Stanislav Tuček

Regulation of acetylcholine synthesis in the brain.

The understanding of the synthesis of acetylcholine (ACh) in cholinergic neurons developed steadily during the 1970s and early 1980s and major progress has been achieved in the investigation of most of its aspects. The discovery and characterization of choline carriers in neuronal membranes, the clarification of the origin of acetyl groups of ACh, the elucidation of the significance of axonal transport for the function of cholinergic nerve terminals, the purification of the synthesizing enzyme, choline acetyltransferase (ChAT), and the production of monoclonal antibodies against it (enabling investigators to identify cholinergic neurons throughout the CNS) are examples of important achievements in this area of research. The interest in the synthesis of ACh and the factors that control or affect it was much stimulated by the discovery that an impairment in the production of ACh plays a crucial role in the pathogenesis of Alzheimer’s disease. Problems of the synthesis of ACh including its regulation have been repeatedly reviewed and the reader is advised to turn to articles by MacIntosh and Collier (1976), Collier (1977), Haubrich and Chippendale (1977), Jope (1979), Jenden (1979a,b), and TuCek (1983a, 1984), and to books written or edited by Goldberg and Hanin (1976), Tutek (1978, 1979), Barbeau et al. (1979), Davis and Berger (1979), and Hanin and Goldberg (1982) for comprehensive summaries of experimental data; Table 1 in TuCek’s (1984) article lists additional reviews concerning specific aspects of the synthesis of ACh. The present article is a commentary or overview rather than a review. It has been written in the belief that time is ripe to summarize (and partly restate) what appears to be, in the author’s view and in the light of data available in mid-1984, the most likely model of how the synthesis of ACh is regulated. It is hoped that such an outline may be of some use to colleagues who became interested in the problems of ACh synthesis only recently and in a way marginally, mainly in connection with the attempts to unravel the pathogenesis and find a treatment for Alzheimer’s disease. Many statements in the article are necessarily of a generalizing and simplifying nature; more complete arguments may be found in TuCek (1984).

The synthesis of acetylcholine in skeletal muscles of the rat

1. The synthesis of acetylcholine (ACh) has been measured in homogenates of the sciatic nerve, normal and denervated extensor digitorum longus (e.d.l.) muscles, and central (innervated) and peripheral (non‐innervated) parts of the diaphragm of the rat. The synthesis proceeded under conditions accepted as optimal for the activity of choline acetyltransferase (ChAT). In view of the finding that cardiac carnitine acetyltransferase (CarAT) is able to acetylate choline (White & Wu, 1973), the possible contribution of CarAT to the synthesis of ACh in the muscles was investigated by using bromoacetylcholine (BrACh) as an inhibitor of ChAT and bromoacetylcarnitine (BrACar) as an inhibitor of CarAT.

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.

PROVENANCE OF THE ACETYL GROUP OF ACETYLCHOLINE AND COMPARTMENTATION OF ACETYL-CoA AND KREBS CYCLE INTERMEDIATES IN THE BRAIN IN VIVO

—The origin of the acetyl group in acetyl‐CoA which is used for the synthesis of ACh in the brain and the relationship of the cholinergic nerve endings to the biochemically defined cerebral compartments of the Krebs cycle intermediates and amino acids were studied by comparing the transfer of radioactivity from intracisternally injected labelled precursors into the acetyl moiety of ACh, glutamate, glutamine, ‘citrate’(= citrate +cis‐aconitate + isocitrate), and lipids in the brain of rats. The substrates used for injections were [1‐14C]acetate, [2‐14C]acetate, [4‐14C]acetoacetate, [1‐14C]butyrate, [1, 5‐14C]citrate, [2‐14C]glucose, [5‐14C]glutamate, 3‐hydroxy[3‐14C]butyrate, [2‐14C]lactate, [U‐14C]leucine, [2‐14C]pyruvate and [3H]acetylaspartate.

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+.

Interaction of the neuromuscular blocking drugs alcuronium, decamethonium, gallamine, pancuronium, ritebronium, tercuronium and d-tubocurarine with muscarinic acetylcholine receptors in the heart and ileum

Summary1.Neuromuscular blocking drugs have a high affinity for muscarinic acetylcholine receptors in the heart atria and ileal smooth muscle. In experiments on homogenates, alcuronium, gallamine, pancuronium, tercuronium and ritebronium inhibited the binding of the muscarinic antagonist (3H)quinuclidinyl, benzilate (QNB) to rat heart atria with IC50 values of 0.15–0.53 μmol · l−1 and to ileal longitudinal muscles with IC50 values of 0.12–0.45 μmol · l−1. d-Tubocurarine and decamethonium inhibited (3H)QNB binding to these tissues with IC50 values of 6.2–8.5 μmol · l−1.2.For each neuromuscular blocking drug, the IC50 values were virtually identical for (3H)QNB displacement in the homogenates of the atria and of the ileal muscle.3.Alcuronium and gallamine differed from the other blocking agents in that they produced less steep (3H)QNB displacement curves both in the atria and the ileal muscle; Hill coefficients for the binding of alcuronium and gallamine to atrial and ileal homogenates were lower than unity.4.On isolated atria, gallamine, pancuronium, ritebronium and tercuronium antagonized the inhibition of tension development caused by the muscarinic agonist, methylfurmethide, with Kd values which were of the same order of magnitude as the IC50 values for the displacement of (3H)QNB binding to homogenates; the Kd of alcuronium was 12.5 times higher. d-Tubocurarine and decamethonium did not antagonize the effects of methylfurmethide at concentrations up to 100 μmol · l−1.5.On isolated ileal longitudinal muscle, gallamine and pancuronium antagonized the effects of methylfurmethide with Kd values that were 53 times and 100 times higher than their respective Kd values in the atria. Alcuronium, d-tubocurarine and decamethonium at concentrations of up to 100 μmol · l−1 did not antagonize the effects of methylfurmethide. Pharmacologically determined Kd values of gallamine and pancuronium were 129 times and 83 times higher in the isolated ileal muscle than were their respective IC50 values for (3H)QNB displacement in ileal homogenates.6.The results indicate that there is a high degree of cardioselectivity in the antimuscarinic action of gallamine, pancuronium and alcuronium, whilst with ritebronium and tercuronium there is little difference between the action on the atria and ileal muscle. The cardioselectivity of gallamine, pancuronium and alcuronium is in a sharp contrast to their equal binding to the homogenates of the atria and ileal smooth muscle. The discrepancy between the binding and pharmacological effect in the ileum suggests that, at low concentrations, gallamine, pancuronium and alcuronium bind to muscarinic receptors in the ileal smooth muscle in such a way that they interfere with the binding of (3H)QNB but not with that of methylfurmethide; the same applies to the binding of d-tuborurarine and decamethonium to muscarinic receptors both in the atria and the ileum. An alternative possibility is that the affinity of muscarinic receptors for neuromuscular blockers is low in intact smooth muscle cells but increases strongly after homogenization.

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.

The effects of brucine and alcuronium on the inhibition of [3H]acetylcholine release from rat striatum by muscarinic receptor agonists.

1 Radioligand binding experiments indicate that the affinity of muscarinic receptors for their agonists may be enhanced by allosteric modulators. We have now investigated if brucine can enhance the inhibitory effects of muscarinic receptor agonists on the electrically evoked release of [3H]acetylcholine ([3H]ACh) from superfused slices of rat striatum. 2 The evoked release of [3H]ACh was inhibited by all agonists tested (i.e., furmethide, oxotremorine‐M, bethanechol and oxotremorine). 3 Brucine enhanced the inhibitory effects of furmethide, oxotremorine‐M and bethanechol on the evoked [3H]ACh release without altering the inhibitory effect of oxotremorine. 4 Alcuronium was applied for comparison and found to diminish the inhibitory effect of furmethide on the evoked [3H]ACh release. 5 The results demonstrate that it is possible both to enhance and diminish the functional effects of muscarinic receptor agonists by allosteric modulators. 6 The direction of the observed effects of brucine and alcuronium on [3H]ACh release fully agrees with the effects of these modulators on the affinities of human M4 receptors for furmethide, oxotremorine‐M, bethanechol and oxotremorine, as described by Jakubík et al. (1997) . This supports the view that the presynaptic muscarinic receptors responsible for the autoinhibition of ACh release in rat striatum belong to the M4 muscarinic receptor subtype.

Testosterone-induced changes of choline acetyl-transferase and cholinesterase activities in rat levator ani muscle

One year after castration the activities of choline acetyltransferase (ChAc) and of cholinesterase (ChE) in the levator ani (LA) muscle of male rats were lowered by 42 and 79% respectively. The weight of the muscle corresponded to 15% of control values. These changes were not accompanied by a decrease in the number of the muscle fibres. Treatment with testosterone rapidly increased the activity of ChAc and the weight of the muscle near to control values; the restoration of ChE was less complete. Testosterone produced an increase in the size of the muscle fibres and increased the histochemically observed activity of ChE in the postsynaptic part of the motor end-plates. In non-castrated rats the administration of testosterone increased the weight ofthe LA muscle, but was not accompanied by an increase of ChAc above control values.

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.