Modulation of synaptosomal high affinity choline transport.

Depolarization of synaptosomes produced by incubation in 35mMK⁺ Krebs Ringer phosphate buffer results in an increased Vmax and no change in K_T of the high affinity transport of [³H]-choline as determined upon re-incubation in normal K⁺ Krebs Ringer phosphate buffer. The high K⁺ induced increase in the uptake of choline appears to be independent of transmitter release. The K⁺ stimulated increase in the Vmax of the high affinity transport of choline is totally blocked by high, 11mM, Mg⁺². The proportion of choline converted to acetylcholine in synaptosomes previously depolarized is the same as those incubated in normal K⁺ Krebs Ringer; thus the absolute rate of acetylcholine synthesis in nerve terminals is increased as a result of prior depolarization.     

Quantitative analysis of acetylcholine release in depolarized hippocampal slices

Time course of the hippocampal slice acetylcholine content and the rate of acetylcholine release were studied during high K⁺-induced depolarization for 4 to 60 min. At the end of the potassium exposure, both the acetylcholine remaining in the tissue and appearing in the incubation medium were quantitatively determined by gas chromatography using a nitrogen-sensitive detector. During prolonged K⁺ incubation, the acetylcholine content of the slices decreased by 60%, reaching a steady state after 16 min. The increase in the acetycholine concentration of the depolarizing medium showed a biphasic pattern, with rate constants of 1.40 and 0.69 nmol/min/g in the early (0–16 min) and late (16–60 min) phase, respectively. K⁺-evoked acetylcholine release was Cal⁺-dependent, but addition of choline did not alter tissue levels of acetylcholine or the pattern of K⁺-evoked acetylcholine release. The rate of acetylcholine release was markedly decreased by inhibition of choline uptake with hemicholinium-3 or by addition of 4-(1-naphthylvinyl)pyridine which inhibits both ACh producing enzyme, choline acetyltransferase and choline uptake mechanism. These data confirm the essential role during depolarization of extracellular choline transport into the cholinergic terminals utilizing choline released by the slices during the incubation. It is concluded that drugs which can influence the processes of choline uptake and acetylcholine sythesis can alter the rate of acetylcholine release measured under similar conditions.     

The ionophore X537A (Lasolocid) transiently increases acetylcholine release from rat brain invitro

The effect of X537A on acetylcholine (ACh) release was examined $\text{in vitro}$ in superfused slices of rat cerebrum and striatum. The ionophore (30 μM) induced a transient release of ACh which was not dependent on calcium in the medium. Also in contrast to K⁺-stimulated release, X537A-induced release was not sustained by 10^?5M choline in the superfusion medium and not inhibited by 5 × 10^?4M pentobarbital. The ionophore did not transport ACh or choline from an aqueous to an organic phase. Both K⁺ and X537A inhibited 1 μM (³H) choline uptake into striatal synaptosomes but this effect of X537A was more extensive and less reversible than that caused by K⁺. X537A did not inhibit choline acetyltransferase activity.     

Choline Uptake and Acetylcholine Synthesis in Synaptosomes: Investigations Using Two Different Labeled Variants of Choline

Abstract: Using sequential incubations in media of different K⁺ composition, we investigated the dynamics of choline (Ch) uptake and acetylcholine (ACh) synthesis in rat brain synaptosomal preparations, using two different deuterated variants of choline and a gas chromatographic-mass spectrometric (GC-MS) assay for ACh and Ch. Synaptosomes were preincubated for 10 min in a Krebs medium with or without high K⁺ and with 2 μM-[²H₉]Ch. At the end of the preincubation all variants of ACh and Ch were measured in samples of the pellet and medium. In the second incubation (4 min) samples of synaptosomes were resuspended in normal or high K⁺ solutions containing [²H₄]Ch (2 μM) and all variants of ACh and Ch were measured in the pellet and medium at the end of this period. This protocol allowed us to compare the effects of preincubation in normal or high K⁺ solution on the metabolism during a second low or high K⁺ incubation of a [²H₉]Ch pool accumulated during the preincubation period. Moreover, we were able to compare and contrast the effects of this protocol on [²H₉]Ch metabolism versus [²H₄]Ch metabolism. The most striking result we obtained was that [²H₉]Ch that had been retained by the synaptosomes after the preincubation was not acetylated during a subsequent incubation in normal or high K⁺ media. This result suggests that if an intraterminal pool of Ch is involved in ACh synthesis, the size of this pool is below the limits of detection of our assay. We have confirmed the observation that a prior depolarizing incubation results in an enhanced uptake of Ch during a second incubation in normal K⁺ Krebs. Moreover, Ch uptake is stimulated by prior incubation under depolarizing conditions relative to normal preincubation when the second incubation is in a high K⁺ solution. These results are discussed in terms of current models of the regulation of ACh synthesis in brain.     

Ionic and Metabolic Requirements for High-Affinity Choline Uptake and Acetylcholine Synthesis in Nerve Terminals at a Neuromuscular Junction

Abstract: We have shown previously that in the chick ciliary nerve-iris muscle preparation Na⁺-dependent high-affinity choline uptake was confined to the nerve terminals. In this paper the sodium-dependent high-affinity choline uptake (SDHACU), which is coupled to acetylcholine (ACh) synthesis, was further characterized by measuring uptake of [³H]choline and its conversion to [³hjach under a variety of ionic and metabolic perturbations. Mannitol equilibration with the extracellular space was found to occur in less than 1 min in this preparation. Na⁺-dependent choline (Ch⁺) uptake was shown to be linear for 16 min and to reach an equilibrium before Na⁺-independent Ch⁺ uptake, which continued to increase for 60 min. Elevated [K⁺]₀ concentrations inhibited Ch⁺ uptake and ACh synthesis. Glycolytic and respiratory inhibitors also reduced both processes, as did ouabain and omission of [K⁺]₀. Incubation conditions that reduce transmitter release had no effect on inhibition by high [K⁺]₀. Reduction of SDHACU and sodium-dependent ACh synthesis by depolarization with high [K⁺]₀ or by inhibition of Na, K-ATPase implies that the electrochemical gradients for Ch⁺ and Na⁺ are important in providing a driving force for high-affinity Ch⁺ uptake. The inhibition by metabolic blockers suggests active transport, but the effects may be indirect, caused by reduced Na, K-ATPase activity and alterations in membrane potential. While most metabolic inhibitors exerted parallel effects on both Ch⁺ uptake and ACh synthesis, in some cases Ch⁺ uptake was more strongly inhibited than ACh synthesis. This occurred in preparations incubated with high [K⁺]₀ and ouabain. Na⁺-dependent Ch⁺ uptake and ACh synthesis were found to be temperature-dependent with a Q₁₀ (20–30°) of 3.6 and 6.6, respectively and a Q₁₀ (30–40°) of 1.3 and 1.0, respectively. Inhibition of acetylcholinesterase by paraoxon increases to 92% the proportion of the Ch⁺ taken up which is converted to ACh. ACh did not reduce Ch⁺ transport when present at 100 μM.     

ACETYLTRIETHYLCHOLINE: A CHOLINERGIC FALSE TRANSMITTER IN CAT SUPERIOR CERVICAL GANGLION AND RAT CEREBRAL CORTEX

The accumulation and metabolism of [¹⁴C]triethylcholine by cat superior cervical ganglia [rested or stimulated (20 Hz)] and by rat cerebral cortex minces was measured. In ganglia, preganglionic nerve stimulation increased the accumulation (2.4 fold) and the acetylation (5.7 fold) of triethylcholine; however the depletion of the ganglion's acetylcholine content was 9.5 times greater than the amount of acetyltriethylcholine synthesized. In the presence of eserine, neither stimulated nor rested ganglia synthesized any extra (surplus) acetyltriethylcholine. It is concluded that the rate-limiting step in acetyltriethylcholine synthesis is the acetylation of triethylcholine by choline acetyltransferase. Subsequent preganglionic nerve stimulation of ganglia, which had been stimulated during the exposure to [¹⁴C]-triethylcholine, caused the increased release of only acetyltriethylcholine; the release was frequency-dependent, required the presence of Ca²⁺, and was blocked by increasing the ratio of Mg²⁺/Ca²⁺ in the perfusion fluid. All of the acetyltriethylcholine which had been accumulated was available for release. Rat cerebral cortex also accumulated triethylcholine and acetylated about 3% of the accumulated choline analogue. Subsequent stimulation by high K⁺ (46 mM)-atropine (3 μM) caused the increased release of acetyltriethylcholine from the cortex and this release required the presence of Ca²⁺. Triethylcholine can therefore form a cholinergic false transmitter in the cat superior cervical ganglion and the rat cerebral cortex.     

EFFECT OF PRETREATMENT UNDER VARIOUS CATIONIC CONDITIONS ON ACETYLCHOLINE CONTENT AND CHOLINE TRANSPORT IN RAT WHOLE BRAIN SYNAPTOSOMES

The effects of different ionic environments were measured on the concentration of acetyl-choline (ACh) from synaptosomes and their effect on subsequent high affinity choline (Ch) transport and ACh synthesis after resuspension of the synaptosomes in the normal Krebs medium. KCl (40 mM) was used to induce ACh release and reduce synaptosomal ACh content. The effects of Na⁺ omission, Ca²⁺ omission, and high Mg²⁺ on spontaneous (KC1: 4.75 mM) and potassium induced (KC1: 40 mM) ACh release and other cholinergic parameters are presented. The high affinity transport of Ch was more highly correlated with the reciprocal of the ACh level (r= 0.934, P= 9.7 × 10^-4) than with the ACh release rate during preincubation (r= 0.792, P= 3.4 × 10^-2). The results are consistent with the view that the consequences of the various ionic conditions on Ch transport and ACh synthesis are dependent on their effects on intrasynaptosomal ACh levels and only secondarily on synaptosomal ACh release.     

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.     

Effect of 2-(4-Phenylpiperidino)cyclohexanol (AH 5183) on the Veratridine-Induced Increase in Acetylcholine Synthesis by Rat Hippocampal Tissue

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

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.     

THE SUBCELLULAR LOCALIZATION OF Ach SYNTHESIS IN RAT STRIATAL SYNAPTOSOMES INVESTIGATED WITH THE USE OF TRITON X-100

—The regulation of [¹⁴C]ACh synthesis was studied in rat striatal synaptosomes incubated in presence of various concentrations of Triton X-100, using [2-¹⁴C]pyruvate or [6-¹⁴C]glucose as precursors. The progressive rupture of the cytoplasmic and mitochondrial compartments induced by the non-ionic detergent was followed by studying the release, into the incubating medium, of lactate dehydrogenase and choline acetyltransferase (ChAc) and of fumarate hydratase, respectively. [³H]Choline uptake (1 μm ) was measured to determine the activity of the high affinity choline permease. ¹⁴CO₂ formation from [2-¹⁴C]pyruvate was used as an index of the Krebs cycle activity. The rate of [¹⁴C]ACh synthesis from [2-¹⁴C] pyruvate was dependent on the Triton X-100 concentration; the ester formation decreased between 0·001% (v/v) and 0·010%, but increased again beyond this concentration of detergent. This last phenomenon was interpreted as the result of an extracellular synthesis of ACh involving pyruvate dehydrogenase and ChAc. At 0·002% Triton X-100 the ¹⁴CO₂ formation was not affected, indicating a normal mitochondrial activity. The decrease of [¹⁴C]ACh synthesis observed up to this detergent concentration could be correlated to the decline of the highaffinity choline permease activity. In these experimental conditions, the ester synthesis could not be restored by the addition of large amounts of choline in the incubating medium suggesting that the molecules of choline must cross the high-affinity choline permease system in order to be acetylated. This could indicate a close association between the permease and choline acetyltransferase.     

High affinity transport of choline into synaptosomes of rat brain

—The accumulation of [³H]choline into synaptosome-enriched homogenates of rat corpus striatum, cerebral cortex and cerebellum was studied at [³H]choline concentrations varying from 0.5 to 100 μm . The accumulation of [³H]choline in these brain regions was saturable. Kinetic analysis of the accumulation of the radiolabel was performed by double-reciprocal plots and by least squares iterative fitting of a substrate-velocity curve to the data. With both of these techniques, the data were best satisfied by two transport components, a high affinity uptake system with K_m. values of 1.4 μM (corpus striatum), and 3.1 μM (ceμ(cerebral cortex) and a low affinity uptake system with respective K_m. values of 93 and 33 μM for these two brain regions. In the cerebellum choline was accumulated only by the low affinity system. When striatal homogenates were fractionated further into synaptosomes and mitochondria and incubated with varying concentrations of [³H]choline, the high affinity component of choline uptake was localized to the synaptosomal fraction. The high affinity uptake system required sodium, was sensitive to various metabolic inhibitors and was associated with considerable formation of [³H]acetylcholine. The low affinity uptake system was much less dependent on sodium, and was not associated with a marked degree of [³H]acetylcholine formation. Hemicholinium-3 and acetylcholine were potent inhibitors of the high affinity uptake system. A variety of evidence suggests that the high affinity transport represents a selective accumulation of choline by cholinergic neurons, while the low affinity uptake system has some less specific function.     

Induced acetylcholine release from active purely cholinergic Torpedo synaptosomes.

Viablse, purely cholinergic synaptosomes were prepared from the electric organ of Torpedo ocellata and partially purified by differential and sucrose density centrifugation. The synaptosomes contain acetylcholine (ACh), synaptic vesicles, cytoplasmic markers and mitochondria. No adherent postsynaptic membranes were detected. K⁺ depolarization as well as the ionophore A23187 mediate Ca²⁺ permeation into the synaptosomes and the consequent release of ACh. Mg²⁺ does not evoke ACh release whereas Sr²⁺ and Ba²⁺ can replace Ca²⁺ in evoking K⁺ depolarization induced ACh secretion. In accordance with the calcium hypothesis of stimulus–secretion coupling, both K⁺ depolarization and the ionophore A23187 seem to mediate the release of the same population of ACh molecules. The mode of action of the ionophore X537A differs from that of A23187. X537A acts independently of Ca²⁺ and induces the release of a larger fraction of the ACh contained in the fractionated nerve terminals. These results demonstrate that the Torpedo synaptosomes contain the neurosecretion apparatus in a functional active state. This preparation extends the utility of synaptosomes for structural and functional biochemical studies of neurotransmission, as it uniquely contains only one neurosecretion system (cholinergic).     

RELEASE OF ATP FROM A SYNAPTOSOMAL PREPARATION BY ELEVATED EXTRACELLULAR K+ AND BY VERATRIDINE

A technique was developed which permitted the release of ATP from synaptosomes by elevated extracellular K⁺ or by veratridine to be directly and continuously monitored. The released ATP interacted with firefly luciferin and luciferase in the incubation medium to produce light which could be detected by a photomultiplier. The assay system was specific for ATP, in that similar concentrations of adenosine, AMP or ADP did not produce chemiluminescence. Moreover, the maximum peak of light emission correlated linearly with the concentrations of ATP present in the medium, so that semiquantitative estimates of ATP release could be made. Elevating the extracellular K⁺ concentration produced a graded release of ATP from synaptosomes. Rb⁺ also released ATP but Na⁺, Li⁺ and choline did not. The response to elevated K⁺ was not blocked by tetrodotoxin (TTX), indicating that this effect was not mediated by the opening of Na⁺-channels in synaptosomal membranes. Veratridine (50 μM) caused a graded release of ATP which was larger and more prolonged than that caused by elevated K⁺. The release of ATP by veratridine was blocked by TTX indicating that the opening of Na⁺-channels was involved. Neither veratridine nor elevated K⁺ released ATP from microsomal or mitochondrial fractions, showing that the release of ATP probably did not originate from microsomal, vesicular or mitochondrial contaminants of the synaptosomal preparation. Release of ATP by elevated K⁺ was diminished in a medium lacking CaCl₊ or when EGTA was added to chelate Ca²⁺. In contrast, release by veratridine appeared to be augmented in Ca²⁺-free media or in the presence of EGTA. The K⁺-induced release of ATP, which is Ca²⁺ dependent, closely resembles the exocytotic release of putative neurotransmitters from presynaptic nerve-terminals. On the other hand, the apparent lack of a Ca²⁺ requirement for veratridine's action suggests that this process could originate from other sites, or involve mechanisms other than conventional neurotransmitter release processes.     

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.     

Amyloid β-Peptide Inhibits High-Affinity Choline Uptake and Acetylcholine Release in Rat Hippocampal Slices

Abstract: The characteristic pathological features of the postmortem brain of Alzheimer's disease (AD) patients include, among other features, the presence of neuritic plaques composed of amyloid β-peptide (Aβ) and the loss of basal forebrain cholinergic neurons, which innervate the hippocampus and the cortex. Studies of the pathological changes that characterize AD and several other lines of evidence indicate that Aβ accumulation in vivo may initiate and/or contribute to the process of neurodegeneration and thereby the development of AD. However, the mechanisms by which Aβ peptide influences/causes degeneration of the basal forebrain cholinergic neurons and/or the cognitive impairment characteristic of AD remain obscure. Using in vitro slice preparations, we have recently reported that Aβ-related peptides, under acute conditions, potently inhibit K⁺-evoked endogenous acetylcholine (ACh) release from hippocampus and cortex but not from striatum. In the present study, we have further characterized Aβ-mediated inhibition of ACh release and also measured the effects of these peptides on choline acetyltransferase (ChAT) activity and high-affinity choline uptake (HACU) in hippocampal, cortical, and striatal regions of the rat brain. Aβ_1–40 (10^?8M) potently inhibited veratridine-evoked endogenous ACh release from rat hippocampal slices and also decreased the K⁺-evoked release potentiated by the nitric oxide-generating agent, sodium nitroprusside (SNP). It is interesting that the endogenous cyclic GMP level induced by SNP was found to be unaltered in the presence of Aβ_1–40. The activity of the enzyme ChAT was not altered by Aβ peptides in hippocampus, cortex, or striatum. HACU was reduced significantly by various Aβ peptides (10^?14 to 10^?6M) in hippocampal and cortical synaptosomes. However, the uptake of choline by striatal synaptosomes was altered only at high concentration of Aβ (10^?6M). Taken together, these results indicate that Aβ peptides, under acute conditions, can decrease endogenous ACh release and the uptake of choline but exhibit no effect on ChAT activity. In addition, the evidence that Aβ peptides target primarily the hippocampus and cortex provides a potential mechanistic framework suggesting that the preferential vulnerability of basal forebrain cholinergic neurons and their projections in AD could relate, at least in part, to their sensitivity to Aβ peptides.     

Effect of scorpion venom, tityustoxin, on the release of acetylcholine from incubated slices of rat brain

Abstract— Purified tityustoxin (TsTX) from the venom of the scorpion, Tityus serrulatus, when incubated in vitro with slices of rat cerebral cortex, increased the amount of free ace-tylcholine (ACh) in the incubation medium and, simultaneously, reduced the amount of bound ACh in the slices. The effect was optimal at pH 7.4 and was dependent upon time of incubation, an energy source and the concentration of toxin. Tityustoxin increased the synthesis of ACh, but this effect seemed to be related to an increase in the release of ACh. The effect of the TsTX was independent of the concentration of K⁺ ion but was dependent on the presence of Na⁺ and Ca²⁺ in the incubation medium. Hexamethonium and hemicholinium reduced the effect of tityustoxin, but cocaine had no effect on the release of ACh stimulated by the TsTX. Tetrodotoxin blocked completely the stimulation caused by the tityustoxin. We suggest that probably both tityustoxin and tetrodotoxin exert different and antagonistic effects competing in the Na⁺ channels.     

Evaluation of acetate uptake and its relative conversion to acetylcholine in Torpedo electric organ synaptosomes under different ionic and metabolic conditions

The uptake of acetate and its incorporation into acetylcholine were measured under various conditions in nerve terminals isolated from the electric organ in order to characterize acetate uptake and to study the relationship between acetate uptake and acetylcholine synthesis in a pure cholinergic preparation. It was found that increasing extracellular choline up to 10^?4 M had no effect on either acetate uptake or the conversion of acetate to ACh, while the addition of hemicholinium-3 to the incubation medium led to decreases in both parameters. Hence, it appears that endogenous levels of choline are sufficient to support ongoing acetylcholine synthesis in this preparation and that this synthesis depends to some extent on the uptake of extracellular choline. Nonetheless, in the absence of choline uptake, both the uptake of acetate and the conversion of acetate to acetylcholine remained substantial, indicating that internal sources of choline as well can be used for acetylcholine synthesis.Acetate uptake displayed a marked requirement for external Na⁺ and was decreased following depolarization of the synaptosomes by an elevated K⁺ concentration. The conversion of acetate to acetylcholine followed a similar pattern, except that a small reduction in acetylcholine synthesis was observed in the absence of external Ca²⁺, while acetate uptake was unaffected. The addition of ATP, AMP-PNP or phosphate to the incubation medium caused an increase in both the uptake and incorporation of acetate, but adenosine had no effect on either of these functions. Choline uptake, meanwhile, was unchanged in the presence of ATP, phosphate or adenosine. Acetate uptake appears to be more closely linked to its intracellular metabolism than to the transmembrane movement of choline itself.The mechanism by which acetate crosses the nerve terminal membrane has not been established, but the possibility that acetate is a substrate for a monocarboxylate transport system such as has been described in other systems can be ruled out as inhibitors of anion permeability do not block acetate uptake in this preparation.     

FURTHER STUDIES ON THE K+-DEPENDENT SWELLING OF PRIMATE CEREBRAL CORTEX IN VIVO: THE ENZYMATIC BASIS OF THE K+-DEPENDENT TRANSPORT OF CHLORIDE

Abstract— The swelling of intact, exposed primate cerebral cortex perfused in vioo under, isosmotic conditions was a linear function of the concentration of K⁺ in perfusate over the range 25–117 mM. The K⁺-dependent swelling was manifested throughout the depth of the cerebral cortex studied and was associated with an increased content of chloride in the swollen tissue, despite the constancy of the concentration of external chloride. The swelling of the cerebral cortex was a linear function of the temperature of the perfusate over the range 15–38°C, despite the constancy of the concentration of external K⁺. Moreover, the content of chloride in the swollen cerebral cortex was a linear function of the temperature of the overlying perfusate, despite the constancy of the external concentration of chloride. The changes in the contents of Na⁺ and K⁺ in the swollen cerebral cortex perfused with solutions containing constant concentrations of external Na⁺ and K⁺ but differing in temperature suggested that the fluid of swelling in the tissue was rich in both K⁺ and CI^-, as had been shown previously in vitro. Perfusion of the exposed, intact cerebral cortex in uiuo with K⁺-rich fluids usually involved the reciprocal reduction of the concentrations of Na+ in the perfusate to maintain isotonicity. When comparable reductions in the concentration of external Na⁺ were achieved by replacement with choline (instead of K⁺), swelling of the perfused, exposed cortex was significantly less than that attributed to isotonic, K⁺-rich but Na⁺-poor fluids. These observations suggested that it was the elevated levels of K⁺ rather than lowered concentrations of Na+ that promoted the swelling of the perfused cerebral cortex. The apparent rate of influx of ³⁶Cl from the perfusate into the underlying exposed and intact monkey cerebral cortex in vivo was a linear function of the concentration of K⁺ in perfusate over the range 25–117 mM and conformed to Michaelis-Menten kinetics when plotted according to Lineweaver and Burk. Moreover, the apparent influx of chloride from perfusate into swollen cerebral cortex was a linear function of the percentage swelling of cerebral cortex over the range 6–30 per cent. However, the apparent rate of influx of chloride from perfusate into unswollen cortex was not consistent with the linear correlation already described for swollen cerebral cortex. One reason for this discrepancy was the reduction in the size of the true (inulin) extracellular space associated with the K⁺-dependent swelling of cerebral cortex in vivo. The anatomical locus for this K⁺-dependent swelling of cerebral cortex was an expanded glial compartment, as demonstrated by electron-microscopy. The parenteral administration (50 mg/kg) or local perfusion (5 mM) of acetazolamide inhibited the K⁺-dependent swelling of cerebral cortex in vivo. Moreover, administration of acetazolamide inhibited the K⁺-dependent increase in content of C1- and the K⁺-dependent rate of influx of ³⁶Cl into swollen cerebral cortex. We have discussed the possible enzymatic basis of these K⁺-dependent alterations in content of fluid and chloride and transport of chloride in mammalian cerebral cortex in viuo.     

Regulation of carrier-mediated and exocytotic release of [3H]GABA in rat brain synaptosomes

In this study we investigated the role of external monovalent cations, and of intracellular Ca²⁺ concentration ([Ca²⁺]i) in polarized and depolarized rat cerebral cortex synaptosomes on the release of [³H]--aminobutyric acid (³H-GABA). We found that potassium-depolarization, in the absence of Ca²⁺, of synaptosomes loaded with³H-GABA releases 7.4±2.1% of the accumulated neurotransmitter, provided that the external medium contains Na⁺, and an additional 19.0±2.5% is released upon adding 1.0 mM CaCl₂ to the exterior. The Ca²⁺-independent release component does not occur in a choline medium and it is only 3.4±0.8% of the³H-GABA accumulated in a Li⁺ medium, but both ions support the Ca²⁺-dependent release of³H-GABA (13.4±0.6% in choline and 15.4±1.5% in Li⁺), which suggests that the exocytotic release is independent of the external monovalent cation present, whereas the carrier-mediated release specifically requires Na⁺ outside. Furthermore, previous release of the cytosolic³H-GABA due to predepolarization in the absence of Ca²⁺ does not influence the amount of³H-GABA subsequently released by exocytosis due to Ca²⁺ addition (19.1±2.5% or 19.1±1.1%, respectively). In choline or Li⁺ medium, the value of the [Ca²⁺]i is raised by Na⁺/Ca²⁺ exchange to 663±75 nM or 782±54 nM, respectively, within three minutes after adding 1.0 mM Ca²⁺, in the absence of depolarization, and parallel release experiments show no release of³H-GABA in the choline medium, but a substantial release (7.1±2.1%) of³H-GABA occurs in the Li⁺ medium without depolarization. Subsequent K⁺-depolarization shows normal Ca²⁺-dependent release of³H-GABA in the choline medium (14.1±2.0%) but only 8.6±1.1% release in the Li⁺ medium, which suggests that raising the [Ca²⁺]i by Na⁺/Ca²⁺ exchange, without depolarization, supports some exocytotic release in Li⁺, but not in choline media. The role of [Ca²⁺]i and of membrane depolarization in the release process is discussed on the basis of the results obtained and other relevant observations which suggest that both Ca²⁺ and depolarization are essential for optimal exocytotic release of GABA.Special issue dedicated to Dr. Santiago Grisolia.