Mobilization of the Readily Releasable Pool of Acetylcholine from a Sympathetic Ganglion by Tityustoxin in the Presence of Vesamicol

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.     

Regulation of the Vesamicol Receptor in Cholinergic Synaptic Vesicles by Acetylcholine and an Endogenous Factor

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.     

Ca2+o-Independent Veratridine-Evoked Acetylcholine Release from Striatal Slices Is Not Inhibited by Vesamicol (AH5183): Mobilization of Distinct Transmitter Pools

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.     

Effect of Protein Kinase C Activation on the Release of [3H]Acetylcholine in the Presence of Vesamicol

Abstract: 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 α-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.     

Vesamicol Binding to Subcellular Membranes That Are Distinct from Catecholaminergic Vesicles in PC 12 Cells

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.     

Specificity of the Rat Vesicular Acetylcholine Transporter

The protein kinase A–deficient PC12 cell line PC12^A123.7 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.     

Parameters not influenced by vesamicol: Membrane potential,calcium uptake,and internal calcium concentration of synaptosomes

In our previous study vesamicol, an inhibitor of the acetylcholine transporter of the cholinergic vesicles, inhibited veratridine-evoked external Ca²⁺-dependent acetylcholine release from striatal slices but did not influence acetylcholine release observed in Ca²⁺-free medium (4). Here we examined if the effect of veratridine on membrane potential, Ca²⁺ uptake, and intracellular Ca²⁺ concentration of synaptosomes was altered by vesamicol in parallel with the inhibition of acetylcholine release. The depolarizing effect of 10 M veratridine (from 67±2.3 mV resting membrane potential to 50.7±2.5 mV) was not significantly influenced by vesamicol (1–20 M). Vesamicol (1–20 M) had no effect on either the overall curve of the veratridine-evoked⁴⁵Ca²⁺ 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 M). The K⁺-evoked (40 mM) increase of Ca²⁺ uptake and of intracellular calcium concentration were also unaltered by vesamicol. In high concentration (50 M) 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 M) 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. The results suggest that vesamicol does not interfere with veratridine-induced changes in isolated nerve terminals other than with the release of acetylcholine, thus further supporting the involvement of a vesamicol-sensitive vesicular transmitter pool in Ca²⁺-dependent veratridine-elicited acetylcholine release.     

Diffusion Pathways to Critical Cysteines in the Vesicular Acetylcholine Transporter of Torpedo

Previous work had demonstrated that organomercurial-mediated modification of two cysteine residues in the vesicular acetylcholine transporter (VAChT) from Torpedo californica inhibits binding of vesamicol. The cysteines are protected by acetylcholine and vesamicol (Keller et al. 2000. J. Neurochem. 74:1739–1748). Modified cysteine 1 is accessible to glutathione from the cytoplasmic surface, whereas modified cysteine 2 is not. Different organomercurials and aqueous environments were used here to characterize diffusion pathway(s) leading to the cysteines. para-Chloromercuriphenylsulfonate modifies VAChT much more slowly than do more hydrophobic p-chloromercuribenzoate and phenylmercury chloride. Permeabilization of vesicles with cholate detergent increases the rate of modification by p-chloromercuriphenylsulfonate. Permeabilization does not affect the ability of glutathione to reverse modification by p-chloromercuriphenylsulfonate. Higher ionic strength causes about four-fold increase in the rate of modification. The results suggest that hydrophobic and electrostatic barriers inhibit modification of Torpedo VAChT by negatively charged organomercurials and glutathione cannot reach cysteine 2 from either side of the membrane.     

Corticotropin-Releasing Factor Administered Centrally, but Not Peripherally, Stimulates Hippocampal Acetylcholine Release

Abstract: In addition to corticotropin-releasing factor's well-known role in mediating hormonal and behavioral responses to stress, this peptide also reportedly affects arousal and cognition, processes that classically have been associated with forebrain cholinergic systems. Corticotropin-releasing factor stimulation of cholinergic neurons might thus provide a mechanism for this peptide's cognitive effects. To examine this possibility, the present experiments characterize the effect of corticotropin-releasing factor on cholinergic neurotransmission, using in vivo microdialysis to measure hippocampal acetylcholine release. Corticotropin-releasing factor (0.5–5.0 µg/rat intracerebroventricularly) was found to increase dialysate concentrations of acetylcholine in a dose-dependent manner in comparison with a control injection, the ovine peptide having a greater effect than the same dose of the human/rat peptide. This effect was found to be centrally mediated, independent of the peripheral effects of an exogenous corticotropin-releasing factor injection; subcutaneous injections of the peptide increased plasma concentrations of corticosterone, the adrenal hormone ultimately secreted in the rat's stress response, to the same level as did the central injections, without affecting hippocampal acetylcholine release. These results demonstrate that corticotropin-releasing factor, acting centrally, regulates hippocampal cholinergic activity, and suggest that corticotropin-releasing factor/acetylcholine interactions may underlie some of the previously identified roles of these neurotransmitters in arousal, cognition, and stress.     

Synthesis of novel 4- and 5-substituted benzyl ether derivatives of vesamicol and in vitro evaluation of their binding properties to the vesicular acetylcholine transporter site

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.     

Drug Effects on the Release of Endogenous Acetylcholine In Vivo: Measurement by Intracerebral Dialysis and Gas Chromatography–Mass Spectrometry

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.     

Characterization of [3H]Vesamicol binding in rat brain preparations

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°C and was reversible (dissociation half-time 4.9 min). At least two binding affinities were found in P₂ fractions from the cerebral cortex (K_d:21 nM and 980 nM), striatum (K_d:28 nM and 690 nM), and cerebellum (K_d:22 nM and 833 nM). High affinity B_max 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 B_max 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 B_max values for [³H]vesamicol-binding were highest in a partially purified synaptic vesicle fraction, followed by puriffied 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 replaement 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.     

Control of synthesis and release of radioactive acetylcholine in brain slices from the rat. Effects of neurotropic drugs

1. Studies of the synthesis and release of radioactive acetylcholine in rat brain-cortex slices incubated in Locke-bicarbonate-[U-(14)C]glucose media, containing paraoxon as cholinesterase inhibitor, revealed the following phenomena: (a) dependence of K(+)-or protoveratrine-stimulated acetylcholine synthesis and release on the presence of Na(+) and Ca(2+) in the incubation medium, (b) enhanced release of radioactive acetylcholine by substances that promote depolarization at the nerve cell membrane (e.g. high K(+), ouabain, protoveratrine, sodium l-glutamate, high concentration of acetylcholine), (c) failure of acetylcholine synthesis to keep pace with acetylcholine release under certain conditions (e.g. the presence of ouabain or lack of Na(+)). 2. Stimulation by K(+) of radioactive acetylcholine synthesis was directly proportional to the external concentration of Na(+), but some synthesis and release of radioactive acetylcholine occurred in the absence of Na(+) as well as in the absence of Ca(2+). 3. The Na(+) dependence of K(+)-stimulated acetylcholine synthesis was partly due to suppression of choline transport, as addition of small concentrations of choline partly neutralized the effect of Na(+) lack, and partly due to the suppression of the activity of the Na(+) pump. 4. Protoveratrine caused a greatly increased release of radioactive acetylcholine without stimulating total radioactive acetylcholine synthesis. Protoveratrine was ineffective in the absence of Ca(2+) from the incubation medium. It completely blocked K(+) stimulation of acetylcholine synthesis and release. 5. Tetrodotoxin abolished the effects of protoveratrine on acetylcholine release. It had blocking effects (partial or complete) on the action of high K(+), sodium l-glutamate and lack of Ca(2+) on acetylcholine synthesis and release. 6. Unlabelled exogenous acetylcholine did not diminish the content of labelled tissue acetylcholine, derived from labelled glucose, suggesting that no exchange with vesicular acetylcholine took place. In the presence of 4mm-KCl it caused some increase in the release of labelled acetylcholine. 7. The barbiturates (Amytal, pentothal), whilst having no significant effects on labelled acetylcholine synthesis in unstimulated brain except at high concentration (1mm), diminished or abolished (at 0.25 or 0.5mm) the enhanced release of acetylcholine, due to high K(+) or lack of Ca(2+). The fall in tissue content of acetylcholine, due to lack of Ca(2+), was diminished or abolished by pentothal (0.25 or 0.5mm) or Amytal (0.25mm).     

Arachidonic Acid Inhibits Choline Uptake and Depletes Acetylcholine Content in Rat Cerebral Cortical Synaptosomes

The effects of arachidonic acid on [3H]choline uptake, on [3H]acetylcholine accumulation, and on endogenous acetylcholine content and release in rat cerebral cortical synaptosomes were investigated. Arachidonic acid (10-150 microM) produced a dose-dependent inhibition of high-affinity [3H]choline uptake. Low-affinity [3H]choline uptake was also inhibited by arachidonic acid. Fatty acids inhibited high-affinity [3H]choline uptake with the following order of potency: arachidonic greater than palmitoleic greater than oleic greater than lauric; stearic acid (up to 150 microM) had no effect. Inhibition of [3H]choline uptake by arachidonic acid was reversed by bovine serum albumin. In the presence of arachidonic acid, there was an increased accumulation of choline in the medium, but this did not account for the inhibition of [3H]choline uptake produced by the fatty acid. Arachidonic acid inhibited the synthesis of [3H]acetylcholine from [3H]choline, and this inhibition was equal in magnitude to the inhibition of high-affinity [3H]choline uptake produced by the fatty acid. A K+-stimulated increase in [3H]acetylcholine synthesis was inhibited completely by arachidonic acid. Arachidonic acid also depleted endogenous acetylcholine stores. Concentrations of arachidonic acid and hemicholinium-3 that produced equivalent inhibition of [3H]choline uptake also produced equivalent depletion of acetylcholine content. In the presence of eserine, arachidonic acid had no effect on acetylcholine release. The results suggest that arachidonic acid may deplete acetylcholine content by inhibiting high-affinity choline uptake and subsequent acetylcholine synthesis. This raises the possibility that arachidonic acid may play a role in the impairment of cholinergic transmission seen in cerebral ischemia and other conditions in which large amounts of the free fatty acid are released in brain.     

Pyrrolovesamicols--synthesis, structure and VAChT binding of two 4-fluorobenzoyl regioisomers

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.     

Increased Acetylcholine Content Induced by Adenosine in a Sympathetic Ganglion and Its Subsequent Mobilization by Electrical Stimulation

Abstract: 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 βγ-methylene ATP, a nonhydrolyzable 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 Ca²⁺-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.     

Tetanus toxin effect on the metabolism and release of acetylcholine in rat central nervous system

The effect of tetanus toxin in doses of 30 mcg/kg on the content, synthesis and release of acetylcholine, and on the activity of choline acetylase and acetylcholine esterase in the central nervous system of the rat was studied. The investigations were carried out after the appearance of tetanus. We found that the tetanus toxin: a) caused no changes in the acetylcholine content in the cerebral cortex and brain stem, and also in the cervical and lumbar parts of the spinal cord; b) stimulated acetylcholine synthesis in the brain stem and in the cervical and lumbar parts of the spinal cord but not in the cerebral cortex; c) activated choline acetylase; d) had no effect on acetylcholine esterase activity; e) released acetylcholine from the neurons in the brain stem and spinal cord. The release could not be inhibited by low concentration of potassium ions in the medium or increased with electrical stimulation.     

Choline increases serum insulin in rat when injected intraperitoneally and augments basal and stimulated aceylcholine release from the rat minced pancreas in vitro.

Intraperitoneal injection of choline (30-90 mg.kg-1) produced a dose-dependent increase in serum insulin, glucose and choline levels in rats. The increase in serum insulin induced by choline (90 mg.kg-1) was blocked by pretreatment with the muscarinic acetylcholine receptor antagonists, atropine (2 mg.kg-1), pirenzepine (2 mg.kg-1) and 4-diphenylacetoxy-N-methylpiperidine (2 mg.kg-1) or the ganglionic nicotinic receptor antagonist, hexamethonium (15 mg.kg-1). The effect of choline on serum insulin and glucose was enhanced by oral glucose administration (3 g.kg-1). Choline administration was associated with a significant (P < 0.001) increase in the acetylcholine content of pancreatic tissue. Choline (10-130 microm) increased basal and stimulated acetylcholine release but failed to evoke insulin release from the minced pancreas at considerably higher concentrations (0.1-10 mm). Hemicholium-3, a choline uptake inhibitor, attenuated the increase in acetylcholine release induced by choline augmentation. Choline (1-32 mm) inhibited [3H]quinuclidinyl benzilate binding to the muscarinic receptors in the pancreatic homogenates. These data show that choline, a precursor of the neurotransmitter acetylcholine, increases serum insulin by indirectly stimulating peripheral acetylcholine receptors through the enhancement of acetylcholine synthesis and release.     

Effect of Increasing Choline, In Vivo and In Vitro, on the Synthesis of Acetylcholine in a Sympathetic Ganglion

Abstract: The experiments described in this paper were designed to test whether increasing choline availability over normal physiological levels increases acetylcholine synthesis in the cat's superior cervical ganglion. When ganglia were perfused with Krebs solution, an increase in the medium's choline concentration over physiological (10⁻³M) levels increased tissue choline but did not increase tissue acetylcholine or the release of acetylcholine from stimulated ganglia. However, increasing plasma choline in the whole animal increased ganglionic acetylcholine levels. The basis for this difference in the effects of in vivo and in Vitro exposure to elevated choline levels on the tissue acetylcholine content was found to involve plasma factor(s), rather than indirect actions of choline, and the acetylcholine content of isolated ganglia was increased when the tissue was perfused with plasma, instead of Krebs solution, containing 10⁻³M-choline. The extra acetylcholine generated by this procedure was associated with a subsequent transient increase in transmitter release during short intervals of stimulation, but most of the extra acetylcholine was not readily available for release from stimulated ganglia. It is concluded that increasing choline available to sympathetic ganglia over physiological concentration does not have a sustained effect on the turnover of releasable transmitter under the conditions of these experiments.     

Nicotinic Stimulation of [3H]Acetylcholine Release from Mouse Cerebral Cortical Synaptosomes

The effects of nicotine and 1,1-dimethyl-4-phenylpiperazinium (DMPP) on the release of newly synthesized [3H]acetylcholine in mouse cerebral cortical synaptosomes were examined. Nicotine and DMPP produced increases in [3H]acetylcholine release, over the level of spontaneous release, of 24% and 30%, respectively, of a maximum depolarization-induced release produced by 50 mM potassium. The maximum effect was achieved at a concentration of 1 X 10(-4) M for both agents. The time course of release indicated a slow onset of action, reaching a maximum effect at 15 min of incubation. Both nicotine and DMPP also produced a slightly greater release of total tritium, measured in the absence of cholinesterase inhibition, than of [3H]acetylcholine. The release induced by nicotine was completely antagonized by hexamethonium and was largely (58%) calcium-dependent. Nicotine also produced an increase in [3H]choline accumulation into synaptosomes. These results indicate that the nicotinic agonists nicotine and DMPP can produce a moderate enhancement of acetylcholine release by a receptor-mediated action on cholinergic nerve terminals in the central nervous system.