RELATIONSHIP BETWEEN CHOLINE AVAILABILITY AND ACETYLCHOLINE SYNTHESIS IN DISCRETE REGIONS OF RAT BRAIN

Abstract— The relationship between choline availability and the synthesis of acetylcholine in discrete brain regions was studied in animals treated with the organophosphorus cholinesterase inhibitor paraoxon. Administration of paraoxon (0.23 mg/kg) inhibited acetylcholinesterase activity by approx 90% in the striatum, hippocampus and cerebral cortex and increased acetylcholine levels to 149%, 124% and 152% of control values, respectively. Free choline levels were unaltered by paraoxon in the hippocampus and cerebral cortex, but were significantly decreased in the striatum to 74% of control. When animals were injected with choline chloride (60 mg/kg), 60 min prior to the administration of paraoxon, the paraoxon-induced choline depletion in the striatum was prevented and the paraoxon-induced acetylcholine increase was potentiated from 149% to 177% of control values. Choline pretreatment had no significant effect in either the hippocampus or cerebral cortex, brain regions that did not exhibit a decrease in free choline levels after paraoxon administration. Results indicate that choline administration, which had no significant effect on acetylcholine levels by itself, increased acetylcholine synthesis in the striatum in the presence of acetylcholinesterase inhibition. However, this effect was not apparent in either the hippocampus or the cerebral cortex at similar levels of enzyme inhibition. It appears that choline generated from the hydrolysis of acetylcholine may play a significant role in the regulation of neurotransmitter synthesis in the striatum, but not in the other brain areas studied. The evidence supports the concept that the regulatory mechanisms controlling the synthesis of acetylcholine in striatal interneurons may differ from those in other brain regions.     

ON THE TURNOVER OF ACETYLCHOLINE IN NERVE ENDINGS OF MOUSE BRAIN IN VIVO

Abstract— Acetylcholine is synthesized and stored in the nerve endings from which the liberation of the nerve transmittor is regulated by the nerve activity. The aim of the present investigation was to measure the in vivo turnover of acetylcholine in this subcellular acetylcholine pool. This has been carried out by injecting labelled choline intravenously and then by measuring at different time intervals the ratio between labelled choline and acetylcholine in the fractions obtained after subcellular fractionation. It was found that the ratio radioactive choline to radioactive acetylcholine was the same (2:1) in whole brain and in the nerve ending fraction 2 to 20 min after injection. Since it was assumed that the same ratio is true also for the endogenous compounds the choline pool in the nerve terminals was considered to make up 13 nmoles/g brain. The results also indicate that plasma choline is rapidly equilibrated with the nerve terminals and transformed to acetylcholine at a rate of about 5 nmoles/g brain/min.     

THE DETERMINATION OF PICOMOLE AMOUNTS OF ACETYLCHOLINE IN MAMMALIAN BRAIN

Abstract— In any assay for the determination of acetylcholine based on the conversion of choline to a product, the immediate problem is the removal of endogenous choline. Other published enzymatic assays have taken advantage of electrophoresis to accomplish this goal. In the assay to be described, this is accomplished by the enzymatic phosphorylation of endogenous choline by choline kinase. Once this reaction is complete, endogenous acetylcholine is simultaneously hydrolysed and then phosphorylated with [³²P]ATP. The labelled product [³²P]phosphorylcholine is separated from the labelled substrate by precipitation of the ATP and further separation is accomplished on microcolumns of ion exchange resin. Using this methodology, picomole amounts of acetylcholine, derived from tissue, can be measured.     

A TECHNIQUE FOR THE STUDY OF ACETYLCHOLINE TURNOVER IN MOUSE BRAIN IN VIVO

Abstract— —A method to measure the rate of acetylcholine turnover in mouse brain in vivo has been developed. It is based on the formation of labelled acetylcholine from intravenously injected labelled choline. The isotopic dilution of choline in the brain has been measured by assaying endogenous choline in the brain by an enzymatic method using tritium-labelled acetyl-CoA and purified choline acetyltransferase.
The rate of acetylcholine turnover in the brain could be calculated at 50 n-moles acetylcholine/g/min in conscious mice. In anaesthetized mice and in mice treated with oxotremorine, a decrease of acetylcholine turnover to about 10 n-moles/g/min was found.     

CHOLINE AND ACETYLCHOLINE IN RATS: EFFECT OF DIETARY CHOLINE

Abstract– The concentration of free choline in peripheral tissues (duodenum, heart, kidney, liver, stomach and plasma) of rats was found to be related to the amount of free choline in the diet. Under steady-state conditions, the concentration of free choline in plasma varied from a minimum of approx 6 nmol/ml (in rats fed a choline-deficient diet) to a maximum value not exceeding 21 nmol/ml. The concentration of plasma choline was elevated above 21 nmol/ml for a short time after parenteral administration of choline chloride or one of its precursors (CDP choline or phosphorylcholine), but was not affected by stress, endocrine manipulations, drug treatments or the time of day when rats were killed. The metabolism of intravenously administered [methyl-³H] choline was accelerated in peripheral tissues (except plasma) of choline-deficient rats, indicating that free choline is not preserved during choline deficiency by a reduction in its rate of turnover. Furthermore, the decrease in concentration of plasma choline that occurred in rats fed a choline-deficient diet was prevented by addition of deanol (dimethylaminoethanol) to the diet. These results indicate that free choline in peripheral tissues of rats is derived from both free choline in the diet, and from precursors of choline present within the diet. In contrast to the effects in peripheral tissues, the concentration of free choline in brain was not reduced by dietary deprivation of free choline; however, the increase in free choline that occurred when rats were decapitated was reduced in brains by deficiency of choline, suggesting a decrease in the concentration of esterified forms of cerebral choline. The concentration of acetylcholine was not reduced in the brain, duodenum, heart, kidney or stomach of 21-week old rats raised from birth on a choline-deficient diet, in the duodenum of rats given a choline-deficient diet for 1, 5 or 11 days, or in brains of rats deprived of free choline for 1 or 11 days. However, the rate of in vivo synthesis of ACh from [methyl-³H]choline was accelerated in cholinergic tissues that were depleted of free choline (i.e. duodenum, heart and stomach).     

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K⁺ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K⁺ media. When labeled choline was present during the release incubation, a‘newly-synthesized’pool of acetylcholine was formed in the tissue. In toxin-treated slices exposed to high K⁺, both the production and the release of this‘newly-synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [³H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.     

IMPAIRED SYNTHESIS OF ACETYLCHOLINE IN BRAIN ACCOMPANYING MILD HYPOXIA AND HYPOGLYCEMIA

Abstract— Lowering the concentration of oxygen or of glucose to which mouse and rat brains were exposed impaired the synthesis of acetylcholine from labelled precursors in vivo. Histotoxic hypoxia induced with KCN or anemic hypoxia induced with NaNO₂ (to oxidize hemoglobin to methemoglobin) reduced incorporation of [²H₄]choline into acetylcholine. This change in acetylcholine metabolism occurred with doses of KCN or NaNO₂ which did not alter the concentrations of ATP or ADP or the adenylate energy charge. Hypoglycemia induced by large doses of insulin also reduced the incorporation of [²H₄]choline into acetylcholine. Both hypoxia and hypoglycemia increased the concentration of choline in the brain. The specific activity of choline did not decrease in hypoxia; it did not decrease enough in hypoglycemia to explain the reduced incorporation of [²H₄]choline into acetylcholine. Pretreatment with the cholinesterase inhibitor physostigmine delayed the onset of both seizures and death in mice after induction of hypoxia by large doses of NaNO₂. Pretreatment with physostigmine also decreased the number of mice dying within 3 h after the induction of hypoglycemia with large doses of insulin. These observations suggest that the effects of hypoxia and hypoglycemia interfere with the synthesis of a critical pool of acetylcholine. The incorporation of labelled precursors into acetylcholine related linearly to both the cytoplasmic redox state (NAD/NADH ratio) and to the NAD/NADH potential across the mitochondrial membrane. The redox potential of NAD/NADH in the cytoplasm was calculated from the [pyruvate]/[lactate] equilibrium and the redox potential of NAD/NADH in the mitochondria from the [NH4][2-oxoglutar-ate]/[glutamate] equilibrium. The potential across the mitochondrial membrane was calculated from the difference. These observations indicate that carbohydrate oxidation is one of the factors on which the synthesis of the neurotransmitter acetylcholine depends closely in mouse and rat brain.     

LABELLING OF ACETYLCHOLINE IN THE BRAIN OF MICE FED ON A DIET CONTAINING DEUTERIUM LABELLED CHOLINE: STUDIES UTILIZING GAS CHROMATOGRAPHY—MASS SPECTROMETRY

—A method to achieve labelling of the acetylcholine stores of the brain under ideal physiological conditions is described. To this end, mice fed on a choline free diet were supplied with deuterium labelled choline in the drinking water. Labelled and unlabelled choline in plasma and in the brain as well as labelled and unlabelled acetyicholine in the brain were measured by a gas chromatographic-mass spectrometric method. It was found that after 1–25 days on the deuterium choline diet, substantial amounts of the plasma choline and brain acetylcholine were displaced by deuterium choline and deuterium acetylcholine, respectively. Already on the first day, the mole ratio of deuterium choline/total choline in plasma was 0·22, and it approached a maximum of 0·57 on the 14th day. The mole ratios of deuterium acetylcholine/total acetylcholine in the brain were slightly but significantly lower than those of deuterium choline/total choline in plasma 1–14 days, but asymptotically approached the mole ratios of deuterium Ch/total Ch in plasma by 25 days. Intact brains submitted to incubation at room temperature for 10 min increased their total choline content by about 500 per cent. Concurrently, in brains from animals kept on a deuterium choline diet for 1–2 days, the level of deuterium choline rose only by 50 per cent after incubation. Deuterium choline levels increased, however, by 200–300 per cent in the brains from animals kept on the deuterium diet for longer time periods. On the basis of these data it is suggested that: (a) choline in plasma is partly supplied from the food and partly from endogenous sources; (b) plasma choline rapidly equilibrates (less than one day) with a pool of Ch in the brain which is responsible for biosynthesis of acetylcholine; (c) the size of this choline pool is in the order of 34–40 nmol/g.     

FURTHER EXPERIMENTS ON THE UPTAKE OF ACETYLCHOLINE AND ATROPINE AND THE RELEASE OF ACETYLCHOLINE FROM MOUSE BRAIN CORTEX SLICES AFTER TREATMENT WITH PHOSPHOLIPASES

—Uptake of acetylcholine (ACh) in mouse brain cortex slices, previously shown with ACh synthesized from tritiated choline is confirmed with acetyl[1-¹⁴C]choline. Radioactivity from tritiated sodium acetate also accumulates in slices, but forms hardly any ACh. Uptake of ACh increases in a Ca²⁺-free medium, decreases again upon addition of a 3 × 10⁵ molar concentration of an anticholinergic benzilate compound and is completely blocked by the same compound at 3 × 10³ m. Slices preloaded with labelled ACh release, after extensive washing, some of their radioactivity into an outer medium free from ACh. Phospholipase, A or C, increases the release of radioactivity from the slices. An equilibrium is reached both with controls and phospholipase-treated slices. Remaining radioactivity seems to be due to bound ACh. Calcium and magnesium ions have no effect on the uptake of tritiated atropine, although low concentrations of Ca²⁺ decrease the effects of phospholipase C on atropine uptake. The inhibitory effect of K⁺ on atropine uptake disappears completely after treatment with small amounts of phospholipase A, but even high concentrations of phospholipase C have no effect.     

THE ORIGIN OF THE ACETYLCHOLINE RELEASED FROM THE SURFACE OF THE CORTEX

—The specific radioactivity of acetylcholine liberated from the surface of the rabbit occipital cortex has been compared with that of the underlying cortical synaptosomal and vesicular acetylcholine at varying times after the administration of [N-Me-³H]choline. Choline was administered by diffusion from solutions placed in cups formed by Perspex cylinders applied to the surface of the cortex. Acetylcholine was collected by diffusion into these cups. The specific radioactivity of the acetylcholine declined progressively. The effect of stimulation of afferent cholinergic pathways was to cause a fall in the specific radioactivity of the released acetylcholine. However this was always higher than that of the synaptosomal or vesicular acetylcholine as represented by fractions P₂ and D of the authors’fractionation scheme. It is concluded that acetylcholine released from the cortex must come from a store or stores more recently synthesized than the endogenous acetylcholine of these subcellular fractions.     

SODIUM-DEPENDENT HIGH AFFINITY CHOLINE UPTAKE: A REGULATORY STEP IN THE SYNTHESIS OF ACETYLCHOLINE

The sodium-dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50-80% reduction in sodium-dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal-hippocampal or habenuleinterpeduncular tracts by lesions (10 min-1 h) also caused a similar, large reduction in sodium-dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively. We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal-hippocampal tract. Stimulation (40 Hz) for 10-15 min completely reversed the depression in sodium-dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal. Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium-de-pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration. We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium-dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake. We examined the conversion of [³H]choline to [³H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60-70% of the total sodium-dependent tritium content was present as [³H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake. A kinetic analysis of sodium-dependent high affinity choline uptake was performed after all treatments. We found changes in V_max, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium-dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium-dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.     

THE EFFECT OF Cl− ON CHOLINE ACETYLTRANSFERASE KINETIC PARAMETERS AND A PROPOSED ROLE FOR Cl− IN THE REGULATION OF ACETYLCHOLINE SYNTHESIS

Abstract— Crude or purified rat brain choline acetyltransferase (ChAc) is activated by anions. Among anions, Cl⁻ is the most effective and may promote an up to 60 fold increase in V _max. In the absence of Cl⁻, at low ionic strength, acetylcholine (ACh) is a good ChAc inhibitor ( K _i= 0.310 m m ). The ACh inhibition becomes negligible when Cl⁻ is increased to 145 m m (ACh K _i= 45 m m ). These results are discussed in terms of regulation of ACh synthesis by nerve terminals. It is proposed that ChAc is part of a presynaptic membrane bound multienzymatic complex under direct control of the ion fluxes promoted by nerve impulses.     

PARTIAL PURIFICATION AND PROPERTIES OF CHOLINE KINASE (EC 2.7.1.32) FROM RABBIT BRAIN: MEASUREMENT OF ACETYLCHOLINE

Choline kinase (EC 2.7.1.32; ATP: choline phosphotransferase) was purified 200-fold from an extract of acetone powder of rabbit brain by a combination of acid precipitation, ammonium sulphate precipitation, DEAE cellulose chromatography, and ultrafiltration. Maximal activity of 243 nmol of phosphorylcholine synthesized. min^?1 mg^?l of protein occurred at pH 9.5–10.0 in the presence of 10 mm MgS0₄, 10 mm choline and 0.005% (w/v) bovine serum albumin. 2-Aminoethanol, 2-methylaminoethanol, and 2-dimethylaminoethanol were also phosphorlyated by the enzyme preparation. The enzyme quantitatively converted low concentrations of choline (2.5–50 μm ) to phosphorylcholine [³²P] in the presence of ATP [y³²P], and may, therefore, be used to measure small amounts of choline acetylcholine. There were two K_m values for choline at pH 9.5; 32 μm and 0.31 mm . At pH 7.4, the higher K_m was not observed and enzyme activity was maximal with 0.1 mm choline. The K_m for ATP was 1.1 mm . Enzyme activity was inhibited by ATP (20 mm ), AMP, ADP, cytidine diphosphocholine (1 or 10 mm ), and activated by choline esters (1.0 mm ), NaCl or KCl(200 mm ).     

ASPECTS OF ACETYLCHOLINE METABOLISM IN THE ELECTRIC ORGAN OF TORPEDO MARMORATA

Abstract— —The synthesis of acetylcholine and its compartmentation were studied in the electric organ of Torpedo marmorata. When electric organ was homogenized in iso-osmotic NaCl-sucrose some 55 per cent of its acetylcholine content was lost unless very potent cholinesterase inhibitors were present. Slices of electric organ incubated in a suitable medium were found to synthesize radioactive-labelled acetylcholine from [ N-Me-³ H] choline. The specific activity of the labelled acetylcholine was higher in the trichloracetic acid extract of the organ slices than in an NaCl-sucrose homogenate. Acetylcholine-containing vesicles isolated from the NaCl-sucrose homogenate contained labelled acetylcholine with about the same specific activity as the parent homogenate. There was thus a fraction of acetylcholine in the incubated tissue of higher specific radioactivity that was lost when the tissue was homogenized. The acetylcholine-containing vesicles lose their acetylcholine when submitted to gel filtration under hypo-osmotic conditions. On standing at 5°C there were only small losses of acetylcholine from the vesicles but at 20°C the losses were substantial. Vesicles containing labelled acetylcholine were studied. On gel filtration under iso-osmotic conditions there was a considerable loss of labelled acetylcholine without a concomitant loss of bio-assayable acetylcholine. The pools of radioactive and bio-assayable acetylcholine are therefore not homogeneous in the vesicles as isolated.     

EFFECTS OF LITHIUM TREATMENT IN VITRO AND IN VIVO ON ACETYLCHOLINE METABOLISM IN RAT BRAIN

Abstract— The effects of LiCl on cholinergic function in rat brain in vitro and in vivo have been investigated. The high affinity transport of choline and the synthesis of acetylcholine in synaptosomes were reduced when part (25-75%) of the NaCl in the buffer was replaced with LiCl or sucrose. This appeared to be due to lack of Na⁺ rather than to Li⁺, as addition of LiCl to normal buffer had little effect. Following an injection of LiCl (10mmol/kg, i.p.) into rats the concentration of a pulsed dose of [²H₄]choline (20 μmol/kg, i.v., 1 min) and its conversion to [²H₄]acetylcholine, and the concentrations of [²H₂]acetylcholine and [²H₀]choline were measured in the striatum, cortex, hippocampus and cerebellum. The [²H₄]choline and [²H₄]acetylcholine were initially (15 min after LiCl) reduced (to ?30% in the cortex) and later (24 h after LiCl) increased (to + 50% in the striatum). There was a corresponding initial increase (to +50% in the cerebellum) and later decrease (to ?30% in the hippocampus) of the endogenous acetylcholine and choline. These results indicate an initial decrease and later increase in the utilization of acetylcholine after acute treatment with LiCl. Following 10 days of treatment with LiCl there was an increased rate of synthesis of [²H₄]acetylcholine from pulsed [²H₄]choline in the striatum, hippocampus and cortex (P < 0.05). The high affinity transport of [²H₄]choline and its conversion to [²H₄]acetylcholine was activated (131% of control; P < 0.01) in synaptosomes isolated from brains of 10-day treated rats. Investigation of synaptosomes isolated from striatum, hippocampus and cortex revealed that only striatal [²H₄]acetylcholine synthesis was significantly stimulated. Kinetic analysis demonstrated that the apparent K_T for choline was decreased by 30% in striatal synaptosomes isolated from rats treated for 10 days with LiCl. Striatal synaptosomes from 10-day treated rats compared to striatal synaptosomes from untreated rats also released acetylcholine at a stimulated rate in a medium containing 35 mM-KCl. These results indicate that LiCl treatment stimulates cholinergic activity in certain brain regions and this may play a significant role in the therapeutic effect of LiCl in neuropsychiatric disorders.     

ACCELERATION OF CHOLINE UPTAKE AFTER DEPOLARIZATION-INDUCED ACETYLCHOLINE RELEASE IN RAT CORTICAL SYNAPTOSOMES

Abstract— Activation of nerve elements in vivo and in vitro is associated with an increased rate of choline uptake by a Na⁺-dependent high affinity transport system. Following the methodology of B arker (1976), rat cortical synaptosomes were depolarized (37°C, 10min) by 25mM-KCl in the presence of CaCl₂ (1 mM) or other divalent cations. After reisolation by centrifugation, the rate of ³H-choline uptake (1.25μM) was measured by Millipore filtration. KCl treatment alone failed to accelerate the rate of uptake in the reisolated synaptosomes. CaCl₂, BaC1₂ or SrCl₂ (but not MgCl₂ or MnCl₂) were necessary (1 mM) to observe the KCl induced acceleration. Moreover, RbCl, but not LiCl or CsCl, also produced the calcium-dependent rate enhancement in the reisolated synaptosomes. The conditions mediating the enhanced rate of choline uptake correlated strongly with those associated with neurotransmitter release. To test this possibility, synaptosomal acetylcholine content was measured in response to the various salt treatments. Treatment with KCI (25 mM) and CaCl₂ (1 mM), but not KCl alone, reduced the synaptosomal acetylcholine content from 154 to 113pmol/mg protein. The respective rates of choline uptake increased about 60%. The increased rate was reversed by incubation with 50 μM-choline followed by synaptosome reisolation. This procedure also normalized the acetylcholine content. In summary, the rate of choline uptake by the high affinity choline uptake system is inversely related to the synaptosomal acetylcholine content.     

CHARACTERISTICS OF ACETYLCHOLINE RELEASE BY SUPERFUSED SLICES OF RAT BRAIN1

—A superfusion system has been used to examine the effects of choline and the utilization of [³H]choline during resting and potassium-stimulated release of ACh from rat cerebrum slices. The rate of ACh release from unstimulated tissue, 0·25 nmol/g per min, increased 8-fold when the concentration of KCl in the superfusing medium was increased from 5 to 50 mm . This rate was not maintained, however, but gradually declined to one-half the peak rate after approx. 30 min. After an initial washout period, choline was released at a rate of 2·5-5 nmol/g per min, which was equal to 1-2 × 10^?6m in the superfusate. The addition of 1 × 10^?5m -choline to the superfusing medium was required to maintain the stimulated ACh release at near peak rates for 90 min. When hemicholinium-3 was added to the 50 mm -KCl medium, the release of ACh reached a peak as usual but then declined to prestimulation rates. After introducing a pulse of radioactive choline in the superfusing medium, the specific radioactivity of choline and ACh in the superfusate was determined before and during stimulation with 50 mm -KCl. The specific radioactivity of released ACh was always greater than that of released choline; it decreased rapidly at the onset of stimulation, and then more gradually as stimulation proceeded. The specific radioactivity of ACh released in the initial minutes of stimulation was higher than that of ACh in the tissue before stimulation. In the last 10-20 min of stimulation the specific radioactivity of the released ACh was lower than that of the tissue ACh at the end of stimulation. The relative contributions of old and newly synthesized ACh to the releasable transmitter pool are discussed.     

SYNTHESIS OF ACETYLCHOLINE IN DIFFERENT COMPARTMENTS OF BRAIN NERVE TERMINALS IN VIVO AS STUDIED BY THE INCORPORATION OF CHOLINE FROM PLASMA AND THE EFFECT OF PENTOBARBITAL ON THIS PROCESS

The time course of the incorporation of choline from plasma into a high and a low molecular weight fraction from mouse brain synaptosomes was studied. The fractions were obtained from lysed synaptosomes by gel filtration on Sephadex G-25. An extremely rapid incorporation of radioactivity into acetylcholine was found in both fractions and in the time interval 0.25-9 min after the intravenous administration of labelled choline, higher specific radioactivities of acetylcholine were found in the high molecular weight fraction than in the low molecular weight fraction. However, the specific radioactivity of choline in the high molecular weight fraction was much lower than that of acetylcholine. It was found that barbiturate anaesthesia caused a marked decrease in the labelling of acetylcholine in the high molecular weight fraction while the incorporation into the low molecular weight fraction was affected to a much smaller extent. Acetylcholine of the high molecular weight fraction showed properties similar to those of vesicle-bound acetylcholine. The recoveries of labelled and endogenous acetylcholine and choline from the brain homogenates were calculated in different steps of the fractionation procedure. In the fraction containing lysed synaptosomes the recovery of radioactive acetylcholine was lower than that of endogenous acetylcholine. This may indicate the presence of two types of bound acetylcholine in the synaptosomes. Different models for the intraneuronal synthesis of acetylcholine are discussed and it is proposed that a site of acetylcholine synthesis in vivo may be closely associated with some constituent of the high molecular weight fraction and directly coupled with the storage of the transmitter.     

ACETYLCHOLINE,CHOLINE AND CHOLINE ACETYLTRANSFERASE ACTIVITY IN THE DEVELOPING BRAIN OF NORMAL AND HYPOTHYROID RATS1

Abstract— Acetylcholine, choline and choline acetyltransferase activity were measured in the whole brains of normal and hypothyroid rats during development. At 1 day postpartum, brain acetylcholine was 73 per cent of adult levels. Propylthiouracil-induced hypothyroidism up to age 20 days did not alter brain acetylcholine concentrations, but at 30 days resulted in significantly decreased levels. At day 1, brain choline was 20 per cent higher than adult levels and decreased between days 8 and 10. In hypothyroid rats this phenomenon did not occur until days 15–20. At day 1 postnatally, choline acetyltransferase activity was only 7 per cent of adult levels, then between days 5 and 20 rose to 77 per cent of adult levels. Beginning at day 8, hypothyroidism resulted in significantly decreased enzyme levels. This effect could be reversed at day 17 by concurrent tri-iodothyronine substitution therapy. In hypothyroid rats, maximum brain choline acetyltransferase activity was 30 per cent less than normal adult levels.     

THE LACK OF SPECIFICITY TOWARDS SALTS IN THE ACTIVATION OF CHOLINE ACETYLTRANSFERASE FROM HUMAN PLACENTA

Abstract— The effects of monovalent and divalent anions on the choline acetyltransferase reaction have been determined at high (5.0 mM) and low (0.58 mM) choline. At 0.58 mM-choline, both monovalent and divalent anions activate the enzyme ±9 fold; however, at 5.0mM-choline, monovalent anions activate the enzyme ±25 fold, while divalent anions activate ±9 fold. Both monovalent and divalent anions show uncompetitive activation with respect to choline. When either dimethylaminoethanol, N -(2-hydroxyethyl)- N -methyl piperidinium iodide, or N -(2-hydroxyethyl)- N -propyl pyrrolidinium iodide was substituted for choline, activation by monovalent or divalent anions was only 2.5-4 fold. With AcCoA as substrate the ChA reaction can be increased ±20 fold by increased salts; however, with acetyl dephosphoCoA as substrate, the reaction is insensitive to the salt concentration. Similar salt effects on the ChA reaction, as measured in the direction of acetylcholine synthesis, have been demonstrated in the reverse reaction. In addition, inhibition of the forward reaction by acetylcholine has been measured as a function of sodium chloride concentration. Although the K₁ for acetylcholine increases with increasing salt, this change in K ₁, parallels the increase in the K _m for choline. These results support the hypothesis that both monovalent and divalent anions activate choline acetyltransferase by the same singular mechanism; which is to increase the rate of dissociation of coenzyme A from the enzyme.