Effects of Aluminum and Calcium on Acetyl-CoA Metabolism in Rat Brain Mitochondria

Abstract: Al complexes are known to accumulate in extra- and intracellular compartments of the brain in the course of different encephalopathies. In this study possible effects of Al accumulation in the cytoplasmic compartment on mitochondrial metabolism were investigated. Al, like Ca, inhibited pyruvate utilization as well as citrate and oxoglutarate accumulation by whole brain mitochondria. Potencies of Ca²⁺_total effects were 10–20 times stronger than those of Al. Al decreased mitochondrial acetyl-CoA content in a concentration-dependent manner, along with an equivalent rise of free CoA level, whereas Ca caused loss of both intermediates from mitochondria. In the absence of P_i in the medium, Ca had no effect on mitochondrial metabolism, whereas Al lost its ability to suppress pyruvate utilization and acetyl-CoA content in Ca-free conditions. Verapamil potentiated, whereas ruthenium red reversed, Ca-evoked suppression of mitochondrial metabolism. On the other hand, in Ca-supplemented medium, Al partially overcame the inhibitory influence of verapamil. Accordingly, verapamil increased mitochondrial Ca levels much more strongly than Al. However, Al partially reversed the verapamil-evoked rise of Ca²⁺_total level. These data indicate that Al accumulated in cytoplasm in the form of the Al(PO₄)OH⁻ complex may inhibit mitochondrial functions by an increase of intramitochondrial [Ca²⁺]_total resulting from the Al-evoked rise of cytoplasmic [Ca²⁺]_free, as well as from inhibitory interference with the verapamil binding site on the Na⁺/Ca²⁺ antiporter.     

Regulation of citrate metabolism and acetycholine synthesis by Ca in rat brain synaptosomes

The relationships between pyruvate and derived citrate metabolism and acetylcholine (ACh) synthesis in synaptosomes were examined. In the presence of 30 mM KCl, 0.1 mM Ca²⁺ caused 31 and 63% inhibition of pyruvate utilization and citrate accumulation, respectively. Verapamil and EGTA (0.5 mM) brought about no change in pyruvate consumption but increased rate of citrate accumulation, and overcame inhibitory effect of Ca²⁺. The rates of citrate accumulation in the presence of verapamil or EGTA were three to six times, respectively, higher than those in the presence of Ca²⁺. (−) Hydroxycitrate increased rate of citrate accumulation under all experimental conditions. The value of this activation appeared to be stable (0.20–0.28 nmol/min/mg of protein) and independent of changes in the basic rate of citrate accumulation. Ca²⁺ caused no significant changes in [¹⁴C]ACh synthesis, but it inhibited ¹⁴CO₂ production by synaptosomes. These activities were inhibited by verapamil by 33 and 60%, respectively. Ca²⁺ did not modify these effects of the drug. On the other hand, (−)hydroxycitrate resulted in 22 and 29% inhibition of [¹⁴C]ACh synthesis in Ca²⁺ free and Ca²⁺ supplemented medium, respectively. These data indicated that rates of acetyl-CoA synthesis in synaptoplasm, via ATP-citrate lyase and probably by another pathways are independent of Ca-evoked changes in pyruvate oxidation and citrate supply from intraterminal mitochondria. This property might play a significant role in maintenance of stable level of ACh in active cholinergic nerve endings.     

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.     

Evidence for the regulatory function of synaptoplasmic acetyl-CoA in acetylcholine synthesis in nerve endings.

Isolated synaptosomes maintained a relatively stable level of acetyl-CoA during their incubation in the presence of 30 mM-KCl. Addition of Ca2+ resulted in inhibition of pyruvate oxidation and slight activation of acetylcholine synthesis. The cation decreased acetyl-CoA in intrasynaptosomal mitochondria, but did not alter its content in synaptoplasm. Verapamil did not affect pyruvate oxidation, but decreased acetyl-CoA in synaptoplasm and inhibited acetylcholine synthesis in synaptosomes. It indicates that Ca2+ might regulate acetylcholine synthesis through changes in the direct transfer of acetyl-CoA from mitochondria to synaptoplasm.     

Inhibition of the synthesis of acetylcholine in rat brain slices by (-)-hydroxycitrate and citrate

Abstract: Slices of rat caudate nucleus were incubated in a solution of 123 mM-NaCl, 5 mM-KCl, 1.2 mM-MgCl₂, 1.2 mM-NaH₂PO₄, 25 mM-NaHCO₃, 0.2 mM-choline chloride, 0.058 mM-paraoxon, 1 mM-EGTA, and oxidizable substrates. (−)-Hydroxycitrate, a specific inhibitor of ATP-citrate lyase (EC 4.1.3.8), used at a concentration of 2.5 mM, inhibited the synthesis of acetylcholine (ACh) from [1,5-¹⁴C]citrate by 82–86%, but that from [U-¹⁴C]glucose by only 33%, from [2-¹⁴C]pyruvate by 24% and from [1-¹⁴C-acetyl]carnitine by 8%; the production of ¹⁴CO₂ from these substrates was not substantially changed. The synthesis of ACh from glucose and pyruvate was in hibited also by citrate; 2.5 mM- and 5 mM-citrate diminished it by 43% and 66%, respectively; the production of from [U-¹⁴C]glucose and from [1-¹⁴C]pyruvate was not affected. The mechanism of the inhibitory effect of citrate on the synthesis of ACh is not clear; the possibility is discussed that citrate alters the intracellular milieu in cholinergic neurons by chelating the intracellular Ca²⁺ and decreases the supply of mitochondrial acetyl-CoA to the cytosol. The results with (−)-hydroxycitrate indicate that the cleavage of citrate by ATP-citrate lyase is not responsible for the supply of more than about one-third of the acetyl-CoA which is used for the synthesis of ACh when glucose or pyruvate are the main oxidizable substrates. This proportion may be even smaller, since (−)-hydroxycitrate possibly affects the synthesis of ACh from glucose and pyruvate by a mechanism (unknown) similar to that of citrate, rather than by the inhibition of ATP-citrate lyase.     

Pharmacological Characterization of Tachykinin Receptors Controlling Acetylcholine Release from Rat Striatum: An In Vivo Microdialysis Study

Abstract: The regulation of striatal cholinergic function by tachykinins was examined in urethane-anesthetized rats by using microdialysis. Substance P (0.01–1 µ M ), [Sar⁹,Met(O₂)¹¹]substance P (1–10 µ M ), septide (0.1–3 µ M ), neurokinin (NK) A (0.1–10 µ M ), and senktide (0.1–10 µ M ) produced concentration-dependent increases in striatal acetylcholine (ACh) release. Septide was the most potent agonist for inducing release of ACh, whereas the stimulating effect of senktide was less pronounced and more progressive in onset. The response to septide was prevented by intraperitoneal administration of the nonpeptide NK₁ antagonist SR 140333 (1–3 mg/kg) but not by the nonpeptide NK₂ receptor antagonist SR 48968, indicating that the effect was mediated specifically by NK₁ receptors. ACh release caused by NKA was reduced by SR 48968 (1–3 mg/kg) and slightly affected by SR 140333, indicating a principal role for NK₂ receptors in the peptide response. The similar efficacy of SR 140333 and SR 48968 in blocking substance P-induced ACh release suggested that the effect of this peptide involves the stimulation of both NK₁ and NK₂ receptors. Finally, our results indicate that the increase in striatal ACh release induced by the D₁ agonist (+)-SKF-38393 (3 µ M ) may be mediated indirectly through local release of NKA or substance P acting at NK₂ receptors.     

Adenosine A2a Receptor-Mediated Modulation of Striatal [3H]GABA and [3H]Acetylcholine Release

Abstract: The ability of adenosine agonists to modulate K⁺-evoked 4D†-[³H]aminobutyric acid ([³H]GABA) and acetylcholine (ACh) release from rat striatal synaptosomes was investigated. The A_2a receptor-selective agonist CGS 21680 inhibited Ca²⁺-dependent [³H]GABA release evoked by 15 m M KCI with a maximal inhibition of 29 ± 4% (IC₅₀ of ∼4 ± 10 ⁻¹² M ). The relative order of potency of three agonists was CGS 21680 ± 5'- N -ethylcarboxamidoadenosine > R-phenylisopropyladenosine (R-PIA), with the inhibition being blocked by A_2a receptor-selective antagonists (CP 66,713 and CGS 15943A) but not by the A₁-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When release of [³H]GABA was evoked by 30 mM KCI, no significant inhibition was observed. In contrast, CGS 21680 stimulated the release of [³H]ACh evoked by 30 m M KCI, with a maximal stimulation of 26 ± 5% (IC₅₀ of ∼10⁻¹¹ M ). This effect was blocked by CP 66,713 but not by DPCPX. The A₁ agonist R -PIA inhibited [³H]ACh release, an effect blocked by DPCPX. It is concluded that adenosine A_2a receptors are present on both GABAergic and cholinergic striatal nerve terminals where they inhibit and stimulate transmitter release, respectively. Key Words : GABA—Acetylcholine—Adenosine receptors—Striatum.     

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.     

Platelet-Activating Factor: Diminished Acetylcholine Release from Rat Brain Slices Is Mediated by a Gi Protein

Abstract: The effect of platelet-activating factor (PAF) on neurotransmitter release from rat brain slices prelabeled with [³H]acetylcholine ([³H]ACh), [³H]norepinephrine ([³H]NE), or [³H]serotonin ([³H]5-HT) was studied. PAF inhibited K⁺ depolarization-induced [³H]ACh release in slices of brain cortex and hippocampus by up to 59% at 10 n M but did not inhibit [³H]ACh release in striatal slices. PAF did not affect 5-HT or NE release from cortical brain slices. The inhibition of K⁺-evoked [³H]ACh release induced by PAF was prevented by pretreating tissues with several structurally different PAF receptor antagonists. The effect of PAF was reversible and was not affected by pretreating brain slices with tetrodotoxin. PAF-induced inhibition of [³H]ACh release was blocked 90 ± 3 and 86 ± 2% by pertussis toxin and by anti-Gαi_1/2 antiserum incorporated into cortical synaptosomes, respectively. The results suggest that PAF inhibits depolarization-induced ACh release in brain slices via a Gαi_1/2 protein-mediated action and that PAF may serve as a neuromodulator of brain cholinergic system.     

THE EFFECT OF THIAMINE DEFICIENCY ON THE ACETYLCOENZYMEA AND ACETYLCHOLINE LEVELS IN THE RAT BRAIN

Abstract— It is shown that transketolase activities in red blood cells and whole brain of normal and thiamine-deficient rats correlate well with heart frequencies.
The effect of thiamine depletion on the levels of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh), and on the activities of pyruvate dehydrogenase, choline acetyl-transferase and acetylcholine esterase was studied in whole brains of thiamine-deficient, thiamine-supplemented ad libitum and pair-fed rats. The concentrations of acetyl-CoA and ACh decreased in thiamine-deficient brains by 42 and 35 per cent, respectively.
Total pyruvate dehydrogenase activity did not change during vitamin B₁ deficiency. The 'resolved' enzyme, reconstituted with thiamine diphosphate, had an association constant of 5.4 × 10⁻⁶ m . Choline acetyltransferase and acetylcholine esterase activities remained unchanged in thiamine deficiency.
Possible mechanisms which could explain the reduced Ach levels in vitamin B₁ deficiency are discussed.     

PYRUVATE METABOLISM IN THE LOBSTER NERVE AS AFFECTED BY THE PARTIAL PRESSURE OF CARBON DIOXIDE: OBSERVATIONS ON THE SYNTHESIS OF ACETYLCHOLINE AND ON METABOLIC COMPARTMENTATION

Abstract— In the lobster nerve the fixation of CO, at various levels of _pCO2 was studied by the incorporation of [l-¹⁴C]pyruvate. Incorporation of ¹⁴C was solely dependent on CO₂ fixation since the C-1 was decarboxylated in the formation of acetyl-CoA. Paired-nerve studies with [2-¹⁴C]pyruvate afforded a study of pyruvate metabolism in the lobster nerve. [I¹⁴C]Pyruvate was incorporated to nearly the same extent at all levels of pCO₂ including zero pCO₂, a finding that suggested metabolic recycling of CO₂. The magnitude of the metabolic recycling of C-1 of pyruvate or pyruvate dismutation was estimated to be nearly 20 per cent of total CO₂ fixation. Re-evaluation of the relative contributions of the CO₂ fixation. and acetyl-CoA pathways on the basis of more extensive data gave a ratio of 2:3.
The pCO₂ affected synthesis of ACh and the level of citrate. With increasing pCO₂, the specific radioactivity of ACh decreased much more than the content of ACh. The decrease in the specific radioactivity of ACh but not that of citrate further suggested metabolic compartmentation. The implication of these findings is discussed.
Alanine functioned as a metabolic sink for the incorporated pyruvate. Pyruvate levels were estimated to be approximately 0.1 nmol/mg of protein.     

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.     

A rapid assay for aluminium phytotoxicity at submicromolar concentrations

Investigations of Al phytotoxicity, including the identification of the Al species responsible for toxicity, require a rapid assay procedure employing very low concentrations of Al and a chemically simple rooting medium. Root elongation in newly germinated red clover ( Trifolium pratense L. cv. Kenland) was inhibited by submicromolar concentrations of Al. Ca²⁺ at concentrations of at least 0.2 m M was essential for optimal elongation in control seedlings. Ca²⁺ also relieved Al toxicity with the net effect that maximum reduction of elongation by 1 μ M Al was achieved at 0.2 m M Ca²⁺. Elongation in control seedlings was at least 90% of maximum from pH 4.5 to 5.7. Increases in pH relieved Al toxicity so that maximum sensitivity to 1 μ M Al occurred at pH 4.7. As a consequence of these experiments and other considerations we chose for our basic assay a medium composed of 0.2 m M CaSO₄ adjusted to pH 4.5 with H₂SO₄, variously supplemented with Al₂(SO₄)₃.
Day-old seedlings were incubated in this aerated medium in the dark at 23°C for one day. No additions of other solutes increased the sensitivity of the assay, but amelioration of Al toxicity was effected by Mg²⁺, F^-, phosphate and citrate. Increases in ionic strength per se had comparatively little effect on the toxic effects of Al. Two barley cultivars ( Hordeum vulgare L. cv. Dayton and Kearney) and two wheat cultivars ( Triticum aestivum L. cv. Hart and Thorne) known to differ in sensitivity to Al were reliably separated at submicromolar Al concentrations by the assay procedure, which was slightly modified. Suggestions for the improvement of the assay and for applications to future research are offered.     

The Release of γ-Aminobutyric Acid, Glutamate, and Acetylcholine from Striatal Slices: A Mass Fragmentographic Study

Abstract: The release processes of endogenous Acetylcholine (ACh), γ-aminobutyric acid (GABA), glutamate (Glu) and glutamine (GLN) were studied in superfused guinea-pig caudatal slices. Basal ACh release remained constant for up to 2 h, while the basal release of GABA, Glu and GLN declined to half or less of its initial values after 1 h of superfusion. Electrical stimulation increased the ACh release by 700–800% and that of GABA by 80% whereas it decreased the output of Glu by 50% and failed to modify the GLN efflux. KCl (25 mM) increased the output of ACh by 400%, that of GABA by approximately 500% and decreased that of Glu by 40%. Substituting of CaCl₂ by MgCl₂ in the superfusion medium reduced the basal ACh release by 70% whereas no differences were observed in the basal efflux of GABA, Glu and GLN. Under these conditions, no evoked release of ACh or of GABA was detected, following electrical or KCl stimulation. Tetrodotoxin 5 × 10^-7 M decreased the basal ACh release by 60% and increased the GABA efflux by 40%. The toxin abolished the stimulus-evoked ACh efflux but scarcely affected that of GABA. These results are consistent with a possible neurotransmitter role of ACh and GABA in the striatum and show some differences in the ionic mechanisms underlying GABA and ACh release.     

Influence of aluminium on phosphorus and calcium localization in roots of beech (Fagus sylvatica)

Beech plants ( Fagus sylvatica L. provenance Maramures) were grown in nutrient solution at low pH (4.2) and exposed to different concentrations of AlCl₃. Uptake and leakage of Ca²⁺(⁴⁵Ca²⁺) and H²PO₄-(³²P) were studied. A high external aluminium concentration (1.0m M ) reduced the uptake and export to the shoot of both calcium and phosphate, while 0.1 m M Al increased the phosphorus level in the roots. To determine the impact of aluminium on the localization of calcium and phosphate, leakage of the elements from both intact plants and plants frozen prior to the leakage experiment was studied. The leakage of Ca²⁺ from intact plants was not affected by prior exposure to 0.1 m M Al. Freezing of the beech plants before the leakage experiment increased leakage of calcium slightly more from roots of control plants than for roots exposed to 0.1 m M Al, indicating that even low concentrations of alminium may impede the influx of calcium across the plasma membrane in the roots. The patterns of Ca²⁺ leakage from roots previously exposed to 1.0 m M Al indicated that very little Ca²⁺ was located extracellularly. The extracellular fraction of phosphate increased with increasing Al concentration in the nutrient solution. Low Al concentration (0.1 m M ) only reduced the intracellular phosphate concentration to a minor extent, while 1.0 m M Al profoundly decreased it. It is concluded that 0.1 m M AlCl₃ has a limited effect upon the localization of Ca²⁺ and phosphate in the roots. At higher levels of Al, 0.1–1.0 m M , there is a more dramatic change in nutrient localization in the free space and uptake over the plasma membrane.     

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.     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

EFFECT OF CHOLINE ON THE RATES OF SYNTHESIS and OF RELEASE OF ACETYLCHOLINE IN THE ELECTRIC ORGAN OF TORPEDO

Abstract— Slices of electric organ of Torpedo marmorata were chopped and incubated in a saline-urea-sucrose medium. This preparation of minced tissue exhibited a relative enrichment in ACh and nerve endings, which was attributed to a loss of electroplaque cytoplasm. Electron microscopic controls showed nerve endings of normal morphology, some of them forming 'chaplets' separated from electro-plaques. Miniature endplate potentials were recorded on sealed fragments also present in this preparation. ACh levels remained unchanged during incubation periods as long as 19 h. The time course of the incorporation of [1-¹⁴C]acetate of [2-¹⁴C]pyruvate into ACh pools was studied. These incorporations were similarly affected by the choline added to the medium. In the presence of increasing choline concentrations (up to 10^-4 m ), the incorporation of [¹⁴C]acetate or [¹⁴C]pyruvate into ACh increased. They both diminished when choline was added above 10^-4M. The ACh content of the tissue was not affected by added choline. From the constancy of ACh levels in the presence of various choline concentrations and from the steady state of our preparation, we can conclude that the release of transmitter varied in parallel to the incorporation rate of the precursor of the acetyl moiety of ACh. This fact was also found using the efflux of [¹⁴C]acetate as an evaluation of ACh release. The values of release calculated by this method were in good agreement with those determined from the incorporations of acetate and pyruvate into ACh. It is suggested that the primary action of choline is on its high affinity carrier system. This triggers a secondary action on the ACh release mechanisms.     

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.     

ACETYLCHOLINE SYNTHESIS FROM [2-14C]PYRUVATE IN RAT STRIATAL SLICES

Abstract— Rat striatal slices were incubated with [2-¹⁴C]pyruvate or [6-¹⁴C]glucose as sole carbon source. The method devised to study the accumulation of labelled ACh in tissues and incubating medium in the presence or absence of eserine 200 μM derived from the previous studies of FONNUM (1969) and H emsworth and M orris (1964). Total ACh was estimated by biological assay.
The specific activity of newly synthesized ACh was found to be equal to that of the precursors, even for short incubation times and low substrates concentrations. When slices were incubated with [2-¹⁴C]pyruvate and eserine, the spontaneous release of ACh occurred at a constant rate, was not modified by the addition of 2 mM-choline in the medium, and consisted only of newly synthesized transmitter.
The initial rate of ACh synthesis was found to be independent of choline concentration, but dependent on the [2-¹⁴C]pyruvate concentration, and reached a maximal value corresponding to about 5 per cent of the measured striatal choline acetyltransferase activity.
The appearance of the so called 'surplus ACh' pool, obtained in the presence of eserine, could be detected only after 30 min and represented 26 per cent of the total tissue ACh content after 180 min of incubation.
In the absence of eserine, tissue ACh levels increased six-fold in 80 min and then remained stable until the end of the incubation period (180 min), if sufficient substrate was provided. The maximal ACh accumulation in slices was independent of both excess of choline and [2-¹⁴C]pyruvate.
The 'ACh plateau' represented the attainment of a new dynamic equilibrium, since ACh synthesis could still be stimulated by 30 mM-K⁺. From these results, it was concluded that ACh synthesis is controlled by a negative feed-back regulation.