiodinated (-)-o-iodovesamicol [(-)-oIV] as a selective vesicular acetylcholine transporter (VAChT) mapping agent. (-)-[125I]oIV exhibited significant accumulation (about 2.8% of the injected dose) in rat brain. The regional brain distribution of radioactivity was similar for both (-)-[125I]olV and (-)-[3H]vesamicol. The accumulation of (-)-[125I]oIV in the brain was significant reduced by post-administration of unlabeled vesamicol (0.5 /micromol/kg(-1)) and (-)-oIV (0.5 micromol/kg(-1)). On the other hand, the post-administration of sigma ligands hardly affected the accumulation of (-)-[125I]oIV in the brain. These studies showed that (-)-[125I]oIV, as well as [3H] vesamicol, bound to VAChT with high affinity in the rat brain. Furthermore, (-)-[125I]oIV binding in the ipsilateral cortex to the lesion was significantly reduced by 17.0%, compared with that in the contralateral cortex in a unilateral NBM-lesioned rat. These results suggested that radioiodinated (-)-oIV may potentially be useful for the diagnosis of cholinergic neurodegenerative disorders.

Topics: Animals; Brain; Carrier Proteins; Ibotenic Acid; Iodine Radioisotopes; Isotope Labeling; Male; Membrane Transport Proteins; Metabolic Clearance Rate; Neurodegenerative Diseases; Organ Specificity; Piperidines; Radionuclide Imaging; Radiopharmaceuticals; Rats; Reproducibility of Results; Sensitivity and Specificity; Tissue Distribution; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

We investigated the binding characteristics of optical isomers of three iodovesamicol analogs to vesicular acetylcholine transporters (VAChT) and to sigma receptors (sigma-1, sigma-2) in rat brains. In competitive inhibition studies, (-)-enantiomers [(-)-ortho-iodovesamicol ((-)-oIV), (-)-meta-iodovesamicol ((-)-mIV), (-)-vesamicol] displayed a higher affinity for VAChT than (+)-enantiomer [(+)-oIV, (+)-mIV, (+)-vesamicol]. (-)-oIV and (-)-mIV showed the same high affinity for VAChT as (-)-vesamicol. For sigma receptors(sigma-1, sigma-2), (-)-oIV (Ki = 62.2 nM (to sigma-1) and 554 nM(to sigma-2)) showed a lower affinity than (-)-mIV (Ki = 4.5 nM (to sigma-1) and 42.9 nM (to sigma-2)). Furthermore, in a saturation binding study, (-)-[125I]-oIV exhibited a Kd of 17.4 +/- 5.1 nM with a maximum number of binding sites, Bmax, of 559 +/- 51 fmol/ mg of protein. These results showed that (-)-oIV binds to vesicular acetylcholine transporters (VAChT) more selectively than (-)-mIV, previously reported as a VAChT mapping agent, and may be suitable for VAChT imaging studies.

Topics: Animals; Binding, Competitive; Brain; Carrier Proteins; Dose-Response Relationship, Drug; Intracellular Membranes; Iodine Radioisotopes; Male; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, sigma; Stereoisomerism; Subcellular Fractions; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

An enantioseparation of racemic vesamicol in human serum by capillary electrophoresis with solid phase extraction and sulfated B-cyclodextrin (S-B-CD) is presented The separation was achieved on an uncoated 72 cm x 50 microm id fused silica capillary maintained at 30 degrees C and + 15 kV applied voltage using a run buffer of 128 micro-B-CD in 50 mM phosphate buffer at pH 5. The detection wavelength was 260 nm. Bond Elut C18 solid phase extraction cartridges were used in the sample preparation of the vesamicol samples from serum. Among the CDs studied, the migration order of the enantiomers was reversed in CM-B-CD compared to S-B-CD. Increases in migration time and differences in time between enantiomers was observed with increasing concentrations of S-B-CD. Baseline separation was achieved in the 2-20 microg/ml range of enantiomer concentration (r > .996). A sample stacking technique was used to improve peak shape and LOD. LODs were 0.5 microg/ml for each enantiomer. Studies of various factors and CE conditions showed the effect of CD type, CD concentration, buffer type, buffer concentration and pH on stability and resolution.

Topics: beta-Cyclodextrins; Cyclodextrins; Electrophoresis, Capillary; Humans; Piperidines; Stereoisomerism; Sulfates

We have recently observed that Die-Huang-Wan has an ability to stimulate the secretion of insulin to decrease the plasma glucose levels in normal rats. In the present study, this effect of Die-Huang-Wan was reversed by the general muscarinic antagonists atropine and scopolamine, but not affected by the ganglionic nicotinic antagonist pentolinium or hexamethonium. Moreover, disruption of synaptically available acetylcholine using an inhibitor of choline uptake, hemicholinium-3, or vesicular acetylcholine transport, vesamicol, abolished the actions induced by Die-Huang-Wan. Mediation of acetylcholine released from nerve terminals by this product can thus be considered. Also, physostigmine at concentration sufficient to inhibit acetylcholinesterase enhanced the effect of Die-Huang-Wan. Blockade of the increase of plasma insulin and plasma glucose lowering action of Die-Huang-Wan by 4-diphenylacetoxy-N-methylpiperdine methiodide (4-DAMP) indicated the mediation of muscarinic M3 receptors. The results suggest that Die-Huang-Wan may enhance the release of acetylcholine from nerve terminals to stimulate the muscarinic M3 receptors for augmenting insulin release to produce plasma glucose lowering action.

Topics: Acetylcholine; Administration, Oral; Animals; Blood Glucose; Cholinergic Agents; Cholinesterase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Drugs, Chinese Herbal; Hemicholinium 3; Insulin; Insulin Secretion; Male; Muscarinic Antagonists; Neuromuscular Depolarizing Agents; Physostigmine; Piperidines; Rats; Rats, Wistar

1. Previous studies have shown that phorbol esters induce protein kinase C (PKC) mediated phosphorylation of the vesicular acetylcholine transporter (VAChT) and change its interaction with vesamicol. However, it is not clear whether physiological activation of receptors coupled to PKC activation can alter VAChT behavior. 2. Here we tested whether activation of kaianate (KA) receptors alters VAChT. Several studies suggest that the cholinergic amacrine cells display KA/AMPA receptors that mediate excitatory input to these neurons. In addition, KA in the chicken retina can generate intracellular messengers with the potential to regulate cellular functions. 3. In cultured chicken retina (E8C11) KA reduced vesamicol binding to VAChT by 53%. This effect was potentiated by okadaic acid, a protein phosphatase inhibitor, and was totally prevented by BIM, a PKC inhibitor. 4. Phorbol myristate acetate (PMA), but not alpha-PMA, reduced in more than 85% the number of L-[3H]-vesamicol-specific binding sites in chicken retina, confirming that activation of PKC can influence vesamicol binding to chicken VAChT. 5. The data show that activation of glutamatergic receptors reduces [3H]-vesamicol binding sites (VAChT) likely by activating PKC and increasing the phosphorylation of the ACh carrier.

Topics: Acetylcholine; Animals; Binding Sites; Carrier Proteins; Chick Embryo; Choline O-Acetyltransferase; Enzyme Inhibitors; Immunohistochemistry; Kainic Acid; Membrane Transport Proteins; Organ Culture Techniques; Phosphorylation; Piperidines; Presynaptic Terminals; Protein Kinase C; Receptors, Kainic Acid; Retina; Synaptic Transmission; Tetradecanoylphorbol Acetate; Up-Regulation; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

We examined presynaptic cholinergic markers and beta-secretase activity during progressive central nervous system amyloidogenesis in Tg2576 Alzheimer mice (transgenic for human amyloid precursor protein Swedish mutation; hAPPswe). At 14, 18, and 23 months of age there were no significant differences between wild-type and transgenic mice in four distinct central nervous system cholinergic indices--choline acetyltransferase and acetylcholinesterase activities, and binding to vesicular acetylcholine transporter and Na(+)-dependent high-affinity choline uptake sites. A novel enzyme-linked immunosorbent assay measuring only the secreted human beta-secretase cleavage product (APPsbetaswe) of APPswe also revealed no change with aging in Tg2576 mouse brain. In contrast, transgenic but not wild-type mice exhibited an age-dependent increase in soluble Abeta40 and Abeta42 levels and progressive amyloid deposition in brain. Thus, aging Tg2576 mice exhibited presynaptic cholinergic integrity despite progressively increased soluble Abeta40 and Abeta42 levels and amyloid plaque density in brain. Older Tg2576 mice may best resemble preclinical or early stages of human Alzheimer's disease with preserved presynaptic cholinergic innervation. Homeostatic APPsbetaswe levels with aging suggest that progressive amyloid deposition in brain results not from increased beta-secretase cleavage of APP but from impaired Abeta/amyloid clearance mechanisms.

Topics: Acetylcholinesterase; Aging; Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Biomarkers; Brain; Choline O-Acetyltransferase; Disease Models, Animal; Endopeptidases; Female; Humans; Male; Mice; Mice, Transgenic; Neuromuscular Depolarizing Agents; Piperidines

Rat VAChT cDNA was stably transfected into PC12 cells to generate cell clones overexpressing different quantities of VAChT protein. Membrane fractions prepared from one cell clone (#3) in which VAChT was highly expressed (as determined by Western blot and [(3)H]vesamicol binding analyses) accumulated approximately two and half times as much [(3)H]ACh during incubation as did membrane fractions prepared from control cells. Vesamicol inhibited this ATP-dependent uptake. Membrane fractions isolated from a second cell clone (#6), which contained considerably less VAChT protein than did clone #3, accumulated no more [(3)H]ACh than did control cells. We compared the accumulation of newly synthesized [(14)C]ACh by a particulate fraction prepared from clones #3 and #6 with untransfected cells when these cells were incubated with either labeled acetate or choline. The results indicated that particulate fractions of clones #3 and #6 did not accumulate any more newly synthesized [(14)C]ACh than did the particulate fraction of untransfected cells. Furthermore, vesamicol reduced the filling of a particulate fraction of untransfected cells with newly synthesized ACh better than it reduced the refilling of a particulate fraction of VAChT-transfected cells. High K(+) depolarization did not release any more newly synthesized [(14)C]ACh from VAChT-transfected than it did from untransfected cells. In related studies, overexpression of VAChT in clone #3 induced a slight but significant increase in ChAT activity. Overall, our results indicate that an increase in the amount of VAChT protein associated with a particulate fraction of PC12 cells does not augment the amount of newly synthesized [(14)C]ACh acquired by that particulate fraction. However, it does reduce the effectiveness of vesamicol in blocking the filling of the particulate fraction with newly synthesized [(14)C]ACh. In summary, our results suggest that the vesicular release of ACh from PC12 cells is not regulated at the level of its uptake into synaptic vesicles.

Topics: Acetates; Acetylcholine; Animals; Brain Chemistry; Carbon Radioisotopes; Carrier Proteins; Central Nervous System; Choline; Choline O-Acetyltransferase; Clone Cells; Cloning, Molecular; Gene Expression Regulation; Membrane Potentials; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; PC12 Cells; Piperidines; Potassium; Presynaptic Terminals; Rats; Subcellular Fractions; Synaptic Membranes; Synaptic Transmission; Synaptic Vesicles; Transfection; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

In the present study, the radiotracer [(18)F] (+)-4-fluorobenzyltrozamicol ((+)-[(18)F]FBT) and positron emission tomography (PET) were used to examine the vesicular acetylcholine transporter and determine if presynaptic cholinergic activity was altered with age in 23 rhesus monkeys that varied in age from 10 to 37 years. Binding of (+)-[(18)F]FBT in the basal ganglia was reduced significantly with increasing age of the monkeys. However, there were individual differences noted in that some middle-aged and aged monkeys demonstrated levels of (+)-[(18)F]FBT binding that were comparable to the binding measured in adult monkeys. These data indicate that presynaptic cholinergic function may decrease with age, but that there may be a differential susceptibility of the cholinergic system to the aging process in different individuals.

Topics: Acetylcholine; Aging; Animals; Basal Ganglia; Carrier Proteins; Cerebellum; Fluorine Radioisotopes; Fluorobenzenes; Macaca mulatta; Male; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Piperidines; Presynaptic Terminals; Tomography, Emission-Computed; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

To evaluate if increased metabolic demand in remaining motor neurons in ALS spinal cord sections can be visualized by 3H-vesamicol binding.. As a presumed marker of the vesicular acetylcholine transporter, 3H-vesamicol was applied in quantitative autoradiography in cervical spinal cord sections from 11 ALS patients and 4 control cases. The regional binding was compared to that of the muscarinic ligand 3H-QNB.. Our results demonstrate the same magnitude of H-vesamicol binding in the ventral horn of ALS spinal cord as compared to controls, despite the profound loss of motor neurons in that specific area in ALS. The specificity of 3H-vesamicol binding for the cholinergic transporter is high in the motor neuron area, and sigma-sites constitute a minor proportion.. The lack of decrease in 3H-vesamicol binding in postmortem ALS spinal cord sections probably reflects an upregulated synthesis of vesicular membranes in remaining and hyperactive motor neurons in vivo.

Topics: Autoradiography; Carrier Proteins; Energy Metabolism; Humans; Membrane Transport Proteins; Motor Neuron Disease; Motor Neurons; Neuromuscular Depolarizing Agents; Piperidines; Reference Values; Spinal Cord; Tritium; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

It is considered that nodosal ganglion grafting improves learning and memory disorder in nucleus basalis magnocellularis lesioned rats, and that [3H]-vesamicol is one of the mapping agents for presynaptic cholinergic neurons. Quantitative autoradiography was performed using high sensitive imaging plate system to evaluate the effect of neurotransplantation on [3H]-vesamicol binding. The affected-side to unaffected-side ratio of [3H]-vesamicol in the nucleus basalis magnocellularis lesioned group was significantly lower than that in the sham-operated group at 1, 2 and 4weeks (P<0.01). In the transplanted group, the ratio was significantly higher than that in the untreated group at 4weeks (P<0.05). [3H]-vesamicol binding is one of the most sensitive indicators of nucleus basalis magnocellularis lesions and the effect of the nodosal ganglion grafting.

Topics: Animals; Autoradiography; Basal Nucleus of Meynert; Image Processing, Computer-Assisted; Male; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Sprague-Dawley; Transplantation, Autologous; Vagus Nerve

Several lines of evidence indicate that nerve growth factor is important for the development and maintenance of the basal forebrain cholinergic phenotype. In the present study, using rat primary embryonic basal forebrain cultures, we demonstrate the differential regulation of functional cholinergic markers by nerve growth factor treatment (24--96 h). Following a 96-h treatment, nerve growth factor (1--100 ng/mL) increased choline acetyltransferase activity (168--339% of control), acetylcholine content (141--185%), as well as constitutive (148--283%) and K(+)-stimulated (162--399%) acetylcholine release, but increased release was not accompanied by increased high-affinity choline uptake. Enhancement of ACh release was attenuated by vesamicol (1 microM), suggesting a vesicular source, and was abolished under choline-free conditions, emphasizing the importance of extracellular choline as the primary source for acetylcholine synthesized for release. A greater proportion of acetylcholine released from nerve growth factor-treated cultures than from nerve growth factor-naïve cultures was blocked by voltage-gated Ca(2+) channel antagonists, suggesting that nerve growth factor modified this parameter of neurotransmitter release. Cotreatment of NGF (20 ng/mL) with K252a (200 nM) abolished increases in ChAT activity and prevented enhancement of K(+)-stimulated ACh release beyond the level associated with K252a, suggesting the involvement of TrkA receptor signaling. Also, neurotrophin-3, neurotrophin-4 and brain-derived neurotrophic factor (all at 5--200 ng/mL) increased acetylcholine release, although they were not as potent as nerve growth factor and higher concentrations were required. High brain-derived neurotrophic factor concentrations (100 and 200 ng/mL) did, however, increase release to a level similar to nerve growth factor. In summary, long-term exposure (days) of basal forebrain cholinergic neurons to nerve growth factor, and in a less-potent fashion the other neurotrophins, enhanced the release of acetylcholine, which was dependent upon a vesicular pool and the availability of extracellular choline.

Topics: Acetylcholine; Animals; Biological Transport; Calcium Channel Blockers; Carrier Proteins; Cells, Cultured; Choline; Choline O-Acetyltransferase; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Membrane Transport Proteins; Nerve Growth Factors; Neuromuscular Depolarizing Agents; Neurons; Piperidines; Potassium; Prosencephalon; Rats; Rats, Sprague-Dawley; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

We investigated how elevated quantal release produced by motor nerve stimulation affects the size of the quanta. The motor nerve was stimulated at 10 Hz in preparations in which excitation-contraction coupling was disrupted. Two hundred stimuli reduced the size of the time integrals of the miniature endplate currents ([integral]MEPCs), measured at the same junction immediately after stimulation, by 16 %. Three thousand stimuli reduced size by 23 %. When the solution contained 10 microM neostigmine (NEO) 3000 stimuli reduced [integral]MEPCs by 60 %, because with acetylcholinesterase (AChE) inhibited, [integral]MEPC size is more sensitive to changes in acetylcholine (ACh) content. Similar decreases in miniature endplate potential size ([integral]MEPP) followed repetitive stimulation of contracting preparations. The depolarization produced by iontophoretic pulses of ACh was scarcely changed by 3000 nerve stimuli at 10 Hz, suggesting that the decreases in miniature sizes are largely due to less ACh released per quantum. Following 3000 stimuli at 10 Hz the sizes of the [integral]MEPCs increased back to pre-stimulus values with a half-time of 8-10 min. Recovery was blocked by (-)-vesamicol (VES), by hemicholinium-3 (HC3) and by nicotinic cholinergic agonists - all of which inhibit ACh loading into synaptic vesicles. The number of quanta in the total store was estimated by releasing them with carbonyl cyanide m-chlorophenylhydrazone (CCCP). CCCP releases fewer quanta after stimulation than from unstimulated controls. After resting for hours following stimulation, the releasable number increased, even when ACh loading inhibitors were present. We conclude that the inhibitors do not block a significant fraction of the ACh loading into reformed reserve vesicles and propose that ACh can be loaded in a series of steps.

Topics: Acetylcholine; Animals; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cholinergic Agents; Cholinesterase Inhibitors; Dimethylphenylpiperazinium Iodide; Electric Stimulation; Hemicholinium 3; Membrane Potentials; Motor Neurons; Neostigmine; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Nicotine; Nicotinic Agonists; Patch-Clamp Techniques; Piperidines; Rana pipiens; Synaptic Vesicles; Uncoupling Agents

To diagnose and investigate neurodegenerative diseases affecting cholinergic neuron density, piperazine derivatives of vesamicol were synthesized and evaluated. Previously, we reported that trans-5-iodo-2-hydroxy-3-[4-phenylpiperazinyl] tetralin (DRC140, 1) possessed high selectivity for vesicular acetylcholine transporter (VAChT). In present study of the effect of alkyl substituents, we observed that the introduction of a methyl group into the ortho or meta positions of the phenyl group of 1 increased affinity for VAChT. trans-5-Iodo-2-hydroxy-3-[4-[2-methylphenyl] piperazinyl]tetralin (2) displayed high affinity and specificity for VAChT. The regional distributions of radioactivity in the rat brain correlated well with known patterns of central cholinergic innervation. [(123)I]2 is a potentially useful compound for SPECT imaging.

Topics: Animals; Autoradiography; Brain; Cholinergic Fibers; Chromatography, High Pressure Liquid; Guinea Pigs; Male; Neuromuscular Depolarizing Agents; Neurons; Piperidines; Radiopharmaceuticals; Rats; Rats, Wistar; Structure-Activity Relationship; Tissue Distribution; Tomography, Emission-Computed, Single-Photon

We examined whether the ACh concentration measured by cardiac microdialysis provided information on left ventricular ACh levels under a variety of vagal stimulatory and modulatory conditions in anesthetized cats. Local administration of KCl (n = 5) and ouabain (n = 7) significantly increased the ACh concentration in the dialysate to 4.3 +/- 0.8 and 7.3 +/- 1.3 nmol/l, respectively, from the baseline value of 0.6 +/- 0.5 nmol/l. Intravenous administration of phenylbiguanide (n = 5) and phenylephrine (n = 6) significantly increased the ACh concentration to 5.4 +/- 0.9 and 6.0 +/- 1.5 nmol/l, respectively, suggesting that the Bezold-Jarisch and arterial baroreceptor reflexes affected myocardial ACh levels. Modulation of vagal nerve terminal function by local administration of tetrodotoxin (n = 6), hemicholinium-3 (n = 6), and vesamicol (n = 5) significantly suppressed the electrical stimulation-induced ACh release from 20.4 +/- 3.9 to 0.6 +/- 0.1, 7.2 +/- 1.9, and 2.7 +/- 0.6 nmol/l, respectively. Increasing the heart rate from 120 to 200 beats/min significantly reduced the myocardial ACh levels during electrical vagal stimulation, suggesting a heart rate-dependent washout of ACh. We conclude that ACh concentration measured by cardiac microdialysis provides information regarding ACh release and disposition under a variety of pathophysiological conditions in vivo.

Topics: Acetylcholine; Animals; Biguanides; Cats; Heart Conduction System; Heart Rate; Heart Ventricles; Hemicholinium 3; Microdialysis; Myocardium; Nerve Endings; Phenylephrine; Piperidines; Potassium; Potassium Chloride; Tetrodotoxin; Vagus Nerve

The physiological interrelationships between cognitive impairments, neurotransmitter loss, amyloid processing and energy metabolism changes in AD, cholinergic dementia and Down's syndrome are largely unknown to date. This report contains novel studies into the association between cognitive function and cerebral metabolism after long-term selective CNS cholinergic neuronal and synaptic loss in a rodent model. We measured local cerebral rates of glucose utilization ((14)C-2-deoxyglucose) throughout the brains of awake rats 4.5 months after bilateral intraventricular injections of a cholinotoxic antibody directed against the low-affinity NGF receptor (p75 NGF) associated with cholinergic neurons (192 IgG-saporin). Permanent cholinergic synapse loss was demonstrated by [(3)H]-vesamicol in vitro autoradiography defining presynaptic vesicular acetylcholine (ACh) transport sites. While other metabolic studies have defined acute and transient glucose use changes after relatively nonspecific lesions of anatomical regions containing cholinergic neurons, our results show sustained reductions in glucose utilization in brain regions impacted by cholinergic synapse loss, including frontal cortical and hippocampal regions, relative to glucose use levels in control rats. In the same animals, impaired cognitive spatial performance in a Morris water maze was correlated with reduced glucose use rates in the cortex and hippocampus at this time point, which is consistent with increased postmortem cortical and hippocampal amyloid precursor protein (APP) levels (45, 46). These results are consistent with the view of cholinergic influence over metabolism, APP processing, and cognition in the cortex and hippocampus.

Topics: Alzheimer Disease; Animals; Antibodies, Monoclonal; Autoradiography; Behavior, Animal; Cerebral Cortex; Cholinergic Agents; Cognition Disorders; Deoxyglucose; Disease Models, Animal; Female; Glucose; Hippocampus; Immunotoxins; In Vitro Techniques; Injections, Intraventricular; Maze Learning; N-Glycosyl Hydrolases; Neurons; Piperidines; Rats; Rats, Sprague-Dawley; Receptor, Nerve Growth Factor; Receptors, Nerve Growth Factor; Ribosome Inactivating Proteins, Type 1; Saporins; Synapses; Wakefulness

Cholinergic neurotransmission depends upon the regulated release of acetylcholine. This requires the loading of acetylcholine into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Here, we identify point mutants in Caenorhabditis elegans that map to highly conserved regions of the VAChT gene of Caenorhabditis elegans (CeVAChT) (unc-17) and exhibit behavioral phenotypes consistent with a reduction in vesicular transport activity and neurosecretion. Several of these mutants express normal amounts of VAChT protein and exhibit appropriate targeting of VAChT to synaptic vesicles. By site-directed mutagenesis, we have replaced the conserved amino acid residues found in human VAChT with the mutated residue in CeVAChT and stably expressed these cDNAs in PC-12 cells. These mutants display selective defects in initial acetylcholine transport velocity (K(m)), with values ranging from 2- to 8-fold lower than that of the wild-type. One of these mutants has lost its specific interaction with vesamicol, a selective inhibitor of VAChT, and displays vesamicol-insensitive uptake of acetylcholine. The relative order of behavioral severity of the CeVAChT point mutants is identical to the order of reduced affinity of VAChT for acetylcholine in vitro. This indicates that specific structural changes in VAChT translate into specific alterations in the intrinsic parameters of transport and in the storage and synaptic release of acetylcholine in vivo.

Topics: Acetylcholine; Amino Acid Sequence; Animals; Biological Transport; Caenorhabditis elegans; Molecular Sequence Data; PC12 Cells; Piperidines; Point Mutation; Rats; Receptors, Cholinergic; Synaptic Vesicles

1. The release of acetylcholine was investigated in the human placenta villus, a useful model for the characterization of the non-neuronal cholinergic system. 2. Quinine, an inhibitor of organic cation transporters (OCT), reduced acetylcholine release in a reversible and concentration-dependent manner with an IC(50) value of 5 microM. The maximal effect, inhibition by 99%, occurred at a concentration of 300 microM. 3. Procaine (100 microM), a sodium channel blocker, and vesamicol (10 microM), an inhibitor of the vesicular acetylcholine transporter, were ineffective. 4. Corticosterone, an inhibitor of OCT subtype 1, 2 and 3 reduced acetylcholine in a concentration-dependent manner with an IC(50) value of 2 microM. 5. Substrates of OCT subtype 1, 2 and 3 (amiloride, cimetidine, guanidine, noradrenaline, verapamil) inhibited acetylcholine release, whereas carnitine, a substrate of subtype OCTN2, exerted no effect. 6. Long term exposure (48 and 72 h) of villus strips to anti-sense oligonucleotides (5 microM) directed against transcription of OCT1 and OCT3 reduced the release of acetylcholine, whereas OCT2 anti-sense oliogonucleotides were ineffective. 7. It is concluded that the release of non-neuronal acetylcholine from the human placenta is mediated via organic cation transporters of the OCT1 and OCT3 subtype.

Topics: Acetylcholine; Amiloride; Cimetidine; Corticosterone; DNA, Antisense; Dose-Response Relationship, Drug; Female; Guanidine; Humans; In Vitro Techniques; Norepinephrine; Organic Cation Transport Proteins; Organic Cation Transporter 1; Organic Cation Transporter 2; Piperidines; Placenta; Pregnancy; Procaine; Quinine; Time Factors; Verapamil

A detailed investigation of endogenous acetylcholine (ACh) release from primary embryonic septal cultures is described in this study. Applications of veratridine (25 microM) or increasing extracellular concentrations of K(+) (6-100 mM) induced robust increases of endogenous ACh release ( approximately 500-15,000 fmol/well/10 min). Release stimulated with K(+) (25 mM) was sustainable and did not differ significantly over 180 min. ACh release was dependent on extracellular choline and decreased proportionally to choline concentrations (0-10 microM). For example, after 30 min of stimulation with K(+) (25 mM), release in the absence of extracellular choline was approximately 25% of that associated with 10 microM choline. The vesicular transport blocker vesamicol (0-5 microM) almost completely prevented stimulated and basal ACh release at the highest concentration evaluated, which suggests a mostly vesicular mode of release in this model. The M(2)-like muscarinic receptor antagonist AF-DX 384 (0-10 microM) enhanced stimulated ACh release ( approximately 150% at the highest concentration evaluated), whereas the nonspecific muscarinic receptor agonist oxotremorine (0-10 microM) decreased stimulated release (approximately 60% at the highest concentration evaluated), suggesting that functional muscarinic autoreceptors exist in primary embryonic septal cultures. Novel findings concerning ACh release from primary embryonic septal cultures are reported herein, and the demonstration of ACh release gives further credit to the use of these cultures for studying cholinergic system functioning and in relation to physiology and pathology.

Topics: Acetylcholine; Animals; Cells, Cultured; Choline; Choline O-Acetyltransferase; Chromatography, High Pressure Liquid; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Muscarinic Agonists; Neuromuscular Depolarizing Agents; Oxotremorine; Parasympatholytics; Piperidines; Pirenzepine; Potassium; Rats; Septum of Brain; Time Factors; Veratridine

The role of proton binding sites in the vesicular acetylcholine transporter was investigated by characterization of the pH dependence for the binding of [3H]vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol] to Torpedo synaptic vesicles. A single proton binds to a site with pKa 7.1 +/- 0.1, which is characteristic of histidine, to competitively inhibit vesamicol binding. The histidine-selective reagent diethylpyrocarbonate causes time-dependent inhibition of [3H]vesamicol binding with a rate constant only about 20-fold lower than for reaction with free histidine. Because its pH titration has a simple, ideal shape, this residue probably controls all pH effects in the transporter between pH 6-8. Inhibition of [3H]vesamicol binding by diethylpyrocarbonate was slowed by vesamicol but not acetylcholine, which binds to a separate site. The data suggest that a critical histidine with a pKa of 7.1 is unhindered when reacting with diethylpyrocarbonate. A conformational model for the histidine is proposed to explain why acetylcholine competes with protons but not with diethylpyrocarbonate. A conserved histidine in transmembrane helix VIII possibly is the histidine detected here.

Topics: Animals; Binding Sites; Carrier Proteins; Cysteine; Diethyl Pyrocarbonate; Electric Organ; Histidine; Hydrogen-Ion Concentration; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Piperidines; Protons; Synaptic Vesicles; Torpedo; Tritium; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.

Topics: Acetylcholine; Animals; Biological Transport; Blotting, Western; Carrier Proteins; Centrifugation, Density Gradient; Glycosylation; Histidine; Membrane Proteins; Membrane Transport Proteins; Mutation; Piperidines; Protein Binding; Protein Structure, Tertiary; Protons; Rats; Sequence Homology, Amino Acid; Static Electricity; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Properties of cysteinyl residues in the vesicular acetylcholine transporter (VAChT) of synaptic vesicles isolated from Torpedo californica were probed. Cysteine-specific reagents of different size and polarity were used and the effects on [3H]vesamicol binding determined. The vesamicol dissociation constant increased 1,000-fold after reaction with p-chloromercuriphenylsulfonate or phenylmercury acetate, but only severalfold after reaction with relatively small methylmercury chloride or methylmethanethiosulfonate (MMTS). Methylmercury chloride, but not MMTS, protected binding from phenylmercury acetate. Thus, two classes of cysteines react to affect vesamicol binding. Class 1 reacts with only organomercurials, and class 2 reacts with both organomercurials and MMTS. Quantitative analysis of the competition between p-chloromercuriphenylsulfonate and VAChT ligands was possible after defining second-order reaction conditions. The results indicate that each cysteinyl class probably contains a single residue. Acetylcholine protects cysteine 1, but apparently does not protect cysteine 2. Vesamicol, which binds to a different site than acetylcholine does, apparently protects both cysteines, suggesting that it induces a conformational change. The relatively large reagent glutathione removes a substituent from cysteine 1, but not cysteine 2, suggesting that cysteine 2 is deeper in the transporter than cysteine 1 is. The complete sequence of T. californica VAChT is given, and possible identities of cysteines 1 and 2 are discussed.

Topics: 4-Chloromercuribenzenesulfonate; Acetylcholine; Amino Acid Sequence; Animals; Base Sequence; Binding Sites; Binding, Competitive; Carrier Proteins; Cysteine; Cytoplasm; Dose-Response Relationship, Drug; Glutathione; Membrane Transport Proteins; Methyl Methanesulfonate; Methylmercury Compounds; Molecular Sequence Data; Neuromuscular Depolarizing Agents; Organomercury Compounds; Phenylmercury Compounds; Piperidines; Protein Structure, Tertiary; Torpedo; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Metabolic labeling of a mutant PC12 cell line, A123.7, expressing recombinant rat vesicular acetylcholine transporter (VAChT) with radiolabeled inorganic phosphate was used to demonstrate phosphorylation of the transporter on a serine residue. Mutational analysis was used to demonstrate that serine 480, which is located on the COOH-terminal cytoplasmic tail, is the sole phosphorylation site. Phosphorylation of serine 480 was attributable to the action of protein kinase C. Using a permanently dephosphorylated form of rat VAChT, S480A rVAChT, it was shown that this mutant displays the same kinetics for the transport of acetylcholine and the binding of the inhibitor vesamicol as does the wild type transporter. However, sucrose gradient density centrifugation showed that, unlike wild type VAChT, the S480A mutant did not localize to synaptic vesicles. These results suggest that phosphorylation of serine 480 of VAChT is involved in the trafficking of this transporter.

Topics: Acetylcholine; Amino Acid Sequence; Animals; Binding Sites; Blotting, Western; Carrier Proteins; Cell Nucleus; Centrifugation, Density Gradient; Cytoplasm; Immunoblotting; Membrane Transport Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Neuromuscular Depolarizing Agents; PC12 Cells; Phosphorylation; Piperidines; Precipitin Tests; Protein Kinase C; Protein Structure, Tertiary; Rats; Recombinant Proteins; Sequence Homology, Amino Acid; Serine; Synaptophysin; Transfection; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

The role of calcium in the modulation of spontaneous [3H]acetylcholine ([3H]ACh) efflux through presynaptic D2 dopamine hetero-receptors was investigated in rat striatal synaptosomes. The kinetic studies of [3H]ACh efflux in the presence or absence of Ca2+ were carried out in nonstimulating conditions. When Ca2+ was omitted from the superfusion medium, a notable and significant (P<0.001) decrease of tritium efflux (39%) was obtained. While [3H]ACh efflux was insensitive to tetrodotoxin (TTX) 1 microM, cadmium (10 microM), a nonselective antagonist of calcium channels, significantly reduced the tritium efflux by 24% (P<0.001), while the L-type calcium antagonist, nifedipine, (30 microM) inhibited the tritium efflux by only 10% (P<0.02). 2-(4-Fenylpiperidine)cyclohexanol (vesamicol), an inhibitor of the vesicular [3H]ACh carrier, significantly depressed the spontaneous tritium efflux in the presence of Ca2+ (60%; P<0.001) and in a low-calcium medium (20%; P<0.001). Although 1 microM of 7-hydroxy-N,N-di-n-propyl-2-aminotetraline (7-OH-DPAT) inhibited spontaneous [3H]ACh efflux in the presence of calcium, this dopaminergic agonist did not modify the neurotransmitter release in either the low-Ca2+ medium or in the presence of vesamicol. These results suggest that the spontaneous [3H]ACh efflux is a process involving a Ca2+-dependent component (39%), sensitive to calcium channel-blockers and vesamicol, in rat striatal synaptosomes. In addition, activation of the D2 dopamine hetero-receptor only modulates the calcium-dependent component of spontaneous [3H]ACh efflux.

Topics: Acetylcholine; Animals; Calcium; Calcium Channel Blockers; Cholinesterase Inhibitors; Corpus Striatum; Dopamine Agonists; Male; Physostigmine; Piperidines; Rats; Rats, Sprague-Dawley; Receptors, Dopamine D2; Synaptosomes; Tetrahydronaphthalenes; Tetrodotoxin; Tritium

m-(Iodobenzyl)trozamicol (MIBT) is a recently discovered vesamicol analogue. It has been shown that radiolabelled [125I]MIBT can be used as a marker of cholinergic innervation in the heart as well as in the brain. The purpose of this study was to analyze the direct effects of MIBT on the atrioventricular and intraventricular conduction in addition to the coronary blood flow using the canine isolated, blood-perfused atrioventricular node preparation. Intracoronary administration of MIBT suppressed the atrioventricular and intraventricular conduction, while it increased the coronary blood flow. The effect and duration of action on the intraventricular conduction was less pronounced compared with other effects. Moreover, the doses of MIBT needed to cause negative dromotropic and coronary vasodilator effects in this study was much greater than those needed for imaging the cardiac cholinergic innervation. Pretreatment of the preparations with a muscarinic receptor antagonist, atropine, did not block these effects of MIBT, suggesting that MIBT may possess muscarinic receptor-independent ion channel activity in the cardiac conduction system and coronary arteries.

Topics: Animals; Atrioventricular Node; Atropine; Cholinergic Antagonists; Coronary Circulation; Dogs; Dose-Response Relationship, Drug; Iodobenzenes; Perfusion; Piperidines; Receptors, Cholinergic

The probes to detect vesicular acetylcholine transporter (VAChT) in vivo are important to evaluate the mapping and function in cholinergic system. To develop high-specific and high-affinity radiotracer for single photon emission computed tomography, we investigated piperazine analogs which replaced the piperidine ring of (-)-vesamicol with a piperazine ring. We found that the piperazine analog of iodobenzovesamicol, trans-5-iodo-2-hydroxy-3-[4-phenylpiperazinyl] tetralin (DRC140), had high affinity for VAChT in rat brain. We carried out binding assay in subcellular fraction of the rat brain. The highest B(max) for [(125)I]-DRC140 binding was observed in the synaptic vesicle fraction (1,751 fmol/mg protein), followed by the crude vesicle (821 fmol/mg protein) and the P2 fraction (187 fmol/mg protein). These K(d) values were similar to the affinity of highly purified synaptic vesicular fraction (K(d) = 0.3 nM) with a one-site model. The possibility that [(125)I]-DRC140 recognizes sigma receptor was excluded by our finding large inhibition constants (K(i) = 849 nM for haloperidol, K(i) = 3,052 nM for 1,3-di(2-tolyl)guanidine). In vivo distribution studies with the [(123)I]-DRC140 in rats showed a rapid brain uptake. The highest brain area was in striatum, followed by frontal cortex, occipital cortex, and hippocampus. The lowest brain area was cerebellum. The radioactivity of high-accumulated areas in ex vivo autoradiography was reduced by a preinjection of (-)-vesamicol and these levels were reduced to the radioactivity in cerebellum. These results show that [(125)I]-DRC140 can provide extremely high specific tracer with excellent brain permeability as a ligand for single photon emission computed tomography.

Topics: Acetylcholine; Animals; Brain; Carrier Proteins; Guinea Pigs; In Vitro Techniques; Iodine Radioisotopes; Male; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Neurons; Piperazines; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Subcellular Fractions; Synaptic Vesicles; Tomography, Emission-Computed, Single-Photon; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

We hypothesized that blockade of synthesis or release of several categories of neurotransmitters would ameliorate opioid neurotoxicity. Rats were randomly assigned to one of six groups in two sequential protocols: vesamicol (VES, n = 10), alpha-fluoromethylhistidine (FMH, n = 10), reserpine (RES, n = 10), BW1003C87 (BW, n = 7), lamotrigine (LAM, n = 10), or one of two control groups (CON, n = 19). Physiologically controlled rats received fentanyl (fen) i.v., loading dose 800 micrograms kg-1 followed by maintenance dose 32 micrograms kg-1 min-1 for 2 h. Drug dosing: CON, isovolemic (between rats) 0.9% saline i.v.; BW, 20 mg kg-1 i.v. 15 min pre-fen; LAM, 16 mg kg-1 i.v. 30 min pre-fen; VES, 2.5 mg kg-1 i.p. 60 min and 30 min pre-fen then infused 3.75 mg kg-1 during fen; FMH, 20 mg kg-1 i.p. 2 h pre-fen; RES, 0.75 mg kg-1 i.p. 18 h pre-fen. Seven days later all rats underwent cerebral perfusion fixation, followed by histologic grading (0-5, 0 = normal). Pathological data was analyzed by Wilcoxen's Signed rank test (two-tailed) for pathologic scores summated across all brain areas (overall severity score) and for scores of areas previously associated with opioid neurotoxicity. Compared to CON, overall severity was decreased by RES (p = 0.05) with an effect suggested by VES (p = 0.10). Compared to CON, lesions were decreased: (a) in the amygdala with VES (p = 0.03) and RES (p = 0.05) with a trend suggested by BW (p = 0.06); (b) in the subiculum by VES (p = 0.02) and RES (p = 0.008) with a trend suggested by FMH (p = 0.06); and (c) in the entorhinal cortex by VES (p = 0.004) and RES (p = 0.008) with a trend suggested by FMH (p = 0.07). The data indicate that brain acetylcholine and catecholamines contribute to opioid neurotoxicity, and suggest a possible role of glutamate and histamine in opioid neurotoxicity.

Topics: Adrenergic Uptake Inhibitors; Analgesics, Opioid; Animals; Blood Pressure; Body Temperature; Brain; Excitatory Amino Acid Antagonists; Fentanyl; Lamotrigine; Male; Neuromuscular Depolarizing Agents; Neuroprotective Agents; Piperidines; Pyrimidines; Rats; Rats, Sprague-Dawley; Reserpine; Triazines

The aim of this study was to investigate spatial memory and quantitative acetylcholine transporter autoradiography using a high-sensitivity imaging plate system in rats treated with beta-amyloid protein, a model of Alzheimer's disease. An eight-arm radial maze was used to evaluate spatial memory. The performance of the eight-arm radial maze task was impaired in beta-amyloid protein-treated rats. In the parietal cortex, [3H]-vesamicol binding to the vesicular acetylcholine transporter was significantly lower in beta-amyloid protein-treated rats than in vehicle-treated rats, and was significantly correlated with the mean number of correct selections in the maze task of the first 5 days in the post-operative state. These results indicate that the reduction in [3H]-vesamicol binding to vesicular acetylcholine transporter is related to memory impairment induced by beta-amyloid protein. Beta-amyloid protein-infused rats with spatial memory impairment may be useful for the development of new radiolabelled vesamicol analogues for the objective evaluation of Alzheimer's disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Autoradiography; Carrier Proteins; Disease Models, Animal; Humans; Immunohistochemistry; Male; Membrane Transport Proteins; Memory Disorders; Piperidines; Quinuclidinyl Benzilate; Radionuclide Imaging; Radiopharmaceuticals; Rats; Rats, Sprague-Dawley; Receptors, Muscarinic; Synaptic Vesicles; Tissue Distribution; Tritium; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

The vesicular acetylcholine transporter (VAChT) is responsible for the transport of the neurotransmitter acetylcholine (ACh) into synaptic vesicles using an electrochemical gradient to drive transport. Rat VAChT has a number of aspartate residues within its predicted transmembrane domains (TM) and cytoplasmic loops, which may play important structural or functional roles in acetylcholine transport. In order to identify functional charged residues, site-directed mutagenesis of rVAChT was undertaken. No effect on ACh transport was observed when any of the five aspartate residues in the cytoplasmic loop were converted to asparagine. Similarly, changing Asp-46 (D46N) in TM1 or Asp-255 (D255N) in TM6 had no effect on ACh transport or vesamicol binding. However, replacement of Asp-398 in TM10 with Asn completely eliminated both ACh transport and vesamicol binding. The conservative mutant D398E retained transport activity, but not vesamicol binding, suggesting this residue is critical for transport. Mutation of Asp-193 in TM4 did not affect ACh transport activity; however, vesamicol binding was dramatically reduced. With mutant D425N of TM11 transport activity for ACh was completely blocked, without an effect on vesamicol binding. Activity was not restored in the conservative mutant D425E, suggesting the side chain as well as the negative charge of Asp-425 is important for substrate binding. These mutants, as well as mutant D193N, clearly dissociated ACh binding and transport from vesamicol binding. These data suggest that Asp-398 in TM10 and Asp-425 in TM11 are important for ACh binding and transport, while Asp-193 and Asp-398 in TM4 and TM10, respectively, are involved in vesamicol binding.

Topics: Acetylcholine; Animals; Aspartic Acid; Carrier Proteins; Cytoplasm; Humans; Membrane Transport Proteins; Mutagenesis, Site-Directed; PC12 Cells; Piperidines; Protein Binding; Rats; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

We investigated whether recycled cholinergic synaptic vesicles, which were not refilled with ACh, would join other synaptic vesicles in the readily releasable store near active zones, dock, and continue to undergo exocytosis during prolonged stimulation. Snake nerve-muscle preparations were treated with 5 microM vesamicol to inhibit the vesicular ACh transporter and then were exposed to an elevated potassium solution, 35 mM potassium propionate (35 KP), to release all preformed quanta of ACh. At vesamicol-treated endplates, miniature endplate current (MEPC) frequency increased initially from 0.4 to >300 s-1 in 35 KP but then declined to <1 s-1 by 90 min. The decrease in frequency was not accompanied by a decrease in MEPC average amplitude. Nerve terminals accumulated the activity-dependent dye FM1-43 when exposed to the dye for the final 6 min of a 120-min exposure to 35 KP. Thus synaptic membrane endocytosis continued at a high rate, although MEPCs occurred infrequently. After a 120-min exposure in 35 KP, nerve terminals accumulated FM1-43 and then destained, confirming that exocytosis also still occurred at a high rate. These results demonstrate that recycled cholinergic synaptic vesicles that were not refilled with ACh continued to dock and undergo exocytosis after membrane retrieval. Thus transport of ACh into recycled cholinergic vesicles is not a requirement for repeated cycles of exocytosis and retrieval of synaptic vesicle membrane during prolonged stimulation of motor nerve terminals.

Topics: Acetylcholine; Animals; Carrier Proteins; Colubridae; Electric Stimulation; Exocytosis; Fluorescent Dyes; In Vitro Techniques; Membrane Transport Proteins; Motor Endplate; Motor Neurons; Nerve Endings; Piperidines; Pyridinium Compounds; Quaternary Ammonium Compounds; Synaptic Membranes; Synaptic Vesicles; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

During partial neuromuscular blockade indirect repetitive nerve stimulation causes fade in the response of the muscle. We studied the intensity of the fade induced by intravenous administration of three steroidal muscle relaxants, and investigated the mechanism of fade by comparing with results obtained during partial blockade with animal toxins and vesamicol.. In 60 cats, we measured the fade in the compound action potentials of the gastrocnemius muscle evoked by repetitive sciatic nerve stimulation at 100 Hz during partial neuromuscular blockade with rocuronium, vecuronium, pancuronium, alpha-bungarotoxin, mu-conotoxin and vesamicol, respectively.. Profound fade was induced by all three non-depolarising muscle relaxants (rocuronium=vecuronium

Topics: Acetylcholine; Action Potentials; Androstanols; Animals; Bungarotoxins; Cats; Conotoxins; Electric Stimulation; Electromyography; Hindlimb; Muscle, Skeletal; Neuromuscular Depolarizing Agents; Neuromuscular Nondepolarizing Agents; Pancuronium; Peptides, Cyclic; Piperidines; Receptors, Cholinergic; Rocuronium; Sodium Channel Blockers; Vecuronium Bromide

Immortalized rat brain endothelial RBE4 cells do not express choline acetyltransferase (ChAT), but they do express an endogenous machinery that enables them to release specifically acetylcholine (ACh) on calcium entry when they have been passively loaded with the neurotransmitter. Indeed, we have previously reported that these cells do not release glutamate or GABA after loading with these transmitters. The present study was set up to engineer stable cell lines producing ACh by transfecting them with an expression vector construct containing the rat ChAT. ChAT transfectants expressed a high level of ChAT activity and accumulated endogenous ACh. We examined evoked ACh release from RBE4 cells using two parallel approaches. First, Ca2+-dependent ACh release induced by a calcium ionophore was followed with a chemiluminescent procedure. We showed that ChAT-transfected cells released the transmitter they had synthesized and accumulated in the presence of an esterase inhibitor. Second, ACh released on an electrical depolarization was detected in real time by a whole-cell voltage-clamped Xenopus myocyte in contact with the cell. Whether cells synthesized ACh or whether they were passively loaded with ACh, electrical stimulation elicited the release of ACh quanta detected as inward synaptic-like currents in the myocyte. Repetitive stimulation elicited a continuous train of responses of decreasing amplitudes, with rare failures. Amplitude analysis showed that the currents peaked at preferential levels, as if they were multiples of an elementary component. Furthermore, we selected an RBE4 transgenic clone exhibiting a high level of ChAT activity to introduce the Torpedo vesicular ACh transporter (VAChT) gene. However, as the expression of ChAT was inactivated in stable VAChT transfectants, the potential influence of VAChT on evoked ACh release could only be studied on cells passively loaded with ACh. VAChT expression modified the pattern of ACh delivery on repetitive electrical stimulation. Stimulation trains evoked several groups of responses interrupted by many failures. The total amount of released ACh and the mean quantal size were not modified. As brain endothelial cells are known as suitable cellular vectors for delivering gene products to the brain, the present results suggest that RBE4 cells genetically modified to produce ACh and intrinsically able to support evoked ACh release may provide a useful tool for improving altered cholinergic function in the CNS.

Topics: Acetylcholine; Animals; Carrier Proteins; Cell Line, Transformed; Cells, Cultured; Cerebrovascular Circulation; Choline O-Acetyltransferase; Endothelium, Vascular; Membrane Potentials; Membrane Transport Proteins; Microcirculation; Muscle, Skeletal; Neuromuscular Depolarizing Agents; Patch-Clamp Techniques; Piperidines; Rats; Recombinant Proteins; Transfection; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins; Xenopus laevis

The laminar binding distribution of three nicotinic receptor agonists, [3H](-)nicotine, [3H]cytisine, and [3H]epibatidine, and their relation to the [3H]vesamicol binding, which is known to represent the vesicular acetylcholine transport sites, was performed employing in vitro autoradiography on the medial temporal cortex (Brodmann area 21). Autopsied brain tissue from nine Alzheimer patients and seven age-matched controls were used. The binding pattern of the three nicotinic ligands in the normal cortex was in general similar, showing binding maxima in the cortical layers I, III and V. The binding of [3H](-)nicotine, [3H]cytisine, and [3H]epibatidine was lower in the older controls and more uniform throughout the layers as compared with younger controls. There was a significant age-related decrease in the binding of the three nicotinic ligands within the controls (age range: 58 to 89 years; P[3H](-)nicotine = 0.002, P[3H]epibatidine = 0.010, P[3H]cytisine = 0.037). In the older controls, the [3H]epibatidine binding was much decreased as compared with that of [3H](-)nicotine and [3H]cytisine. This may indicate a higher selectivity of [3H]epibatidine for a nicotinic receptor subtype that is particularly affected by aging. The laminar binding pattern of [3H]vesamicol showed one maximum in the outer cortical layers II/III. The [3H]vesamicol binding did not change with aging. The binding of all ligands was significantly decreased in all layers of the temporal cortex in Alzheimer's disease, but the [3H]vesamicol binding decreased only half as much as the nicotinic receptors. Also, choline acetyltransferase activity was percentually more reduced than [3H]vesamicol binding in Alzheimer's disease. The cortical laminar binding pattern of all 3H-ligands was largely absent in the Alzheimer's disease cases. The less severe loss of vesicular acetylcholine transport sites as compared with the loss of the nicotinic receptors and choline acetyltransferase activity may suggest that vesamicol binding sites might be more preserved in presynaptic terminals still existing and thereby expressing compensatory capacity to maintain cholinergic activity.

Topics: Acetylcholine; Age Factors; Aged; Aged, 80 and over; Alkaloids; Alzheimer Disease; Autoradiography; Azocines; Bridged Bicyclo Compounds, Heterocyclic; Carrier Proteins; Female; Humans; Male; Membrane Transport Proteins; Middle Aged; Nicotine; Piperidines; Pyridines; Quinolizines; Reference Values; Temporal Lobe; Tritium; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Four isomeric N-(halophenyl)trozamicol analogues (6a-d) were synthesized and evaluated as potential vesicular acetylcholine transporter (VAChT) ligands. Of the four compounds, N-(3-bromophenyl) trozamicol (6b) and N-(3-iodophenyl)trozamicol (6d) displayed the highest affinity for the VAChT in vitro, whereas the para-substituted compound 6c showed the lowest affinity for this transporter. Tissue distribution studies of N-(3-[125I]iodophenyl)trozamicol ([125I]6d, [125I)IPHT) suggest that the central distribution of the latter is consistent with cholinergic innervation. However, only moderate target-to-background ratios were obtained, suggesting little improvement over the N-(halobenzyl)trozamicols described previously.

Topics: Acetylcholine; Animals; Binding, Competitive; Brain; Carrier Proteins; Indicators and Reagents; Iodine Radioisotopes; Iodobenzenes; Isomerism; Kinetics; Ligands; Male; Membrane Transport Proteins; Molecular Structure; Piperazines; Piperidines; Rats; Rats, Wistar; Receptors, sigma; Structure-Activity Relationship; Tissue Distribution; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Topics: Acetylcholine; Alzheimer Disease; Brain; Carbon Radioisotopes; Carrier Proteins; Humans; Membrane Glycoproteins; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Neurons; Neuropeptides; Neurotransmitter Agents; Parkinson Disease; Piperidines; Tetrabenazine; Tomography, Emission-Computed; Tomography, Emission-Computed, Single-Photon; Tritium; Vesicular Acetylcholine Transport Proteins; Vesicular Biogenic Amine Transport Proteins; Vesicular Transport Proteins

1. Calcitonin gene-related peptide (CGRP) is found in dense-cored vesicles in the motor nerve terminal. 2. Exogenous CGRP increased the size of the quanta. The increase in size reached a maximum after about 40 min. The lowest effective concentration of human CGRP (hCGRP) was 0.8 nM. The action of hCGRP was antagonized by (-)-vesamicol, a drug that blocks active acetylcholine (ACh) uptake into synaptic vesicles, so it appears that hCGRP increases size by adding more ACh to the quanta. The action of hCGRP was antagonized by drugs that block the activation of protein kinase A (PKA). (In other preparations CGRP also activates PKA.) 3. The hCGRP effect was not blocked by fragment 8-37, an antagonist of one class of CGRP receptor. 4. hCGRP increases evoked quantal output and miniature endplate potential (MEPP) frequency, again by activating PKA. 5. CGRP release was measured by radioimmunoassay. Release was increased by depolarization with elevated K+, but the amounts released appear to be below those needed to affect quantal size or output. Moreover, although elevated K+ can increase quantal size it acts by a pathway that does not involve PKA. We suggest that the most likely target of endogenously released CGRP is the regulation of circulation of the muscle.

Topics: Acetylcholine; Animals; Arachidonic Acids; Calcitonin Gene-Related Peptide; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Endocannabinoids; Enzyme Inhibitors; Evoked Potentials; Humans; In Vitro Techniques; Kinetics; Motor Neurons; Muscle, Skeletal; Neuromuscular Junction; Patch-Clamp Techniques; Peptide Fragments; Piperidines; Polyunsaturated Alkamides; Quantum Theory; Rana pipiens; Thionucleotides

The present experiments investigated the release of [3H]acetylcholine ([3H]ACh) from the guinea pig myenteric plexus treated with 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a drug that impairs ACh accumulation by synaptic vesicles. Ouabain, an Na+-K+ ATPase inhibitor, released [3H]ACh synthesised in the presence of (-)-vesamicol, while electrical field stimulation or KCl depolarisation were not effective to release the transmitter in this condition. The effect of ouabain was Ca2+-dependent and in the presence of (-)-vesamicol it was blocked by calphostin C, an inhibitor of protein kinase C (PKC). In addition, stimulation of kinase C activity by a phorbol ester, but not by its inactive isomer, prevented (-)-vesamicol from interfering with the release of [3H]ACh in electrically-stimulated myenteric plexus, similar to the effect of ouabain. We conclude that release of [3H]ACh induced by ouabain in the presence of (-)-vesamicol depends on PKC activation.

Topics: Acetylcholine; Animals; Calcium; Electric Stimulation; Enzyme Inhibitors; Female; Guinea Pigs; Kinetics; Male; Myenteric Plexus; Naphthalenes; Neuromuscular Depolarizing Agents; Ouabain; Piperidines; Potassium Chloride; Protein Kinase C; Synaptic Vesicles; Tritium

1. Acetylcholine (ACh) is important as the transmitter responsible for neuromuscular transmission. Here we report the non-quantal release of ACh from embryonic myocytes. 2. Co-cultures of spinal neurons and myotomal muscle cells were prepared from 1-day-old Xenopus embryos. Single channel currents were recorded in the non-innervated myocytes. When the patch pipette was filled with Ringer solution alone, spontaneous single channel currents occurred, which were inhibited by d-tubocurarine (d-Tc). 3. The channel conductance appearing in Ringer solution (37.3 pS) was similar to that of an embryonic-type ACh channel (36.9 pS), indicating that ACh is probably released from myocytes in normal Ringer solution. 4. When the patch pipette was filled with anticholinesterase alone to prevent hydrolysis of ACh released from myocytes, both physostigmine and neostigmine in a concentration-dependent manner increased channel open probability; it was reduced by d-Tc or alpha-bungarotoxin. 5. Vesamicol and quinacrine, vesicular transporter inhibitors, reduced the channel open probability caused by ACh released from myocytes in the presence of neostigmine or physostigmine. 6. Intracellular alkalinization with NH4Cl inhibited the ACh release from myocytes, whereas, extracellular alkalinization, brought about by replacing normal Ringer solution, with pH 8.6 Ringer solution enhanced ACh release. 7. The immunocytochemistry of choline acetyltransferase (ChAT) showed that ChAT exists in both myocytes and neuronal cells but not in fibroblasts. 8. These results suggest that embryonic myocytes are capable of synthesizing and releasing ACh in a non-quantal manner. Extracellular alkalinization enhanced and intracellular alkalinization inhibited ACh release from myocytes.

Topics: Acetylcholine; Ammonium Chloride; Animals; Cells, Cultured; Choline O-Acetyltransferase; Coculture Techniques; Embryo, Nonmammalian; Heart; Membrane Potentials; Muscles; Myocardium; Neurons; Piperidines; Quinacrine; Receptors, Cholinergic; Spinal Cord; Tubocurarine; Xenopus

Mediatophore is a protein that translocates acetylcholine (ACh) on calcium action. It is a homopolymer of a 15-kDa proteolipid that is also a constituent of the membrane sector of vacuolar H+-adenosine trisphosphatase (V-ATPase; vacuolar proton pump). Experiments on neuroblastoma cell lines (N18TG-2) that are deficient for ACh release and on cells that are competent for release, such as the glioma C6BU-1 or the N18TG-2/C6BU-1 fusion product NG108-15, show that there is a correlation between ACh release and the 15-kDa proteolipid content of the cell membrane. In another cell line, L-M(TK-), it has been possible to up-regulate ACh release and the membrane proteolipid content after treating the cells with dibutyryl-cyclic AMP or dexamethasone. As mediatophore translocates ACh and as V-ATPase may help vesicular ACh storage, it was interesting to determine the respective role of the two proteins in the observed correlation between release and proteolipid content. After blocking vesicular loading with vesamicol, we did not affect release from these cells, suggesting that the observed correlation may be attributed to mediatophore. The acquisition of an ACh release mechanism would then depend on the process that guides the proteolipid to the plasma membrane of the cell.

Topics: Acetylcholine; Animals; Anti-Inflammatory Agents; Biological Transport; Cell Membrane; Dexamethasone; Glioma; Hybrid Cells; Mice; Nerve Tissue Proteins; Neuromuscular Depolarizing Agents; Piperidines; Proteolipids; Proton Pumps; Proton-Translocating ATPases; Rats; Vacuolar Proton-Translocating ATPases

Topics: Acetylcholine; Animals; Carrier Proteins; Cell Membrane; CHO Cells; COS Cells; Cricetinae; Cytoplasmic Granules; Kinetics; Membrane Transport Proteins; Neurons; Neurotransmitter Agents; Piperidines; Recombinant Proteins; Tetrabenazine; Transfection; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Chimeras between the human vesicular acetylcholine transporter (hVAChT) and the neuronal isoform of the human vesicular monoamine transporter (hVMAT2) have been constructed and stably expressed in a rat pheochromocytoma cell line (PC12) in an effort to identify cholinergic-specific domains of VAChT. Examination of the transport properties of a chimera in which the N-terminal portion (up to putative transmembrane domain II and including the lumenal glycosylated loop) of hVAChT was replaced with hVMAT2 sequences (2/V@NheI) revealed that its apparent affinity for acetylcholine (ACh) was reduced approximately seven-fold compared to wild-type. However, the affinity of this chimera for vesamicol did not significantly differ from hVAChT. Similarly, the 2/V@NheI chimera retained its preferential targeting to the small synaptic-like vesicles found in PC12 cells in agreement with our recently reported observations that the synaptic vesicle targeting domain resides in the cytoplasmic tail of VAChT.

Topics: Acetylcholine; Adrenal Gland Neoplasms; Animals; Binding Sites; Biological Transport; Carrier Proteins; Humans; Kinetics; Membrane Glycoproteins; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Neuropeptides; PC12 Cells; Pheochromocytoma; Piperidines; Rats; Recombinant Fusion Proteins; Synaptic Vesicles; Transfection; Vesicular Acetylcholine Transport Proteins; Vesicular Biogenic Amine Transport Proteins; Vesicular Monoamine Transport Proteins; Vesicular Transport Proteins

BeWo choriocarcinoma cells were cultured onto solid microcarrier beads, packed into syringe barrels and superfused. The unidirectional choline uptake across the microvillous membrane of the cells was measured by a rapid single-circulation paired-tracer dilution procedure using methyl[3H]choline with D-[14C]mannitol as the extracellular reference molecule. Choline influx was saturable with a K(t) of 214+/-15 microM and a V(max) of 45.29+/-0.94 nmol/min/mg of cell protein. Uptake of labelled choline was partially inhibited by nicotine, strongly inhibited by hemicolinium-3, and was reduced by about 50 per cent in sodium-free perfusates. A range of agents was added to the stirrer flasks 24 h prior to the experiments to determine if intracellular or extracellular levels of choline or its metabolic product, acetylcholine, regulated choline uptake. Pre-incubation with 2 mM choline reduced the choline maximal uptake by half, while pre-incubation with 100 microM alpha-NETA [2-(alpha-naphthoyl)ethyltrimethyl-ammonium] reduced the influx by 77 per cent. Choline influx was also reduced to about half in the presence of 100 microM vesamicol, bethanecol or neostigmine. It is concluded that BeWo cells possess a choline transporter similar to that described in isolated cytotrophoblasts and syncytiotrophoblast microvillous membrane preparations, and that uptake appeared to be regulated by both intracellular and extracellular concentrations of choline and acetylcholine. Therefore, these cells provide a novel model for studying the role of acetylcholine in human placenta.

Topics: Bethanechol; Biological Transport; Choline; Choriocarcinoma; Drug Carriers; Female; Hemicholinium 3; Humans; Microvilli; Naphthalenes; Neostigmine; Nicotine; Physostigmine; Piperidines; Quaternary Ammonium Compounds; Trophoblasts; Tumor Cells, Cultured; Uterine Neoplasms

The release of acetylcholine (ACh) from airway parasympathetic nerves was studied in rat trachea. We established stimulus parameters, examined the role of extracellular Ca2+, and investigated the origin of the released ACh by use of vesamicol, an inhibitor of ACh uptake in synaptic vesicles. The role of muscarinic autoreceptors and prostanoids on ACh release was also studied. Tracheal rings were incubated in Krebs-Henseleit solution containing neostigmine and guanethidine with or without atropine. ACh release was measured by high-performance liquid chromatography with electrochemical detection. ACh release was dependent on frequency (0.5-16 Hz), voltage (10-25 V), and pulse duration (0.5-4 ms). At 4 Hz, one-fifth of electrical field stimulation-induced ACh release was extracellular Ca2+ independent and vesamicol resistant, indicating its nonvesicular origin. Three-fifths were Ca2+ dependent and vesamicol sensitive, indicating that it was newly synthesized, and one-fifth was Ca2+ dependent but vesamicol resistant, indicating its origin from prestored vesicles. At 16 Hz, two-fifths were nonvesicular and three-fifths were newly synthesized. Blockade of the muscarinic autoreceptor by atropine potentiated the release of ACh four- to fivefold. Neither of the cyclooxygenase inhibitors indomethacin or meclofenamate nor exogenous prostaglandin E2 affected ACh release, indicating that inhibitory prostanoids do not modulate ACh release.

Topics: Acetylcholine; Animals; Atropine; Calcium; Electric Stimulation; Extracellular Space; Female; Neuromuscular Depolarizing Agents; Parasympathetic Nervous System; Piperidines; Prostaglandins; Rats; Rats, Sprague-Dawley; Tetrodotoxin; Tissue Distribution; Trachea

We tested whether D2 ligands inhibit basal and forskolin-stimulated [3H]ACh release from dissociated striata, as opposed to striatal slices. Quinpirole inhibited both basal (40% maximal inhibition; IC50 approximately 50 nM) and 10 microM forskolin-stimulated release (80% inhibition; IC50 approximately 25 nM quinpirole) and both actions were blocked by a D2 antagonist. Vesamicol prevented the quinpirole and forskolin actions. The ability of D2 agonists to inhibit basal and cyclase-stimulated acetylcholine release emanating from vesamicol-sensitive vesicles appears to be tonically suppressed by inhibitory elements within striatal circuitry.

Topics: Acetylcholine; Adenylyl Cyclases; Animals; Colforsin; Dopamine Agonists; Dopamine Antagonists; In Vitro Techniques; Interneurons; Male; Neostriatum; Neuromuscular Depolarizing Agents; Parasympathetic Nervous System; Piperidines; Quinpirole; Rats; Receptors, Dopamine D2; Salicylamides

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 (sigma 1 and sigma 2) receptors. (+)-FBT was found to have a high affinity (Ki = 0.22 nM) for the VAcChT and lower affinities for sigma 1 (21.6 nM) and sigma 2 (35.9 nM) receptors, whereas (-)-FBT had similar affinities for the VAcChT and sigma 1 receptors (approximately 20 nM) and a lower affinity for sigma 2 (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.

Topics: Animals; Basal Ganglia; Brain; Carrier Proteins; Cerebellum; Fluorine Radioisotopes; Fluorobenzenes; Frontal Lobe; Macaca mulatta; Magnetic Resonance Imaging; Male; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Occipital Lobe; Organ Specificity; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, sigma; Stereoisomerism; Synaptic Vesicles; Temporal Lobe; Tomography, Emission-Computed; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

It is well established that extracellular choline is transported into central cholinergic nerve terminals by 'high' and 'low' affinity processes to form the neurotransmitter acetylcholine (ACh). The intent of the present investigation was to ascertain whether extracellular acetate might also be transported into central cholinergic nerve terminals to form ACh. To test this possibility, rat hippocampal tissue was incubated with varying concentrations of extracellular [1-(14)C]acetate (0.1-100 microM) and the uptake of [1-(14)C]acetate and the amount of [14C]ACh formed by the tissue determined. The results indicated that the uptake of extracellular [1-(14)C]acetate was temperature-dependent and saturable having an apparent Michaelis constant (Km) of 22 microM. The formation of [14C]ACh in the tissue as a function of extracellular [1-(14)C]acetate appeared to occur by both 'high' and 'low' affinity processes with apparent Km values of 0.5 and 19.6 microM, respectively. In other experiments, three inhibitors (lithium, allicin and sodium) of acetyl CoA synthetase (EC 6.2.1.1 acetate: CoA ligase), the enzyme which converts acetate to acetyl CoA when ATP and CoA are present, inhibited [1-(14)C]acetate uptake and the amount of [14C]ACh formed from that [1-(14)C]acetate. Additionally, vesamicol, an inhibitor of ACh transport into synaptic vesicles, blocked the filling of a synaptic vesicle-enriched fraction of hippocampal tissue with newly synthesized [14C]ACh formed from extracellular [1-(14)C]acetate. High K+ depolarization of hippocampal tissue loaded with extracellular [1-(14)C]acetate not only increased the synthesis but also the release of [14C]ACh. These results suggest that extracellular acetate is recycled by rat hippocampal cholinergic nerve terminals for the formation and release of ACh. They also suggest that the enzyme acetyl CoA synthetase mediates extracellular acetate uptake into hippocampal cholinergic nerve terminals by metabolizing it to acetyl CoA and thereby creating a diffusion gradient for it to follow.

Topics: Acetate-CoA Ligase; Acetates; Acetylcholine; Animals; Biological Transport; Cholinesterase Inhibitors; Hippocampus; Male; Membrane Potentials; Nerve Endings; Paraoxon; Piperidines; Potassium; Rats; Rats, Inbred Strains; Temperature

A putative vesicular acetylcholine transporter (VAChT) was overexpressed in developing Xenopus spinal neurons by injection of rat VAChT cDNA or synthetic mRNA into Xenopus embryos. This resulted in a marked increase in the amplitude and frequency of miniature excitatory postsynaptic currents at neuromuscular synapses, reflecting an over 10-fold increase in the vesicular packaging of acetylcholine (ACh). The effect appeared in developing neurons even before synaptogenesis and was blocked by L-vesamicol, a specific blocker of ACh uptake into synaptic vesicles. Mutational studies showed that two highly conserved aspartate residues within putative transmembrane domains 4 and 10 are essential for the transport activity. These results provide direct evidence for the physiological function of a putative VAChT and demonstrate that quantal size can be regulated by changes in vesicular transporter activity.

Topics: Acetylcholine; Animals; Aspartic Acid; Blastomeres; Carrier Proteins; Cells, Cultured; Embryo, Nonmammalian; Female; Gene Expression Regulation, Developmental; Membrane Transport Proteins; Microinjections; Muscles; Mutagenesis; Nervous System; Neuromuscular Depolarizing Agents; Neurons; Patch-Clamp Techniques; Piperidines; Rats; Synapses; Synaptic Transmission; Synaptophysin; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins; Xenopus laevis

Vesamicol is known to inhibit the transport of acetylcholine (ACh) into synaptic vesicles in vitro, but much less is known about its effects in the brain in vivo. To assess the effect of vesamicol in vivo, we examined cholinergic parameters, such as the subcellular distribution of ACh, activities of enzymes, uptake of choline, and muscarinic receptor binding in the striatum, hippocampus, and cerebral cortex of rats 30 and 60 min after intraperitoneal injection of vesamicol (3 mg/kg) or of vesamicol in combination with DDVP (5 mg/kg), which was administered 10 min before vasamicol. The levels of cytosolic ACh increased in all regions of the brain after injection of vesamicol, while those of vesicular ACh decreased in all regions except for the striatum. The increase in the levels of extracellular ACh and cytosolic ACh in the striatum induced by DDVP was generally enhanced after injection of vesamicol, Vesamicol did not reduce the level of vesicular ACh when DDVP had been injected previously. Vesamicol did not induce any significant changes in the activities of enzymes, choline uptake, or binding of [6H]quinuclidinyl benzilate to the muscarinic ACh receptors in the three regions. Changes in the cholinergic parameters caused by DDVP were not reversed by the combined administration of DDVP with vesamicol. The present results indicate that vesamicol can inhibit the transport of ACh into synaptic vesicles in the brain tissue in vivo, although it cannot reverse the effects of DDVP that has been injected prior to vesamicol.

Topics: Acetylcholine; Acetylcholinesterase; Animals; Brain; Choline; Choline O-Acetyltransferase; Dichlorvos; Injections, Intraperitoneal; Male; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Wistar; Receptors, Muscarinic; Subcellular Fractions

The present work tested whether pharmacological activation of protein kinase C (PKC) influences the release of [3H]acetylcholine ([3H]ACh) synthesized in the presence of vesamicol, an inhibitor of the vesicular acetylcholine transporter (VAChT). Newly synthesized [3H]ACh was released from hippocampal slices by field stimulation (15 Hz) in the absence of vesamicol, but as expected [3H]ACh synthesized during exposure to vesamicol was not released significantly by stimulation. Treatment of slices with the PKC activator phorbol myristate acetate (PMA) decreased the inhibitory effect of vesamicol on [3H]ACh release. The effect of PMA was dose-dependent, was sensitive to calphostin C, a PKC-selective inhibitor, and could not be mimicked by alpha-PMA, an inactive phorbol ester. PMA did not alter the release of [3H]ACh in the absence of vesamicol, suggesting that the site of PKC action could be related to the VAChT. In agreement with this observation, immunoprecipitation of VAChT from 32P-labeled synaptosomes showed that phosphorylation occurs and that incorporation of 32P in the VAChT protein increases in the presence of PMA. We suggest that PKC alters the output of [3H]ACh formed in the presence of vesamicol and also provide circumstantial evidence for a role of phosphorylation of VAChT in this process.

Topics: Acetylcholine; Animals; Carrier Proteins; Electric Stimulation; Enzyme Activation; Female; Hippocampus; In Vitro Techniques; Male; Membrane Transport Proteins; Phosphorylation; Piperidines; Protein Kinase C; Rats; Rats, Wistar; Synaptosomes; Tetradecanoylphorbol Acetate; Tritium; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Both human essential hypertension and genetically induced hypertension in rats have been associated with a range of impairments of cognitive ability. The spontaneous hypertensive rat (SHR) previously has been shown to exhibit a decrease in the expression of brain nicotinic acetylcholine receptors, a factor that could play a role in the impaired ability of this strain in the performance of learning and memory-related tasks. The purpose of this study was to help determine whether task impairment by SHR was related to the reduced expression of central nicotinic acetylcholine receptors. Twelve-week-old SHR were tested in two phases of a water maze (spatial memory) task, and their performance was compared with that of two age-matched normotensive strains, Wistar Kyoto (WKY) and Wistar rats. During Phase 1, SHR exhibited significantly increased latencies to locate a hidden platform as compared with either WKY or Wistar rats. During Phase 2 (subsequent series of trials after a 4-day inter-phase period), where rats were required to find a new platform location, SHR again exhibited significantly impaired performance compared to the normotensive strains. In a single trial passive avoidance paradigm, SHR again displayed significantly reduced avoidance behavior as compared with both WKY and Wistar rats. In consecutive coronal sections, the density of [3H]cytisine binding sites was decreased in SHR by up to 25% in about half of the brain regions examined, with the deficits particularly apparent in cephalic regions. The binding of [125I]alpha-bungarotoxin to brain sections also was decreased in SHR; however, only certain brain areas exhibited significant interstrain differences. These alterations in the expression of putative nicotinic receptor subtypes in SHR were not due to changes in the density of cholinergic neurons since there were no interstrain differences in the binding densities for [3H]vesamicol, which labels the vesicular acetylcholine transporter. Moreover, the magnitude of nicotine-stimulated rubidium efflux from cortical and striatal synaptosomes in vitro was significantly reduced in samples derived from SHR as compared with those from normotensive rats. These results are consistent with the possibility that a reduction in the expression of cortical nicotinic receptors in SHR plays a role in this strain's impaired performance of both spatial and non-spatial learning and memory-related tasks.

Topics: Alkaloids; Animals; Autoradiography; Avoidance Learning; Azocines; Blood Pressure; Brain; Bungarotoxins; Carrier Proteins; Cognition Disorders; Corpus Striatum; Frontal Lobe; Humans; Hypertension; Iodine Radioisotopes; Male; Maze Learning; Membrane Transport Proteins; Organ Specificity; Piperidines; Quinolizines; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Rats, Wistar; Receptors, Nicotinic; Rubidium; Tritium; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

As part of our ongoing structure-activity studies of the vesicular acetylcholine transporter ligand 2-(4-phenylpiperidino)cyclohexanol (vesamicol, 1), 22 N-hydroxy(phenyl)alkyl derivatives of 3 beta-phenyltropane, 6, and 1-methylspiro[1H-indoline-3,4'-piperidine], 7, were synthesized and tested for binding in vitro. Although a few compounds displayed moderately high affinity for the vesicular acetylcholine transporter, no compound was more potent than the prototypical vesicular acetylcholine transporter ligand vesamicol. However, a few derivatives of 6 displayed higher affinity for the dopamine transporter than cocaine. We conclude that modification of the piperidyl fragment of 1 will not lead to more potent vesicular acetylcholine transporter ligands.

Topics: Anesthetics, Local; Animals; Carrier Proteins; Cocaine; Dopamine Plasma Membrane Transport Proteins; Guinea Pigs; Kinetics; Male; Membrane Glycoproteins; Membrane Transport Proteins; Molecular Conformation; Nerve Tissue Proteins; Neuromuscular Depolarizing Agents; Neurotransmitter Agents; Piperidines; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Tropanes; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

The vesamicol analogue, meta-[(125)I]iodobenzyltrozamicol [(+)-[(125)I]MIBT] was evaluated as a probe for the in vitro labeling of the vesicular acetylcholine transporter in primate brain. In the striatum, (+)-[(125)I]MIBT bound a single high-affinity site with a Kd value of 4.4 +/- 0.7 nM. Competition for (+)-[(125)I]MIBT binding to the striatum by a group of vesamicol analogues displayed a pharmacological profile similar to the rank order of potency previously observed for the vesicular acetylcholine transporter on Torpedo synaptic vesicles. High-affinity binding of (+)-[(125)I]MIBT in the occipital cortex was characterized by a Kd value of 4.6 +/- 1.1 nM. However, the rank order of potency for inhibition of (+)-[(125)I]MIBT binding to the occipital cortex by the same test compounds differed from that observed in the striatum. The results suggest that (+)-[(125)I]MIBT is a reliable probe of the vesicular acetylcholine transporter in primate striatum, but its binding in primate occipital cortex is more complex.

Topics: Acetylcholine; Animals; Brain Chemistry; Carrier Proteins; Iodobenzenes; Ligands; Macaca mulatta; Male; Membrane Transport Proteins; Neostriatum; Neuromuscular Depolarizing Agents; Piperidines; Radioligand Assay; Synaptic Vesicles; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Presynaptic cholinergic markers could be used for estimating the integrity of the cholinergic systems in the human brain with brain imaging techniques such as Single-photon emission computed tomography (SPECT). Vesamicol, an inhibitor of the vesicular acetylcholine transporter, and some of its derivatives have been suggested as potential ligands for this purpose. However, vesamicol binds not only to acetylcholine transporters but also to sigma binding sites. In the present study, we estimated the contribution of sigma site labelling to [3H](-)-vesamicol binding in different rat brain regions by selectively labelling the acetylcholine transporter, using [3H](-)-vesamicol in the presence of the sigma-ligand 1,3-di(2-tolyl)guanidine to occlude the sigma binding sites. The contribution of sigma site labelling was substantial in all brain regions and ranged from 25% in the striatum to 60% in the medulla. In addition, we investigated, in various experimental set ups, the affinities of several vesamicol derivatives for acetylcholine transporters and sigma binding sites. All vesamicol derivatives used displayed a higher affinity for the sigma1 site than for the acetylcholine transporter and also displayed a high sigma2 site affinity. This poor selectivity limits the usefulness of these compounds as selective cholinergic markers for brain imaging studies.

Topics: Acetylcholine; Animals; Anticonvulsants; Binding, Competitive; Brain Chemistry; Carrier Proteins; Cells, Cultured; Female; Guanidines; Guinea Pigs; In Vitro Techniques; Kinetics; Ligands; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Wistar; Receptors, sigma; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

At least two distinguishable forms of synaptic vesicles exist, the active and reserve, but the reserve form is studied most because it has been difficult to purify the active vesicles. In the work reported here the active vesicles (termed VP2) were highly enriched from the electric organ of Torpedo californica by an improved method developed for the reserve vesicles (termed VP1) with the addition of density gradient centrifugation based on Percoll. No significant differences between the vesicular types were found in the amounts of SV1, SV2, and SV4 epitopes and P-type and V-type ATPase activities. The buoyant densities (g/ml) of VP1 and VP2 vesicles were determined by centrifugation in isosmotic sucrose (1.051, 1.069), Percoll (1.034, 1.040), and glycerol (1.087, 1.090) gradients. The radii were determined by dynamic quasi-elastic laser light-scattering to be (56.6 +/- 10.8) nm and (55.0 +/- 12.7) nm. For both vesicular types the volume of excluded sucrose is only about 37% of the volume of excluded Percoll, indicating that the surfaces are rough. Approx. 51% of the VP1 and 32% of the VP2 vesicular volumes are 'osmotically active' water that is exchangeable with glycerol. The different buoyant densities and amounts of osmotically active water in VP1 and VP2 vesicles probably are due to the different internal solutes. Previously observed differences in acetylcholine active transport and vesamicol binding by VP1 and VP2 synaptic vesicles cannot be explained by major alterations in the protein composition or conformation of the membranes in the two types of vesicles.

Topics: Animals; Binding Sites; Cell Fractionation; Centrifugation, Density Gradient; Electric Organ; Electrophoresis, Polyacrylamide Gel; Glycerol; Light; Molecular Weight; Neuromuscular Depolarizing Agents; Peptides; Piperidines; Povidone; Scattering, Radiation; Silicon Dioxide; Synaptic Vesicles; Torpedo

Release-regulating heterocarriers exist on brain nerve endings. We have investigated in this study the mechanisms involved in the neurotransmitter release evoked by GABA heterocarrier activation. GABA increased the basal release of [3H]acetylcholine and [3H]noradrenaline from rat hippocampal synaptosomes and of [3H]dopamine from striatal synaptosomes. These GABA effects, insensitive to GABA receptor antagonists, were prevented by inhibiting GABA uptake but not by blocking noradrenaline, choline, or dopamine transport. Lack of extracellular Ca2+ or addition of tetrodotoxin selectively abolished the GABA-evoked release of [3H]noradrenaline, leaving unaffected that of [3H]acetylcholine or [3H]dopamine. 1,2-Bis(2-aminophenoxyl)-ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) or vesamicol attenuated the release of [3H]acetylcholine elicited by GABA. Reserpine, but not BAPTA-AM, prevented the effect of GABA on [3H] dopamine release. Autoreceptor activation inhibited the GABA-evoked release of [3H]noradrenaline but not that of [3H]acetylcholine or [3H]dopamine. It is concluded that (a) the release of [3H]noradrenaline consequent to activation of GABA heterocarriers sited on noradrenergic terminals meets the criteria of a conventional exocytotic process, (b) the extracellular [Ca2+]-independent releases of [3H]acetylcholine and [3H]dopamine appear to occur from vesicles possibly through involvement of intraterminal Ca2+, and (c) autoreceptor activation only affects heterocarrier-mediated vesicular release linked to entry of extracellular Ca2+.

Topics: Acetylcholine; Adrenergic alpha-Agonists; Adrenergic alpha-Antagonists; Animals; Calcium; Carrier Proteins; Chelating Agents; Choline; Clonidine; Dopamine; Dopamine Agonists; Egtazic Acid; Ergolines; GABA Modulators; Hippocampus; Male; Muscarinic Agonists; Neostriatum; Neuromuscular Depolarizing Agents; Neurons; Neurotransmitter Agents; Norepinephrine; Oxotremorine; Piperidines; Presynaptic Terminals; Quinpirole; Rats; Rats, Sprague-Dawley; Synaptosomes; Tritium; Yohimbine

We incorporated bromoacetylcholine (an inhibitor of choline acetyltransferase), acetylcholinesterase, or both into liposomes made of phosphatidylcholine. Frog sartorius muscles were exposed to these liposomes for 30-60 min. The liposome treatment itself did not decrease the size of the quanta compared to untreated controls. Then the preparations were exposed for 10-20 min to a hypertonic solution, which increases the rate of spontaneous quantal release and elicits an increase in the amount of acetylcholine released per quantum. Following the hypertonic treatment the quanta were significantly smaller in the liposome-treated preparations. Most of the difference occurred because in the preparations not exposed to the liposomes quantal size increased following the hypertonic treatment. This increase is thought to be due to the incorporation of more acetylcholine into each quantum. Our conclusions are that the treatments decreased the acetylcholine concentration in the cytoplasm, and that the increase in size occurs because additional acetylcholine is added to the vesicles containing the quanta from the cytoplasm.

Topics: Acetylcholine; Acetylcholinesterase; Animals; In Vitro Techniques; Liposomes; Membrane Potentials; Motor Neurons; Nerve Endings; Neuromuscular Depolarizing Agents; Phosphatidylcholines; Piperidines; Rana pipiens

The decline in choline acetyltransferase activity has been identified previously within the brains of patients with Rett syndrome and Alzheimer's disease. The level of [3H]vesamicol binding to a terminal vesicular acetylcholine transporter is inversely related to the decline in cortical choline acetyltransferase activity in Alzheimer's disease, which may be due to compensatory processes within surviving cholinergic terminals. In order to investigate whether similar cholinergic compensatory processes are present in the Rett syndrome brain and are altered by normal aging, we investigated the density of cholinergic vesicular transporters in (i) the brains of Rett syndrome patients, and (ii) young and old rats with experimentally-induced cholinergic cell loss. In Rett syndrome, a significant decline in choline acetyltransferase activity within the putamen and thalamus was directly correlated with a decline in [3H]vesamicol binding. In both young and old rats, basal forebrain lesions decreased cortical choline acetyltransferase activity significantly, while [3H]vesamicol binding was unchanged. In contrast to young and old lesioned rats and patients with Alzheimer's disease, cholinergic cells in the brains of patients with Rett syndrome do not compensate for the loss of cholinergic cells by increasing acetylcholine vesicular storage.

Topics: Acetylcholine; Adolescent; Adult; Aging; alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid; Animals; Basal Ganglia; Cerebral Cortex; Child; Child, Preschool; Choline O-Acetyltransferase; Excitatory Amino Acid Agonists; Female; Humans; Neuromuscular Depolarizing Agents; Piperidines; Putamen; Rats; Rats, Inbred F344; Rett Syndrome; Thalamus

We investigated the binding characteristics of 125I-(-)-m-iodovesamicol (125I-(-)-mIV), radioiodinated at the meta position of the 4-phenylpiperidine moiety, in cerebral membranes of the rat brain. The receptor binding affinity of (-)-mIV (Ki = 37 nM) was comparable to that of (-)-vesamicol (Ki = 30 nM). The stereoselectivity of (-)-mIV and (-)-vesamicol for the vesamicol receptor was very high [both (-)-mIV and (-)-vesamicol binding more than 20-fold more active than their (+)isomers], and 125I-(-)-mIV had low affinity for the sigma, dopamine, serotonin, adrenaline and acetylcholine receptors. Furthermore, in a saturation binding study using cerebral membrane preparations, (-)-mIV exhibited a Kd of 18.2 nM with maximum number of binding site Bmax of 660 fmol/mg of protein. These results showed that the characteristics of binding between (-)-mIV and (-)-vesamicol to cholinergic binding sites in the rat brain were similar.

Topics: Animals; Binding, Competitive; Brain; Iodine Radioisotopes; Male; Neuromuscular Depolarizing Agents; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley

We synthesized the (-)-enantiomer of radioiodinated 2-[4-(3-iodophenyl) piperidino] cyclohexanol ((-)-[125I]-m-iodovesamicol [(-)-[125I]mIV]) and evaluated its in vivo binding specificity and affinity for the vesamicol receptor in the rat brain. Significant amounts (approximately 3% of the injected dose) of (-)-mIV accumulated and retention was prolonged. The accumulation of (-)-[125I]mIV in the brain was significantly reduced by the pre- and post-administration of unlabelled vesamicol (0.5 mumol kg -1). Blocking and displacement studies in vivo showed that the binding affinity of (-)-[125I]mIV for the vesamicol receptor was very high, but that for other receptors was very low. Autoradiographic studies showed that the regional distribution of radioactivity, as revealed by coronal sections of the rat brain, was similar for both (-)-[125I]mIV and (-)-[3H]vesamicol. The results demonstrated that the characteristics of (-)-[125I]mIV for binding sites in rat brain were similar in vivo and in vitro. Radioiodinated (-)-mIV may potentially be useful for studying presynaptic cholinergic neurons in the brain.

Topics: Animals; Autoradiography; Binding, Competitive; Brain; Iodine Radioisotopes; Male; Neurons; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Tissue Distribution; Tritium

Monoethylcholine (MECH) enters motor nerve terminals where it is made into acetylmonoethylcholine (AMECH). AMECH opens endplate channels for about half of the average duration observed where they are opened by acetylcholine (ACH). Therefore when AMECH is present in a quantum the endplate currents decay more rapidly. MECH has been used to measure quantal turnover in motor nerve terminals. We find that the incorporation of AMECH into quanta is blocked by vesamicol, an inhibitor of ACH transport into synaptic vesicles. AMECH is incorporated more rapidly when acetylcholinesterase is inhibited, when the choline uptake inhibitor, hemicholinium-3, is present or when extracellular Na+ (required for active CH uptake) is replaced with methylamine. This suggests that in the absence of these inhibitors CH obtained from released ACH is recycled. Therefore, experiments on the rate of incorporation of MECH are misleading unless CH recycling is prevented. Previous work also suggested that MECH is incorporated at a faster rate into those quanta which are released by stimulation than into those released spontaneously. We conclude that quanta released spontaneously and following nerve stimulation probably come from the same pool. The distribution of t1/2's during the incorporation of MECH can be accounted for in the framework of recent studies of the recycling of synaptic vesicles. We conclude that false transmitter is a valuable tool for studying the loading of quanta, but that there are several complications to be considered when trying to use it to measure the turnover of the population of quanta.

Topics: Animals; Choline; Cholinesterase Inhibitors; Edrophonium; Electric Stimulation; Hemicholinium 3; Methylamines; Mice; Mice, Inbred Strains; Neostigmine; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Neurotransmitter Agents; Neurotransmitter Uptake Inhibitors; Piperidines; Prodrugs; Rana pipiens

The existence of possible relationships among the developmental profile of various cholinergic markers in the main olfactory bulb (OB) was assessed by using in vitro quantitative autoradiography. Muscarinic receptors were visualized with [3H]pirenzepine (muscarinic M1-like sites) and [3H]AF-DX 384 (muscarinic M2-like sites); nicotinic receptors by using [3H]cytisine (nicotinic 42-like subtype) and [125I] alpha-bungarotoxin (nicotinic 7-like subtype); cholinergic nerve terminals by using [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites). These various cholinergic markers exhibited their lowest levels at birth and reached adult values by the end of the 4-5 postnatal weeks. However, the density of presynaptic cholinergic markers and nicotinic receptors at postnatal day 2 represented a large proportion of the levels observed in adulthood, and displays a transient overexpression around postnatal day 20. In contrast, the postnatal development of cholinergic muscarinic M1-like and M2-like receptors is apparently regulated independently of the presynaptic cholinergic markers and nicotinic receptors. Two neurochemically and anatomically separate olfactory glomeruli subsets were observed in the posterior OB of the developing rat. These atypical glomeruli expressed large amounts of [3H]vesamicol-and [3H]hemicholinium binding sites without significant amounts of muscarinic M1, M2, or nicotinic alpha 4 beta 2 receptor binding sites. A significant density of [125I] alpha-bungarotoxin binding sites could be detected only at early postnatal ages. A few olfactory glomeruli specifically restricted to the dorsal posterior OB expressed a high density of [3H]cytisine binding sites but lacked significant binding of the two presynaptic cholinergic markers used here, suggesting their noncholinergic but cholinoceptive nature.

Topics: Alkaloids; Animals; Autoradiography; Azocines; Biomarkers; Bungarotoxins; Female; Hemicholinium 3; Male; Olfactory Bulb; Piperidines; Pirenzepine; Presynaptic Terminals; Quinolizines; Radioligand Assay; Rats; Rats, Wistar; Receptors, Muscarinic; Receptors, Nicotinic

1. This investigation was undertaken to explore an unexpected effect of vesamicol, an agent that inhibits active acetylcholine (ACh) uptake into isolated synaptic vesicles. Previous studies at the neuromuscular junction showed that vesamicol makes miniature end-plate currents (MEPCs) smaller only after tens of thousands of quanta have been released. Inhibiting acetylcholinesterase (AChE) makes the MEPCs larger than normal. Our unexpected finding was that with the AChE inhibitor present, adding 2 microM (-)-vesamicol decreases the size of the MEPCs by approximately 30%. The decrease was apparent within 15-30 min, during which only a few thousand quanta had been released. 2. Experimental tests showed that the (-)-vesamicol treatment is unlikely to be acting postsynaptically. For example, it did not slow the rise of MEPCs, which would occur if the endplate receptors were blocked. 3. When AChE was inhibited, three treatments expected to block active choline (Ch) uptake into the presynaptic terminals decreased MEPC size: 1) elevating extracellular K+ to diminish the Na+ electrochemical gradient required for Ch uptake; 2) replacing extracellular Na+ with methylamine+; and 3) adding hemicholinium-3 (HC-3), an inhibitor of the Ch transporter. These treatments did not act by reactivating AChE, blocking the endplate ACh receptor, or by enhancing the desensitization of the ACh receptor. 4. Previous evidence suggests that synaptic vesicles are formed and partially filled with ACh in the cytoplasm and then receive additional ACh when they attach to the active zones, a process that is called second-stage loading. We conclude that the MEPCs are becoming smaller when second-stage loading is blocked by (-)-vesamicol or when the supply of ACh in the cytoplasm of the motor nerve terminal is depleted. 5. To follow the time course of second-stage loading, we used the false transmitter precursor monoethylcholine (MECh). It enters the terminal and is transformed into acetylmonoethylcholine (AMECh). When 200 microM MECh was placed in the extracellular solution and the AChE was inhibited, MEPC size was significantly smaller after 10 min. MEPC size increased once again over a period of time when MECh was removed from the extracellular solution and replaced with Ch. 6. We conclude that at the neuromuscular junction second-stage loading is responsible for loading a significant fraction of the ACh into the quanta.

Topics: Acetylcholine; Analysis of Variance; Animals; Choline; Cholinesterase Inhibitors; Motor Endplate; Neostigmine; Neuromuscular Depolarizing Agents; Piperidines; Rana pipiens

The characteristics of ATP-dependent transport of acetylcholine (ACh) in homogenates of pheochromocytoma (PC-12) cells stably transfected with the human vesicular acetylcholine transporter (VAChT) cDNA are described. The human VAChT protein was abundantly expressed in this line and appeared as a diffuse band with a molecular mass of approximately 75 kDa on Western blots. Vesicular [3H]ACh accumulation increased approximately 20 times over levels attained by the endogenous rat VAChT, expressed at low levels in control PC-12 cells. The transport of [3H]ACh by human VAChT was dependent upon the addition of exogenous ATP at 37 degrees C. Uptake was abolished by low temperature (4 degrees C), the proton ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (2.5 microM) and bafilomycin A1 (1 microM), a specific inhibitor of the vesicular H+-ATPase. The kinetics of [3H]ACh uptake by human VAChT were saturable, exhibiting an apparent Km of 0.97 +/- 0.1 mM and Vmax of 0.58 +/- 0.04 nmol/min/mg. Maximal steady-state levels of vesicular [3H]ACh accumulation were directly proportional to the concentration of substrate present in the medium with saturation occurring at approximately 4 mM. Uptake was stereospecifically inhibited by L-vesamicol with an IC50 of 14.7 +/- 1.5 nM. The apparent affinity (Kd) of [3H]vesamicol for human VAChT was 4.1 +/- 0.5 nM, and the Bmax was 8.9 +/- 0.6 pmol/mg. The turnover (Vmax/Bmax) of the human VAChT was approximately 65/min. This expression system should prove useful for the structure/function analysis of VAChT.

Topics: Acetylcholine; Adrenal Gland Neoplasms; Animals; Biological Transport, Active; Carrier Proteins; Cytosol; Humans; Kinetics; Membrane Transport Proteins; Neuromuscular Depolarizing Agents; PC12 Cells; Pheochromocytoma; Piperidines; Rats; Recombinant Proteins; Substrate Specificity; Tetrabenazine; Transfection; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

The aim of the present experiments was to test whether vesamicol alters the evoked release of ATP from nerve terminals. Continuous or cumulative release of ATP evoked by 33 mM KC1 from rat cerebrocortical synaptosomes was largely calcium-dependent. Vesamicol interfered with release of ATP from synaptosomes depolarized with KCl (33 mM) in a dose-dependent and stereoselective way. The (-)-vesamicol decreased the output of ATP in doses much lower than (+)-vesamicol. The release of the major excitatory neurotransmitter glutamate from depolarized nerve endings was not impaired by vesamicol. We suggest that vesamicol may alter the release of ATP specifically, probably by interacting with a protein similar to the vesamicol receptor found in cholinergic synaptic vesicles.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Cerebral Cortex; Female; In Vitro Techniques; Male; Neuromuscular Depolarizing Agents; Piperidines; Potassium Chloride; Rats; Synaptosomes

Vesamicol (AH5183) is an inhibitor (IC50, 50 nM) of acetylcholine (ACh) vesicle packaging. Vesamicol increases the phosphorylation pattern of synaptophysin (p38), identified as a vesicle-specific phosphoprotein involved in vesicle-mediated neurotransmitter release. Percoll fractionation of the rat cortex yielded a cholinergic-enriched synaptosomal Fraction 4. Fraction 4 contained the highest enrichment of cholineacetyl-transferase activity (86 +/- 4.6 mumole AcCh/g protein/hr.) in the Percoll gradient. Fraction 4 demonstrated oxygen consumption (108 +/- 23.4 nmole/mg protein), levels of adenosine triphosphate, ATP, (10.29 +/- 0.45 nmole/mg protein) and adenosine diphosphate, ADP, (10.54 +/- 2.72 nmole/mg protein), energy potential (ATP/[ADP] [Pi], (0.49) phosphate uptake (65-80 nmoles phosphate/mg tissue), 32Pi labelling (130 +/- 12 x 10(5) DPM/mg tissue; 74 +/- 9.8 x 10(2) nmoles phosphate/mg tissue). Synaptophysin was identified by Western blotting and confirmed by qualitative immunoprecipitation. Synaptophysin phosphorylation was confirmed by autoradiograph. Synaptophysin phosphorylation increased (225%) in the presence of vesamicol (ED50, 1 nM) in Fraction 4. Vesamicol (50 nM) and vanadate (54 microM) were compared for their effects on synaptophysin. This study suggests that during the inhibition of acetylcholine packaging by vesamicol that synaptophysin is phosphorylated. Therefore, the phosphorylation and dephosphorylation of synaptophysin may be involved in the transport of acetylcholine in or out of the synaptic vesicle.

Topics: Acetylcholine; Animals; Cerebral Cortex; Dichloroacetic Acid; Energy Metabolism; Male; Neuromuscular Depolarizing Agents; Oxygen; Phosphates; Phosphorylation; Piperidines; Rats; Rats, Sprague-Dawley; Synaptophysin; Synaptosomes

Topics: Acetyl Coenzyme A; Acetylcholine; Animals; Azepines; Carotid Body; Carrier Proteins; Cats; Cell Hypoxia; Choline; Choline O-Acetyltransferase; Enzyme Inhibitors; Glossopharyngeal Nerve; Hemicholinium 3; Hypoxia; Membrane Transport Proteins; Models, Neurological; Naphthylvinylpyridine; Nerve Tissue Proteins; Piperidines; Pyridines; Receptors, Presynaptic; Signal Transduction; Synaptic Vesicles

Stimulation of mammalian motor neurons can elicit Ca(2+)-dependent regenerative release of acetylcholine and prolonged endplate depolarization when the enzymatic degradation of the neurotransmitter is inhibited. Unlike physiological phasic release of acetylcholine, the regenerative release is sensitive to L-type Ca2+ channel blockers. We studied the effects of vesamicol (an inhibitor of active transport of acetylcholine into synaptic vesicles) and omega-agatoxin IVA (a blocker of the motor nerve P-type Ca2+ channel) on these two types of acetylcholine release to compare the vesicle pools and Ca2+ channels responsible for the release. When coupled with repetitive stimulations, vesamicol decreased mean amplitude of miniature endplate potentials, resulting in a skewed distribution to lower amplitude, reduced quantal content of endplate potentials and decreased immediate available pool of acetylcholine. omega-Agatoxin IVA had no effect on miniature endplate potential but inhibited quantal content of endplate potential. The mean inhibitory concentration was around 5-10 nM. Vesamicol and omega-agatoxin IVA decreased the probability of triggering regenerative release. However, the magnitude and duration of regenerative release, once triggered, were not depressed by either agent. It appears that the majority of Ca2+ necessary for regenerative release is translocated via omega-agatoxin IVA-insensitive Ca2+ channels, which can be activated by prolonged depolarization of nerve terminals induced by accumulated acetylcholine. The results suggest that different Ca2+ channels are activated in the regenerative (L-type) and phasic (P-type) acetylcholine release, which utilize different pools of synaptic vesicles.

Topics: Acetylcholine; Animals; Calcium Channel Blockers; Diaphragm; Electric Stimulation; Evoked Potentials; In Vitro Techniques; Mice; Mice, Inbred ICR; Motor Endplate; Motor Neurons; Neostigmine; Nerve Regeneration; Neuromuscular Depolarizing Agents; omega-Agatoxin IVA; Phrenic Nerve; Piperidines; Spider Venoms

Three conformationally restricted analogs of vesamicol, 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-spirol[1H-i nde ne-1,4'- piperidine] (5), 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl]-3,4- dihydrospiro[indene-1,4'-piperidine] (6) and 1'-[1-(3-iodobenzyl)-4-hydroxypiperidin-3-yl)-3,4- dihydrospiro[naphthalene-1(2H),4'-piperidine] (7), were labelled with iodine-125 and evaluated as potential radioligands for mapping vesamicol receptor (VR) density and cholinergic function in vivo. All compounds showed similar kinetics in most tissues. However, differences were observed in the brain. Although comparable levels of each corresponding enantiomeric pair were obtained initially in the brain, the levels of the dextrorotatory enantiomers (+)-5, (+)-6 and (+)-7 were found to decrease by 72-82% over a period of 3 h. In contrast, the brain levels of the corresponding levorotatory isomers were maintained throughout the duration of the experiment. Among the dextrorotatory isomers, (+)-6 showed the highest brain extraction, while (+)-7 showed the lowest. In tissue dissection experiments, the levels of (+)-5, (+)-6 and (+)-7 were highest in the striatum and moderate to low in the cortex and cerebellum. Co-administration of haloperidol with (+)-6 decreased the levels of the latter in the striatum by 27%, while the levels in the cortex and cerebellum were each reduced by 60%. In addition, haloperidol failed to affect the regional distribution of (+)-7 in the brain. However, both haloperidol and spiperone increased the striatal levels of (+)-5 by 67 and 76%, respectively, suggesting that the binding of this radioligand is related to cholinergic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Analysis of Variance; Animals; Iodine Radioisotopes; Male; Molecular Conformation; Neuromuscular Depolarizing Agents; Piperidines; Radioligand Assay; Rats; Rats, Wistar; Receptors, Cholinergic; Spiro Compounds; Structure-Activity Relationship; Tissue Distribution

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.

Topics: Animals; Molecular Conformation; Neuromuscular Depolarizing Agents; Piperidines; Receptors, Cholinergic; Receptors, sigma; Structure-Activity Relationship; Torpedo

Veratridine produces a marked elevation in spontaneous quantal release from nerve endings through its ability to enhance sodium-channel activity, leading to sustained membrane depolarization. In the course of an electrophysiological investigation into the effects of vesamicol, an inhibitor of the synaptic vesicle acetylcholine transporter, on veratridine-induced acetylcholine release from rat motor nerve terminals we observed that veratridine itself has an effect on miniature endplate current amplitude distributions suggestive of an effect of the compound on the filling of cholinergic synaptic vesicles with acetylcholine. This effect of veratridine is release-dependent, being inhibited by either removal of extracellular calcium ions or by the addition of the sodium channel blocking toxin, tetrodotoxin. Biochemical studies using synaptic vesicles isolated from Torpedo electroplaque confirmed the ability of veratridine to directly inhibit the vesicular transport of acetylcholine. This appears to be a consequence of its ability to dissipate the trans-vesicular membrane proton gradient, which normally drives the active transport of acetylcholine into synaptic vesicles. We discuss how such an action of veratridine could lead to the observed release-dependent effects of the compound on electrophysiologically monitored spontaneous quantal acetylcholine release. The action of veratridine on cholinergic synaptic vesicles could be of considerable import when using this agent to elicit neurotransmitter release from either peripheral or central nerve endings.

Topics: Acetylcholine; Animals; Electric Organ; Electrophysiology; Hydrogen-Ion Concentration; In Vitro Techniques; Male; Membrane Potentials; Motor Endplate; Muscle Fibers, Skeletal; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Piperidines; Rats; Rats, Sprague-Dawley; Synaptic Vesicles; Tetrodotoxin; Torpedo; Veratridine

Three iodovesamicol analogs, iodinated at the ortho, meta, and para positions of the 4-phenylpiperidine moiety, were synthesized and labeled with 125I by isotopic exchange reaction. Their potencies as a vesamicol-like drug were evaluated with competitive inhibition studies using (-)[3H]vesamicol. The radiochemical yields were 40-85%, the radiochemical purities exceeded 95% and their specific activities were 370-740 GBq/mmol. The descending order of binding affinity of the tested compounds against the vesamicol receptor was m-iodovesamicol > o-iodovesamicol > p-iodovesamicol. The receptor binding affinity of m-iodovesamicol (IC50 = 133 nM) was comparable with that of vesamicol (IC50 = 109 nM). Therefore, the meta position of the 4-phenylpiperidinyl fragment of vesamicol was the optimum site for iodination, and radioiodinated m-iodovesamicol may serve as a useful radiopharmaceutical for in vitro and in vivo studies of presynaptic cholinergic neurons in rats.

Topics: Animals; Cell Membrane; Cerebral Cortex; Iodine Radioisotopes; Isotope Labeling; Kinetics; Male; Neuromuscular Depolarizing Agents; Piperidines; Radioligand Assay; Rats; Rats, Sprague-Dawley; Receptors, Cholinergic; Structure-Activity Relationship

The Ewing's sarcoma cell line ICB 112 was examined in detail for a cholinergic phenotype. Choline acetyltransferase activity (12.3 +/- 2.9 nmol/h/mg of protein) was associated with the presence of multiple mRNA species labeled with a human choline acetyltransferase riboprobe. Choline was taken up by the cells by a high-affinity, hemicholinium-3-sensitive transporter that was partially inhibited when lithium replaced sodium in the incubation medium; the choline taken up was quickly incorporated into both acetylcholine and phosphorylcholine. High-affinity binding sites for vesamicol, an inhibitor of vesicular acetylcholine transport, were also present. The mRNAs for synaptotagmin (p65) and the 15-kDa proteolipid were readily detected and were identical in size to those observed in cholinergic regions of the human brain. Cumulative acetylcholine efflux was increased by raising the extracellular potassium level or the addition of a calcium ionophore, but the time course of stimulated efflux was slow and persistent. These results show that this morphologically undifferentiated cell line is capable of acetylcholine synthesis and expresses markers for synaptic vesicles as well as proteins implicated in calcium-dependent release but lacks an organized release mechanism.

Topics: Acetylcholine; Adult; Calcium-Binding Proteins; Choline; Choline O-Acetyltransferase; Cholinergic Fibers; Humans; Male; Membrane Glycoproteins; Nerve Tissue Proteins; Neuromuscular Depolarizing Agents; Neurons; Piperidines; Proteolipids; RNA, Messenger; Sarcoma, Ewing; Synaptotagmin I; Synaptotagmins; Tritium; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Uptake of acetylcholine (ACh) by synaptic vesicles isolated from the electric organ of Torpedo was induced with an artificially imposed proton gradient. The gradient was formed by hyposmotic lysis and resealing of vesicles in a low pH buffer to form vesicular ghosts followed by sudden elevation of the pH of the ghost suspension. [3H]ACh accumulated rapidly, the proton gradient collapsed spontaneously within 5 min as monitored by [14C]methylamine uptake, and the accumulated ACh leaked out of the ghosts after 5 min. Vesamicol blocked both uptake and efflux of the [3H]ACh, demonstrating that both processes are mediated by the ACh transporter. The protonophore nigericin also blocked uptake very potently. Specific uptake was titrated with variable concentrations of [3H]ACh. It exhibited Km and Vmax values of approximately 200-500 microM and 7-30 nmol [3H]ACh/mg at 5 min, respectively, which are values close to those commonly observed for ATP-dependent uptake by intact vesicles. Specific uptake by ghosts was titrated with variable internal pH and constant external pH. It exhibited maximal uptake between internal pH 4.5 and 5.5. The dependence was very steep and could be fit best by assuming that the active form of the transporter requires protonation of two internal sites of apparent pK value of 5.3 +/- 0.2. A similar result was obtained when the uptake was titrated with variable internal pH with a constant thermodynamic driving force maintained by keeping the external pH approximately 2.6 units higher. The origin of the transport inhibition that sets in at very low internal pH values is not clear.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Biological Transport, Active; Carrier Proteins; Hydrogen-Ion Concentration; In Vitro Techniques; Membrane Potentials; Membrane Transport Proteins; Methylamines; Nigericin; Piperidines; Synaptic Vesicles; Torpedo; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

Normal aged rats (26 months) displayed significant impairments in learning the Morris water maze task as compared with young adult rats (3 months). The learning deficits of aged basal forebrain (BF)-lesioned rats (26 months; ethylcholine aziridinium ion was injected into the bilateral basal forebrain at 3 months age) were more severe than those of normal aged rats. Choline acetyltransferase (ChAT) in the frontal cortex of aged BF-lesioned rats activity was significantly reduced, but not in normal aged rats which level was almost the same as that in young adult rats. Histological examination showed that cholinergic fibers (acetylcholinesterase staining) in the frontal cortex reduced in aged BF-lesioned rats, but not in normal aged rats. The number of binding sites (Bmax) for [3H]vesamicol, a ligand for the vesicular acetylcholine transporter, in the frontal cortex of normal aged rats was significantly less than that in young adult rats, while the Bmax of aged BF-lesioned rats was higher than that of normal aged rats. The levels of monoamines and their metabolites in the frontal cortex and striatum but not hippocampus of aged BF-lesioned rats were markedly reduced as compared with those of normal aged and young adult rats. These results taken together indicate that normal aged and aged BF-lesioned rats exhibit learning deficits and that the differences of the severity of spatial learning deficits between normal aged and aged BF-lesioned rats may be due to, at least in part, the different properties of cathecolaminergic, serotonergic and cholinergic dysfunctions in the discrete brain sites.

Topics: Acetylcholinesterase; Aging; Animals; Aziridines; Cerebral Cortex; Choline; Choline O-Acetyltransferase; Coloring Agents; Kinetics; Male; Maze Learning; Neuromuscular Blocking Agents; Piperidines; Prosencephalon; Rats; Rats, Inbred F344; Space Perception

Iodovesamicol analogs, radioiodinated at the ortho (1), meta(2) and para(3) positions of the 4-phenylpiperidine moiety, were evaluated as potential presynaptic cholinergic neuron mapping agents. Significant accumulation of m-[125I]iodovesamicol (mIV(2)) (about 3% of the injected dose) was noted in the rat brain with prolonged retention times. The accumulation of mIV(2) in the rat brain was decreased by 67% by 5 min pre-injection of dl-vesamicol(1 mumol/kg). Pre-injection of (+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperdine[(+)-3-PPP](0.75 mumol/kg) did not markedly decrease the levels of radiotracers (oIV(1) and mIV(2)) in the rat brain. These results suggest that radioidinated m-iodovesamicol (mIV(2)) is suitable for use in presynaptic cholinergic neuron mapping.

Topics: Animals; Autoradiography; Brain; Iodine Radioisotopes; Kinetics; Male; Neuromuscular Depolarizing Agents; Piperidines; Radioligand Assay; Radionuclide Imaging; Rats; Rats, Sprague-Dawley; Structure-Activity Relationship; Time Factors; Tissue Distribution

To provide anatomical information on the complex effects of acetylcholine (ACh) in the olfactory bulb (OB), the distribution of different cholinergic muscarinic and nicotinic receptor sub-types was studied by quantitative in vitro autoradiography. The muscarinic M1-like and M2-like sub-types, as well as the nicotinic bungarotoxin-insensitive (alpha 4 beta 2-like) and bungarotoxin-sensitive (alpha 7-like) receptors were visualized using [3H]pirenzepine, [3H]AF-DX 384, [3H]cytisine and [125I] alpha-bungarotoxin (BTX), respectively. In parallel, labelling patterns of [3H]vesamicol (vesicular acetylcholine transport sites) and [3H]hemicholinium-3 (high-affinity choline uptake sites), two putative markers of cholinergic nerve terminals, were investigated. Specific labelling for each cholinergic radioligand is distributed according to a characteristic laminar and regional pattern within the OB revealing the lack of a clear overlap between cholinergic afferents and receptors. The presynaptic markers, [3H]vesamicol and [3H]hemicholinium-3, demonstrated similar laminar pattern of distribution with two strongly labelled bands corresponding to the glomerular layer and the area around the mitral cell layer. Muscarinic M1-like and M2-like receptor sub-types exhibited unique distribution with their highest levels seen in the external plexiform layer (EPL). Intermediate M1-like and M2-like binding densities were found throughout the deeper bulbar layers. In the glomerular layer, the levels of muscarinic receptor subtypes were low, the level of M2-like sites being higher than M1. Both types of nicotinic receptor sub-types displayed distinct distribution pattern. Whereas [125I] alpha-BTX binding sites were mostly concentrated in the superficial bulbar layers, [3H]cytisine binding was found in the glomerular layers, as well as the mitral cell layer and the underlying laminae. An interesting feature of the present study is the visualization of two distinct cholinoceptive glomerular subsets in the posterior OB. The first one exhibited high levels of both [3H]vesamicol and [3H]hemicholinium-3 sites. It corresponds to the previously identified atypical glomeruli and apparently failed to express any of the cholinergic receptors under study. In contrast, the second subset of glomeruli is not enriched with cholinergic nerve terminal markers but displayed high amounts of [3H]cytisine/nicotinic binding sites. Taken together, these results suggest that although muscarinic receptors

Topics: Alkaloids; Animals; Autoradiography; Azocines; Biomarkers; Brain Mapping; Bungarotoxins; Cholinergic Agents; Hemicholinium 3; Iodine Isotopes; Male; Neuromuscular Depolarizing Agents; Olfactory Bulb; Piperidines; Quinolizines; Rats; Rats, Wistar; Receptors, Muscarinic; Receptors, Nicotinic; Tritium

Vesamicol inhibits acetylcholine uptake in presynaptic vesicles and reduce its release. The present study was performed in order to test the effects of this drug in a cholinergic related function as rapid eye movement (REM) sleep. Wistar male rats were implanted for sleep recordings. In addition, a stainless steel cannula was implanted into the left lateral ventricle for intracerebroventricular (ICV) injections. In experiment 1, a dose-response curve was performed. Saline or vesamicol (20, 40, 80 and 100 micrograms) were injected. Following the ICV injections, animals' sleep was recorded for 8 h. In experiment 2, after adaptation and baseline recordings, animals received 50 micrograms vesamicol ICV at 1000 hours. every 24 h for 2 consecutive days. After each injection an 8-h sleep recording session was performed. Two subsequent recovery recordings were allowed. Results obtained in experiment 1 showed a dose-response reduction of REM sleep with significant values at 80 micrograms and 100 micrograms of vesamicol. The main findings in experiment 2 were a reduction in REM sleep time and an increase in REM sleep latency. On the recovery days, a dramatic rebound of REM sleep was observed. Vesamicol behaved as an anticholinergic drug. It produced a reduction in REM sleep time and a rebound of this sleep stage after its withdrawal.

Topics: Analysis of Variance; Animals; Dose-Response Relationship, Drug; Male; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Wistar; Sleep; Sleep, REM

Vesamicol (AH5183), a potent and specific inhibitor of the vesicular acetylcholine uptake system, reduces at relatively high doses the slope of the log-linear frequency augmentation-potentiation (FAP) relation observed between the mean quantal content of endplate potential and the stimulation frequency. Recent biochemical studies identified two binding sites for vesamicol; one is the vesicular acetylcholine uptake system and the other is the vesamicol binding protein localized in the nerve terminal membrane. The present experiments were designed to determine the exact site of action of vesamicol on the FAP relation. Studies using epsilon-maleimidocaproic acid and picrylsulfonic acid, both membrane-impermeant protein-modifying reagents, revealed that these reagents diminished the vesamicol effect concomitant with an impairment of the functioning site for FAP. Because of the membrane-impermeant nature of the reagents, the present results were interpreted to mean that a vesamicol binding protein or a related component, localized in the nerve terminal membrane, is the target of action by vesamicol. This vesamicol binding component seems to play an important role in the stimulation frequency-dependent modulation of the evoked release of transmitter quanta observable as FAP.

Topics: Animals; Binding Sites; Dose-Response Relationship, Drug; Electric Stimulation; Evoked Potentials; Magnesium; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Piperidines; Receptors, Cholinergic; Time Factors

Cholinergic transmission may be part of the normal neurochemical processes that support spontaneous ventilation. If this is true, perturbations in acetylcholine (ACh) turnover should alter ventilatory output in a predictable manner. With the use of the isolated perfused brain stem-spinal axis from the neonatal rat, the effects of modifiers of ACh release and blockers of muscarinic receptors on spontaneous C4 (phrenic) output were determined. Vesamicol and cetiedil, inhibitors of ACh release, caused depression and cessation of the C4 output in a dose-dependent manner when added to the perfusate. Muscarinic blockers, particularly M1 and M3 blockers, caused a similar depression. 4-Aminopyridine and tetraethylammonium chloride, facilitators of ACh release, caused stimulation of C4 (phrenic) output. The depressive effects of the blockers and inhibitors were reversible with facilitation of ACh release except in the case of cetiedil. These findings are consistent with the view that the synaptic turnover of endogenous ACh is an important part of the normal neurochemical process that supports and modulates ventilation.

Topics: Acetylcholine; Animals; Azepines; Brain Stem; Cholinergic Antagonists; In Vitro Techniques; Muscarinic Antagonists; Phrenic Nerve; Piperidines; Rats; Receptors, Muscarinic; Respiration; Respiratory Muscles

Fluctuation analysis was used to estimate the mean single-channel conductance and the mean channel duration of opening. Miniature endplate currents (MEPCs) were measured with the voltage-clamp technique. The timing of endplate channel opening during the generation of the MEPC was estimated by a deconvolution method. Often all of the channels opened during the rise of the MEPC, but in about half of the examples some 10% of the channels opened after the peak. We studied the effects of acetylcholinesterase (AChE) inhibition with neostigmine, diisopropyl fluorophosphate (DFP) and fasciculin-2. With AChE largely inhibited, the number of channels opening increased as much as fourfold, largely by channels opening in the "tail" that follows the peak of the MEPC. The results were compared to models of MEPC generation. Models did not account well for the pattern of channel opening, particularly after AChE inhibition. In the presence of fasciculin-2, the addition of 2 microM (-)-vesamicol reduced the number of channels opening and shortened the period over which channels were open. One interpretation is that quantal ACh release is not almost instantaneous, but that some of the ACh is released over a period of a millisecond or more and that some of the release is blocked by (-)-vesamicol.

Topics: Acetylcholine; Animals; Cholinesterase Inhibitors; Elapid Venoms; Female; In Vitro Techniques; Ion Channel Gating; Isoflurophate; Male; Mathematics; Membrane Potentials; Mice; Models, Neurological; Motor Endplate; Piperidines; Rana pipiens

Vesamicol inhibits the vesicular loading of acetylcholine molecules. The effects of vesamicol and similarly acting compounds on neuromuscular transmission in frogs were investigated to determine whether these inhibitors-inhibit the frequency augmentation-potentiation of transmitter release. Various vesicular acetylcholine transport blockers suppressed the stimulation frequency-related release parameter, k, in a dose-dependent manner. Artane, cetiedil, chloroquine, ethodin, quinacrine, vesamicol and its benzyl-analogue, 2-(4-benzylpiperidino)cyclohexanol, had strong effects, while those of aminacrine, chlorpromazine, fluphenazine, imipramine, pyrilamine and thioridazine were weak. A significant correlation was observed between the biochemically reported values of IC50 and the electrophysiological inhibitory potencies on k at 20 microM. Contrary to expectations from the biochemical data, however, vesamicol and its benzyl-analogue showed equipotent inhibitory actions on the electrophysiological frequency augmentation-potentiation relation. Low sensitivity and low selectivity of the frequency augmentation-potentiation for vesamicol and its benzyl-analogue lead us to conclude that the vesicular acetylcholine transporter is not the site of the electrophysiological action of vesamicol and similarly acting chemicals.

Topics: Acetylcholine; Animals; Antidepressive Agents; Azepines; Chloroquine; Ethacridine; Evoked Potentials; In Vitro Techniques; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Piperidines; Pyrilamine; Quinacrine; Ranidae; Regression Analysis; Synaptic Transmission

Uptake of acetylcholine was studied in a synaptic vesicle fraction isolated from rat brain. Hyposmotically treated P3 vesicles took up acetylcholine (ACh) in the presence of MgATP, and the uptake was inhibited by low temperature, ammonium ions, the protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) and bafilomycin A1, a specific inhibitor of the vacuolar H(+)-ATPase. Uptake was also inhibited by drugs that bind allosterically to the ACh transporter, namely vesamicol (IC50 value of 170 nM) and 4-aminobenzovesamicol (IC50 value of 25 nM). KM and Vmax values for ACh active transport were estimated to be 5 mM and 4 nmol min-1 mg-1 of cholinergic vesciles, respectively. Active transport of ACh by synaptic vesicles partially purified from brain is mediated by a vesamicol-sensitive transporter and is dependent on a proton gradient generated by the vesicular H(+)-ATPase.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Biological Transport, Active; Brain; Piperidines; Rats; Synaptic Vesicles

The direct optical resolution of the vesicular acetylcholine uptake inhibitors vesamicol and benzovesamicol and nine benzovesamicol analogues were performed by HPLC on a commercially available cellulose tris(3,5-di-methylphenyl carbamate) chiral stationary phase. Separation of each enantiomeric pair was optimized with respect to solvent strength and flow-rate, using mobile phase mixtures of hexane-2-propanol-diethylamine. The method has been successfully applied to the analysis of the optical purity of benzovesamicol intermediates and products, including (-)-5-[123I]iodobenzovesamicol which is currently undergoing clinical evaluation as a tracer for mapping central cholinergic neurons, and the purification of both antipodes of (+/-)-7-[125I]iodobenzovesamicol.

Topics: 2-Naphthylamine; Cellulose; Chromatography, High Pressure Liquid; Indicators and Reagents; Iodine Radioisotopes; Phencyclidine; Piperidines; Spectrophotometry, Ultraviolet; Stereoisomerism; Tetrahydronaphthalenes

In an effort to develop selective inhibitors of vesicular acetylcholine storage, we have synthesized a series of semirigid vesamicol receptor ligands based on the structure of 2-(4-phenylpiperidino)-cyclohexanol (vesamicol, AH5183, 1). In these compounds, the planes of the phenyl and piperidyl moieties of the parent ligand 1 are held at right angles by vinyl, ethylene, and propylene bridges to form N-substituted derivatives of spiro[indene-1,4'-piperidine], 2,3-dihydrospiro[indene-1,4'-piperidine], and 3,4-dihydrospiro[naphthalene-1(2H),4'-piperidine], respectively. Preliminary evaluation of these compounds in electric organ synaptic vesicles revealed several potent vesamicol receptor ligands, such as 1'-(2-hydroxy-1,2,3,4-tetrahydronaphth-3-yl)spiro[1H-indene-1,4'-p iperidine (11b) and 1'-(2-hydroxy-1,2,3,4-tetrahydronaphth-3-yl)spiro[2-bromo-1H-in den e- 1,4'-piperidine] (14), which display subnanomolar affinity for this receptor. In general, the vinyl and ethylene bridges yielded the most potent analogs while the propylene-bridged analogs were among the least potent compounds. The increased rigidity of these spiro-fused compounds, relative to the corresponding simple 4-phenylpiperidine derivatives of vesamicol, is expected to confer greater selectivity for the vesamicol receptor.

Topics: Acetylcholine; Alkenes; Animals; Electric Organ; Ethylenes; Indenes; Molecular Conformation; Molecular Structure; Neuromuscular Depolarizing Agents; Piperidines; Receptors, Cholinergic; Structure-Activity Relationship; Synaptic Vesicles; Vinyl Compounds

Complementary DNA clones corresponding to a messenger RNA encoding a 56 kDa polypeptide have been obtained from Torpedo marmorata and Torpedo ocellata electric lobe libraries, by homology screening with a probe obtained from the putative acetylcholine transporter from the nematode Caenorhabditis elegans. The Torpedo proteins display approximately 50% overall identity to the C. elegans unc-17 protein and 43% identity to the two vesicle monoamine transporters (VMAT1 and VMAT2). This family of proteins is highly conserved within 12 domains which potentially span the vesicle membrane, with little similarity within the putative intraluminal glycosylated loop and at the N- and C-termini. The approximately 3.0 kb mRNA species is specifically expressed in the brain and highly enriched in the electric lobe of Torpedo. The Torpedo protein, expressed in CV-1 fibroblast cells, possesses a high-affinity binding site for vesamicol (Kd = 6 nM), a drug which blocks in vitro and in vivo acetylcholine accumulation in cholinergic vesicles.

Topics: Amino Acid Sequence; Animals; Base Sequence; Brain; Caenorhabditis elegans; Caenorhabditis elegans Proteins; Carrier Proteins; Cloning, Molecular; Glycoproteins; Helminth Proteins; Membrane Glycoproteins; Membrane Transport Proteins; Molecular Sequence Data; Neuropeptides; Piperidines; Receptors, Cholinergic; RNA, Messenger; Sequence Alignment; Sequence Homology, Amino Acid; Torpedo; Vesicular Acetylcholine Transport Proteins; Vesicular Biogenic Amine Transport Proteins; Vesicular Monoamine Transport Proteins; Vesicular Transport Proteins

As possible presynaptic tracers for cholinergic function in humans, three 18F-labelled vesamicol analogs were synthesized for use in positron emission tomography (PET): cis-[18F]-4-fluoromethylvesamicol (FMV), [18F]-N-fluoroacetamidobenzovesamicol (FAA) and [18F]-N-ethyl-N-fluoroacetamidobenzovesamicol (NEFA). Radiolabelling was accomplished using [18F]fluoride and the corresponding tosylates, the syntheses of which are also described. Yields were on the order of 40-60, 5 and 40-60%, respectively. Dynamic studies of the biodistribution in rats of [18F]FAA and [18F]NEFA using PET were compared with those previously reported for [18F]FMV. Due to probable rapid metabolism, [18F]FAA was considered unsuitable as a ligand for in vivo imaging. [18F]NEFA, similar to [18F]FAA, displayed a more moderate cerebral uptake than that of [18F]FMV (2 vs 20-30%). Pretreatment with vesamicol blocked the cerebral uptake, indicating a specific interaction with the vesamicol binding site. The biodistribution of high specific activity [18F]NEFA with time could be described with a three-compartmental model. The evaluation of [18F]NEFA as a tracer for cholinergic function is currently being pursued in monkeys and humans.

Topics: Animals; Evaluation Studies as Topic; Fluorine Radioisotopes; Male; Molecular Structure; Piperidines; Rats; Rats, Wistar; Tomography, Emission-Computed

The acetylcholine transporter exhibits such low affinity and specificity for acetylcholine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [3H]serotonin, [3H]noradrenaline, or [3H]glutamate occurred. Serotonin, noradrenaline, and N-acetyl-aspartyl glutamate inhibited active transport of [3H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and gamma-aminobutyric acid were shown here not to inhibit active transport of [3H]-acetylcholine. Noradrenaline was competitive with respect to [3H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [3H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.

Topics: Acetylcholine; Animals; Biological Transport; Electric Organ; Glutamates; Glutamic Acid; Neurotransmitter Agents; Norepinephrine; Piperidines; Serotonin; Synaptic Vesicles; Torpedo

1. Local contraction of the endplate region in response to nerve stimulation was studied in isolated mouse diaphragms. In normal preparations, muscle contractions involved the whole length of the muscle fibre with rise and decay times in the order of tens of ms whether evoked with a single or train of pulses. 2. When acetylcholinesterase was inhibited with neostigmine, tetanic stimulation produced a twitch-like phasic contraction and a delayed tonic contracture. A brief train of pulse (10 ms, 300 Hz) was enough to trigger a full size tonic contracture which reached an amplitude about one tenth that of control tetanus and had a duration of about 4 s. 3. Tetanic stimulation evoked a non-propagating prolonged depolarization at the endplate region lasting for about 1 s following a few muscle action potentials. 4. mu-Conotoxin, a specific inhibitor of muscle Na+ channel, selectively abolished the phasic contraction and the muscle action potentials leaving the tonic contracture and the prolonged depolarization unaffected. 5. Both the tonic contracture and the prolonged depolarization were highly sensitive to blockade by tubocurarine (IC50 0.05-0.1 microM) and vesamicol (1 microM, an inhibitor of packaging acetylcholine into synaptic vesicles), were attenuated by increasing Ca2+ concentration and were prolonged by decreasing Ca2+. 6. The results suggest that prolonged activation of endplate nicotinic receptors by endogenously released transmitter can produce substantial contractions of the endplate region when acetylcholinesterase are inhibited. The source of Ca2+ for the contraction seems to come mainly from intracellular stores.

Topics: Action Potentials; Animals; Calcium; Cholinesterase Inhibitors; Diaphragm; Electric Stimulation; Formamides; In Vitro Techniques; Mice; Mice, Inbred ICR; Motor Endplate; Muscle Contraction; Neostigmine; Neuromuscular Depolarizing Agents; Neurotransmitter Agents; omega-Conotoxins; Peptides; Phrenic Nerve; Piperidines; Respiratory Muscles; Tubocurarine

Electrophysiology and binding studies were used to determine the actions of vesamicol [2-(4-phenylpiperidino)cyclohexanol (AH5183)] on an alpha-bungarotoxin-sensitive, neuronal nicotinic acetylcholine receptor in the nervous system of the cockroach, Periplaneta americana. Electrophysiological studies on an identified motor neurone revealed a reversible blocking action of (+/-)-vesamicol on the response to ionophoretically applied acetylcholine with an IC50 value of 8.0 x 10(-6) mol1(-1). The block was weakly voltage-dependent over the membrane potential range of -50 mV to -90 mV, and appeared to be non-competitive. No difference in potency was observed between the resolved stereoisomers. (+/-)-Vesamicol was found to suppress specific binding of 125I-labelled alpha-bungarotoxin to cockroach nervous tissue with an IC50 value of 5.1 x 10(-3) mol1(-1) and an estimated Hill coefficient of 0.73. Differences in the Hill coefficients were found when the resolved stereoisomers were tested separately. These data provide the first demonstration of a blocking action by vesamicol of a neuronal nicotinic acetylcholine receptor.

Topics: Animals; Bungarotoxins; In Vitro Techniques; Male; Membrane Potentials; Motor Neurons; Nicotinic Antagonists; Parasympatholytics; Periplaneta; Piperidines; Receptors, Nicotinic; Sensory Receptor Cells; Stereoisomerism

We investigated the binding of [3H]AH5183 (2-(4-phenylpiperidino)cyclohexanol) to rat brain synaptosomes and subcellular fractions. A high content of specific binding was observed in crude synaptosomes obtained from the striatum, cerebral cortex and hippocampus. The highest density of subcellular binding sites was observed in the synaptic vesicle-rich fraction. The affinities of AH5183 binding to crude synaptosomes and the synaptic vesicle-rich fraction were almost equivalent, but the density of binding sites was higher in the synaptic vesicle fraction. The present findings indicate that [3H]AH5183 binding to both synaptosomes and the synaptic vesicle-rich fraction is useful as a cholinergic marker, and that for quantitative studies, binding to the latter fraction is more preferable.

Topics: Animals; Brain; In Vitro Techniques; Kinetics; Male; Nerve Tissue Proteins; Neuromuscular Depolarizing Agents; Piperidines; Protein Binding; Rats; Rats, Wistar; Subcellular Fractions; Synaptosomes

Topics: Acetylcholine; Animals; Calcium; Cholinesterase Inhibitors; Membrane Potentials; Motor Neurons; Nerve Endings; Piperidines; Rats; Receptors, Nicotinic; Secretory Rate; Tetrodotoxin; Tubocurarine

The binding of (1)-[3H]vesamicol was characterized in several subcellular fractions and brain regions of the rat. Binding to a lysed P2 fraction from the rat cerebral cortex reached equilibrium within 4 min at 37 degrees C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P2 fractions from the cerebral cortex (Kd: 21 nM and 980 nM), striatum (Kd: 28 nM and 690 nM), and cerebellum (Kd: 22 nM and 833 nM). High affinity Bmax values were highest in striatum (1.17 pmol/mg protein), followed by cerebellum (0.67 pmol/mg protein), and cerebral cortex (0.38 pmol/mg protein). Low affinity Bmax values were highest in cerebellum (5.2 pmol/mg protein), with similar values for cerebral cortex (3.7 pmol/mg protein) and striatum (3.8 pmol/mg protein). High affinity but not low affinity binding in each brain region was stereospecific. Another inhibitor of vesicular ACh-transport also displaced 1-vesamicol binding potently (IC50: 17 nM) and efficaciously (over 90%). Both high affinity and low affinity Bmax values for [3H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by purified synaptosomes, crude membranes and P2 fractions. Specific binding was not observed in a mitochondria-enriched fraction. Crude membrane preparations of primary, neuron-enriched whole brain cultures also exhibited high (64 nM) and low affinity (1062 nM) [3H]vesamicol binding. Isoosmotic replacement of 0.18 M KCl in the binding-buffer with NaCl had no effect on binding. These results suggest that at least some high affinity [3H]vesamicol binding in rat brain preparations may be associated with synaptic vesicles, some of which may not be cholinergic in origin.

Topics: Animals; Brain; Cell Membrane; Cells, Cultured; Cerebellum; Cerebral Cortex; Corpus Striatum; Hypotonic Solutions; Male; Piperidines; Rats; Rats, Sprague-Dawley; Synaptic Vesicles; Synaptosomes; Tritium

On the basis of the high-affinity vesamicol analog 4-aminobenzovesamicol (ABV), a tritiated, arylazido ligand (azidoABV) of the vesamicol receptor (VR) in cholinergic synaptic vesicles was synthesized. azidoABV is an inhibitor of acetylcholine (AcCh) active transport, and it binds to the VR with higher affinity than vesamicol. The rate of dissociation of azidoABV from synaptic vesicles is 0.058 +/- 0.003 min-1 at 20 degrees C (about 3-fold slower than that of vesamicol), and the equilibrium dissociation constant is 2 nM (about 4-fold lower than that of vesamicol). Photolysis of [3H]azidoABV in the presence of a stoichiometric excess of the VR led to incorporation of 28% of the radiolabel, of which 57% was blocked by 50 microM vesamicol. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the labeled vesicles revealed, after autofluorography, specific labeling over a broad molecular weight range that extended from about 50 to 200 kDa. This labeling pattern was essentially the same as that obtained with an azido analog of AcCh that was used to label the AcCh transporter (Rogers, G.A., & Parsons, S. M. (1992) Biochemistry 31, 5770-5777). In addition, about 6% of the radioligand that was specifically incorporated into proteins with M(r) greater than 12 kDa labeled four polypeptides that corresponded to bands in the Coomassie image at M(r) = 23, 33, 35, and 38 kDa. The results suggest that the VR exists as part of a complex system of subunits.

Topics: Acetylcholine; Affinity Labels; Animals; Autoradiography; Binding, Competitive; Biological Transport, Active; Electric Organ; Electrophoresis, Polyacrylamide Gel; Kinetics; Molecular Weight; Neuromuscular Depolarizing Agents; Piperidines; Receptors, Cholinergic; Synaptic Vesicles; Torpedo; Tritium

The present study compares the effects of chronic administration of basic fibroblast growth factor (bFGF) and nerve growth factor (NGF) on various hippocampal cholinergic parameters in rats with partial unilateral fimbrial transections. Lesions resulted in marked reductions of several presynaptic cholinergic parameters: choline acetyltransferase (ChAT) activity (by 50%), [3H]-acetylcholine ([3H]ACh) synthesis (by 59%), basal and veratridine (1 microM)-evoked [3H]ACh release (by 44 and 57%, respectively), and [3H]vesamicol binding site densities (by 35%). In addition, [3H]AF-DX 116/muscarinic M2 binding site densities were also modestly decreased (by 23%). In contrast, [3H]pirenzepine/muscarinic M1 and [3H]AF-DX 384/muscarinic M2/M4 binding site densities were not altered by the lesions, nor were they affected by any of the treatments. Intracerebroventricular administration of bFGF (10 ng, every other day, for 21 days) partially prevented the lesion-induced deficit in hippocampal ChAT activity, an effect that was not markedly different from that measured in the NGF-treated (1 microgram, intracerebroventricularly, every other day, for 21 days) rats. In rats treated with a combination of bFGF and NGF, ChAT activity was not different from that in rats treated with the individual factors alone. In contrast, the lesion-induced deficits in the other cholinergic parameters were not attenuated by bFGF treatment, although they were at least partially prevented by NGF administration. To determine whether higher concentrations of bFGF are necessary to affect cholinergic parameters other than hippocampal ChAT activity, rats were treated with 1 microgram (every other day, 21 days) of the growth factor. In this group of rats, detrimental effects of bFGF, manifested by an increased death rate (46%), and marked reductions in body weight of the survivors, were observed. In addition, this concentration of bFGF appeared to exacerbate the lesion-induced reduction in [3H]ACh synthesis by hippocampal slices; [3H]ACh synthesis in lesioned hippocampi represented 36 and 52% of that in contralateral unlesioned hippocampi for the bFGF-treated and control groups, respectively. In conclusion, although bFGF administration attenuates the deficit in hippocampal ChAT activity induced by partial fimbrial transections, this does not appear to translate into enhanced functional capacity of the cholinergic terminals. This is clearly in contrast to NGF, which enhances not only hippocampal ChAT

Topics: Acetylcholine; Animals; Binding Sites; Choline O-Acetyltransferase; Denervation; Female; Fibroblast Growth Factor 2; Hippocampus; Muscarine; Nerve Growth Factors; Parasympathomimetics; Piperidines; Rats; Rats, Wistar; Receptors, Muscarinic; Synapses; Time Factors

Previous studies from this laboratory characterized 83 analogs of vesamicol by their potencies for inhibition of acetylcholine active transport by synaptic vesicles isolated from Torpedo electric organ. Examination of the more potent of these compounds, plus five new analogs, by kinetic and equilibrium measurements on complexes with the vesamicol receptor (VR) revealed nine analogs that are significantly more potent than vesamicol. Equilibrium measurements were performed at very low protein concentrations and extended incubation times, which allowed the characterization of very high affinity analogs. Better understanding of the structural binding requirements of the VR has resulted, and a spatial map of allowed hydrophobicity has been clearly established. Three analogs were resolved, and they displayed enantioselectivity ratios as high as 260 for binding to the VR (10-times higher than that of vesamicol). The most potent analog, 4-aminobenzovesamicol (ABV), was synthesized in tritiated form and shown to dissociate from the VR with a half-life of about 14 hr at 20 degrees. The estimated dissociation constant is < or = 6.5 +/- 0.5 pM. By reciprocal kinetic experiments with vesamicol and ABV, coincidence of the two binding sites on vesicles was established. The high affinity and enantioselectivity of ABV and other similar analogs, coupled with good chemical and radiochemical stability, make these ligands attractive for the study of the VR in complex tissues. The observed difference between the equilibrium dissociation constant for the vesamicol-VR complex as estimated by titration with [3H]vesamicol (7.6 nM) and by displacement of subsaturating [3H]vesamicol by nonlabeled vesamicol (1.0 nM) suggests that high and low affinity populations of the VR exist.

Topics: Animals; Binding, Competitive; In Vitro Techniques; Kinetics; Ligands; Models, Chemical; Piperidines; Receptors, Cholinergic; Stereoisomerism; Structure-Activity Relationship; Synaptic Vesicles; Torpedo

The effects of ibotenic acid-induced basal forebrain lesions and treatment with the triazole MDL 26,479 on the acquisition of an operant visual conditional discrimination task and on [3H]hemicholinium-3 and [3H]vesamicol binding were examined. Lesioned animals required more training sessions to acquire the stimulus-response rules of this task. They also showed longer response latencies throughout the experiment. The effects of the treatment with MDL 26,479 (5 mg/kg; i.p. 60 min before each training session) interacted with the effects of the lesion, producing a decrease in the number of sessions required to perform above chance-level in lesioned but not in control animals. MDL 26,479 did not seem to produce immediate performance effects but interacted with the learning process. The lesions destroyed the cell bodies in the area of the substantia innominata, basal nucleus of Meynert, and the globus pallidus. The number of frontocortical cholinergic terminals as primarily indicated by hemicholinium-3 binding was reduced in lesioned animals; however, another measure of cholinergic terminals, vesamicol binding, was unchanged. Behavioral performance of animals correlated significantly with hemicholinium binding in the frontal cortex of the right hemisphere. The fact that the lesion delayed but did not block the acquisition of the task may have been a result of compensatory mechanisms in remaining cholinergic terminals as indicated by stable vesamicol binding. These data allow assumptions about the conditions for the demonstration of beneficial behavioral effects of MDL 26,479. They also suggest that the long-term effects of basal forebrain lesions on cortical cholinergic transmission remain unsettled.

Topics: Animals; Antidepressive Agents; Autoradiography; Biomarkers; Body Weight; Choline O-Acetyltransferase; Conditioning, Operant; Hemicholinium 3; Ibotenic Acid; Immunohistochemistry; Learning; Neuromuscular Depolarizing Agents; Parasympathetic Nervous System; Piperidines; Prosencephalon; Rats; Rats, Sprague-Dawley; Stereotaxic Techniques; Synaptic Transmission; Triazoles

We have examined the question of how regenerating sympathetic neurons that are concomitantly induced to become cholinergic regulate choline allocation between ACh and the phospholipid synthetic pathway. The allocation of choline into ACh increased parabolically with time in culture, and by 3 weeks, cultures with neurites of approximately 6 mm length were incorporating over 85% of the choline locally in the neurites into four major metabolites: ACh, phosphorylcholine, cytidine diphosphocholine, and phosphatidylcholine. The near-equivalent distribution of labeled choline between intracellular choline, ACh, and phosphorylcholine was independent of time (5 min to 6 hr) and choline concentration (0.125-30 microM), phosphatidylcholine being the sole metabolite whose level in the neurites increased steadily with incubation time. Relative choline distribution into ACh and phosphorylcholine was unaltered even after a brief depolarizing prepulse, which caused a two- to fourfold enhancement in the total choline incorporated. These observations, allied with the similar half-saturation constants and Vmax values of CAT and choline kinase for intracellular choline, suggest that growing sympathetic neurons are poised to allocate choline symmetrically between the synthesis of ACh and phosphatidylcholine in the neurites. When, however, the supply of choline was limited either by replacement of Na+ in the medium with N-methyl-D-glucamine, or by vesamicol, a 90-97% reduction in intracellular choline caused a similar decline in ACh levels but synthesis of metabolites of the phosphatidylcholine pathway was maintained unperturbed, as if no drug was present. We suggest that this can be accounted for by a 10-fold increase in choline kinase activity. Thus, growing sympathetic neurons that express cholinergic properties not only maintain their chief cellular phosphatidylcholine-synthesizing activity concomitantly with ACh synthesis in the neurites, but may also preserve phosphatidylcholine synthesis more effectively than ACh synthesis when the supply of choline is perturbed. Relinquishing ACh synthesis during growth may be one way of conserving and encouraging neurite regeneration.

Topics: Acetylcholine; Animals; Animals, Newborn; Biological Transport; Cells, Cultured; Choline; Choline O-Acetyltransferase; Ganglia, Sympathetic; Kinetics; Meglumine; Neurites; Neuromuscular Depolarizing Agents; Neurons; Organ Culture Techniques; Phosphatidylcholines; Piperidines; Potassium; Rats; Time Factors; Tritium

Quantitative in situ hybridization and northern blot analysis techniques were used to determine the effects of removal of the cholinergic input on levels and topographical distribution of brain-derived neurotrophic factor mRNA in the hippocampus of adult rats. First, the effects of partial and full fimbrial transections, which result in partial and near-total cholinergic deafferentation respectively, were compared. Twenty-one days after partial unilateral fimbrial transections, there were significant decreases in brain-derived neurotrophic factor mRNA expression throughout the hippocampal formation. Decreased expression of brain-derived neurotrophic factor mRNA was evident in all areas of localization within the hippocampal formation. The decreases amounted to 22-36% reductions compared with unlesioned control animals. Brain-derived neurotrophic factor mRNA levels were decreased to a greater extent (50-69%) following full unilateral fimbrial transections. Quantitative northern blot analysis indicated that hippocampal BDNF mRNA was decreased by 29 and 68%, three weeks after partial or full unilateral fimbrial transections, respectively. The extent of the reductions in brain-derived neurotrophic factor mRNA levels correlated with reductions in acetylcholinesterase staining density and cholinergic terminal density determined by quantitative autoradiographic analysis of [3H]vesamicol binding sites. Second, we found that chronic treatment with atropine (20 mg/kg per day for 14 days) decreased (by 54%) brain-derived neurotrophic factor mRNA levels in all areas of localization within the hippocampus. In contrast, chronic treatment with nicotine (1.18 mg/kg per day for 14 days), a treatment known to desensitize nicotinic receptors, did not affect brain-derived neurotrophic factor mRNA expression in the hippocampal formation. The findings provide evidence for cholinergic muscarinic regulation of brain-derived neurotrophic factor mRNA expression in the adult rat hippocampal formation and they suggest the existence of a tonic stimulation of brain-derived neurotrophic factor synthesis by the cholinergic afferents.

Topics: Acetylcholine; Acetylcholinesterase; Animals; Atropine; Blotting, Northern; Brain; Brain-Derived Neurotrophic Factor; Female; Gene Expression Regulation; Hippocampus; Nerve Growth Factors; Nerve Tissue Proteins; Neuromuscular Depolarizing Agents; Nicotine; Piperidines; Pyramidal Tracts; Rats; Rats, Wistar; RNA, Messenger; Stereotaxic Techniques

Introduction of a nitrogen atom into the cyclohexane ring of 2-(4-phenylpiperidinyl)cyclohexanol (vesamicol, AH5183) yielded two positional isomers, 5-azavesamicol (5, prezamicol) and 4-azavesamicol (6, trozamicol). As inhibitors of vesicular acetylcholine transport, 5 and 6 were found to be 147 and 85 times less potent than vesamicol. N-Benzoylation of 5 (to yield 9a) increased the potency 3-fold. In contrast, 10a, a compound derived from N-benzoylation of 6, was 50 times more potent than the latter and almost equipotent with vesamicol, thereby suggesting a preference for the 4-azavesamicol series. Although (-)-vesamicol is more potent than its dextrorotary isomer, (+)-10a was found to be 3 times more potent than (-)-10a, suggesting a reversal of the sign of rotation in the azavesamicol series. Reduction of 9a and 10a (to yield the corresponding N-benzyl derivatives 11a and 12a) increased potency 20- and 2-fold, respectively, indicating a preference for a basic nitrogen. The reaction of 5 or 6 with substituted benzyl halides yielded several potent inhibitors of vesicular acetylcholine transport, including N-(p-fluorobenzyl)trozamicol, 12d, which is twice as potent as vesamicol. Thus the introduction of a nitrogen atom into the cyclohexane ring of vesamicol provides opportunities for developing a new class of anticholinergic agents.

Topics: Acetylcholine; Animals; Crystallography; Neuromuscular Depolarizing Agents; Piperidines; Stereoisomerism; Structure-Activity Relationship; Synaptic Vesicles; Torpedo

The present study was initiated to examine the effects of ATP on acetylcholine (ACh) synthesis. The exposure of superior cervical ganglia to ATP increased ACh stores by 25%, but this effect was also evident with ADP, AMP, and adenosine, but not with beta gamma-methylene ATP, a nonhdydrolyzable analogue of ATP, or with inosine, the deaminated product of adenosine. Thus, we attribute the enhanced ACh content caused by ATP to the presence of adenosine derived from its hydrolysis by 5'-nucleotidase. The adenosine-induced increase of tissue ACh was not the consequence of an adenosine-induced decrease of ACh release. The extra ACh remained in the tissue for more than 15 min after the removal of adenosine, but it was not apparent when ganglia were exposed to adenosine in a Ca(2+)-free medium. Incorporation of radiolabelled choline into [3H]ACh was also enhanced in the presence of adenosine, suggesting an extracellular source of precursor. Moreover, the synthesis of radiolabelled forms of phosphorylcholine and phospholipid was not reduced in adenosine's presence, suggesting that the extra ACh was not likely derived from choline destined for phospholipid synthesis. Aminophylline did not prevent the adenosine effect to increase ACh content; this effect was blocked by dipyridamole, but not by nitrobenzylthioinosine (NBTI). In addition, two benzodiazepine stereoisomers known to inhibit stereoselectively the NBTI-resistant nucleoside transporter displayed a similar stereoselective ability to block the effect of adenosine. Together, these results argue that adenosine is transported through an NBTI-resistant nucleoside transporter to exert an effect on ACh synthesis. The extra ACh accumulated as a result of adenosine's action was releasable during subsequent preganglionic nerve stimulation, but not in the presence of vesamicol, a vesicular ACh transporter inhibitor. We conclude that the mobilization of ACh is enhanced as a result of adenosine pretreatment.

Topics: Acetylcholine; Adenine Nucleotides; Adenosine; Adenosine Triphosphate; Animals; Cats; Dose-Response Relationship, Drug; Electric Stimulation; Female; Ganglia, Sympathetic; Kinetics; Male; Neuromuscular Depolarizing Agents; Perfusion; Phospholipids; Phosphorylcholine; Piperidines

The effect of vesamicol on the ability of recycling cholinergic synaptic vesicles to recover, during a period of post-stimulation rest, the biophysical properties of the reserve pool was studied in prestimulated perfused blocks of the electric organ of the electric ray, Torpedo marmorata, a tissue rich in cholinergic synapses. The effect of the drug was analysed by high-resolution centrifugal density-gradient fractionation in a zonal rotor of the extracted vesicles. The two vesicle fractions were identified by their ATP and acetylcholine content and the recycled vesicles by their acquisition of [3H]acetylcholine derived from [3H]acetate in the perfusate. Vesamicol (10 microM) blocked the uptake of tritiated acetylcholine by recycled vesicles and also prevented them from rejoining the reserve pool. This is consistent with a previously formulated model of the recovery process, whereby the increase in the acetylcholine and ATP content of the recycled vesicles which takes place during a post-stimulus period of rest increases their osmotic load and thus their content of free water. Vesamicol, by blocking acetylcholine uptake, also blocks rehydration of the recycled vesicles and thus the accompanying decrease in their density to the value characteristic of fully charged vesicles.

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Cholinergic Fibers; Female; Neuromuscular Depolarizing Agents; Piperidines; Synaptic Vesicles; Torpedo

Racemic (m-iodobenzyl)trozamicol (6, MIBT), a high-affinity vesamicol receptor ligand, was radiolabeled, resolved, and evaluated in rats. Following iv injection, (+)- and (-)-[125I]MIBT achieved initial brain levels of 0.57 and 0.92% dose/g of tissue, respectively. The level of (+)-[125I]MIBT subsequently declined by 74% within 3 h, while that of (-)-[125I]MIBT remained stable for the duration. Ex vivo autoradiographic mapping of (-)-[125I]MIBT distribution in rat brain revealed a pattern which was inconsistent with central cholinergic innervation. However, high levels of (+)-[125I]MIBT were observed over the amygdala, striatum, nucleus accumbens, olfactory tubercle, and nuclei of the fifth and seventh cranial nerves, while moderate to low levels were detected within the cortex, hippocampus, and cerebellum. Thus, the distribution of (+)-[125I]MIBT parallels that of other presynaptic cholinergic markers. Co-injection of (+)-[125I]MIBT with 4-aminobenzovesamicol (2b), a potent vesamicol receptor ligand, reduced the levels of radiotracer in the striatum, cortex, and cerebellum by 58, 35, and 9%, respectively. Thus, (+)-[125I]MIBT binds to vesamicol receptors in vivo. In contrast, coadministration of (+)-[125I]MIBT with haloperidol (0.5 mumol/kg), reduced radiotracer levels in the cortex and cerebellum by 34 and 59%, respectively, while increasing the levels in the striatum by 32%. We conclude that although the distribution of (+)-[125I]MIBT qualitatively reflects cholinergic innervation, a fraction of radiotracer in the cortex and cerebellum is bound to sigma receptors.

Topics: Animals; Autoradiography; Brain; Cerebellum; Cerebral Cortex; Choline; Corpus Striatum; Iodine Radioisotopes; Iodobenzenes; Isotope Labeling; Kinetics; Male; Molecular Structure; Piperidines; Rats; Rats, Wistar; Receptors, sigma; Tissue Distribution

These experiments investigate the release of transmitter from the perfused superior cervical ganglia of cats induced by ouabain in the absence or presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol), a blocker of acetylcholine (ACh) uptake. Ouabain, perfused through the ganglia, released ACh in a Ca(2+)-dependent way. Vesamicol caused some inhibition of the release of ACh by ouabain; however, under this condition, the Na+,K(+)-ATPase inhibitor released five times more transmitter than did preganglionic stimulation at 5 Hz. Also, when ganglia exposed to vesamicol were depleted of the impulse-releasable pool of ACh, subsequent perfusion with ouabain released ACh, and this included ACh newly synthesized in the presence of vesamicol; this phenomenon could be inhibited by the lack of Ca2+ and presence of EGTA, and was completely abolished by perfusion with a medium containing 18 mM Mg2+. To test whether the release of this vesamicol-insensitive Ca(2+)-dependent pool by ouabain is associated with a decrease in the number of synaptic vesicles, ganglia treated with the ATPase inhibitor after the depletion of the impulse-releasable pool of ACh were fixed for electron microscopy. In the presence of Ca2+, coincident with the release of the vesamicol-insensitive pool of ACh, nerve terminals were almost depleted of synaptic vesicles; ganglia treated similarly, but with medium containing 18 mM Mg2+ instead of Ca2+, were not depleted of synaptic vesicles. These results suggest that ouabain releases a vesamicol-insensitive pool of ACh from the sympathetic ganglion and also support the notion that this compartment is vesicular and its exocytosis depends on extracellular Ca2+. It is suggested that empty-vesicle recycling in the presence of vesamicol restricts mobilization of full vesicles to release sites.

Topics: Acetylcholine; Animals; Cats; Drug Resistance; Female; Ganglia, Sympathetic; Male; Ouabain; Piperidines; Synaptic Vesicles

1. Rat hemidiaphragms were incubated in a physiological low-K+ medium without stimulation and the amount of acetylcholine (ACh) released was measured radioenzymatically. Cholinesterases were inhibited by paraoxon. 2. In the presence of 1 microM tetrodotoxin (TTX), the amount of ACh released during a 2 h incubation was lowered by 40%. A similar decrease was observed in the absence of Ca2+ and in the presence of 10 microM-d-tubocurarine (dTC). The effects of TTX combined with Ca2+ removal, and of TTX combined with dTC were no greater than those of TTX, dTC or Ca2+ removal alone. TTX and dTC had no effect on the release of ACh from diaphragms 4 days after denervation. 3. The reduction of spontaneous ACh release observed in the presence of TTX or dTC or in the absence of Ca2+ is best interpreted on the assumption that about 40% of the ACh release was due to the impulse activity known to be generated in intramuscular motor nerve branches by the ACh which accumulates after the inhibition of cholinesterases. 4. In the presence of 1 and 10 microM vesamicol (AH5183, 2-(4-phenylpiperidino)-cyclohexanol), the release of ACh was also diminished by approximately 40%. Vesamicol did not augment the inhibition of release produced by TTX or by the omission of Ca2+.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Calcium; Cholinesterases; Denervation; Depression, Chemical; Diaphragm; In Vitro Techniques; Male; Muscles; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Wistar; Tetrodotoxin; Time Factors; Tubocurarine

Vesamicol is a highly potent inhibitor of active acetylcholine transport into isolated cholinergic vesicles from Torpedo. On the basis of transport kinetics and vesamicol sensitivity, we have shown that the acetylcholine transporter could be in an activated state even in the absence of a stimulated ATPase. In this preparation, N,N'-dicyclohexylcarbodiimide (DCCD), an hydrophobic carbodiimide, inactivates both ACh transport and vesamicol binding. Inhibition of vesamicol binding by DCCD is time dependent, saturable and prevented by vesamicol. DCCD first affected the affinity constant for vesamicol. Ki-value for DCCD lies in the micromolar range. These results imply that there is a DCCD reactive site within the ACh transporter and that it is located in an hydrophobic environment near the vesamicol binding site. SDS-gel electrophoresis after labelling of the vesicle membrane proteins with [14C]DCCD shows that radioactivity is mainly incorporated in a 15 kDa subunit. Time-course and concentration dependence of [14C]DCCD labelling and vesamicol inhibition do not coincide. Hence, the two processes are probably unrelated and the result rather points to another inactivation mechanism which can be an intramolecular cross link.

Topics: Acetylcholine; Animals; Biological Transport; Dicyclohexylcarbodiimide; Dose-Response Relationship, Drug; Electric Organ; Electrophoresis, Polyacrylamide Gel; Hydrogen-Ion Concentration; Kinetics; Membrane Proteins; Molecular Weight; Neuromuscular Depolarizing Agents; Piperidines; Protein Binding; Synaptic Vesicles; Torpedo

The ligand binding relationship between the acetylcholine transporter (AcChT) and the vesamicol receptor (VR) and the kinetics of active transport were studied in synaptic vesicles purified from the Torpedo electric organ using analogues of AcCh and vesamicol. Methoxyvesamicol, which should exhibit better equilibration properties for kinetics measurements than the more potent parent, inhibits active transport in a nonlinear noncompetitive manner. AcCh analogues competitively inhibit binding of [3H]vesamicol with higher affinity in hyposmotically lysed vesicle ghosts than in intact vesicles, apparently due to removal of a competing internal, osmotically active factor. AcCh and actively transported analogues of AcCh that are up to 57% larger in van der Waals volume exhibit up to a 200-fold ratio for the dissociation constant measured by inhibition of vesamicol binding to ghosts (KIAg) compared to the Michaelis constant for transport (KM) or the IC50 value for inhibition of [3H]AcCh active transport. In contrast, two AcCh analogues that are about 120% larger and that almost surely are not transported exhibit a KIAg/IC50 ratio of about 1. The data demonstrate that the vesamicol family of compounds binds to an allosteric site in the AcChT. Initiation of active transport has no apparent effect on the affinities of vesamicol and AcCh analogues, which suggests that most of the AcChT-VR in purified vesicles is transport incompetent. Vesicle ghosts actively transport [3H]AcCh nearly as well as intact vesicles, which suggests that internal factor does not affect transport-competent AcChT-VR. A kinetics model is proposed that predicts that AcCh analogues exhibiting a KIAg/IC50 ratio significantly greater than 1 are actively transported. Some of the microscopic constants in the model are estimated. The AcChT binds AcCh very weakly with a dissociation constant of about 20-50 mM, but it transports substrates rapidly in a process exhibiting remarkably little selectivity for the detailed shape and volume of the transported ion.

Topics: Acetylcholine; Allosteric Site; Animals; Binding, Competitive; Biological Transport, Active; Carrier Proteins; Electric Organ; Kinetics; Membrane Transport Proteins; Piperidines; Receptors, Neurotransmitter; Receptors, Phencyclidine; Synaptic Vesicles; Torpedo; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

The acetylcholine (AcCh) binding site in the AcCh transporter-vesamicol receptor (AcChT-VR) present in synaptic vesicles isolated from the electric organ of Torpedo was characterized. A high-affinity analogue of AcCh containing an aryl azido group, namely, cyclohexylmethyl cis-N-(4-azidophenacyl)-N-methylisonipecotate bromide (AzidoAcCh), was synthesized in nonradioactive and highly tritiated forms. AzidoAcCh was shown to be a competitive inhibitor of [3H]AcCh active transport and binding of [3H]-vesamicol to the allosteric site. The [3H]AzidoAcCh saturation curve was determined. In all cases the AcChT.AzidoAcCh complex exhibited an inhibition or dissociation constant of about 0.3 microM. Binding of [3H]AzidoAcCh was inhibited by vesamicol and AcCh. AzidoAcCh irreversibly blocked greater than 90% of the [3H]vesamicol binding sites after multiple rounds of photolysis and reequilibration with fresh ligand. Autofluorographs of synaptic vesicles photoaffinity-labeled with [3H]AzidoAcCh showed specific labeling of material exhibiting a continuous distribution from 50 to 250 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The result demonstrates that the AcChT has an unexpected structure highly suggestive of the synaptic vesicle proteoglycan.

Topics: Acetylcholine; Affinity Labels; Animals; Azides; Binding Sites; Binding, Competitive; Biological Transport, Active; Carrier Proteins; Electric Organ; Electrophoresis, Polyacrylamide Gel; Kinetics; Membrane Transport Proteins; Photochemistry; Piperidines; Receptors, Neurotransmitter; Receptors, Phencyclidine; Spectrophotometry, Ultraviolet; Synaptic Vesicles; Torpedo; Tritium; Vesicular Acetylcholine Transport Proteins; Vesicular Transport Proteins

1. The effects of vesamicol, an inhibitor of vesicular acetylcholine (ACh) storage, were studied on trains of endplate currents (e.p.cs) in the cut rat hemidiaphragm nerve-muscle preparation and on trains of focally recorded nerve terminal current waveforms in the mouse triangularis sterni nerve-muscle preparation. 2. In the rat, 0.1 and 1 microM (-)-vesamicol produced an enhancement of the rundown of e.p.c. amplitudes during trains of high frequency (50 Hz) nerve stimulation. However, 1 microM (+)-vesamicol had no effect on the rundown of e.p.c. amplitudes. 3. In the mouse, high concentrations of (-)-vesamicol (10-100 microM) produced a concentration- and stimulation-dependent decrease in the amplitude of the second negative-going deflection of focally recorded nerve terminal current waveforms. 4. At 1 mM, (-)-vesamicol produced a stimulation-independent decrease in the amplitude of the first negative-going deflection of the nerve terminal current waveforms, an increase in signal delay and evidence of nerve conduction failure. These all indicate a local anaesthetic-like block of nodal Na(+)-channels. 5. In contrast to its effects on trains of e.p.cs, the effects of vesamicol on the nerve terminal current waveforms were not stereoselective, the (+)-isomer being equipotent with the (-)-isomer. 6. Low concentrations of the Na(+)-channel blocking toxin, tetrodotoxin (15-60 nM), produced similar changes in the focally recorded nerve terminal current waveforms to those seen with vesamicol. 7. It is concluded that the stereoselective rundown of e.p.c. amplitudes produced by (-)-vesamicol is due to an effect, either direct or indirect, on ACh mobilization within motor nerve terminals. Furthermore, in mammalian species, the inhibitory effects of vesamicol on nodal Na+-channels which are seen at high concentrations do not contribute to the principal neuromuscular effects of the compound.

Topics: Animals; Electric Stimulation; Ion Channels; Male; Motor Endplate; Nerve Endings; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Inbred Strains; Tetrodotoxin

The vesamicol receptor (VR) present in cholinergic synaptic vesicles isolated from the electric organ of Torpedo was solubilized in cholate detergent and stabilized with glycerol and a phospholipid mixture. The receptor was purified in 7% yield by hydroxylapatite, wheat germ lectin affinity, DEAE anion-exchange, and size exclusion chromatographies based on a [3H]vesamicol binding assay. A final specific binding of 4400 pmol/mg of protein was obtained. The cholate-solubilized VR complex exhibited variable aggregation states with particle molecular masses of 210-3500 kDa in different experiments. The purified VR exhibited very heterogeneous electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with very diffuse protein staining at about 240 kDa. No "classical" polypeptide or glycopeptide band was detected. One form of the SV1 epitope, which is characteristic of cholinergic synaptic vesicle proteoglycan, copurified precisely with the VR. The SV2 epitope, which is found in most neuronal and endocrine secretory vesicles, also closely purified with the VR. Substantially purified VR retained both enantioselectivity for (-)-vesamicol and a linked AcCh-binding site. This confirms the allosteric model for the VR in the AcCh transporter. The physicochemical properties of the VR and copurification of it with the SV1 epitope strongly suggest that the VR is associated with cholinergic vesicle proteoglycan. A second proteoglycan that is not associated with the VR but which carries the SV1 and SV2 epitopes also was observed.

Topics: Acetylcholine; Allosteric Site; Animals; Chromatography; Detergents; Electric Organ; Electrophoresis, Polyacrylamide Gel; Epitopes; Piperidines; Receptors, Neurotransmitter; Receptors, Phencyclidine; Solubility; Synaptic Vesicles; Torpedo

Endplate preparations of the left rat hemidiaphragm were incubated with [3H]choline to label neuronal acetylcholine stores. Elevation of the concentration (13.5-135 mmol/l) of extracellular potassium chloride (KCl) stimulated the release of [3H]acetylcholine in a concentration-dependent manner. KCl (27 mmol/l) still caused a significant efflux of [3H]acetylcholine in a Ca(2+)-free medium. Inhibitors of cholinesterase (physostigmine, diisopropylfluorophosphate) suppressed by 80% this Ca(2+)-independent efflux of [3H]acetylcholine. Vesamicol (10 mumol/l), the blocker of the vesicular acetylcholine carrier, also suppressed the stimulated, Ca(2+)-independent efflux of [3H]acetylcholine. The inhibitory effect of physostigmine was not prevented by muscarine or nicotine receptor antagonists, but the inhibitory effect was lost when the stimulus strength was increased (81 mmol/l KCl). The present experiments showed cholinesterase inhibition to suppress a Ca(2+)-independent efflux of [3H]acetylcholine, probably by interference with a membrane-bound acetylcholine carrier.

Topics: Acetylcholine; Animals; Calcium; Choline; Cholinesterase Inhibitors; Diaphragm; Female; In Vitro Techniques; Male; Neuromuscular Junction; Physostigmine; Piperidines; Potassium Chloride; Rats; Rats, Sprague-Dawley; Tritium

Synthesis and release of acetylcholine (ACh) in the rat hippocampal slices were examined to clarify the mechanism of modulation of ACh synthesis. Treatment with 2-(4-phenylpiperidino)cyclohexanol (AH5183, 50 microM), an inhibitor of ACh transport from cytosol to synaptic vesicles, inhibited the increase in ACh content of the membrane-bound fraction which is readily releasable, but did not affect the cytosolic ACh content. Under these conditions, the total ACh content reached a plateau value. These results indicate that ACh synthesis is modulated by cytosolic ACh content but not by the vesicular fraction.

Topics: Acetylcholine; Animals; Calcium; Cytosol; Hippocampus; In Vitro Techniques; Male; Membranes; Neuromuscular Depolarizing Agents; Perfusion; Piperidines; Potassium; Radioimmunoassay; Rats; Rats, Wistar

A series of novel lipophilic amino alcohols, analogs of the anticholinergic drug vesamicol, were evaluated for Ca2+ channel blocking activity. The effects of these drugs on depolarization-induced intracellular free Ca2+ concentration ([Ca2+]i) transients were examined in single NG108-15 cells and dorsal root ganglion (DRG) neurons in culture. [Ca2+] was recorded with the Ca2+ indicator Indo-1 and a dual emission microfluorimeter. Structure-activity studies indicated that features required for Ca2+ channel blocking activity were distinct from those required for anticholinergic activity. In particular, the Ca2+ channel blocking activity was insensitive to the configuration at the chiral center, whereas the anticholinergic activity was clearly enantioselective. One of the most active compounds, 3-(3-bromophenyl)-2-hydroxy-1-[1-(4-phenylpiperidinyl)]propane (2b), was characterized in more detail. This compound inhibited the dihydropyridine-sensitive Ca2+ channel response in NG108-15 cells, evoked by depolarization with 50 mM K+, with an IC50 of 5 microM. Field potential stimulation of DRG neurons elicited [Ca2+]i transients mediated by at least three Ca2+ channel subtypes; compound 2b inhibited the entire Ca2+ channel response with an IC50 of 1 microM. A key element required for Ca2+ channel blocking activity was the presence of an electron withdrawing substituent on the pendant phenyl ring. Modification of the amino alcohol structure may lead to more potent compounds with broad spectrum Ca2+ channel blocking activity. These structures provide a new chemical starting point for the development of Ca2+ antagonists.

Topics: Amino Alcohols; Animals; Calcium Channel Blockers; Dose-Response Relationship, Drug; Neurons; Piperidines; Rats; Rats, Sprague-Dawley; Torpedo; Tumor Cells, Cultured

Acetylcholine efflux at the rat neuromuscular junction was assayed following blockage of ACh transport into synaptic vesicles by 2-(4-phenylpiperidino) cyclohexanol (AH5183). [2H4]Choline was used as a labeled precursor. AH5183 completely blocked ACh efflux from the cytosolic compartment but had comparatively less effect on release from the unlabeled vesicular pool. Tissue [2H4]ACh levels increased after AH5183 addition due to cytosolic ACh retention. Thus, ACh in the non-vesicular pool (calculated to be 34% of the total ACh) may efflux solely via the AH5183-sensitive ACh transporter inserted into the terminal membrane. ACh released from the vesicular fraction was about 100-fold more than could be accounted for by miniature end-plate potentials; possible causes of this overestimate are discussed.

Topics: Acetylcholine; Animals; Cytosol; Dose-Response Relationship, Drug; In Vitro Techniques; Kinetics; Motor Neurons; Muscles; Nerve Endings; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Inbred F344; Synaptic Vesicles

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca(2+)-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca(2+)-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca(2+)-uptake, and intracellular Ca2+ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 microM veratridine (from 67 +/- 2.3 mV resting membrane potential to 50.7 +/- 2.5 mV) was not significantly influenced by vesamicol (1-20 microM). Vesamicol (1-20 microM) had no effect on either the overall curve of the veratridine-evoked 45Ca2+ uptake or the amount of Ca2+ taken up by synaptosomes. Veratridine caused a rise in intrasynaptosomal Ca2+ concentration as measured by Fura2 fluorescence, and the same increase both in characteristics and in magnitude was observed in the presence of vesamicol (20 microM). The K(+)-evoked (40 mM) increase of Ca2+ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 microM) vesamicol inhibited both the fall in membrane potential and the elevated Ca2+ uptake by veratridine, indicating a possible nonspecific effect on potential-dependent Na+ channels at this concentration. Vesamicol, in lower concentration (20 microM) when neither of the above parameters was changed, completely prevented veratridine-evoked increase of [14C]acetylcholine release. This was observed only when vesamicol was present in the media throughout the experiment after loading the preparation with [14C]choline.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Calcium; Fura-2; Guinea Pigs; Membrane Potentials; Neuromuscular Depolarizing Agents; Piperidines; Potassium; Sodium; Synaptosomes; Veratridine

Radioiodinated 2-hydroxy-3-(4-iodophenyl)-1-(4-phenylpiperidinyl)propane, 5 (4-HIPP), was synthesized and evaluated as a simple vesamicol-like radiotracer for mapping cholinergic pathways in the brain. Both enantiomers of 5 exhibit significant accumulation (approx. 2% of injected dose) and prolonged retention (t1/2 greater than 3 h) within the rat brain. The accumulation of radioiodinated 5 in the rat brain was reduced by up to 70% in the presence of vesamicol and its analogs. The levorotary isomer (-)-4-[123I]HIPP exhibits significant accumulation in the monkey brain, with a half-life of about 9 h. Radioiodinated 5 may therefore be a useful tool for studying cholinergic pathways in the brain.

Topics: Animals; Autoradiography; Brain; Cholinergic Fibers; Iodine Radioisotopes; Iodobenzenes; Male; Myocardium; Neuromuscular Depolarizing Agents; Piperidines; Radionuclide Imaging; Rats; Rats, Inbred Strains; Stereoisomerism; Tissue Distribution

The present experiments tested whether preganglionic stimulation and direct depolarization of nerve terminals by tityustoxin could mobilize similar or different pools of acetylcholine (ACh) from the cat superior cervical ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (vesamicol, AH5183), an inhibitor of ACh uptake into synaptic vesicles. In the absence of vesamicol, both nerve stimulation and tityustoxin increased ACh release. In the presence of vesamicol, the release of ACh induced by tityustoxin was inhibited, and just 16% of the initial tissue content could be released, a result similar to that obtained with electrical stimulation under the same condition. When the impulse-releasable pool of ACh had been depleted, tityustoxin still could release transmitter, amounting to some 10% of the ganglion's initial content. This pool of transmitter seemed to be preformed in the synaptic vesicles, rather than synthesized in response to stimuli, as tityustoxin could not release newly synthesized [3H]ACh formed in the presence of vesamicol, and hemicholinium-3 did not prevent the toxin-induced release. In contrast to the results with tityustoxin, preganglionic stimulation could not release transmitter when impulse-releasable or toxin-releasable compartments had been depleted. Our results confirm that vesamicol inhibits the mobilization of transmitter from a reserve to a more readily releasable pool, and they also suggest that, under these experimental conditions, there might be some futile transmitter mobilization, apparently to sites other than nerve terminal active zones.

Topics: Acetylcholine; Animals; Cats; Choline; Electric Stimulation; Female; Ganglia, Sympathetic; Male; Neuromuscular Depolarizing Agents; Piperidines; Scorpion Venoms; Tritium

We have examined PC12 cells for the localization of binding sites for vesamicol [l-2-(4-phenylpiperidino) cyclohexanol], a compound that has previously been shown to bind to cholinergic vesicles and to inhibit the uptake of acetylcholine. Initial studies presented in this article demonstrate the existence of a specific, saturable vesamicol binding site in PC12 cells. Subsequent experiments show that these binding sites reside in a membrane population that is distinct from catecholamine-containing compartments with respect to density and antigenic composition. In particular, vesamicol binding compartments have a lower density than catecholaminergic vesicles and, unlike these latter vesicles, do not appear to contain the vesicle-specific proteins synaptophysin and SV2 as part of the same membrane. These results suggest that vesicular transport proteins for acetylcholine and catecholamines are differentially sorted to distinct membrane compartments in PC12 cells.

Topics: Animals; Binding Sites; Catecholamines; Intracellular Membranes; PC12 Cells; Piperidines; Precipitin Tests; Proteins; Subcellular Fractions; Tissue Distribution

Synaptic transmission in intact pieces of the Torpedo electric organ treated with vesamicol (2-(4-phenylpiperidino)cyclohexanol, formerly AH5183) was elicited by trains of repetitive electrical stimulation at different frequencies. When the frequency of stimulation was increased from 10 to 50 or 100 Hz, micromolar concentrations of vesamicol enhanced the tetanic rundown of the successive tissue responses. This effect was already detectable with 10 microM vesamicol. It was dramatically potentiated with concentrations of 50 or 100 microM vesamicol, which caused complete failure of transmission after usually less than 10 responses. The drug was unequivocally demonstrated to act by depressing the evoked release of acetylcholine as a consequence of a highly frequency- and concentration-dependent impairment of Na+ channel function in afferent axons. It is concluded that, in the electric organ, vesamicol blocks transmission by acting as a local anaesthetic. This action of micromolar concentrations of vesamicol must be taken into account especially during high-rate nerve activity.

Topics: Acetylcholine; Anesthetics, Local; Animals; Depression, Chemical; Dose-Response Relationship, Drug; Electric Organ; Electric Stimulation; Piperidines; Synapses; Synaptic Transmission; Torpedo

A protein that binds vesamicol has been purified from a soluble fraction of the Torpedo electric organ homogenate that does not contain synaptic vesicles. The purified vesamicol-binding protein (VBP) has a molecular mass of 470 kDa composed of 30- and 24-kDa subunits. Chemical deglycosylation yielded a single, heterogeneous protein of 24 kDa. The 30-kDa subunit is also sensitive to endo-beta-galactosidase. The dissociation constant of the VBP.vesamicol complex is 0.9 microM, and the Bmax is 5,500 pmol/mg. Antiserum raised to the 30-kDa subunit cross-reacts with the 24-kDa subunit, but not with synaptic vesicles. Drug binding studies and Western blot analysis show that VBP is present in other Torpedo tissues as well as mammalian brain. Immunofluorescence microscopy demonstrates that VBP-like immunoreactivity is not localized exclusively to the nerve terminal regions of the electric organ. Thermal stability, the pH dependence of vesamicol binding, and pharmacological comparisons demonstrate that the VBP is not the cholinergic synaptic vesicle receptor for vesamicol. The implications of this finding for current efforts to develop in vivo diagnostics of cholinergic nerve terminal status based on vesamicol are discussed.

Topics: Animals; Binding, Competitive; Blotting, Western; Brain; Cattle; Chromatography, Affinity; Chromatography, Gel; Chromatography, High Pressure Liquid; Chromatography, Ion Exchange; Cross Reactions; Electric Organ; Electrophoresis, Polyacrylamide Gel; Immune Sera; Macromolecular Substances; Molecular Weight; Neuromuscular Depolarizing Agents; Piperidines; Rats; Receptors, Neurotransmitter; Receptors, Phencyclidine; Torpedo

The effect of the vesicular acetylcholine (ACh) transport blocker trans-2-(4- phenyl-piperidino)-cyclohexanol (AH5183) was studied in bronchial smooth muscle during activation of the vagus nerve. AH5183 inhibited in a dose-dependent manner the Ca(2+)-sensitive electrically induced smooth muscle contractions in vitro with a half-inhibitory concentration (IC50) of 1.6 +/- 0.4 microM. The inhibition was complete within 68 +/- 1 min (n = 8) at approximately 20 microM AH5183 and was partly reversible after washing of the preparations. AH5183 (20 microM) reduced the level of endogenous ACh by 47.4 +/- 7.6% (n = 4) during this time period. The effect of AH5183 is most likely prejunctional, since the contractions induced post-junctionally by carbachol were not altered by AH5183. The irreversible anticholinesterase, soman, increased the tonus of airway smooth muscle as a result of accumulation of spontaneously released ACh from prejunctional leakage. AH5183 had no effect on this increase of muscle contraction. The present results show that the nerve-evoked release of ACh comes from an AH5183-sensitive pool, probably a vesicular pool, whereas leakage of ACh presumably comes from the cytoplasmic pool in airway smooth muscle.

Topics: Acetylcholine; Animals; Bronchi; Butyrylcholinesterase; Cholinergic Fibers; Electric Stimulation; Male; Muscle Contraction; Muscle, Smooth; Neuromuscular Depolarizing Agents; Piperidines; Rats; Rats, Inbred Strains; Synaptic Transmission

Experiments examined the effects of peripheral and central administration of the vesicular acetylcholine transport blocker vesamicol (AH5183) on the content, synthesis, and release of acetylcholine in the rat brain in vivo. In time course studies, a single intraperitoneal dose of DL-vesamicol (5 mg/kg) rapidly and reversibly (within 2 h) doubled the content of acetylcholine in the striatum and hippocampus, without affecting choline levels or the rate of transmitter synthesis. In microdialysis experiments, the same peripheral dose of drug produced a reversible 55% reduction in endogenous striatal acetylcholine release. A similar inhibitory effect was produced by direct intrastriatal perfusion with vesamicol. Moreover, this effect of vesamicol was (a) concentration-dependent and saturable (EC50 = 68 nM), (b) rapidly reversible, (c) stereospecific for the L-isomer, and (d) poorly mimicked by a vesamicol analog with lower plasma membrane permeability. This profile of effects is consistent with an interaction with a specific vesamicol receptor as defined by previous in vitro binding studies. These results support a functional role for vesamicol receptors in modulating central cholinergic transmission in vivo.

Topics: Acetylcholine; Animals; Brain; Corpus Striatum; Dialysis; Dose-Response Relationship, Drug; Male; Neuromuscular Depolarizing Agents; Piperidines; Rats; Receptors, Neurotransmitter; Receptors, Phencyclidine

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.

Topics: Acetylcholine; Animals; In Vitro Techniques; Motor Endplate; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Piperidines; Pyrrolidines; Rats; Rats, Inbred Strains; Synaptic Transmission; Synaptic Vesicles

The side-effects of vesamicol, an inhibitor of acetylcholine storage, on alpha 1- and alpha 2-adrenoceptors have been studied in the isolated rat vas deferens. Antagonism of alpha 1-adrenoceptors was determined from the ability of vesamicol to reduce contractions elicited by exogenous noradrenaline. Antagonism of alpha 2-adrenoceptors was determined from the ability of vesamicol to block the inhibitory effects of the alpha 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 uptake1. In the presence of noradrenaline uptake blockers, (-)-vesamicol produced a competitive, non-selective block of both alpha 1- and alpha 2-adrenoceptors with a Kd of around 40 microM (pA2 4.4). (+)-Vesamicol, the isomer that has no activity at cholinergic synapses, was equipotent with the (-)-isomer for blocking alpha 2-adrenoceptors. In addition to its alpha-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 alpha-adrenoceptor blocking activity of vesamicol is a function of the phenylpiperidino moiety of the molecule.

Topics: Adrenergic alpha-Antagonists; Animals; Clonidine; Corticosterone; Desipramine; Dioxanes; Electric Stimulation; Idazoxan; Male; Muscle Contraction; Muscle, Smooth; Norepinephrine; Piperidines; Rats; Rats, Inbred Strains; Receptors, Adrenergic, alpha; Stereoisomerism; Vas Deferens

1. In frog a variety of treatments have been shown to increase quantal size at the neuromuscular junction (NMJ), apparently by releasing more acetylcholine (ACh) per quantum. The present experiments were undertaken to see whether similar changes occur at the mouse NMJ. 2. None of the hormones tested, adrenaline, insulin or corticosteroids, significantly increased quantal size at the mouse NMJ. 3. Soaking diaphragms in hypertonic solution (about 475 mosmol/kg) for 15-30 min roughly doubled the size of miniature endplate potentials (MEPPs), miniature endplate currents (MEPCs), and uniquantal endplate potentials (EPPs). We will refer to these as 'large quanta'. Note that all of the measurements were made after returning the preparation to normal (Tyrode) solution. 4. In frog hypertonic solution made with sodium gluconate replacing NaCl increases quantal size four- rather than two-fold. In mouse there was little difference in the effects of solutions made with the two anions. In Cl(-)-free hypertonic solution, made with sodium gluconate and SO4(2-) solutions, quantal size increase is somewhat less, so there may be a role for Cl- in enlarging quantal size. 5. After hypertonic treatment, quantal size remained elevated for at least 1 h and then gradually declined back to usual levels. The data suggest a gradual decrease in mean quantal size, rather than the appearance of a new subpopulation of smaller quanta. 6. Hypertonic treatment did not change the endplate depolarization in response to ionophoretically applied ACh. This suggests that the increased quantal size is not due to a postsynaptic change. Large MEPP's disappear in the presence of tubocurarine and reappear when the drug is washed away. 7. Vesamicol is an inhibitor of active ACh uptake into isolated synaptic vesicles. 5 microM-vesamicol has no detectable postjunctional effect. However, when vesamicol was included in the Tyrode and the hypertonic solutions the increase in quantal size was blocked. This is further evidence that the large quanta are produced by the release of more ACh per quantum. 8. Even when added after the hypertonic treatment, vesamicol soon decreased quantal size back to the normal level. Two other inhibitors of active ACh uptake into vesicles, tetraphenylboron (TPB) and hexanitrodiphenylamine (HNPA) also decreased quantal size after hypertonic treatment, apparently by a presynaptic action. This suggests that the additional ACh that produces large miniatures may be incorporated into

Topics: Acetylcholine; Animals; Anura; Choline; Curare; Electric Stimulation; Female; Hormones; Hypertonic Solutions; In Vitro Techniques; Kinetics; Male; Mice; Models, Neurological; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Piperidines; Sodium; Species Specificity; Synapses

The effect of lead ions on the release of acetylcholine (ACh) was investigated in intact and digitonin-permeabilized rat cerebrocortical synaptosomes that had been prelabeled with [3H]choline. Release of ACh was inferred from the release of total 3H label or by determination of [3H]ACh. Application of 1 microM Pb2+ to intact synaptosomes in Ca2(+)-deficient medium induced 3H release, which was enhanced by K+ depolarization. This suggests that entry of Pb2+ into synaptosomes and Pb2(+)-induced ACh release can be augmented by activation of the voltage-gated Ca2+ channels in nerve terminals. The lead-induced release of [3H]ACh was blocked by treatment of synaptosomes with vesamicol, which prevents uptake of ACh into synaptic vesicles without affecting its synthesis in the synaptoplasm. This indicates that Pb2+ selectively activates the release of a vesicular fraction of the transmitter with little or no effect on the leakage of cytoplasmic ACh. Application of 1-50 nM (EC50 congruent to 4 nM) free Pb2+ to digitonin-permeabilized synaptosomes elicited release of 3H label that was comparable with the release induced by 0.2-5 microM (EC50 congruent to 0.5 microM) free Ca2+. This suggests that Pb2+ triggers transmitter exocytosis directly and that it is a some 100 times more effective activator of exocytosis than is the natural agonist Ca2+.

Topics: Acetylcholine; Animals; Calcium; Calcium Channels; Cell Membrane Permeability; Cerebral Cortex; Digitonin; Exocytosis; Lead; Male; Piperidines; Potassium; Rats; Rats, Inbred Strains; Synaptosomes

Several 1,3-disubstituted propan-2-ols and one alpha,beta-disubstituted ethanol (11i) were synthesized and evaluated as potential acyclic mimics of the vesicular acetylcholine transport inhibitor 2-(4-phenylpiperidinyl)cyclohexanol (1, vesamicol, AH5183). Analogues containing the 4-phenylpiperidyl fragment (11a, 11b) were more potent than those containing the 4-phenylpiperazyl moiety (11e, 11f). Substitution at the second terminal carbon of the propyl (or ethyl) fragment with simple lipophilic aryl substituents yielded potent inhibitors of vesicular acetylcholine storage, including (-)-11a and d-11i, which are equipotent with vesamicol. However, the activity of analogues containing bicyclic aryl groups was susceptible to aryl substitution patterns (11g vs 11h), indicating a definite receptor site topography. In addition, the inhibitory activity of these acyclic analogues was enantioselective, exhibiting a preference, similar to the parent vesamicol, for the levorotatory isomer [(-)-11a vs (+)-11a]. Therefore, the simple lipophilic acyclic vicinal amino alcohols may successfully mimic the biological activity of vesamicol.

Topics: Acetylcholine; Biological Transport; Chemical Phenomena; Chemistry; Molecular Conformation; Molecular Structure; Parasympatholytics; Piperidines; Structure-Activity Relationship

Vesamicol (AH5183) inhibits the uptake of acetylcholine into cholinergic neuronal storage vesicles. Earlier in vitro studies have demonstrated that such inhibition can lead to a failure of transmission, particularly in peripheral cholinergic tissues. The present study was designed to determine whether vesamicol could inhibit central cholinergic transmission in conscious freely moving rats. Central (lateral cerebroventricular) injection of 20 micrograms of vesamicol significantly reduced the hypertensive and bradycardic response to subsequent central injection of physostigmine in spontaneously hypertensive rats. Inhibition of the pressor response was greatest when physostigmine was administered 1 hr after vesamicol. Acetylcholine and choline levels were determined in three brain regions derived from rats treated one hr earlier with either vehicle or vesamicol. Acetylcholine levels were found to be unaltered after vesamicol treatment, although choline levels were significantly higher in two brain regions. These results are consistent with the ability of vesamicol to inhibit the function of central cholinergic cardiovascular regulatory neurons. The mechanism for this inhibition is not related to depletion of total brain acetylcholine content but may be due to depletion of a small critical pool of transmitter.

Topics: Acetylcholine; Animals; Biological Transport; Blood Pressure; Brain; Heart Rate; Male; Neurons; Parasympathetic Nervous System; Physostigmine; Piperidines; Rats; Rats, Inbred SHR

I investigated the efficacy of 10 drugs with respect to reducing the lethality in mice of the following venoms and their respective neurotoxins: Bungarus caeruleus venom; Bungarus multicinctus venom, alpha-bungarotoxin, beta-bungarotoxin; Crotalus durissus terrificus venom, crotoxin: Notechis scutatus scutatus venom; Oxyuranus scutellatus venom, taipoxin. The drugs diltiazem, nicergoline, primaquine, verapamil and vesamicol protected mice from the lethality of B. caeruleus venom, B. multicinctus venom, and/or beta-bungarotoxin. Dexamethasone provided protection from B. multicinctus venom, beta-bungarotoxin, crotoxin, O. scutellatus venom and taipoxin. Protective activity resided in amphiphilic drugs and correlated with the charge on the drug at physiological pH. Protection from lethality was maximal when the drugs were administered immediately after injection of the venom or toxin. Nifedipine, piracetam and reserpine provided no protection from any of the venoms or toxins tested.

Topics: Animals; Dexamethasone; Diltiazem; Female; Lethal Dose 50; Mice; Mice, Inbred ICR; Neuromuscular Depolarizing Agents; Neurotoxins; Nicergoline; Nifedipine; Piperidines; Piracetam; Primaquine; Reserpine; Snake Venoms; Snakes; Verapamil

Dually perfused human placental lobules were employed to study the effects of vesamicol (an inhibitor of acetylcholine (ACh) storage) and physostigmine (an inhibitor of cholinesterase breakdown of ACh) on ACh output from fetal vessels and on materno-fetal transfer of the nonmetabolizable amino acid alpha-aminoisobutyric acid (AIB). Bilateral perfusion with vesamicol (100 mumol L-1) or omission of physostigmine (2 x 7 mumol L-1) from the perfusate significantly reduced the output of ACh. However, neither drug had any significant effect on AIB transfer. Thus, the results of this study do not provide evidence for a close relationship between the placental cholinergic system and materno-fetal AIB transfer.

Topics: Acetylcholine; Aminoisobutyric Acids; Biological Transport; Female; Humans; Maternal-Fetal Exchange; Perfusion; Physostigmine; Piperidines; Placenta; Pregnancy

Human placental explants were incubated in the presence of physostigmine (3.08 microM), and release of acetylcholine (ACh) and prostaglandin (PG) were measured in the fourth hour by bioassay and radioimmunoassay, respectively. The choline acetyltransferase inhibitor (2-benzoylethyl)trimethylammonium (100 microM, n = 6) significantly reduced ACh release by 36 +/- 6%, PGE2 release by 23 +/- 8% and PGF2 alpha release by 29 +/- 10%. The inhibitor of vesicular acetylcholine storage, vesamicol (100 microM, n = 7), significantly reduced ACh release by 22 +/- 4%, PGE2 release by 46 +/- 13% and PGF2 alpha release by 32 +/- 9%. In the absence of physostigmine, ACh release was reduced by 89 +/- 2%, whereas PG release did not change compared with that in the presence of physostigmine. The presence of atropine (14.4 microM) did not affect PG release. These results suggest that if there is a relationship between human placental production of ACh and PGs, it does not appear to depend on muscarinic receptor activation or the activity of placental cholinesterase.

Topics: Acetylcholine; Dinoprost; Dinoprostone; Female; Humans; Organ Culture Techniques; Physostigmine; Piperidines; Placenta; Pregnancy

The effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol), a compound known to block the uptake of acetylcholine (ACh) into cholinergic synaptic vesicles, on the release of endogenous and [14C]ACh from slices of rat striatum was investigated. ACh release was evoked either by electrical stimulation or by veratridine. The effect of electrical stimulation was entirely dependent on external Ca2+. By contrast, veratridine (40 microM) also enhanced ACh release in the absence of Ca2+. Indeed, with veratridine two components were clearly distinguished: one dependent on external Ca2+ and the other not. Vesamicol inhibited [14C]ACh release evoked by both veratridine and electrical stimulation in the presence of external Ca2+, provided it was added to the tissue prior to loading with [14C]choline. With the same treatment vesamicol only slightly affected the release of endogenous ACh. Under the same conditions the Ca2(+)-independent [14C]ACh release evoked by veratridine was not prevented by vesamicol. The differential responsiveness to vesamicol suggests that ACh pools involved in Ca2+o-dependent ACh release are different from those mobilized during Ca2+o-independent ACh release.

Topics: Acetylcholine; Animals; Calcium; Carbon Radioisotopes; Corpus Striatum; Electric Stimulation; In Vitro Techniques; Male; Perfusion; Piperidines; Rats; Rats, Inbred Strains; Veratridine

Although the exocytotic mechanism for quantal acetylcholine (ACh) release has been widely accepted for many years, it has repeatedly been challenged by reports that ACh released upon stimulation originates from the cytosol rather than synaptic vesicles. In this report, two independent experimental approaches were taken to establish the source of ACh released from the electromotor system of Narcine brasiliensis. Since ATP is colocalized with ACh in the cholinergic vesicle, the exocytotic theory predicts the corelease of these two components with a stoichiometry identical to that of the vesicle contents. The stimulated release of ATP from isolated synaptosomes could be accurately quantitated in the presence of the ATPase inhibitor adenosine 5'-[alpha, beta-methylene]triphosphate (500 microM), which prevented degradation of the released ATP. Various concentrations of elevated extracellular potassium (25-75 mM), veratridine (100 microM), and the calcium ionophore ionomycin (5 microM) all induced the corelease of ACh and ATP in a constant molar ratio of 5-6:1 (ACh/ATP), a stoichiometry consistent with that established for the vesicle content. In parallel to these stoichiometry studies, the compound 2-(4-phenylpiperidino)cyclohexanol (AH5183) was used to inhibit specifically the vesicular accumulation of newly synthesized (radiolabeled) ACh without affecting cytosolic levels of newly synthesized ACh in cholinergic nerve terminals. Treatment with AH5183 (10 microM) was shown to inhibit the release of newly synthesized ACh without markedly affecting total ACh release; thus, the entry of newly synthesized ACh into the synaptic vesicle is essential for its release. We conclude that ACh released upon stimulation originates exclusively from the vesicular pool and is coreleased stoichiometrically with other soluble vesicle contents.

Topics: Acetylcholine; Adenosine Triphosphatases; Adenosine Triphosphate; Animals; Calcimycin; Electric Organ; Exocytosis; Ionomycin; Kinetics; Motor Neurons; Nerve Endings; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Potassium; Subcellular Fractions; Synaptosomes; Torpedo; Veratridine

The effect of vesicular acetylcholine (ACh) transport blocker 2-(4-phenylpiperidino)cyclohexanol (AH-5183) on the subcellular storage and release of ACh was studied in rat brain cortical slices. AH-5183 reduced the release of ACh from cortical slices stimulated by tityustoxin and ouabain. Tissue stimulated in the presence of AH-5183 contained more ACh in both the nerve terminal synaptic vesicles and cytoplasmic fraction than did tissue stimulated in drug's absence. Thus, AH-5183 blocked the tityustoxin and ouabain induced release of ACh from both cytoplasmic and vesicular pools. AH-5183 also depressed the spontaneous release of ACh from incubated slices and, in this condition, the drug had no effect in the subcellular distribution of ACh. It is suggested that AH-5183 interferes with the process of ACh release independent of its blocking action on ACh transport into the synaptic vesicles.

Topics: Acetylcholine; Animals; Cerebral Cortex; In Vitro Techniques; Neurotransmitter Uptake Inhibitors; Ouabain; Phencyclidine; Piperidines; Rats; Rats, Inbred Strains; Scorpion Venoms

The actions of the active L-isomer of vesamicol, an inhibitor of the vesicular storage of acetylcholine, has been studied on spontaneous and evoked acetylcholine release at the snake neuromuscular junction. Miniature endplate currents and endplate currents were recorded from cut muscle fibres of the garter snake, Thamnophis sirtalis. In controls, prolonged periods of high frequency nerve stimulation produced a bimodal distribution of miniature endplate current amplitudes. The stimulation induced "small-mode" miniature endplate currents had a mean amplitude of around 40-55% of the pre-stimulation miniature endplate current. Relative to the normal-sized post-stimulation miniature endplate current, the proportion and, to a lesser extent, amplitude of the small-mode miniature endplate currents was related to both the frequency and duration of nerve stimulation and to the extracellular calcium ion concentration. In unstimulated preparations, L-vesamicol (2-5 microM) did not affect either endplate current quantal content or miniature endplate current amplitude or frequency. However, at these doses, the mean amplitude of the stimulation-induced, small-mode miniature endplate current was reduced by L-vesamicol in a concentration-dependent manner such that they were not visible at the highest dose. L-Vesamicol had no affect on the mean or coefficient of variance of amplitude of the larger, normal-sized miniature endplate current. Additionally, the stimulation-induced increase in overall miniature endplate current frequency seen in controls was abolished by 5 microM L-vesamicol. After prolonged 10 Hz nerve stimulation endplate current amplitude was markedly reduced in both controls (by 94%) and in the presence of 5 microM L-vesamicol (by 98%). Analysis of endplate current amplitude variance showed that in control the decrease was due to reductions in both quantal content and quantal size while in L-vesamicol the decrease was due entirely to a change in quantal content with no change in quantal size. Thus, we have observed that L-vesamicol selectively reduces the amplitude of a population of stimulation-induced small-mode quanta both as miniature endplate currents and as constituents of endplate currents. We suggest that these quanta are derived from a highly active, readily releasable pool. An action of L-vesamicol on this labile pool is consistent with previous observations on its ability to inhibit the vesicular storage of acetylcholine.

Topics: Acetylcholine; Action Potentials; Animals; Electric Stimulation; Neuromuscular Junction; Neurotransmitter Uptake Inhibitors; Phencyclidine; Piperidines; Snakes

We measured the binding of the vesicular acetylcholine transport blocker [3H]vesamicol (2-[4-phenylpiperidino] cyclohexanol; AH-5183) to autopsied frontal cortex and amygdala of patients from 4 disorders having a marked brain cholinergic reduction, namely Alzheimer's disease, Parkinson's disease with dementia, dominantly inherited olivopontocerebellar atrophy and Down's syndrome. Although mean activity of the specific cholinergic marker enzyme choline acetyltransferase (ChAT) was markedly reduced by about 60% in frontal cortex in the 4 patient groups and by 80% or greater in amygdala of the Alzheimer's and Down's syndrome patients, [3H]vesamicol binding density was, on average, either normal or only slightly reduced as compared with the controls. This discrepancy suggests that in human brain [3H]vesamicol binding is either not preferentially localized to cholinergic nerve endings or, in these cholinergic deficiency syndromes, a substantial proportion of the vesamicol binding sites persist on cholinergic nerve terminals despite loss of ChAT activity.

Topics: Acetylcholine; Adult; Aged; Alzheimer Disease; Amygdala; Autonomic Nervous System Diseases; Brain Chemistry; Cerebral Cortex; Choline O-Acetyltransferase; Down Syndrome; Humans; Middle Aged; Neuromuscular Depolarizing Agents; Olivopontocerebellar Atrophies; Parasympathetic Nervous System; Parkinson Disease; Piperidines

[3H]Vesamicol binding was characterized in human brain post mortem. The number of binding sites was then determined in parallel with choline acetyltransferase activity in the temporal cortex of patients with Alzheimer's disease, demented and non-demented patients with Parkinson's disease, and in the cerebral cortex of rats with quisqualic acid lesions of the nucleus basalis magnocellularis. Whereas choline acetyltransferase activity decreased in patients with Alzheimer's or Parkinson's disease indicating loss of cholinergic innervation, the number of binding sites for [3H]vesamicol was the same as or higher than in controls. Similar results were obtained with the lesioned rats. It is suggested that the increase in binding sites may reflect compensatory regulation of the spared neurons at the level of the synaptic vesicle.

Topics: Aged; Aged, 80 and over; Alzheimer Disease; Animals; Brain Injuries; Choline O-Acetyltransferase; Female; Humans; Hydroxydopamines; Kinetics; Male; Neuromuscular Depolarizing Agents; Oxadiazoles; Oxidopamine; Parkinson Disease; Phencyclidine; Piperidines; Quisqualic Acid; Rats; Rats, Inbred Strains; Receptors, Neurotransmitter; Receptors, Phencyclidine; Reference Values; Substantia Nigra; Temporal Lobe

A fundamental study was performed on the nuclear medicine imaging of cholinergic innervation in the brain. In a cholinergic denervation model prepared by producing an unilateral basal forebrain lesion in the rat, which is reported to be one of animal models of Alzheimer's disease, quantitative determination of acetylcholine in parietal cortices revealed statistically significant 31% decrease on an average in the ipsilateral side relative to the contralateral side to the lesion. In vitro receptor autoradiography showed no significant differences in total, M1 and M2 muscarinic acetylcholine receptors between the ipsilateral and contralateral cortices to the lesion. Simultaneous mapping of presynaptic cholinergic innervation using 3H-2-(4-phenylpiperidino) cyclohexanol (AH5183) demonstrated significant 14% decrease of AH5183 binding on an average in the ipsilateral relative to the contralateral fronto-parieto-temporal cortices to the lesion. These results suggest that AH5183 is a promising ligand for mapping cholinergic innervation in nuclear medicine imaging.

Topics: Acetylcholine; Alzheimer Disease; Animals; Autoradiography; Brain; Disease Models, Animal; Piperidines; Rats; Receptors, Cholinergic

Intracerebral microdialysis was combined with a sensitive and specific gas chromatographic-mass spectrometric assay to measure the release of endogenous acetylcholine in the rat striatum in vivo. In rats anesthetized with urethane (1.2 g/kg i.p.), the levels of striatal acetylcholine dialyzed into a Ringer's perfusate were: (a) reliably measurable only in the presence of physostigmine; (b) stable at between 3 and 8 h of perfusion (30-75 pmol/20 min in the presence of 75 microM physostigmine); (c) reduced by calcium-free Ringer's solution, tetrodotoxin (0.1 microM), and vesamicol (1.0 microM); and (d) increased by elevated potassium (100 mM), atropine (3-300 microM), and haloperidol (0.75 mg/kg i.p.). In conscious unrestrained rats, the spontaneous release of striatal acetylcholine was not altered significantly following the administration of urethane. The changes in acetylcholine release observed in this study are consistent with the known actions of some drugs or ionic conditions on striatal cholinergic neurotransmission and are evident under the condition of urethane anesthesia. The present results demonstrate the sensitivity and suitability of this method for monitoring endogenous striatal acetylcholine release in vivo.

Topics: Acetylcholine; Animals; Atropine; Chromatography, Gas; Consciousness; Corpus Striatum; Dialysis; Haloperidol; Magnesium; Male; Mass Spectrometry; Phencyclidine; Physostigmine; Piperidines; Potassium; Rats; Rats, Inbred Strains; Tetrodotoxin

Topics: Acetylcholine; Animals; Brain; Cerebellum; Cerebral Cortex; Choline; Corpus Striatum; Female; Iodine Radioisotopes; Kinetics; Mice; Molecular Structure; Neurons; Phencyclidine; Piperidines; Stereoisomerism; Tetrahydronaphthalenes; Tritium

We examined the effects of two drugs, AH5183 and cetiedil, demonstrated to be potent inhibitors of acetylcholine (ACh) transport by isolated synaptic vesicles on cholinergic functions in Torpedo synaptosomes. AH5183 exhibited a high specificity toward vesicular ACh transport, whereas cetiedil was shown to inhibit both high-affinity choline uptake and vesicular ACh transport. Choline acetyltransferase was not affected by either drug. High external choline concentrations permitted us to overcome cetiedil inhibition of high-affinity choline transport, and thus synthesis of [14C]ACh in treated preparations was similar to that in controls. We then tested evoked ACh release in drug-treated synaptosomes under conditions where ACh translocation into the vesicles was blocked. We observed that ACh release was impaired only in cetiedil-treated preparations; synaptosomes treated with AH5183 behaved like the controls. Thus, this comparative study on isolated nerve endings allowed us to dissociate two steps in drug action: upstream, where both AH5183 and cetiedil are efficient blockers of the vesicular ACh translocation, and downstream, where only cetiedil is able to block the ACh release process.

Topics: Acetylcholine; Animals; Azepines; Biological Transport; Calcimycin; Choline; Electric Organ; Hydrogen-Ion Concentration; Kinetics; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Synaptic Vesicles; Synaptosomes; Torpedo

Synaptic vesicles purified on a sucrose-KCl sedimentation gradient were tested for their ability to accumulate [1-14C]acetylcholine ([1-14C]ACh) in the absence and in the presence of AH5183 and cetiedil. Kinetic studies of ACh transport showed that it was time dependent and saturable as a function of ACh concentration, with a KT of 1.2 mM. The protein-modifying agents N-ethylmaleimide and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole were powerful inhibitors of ACh uptake. In agreement with other studies, AH5183 was found to be a potent inhibitor of ACh uptake by synaptic vesicles. Inhibition was of the mixed noncompetitive type, and the inhibition constant was 45.2 +/- 3.4 nM. Cetiedil, a drug that resembles ACh, was previously shown on intact nerve endings to inhibit the translocation of newly synthesized ACh into the synaptic vesicle compartment, and we demonstrate here that cetiedil is indeed an efficient blocker of ACh uptake by isolated synaptic vesicles. It acted as a competitive inhibitor, with a Ki of 118.5 +/- 9.5 nM. Neither ATP-dependent calcium uptake nor Mg2+-ATPase activity was affected by the drugs, a finding showing their specificity toward the ACh uptake process. The binding of L-[3H]AH5183 to intact vesicles was characterized in the absence or the presence of ACh or cetiedil. Saturation experiments showed a total binding capacity of approximately 126 pmol/mg of vesicular protein and a dissociation constant of 19.9 +/- 4.1 nM under control conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Adenosine Triphosphate; Animals; Azepines; Binding, Competitive; Biological Transport; Ca(2+) Mg(2+)-ATPase; Calcium; Electric Organ; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Synaptic Vesicles; Torpedo

Cholinergic synaptic vesicles obtained from Torpedo electric organ have an active transport system for acetylcholine (ACh). Linked to ACh transport is a cytoplasmically oriented receptor for the inhibitory drug (-)-trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183). Storage of freshly isolated vesicles for several days leads to more vesamicol binding. This can be induced immediately by hyposmotic lysis of the vesicles, which reseal to form right-side-out ghosts. The increased drug binding was due to a twofold increase in the affinity and a 20% increase in the amount of the receptor expressed, probably as a result of the release of an endogenous factor. Binding of vesamicol to ghosts was specifically inhibited by exogenous ACh acting with a dissociation constant of 18 mM. This suggests that the vesamicol binding site probably is linked to a low-affinity ACh binding site that is different from the higher affinity transport binding site. Equilibrium and kinetic attempts to determine whether exogenous ACh acts on the outside or the inside of the ghost membrane to inhibit vesamicol binding failed because of rapid equilibration of exogenous ACh across the ghost membrane. It is argued that the endogenous factor released by hyposmotic lysis might be ACh. Potential roles for such a transmembrane signal regulating the vesamicol receptor are discussed.

Topics: Acetylcholine; Animals; Binding, Competitive; Biological Transport, Active; Electric Organ; Kinetics; Neuromuscular Depolarizing Agents; Osmolar Concentration; Phencyclidine; Piperidines; Receptors, Neurotransmitter; Receptors, Phencyclidine; Synaptic Vesicles; Torpedo

Vesamicol [(-)-(trans)-2-(4-phenylpiperidino)cyclohexanol] receptor binding and inhibition of acetylcholine (AcCh) active transport by cholinergic synaptic vesicles that were isolated from Torpedo electric organ were studied for 23 vesamicol enantiomers, analogues, and other drugs. Use of trace [3H]vesamicol and [14C]AcCh allowed simultaneous determination of the concentrations of enantiomer, analogue, or drug required to half-saturate the vesamicol receptor (Ki) and to half-inhibit transport (IC50), respectively. Throughout a wide range of potencies for different compounds, the Ki/IC50 ratios varied from 1.5 to 24. Compounds representative of the diverse structures studied, namely deoxyvesamicol, chloroquine, and levorphanol, were competitive inhibitors of vesamicol binding. It is concluded that many drugs can bind to the vesamicol receptor and binding to only a small fraction of the receptors can result in AcCh active transport inhibition. Possible mechanisms for this effect are discussed.

Topics: Acetylcholine; Animals; Binding, Competitive; Biological Transport, Active; In Vitro Techniques; Phencyclidine; Piperidines; Receptors, Neurotransmitter; Receptors, Phencyclidine; Structure-Activity Relationship; Synaptic Vesicles; Torpedo

Topics: Animals; Electric Stimulation; In Vitro Techniques; Motor Neurons; Nerve Endings; Neuromuscular Depolarizing Agents; Neurotransmitter Agents; Phencyclidine; Piperidines; Rats

1. The effects of optical isomers of vesamicol (2-(4-phenylpiperidino) cyclohexanol), an inhibitor of acetylcholine (ACh) storage, on stimulation-evoked release of [3H]-acetylcholine [( 3H]-ACh) from the neuromuscular junction have been studied in the region of the mouse hemidiaphragm which contains the motor endplates, and which can easily be loaded with [3H]-choline. This method made it possible to detect exclusively the Cao-dependent release of [3H]-ACh in response to stimulation, and therefore to test the vesicular hypothesis. 2. (-)-Vesamicol was approximately 20 times more potent than (+)-vesamicol in reducing stimulation-evoked release of [3H]-ACh. 3. 4-Aminopyridine, a potassium channel blocker, enhanced the release of ACh in response to stimulation, but failed to increase release from hemidiaphragm which had been pretreated with (-)-vesamicol. 4. The fact that (-)-vesamicol inhibited the release of [3H]-ACh in response to electrical stimulation only when it was administered prior to the loading of the tissue with [3H]-choline, and had no effect when the stores had already been filled with labelled [3H]-ACh indicates that the stimulation-evoked release of [3H]-ACh is of vesicular origin and (-)-vesamicol has no effect on the release process. This is the first neurochemical evidence for the vesicular origin of stimulation-evoked release of ACh from the neuromuscular junction.

Topics: Acetylcholine; Animals; Calcium; Choline; Diaphragm; Electric Stimulation; In Vitro Techniques; Male; Mice; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Phencyclidine; Phrenic Nerve; Piperidines

The present experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183 or vesamicol) and/or picrylsulfonic acid (TNBS), two compounds known to have the ability to block the uptake of ACh by cholinergic synaptic vesicles in vitro. We confirmed that, in stimulated (5 Hz) perfused (30 min) ganglia, AH5183 depressed ACh release and ACh tissue content increased by 86 +/- 6% compared to contralateral ganglia used as controls. Preganglionic activity increased ACh release by a similar amount in the presence (19.9 +/- 1.0 pmol/min) or absence (20.5 +/- 2.4 pmol/min) of TNBS. The final tissue ACh content was also similar in the presence (1,668 +/- 166 pmol) or absence (1,680 +/- 56 pmol) of TNBS. However, the AH5183-induced increase of tissue ACh content (86 +/- 6%) was abolished completely when AH5183 was perfused with 1.5 mM TNBS (-3.0 +/- 1.0%). This inhibition of ACh synthesis, observed in TNBS-AH5183-perfused ganglia, was not dependent upon further inhibition of ACh release beyond that caused by AH5183 alone, because 14.0 +/- 1.9% of the transmitter store was released by preganglionic nerve stimulation in the presence of TNBS plus AH5183 and this was similar in the presence of AH5183 without TNBS (14.0 +/- 0.6%). Moreover, when ganglia were first treated with TNBS and then stimulated in the presence of AH5183, an increase of 64 +/- 6% of the ganglionic ACh content occurred, and this increase was not statistically different from the increase measured with AH5183 alone (86 +/- 6%).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Cats; Choline; Drug Combinations; Electric Stimulation; Female; Ganglia, Sympathetic; Male; Neuromuscular Depolarizing Agents; Nitrobenzenes; Osmolar Concentration; Phencyclidine; Piperidines; Trinitrobenzenesulfonic Acid

Eighty-four analogues and derivatives of the acetylcholine-storage-blocking drug trans-2-(4-phenylpiperidino)-cyclohexanol (vesamicol) were synthesized, and their potencies were evaluated with the acetylcholine active-transport assay utilizing purified synaptic vesicles from Torpedo electric organ. The parent drug exhibits enantioselectivity, with (-)-vesamicol being 25-fold more potent than (+)-vesamicol. The atomic structure and absolute configuration of (+)-vesamicol were determined by X-ray crystallography. The absolute configuration of (-)-vesamicol is 1R,2R. Structure-activity evidence indicates that (-)-vesamicol does not act as an acetylcholine analogue. Alterations to all three rings can have large effects on potency. Unexpectedly, analogues locking the alcohol and ammonium groups trans-diequatorial or trans-diaxial both exhibit good potency. A potent benzovesamicol family has been discovered that is suitable for facile elaboration of the sort useful in affinity labeling and affinity chromatography applications. A good correlation was found between potencies as assessed by the acetylcholine transport assay and LD50 values in mouse.

Topics: Acetylcholine; Animals; Biological Transport; Chemical Phenomena; Chemistry; Electric Organ; Lethal Dose 50; Mice; Molecular Structure; Narcotics; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Stereoisomerism; Structure-Activity Relationship; Synaptic Vesicles; Torpedo; X-Ray Diffraction

The ability of nicotine to induce antinociception after subcortical administration was investigated in the rat. Adult male Sprague-Dawley rats were implanted unilaterally with guide cannulas aimed at the pedunculopontine tegmental nucleus of the mesopontine tegmentum. After 1 week, nicotine was injected in 0.5 microliter of 0.2 M pH 7.4 phosphate buffer. Antinociception was assessed using the 52 degrees C hot-plate test and the tail-flick method; for the most part, the results in the hot-plate test parallelled those in the tail-flick test. Nicotine inhibited nociceptive responses at a median effective antinociceptive dose (A5O) of 1.6 nmol in the hot-plate test and 3.4 nmol in the tail-flick test. Mecamylamine, 0.8 nmol coadministered with nicotine, antagonized nicotine antinociception as evidenced by 5- to 8-fold increases in the nicotine A5O. Nicotine antinociception was also antagonized by coadministrations of either 0.8 nmol of (-)-scopolamine or 0.4 nmol of the M1 antagonist pirenzepine by over 12-fold in the hot-plate test and 5-fold in the tail-flick test. The M2 antagonist methoctramine had antinociceptive effects of its own when injected into the mesopontine tegmentum at a dose of 0.1 nmol; when coinjected with nicotine, the effects of the methoctramine-nicotine combination appeared to be additive. One hour preinjection into the mesopontine tegmentum with 13.5 nmol of (-)-vesamicol, an agent which interferes with acetylcholine storage and/or release, markedly inhibited nicotine antinociception; only a 24% antinociceptive response could be elicited by nicotine in the hot-plate test whereas the nicotine A5O was increased 3-fold in the tail-flick test. Pretreatment with the inactive isomer (+)-vesamicol had no effect. In other experiments, mesopontine tegmental injection of (+)-cis-dioxolane, a high affinity muscarinic cholinergic agonist, elicited strong antinociceptive responses which were potently antagonized by coadministration with 0.5 nmol of pirenzepine but not by 0.8 nmol of mecamylamine. The data indicate that nicotine-induced antinociception may depend upon intact neurotransmission at M1 sites in addition to nicotinic sites within the mesopontine tegmentum.

Topics: Analgesia; Animals; Male; Microinjections; Nicotine; Phencyclidine; Piperidines; Pirenzepine; Pons; Rats; Rats, Inbred Strains; Receptors, Muscarinic; Scopolamine; Tegmentum Mesencephali

Cholinergic synaptic vesicles isolated from Torpedo electric organ contain a receptor for the compound l-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183), which when occupied blocks storage of acetylcholine (AcCh). The inside or outside orientation of the receptor and its chemical and ligand binding kinetics characteristics were studied. Binding of [3H]vesamicol to the receptor is inhibited efficiently by the protein modification reagents 4-(chloromercuri)benzenesulfonate and N,N'-dicyclo-hexylcarbodiimide and by protease treatment of cholate-solubilized receptor. The receptor in native vesicles is resistant to irreversible inactivation by proteases, elevated temperature, or pH extremes. [3H]Vesamicol binding depends on deprotonation of a group of pKa1 = 6.26 +/- 0.03 and protonation of a group of pKa2 = 10.60 +/- 0.04, which is probably the tertiary amine of the drug molecule itself. The membrane-impermeant zwitterionic vesamicol analogue dl-trans-4-oxo-4-[5,6,7,8-tetrahydro-6-hydroxy-7-(4-phenyl-1-piperidinyl )-1- naphthalenyl]amino]butanoic acid (TPNB) is an effective inhibitor of AcCh active transport with an IC50 value of (51 +/- 8) x 10(-9) M. At 23 degrees C, [3H]vesamicol bound to the receptor at a rate of (1.74 +/- 0.06) x 10(5) M-1 s-1, and excess unlabeled vesamicol displaced a low concentration of bound [3H]vesamicol at 0.29 +/- 0.01 min-1. At 0 degrees C, 10 microM unlabeled vesamicol displaced 36 +/- 2% of a low concentration of bound [3H]vesamicol at 0.16 +/- 0.02 min-1 and 64 +/- 2% at 0.013 +/- 0.001 min-1. One micromolar unlabeled vesamicol behaved similarly.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Algorithms; Animals; Cholinergic Fibers; Electric Organ; Hydrogen-Ion Concentration; Kinetics; Phencyclidine; Piperidines; Receptors, Neurotransmitter; Receptors, Phencyclidine; Synaptic Vesicles; Time Factors; Torpedo

1. By applying electrophysiological techniques such as frequency facilitation, tetanic run-down and depression, recovery from depression and post-tetanic potentiation (PTP) of the end-plate potential (EPP), the effects on frog neuromuscular transmission of 2-(4-phenylpiperidino)cyclohexanol (AH5183), a compound known to inhibit specifically the loading of newly synthesized acetylcholine (ACh) molecules into synaptic vesicles, and Ba2+, a selective activator of the augmentation phase of PTP, were investigated to elucidate whether these were related to ACh turnover. 2. Effects of AH5183 and Ba2+ ions were frequency dependent. At low frequency of stimulation, both agents showed essentially no effects on the EPPs recorded from Mg2+-blocked preparations. 3. AH5183 pivoted the log-linear frequency facilitation relation clockwise without altering the intercept on the ordinate, whereas Ba2+ ions did so counter-clockwise. As is the case with Ca2+ ions, Sr2+ ions shifted the relation upwards leaving its slope unaffected. 4. AH5183 selectively depressed the component of potentiation in PTP while the effect of Ba2+ ions was a specific increase in the augmentation phase of PTP. 5. Ba2+ ions increased the amplitude of EPPs in the late depressed phase during the tetanic run-down and depression experiment, but 4-aminopyridine and Ca2+ ions failed to do so. 6. AH5183 increased, Ba2+ ions reduced but Ca2+ ions did not change the constant of recovery from depression of the EPP measured on curarized preparations. 7. The present results suggested that mobilization of the ACh quanta readily available for release might be a common mechanism underlying both frequency facilitation and two components of PTP (augmentation and potentiation). The term 'frequency facilitation' would be more comprehensive if it were re-termed 'frequency augmentation' or 'frequency potentiation'.

Topics: 4-Aminopyridine; Action Potentials; Aminopyridines; Animals; Barium; Calcium; In Vitro Techniques; Magnesium; Motor Endplate; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Phencyclidine; Piperidines; Ranidae; Strontium; Synaptic Transmission

An autoradiographic analysis of high-affinity binding sites for the vesicular acetylcholine transport blocker [3H]vesamicol (2-(4-phenylpiperidino) cyclohexanol; AH 5183) was conducted in rat brain. [3H]Vesamicol binding was displaced 52-99% by DPPN [( 2,3,4,8]-decahydro-3-(4-phenyl-1-piperidinyl)-2-napthalenol) (IC50 = 14 nM) and by ketanserin (500 nM), haloperidol (43 nM), and vesamicol analogs, but not by drugs selective for adenosine, adrenergic, amino acid, calcium channel, monoaminergic, opioid, PCP, sigma, or several other receptor classes. [3H]Vesamicol binding was most concentrated in the interpeduncular nucleus and fifth and seventh cranial nerve nuclei. Moderate binding was found in the lateral caudate-putamen, medial nucleus accumbens, olfactory tubercle, vertical and horizontal diagonal bands of Broca, and basolateral amygdala. The distribution of [3H]vesamicol binding was similar to distributions of acetylcholine (r = 0.88), acetylcholine esterase (r = 0.97), choline acetyltransferase (ChAT) (r = 0.97), and [3H]hemicholinium-3 binding sites (r = 0.95-0.99). Lower correlations were obtained between [3H]vesamicol and muscarinic receptor densities (r = 0.50-0.70). Few exceptions to the match between binding and cholinergic neuronal markers were found, e.g., the molecular layer of the cerebellum and the thalamus. Lesions of cholinergic neuronal projections to the neocortex or hippocampus reduced [3H]vesamicol binding in each of these regions, but to a lesser extent than reductions in ChAT. [3H]Vesamicol binding sites appear to be anatomically associated with brain cholinergic neurons, a locus that is consistent with the control by this site of vesicular acetylcholine uptake.

Topics: Acetylcholine; Acetylcholinesterase; Animals; Autoradiography; Brain; Cholinergic Fibers; Frontal Lobe; Haloperidol; Hippocampus; Image Processing, Computer-Assisted; Ketanserin; Male; Neural Pathways; Neurotransmitter Uptake Inhibitors; Phencyclidine; Piperidines; Rats; Rats, Inbred Strains

Crude cholinergic synaptic vesicles from a homogenate of the electric organ of Torpedo californica were centrifuged to equilibrium in an isosmotic sucrose density gradient. The classical VP1 synaptic vesicles banding at 1.055 g/mL actively transported [3H]acetylcholine (AcCh). An organelle banding at about 1.071 g/mL transported even more [3H]AcCh. Transport by both organelles was inhibited by the known AcCh storage blockers trans-2-(4-phenylpiperidino)cyclohexanol (vesamicol, formerly AH5183) and nigericin. Relative to VP1 vesicles the denser organelle was slightly smaller as shown by size-exclusion chromatography. It is concluded that the denser organelle corresponds to the recycling VP2 synaptic vesicle originally described in intact Torpedo marmorata electric organ [Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 695-714; Zimmermann, H., & Denston, C.R. (1977) Neuroscience (Oxford) 2, 715-730]. The properties of the receptor for vesamicol were studied by measuring binding of [3H]vesamicol, and the amount of SV2 antigen characteristic of secretory vesicles was assayed with a monoclonal antibody directed against it. Relative to VP1 vesicles the VP2 vesicles had a ratio of [3H]AcCh transport activity to vesamicol receptor concentration that typically was 4-7-fold higher, whereas the ratio of SV2 antigen concentration to vesamicol receptor concentration was about 2-fold higher. Based on an antibody standardization, in a typical preparation the VP1 vesicles contained 237 +/- 15 pmol of receptor/mg of protein whereas VP2 vesicles contained 102 +/- 3 pmol of receptor/mg of protein, and VP2 vesicles transported AcCh 2-3-fold more actively than VP1 vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylcholine; Animals; Biological Transport, Active; Cholinergic Fibers; Electric Organ; Nigericin; Phencyclidine; Piperidines; Synaptic Vesicles; Torpedo

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.

Topics: Anesthetics, Local; Animals; Electrophysiology; In Vitro Techniques; Male; Motor Endplate; Muscle Contraction; Muscles; Neuromuscular Depolarizing Agents; Neurons; Phencyclidine; Phrenic Nerve; Piperidines; Rana pipiens; Rats; Stereoisomerism

1. Following the addition of crotoxin (250 nM) at the frog neuromuscular junction, there was an initial fall in frequency of miniature endplate potentials (m.e.p.ps), followed by a secondary rise which was characterized by the appearance of large spontaneous potentials (giants, g.m.e.p.ps) and an occasional large potential of the burst type. 2. In the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183, vesicamol), an inhibitor of vesicular acetylcholine uptake, the frequency of g.m.e.p.ps induced by crotoxin was reduced. 3. The characteristic changes in m.e.p.p. frequency and amplitude distribution were absent with crotoxin in Sr-EGTA Ringer. In the presence of high concentrations of Mn (3.6 or 5.4 mM with 0.9 mM Ca), the crotoxin-induced initial fall and the onset of the secondary rise in m.e.p.p. and g.m.e.p.p. frequencies were slower. The timing of these phases was unaffected by Ca concentrations ranging from 6.3 to 0.9 mM. 4. High concentrations of Mn ions partially inhibited the phospholipase A2 activity of crotoxin on artificial phospholipid membranes. This also supports the involvement of the Ca-dependent phospholipase A2 subunit in both phases of the physiological action of the toxin. 5. G.m.e.p.ps were associated with a moderate increase in m.e.p.p. frequency (2-3 s-1) and were of a time-course similar to that of m.e.p.ps. They persisted after washing with medium lacking Ca ions and in the presence of Ca-Mn Ringer that blocked evoked responses. 6. It is concluded that crotoxin, acting through its phospholipase A2 subunit, produces specific disturbances of synaptic exocytosis and vesicle formation in the axolemma of the motor nerve terminal which lead to biphasic changes in m.e.p.p. frequency and the onset of large spontaneous potentials.

Topics: Animals; Calcium; Chlorides; Crotalid Venoms; Crotoxin; In Vitro Techniques; Manganese; Membrane Potentials; Motor Endplate; Muscles; Phencyclidine; Phospholipases A; Phospholipases A2; Piperidines; Rana pipiens

The drug 2-(4-phenylpiperidino)cyclohexanol (AH5183), which potently inhibits the active transport of acetylcholine (ACh) into synaptic vesicles, was used as a pharmacological tool to study the functional role of synaptic vesicles in quantal transmitter release. Using microelectrode recording techniques, miniature endplate potentials (mepps) and nerve-evoked endplate potentials (epps) were recorded from frog cutaneous pectoris neuromuscular junctions in low Ca2+/high Mg2+ Ringer solution, and in normal Ringer with added D-tubocurarine (D-TC). Stimulation in the presence of AH5183 caused a 40% reduction in quantal size (mepp amplitude), depressed tetanic potentiation, and decreased the number of quanta released with each nerve impulse in the presence of D-TC. All of these effects appeared gradually and only after extended stimulation of the nerve, during which several hundred thousand quanta were released. Consequently, these findings suggest a serial one-time usage of vesicles, with little or no re-entry of recycled vesicles until after a large fraction of the original vesicles has been exhausted. The results primarily show that filling of synaptic vesicles with ACh is crucial for sustaining synaptic transmission, and gives further evidence that the ACh released by nerve impulses originates from these organelles.

Topics: Acetylcholine; Animals; Calcium; Electric Stimulation; Evoked Potentials; In Vitro Techniques; Magnesium; Motor Endplate; Neostigmine; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Phencyclidine; Piperidines; Rana temporaria; Synaptic Vesicles; Tubocurarine

The present experiments measured the release of acetylcholine (ACh) by the cat superior cervical ganglia in the presence of, and after exposure to, 2-(4-phenylpiperidino)cyclohexanol (AH5183), a compound known to block the uptake of ACh by cholinergic synaptic vesicles. We confirmed that AH5183 blocks evoked ACh release during preganglionic nerve stimulation when approximately 13-14% of the initial ganglial ACh stores had been released; periods of rest in the presence of the drug did not promote recovery from the block, but ACh release recovered following the washout of AH5183. ACh was synthesized in AH5183-treated ganglia, as determined by the synthesis of [3H]ACh from [3H]choline, and this [3H]ACh could be released by stimulation following drug washout. The specific activity of the released ACh matched that of the tissue's ACh, and thus we conclude that ACh synthesized in the presence of AH5183 is a releasable as pre-existing ACh stores once the drug is removed. We tested the relative releasability of ACh synthesized during AH5183 exposure (perfusion with [3H]choline) and that synthesized during recovery from the drug's effects (perfusion with [14C]choline: the ratio of [3H]ACh to [14C]ACh released by stimulation was similar to the ratio in the tissue. These results suggest that the mobilization of ACh for release by ganglia during recovery from an AH5183-induced block is independent of the conditions under which the ACh was synthesized. Unlike nerve impulses, black widow spider venom (BWSV) induced the release of ACh from AH5183-blocked ganglia, even in the drug's continued presence. Venom-induced release of ACh from AH5183-treated ganglia was not less than the venom-induced release from tissues not exposed to AH5183. This effect of BWSV was attributed to the action of the protein, alpha-latrotoxin, because an anti-alpha-latrotoxin antiserum blocked the venom's action. ACh synthesized during AH5183 exposure was labelled from [3H]choline, and subsequent treatment with BWSV released [3H]ACh with the same temporal pattern as the release of total ACh. To exclude a nonexocytotic origin for the [3H]ACh released by BWSV, ganglia were preloaded with [3H]diethylhomocholine to form [3H]acetyldiethylhomocholine, an ACh analogue excluded from vesicles; the venom did not increase the rate of [3H]acetyldiethylhomocholine efflux. It is concluded that a vesicular ACh pool insensitive to the inhibitory action of AH5183 might exist and that this vesicular pool is not mo

Topics: Acetylcholine; Animals; Black Widow Spider; Cats; Choline; Electric Stimulation; Female; Ganglia, Sympathetic; Kinetics; Male; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Spider Venoms; Tritium

The effects of vesamicol (AH5183), a blocker of acetylcholine transport, on the voltage-clamped neuromuscular junction of the frog were studied. Vesamicol (15-30 microM) reduced the peak height of the ionophoretically applied acetylcholine-induced current. The amplitude of the evoked endplate current was also decreased in the presence of vesamicol (30 microM). The endplate current was reduced further when the nerve was tetanically stimulated. The reduction of the endplate current after tetanic stimulation in the presence of vesamicol was due to a decrease in the mean quantal content. The decay time constants of the evoked endplate current and the miniature endplate current were also decreased by vesamicol. It was concluded that vesamicol acts as a postsynaptic blocker at the endplate. This neurotoxin could also decrease the immediately available quanta in the presynaptic nerve terminal, but the mechanism of action of vesamicol on transmitter release remains obscure.

Topics: Acetylcholine; Animals; Dimethyl Sulfoxide; Electric Stimulation; In Vitro Techniques; Iontophoresis; Muscle Contraction; Muscles; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Phencyclidine; Piperidines; Ranidae; Sciatic Nerve

The effect of L- and D-stereoisomers of 2-(4-phenylpiperidino) cyclohexanol (AH 5183) on the spontaneous release of acetylcholine (ACh) from rat hippocampal tissue was studied. L-AH 5183 was approximately 100 times more potent than was D-AH 5183 in reducing spontaneous ACh release. Spontaneous ACh release was also temperature dependent. These results may suggest that the spontaneous release of ACh from brain tissue is carrier-mediated.

Topics: Acetylcholine; Animals; Hippocampus; In Vitro Techniques; Kinetics; Male; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Rats; Rats, Inbred Strains; Reference Values; Stereoisomerism

The intent of this study was to determine whether the drug 2-(4-phenylpiperidino)cyclohexanol (AH 5183 or vesamicol) might inhibit the veratridine-induced increase in acetylcholine (ACh) synthesis by reducing the veratridine-induced activation of a detergent-soluble choline-O-acetyltransferase (EC 2.3.1.6; ChAT) fraction associated with a vesicle-bound store of ACh. When minces of rat hippocampal tissue were loaded with [14C]choline and subsequently depolarized with veratridine, an increase in the synthesis of [14C]ACh occurred that could be abolished by L-AH 5183 (75 nM). When minces were depolarized with veratridine in the presence of L-AH 5183 (75 nM), the depolarization-induced activation of a detergent-soluble ChAT fraction associated with a vesicle-bound store of ACh was blocked. Conversely, the veratridine-induced activation of a water-soluble ChAT fraction believed to be cytosolic was not. AH 5183 also blocked the repletion of the vesicle-bound store with newly synthesized ACh following veratridine-induced depletion of ACh, a result that appeared to be mediated by an effect on the synthesis of ACh at the vesicular surface. These results suggest that veratridine depolarization of rat hippocampal nerve terminals stimulates the synthesis of ACh by activating a detergent-soluble fraction of ChAT closely associated with synaptic vesicle release sites. ACh synthesis and transport at the vesicular surface may be influenced by a common AH 5183-sensitive regulatory protein.

Topics: Acetylcholine; Animals; Choline; Choline O-Acetyltransferase; Hippocampus; Isotonic Solutions; L-Lactate Dehydrogenase; Male; Naphthylvinylpyridine; Octoxynol; Phencyclidine; Piperidines; Polyethylene Glycols; Rats; Rats, Inbred Strains; Solubility; Subcellular Fractions; Veratridine; Veratrine

Topics: Acetylcholine; Adenosine Triphosphatases; Animals; Biological Transport, Active; Electric Organ; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Receptors, Neurotransmitter; Receptors, Phencyclidine; Synaptic Vesicles; Torpedo

2-(4-Phenylpiperidino)cyclohexanol (AH5183) is a noncompetitive and potent inhibitor of high-affinity acetylcholine transport into cholinergic vesicles. It is reported here that [3H]AH5183 binds specifically and saturably to slide-mounted sections of the rat forebrain (Kd = 1.1 to 2.2 X 10(-8) M; Bmax = 286 to 399 fmol/mg of protein). The association and dissociation rate constants for [3H]AH5183 binding are 8.6 X 10(6) M-1 X min-1 and 0.18 min-1, respectively. Bound [3H]AH5183 can be displaced by nonradioactive AH5183 and by the structural analog (2 alpha,3 beta,4A beta,8A alpha)-decahydro-3-(4-phenyl-1-piperidinyl)-2- naphthalenol but not by 10 microM concentrations of the cholinergic drugs acetylcholine, choline, atropine, hexamethonium, eserine, or hemicholinium-3 or by the structurally related compounds 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridine, (+/-)-N-allylnormetazocine (SKF 10,047), levoxadrol, or dexoxadrol. Quantitative autoradiography reveals that [3H]AH5183 binding sites are distributed heterogenously throughout the rat forebrain and are highly localized to cholinergic nerve terminal regions. At the level of the caudate nucleus-putamen, the highest concentrations of saturable [3H]AH5183 binding (713-751 fmol/mg of protein) are found in the vertical limb of the diagonal band and the olfactory tubercle, with lesser amounts (334-516 fmol/mg of protein) in the caudate-putamen, nucleus accumbens, superficial layers of the cerebral cortex, and the primary olfactory cortex. At day 7 after transsection of the left fimbria, [3H]AH5183 binding and choline acetyltransferase activity in the left hippocampus were reduced by 33 +/- 6% and 61 +/- 7%, respectively. These findings indicate that [3H]AH5183 binds to a unique recognition site in rat brain that is topographically associated with cholinergic nerve terminals.

Topics: Acetylcholine; Animals; Autoradiography; Brain; Kinetics; Male; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Rats; Rats, Inbred Strains; Receptors, Cholinergic; Tritium

These experiments measured the release and the synthesis of acetylcholine (ACh) by cat sympathetic ganglia in the presence of 2-(4-phenylpiperidino) cyclohexanol (AH5183), an agent that blocks the uptake of ACh into synaptic vesicles. Evoked transmitter release during short periods of preganglionic nerve stimulation was not affected by AH5183, but release during prolonged stimulation was not maintained in the drug's presence, whereas it was in the drug's absence. The amount of ACh releasable by nerve impulses in the presence of AH5183 was 194 +/- 10 pmol, which represented 14 +/- 1% of the tissue ACh store. The effect of AH5183 on ACh release was not well antagonized by 4-aminopyridine (4-AP), and not associated with inhibition of stimulation-induced calcium accumulation by nerve terminals. It is concluded that AH5183 blocks ACh release indirectly, and that the proportion of stored ACh releasable in the compound's presence represents transmitter in synaptic vesicles available to the release mechanism. The synthesis of ACh during 30 min preganglionic stimulation in the presence of AH5183 was 2,448 +/- 51 pmol and in its absence it was 2,547 +/- 273 pmol. Thus, as the drug decreased ACh release it increased tissue content. The increase in tissue content of ACh in the presence of AH5183 was not evident in resting ganglia; it was evident in stimulated ganglia whether or not tissue cholinesterase was inhibited; it was increased by 4-AP and reduced by divalent cation changes expected to decrease calcium influx during nerve terminal depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 4-Aminopyridine; Acetylcholine; Aminopyridines; Animals; Calcium; Cats; Electric Stimulation; Female; Ganglia, Sympathetic; Kinetics; Male; Phencyclidine; Physostigmine; Piperidines

AH5183 was studied because it inhibits acetylcholine transport into synaptic vesicles. The drug apparently is a slow-acting anti-cholinesterase, so further experiments were performed with this enzyme inhibited. Soaking for hours in AH5183 in Ringer does not decrease quantal size. However, a few minutes of tetanic nerve stimulation results in a marked decrease in quantal size. Quantal size also decreased after hours in a hypertonic Ringer containing the drug.

Topics: Acetylcholine; Animals; Depression, Chemical; In Vitro Techniques; Membrane Potentials; Motor Endplate; Neuromuscular Junction; Phencyclidine; Piperidines; Rana pipiens; Synaptic Transmission

A post-stimulation synthesis of acetylcholine (ACh), its incorporation into a 'stable-bound' (vesicular) compartment and subsequent release, were compared in K+-stimulated synaptosomes, in the absence and presence of 10 microM AH5183. The drug depressed by 16% the net intrasynaptosomal formation of ACh from 1 microM [3H]choline (Ch) in the medium, by competitively inhibiting (Ki approximately equal to 20 microM) the high-affinity Ch transport, but it had no direct effect on the intraterminal synthesis of ACh per se. The drug reduced incorporation of newly synthesized [3H]ACh into synaptic vesicles by 55% and subsequent K+-depolarization-induced release of [3H]ACh by 83%, although it had no effect on Ca2+ influx into synaptosomes. These results are consistent with the hypothesis that AH5183 blocks cholinergic neurotransmission presynaptically by interfering with recharging of synaptic vesicles with ACh. Since the reduction of ACh release in the presence of AH5183 had no direct effect on ACh synthesis, these results also suggest that the transmitter release is not prerequisite for enhancement of Ch uptake and ACh synthesis in stimulated nerve terminals.

Topics: Acetylcholine; Animals; Calcium; Cerebral Cortex; Choline; Cholinergic Fibers; Phencyclidine; Piperidines; Potassium; Rats; Synaptic Transmission; Synaptosomes

Transport and storage of acetylcholine by purified Torpedo electric organ synaptic vesicles is blocked by the drug L-trans-2-(4-phenylpiperidino)cyclohexanol (AH-5183). This study sought evidence of a specific receptor for the drug. Highly tritiated L-trans-2-(4-phenyl [3,4-3H] piperidino)-cyclohexanol (L-[3H] AH5183) was synthesized. An excess of nonradioactive L-isomer competed with L-[3H]AH5183 for binding to purified Torpedo synaptic vesicles whereas nonradioactive D-isomer did so poorly. Dissociation of bound L-[3H]AH5183 was first order with a rate constant at 23 degrees C of 0.23 +/- 0.03 min-1, and association was too rapid to study. At equilibrium the amount of L-[3H]AH5183 bound at saturation varied in different vesicle preparations, but in one typical preparation specific binding of 181 +/- 15 pmol L-[3H]AH5183 per mg of synaptic vesicle protein was observed with a dissociation constant of 34 +/- 6 nM. Neither acetylcholine nor choline compete effectively with L-[3H]AH5183 for binding. The evidence suggests that about 3.7 +/- 0.3 enantioselective receptors for L-[3H]AH5183 are typically present in each cholinergic synaptic vesicle, and the L-AH5183 binding site does not recognize acetylcholine.

Topics: Acetylcholine; Animals; Binding, Competitive; Choline; Electric Organ; Kinetics; Phencyclidine; Piperidines; Receptors, Drug; Stereoisomerism; Synaptic Vesicles; Torpedo

1. To study the nature and origin of slow-rising, Ca2+-insensitive miniature end-plate potentials (m.e.p.p.s) in mammalian muscle we used intracellular recording techniques and drugs which block acetylcholine (ACh) synthesis or the uptake of ACh into synaptic vesicles. Slow m.e.p.p.s were induced in vivo by paralysing the extensor digitorum longus muscle of the rat with botulinum toxin type A or in vitro by the application of 4-aminoquinoline to the mouse diaphragm nerve-muscle preparation. 2. Hemicholinium-3, which blocks ACh synthesis, reduced the amplitude of all synaptic potentials including slow m.e.p.p.s, but only if the nerve was stimulated. 3. 2(4-phenylpiperidino)cyclohexanol (AH-5183), which blocks the active uptake of ACh into synaptic vesicles, reduced both the frequency and the amplitude of slow m.e.p.p.s and did so without requiring nerve stimulation. 4. No correlation was observed between the molecular leakage of ACh from the motor nerve and the frequency and amplitude of slow m.e.p.p.s. 5. We conclude that slow m.e.p.p.s are caused by the release of ACh from the nerve terminal, possibly from a small pool of synaptic vesicle-like structures.

Topics: Acetylcholine; Action Potentials; Aminoquinolines; Animals; Botulinum Toxins; Calcium; Hemicholinium 3; In Vitro Techniques; Mice; Motor Endplate; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Phencyclidine; Piperidines; Rats; Rats, Inbred Strains; Temperature; Time Factors

The rate of translocation of newly synthesized acetylcholine (ACh) from the presynaptic cytosol of Torpedo electric organ nerve terminals into synaptic vesicles and the extent to which ACh release from these neurons is mediated by a vesicular mechanism were investigated. For this purpose the compound 2(4-phenylpiperidino)cyclohexanol (AH5183), which inhibits the active transport of ACh into isolated cholinergic synaptic vesicles, was employed. Preincubation of purified Torpedo nerve terminals (synaptosomes) with AH5183 does not affect the intraterminal synthesis of [3H]ACh but results in a marked inhibition (85%) of its Ca2+-dependent K+-evoked release. By contrast, the evoked release of the endogenous nonlabeled ACh is not affected by this compound. When AH5183 is added during radiolabeling, it causes a progressively smaller inhibition of [3H]ACh release which is completely abolished if the drug is added after the preparation has been labeled. These findings suggest that most of the newly synthesized synaptosomal [3H]ACh (85%) is released by a vesicular mechanism and that some [3H]ACh (15%) may be released by a different process. The translocation of cytosolic [3H]ACh into the synaptic vesicles was monitored by determining the time course of the loss of susceptibility of [3H]ACh release to AH5183. It was found not to be coupled kinetically to [3H]ACh synthesis and to lag behind it. The nature of the intraterminal processes underlying this lag is discussed.

Topics: Acetylcholine; Animals; Biological Transport; Cytosol; Electric Organ; Kinetics; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Synaptic Vesicles; Synaptosomes; Torpedo

These experiments measured the effect of 2-(4-phenylpiperidino)cyclohexanol (AH5183) on the release of acetylcholine (ACh) and its subcellular distribution in slices of rat striatum incubated in vitro. The AH5183, a drug that blocks the uptake of ACh by isolated synaptic vesicles, reduced the release of ACh from slices stimulated to release transmitter in response to K+ depolarization. Tissue stimulated in the presence of AH5183 contained more ACh in a nerve terminal cytoplasmic fraction than did tissue stimulated in the drug's absence, but stimulation in AH5183's presence reduced the amount of ACh measured in fractions containing synaptic vesicles. The depletion of ACh caused by stimulating tissue in the presence of AH5183 was more evident in the fraction of nerve terminal ACh occluded within synaptic vesicles as isolated by gradient centrifugation (fraction D) than it was in other nerve terminal occluded stores. It is concluded that the synaptic vesicles isolated as fraction D under the present experimental conditions likely contain releasable transmitter. The AH5183 also depressed the spontaneous release of ACh from incubated slices of striatum and this effect was evident in the presence or the absence of medium Ca2+. It is suggested that this effect might indicate that the process of spontaneous ACh release measured neurochemically results, in part, from an AH5183-sensitive carrier-mediated process.

Topics: Acetylcholine; Animals; Calcium; Corpus Striatum; Male; Phencyclidine; Piperidines; Potassium; Rats; Rats, Inbred Strains; Subcellular Fractions; Tissue Distribution

Isolated unparalysed mouse phrenic nerve-hemidiaphragm preparations were indirectly stimulated in the presence of 2-(4-phenylpiperidino)cyclohexanol (AH5183, vesamicol). Spontaneous miniature endplate potentials were subsequently studied. They exhibited a large depression of amplitude which was more profound at higher stimulation frequencies and drug concentration. No post-junctional effects of the drug were observed. Since the drug blocks storage of acetylcholine by isolated synaptic vesicles, it is argued that the results support the theory of vesicular release of acetylcholine.

Topics: Acetylcholine; Animals; Carbachol; In Vitro Techniques; Membrane Potentials; Mice; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Phencyclidine; Piperidines; Receptors, Cholinergic; Synaptic Vesicles

The effects of 2-(4-phenylpiperidino)cyclohexanol (AH5183) and quinacrine, two potent inhibitors of acetylcholine transport into vesicles isolated from Torpedo electric organ, were examined on acetylcholine metabolism in rat cortical slices. K+-stimulated acetylcholine release was reduced in a concentration-dependent manner by AH5183 and quinacrine, with IC50 values of 1 microM and 50 microM, respectively. Both drugs also reduced the synthesis of acetylcholine in slices and inhibited synaptosomal high affinity choline transport. The inhibitory effect of AH5183 appears to be directed primarily on the release of acetylcholine while the major effect of quinacrine is on the synthesis of acetylcholine. Examination of the subcellular distribution of acetylcholine in brain slices incubated in high K+ showed that AH5183 increased S3 (cytoplasmic) acetylcholine levels but did not alter P3 (vesicular) acetylcholine levels. P3 acetylcholine levels were reduced by AH5183 in a low K+ media while the S3 acetylcholine levels were the same as controls. These results are consistent with the concept that there is a small, active, highly labile fraction of vesicles that are the source of the released acetylcholine and that the loading of these vesicles is blocked by AH5183.

Topics: Acetylcholine; Animals; Biological Transport; Brain; Choline; In Vitro Techniques; Kinetics; Male; Neuromuscular Depolarizing Agents; Phencyclidine; Piperidines; Potassium; Quinacrine; Rats; Rats, Inbred Strains

Highly purified Torpedo electric organ synaptic vesicles form a 49 nM suspension at 1 mg protein/ml. Under active transport conditions hundreds of molecules of [3H]acetylcholine ([3H]ACh) can be accumulated per vesicle, which requires the ACh transporter to undergo multiple turnovers. The transport blocker trans-2-(4-phenylpiperidino)cyclohexanol (AH5183) has no effect on storage of endogenous ACh by vesicles. In contrast, AH5183, other blocking drugs, and nonradioactive ACh caused a rapid release of at least 30-63 molecules of newly transported [3H]ACh per vesicle. Thus AH5183 distinguishes recently transported "new" vesicular ACh from "old" endogenous ACh. l-AH5183 inhibits transport of ACh with a half-inhibitory concentration of 16 +/- 7 nM at 12 nM vesicles and 115 +/- 34 nM at 120 nM vesicles. With the assumption that AH5183 acts on a receptor in an unamplified manner about 2.7 or fewer receptors per vesicle need to be occupied to cause inhibition of ACh transport. The apparent amplification in the number of [3H]ACh molecules per vesicle that are released by AH5183 suggests that AH5183 inhibits ACh storage by an indirect mechanism that distinguishes new from old ACh.

Topics: Acetylcholine; Animals; Biological Transport, Active; Electric Organ; Hemicholinium 3; Phencyclidine; Piperidines; Quinacrine; Synaptic Vesicles; Torpedo

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.

Topics: Acetylcholine; Allosteric Regulation; Animals; Biological Transport, Active; Electric Organ; Kinetics; Phencyclidine; Piperidines; Synaptic Vesicles; Torpedo

Experiments were performed to investigate the effects on the spontaneous, nonquantal release of acetylcholine (AcCho) from motor nerve terminals of substances known to inhibit the AcCho transport system present in cholinergic synaptic vesicles. In mouse diaphragms, the hyperpolarization normally produced by d-tubocurarine in the endplate area of muscle fibers that had been treated by an anticholinesterase was partly or completely blocked by 2-(4-phenylpiperidino)cyclohexanol (AH5183, 0.1-1 microM), quinacrine (0.1 microM), and tetraphenylborate (1 microM). Since the sensitivity of the subsynaptic area to AcCho was not changed, the block of the hyperpolarizing action of d-tubocurarine indicated in inhibition of the spontaneous, nonquantal release of AcCho. This was confirmed in experiments on rat diaphragm using direct radioenzymatic measurement of the AcCho released into the incubation medium. The release of AcCho from the innervated diaphragm was decreased by about 50% in the presence of AH5183 (0.01-1 microM) and by 42% in the presence of quinacrine (0.1-1 microM). The AcCho released was presumably neural, since the release of AcCho from 4-day denervated diaphragms was not diminished by either AH5183 or quinacrine. The results indicate that the spontaneous release of AcCho from the motor nerve terminals is highly sensitive to low concentrations of specific inhibitors and is probably mediated by a carrier. It is proposed that spontaneous release is due to the incorporation into the membrane of the nerve terminal during exocytosis of the vesicular transport system responsible for moving AcCho into the vesicle.

Topics: Acetylcholine; Animals; Female; Hydrogen-Ion Concentration; Male; Membrane Potentials; Mice; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Phencyclidine; Piperidines; Quinacrine; Rats; Secretory Rate

Superior cervical ganglia of the cat perfused with [14C]diethylhomocholine [( 14C]DEHCh) synthesized acetyldiethylhomocholine (ADEHCh), but rather little of this ester was released by subsequent preganglionic nerve stimulation. Stimulation evoked the release of an appreciable amount of unchanged DEHCh when ganglia had been exposed to the analogue in the absence of choline (Ch), but did not do so when exposed to both Ch and DEHCh. The release of DEHCh was Ca2+ dependent, and was not the result of the release and subsequent hydrolysis of ADEHCh. This is the first clear demonstration of the release of an unacetylated compound from mammalian tissue; therefore, the characteristics of the transmitter release mechanism are further defined. The effect of preganglionic nerve stimulation on the uptake and acetylation of DEHCh was also measured. Stimulated ganglia accumulated approximately 4 times more labeled analogue and synthesized 7.5 times more ADEHCh than did rested ganglia. Stimulated ganglia perfused with 2-(4-phenylpiperidino)cyclohexanol, a compound considered to inhibit acetylcholine (ACh) release by inhibiting its transport into synaptic vesicles, accumulated 3.4 times as much and acetylated 6 times as much DEHCh as did rested ganglia. When the concentration of Mg2+ in the perfusion medium was increased to block ACh release, accumulation of the labelled analogue was enhanced by stimulation, but its acetylation was increased much less than during perfusion with normal medium. It is concluded that the synthesis of ADEHCh is subject to the same regulation as is ACh synthesis and that the activation of ester synthesis during activity can be dissociated from ester release.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Acetylation; Acetylcholine; Animals; Calcium; Cats; Choline; Electric Stimulation; Female; Ganglia, Sympathetic; Magnesium; Male; Neuromuscular Depolarizing Agents; Phencyclidine; Physostigmine; Piperidines

The nature of the intraterminal compartments from which acetylcholine (ACh) is released following presynaptic stimulation was investigated. This was pursued by examining the effects of the anticholinergic drug 2-(4-phenylpiperidino)cyclohexanol (AH5183) on the release of newly synthesized [3H]ACh and of endogenous ACh from purified cholinergic nerve terminals (synaptosomes) which were isolated from the electric organs of Torpedo. Preincubation of the synaptosomes, with AH5183 (1-10 microM), does not affect either the intraterminal synthesis of [3H]ACh or the uptake of its precursors, but results in a marked inhibition (85%) of the release of the newly synthesized [3H]ACh. However, when AH5183 is added following the accumulation of [3H]ACh in the nerve terminals, it does not affect [3H]ACh release. AH5183 also has no effect on the release of preformed endogenous ACh. These findings, together with the previous in vitro demonstrations that AH5183 is a potent inhibitor of ACh uptake into isolated cholinergic vesicles, suggest that most of the synaptosomal ACh is secreted by a vesicular mechanism.

Topics: Acetates; Acetylcholine; Animals; Electric Organ; In Vitro Techniques; Phencyclidine; Piperidines; Synaptic Vesicles; Synaptosomes; Torpedo

The drug 2(4-phenylpiperidine)cyclohexanol (AH 5183) caused hyperpolarization by 1.8 +/- 0.6 mV in an end-plate zone of mouse diaphragm fibers without any change in the amplitude of miniature end-plate potentials. This supports the idea that the drug inhibits the non-quantal leakage from motor nerve terminals, probably at those parts of the nerve terminals which were incorporated into the terminal membrane after vesicle exocytosis.

Topics: Acetylcholine; Animals; Diaphragm; Female; Membrane Potentials; Mice; Motor Neurons; Muscles; Neural Inhibition; Neuromuscular Depolarizing Agents; Neuromuscular Junction; Phencyclidine; Piperidines

The effect of the acetylcholine (ACh) transport blocker 2-(4-phenylpiperidino) cyclohexanol (AH5183) on the subcellular storage and release of acetylcholine was studied in mouse forebrain. Results indicated that AH5183 reduced the amount of ACh released from mouse forebrain minces by high K+ and veratridine over the identical concentration range as it inhibits the active transport of ACh into synaptic vesicles isolated from the electric organ of Torpedo. However, AH5183 did not block the K+- or veratridine-induced reduction of cytoplasmic (S3) ACh. Also, it did not block the loss of vesicular (P3) ACh caused by these depolarizing agents. It did, however, cause a disappearance of nerve ending ACh which was partially matched by a selective gain in the choline content of the P3 fraction. When minces of mouse forebrain were pretreated in high K+ to deplete the S3 and P3 fractions of their ACh content and then subsequently incubated in normal Krebs with [14C]choline, AH5183, at a concentration which reduces ACh release by 50%, did not affect the repletion of P3 stores with newly synthesized [14C]ACh. At somewhat higher concentrations, however, AH5183 reduced the amount of [14C]ACh in the P3 fraction without affecting the amount of [14C]ACh in the S3 fraction. At these concentrations it did not inhibit extracellular choline transport or ChAT activity. These results suggest that AH5183 may reduce the amount of ACh released from central cholinergic nerve terminals in response to depolarization through a combination of effects: (1) it may facilitate the breakdown or loss of ACh stored in the vesicular fraction; (2) it may also block the transport of newly synthesized ACh into the vesicular fraction.

Topics: Acetylcholine; Animals; Biological Transport; Brain; Electric Organ; Mice; Mice, Inbred ICR; Phencyclidine; Piperidines; Potassium; Synaptic Vesicles; Torpedo; Veratridine

The cholinergic vesicular uptake blocker, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183), had several effects on presynaptic cholinergic function that depended on the duration of treatment and dose. The synthesis, storage and release of newly synthesized [3H]ACh were monitored because the vesicular uptake of this pool of transmitter may be preferentially affected by the drug. Initially, high concentrations of AH5183 (over 10 microM) increased the spontaneous release but decreased the K+ depolarization-induced release of newly synthesized transmitter. [3H]Choline efflux was not altered by the drug. High affinity choline uptake was slightly (10-20%) inhibited by AH5183 in an apparently competitive but time-dependent manner. In contrast to its initial effects on [3H]ACh release, AH5183 (50nM-100 microM) very potently inhibited both the spontaneous and K+-induced release of [3H]ACh but not of [3H]choline after a 60 min preincubation. [3H]ACh levels in cytoplasmic (S3) and crude membrane (P3) fractions were not affected by a 2-min incubation with 10 microM AH5183. After a 60-min preincubation with this drug dose, however, the P3 and S3 levels of newly synthesized transmitter were decreased and increased, respectively. Subsequent fractionation of synaptosomes by sucrose-density gradient centrifugation revealed that these reductions in P3 [3H]ACh-levels were referable to reductions in two subfractions D and H that have been reported to contain low density vesicles and denser vesicles associated with plasma membranes, respectively.

Topics: Acetylcholine; Animals; Cerebral Cortex; Choline; In Vitro Techniques; Male; Phencyclidine; Piperidines; Rats; Rats, Inbred Strains; Synaptosomes

Treatment of PC12 cells with AH5183 at concentrations of 40 nM to 40 microM inhibited the loading of newly synthesized acetylcholine into storage vesicles, but it had little effect on choline uptake, acetylcholine synthesis, or the vesicular content of previously loaded acetylcholine. AH5183 at 4 microM inhibited the Ba2+-evoked release of newly synthesized acetylcholine but not of older stores of acetylcholine. These data indicate that the vesicles are the source of the acetylcholine released from stimulated cells. AH5183 had little effect on the vesicular loading of dopamine and on the evoked release of dopamine, but at concentrations of 4-40 microM it did inhibit dopamine uptake by the cells.

Topics: Acetylcholine; Animals; Cells, Cultured; Cytoplasmic Granules; Dopamine; Phencyclidine; Pheochromocytoma; Piperidines; Rats; Secretory Rate

A wide variety of pharmacologically active compounds was surveyed for effects on active transport of [3H]acetylcholine by synaptic vesicles isolated from the electric organ of Torpedo californica. In over 80 compounds tested, inhibitors of a wide range of potencies were found. The most potent inhibitor was 2-(4-phenylpiperidino)cyclohexanol (AH5183), which half-inhibited transport at 40 nM. This compound had been predicted by Marshall [Br. J. Pharmacol. 38:503-516 (1970)] to block acetylcholine storage by vesicles in vivo. The synaptic vesicle active transport system is shown to be pharmacologically distinct from other cholinergic systems. The site of action of AH5183 and other potent inhibitors is not certain, but the possibility of trivial action on the vesicle ATPase or a vesicle proton gradient was eliminated. The results constitute new evidence supporting vesicle exocytosis as the source of evoked acetylcholine release by nerve terminals. AH5183 appears to be the prototype for a new family of anticholinergics. The possibility that some drugs that exhibit secondary anticholinergic effects act in part by antagonizing acetylcholine storage is discussed.

Topics: Acetylcholine; Animals; Binding Sites; Biological Transport, Active; Electric Organ; In Vitro Techniques; Phencyclidine; Piperidines; Receptors, Cholinergic; Synaptic Vesicles; Torpedo