Compartmentation and regulation of acetylcholine synthesis at the synapse.
ABSTRACT: Acetylcholine and choline release was measured by using an automated and modified version of the chemiluminescence technique of Israel & Lesbats [(1981) Neurochem. Int. 3, 81-90]. A comparison of acetylcholine and choline release from synaptosomes demonstrated that acetylcholine release was K+-stimulated and inhibited by the Ca2+ ionophore A23187 and cyanide. Choline release, however, did not vary markedly under different conditions, suggesting that it is not associated with acetylcholine release at the nerve ending. Total acetylcholine synthesis in synaptosomal preparations was measured concurrently with the incorporation of [14C]acetyl and [3H]choline moieties by using the chemiluminescence method. Under sub-optimal glucose concentrations or in the absence of treatment of the synaptosomes with the acetylcholinesterase inhibitor phospholine, the incorporation of radioactivity exceeded total synthesis, indicating that cycling between acetylcholine and its precursors may occur. After treatment with phospholine, acetyl-group incorporation from D-[U-14C]glucose occurred without dilution of the precursor at optimal (1.0 mM) and low (0.1 mM) glucose concentrations; however, at very low (0.01 mM) glucose concentrations, dilution by a small endogenous pool occurred. [14C]Acetyl incorporation into acetylcholine was compared with various metabolic parameters. A closer correlation was observed between [14C]acetyl-group incorporation into acetylcholine and the calculated acetyl-carrier efflux from the mitochondria than with the calculated pyruvate-dehydrogenase-complex flux. The results are discussed with respect to the regulation of acetylcholine concentrations at the synapse and the mechanism whereby turnover occurs.
Project description:1. Cerebral-cortex synaptosomes were shown to synthesize (14C)acetylcholine after incubation with (14C)choline, and 25mM-KCl released (14C)acetylcholine (but not (14C)choline) into the medium by a Ca2+-dependent and Mg2+-sensitive process. 2. The K+-stimulated release of (14C)acetylcholine was inhibited by more than 80% after preincubation of the synaptosomes with 10(5) mouse lethal doses of botulinum toxin/ml. (14C)choline uptake, (14C)acetylcholine synthesis, intrasynaptosomal K+ and occluded lactate dehydrogenase were unaffected by the toxin. It also failed to prevent the K+-stimulated release of (3H)noradrenaline and (14C)glycine from synaptosomes. 3. Fractionation of hypo-osmotically shocked synaptosomes revealed that more than 75% of the radioactive acetylcholine was in the cytoplasmic compartment, although the vesicle pellet contained more total acetylcholine than the cytoplasmic pool. Consequently the specific radioactivity of acetylcholine in the cytoplasmic pool was almost 5 times that of the vesicles. This distribution was unaffected by preincubation with botulinum toxin. It is concluded that the toxin acts directly on the release of acetylcholine, rather than influencing its storage. 4. After K+-stimulation, toxin-inhibited synaptosomes contained increased amounts of total acetylcholine, which suggests that its rate of synthesis is controlled by depolarization rather than release.
Project description:Synaptosomes from several regions of the rat brain were found to exhibit half-maximal rates of 14CO2 output and [14C]acetylcholine synthesis from D-[U-14C]glucose at glucose concentrations approx. 50-fold lower than those required by the brain in situ. However, synaptosomal acetylcholine synthesis was found not to be directly proportional to substrate oxidation as measured by 14CO2 output. When synaptosomes had been exposed to anoxia in vitro, their metabolic indices (14CO2 and [14C]acetylcholine synthesis, and adenine nucleotide levels) were found not to be significantly different from control aerobic values, unless they had been subjected to veratridine depolarization. This is in accord with previous findings that neither the absolute metabolic rates nor the vulnerability to hypoxic damage exhibited by brain in situ is reflected by brain slices in vitro, unless these are stimulated by depolarization. The use of synaptosomes as a model for synaptic damage in vivo is discussed.
Project description:Slices of rat caudate nuclei were incubated in vitro in media containing, among other constituents, three different concentrations of glucose (0.5, 2 and 10 mM), 0.2 mM-choline, paraoxon as an inhibitor of cholinesterase, and 5 mM- or 30 mM-K+. After 30 and 60 min of incubation, the concentrations of acetyl-CoA, acetylcholine and choline in the tissue and of acetylcholine in the incubation medium were measured. The content of acetyl-CoA in the sliced varied in direct relation to the concentration of glucose in the incubation medium. The content of acetylcholine in the slices and, in experiments with high K+, also the amount of acetylcholine released into the incubation medium varied in direct relation to the concentration of glucose in the incubation medium and to the concentration of acetyl-CoA in the slices; the relation between the concentrations of acetyl-CoA and of acetylcholine in the slices was linear. It was concluded that the availability of acetyl-CoA had a decisive influence on both the rate of synthesis of acetylcholine and its steady-state concentration. The observations accord with the view that, at the ultimate level, the synthesis of acetylcholine is controlled by the Law of Mass Action.
Project description:Menadione bisulphite increased endogenous oxygen-radical production by rat brain synaptosomes, as indicated by H2O2 generation. Increased oxygen-radical production was also demonstrated in synaptosomes prepared from menadione-treated rats and synaptosomes reoxygenated after an anoxic insult. Acetylcholine synthesis de novo was inhibited in synaptosomes incubated with menadione in vitro, in synaptosomes prepared from menadione-treated animals in vivo, and in depolarized post-anoxic synaptosomes. Intrasynaptosomal free Ca2+ was increased by menadione in vitro (50 microM), but this increase was not due to stimulation of Ca2+ entry into the nerve terminals. Acetylcholine release was stimulated by menadione in vitro, possibly as a consequence of the elevated intrasynaptosomal Ca2+ content. The Ca2+ contents of synaptosomes prepared from menadione (10 mg/kg)-treated animals in vivo and synaptosomes reoxygenated after anoxia were unchanged. In synaptosomes prepared from menadione-treated animals, acetylcholine release was no longer significantly stimulated by K+, whereas it was unchanged from control (normoxic) values in synaptosomes reoxygenated after anoxia. None of these treatments caused any measurable damage to the synaptic plasma membrane (as judged by the release of lactate dehydrogenase), or to synaptosomal phospholipases (as judged by choline release from membrane phospholipids). Synaptosomes prepared from menadione-treated rats were found to be a good model for the study of post-anoxic damage to nerve-terminal function.
Project description:Isolated alveolar epithelial type II cells were exposed to paraquat and to hyperoxia by gas diffusion through the thin Teflon bottom of culture dishes. After exposure, type II cells were further incubated in the presence of labelled substrates to assess their capacity to synthesize lipids. Hyperoxia alone (90% O2; 5 h) had minor effects on lipid metabolism in the type II cells. At low paraquat concentrations (5 and 10 microM), hyperoxia enhanced the paraquat-induced decrease of [Me-14C]choline incorporation into phosphatidylcholines. The incorporation rates of [Me-14C]choline, [1-14C]palmitate, [1-14C]glucose and [1,3-3H]glycerol into various phospholipid classes and neutral lipids were decreased by paraquat, depending on the concentration and duration of the exposure. The incorporation of [1-14C]acetate into phosphatidylcholines, phosphatidylglycerols and neutral lipids appeared to be very sensitive to inactivation by paraquat. At 5 microM-paraquat the rate of [1-14C]acetate incorporation was decreased to 50% of the control values. The rate of [1-14C]palmitate incorporation into lipids was much less sensitive; it even increased at low paraquat concentrations. At 10 microM-paraquat both NADPH and ATP were significantly decreased. It is concluded that lipid synthesis in isolated alveolar type II cells is extremely sensitive to paraquat. At low concentrations of this herbicide, lipid synthesis, and particularly fatty acid synthesis, is decreased. The effects on lipid metabolism may be partly related to altered NADPH and ATP concentrations.
Project description:Previous studies with electropermeabilized cells have suggested the occurrence of metabolic compartmentation and Ca2+-dependent channeling of intermediates of phosphatidylcholine (PC) biosynthesis in C6 rat glioma cells. With a more accessible permeabilization technique, we investigated whether this is a more general phenomenon also occurring in other cell types and whether channeling is involved in phosphatidylethanolamine (PE) synthesis as well. C6 rat glioma cells, C3H10T12 fibroblasts and rat hepatocytes were permeabilized with Staphylococcus aureus alpha-toxin, and the incorporation of the radiolabelled precursors choline, phosphocholine (P-choline), ethanolamine and phosphoethanolamine (P-EA) into PC and PE were measured both at high and low Ca2+ concentrations. In glioma cells, permeabilization at high Ca2+ concentration did not affect [14C]choline or [14C]P-choline incorporation into PC. However, reduction of free Ca2+ in the medium from 1.8 mM to <1 nM resulted in a dramatic increase in [14C]P-choline incorporation into permeabilized cells, whereas [14C]choline incorporation remained unaffected. Also, in fibroblasts, reduction of extracellular Ca2+ increased [14C]P-choline and [14C]P-EA incorporation into PC and PE respectively. In hepatocytes, a combination of alpha-toxin and low Ca2+ concentration severely impaired [14C]choline incorporation into PC. Therefore, alpha-toxin-permeabilized hepatocytes are not a good model in which to study channeling of intermediates in PC biosynthesis. In conclusion, our results indicate that channeling is involved in PC synthesis in glioma cells and fibroblasts. PE synthesis in fibroblasts is also at least partly dependent on channeling.
Project description:Increasing concentrations of CDP-choline progressively inhibit the measured incorporation of CDP-[2-14C]ethanolamine into phosphatidylethanolamine catalysed by the ethanolaminephosphotransferase present in endoplasmic-reticulum membranes isolated from castor-bean endosperm cells. This inhibition parallels that observed during CDP-[Me-14C]choline incorporation and suggests that a single enzyme utilizes both these substrates.
Project description:The relation between pyruvate utilization and acetylcholine synthesis was investigated in minces of adult rat brain. The flux of pyruvate to acetylcholine was less than 1% of that to CO2; nevertheless, a number of agents which inhibited conversion of [1-14C]-pyruvate or [2-14C]pyruvate into 14CO2 were associated with corresponding decreases in the conversion of [2-14C]pyruvate into acetylcholine. The amount of acetylcholine produced by minces of whole rat brain, measured by g.l.c.-mass spectrometry, decreased similarly. Among the inhibitory compounds tested were 3-bromopyruvate, an irreversible inhibitor of pyruvate dehydrogenase; 2-oxobutyrate, a competitive inhibitor of pyruvate dehydrogenase; other 2-oxo acids; and amobarbital and pentobarbital. Linear-regression equations relating CO2 production to acetylcholine synthesis gave correlation coefficients of 0.89-0.93 for the combined observations. The inhibition of acetylcholine synthesis could not be attributed to inhibition of choline acetyltransferase. Incorporation of [2-14C]pyruvate into lipids, proteins and nucleic acids was effected less than that into acetylcholine. Under these experimental conditions, it was shown that pyruvate utilization can limit acetylcholine synthesis.
Project description: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.
Project description:Formation of acetyl-CoA through acetyl-CoA synthetase (forward reaction) and through choline acyltransferase (backward reaction) was investigated in tissue extract from the electric organ of Torpedo marmorata. When the tissue extract was submitted to gel filtration on Sephadex G-25, the formation of acetyl-CoA by acetyl-CoA synthetase appeared fully dependent on ATP and CoA and partially dependent on acetate (an endogenous supply of acetate is discussed). Choline acetyltransferase was a potent source of acetyl-CoA, only requiring acetylcholine and CoA, and was much more efficient than acetyl-CoA synthetase for concentrations of acetylcholine likely to be present in nerve endings.