Acetylcoenzyme A and Acetylcholine in Slices of Rat Caudate Nuclei Incubated with (-)-Hydroxycitrate, Citrate, and EGTA

Abstract: The effects of (-)-hydroxycitrate (OHC) and citrate on the concentration of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh) in the tissue and on the release of ACh into the medium were investigated in experiments on slices of rat caudate nuclei incubated in media with 6.2 or 31.2 m M K⁺, 0 or 2.5 mM Ca²⁺, and 0, 1, or 10 m M EGTA. OHC diminished the concentration of acetyl-CoA in the slices under all conditions used: in experiments with 2.5 m M OHC, the concentration of acetyl-CoA was lowered by 25-38%. Citrate, in contrast, had no effect on the level of acetyl-CoA in the tissue. Although both OHC and citrate lowered the concentration of ACh in the slices during incubations with 6.2 m M K⁺ and 1 m M EGTA, they had different effects on the content of ACh during incubations in the presence of Ca²⁺. The concentration of ACh in the slices was increased by citrate during incubations with 2.5 mM Ca²⁺ and 31.2 or 6.2 m M K⁺, but it was lowered or unchanged by OHC under the same conditions. The release of ACh into the medium was lowered or unchanged by OHC and lowered, unchanged, or increased by citrate. It is concluded that most effects of OHC on the metabolism of ACh can be explained by the inhibition of ATP-citrate lyase; with glucose as the main metabolic substrate, ATP-citrate lyase appears to provide about one-third of the acetyl-CoA used for the synthesis of ACh. Experiments with citrate indicate that an increased supply of citrate may increase the synthesis of ACh. The inhibitory effect of citrate on the synthesis of ACh, observed during incubations without Ca⁺², is interpreted to be a consequence of the chelation of intracellular Ca²⁺; this interpretation is supported by the observation of a similar effect caused by 10 m M EGTA.     

Ca2+ UPTAKE BY SYNAPTOSOMES AND ITS EFFECT ON THE INHIBITION OF ACETYLCHOLINE RELEASE BY BOTULINUM TOXIN

Abstract— ⁴⁵Ca²⁺ uptake by cerebral cortex synaptosomes was determined by gel filtration, glass fibre disc filtration under suction and by centrifugation with EGTA present. The filtration methods gave comparable results which were higher than values obtained by the centrifugation method. Uptake was increased by 25mM-K⁺ at all times investigated. The accumulated ⁴⁵Ca²⁺ was bound within the synaptosome. ⁴⁵Ca²⁺-ionophore A23187 stimulated uptake only during the first min; levels of intra-synaptosomal ⁴⁵Ca²⁺ then returned to control values. A23187 also increased intra-synaptosomal Na⁺ and Cl⁻ contents. Botulinum toxin inhibits the K.⁺-stimulated release of [¹⁴C]ACh from synaptosomes but the ionophore released [¹⁴C]ACh from both normal and botulinum-treated preparations in a Ca²⁺-dependent manner. However, it also elicited Ca²⁺-dependent release of [choline. Increased extracellular Ca²⁺ (10 mM and 20 mM) released [¹⁴C]ACh (but not [¹⁴C]choline) from both normal and botulinum-treated synaptosomes. It is concluded that botulinum toxin interferes with the provision of Ca²⁺ essential for the mechanism of ACh release.     

Temporal Characteristics of Potassium-Stimulated Acetylcholine Release and Inactivation of Calcium Influx in Rat Brain Synaptosomes

Abstract: The time course of Ca²⁺-dependent [³H]acetylcholine ([³H]ACh) release and inactivation of ⁴⁵Ca²⁺ entry were examined in rat brain synaptosomes depolarized by 45 m M [K⁺]_o. Under conditions where the intrasynaptosomal stores of releasable [³H]ACh were neither exhausted nor replenished in the course of stimulation, the K⁺-evoked release consisted of a major (40% of the releasable [³H]ACh pool), rapidly terminating phase ( t _1/2 = 17.8 s), and a subsequent, slow efflux that could be detected only during a prolonged, maintained depolarization. The time course of inactivation of K⁺-stimulated Ca²⁺ entry suggests the presence of fast-inactivating, slow-inactivating, and noninactivating, or very slowly inactivating, components. The fast-inactivating component of the K⁺-stimulated Ca²⁺ entry into synaptosomes appears to be responsible for the rapidly terminating phase of transmitter release during the first 60 s of K⁺ stimulus. The noninactivating Ca²⁺ entry may account for the slow phase of transmitter release. These results indicate that under conditions of maintained depolarization of synaptosomes by high [K⁺]_o the time course and the amount of transmitter released may be a function of the kinetics of inactivation of the voltage-dependent Ca channels.     

ACETYLCHOLINE TRANSLOCATION IN SYNAPTIC VESICLE GHOSTS IN VITRO

Abstract— Translocation of acetylcholine (ACh) into cholinergic synaptic vesicles depleted of ACh and ATP was studied by Sephadex gel filtration. The hypo-osmotically shocked vesicles become transiently leaky, but retain ACh under iso-osmotic conditions. Intravesicular accumulation of [³H]ACh is due to a simple diffusional equilibration. Addition of 2mM-ATP and Mg²⁺ to the incubation medium is without effect. When acetylcoenzyme A (AcCoA) and choline (Ch ⁺) are used in place of preformed ACh, the intravesicular concentration of ACh does not exceed that of the newly synthesized, extravesicular ACh. However, in the absence of Na ⁺ the quantity of [³H]ACh associated with the vesicles increased, presumably due to ACh binding to ion-exchanger sites in the vesicles.     

KINETICS OF [3H]ACETYLCHOLINE SYNTHESIS AND RELEASE IN PRIMARY CELL CULTURES FROM MAMMALIAN CNS

Abstract— The present study was undertaken to characterize the cholinergic system of primary cell cultures of mouse and rat CNS.
In confirmation of previous reports, primary cultures were found to contain choline acetyltransferase (ChAc). Furthermore they contain acetylcholine (ACh) as measured by two different bioassays. They also synthesize [³H]ACh from [³H]Choline offered to the cultures.
The formation of [³H]ACh is inhibited in the presence of hemicholinium-3 (10⁻⁶ m ) to 50% or ouabain (10⁻³ m ) to 20% of the values found in untreated cultures. Omission of Na ⁺ from the incubation solution also diminishes the [³H]ACh formation of the cells.
[³H]ACh is released upon depolarisation by K⁺ ions in a concentration dependent manner. The release can be prevented by lack of Ca²⁺ ions in the incubation solution.     

THE FORMATION OF CHOLINE AND OF ACETYLCHOLTNE BY BRAIN IN VITRO

Abstract— Free choline and acetylcholine (ACh) in mouse or rat brain were assayed biologically. The subcellular distribution of ACh in brain slices that had been incubated in the presence of eserine was compared to that in control brain; during incubation, the ACh outside nerve endings increased four-fold, the ACh released from synaptosomes by osmotic shock doubled but the ACh bound firmly within nerve endings did not increase. The two nerve ending stores of ACh were labelled to similar specific radioactivities when slices were incubated with [³H]choline, but the specific radioactivity of the ACh formed was much lower than that of the added choline. Tissue incubated in the presence of eserine released choline and ACh into the medium and the tissue levels of both substances increased. Brain tissue exposed to Na⁺-free medium lost 84 per cent of its ACh and 66 per cent of its free choline; the amounts of both substances returned towards control values during subsequent incubation in a normal-Na⁺ medium (choline-free). Both the ACh outside nerve endings and the ACh associated with synaptosomes were depleted when tissue was incubated in Na⁺-free medium.     

Differential Labeling of Depot and Active Acetylcholine Pools in Nondepolarized and Potassium-Depolarized Rat Brain Synaptosomes

Abstract: To test the hypothesis that a pool of newly synthesized acetylcholine (ACh) turns over independently of preformed ACh, compartmentation and K⁺ -evoked release of ACh were examined in perfused synaptosomal beds intermittently stimulated by 50 m M K⁺. In resting synaptosomes, endogenous and labeled ACh was distributed between synaptic vesicles and the cytoplasm in a dynamic equilibrium ratio of 4:6. In the absence of new ACh synthesis, five sequential K⁺ -depolarizations caused a decremental release of preformed labeled ACh totaling 30% of the initial transmitter store. Further depolarization evoked little additional release, despite the fact that 60% of the labeled ACh remained in these preparations. Release of the preformed [¹⁴C]ACh was unaltered while new ACh was being synthesized from exogenous [³H]choline. Since the evoked release of [³H]ACh was maintained while that of [¹⁴C]ACh was decreasing, the [³H]ACh/[¹⁴C]ACh ratio in perfusate increased with each successive depolarization. This ratio was six to ten times higher than the corresponding ratio in vesicles or cytoplasm. These results indicate that the newly synthesized ACh did not equilibrate with either the depot vesicular or cytoplasmic ACh pools prior to release.     

Effects of Calcium on the Binding of Phencyclidine to Acetylcholine Receptor-Rich Membrane Fragments from Torpedo californica Electroplaque

Abstract: The influence of calcium on the binding of phencyclidine (PCP) to acetylcholine (ACh) receptor-rich membrane fragments was investigated. Calcium decreased the equilibrium affinity for PCP in the presence, but not in the absence, of the cholinergic agonist carbamylcholine. The effect of calcium was rapidly reversible by EGTA, indicating that it was not attributable to a calcium-activated protease or a phospholipase. Following detergent solubilization of the nicotinic ACh receptor, the calcium effect on PCP remained, suggesting that calcium may interact directly with the receptor to exert its effect. Other divalent cations (Mn²⁺, La²⁺ Co²⁺, Mg²⁺) had similar effects. A correlate of "desensitization" of the ACh receptor can be observed using PCP binding, and a two-step "desensitization" process can be observed. Calcium seemed to increase the amplitude of a rapid component of receptor "desensitization." The results presented in this paper suggest that calcium may play a role in the modulation of the nicotinic ACh receptor.     

Increased acetylcholine synthesis and release following presynaptic activity in a sympathetic ganglion

Abstract: The acetylcholine (ACh) content of sympathetic ganglia increases above its normal level following a period of preganglionic nerve stimulation. In the present experiments, this extra ACh that accumulates following activity was labeled radioactively from [³H]choline and its specific activity was compared with that of ACh subsequently released during preganglionic nerve stimulation. The specific activity of the released ACh was similar to that of the total tissue ACh, suggesting that the extra ACh mixes fully with endogenous stores. The present experiments also show that transmitter release during neuronal stimulation is necessary for the poststimulation increase in transmitter store. However, the increase was not evident when transmitter release was induced by K⁺. It is concluded that both transmitter release and impulse invasion of the nerve terminals are necessary for the adaptive phenomenon to manifest itself. The role of choline delivery and choline acetyltransferase activity in generating the poststimulation increase in transmitter store was tested. When choline transport activity measured as choline analogue (homocholine) accumulation increased, ACh synthesis was increased and when transport activity was not increased, neither was ACh synthesis. There was no poststimulation increase in measured choline acetyltransferase activity.     

Effect of Chronic Lead Ingestion by Rats on Glucose Metabolism and Acetylcholine Synthesis in Cerebral Cortex Slices

Abstract: The effect of chronic low-level lead (Pb²⁺) ingestion on the metabolic pathways leading to the acetyl moiety of acetylcholine (ACh) was examined. Cerebral cortex slices, prepared from untreated or Pb²⁺-exposed rats (600 ppm lead acetate in the drinking water for 20 days), were incubated in Krebs-Ringer bicarbonate buffer with 10 m M glucose and tracer amounts of [6-³H]glucose and either [6-¹⁴C]glucose or [3-¹⁴C] β -hydroxybutyrate. Altering the concentration of Pb²⁺ in the drinking water produced a dose-related increase in blood and brain lead levels. When tissue from Pb²⁺-exposed rats was incubated with mixed-labeled glucose, incorporation into lacate, citrate, and ACh was considerably decreased, although no changes occurred in the ³H/¹⁴C ratios. Similar effects of Pb²⁺ were found when ¹⁴C-labeled β -hydroxy-butyrate was substituted for the [¹⁴C]glucose. It appears from these data that Pb²⁺ exerts a generalized effect on energy metabolism and not on a specific step in glucose metabolism. The impairment of glucose metabolism may explain partially the Pb²⁺-induced changes observed in cholinergic function.     

Inhibition of Na+,K+-ATPase by Ouabain Opens Calcium Channels Coupled to Acetylcholine Release in Guinea Pig Myenteric Plexus

Abstract: Ouabain, an Na⁺,K⁺-ATPase inhibitor, increases the release of acetylcholine (ACh) from various preparations in a Ca²⁺-independent way. However, in other preparations the release of ACh evoked by ouabain is dependent on the presence of extracellular calcium. In the present study, we have labeled the ACh of myenteric plexus longitudinal muscles of guinea pig ileum and compared the effect of calcium channel blockers on ouabain-evoked release of [³H]ACh. Release of [³H]ACh evoked by ouabain is dose dependent and decreased markedly in the absence of calcium or in the presence of cadmium, a nonspecific calcium channel blocker. N-type calcium channel blockage by the ω-conotoxins GVIA (selective N-type calcium channel blocker) and MVIIC (a nonselective calcium channel blocker) inhibited by 45 and 55%, respectively, the release of [³H]ACh. L-type calcium channel suppression by low concentrations of verapamil, nifedipine, and diltiazem had no effect on the release of [³H]ACh. The release of transmitter was also not affected significantly by nickel, a T-type calcium channel blocker. In addition, ω-agatoxin-IVA, at concentrations that block P- and Q-type calcium channels, did not affect significantly the release of [³H]ACh. Thus, extracellular Ca²⁺ is essential for the release of ACh induced by ouabain from guinea pig ileum myenteric plexus. In this preparation, the N-type calcium channel plays a dominant role in transmitter release evoked by inhibition of Na⁺,K⁺-ATPase, but other routes of calcium entry in addition to these channels can also support the release of neurotransmitter induced by ouabain.     

THE RELATIONSHIP BETWEEN ACETYLCHOLINE RELEASE FROM BRAIN SLICES AND THE ACETYLCHOLINE CONTENT OF SUBCELLULAR FRACTIONS PREPARED FROM BRAIN

Abstract— Acetylcholine (ACh) release from sliced cerebral cortex of rats was measured when the tissue was incubated in a high K⁺ (46 m m ) medium containing eserine. In the absence of hemicholinium (HC-3), ACh release was well maintained, but in the presence of HC-3, ACh release declined within 15–20 min. Subcellular fractions representing nerve-ending free (cytoplasmic) ACh and nerve-ending bound ACh were prepared from slices that had been stimulated to release ACh in the presence of HC-3. Both nerve-ending stores of ACh were depleted when their content was compared to tissue that had not been stimulated and there was no demonstrable difference in the rate of depletion of either of the two fractions. Stimulating slices with K⁺ in the absence of HC-3 also depleted cytoplasmic and vesicle-bound ACh. It is concluded that, under these experimental conditions, both nerve ending stores of ACh are available for release and that, in the absence of HC-3, ACh synthesis can maintain ACh release, but cannot maintain tissue ACh content.     

Novel Pharmacological Sensitivity of the Presynaptic Calcium Channels Controlling Acetylcholine Release in Skate Electric Organ

Abstract: The presynaptic terminals of skate ( Raja montagui ) electric organ were tested for their sensitivity to calcium channel antagonists. Acetylcholine (ACh) release and the elevation of intraterminal Ca²⁺ concentrations triggered by K⁺ depolarisation were studied. ACh release was measured as ³H efflux from slices of organ prelabelled with [³H]choline. Depolarisation caused a marked, Ca²⁺-dependent increase in ³H efflux that was completely blocked by 100 µ M Cd²⁺ and by 300 n M ω-conotoxin-MVIIC (MVIIC). Inhibition by MVIIC was concentration dependent (IC₅₀ of ∼20 n M ) and reversible. No inhibition was seen with nifedipine (5 µ M ) or the two other peptide antagonists studied: ω-conotoxin-GVIA (GVIA) at 5 µ M and ω-agatoxin-IVA (Aga-IVA) at 1 µ M . In a "nerve plate" preparation (a presynaptic plexus of nerve fibres, Schwann cells, and nerve terminals) changes in intraterminal Ca²⁺ concentrations were measured by microfluorimetry using fluo-3. An increase in fluorescence, indicating a rise in the free [Ca²⁺], rapidly followed K⁺ depolarisation, and this change was restricted to the nerve terminals. This response was insensitive to nifedipine (5 µ M ), GVIA (5 µ M ), and Aga-IVA (300 n M ) but almost completely abolished by MVIIC (1 µ M ). MVIIC inhibition was concentration dependent and partially reversible. These results show that the nerve terminals in skate electric organ have calcium channels with a pharmacological sensitivity that is markedly different from the established L, N, and P types in other systems but shares some, but not all, of the features of the recently described Q type.     

Modulation of [3H]Acetylcholine Release from Cultured Amacrine-Like Neurons by Adenosine A1 Receptors

Abstract: We have investigated the effect of endogenous adenosine on the release of [³H]acetylcholine ([³H]ACh) in cultured chick amacrine-like neurons. The release of [³H]ACh evoked by 50 m M KCl was mostly Ca²⁺ dependent, and it was increased in the presence of adenosine deaminase and in the presence of 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), an adenosine A₁ receptor antagonist. The effect of adenosine on [³H]ACh release was sensitive to pertussis toxin (PTX) and was due to a selective inhibition of N-type Ca²⁺ channels. Ligand binding studies using [³H]DPCPX confirmed the presence of adenosine A₁ receptors in the preparation. Using specific inhibitors of the plasma membrane adenosine carriers and of the ectonucleotidases, we found that the extracellular accumulation of adenosine in response to KCl depolarization was due to the release of endogenous adenosine per se and to the extracellular conversion of released nucleotides into adenosine. Activation of adenosine A₁ receptors was without effect on the intracellular levels of cyclic AMP under depolarizing conditions, but it inhibited the accumulation of inositol phosphates. Our results indicate that in cultured amacrine-like neurons, the Ca²⁺-dependent release of [³H]ACh evoked by KCl is under tonic inhibition by adenosine, which activates A₁ receptors. The effect of adenosine on the [³H]ACh release may be due to a direct inhibition of N-type Ca²⁺ channels and/or secondary to the inhibition of phospholipase C and involves the activation of PTX-sensitive G proteins.     

Cd2+ uptake, Cd2+ binding and loss of cell K+ by a Cd-sensitive and a Cd-resistant strain of Saccharomyces cerevisiae

Abstract Uptake of Cd²⁺ into Cd-resistant cells was approximately four times lower than in Cd-sensitive cells of Saccharomyces cerevisiae . Binding of Cd²⁺ to the yeast cells increased during incubation of the cells in the presence of Cd²⁺. The increase in the binding was much higher for wild-type cells than for Cd-resistant cells. This increased binding is ascribed to permeabilization of part of the cells. There is no single relation between the relative rate of K⁺ efflux and the cellular Cd content as has been found previously for wild-type cells. The rates of K⁺ efflux were much less than those found for the wild-type cells. Only with short incubation periods of the cells with Cd²⁺ was the same dependence found between the efflux of K⁺ and the cellular Cd content for both types of cell. The discrepancies found after extended incubation of the cells with Cd²⁺ are ascribed to the fact that Cd-provoked K⁺ release proceeds via an all-or-nothing process and that K⁺ released from permeabilized cells can be reaccumulated in still intact cells. The latter proceeds more efficiently in Cd-resistant cells than in wild-type cells.     

Net Glutamate Release from Astrocytes Is Not Induced by Extracellular Potassium Concentrations Attainable in Brain

Abstract: Elevated extracellular potassium concentration ([K⁺]_e) has been shown to induce reversal of glial Na⁺-dependent glutamate uptake in whole-cell patch clamp preparations. It is uncertain, however, whether elevated [K⁺]_e similarly induces a net glutamate efflux from intact cells with a physiological intracellular milieu. To answer this question, astrocyte cultures prepared from rat and mouse cortices were incubated in medium with elevated [K⁺]_e (by equimolar substitution of K⁺ for Na⁺), and glutamate accumulation was measured by HPLC. With [K⁺]_e elevations to 60 m M , medium glutamate concentrations did not increase during incubation periods of 5–120 min. By contrast, 45 min of combined inhibition of glycolytic and oxidative ATP production increased medium glutamate concentrations 50–100-fold. Similar results were obtained in both rat and mouse cultures. Studies were also performed using astrocytes loaded with the nonmetabolized glutamate tracer d -aspartate, and parallel results were obtained; no increase in medium d -aspartate content resulted from [K⁺]_e elevation up to 90 m M , whereas a large increase occurred during inhibition of energy metabolism. These results suggest that a net efflux of glutamate from intact astrocytes is not induced by any [K⁺]_e attainable in brain.     

Ion Channels and Membrane Potential in Stimulus-Secretion Coupling in Adrenal Medulla Cells

Abstract: The role of Na⁺ channels and membrane potential in stimulus secretion coupling in adrenal medulla cell cultures was investigated. Veratridine, aconitine, batrachotoxin (BTX), and scorpion venom, which increase the flux of ions through tetrodotoxin(TTX)-sensitive Na⁺ channels, all evoke secretion of catecholamines that is blocked by TTX. TTX partially inhibits secretion induced by low concentrations of nicotine in Locke's solution but has no effect on high concentrations of nicotine (20 μM). In Ca²⁺-sucrose media TTX has no effect on secretion at either high or low concentrations of nicotine. Replacement of Na⁺ with Li⁺ in Locke's solution reduces the response to nicotine and to veratridine. Complete replacement of Na⁺ with hydrazine, diethanolamine, TRIS, and choline completely inhibits the response to nicotine and almost completely inhibits the response to veratridine. Following exposure of cells to 50 mM-100 mM-K⁺, nicotine does not stimulate catecholamine secretion unless the cells are resuspended in media containing less than 50 mM-K⁺. Neither dibutyryl-cyclic AMP nor dibutyryl-cyclic GMP evokes secretion. α-Bungarotoxin (1 μM) did not inhibit nicotine-induced secretion. These studies indicate that Na⁺ channels and acetylcholine (ACh) receptor ion channels are independently coupled to the influx of Ca²⁺. The membrane potential appears to affect nicotine- and veratridine-evoked secretion.     

Acetylcholine Turnover and Compartmentation in Rat Brain Synaptosomes

Abstract: The turnover of acetylcholine (ACh) in rat brain synaptosomes and its compartmentation in the labile bound and stable bound pools were investigated. The P₂ fraction from rat brain was subjected to three sequential incubations, each terminated by centrifugation followed by determination of ACh concentrations by gas chromatography-mass spectrometry (GCMS): (1) Depletion phase: Incubation of synaptosomes at 37°C for 10 min in Na⁺-free buffer containing 35 mM-KCl reduced the content of both labile bound and stable bound ACh by 40%. (2) Synthesis phase: Incubation at 37°C with 2 μ M -[²H₄]choline resulted in accumulation of labeled and unlabeled ACh in both compartments. Addition of an anticholinesterase had little effect on stable bound ACh but greatly increased the content of labile bound ACh. This excess accumulated ACh was probably due to inhibition of intracellular acetylcholinesterase (AChE), because negligible uptake of ACh from the medium was observed. The effects on ACh synthesis of altered cation concentrations and metabolic inhibitors were examined. (3) Release phase: The tissue was incubated in the presence of 35 mM-KCl, 40 μM-paraoxon, and 20 μM-hemicholinium-3 (HC-3) (to inhibit further synthesis of ACh). Measurements of the compartmental localization of ACh at several time points indicated that ACh was being released from the labile bound fraction. In support of this conclusion, 20 mM-Mg²⁺ reduced ACh release and increased the labile bound ACh concentration.     

THE ACCUMULATION OF RADIOACTIVE ACETYLCHOLINE BY A SYMPATHETIC GANGLION AND BY BRAIN: FAILURE TO LABEL ENDOGENOUS STORES

Abstract— The accumulation of radioactively labelled acetylcholine (ACh) by perfused superior cervical ganglia of cats and by incubated brain slices from rats was studied in the presence of diisopropylphosphorofluoridate. Ganglia accumulated more labelled ACh than an extracellular marker (inulin), but the amount of ACh accumulated did not increase when ACh turnover was increased by preganglionic nerve stimulation. The ACh that accumulated in ganglia was not released when the preganglionic nerve was subsequently stimulated. Sliced cerebral cortex also accumulated labelled ACh but this was not released when the tissue was subsequently exposed to a high K⁺ medium. Thus accumulated ACh does not appear to mix with releasable transmitter stores. Chronically (7 days) decentralized ganglia lost most of their transmitter store but retained their ability to accumulate labelled ACh. Uptake of ACh by sliced cerebellum was not less than uptake of ACh by sliced cerebral cortex and the amount of ACh accumulated by synaptosomes isolated from cerebellum was similar to the amount of ACh accumulated by synaptosomes isolated from cerebral cortex. It is concluded that ACh uptake is not specifically into cholinergic nerve endings. Hexamethonium reduced ACh uptake by cerebral cortex slices but did not increase the amount of ACh collected from slices stimulated by raised K⁺.     

RELATED CHANGES IN AMOUNTS OF ACh AND ATP IN RESTING AND ACTIVE TORPEDO NERVE ELECTROPLAQUE SYNAPSES

Abstract— Closely related changes in the levels of acetylcholine (ACh) and adenosine triphosphate (ATP) in the electric organ of Torpedo exist during rest and synaptic activity. The present work clarifies these relations by showing:

• (1) 

  That both substances are involved in an oscillatory process induced by nerve stimulation.
• (2) 

  Both substances are present in synaptic vesicles; the size of the bound pool of ACh is Ca²-dependent and is large when Ca²⁺ is low. Free ACh and transmission are restored when Ca²⁺ is present in the incubation medium.
• (3) 

  The amount of ATP in the tissue is also Ca²⁺-dependent but is low when Ca²⁺ is omitted. The addition of Ca²⁺ to the physiological medium restores the amount of ATP in the tissue.
• (4) 

  There is a postsynaptic release of ATP triggered by transmitter depolarization. This release was measured after single nerve impulses.
• (5) 

  When added to the incubation medium, nucleotides strongly inhibit transmitter release. It is suggested that the postsynaptic release of ATP regulates transmitter release.