The pharmacological characteristics of two types of cholinoreceptors in the membrane of dialyzed neurons

1. The ramped voltage clamp technique was developed as a rapid means of studying the effects of certain nicotinic and muscarinic agents on ionic involvement and conductance changes during acetylcholine (ACh) responses of Helix pomatia neurons. 2. Atropine was found to be a potent cholinolytic on A-type neurons, ACh responses of which are blocked by ouabain and mediated by Na+ and Cl- permeabilities, while d-tubocurarine blocked B-type ACh responses which are insensitive to ouabain and mediated by Na+ and K+ permeabilities. 3. Nicotinic agent butyrylcholine was found to be a potent cholinomimetric on B-type cells. 4. The results suggest that ACh receptors on A-type cells are more "muscarinic" while those on B-type cells are more "nicotinic". 5. It was also suggested that both muscarinic and nicotinic ACh receptors may coexist in the Helix neuronal membrane and the possibility of ACh interacting with one of them is determined by the level of phosphorylation of the membrane proteins.     

Further study of the correlation between na-pump activity and membrane chemosensitivity

Using internally dialyzed neurons of Helix, we have examined the effects of sodium-pump activity and intracellular ATP concentration on transmembrane currents induced by acetylcholine (ACh) and gamma-aminobutyric acid (GABA). We also report on the effects of pump activity and levels of intracellular ATP on binding by Helix ganglia of 3H-alpha-bungarotoxin (3H-alpha-BT) and 3H-GABA. Both ouabain-containing and potassium-free solutions depressed the neurotransmitter-induced transmembrane current of one type of dialyzed neurons. An increase in the intracellular ATP concentration led to a depression of ACh-induced currents and to the disappearance of the blocking effect of ouabain on these currents. Intracellular ADP had a similar but smaller effect on transmitter-induced currents, and intracellular AMP was ineffective. The depressing effect of internal ATP on ACh-induced currents was absent in the presence of an inhibitor of membrane phosphorylation (dinitrophenol). The binding of tritium-labeled alpha-BT and GABA to the membranes was depressed by both ouabain-containing and K-free solutions and also by compounds (theophylline and NaF) which increase the levels of intracellular ATP. The results suggest that the Na pump modulates the affinity of ACh and GABA membrane receptors by the regulation of the phosphorylated state of membrane receptors.     

Acetylcholine Mobilization in a Sympathetic Ganglion in the Presence and Absence of 2-(4-Phenylpiperidino)Cyclohexanol (AH5183)

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 mobilized by electrical stimulation to exocytose in the presence of AH5183, but it is by BWSV.     

GAS CHROMATOGRAPHIC ESTIMATION OF ACETYLCHOLINE IN THE RABBIT HEART USING A NITROGEN SELECTIVE DETECTOR

The distribution of ACh in the rabbit heart was investigated by a modified gas chromatographic estimation method. ACh was extracted with perchloric acid, precipitated as reineckate and demethylated with sodium benzenethiolate. The tertiary amines derived from ACh and other choline esters were concentrated by a microdistillation procedure. Gas chromatography was performed using a nitrogen selective detector. In the range of concentrations between 0.4 and 2.5 nmol ACh per tissue sample the coefficient of variation was 5.2 per cent. The recovery of ACh added to heart extracts was 101 per cent. Evidence for the identity of the choline ester isolated from rabbit hearts and authentic ACh was obtained by equal retention times and by correspondence of the ratio N/C of the respective tertiary amines. Parallel measurements using gas chromatography and bioassay on the rat blood pressure yielded closely corresponding values of ACh levels in the rabbit heart. The concentration of ACh was much higher in the atria than in the ventricles. In both atria, and ventricles the ACh concentration was higher in the right than in the left part of the rabbit heart. Endogenous propionylcholine or butyrylcholine were not detected.     

A Comparison of Responses to Raised Extracellular Potassium and Endothelium-Derived Hyperpolarizing Factor (EDHF) in Rat Pressurised Mesenteric Arteries

The present study examined the hypothesis that potassium ions act as an endothelium-derived hyperpolarizing factor (EDHF) released in response to ACh in small mesenteric arteries displaying myogenic tone. Small mesenteric arteries isolated from rats were set up in a pressure myograph at either 60 or 90 mmHg. After developing myogenic tone, responses to raising extracellular potassium were compared to those obtained with ACh (in the presence of nitric oxide synthase and cyclo-oxygenase inhibitors). The effects of barium and oubain, or capsaicin, on responses to raised extracellular potassium or ACh were also determined. The effects of raised extracellular potassium levels and ACh on membrane potential, were measured using sharp microelectrodes in pressurised arteries. Rat small mesenteric arteries developed myogenic tone when pressurised. On the background of vascular tone set by a physiological stimulus (i.e pressure), ACh fully dilated the small arteries in a concentration-dependent manner. This response was relatively insensitive to the combination of barium and ouabain, and insensitive to capsaicin. Raising extracellular potassium produced a more inconsistent and modest vasodilator response in pressurised small mesenteric arteries. Responses to raising extracellular potassium were sensitive to capsaicin, and the combination of barium and ouabain. ACh caused a substantial hyperpolarisation in pressurized arteries, while raising extracellular potassium did not. These data indicate that K⁺ is not the EDHF released in response to ACh in myogenically active rat mesenteric small arteries. Since the hyperpolarization produced by ACh was sensitive to carbenoxolone, gap junctions are the likely mediator of EDH responses under physiological conditions.     

Unique ionotropic receptors for D-aspartate are a target for serotonin-induced synaptic plasticity in Aplysia californica

The non-L-glutamate (L-Glu) receptor component of D-aspartate (D-Asp) currents in Aplysia californica buccal S cluster (BSC) neurons was studied with whole cell voltage clamp to differentiate it from receptors activated by other well-known agonists of the Aplysia nervous system and investigate modulatory mechanisms of D-Asp currents associated with synaptic plasticity. Acetylcholine (ACh) and serotonin (5-HT) activated whole cell excitatory currents with similar current voltage relationships to D-Asp. These currents, however, were pharmacologically distinct from D-Asp. ACh currents were blocked by hexamethonium (C6) and tubocurarine (D-TC), while D-Asp currents were unaffected. 5-HT currents were blocked by granisetron and methysergide (MES), while D-Asp currents were unaffected. Conversely, while (2S,3R)-1-(Phenanthren-2-carbonyl)piperazine-2,3-dicarboxylic acid(PPDA) blocked D-Asp currents, it had no effect on ACh or 5-HT currents. Comparison of the charge area described by currents induced by ACh or 5-HT separately from, or with, D-Asp suggests activation of distinct receptors by all 3 agonists. Charge area comparisons with L-Glu, however, suggested some overlap between L-Glu and D-Asp receptors. Ten minute exposure to 5-HT induced facilitation of D-Asp-evoked responses in BSC neurons. This effect was mimicked by phorbol ester, suggesting that protein kinase C (PKC) was involved.     

The measurement of nanogram amounts of acetylcholine in tissues by pyrolysis gas chromatography

Abstract— A method previously described for measuring ACh in biological effluents has been simplified and extended for use with tissues. The tissue is homogenized in acetonitrile containing propionylcholine as the internal standard and after centrifugation the acetonitrile is removed by shaking with toluene. To the aqueous solution is added a solution of KI-I₂ to precipitate the quaternary compounds. The precipitate is dissolved in aqueous acetonitrile and then drawn through a small column of ion-exchange resin to convert the periodides of the quaternary compounds to chlorides which are then simultaneously pyrolysed and gas chromatographed. On the column the pyrolytic product of choline has a slower retention time than that of acetylcholine; under these circumstances the choline present in tissues does not obscure the measurement of acetylcholine. Specificity was demonstrated by several procedures including mass spectroscopy. The method can measure 25 ng (171 pmoles) of acetylcholine in extracts of brain, simply, and with high reproducibility. With the usual gas chromatograph, 16 samples can be run in a working day. The content of acetylcholine in rat brain was 26.4 nmol/g or almost precisely the values found with other gas chromatographic methods. The pyrolytic method was shown to be applicable to the detection of biologically interesting substances other than choline esters, including betaine, carnitine and the non- quaternary compound, ?-aminobutyric acid, which is readily converted to a volatile compound (probably its methyl ester) when pyrolysed in the presence of tetramethylammonium hydroxide. Of additional general interest is the demonstration of the advantages of acetonitrile as a solvent for extracting water-soluble compounds from tissues.     

Sodium currents in muscles incubated in potassium-free Ringer's solution. Effect of ouabain on unidirectional sodium currents

Frog sartorius muscles subjected to loading with Na in K-free Ringer solution in the cold were subsequently labelled with 22Na. The uptake of 22Na is not sensitive to ouabain (10(-4) M) while sodium efflux is decreased by oubain. It is concluded that ouabain-sensitive Na-for Na interchange is not present in this condition. Possibly ouabain-sensitive sodium efflux is partly or completely potassium-requiring fraction since some K (approximately 10 microM) is inevitably present in K-free solution. The increase in the rate constant for potassium loss in the presence of ouabain favours this supposition.     

Direct measurement of the concentration- and time-dependent open probability of the nicotinic acetylcholine receptor channel.

Using the outside-out patch clamp recording technique together with a rapid solution exchange system, we measured ionic currents through nicotinic acetylcholine (ACh) receptor channels from BC3H-1 cells in response to rapid applications of 0.3-1,000 microM ACh. We used nonstationary fluctuation analysis of ensembles of responses to deduce the number of channels in the patch, the maximum open channel probability as a function of ACh concentration and the time course of a fast desensitization process. We found that: (a) Excised patches from BC3H-1 cells typically contain between 50 and 150 functional ACh receptor ion channels. (b) The open channel probability is proportional to [ACh]1.95 at low concentrations of ACh, is half-maximal at 20 microM ACh and saturates above 100 microM ACh. (c) ACh is a very efficacious agonist; 100 microM ACh opens at least 90% of the available channels. This estimate of efficacy is model-independent. (d) The rate of decay of the agonist-induced current is concentration-dependent. In the presence of 100 microM ACh the current decays with a time constant of 50-100 ms. It decays more slowly in the presence of lower concentrations of agonist but is relatively insensitive to voltage.     

The Na,K pump regulates a decrease in the cholinosensitivity of the neurons in the snail Helix lucorum taurica Kryn in a cellular analog of habituation: its dependence on intracellular calcium

In Helix lucorum snail we studied the effects of ouabain, inhibitor of Na,K-pump, on the depression of cholinosensitivity in command neurons of withdrawal behavior and the role of the intracellular free Ca2+. The cellular analog of the negative learning (habituation) was used Transmembrane integral inward currents were recorded from the identified LPa2, LPa3, RPa3, and RPa2 neurons in ganglia preparation using two-electrode voltage clamp technique. Acetylcholine (ACh) was locally applied iontophoretically. Reduction of neuronal cholinosensitivity was estimated as a depth of depression of the ACh-induced inward current during rhythmic local application of ACh (interstimulus interval of 1-3 min) onto the somatic membrane. Bath application of ouabain (0.1 mM) produced an increase in depression in one group of neurons and its decrease in another group. After 60-150 min of spontaneous diffusion of a calcium ion chelator BAPTA (1 mM) from the intracellular microelectrode, ouabain produced only the increase in depression. If CaCl2 (100 mM) was added to the solution of the voltage-recording intracellular microelectrode, 60 min later ouabain produced only the reduction of the depression of the ACh current. The conclusion is drawn that the inhibition of the Na,K-pump by ouabain modifies the depression of neuronal cholinosensitivity in the cellular analog of habituation. The direction of the modulatory effect depends on the basal concentration of the intracellular free Ca2+.     

Isolation of choline esters from aqueous solutions by extraction with sodium tetraphenylboron in organic solvents

1. The method is based on the observation that choline esters and sodium tetraphenylboron (Kalignost) form complexes that are insoluble in water but soluble in organic solvents such as nitriles, higher ketones and benzyl alcohol. 2. The extraction procedure is an example of liquid cation exchange where tetraphenylboron is the cation-exchange group. 3. The proportion of choline esters extracted depends on the type and total amount of cation in the aqueous phase and the amount of sodium tetraphenylboron in the organic solvent. 4. The proportion of choline esters extracted is independent of the choline ester concentration, the pH (between 8 and 3) and the relative volumes of the two phases. 5. The affinity of sodium tetraphenylboron for choline esters increases with an increase in the size of the acyl group. 6. The choline ester extracted can be released into an aqueous solution by treatment with strong acids, silver salts and anion-exchange resins.     

Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

Endogenous acetylcholine release and labelled acetylcholine formation from [3H]choline in the myenteric plexus of the guinea-pig ileum.

The spontaneous release of acetylcholine (ACh) from the guinea-pig myenteric plexus - longitudinal muscle preparation superfused at a constant rate in the presence of physostigmine was 10 nmol-g-1-h-1. This release was decreased to one-third by tetradotoxin or by MnCl2 and increased 2.5 times by 0.1 Hz and 20 times by 16 Hz stimulation. The formation of [3H]ACh from [3H]choline increased from 3 to 33 nmol-g(-1)-h(-1) when the concentration of [3H]choline was increased from 1 muM to 50 muM. The rate of [3H]ACh formation was not affected by tetrodotoxin, MnCl2, or physostigmine in the absence of stimulation. It was increased by 50% by 0.1 Hz and by 100% by 16 Hz stimulation during the first 9 min of exposure to [3H]choline but not subsequently. The myenteric plexus - longitudinal muscle preparation contains 200 nmol/g choline. Results suggest that the apparent small [3H]ACh formation from low concentrations of [3H]choline is due to the dilution of [3H]choline by endogenous choline. The major part of [3H]ACh formation appears to be due to the intracellular turnover of ACh while the evoked release of [3H]ACh appears to originate from a small pool.     

Mechanism of hydrolysis of dicholine esters with long polymethylene chain by human butyrylcholinesterase

Human butyrylcholinesterase hydrolyzes long chain dicholine esters more rapidly than short chain dicholine esters. The active site of butyrylcholinesterase is deeply buried within the enzyme molecule and there is limited space for binding of large compounds. Our goal was to understand how butyrylcholinesterase accommodates long chain dicholine esters to make them better substrates than short chain dicholine esters. For this purpose we studied the rate of hydrolysis of adipyldicholine (n=4) and sebacyldicholine (n=8) with mass spectrometry, a method that allowed monitoring the dicholine substrates, the monocholine intermediates, the dicarboxylic acid and choline products. It was shown that hydrolysis of adipyldicholine involves two consecutive steps, dicholine ester hydrolysis followed by relatively slow monocholine ester hydrolysis. However, sebacyldicholine was hydrolyzed at both choline ester sites, though hydrolysis of dicholine was faster than hydrolysis of monocholine. Sebacyldicholine was completely converted to sebacic acid and choline within 90 min, whereas only 15% of the adipyldicholine was converted to adipic acid in this time. Molecular modeling indicated that these dicholine esters can bind to butyrylcholinesterase in two energetically equivalent alternative conformations that may theoretically lead to hydrolysis. The long chain dicholine ester makes closer contact than the short chain ester between one of its carbonyl carbons and the catalytic Ser198, thus explaining why long-chain dicholine esters are hydrolyzed more rapidly by butyrylcholinesterase.     

The Effect of Ouabain on the Release of [14C]Acetylcholine and Other Substances from Synaptosomes

Abstract: The effect of ouabain and dihydroouabain on Na⁺-K⁺ ATPase, ⁸⁶Rb uptake and the release of [¹⁴C]ACh (acetylcholine) from synaptosomal preparations of guinea pigs was compared. At low concentrations of glycoside (<50 μm ) there was a good correlation between the potency of ouabain and of dihydroouabain in inhibiting Na⁺-K⁺ ATPase and in causing the release of [^l4C]ACh in a nondepolarising medium. Ouabain (200 μM) increased the release of [¹⁴C]ACh evoked by 25 mm -KCl, but not that evoked by 100μm -veratrine. The enhancement of release was independent of the presence of calcium. It was observed that in addition to [¹⁴C]ACh release, choline efflux was also stimulated by ouabain, independently of the presence of Ca²⁺. Experiments with hemicholinium-3 showed that the ouabain-induced increase in choline efflux was not due to an inhibition of reuptake. The effect of ouabain on intrasynaptosomal K⁺ concentration was measured in order to investigate the degree of depolarisation it caused. The decrease in K⁺ was found to be similar in magnitude and time course to that caused by veratrine. It was shown that ouabain-induced depolarisation caused an increased efflux of another positive ion (dibenzyldimethylammonium chloride) and retention of a negatively charged ion (chloride), as would be expected from the operation of the electrochemical potential gradient changing as a result of depolarisation. It is suggested that ouabain acts to stimulate ACh release from synaptosomes as follows: following blockage of the Na⁺-K⁺ ATPase there is rapid depolarisation which, if Ca²⁺ is present, provokes the normal Ca²⁺-dependent transmitter release process to occur. In addition, depolarisation accelerates the leakage of positive ions down their electrochemical potential gradient, but causes a retention of negative ions. Such an action does not depend on the presence of Ca²⁺, nor is it specific to transmitters.     

REGIONAL BIOSYNTHESIS OF ACETYLCHOLINE IN BRAIN FOLLOWING FORCED ORAL CHRONIC BARBITONE TREATMENT TO RAT

Rats received a solution of sodium barbitone as their only drinking fluid for 33 and 42–44 weeks. In three groups (A3, A12 and A30) the barbitone solution was withheld and replaced by water 3, 12 and 30 days respectively before death. Two other groups consisted of animals drinking barbitone until death (B) and untreated controls (C). Abstinence convulsions were recorded by jiggle cages. Thirty nmol of tritium-labelled choline ([³H]Ch) were injected i.v. and the rats were killed by decapitation 1 min later. A significantly higher content of tritium-labelled acetylcholine ([³H]ACh) was found in the cerebellum + medulla oblongata + midbrain of rats receiving barbitone until death (group B) (+22%) and abstinent for 3 days (+54%) (group A3) compared with group C. The [³H]ACh content was also significantly increased in the hippocampus + cortex of rats abstinent for 3 days (+23%). In the striatum no significant effect on [³H]ACh content was found in any of the groups. The ratio [³H]ACh/[³H]Ch was significantly increased in the cerebellum + medulla oblongata + midbrain of rats in group B and A3 and in the hippocampus + cortex in group A3. These results might indicate an increased turnover of ACh. The effect of long-term barbitone treatment on the enzyme activities of brain choline acetyltransferase and acetylcholinesterase was also studied but no significant effect was found.     

Discrimination Between A-type and B-type Conformations of Double Helical Nucleic Acid Fragments in Solution by Means of Two-dimensional Nuclear Overhauser Experiments

Abstract

The solution structure of two double helical nucleic acid fragments, viz. r(CGCGCG) and d(CGCGCG), was probed by means of two-dimensional nuclear Overhauser effect spectroscopy. The two compounds were selected as models for the A-type and B-type double helical conformations, respectively, and it is shown that for each of the two model compounds the intensities of the NOE cross peaks between base- and H2′ (deoxy)ribose proteins are qualitatively in correspondence with the relative NOE intensities expected on basis of the supposed duplex conformations. Thus our results indicate that NOE-data can be used to differentiate between A- and B-type double helical conformations in solution.

Coupling constant data show that, except for G(6), all ribose rings in r(CGCGCG) adopt pure N (C3′-endo) conformations thereby manifesting that this molecule takes up a regular A-type double helical conformation in solution. In contrast, the deoxyribose rings in d(CGCGCG) retain conformational freedom in the duplex state, albeit that the N/S- equilibrium is biased towards the S (C2′-endo) sugar conformation. This finding indicates that in solution the B-DNA backbone is highly dynamic.     

Choline availability and acetylcholine synthesis in the hippocampus of acetylcholinesterase-deficient mice

Mice deficient for acetylcholinesterase (AChE) have strongly increased extracellular levels of acetylcholine (ACh) in the dorsal hippocampus [Hartmann, J., Kiewert, C., Duysen, E.G., Lockridge, O., Greig, N.H., Klein, J., 2007. Excessive hippocampal acetylcholine levels in acetylcholinesterase-deficient mice are moderated by butyrylcholinesterase activity. J. Neurochem. 100, 1421-1429]. Using microdialysis, we found that increased ACh levels are accompanied by decreased levels of extracellular choline which were 1.60 microM in AChE-deficient mice and 4.36 microM in wild-type mice. Addition of choline (10 microM) to the perfusion fluid, while ineffective in wild-type animals, more than doubled extracellular ACh levels in AChE-deficient mice. High-affinity choline uptake (HACU), as measured ex vivo in corticohippocampal synaptosomes, was more than doubled in AChE-deficient mice. Inhibition of HACU by hemicholinium-3 (HC-3) in vivo reduced extracellular levels of ACh by 60% in wild-type mice but by more than 90% in AChE-deficient mice. Decreased ACh levels caused by infusion of HC-3 or tetrodotoxin (TTX) were accompanied by increased levels of free choline. Infusion of scopolamine (1 microM) caused a fivefold increase of ACh levels in wild-type animals but only a 50% increase in AChE-deficient mice. In conclusion, absence of AChE causes dynamic changes in the ratio of choline to ACh. High levels of extracellular ACh are accompanied by reduced levels of extracellular choline, and ACh release becomes strongly dependent on choline availability. Similar changes may take place in patients chronically exposed to AChE inhibitors.     

On the Mechanism of Ouabain-Induced Release of Acetylcholine from Synaptosomes

Ouabain (5 x 10(-8)-5 x 10(-4) M) was confirmed to cause a dose-dependent increase in [3H]acetylcholine ([3H]ACh) release, cytosolic free Ca2+ concentration ([Ca2+]i), and 22Na+ uptake in cerebrocortical synaptosomes of rats in the presence of extracellular Ca2+. Ouabain also caused a dose-dependent decrease in membrane potential. In a low-Na+ (10 mM) medium, ouabain failed to increase [3H]ACh release and [Ca2+]i. Tetrodotoxin (10(-6) M) had no effect on the ouabain-induced increase in both [3H]ACh release and [Ca2+]i but abolished the increase in 22Na+ uptake and partially inhibited the depolarizing effect. Verapamil (10(-6)-5 x 10(-4) M) inhibited the ouabain-induced increase in both [3H]ACh release and [Ca2+]i in a dose-dependent manner. Removal of extracellular Ca2+ abolished the effect of ouabain on [Ca2+]i but not on [3H]ACh release and 22Na+ uptake, regardless of the presence or absence of EGTA. In the absence of extracellular Ca2+, 10 mM Mg2+ blocked ouabain-induced [3H]ACh release, which was resistant to verapamil. These results suggest that ouabain can increase ACh release from synaptosomes without the preceding increases in intracellular Ca2+ and/or Na+ content. It seems likely that the removal of extracellular Ca2+ unmasks mechanisms of ouabain action different from those operating in the presence of Ca2+.     

The source of choline for acetylcholine synthesis in brain

More is known about the synthesis and metabolism of acetylcholine (ACh) than other choline (Ch) containing compounds in the brain in spite of the fact that ACh represents only a small fraction of the total Ch esters. This review will attempt to summarize the evidence for the source of Ch in the brain and its relation to the turnover of ACh. Ch is a precursor not only for ACh but also for phosphoryl Ch and phospholipids. It appears that in the rat a bound form of Ch in the brain can produce free Ch which can leave the brain, be converted to ACh or be reutilized for phospholipid synthesis. There is evidence that one of the sources of free Ch that is utilized for ACh synthesis is outside the cholinergic nerve terminal.