Vladimír Doležal

Utilization of Citrate, Acetylcarnitine, Acetate, Pyruvate and Glucose for the Synthesis of Acetylcholine in Rat Brain Slices

Abstract: Slices of rat caudate nuclei were incubated in saline media containing choline, paraoxon, unlabelled glucose, and [1,5‐14C]citrate, [1‐14C‐acetyl]carnitine, [1‐14C]acetate, [2‐14C]pyruvate, or [U‐14C]glucose. The synthesis of acetyl‐labelled acetylcholine (ACh) was compared with the total synthesis of ACh. When related to the utilization of unlabelled glucose (responsible for the formation of unlabelled ACh), the utilization of labelled substrates for the synthesis of the acetyl moiety of ACh was found to decrease in the following order: [2‐14C]pyruvate > [U‐14C]glucose > [1‐14C‐acetyl]carnitine > [1,5‐14C]citrate > [1‐14C]acetate. The utilization of [1,5‐14C]citrate and [1‐14C]acetate for the synthesis of [14C]ACh was low, although it was apparent from the formation of and 14C‐labelled lipid that the substrates entered the cells and were metabolized. The utilization of [1,5‐14C]citrate for the synthesis of [14C]ACh was higher when the incubation was performed in a medium without calcium (with EGTA); that of glucose did not change, whereas the utilization of other substrates for the synthesis of ACh decreased. The results indicate that earlier (indirect) evidence led to an underestimation of acetylcar‐nitine as a potential source of acetyl groups for the synthesis of ACh in mammalian brain; they do not support (but do not disprove) the view that citrate is the main carrier of acetyl groups from the intramitochondrial acetyl‐CoA to the extramitochondrial space in cerebral cholinergic neurons.

The effects of 4-aminopyridine and tetrodotoxin on the release of acetylcholine from rat striatal slices.

Summary1.Rat striatal slices were incubated in the presence of various concentrations of Ca2+ and K+ and of 4-aminopyridine and tetrodotoxin and the release of acetylcholine (ACh) into the medium was measured. In the presence of 2.5 mM Ca2+ and 5 mM K+, the release of ACh was increased by 230–410% when 0.1 mM 4-aminopyridine had been added to the medium. The effect of 4-aminopyridine was lower when the concentration of Ca2+ was diminished and became insignificant when Ca2+ had been omitted; it was also lower when the concentration of K+ was raised.2.The release of ACh, measured at 5 mM K+ and 2.5 mM Ca2+, was inhibited by only 15% when 1 μM tetrodotoxin had been added to the medium, an observation indicating that there was little conducted impulse activity in the slices. In the presence of 4-aminopyridine, however, tetrodotoxin inhibited the release of ACh by more than 60%. When expressed in absolute values of nmoles of ACh released, the inhibitory effect of tetrodotoxin on the release of ACh was 12–19 times as high in the presence as in the absence of 4-aminopyridine. This indicates that the stimulation of the release of ACh produced by 4-aminopyridine was associated with a substantial increase in the impulse activity in the slices.3.Most of the 4-aminopyridine-induced increase in the release of ACh from the slices incubated in 5 mM K+ can be explained by the increase in impulse activity and by a prolongation of nerve terminal depolarizations. However, 4-aminopyridine increased the release of ACh by more than 50% even in the presence of 1 μM tetrodotoxin. Available data do not permit to decide whether this part of the effect of 4-aminopyridine was due to its direct action on the Ca2+ channels in the nerve terminals or to its action on the K+ channels, only secondarily promoting the appearance of regenerative inward currents of Ca2+.

The synthesis and release of acetylcholine in normal and denervated rat diaphragms during incubation in vitro

1. Normal and denervated rat diaphragms and neural (central) and aneural (peripheral) parts of normal diaphragms were incubated under several different conditions likely to affect the metabolism of acetylcholine (ACh), with the aim of discovering specific features of the control of neural and aneural ACh in the muscle. The concentrations of ACh in the tissue and the medium were measured at the end of the incubations using a radioenzymatic assay, and the amount of ACh synthesized during the incubations was calculated by subtracting the initial amount of ACh present in the tissue from that found in the tissue plus the medium at the end of the incubations.

Activation of muscarinic receptors stimulates the release of choline from brain slices

The effect of several agents known to interact with muscarinic acetylcholine receptors on the release of choline from slices of rat corpus striatum into the incubation medium has been investigated. The amount of released choline was increased if choline, acetylcholine, or oxotremorine had been added to the incubation medium. Atropine blocked the effects of acetylcholine and oxotremorine; it was not tested with choline. It is proposed that the increased release of choline is due to an increased hydrolysis of phosphatidylcholine, brought about by the activation of muscarinic acetylcholine receptors.

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.

Failure of the calcium channel activator, Bay K 8644, to increase the release of acetylcholine from nerve terminals in brain and diaphragm

1 The calcium channel activator Bay K 8644 did not increase the release of acetylcholine from rat brain cortex prisms incubated in the presence of 3 mmol l−1 or 25 mmol l−1 K+ nor from rat diaphragms incubated in the presence of 5 mmol l−1 or 25 mmol l−1 K+. It also did not influence the release of acetylcholine from cortex prisms incubated in the presence of 25 mmol l−1 K+ and of lowered concentrations of Ca2+ ions. 2 It is concluded that the voltage‐dependent Ca2+ channels in the nerve terminals, responsible for the depolarization‐induced influx of Ca2+ ions into the nerve terminals and thus for the depolarization‐evoked release of acetylcholine from the nerve terminals, are different from the voltage‐dependent Ca2+ channels in the heart and smooth muscle cells.

Negative Effects of Tacrine (Tetrahydroaminoacridine) and Methoxytacrine on the Metabolism of Acetylcholine in Brain Slices Incubated Under Conditions Stimulating Neurotransmitter Release

Abstract: The effects of tacrine (1,2,3,4‐tetrahydro‐9‐aminoacridine) and 7‐methoxytacrine on the metabolism of brain acetylcholine were investigated in experiments in which acetylcholine turnover was stimulated by tissue depolarization or by 4‐aminopyridine. Rat cerebrocortical prisms were preincubated under “resting”conditions (Krebs‐Ringer buffer with 3 mmol/L K+ and with paraoxon to inhibit cholinesterases) and then incubated in the presence of tacrine or methoxytacrine and of 50 mmol/L K+. Both drugs diminished the amount of acetylcholine released by depolarization and the amount of acetylcholine synthesized during incubation; in experiments in which [14]choline was present in the incubation medium simultaneously with tacrine or methoxytacrine, the drugs diminished the uptake of [14C]choline by the tissue and the amount of [14C]‐acetylcholine synthesized and released into the medium. In these experiments, it was not possible to distinguish whether the inhibitory effects of tacrine and methoxytacrine were primarily on the process of acetylcholine synthesis (particularly on the uptake of choline), or whether the drugs also acted directly on the process of neurotransmitter release. In subsequent experiments the prisms were preincubated with [14C]choline and only then subjected to a short depolarization in the presence of hemicholinium‐3 and tacrine or methoxytacrine. Both drugs severely inhibited the release of preformed [14C]acetylcholine and prevented the diminution of tissue [14C]acetylcholine stores. Methoxytacrine was also found to diminish the release of acetylcholine induced by 4‐aminopyridine while increasing the content of acetylcholine in the tissue. Tacrine and methoxytacrine had no effect on the activity of choline acetyltransferase (EC 2.3.1.6). Our observations indicate that, in addition to reducing the uptake of choline, tacrine and methoxytacrine inhibit the stimulated release of acetylcholine by acting directly on the process of neurotransmitter liberation. Most effects of tacrine and methoxytacrine were observed at a concentration of 100 μmol/L, but some were already apparent at a concentration of 10 μmol/L. The molecular mechanism of the direct inhibitory action of tacrine and methoxytacrine on the stimulated release of acetylcholine is not clear.

Positive and Negative Effects of Tacrine (Tetrahydroaminoacridine) and Methoxytacrine on the Metabolism of Acetylcholine in Brain Cortical Prisms Incubated Under “Resting”Conditions

Abstract: The effects of tacrine (1,2,3,4‐tetrahydro‐9‐aminoacridine) and 7‐methoxytacrine on the metabolism of acetylcholine were investigated in experiments on prisms of rat cerebral cortex incubated in vitro in low‐potassium (3 mmol/L K+) media; cholinesterases were inactivated by paraoxon to avoid any action of tacrine and methoxytacrine via their inhibition. Under “resting”conditions, tacrine and methoxytacrine increased the synthesis of unlabeled acetylcholine in the prisms; at the same time, they inhibited the uptake of [14C]choline from the medium and the synthesis of [14C]acetylcholine. The concentration of free choline was not increased by tacrine or methoxytacrine in either the tissue or the medium. The contradiction between the increased synthesis of unlabeled and the diminished synthesis of labeled acetylcholine indicates that the utilization of intracellular choline (which is presumably mobilized from intracellular choline esters) for the synthesis of acetylcholine is increased by tacrine and methoxytacrine. This conclusion is supported by the observation that the inhibition of acetylcholine synthesis during incubation with hemicholinium‐3 (an inhibitor of choline transport into cholinergic nerve terminals) was overcome when tacrine was present simultaneously with hemicholinium‐3. When the prisms were preincubated with [14C]choline and incubated with tacrine or methoxytacrine only after this, the amount of [14C]acetylcholine recovered in the tissue plus the medium was higher at the end of incubation with tacrine or methoxytacrine than without them, again suggesting that the drugs were able to increase the utilization of intracellular [14C]choline or its esters for acetylcholine synthesis. Most experiments were performed with tacrine and methoxytacrine at concentrations of 100 μmol/L, but a positive action of tacrine on the synthesis of acetylcholine was apparent at a concentration as low as 10 μmol/L. The mechanism by which tacrine and methoxytacrine increase the utilization of intracellular choline or its esters for the synthesis of acetylcholine has not been identified.

Effects of atropine on the release of newly synthesized acetylcholine from rat striatal slices at various concentrations of calcium ions

The release of acetylcholine (ACh) from brain tissue is known to be inhibited by muscarinic autoreceptors on cholinergic nerve terminals but the mechanism of the inhibition is not understood. Atropine brings about an increase of ACh release by removing the inhibitory action of autoreceptors. We investigated whether the effect of atropine on the release of [14C]ACh newly synthesized during incubations from [U-14C] glucose depends on the concentration of Ca2+ in the medium. In rat striatal slices incubated in the presence of an inhibitor of cholinesterases and of 30 mmol/l K+, significant increases in the release of [14C]ACh elicited by atropine were only observed during incubations with very low concentrations of Ca2+. This finding supports the view that the activation of presynaptic muscarinic autoreceptors in the brain affects the release of ACh by reducing the availability of Ca2+ that is required for transmitter liberation.

Tacrine (Tetrahydroaminoacridine) and the Metabolism of Acetylcholine and Choline

Tacrine (9-amino-1,2,3,4-tetrahydroacridine) is a reversible noncompetitive inhibitor of cholinesterases (Shaw and Bentley, 1953; Heilbronn, 1961; Patocka et al., 1976; Bajgar et al., 1979; Marquis, 1990) which has been described to have a marked positive effect in the treatment of patients with Alzheimer’s disease (Summers et al., 1986). Since the attempts to treat such patients with other potent inhibitors of cholinesterases had been less successful or not successful at all, the possibility appears likely that tacrine acts not only by inhibiting cholinesterases, but also by producing some additional pharmacological effects.