Phospholipid-derived choline intermediates and acetylcholine synthesis in mouse brain synaptosomes

Endogenous free choline levels and acetylcholine (ACh) synthesis in nerve terminals were investigated using cerebral cortical synaptosomes of C57BL/6 mice. Endogenous choline was produced at a rate ten-fold faster than ACh to provide levels adequate for the formation of the latter. The combined pool size of the water-soluble intermediates derived from phosphatidylcholine (PhC), such as glycerophosphorylcholine (GpCh) and phosphorylcholine (PCh), increased significantly during the first 10-15 min of incubation and was always higher than that of free choline. These results most likely indicate an effective degradation of PhC by the combined action of phospholipase A2/lysophospholipase, as well as by phospholipase C in synaptosomes. ACh synthesis proceeded at a constant rate in the presence or absence of exogenous free choline (0-10 microM) and was almost entirely abolished in the presence of 10(-6) M hemicholinium-3. These results suggest that ACh is effectively synthesized by free choline generated in synaptosomes by a coupling mechanism involving the high-affinity choline uptake system. No changes in the production rates of choline and ACh were observed between adult and aged mice.     

Stimulation of choline acetyl transferase activity by l- and d-carnitine in brain areas of neonate rats

Acetyl-CoA supply to the cytosol and its regulatory influence on acetylcholine biosynthesis is still an unsolved question. Acetylcarnitine through the carnitine acetyl transferase (CarAT) system has been proposed to be the acetyl donor in this process. Carnitine isomers were injected into rat developing brains every day for 14 days after birth. Results showed that carnitine and its associated forms produced a choline acetyl transferase (ChAT) activity increase in the striatum and the hippocampus. Carnitine acetyl transferase activity was stimulated by the treatment of 1-carnitine in the hippocampus but it remained unchanged in the striatum and the cerebral cortex. These results suggest that ChAT and CarAT activities might be modulated by Acetyl-CoA derived preferentially from acetylcarnitine. It is suggested that ChAT activity enhancement depends on intrinsic and extrinsic cholinergic afferents to these brain areas. © 1995 Wiley-Liss, Inc.     

High affinity choline transport and acetylCoA production in brain and their roles in the regulation of acetylcholine synthesis

This review describes recent advances made in the understanding of the regulation of acetylcholine synthesis in brain with regard to the availability of its two precursors, choline and acetylCoA. Choline availability appears to be regulated by the high affinity choline transport system. Investigations of the localization and inhibition of this system are reviewed. Procedures for measuring high affinity choline transport and their shortcomings are described. The kinetics and effects of previous in vivo and in vitro treatments on high affinity choline transport are reviewed. Kinetic and direct coupling of the transport and acetylation of choline are discussed.Recent investigations of the source of acetylCoA used for the synthesis of acetylcholine are reviewed. Three sources of acetylCoA have recently received support: citrate conversion catalyzed by citrate lyase, direct release of acetylCoA from mitochondria following its synthesis from pyruvate catalyzed by pyruvate dehydrogenase, and production of acetylCoA by cytoplasmic pyruvate dehydrogenase. Investigations indicating that acetylCoA availability may limit acetylcholine synthesis are reviewed. A model for the regulation of acetylcholine synthesis which incorporates most of the reviewed material is presented.     

Choline and phospholipid metabolism and the synthesis of acetylcholine in rat brain.

The metabolism of choline by rat brain, plasma, and liver was investigated using combined gas chromatography mass spectrometry following microwave irradiation and treatment with deuterium-labeled choline. Methods were established to measure simultaneously the concentrations of six choline-containing compounds and the incorporation of labeled choline into each of them. Intravenous injection of [2H4]-choline led to initial labeling of choline, acetylcholine, and phosphocholine in rat brain, with all of the label eventually entering the phosphocholine pool. When labeled choline was administered in the diet its rate of incorporation into choline, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the plasma and liver and into choline, acetylcholine, phosphocholine, glycerophosphorylcholine, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the brain were determined. Choline, phosphatidylcholine, and combined choline plasmalogen and lysophosphatidylcholine in the plasma had similar specific activities. In the cortex and the striatum, choline and combined choline plasmalogen and lysophosphatidylcholine fraction generally had the highest specific activities. The time course of the post-mortem release of choline by the brain was measured, and the sources of this choline were, sequentially, acetylcholine, glycerophosphoryl-choline, and phospholipids.     

Inhibitors of serine/threonine phosphatases increase membrane-bound choline acetyltransferase activity and enhance acetylcholine synthesis

The present investigation examines the effects of phosphatase inhibition on short-term regulation of cholinergic function, with particular emphasis on choline acetyltransferase, the enzyme which synthesizes acetylcholine. Rat hippocampal synaptosomes were treated with either okadaic acid (10 nM) or calyculin-A (50 nM) to inhibit protein phosphatases 1 and 2A for 20 min prior to subfractionation of nerve terminals and measurement of choline acetyltransferase activity, or quantification of high-affinity choline transport and acetylcholine synthesis. Inhibition of synaptosomal phosphatases did not alter total or salt-soluble choline acetyltransferase activity, but membrane-bound and water-soluble forms of the enzyme were selectively increased in okadaic acid-treated nerve terminals to 129±11% and 137±10% of control, respectively. High-affinity choline transport was reduced to 77±6% and 76±7% of control in calyculin-A- and okadaic acid-treated nerve terminals, respectively. Acetylcholine synthesis was reduced to 73±6% of control in calyculin-A-treated synaptosomes only; acetylcholine synthesis was at control levels in okadaic acid-treated cultures correlating with enhanced choline acetyltransferase activity in the water-soluble and nonionically membrane-bound fractions. These investigations indicate a role for phosphoprotein phosphatases in the regulation of acetylcholine synthesis in the cholinergic nerve terminal. The observed increases in choline acetyltransferase activity in two subcellular fractions appears to compensate for decreased choline precursor availability, allowing acetylcholine synthesis to be maintained at control levels. The uncoupling of choline transport and acetylcholine synthesis in this situation represents a unique functional role for a subfraction of choline acetyltransferase.     

Veratridine-induced release of acetylcholine from mouse forebrain minces: Dependence on the hydrolysis of cytoplasmic acetylcholine for a source of choline

The importance of depolarization induced hydrolysis of cytoplasmic acetylcholine (ACh) in providing choline for the veratridine- and high K⁺-induced release of acetylcholine was studied in mouse forebrain minces. Results indicated that a loss of hydrolyzable cytoplasmic ACh prior to depolarization reduced the amount of ACh released by veratridine but not the amount released by high K⁺. The reduction in the veratridine-induced release of ACh did not occur during the first 5 min of incubation. Loss of vesicular ACh prior to depolarization reduced both the veratridine- and K⁺-induced release of ACh during the first 5 min of incubation. Blockade of extra-cellular choline transport by hemicholinium (HC-3) did not affect the veratridine-induced release of ACh during a 10 min incubation period unless the cytoplasmic pool of ACh had first been depleted and was unavailable as a source of choline. In contrast, HC-3 reduced the K⁺-induced release of ACh from brain tissue with normal stores of cytoplasmic ACh. These results indicate that both depolarizing agents primarily stimulate the release of preformed ACh from a vesicular fraction during the first 5 min of mince incubation. Thereafter, they both stimulate the release of newly synthesized ACh, however, they differ in one important respect. The principal source of choline for the veratridine-induced release of newly synthesized ACh appears to be the cytoplasmic pool of ACh, whereas the major source of choline for the K⁺-induced release of newly synthesized ACh appears to be extracellular choline.     

Metabolism of choline in brain of the aged CBF-1 mouse

In order to quantify the changes that occur in the cholinergic central nervous system with aging, we have compared acetylcholine (Ach) formation in brain cortex slice preparations from 2-year-old aged CBF-1 mouse brains and compared the findings with those in 2-4-month-old young adult mouse brain slices. Incorporation of exogenous radioactively labelled choline (31 nM [3H] choline) into acetyl choline in incubated brain slices was linear with time for 90 min. Percentage of total choline label distributed into Ach remained constant from 5 min after starting the incubation to 90 min. In contrast, distribution of label into intracellular free choline (Ch) and phosphorylcholine (Pch) changed continuously over this period suggesting that the Ch pool for Ach synthesis in brain cortex is different from that for Pch synthesis. Incorporation of radioactivity into Ach was not influenced by administration of 10 microM eserine, showing that the increment of radioactivity in Ach reflects rate of Ach formation, independently from degradation by acetylcholine esterases. Under our experimental conditions, slices from cortices of aged 24-month-old mouse brain showed a significantly greater (27%) incorporation of radioactivity into intracellular Ach than those from young, 2-4-month-old, brain cortices. Inhibitors of Ach release, 1 mM ATP or GABA, had no effect. Since concentration of radioactive precursor in the incubation medium was very low (31 nM), the Ch pool for Ach synthesis in slices was labelled without measurably changing the size of the endogenous pool. These data suggest a compensatory acceleration of Ach synthesis or else a smaller precursor pool specific for Ach synthesis into which labelled Ch migrated in aged brain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of electrical stimulation on acetylcholine synthesis in cat caudate nucleus

The concentration of endogenous- and deuterium-labeled acetylcholine (ACh) in the cat caudate nucleus was determined after stimulation of either the substantia nigra or the precruciate cortex. In this procedure the caudate nucleus is exposed surgically, and a coring device is used to obtain biopsy specimens which are immediately frozen in liquid nitrogen. Samples are collected at rest, 5 min after stimulation, and again 5 min after a resting period. An infusion of 2H9-choline is maintained during these manipulations to provide a label for ACh synthesis. Electrical stimulation of the substantia nigra, which increases the release of dopamine, produced a decrease in endogenous ACh and the newly synthesized deuterium-labeled ACh. Stimulation of the precruciate cortex produced no significant effect on the levels or synthesis of ACh, but attenuated the effect of subsequent nigral stimulation. These preliminary results indicate that stimulation of the substantia nigra has a net excitatory effect on ACh synthesis in the caudate. This stimulation apparently is modulated by input from the cortex.     

Effects of 4-(2-Hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183) on rat cortical synaptosome choline uptake, acetylcholine storage and release

The cholinergic vesicular uptake blocker, 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (AH5183), had several effects on presynaptic cholinergic function that depended on the duration of treatment and dose. The synthesis, storage and release of newly synthesized [3H]ACh were monitored because the vesicular uptake of this pool of transmitter may be preferentially affected by the drug. Initially, high concentrations of AH5183 (over 10 microM) increased the spontaneous release but decreased the K+ depolarization-induced release of newly synthesized transmitter. [3H]Choline efflux was not altered by the drug. High affinity choline uptake was slightly (10-20%) inhibited by AH5183 in an apparently competitive but time-dependent manner. In contrast to its initial effects on [3H]ACh release, AH5183 (50nM-100 microM) very potently inhibited both the spontaneous and K+-induced release of [3H]ACh but not of [3H]choline after a 60 min preincubation. [3H]ACh levels in cytoplasmic (S3) and crude membrane (P3) fractions were not affected by a 2-min incubation with 10 microM AH5183. After a 60-min preincubation with this drug dose, however, the P3 and S3 levels of newly synthesized transmitter were decreased and increased, respectively. Subsequent fractionation of synaptosomes by sucrose-density gradient centrifugation revealed that these reductions in P3 [3H]ACh-levels were referable to reductions in two subfractions D and H that have been reported to contain low density vesicles and denser vesicles associated with plasma membranes, respectively.     

蓝斑核注射乙酰胆碱对大鼠蓝斑核痛反应神经元电活动的影响

[摘要] 目的 观察蓝斑核（LC）注射乙酰胆碱(ACh)后,蓝斑核（LC）中痛反应神经元的放电变化,研究ACh与LC在痛觉信息通路中的作用。 方法 以电脉冲刺激坐骨神经作为伤害性刺激,用玻璃微电极引导LC中痛反应神经元的电变化。结果 ① LC内注入ACh能够使大鼠LC中痛兴奋神经元（PEN）痛诱发放电频率增加、潜伏期缩短;痛抑制神经元（PIN）痛诱发放电频率减少、完全抑制时程延长;② LC内注入ACh 的M受体拮抗剂阿托品能够阻断ACh的上述效应。结论 ACh可使正常大鼠LC中痛反应神经元对伤害性刺激的反应增强,表现为致痛效应;揭示了ACh和LC在痛觉调制中具有非常重要的作用。     

Investigation of the mechanism of the effect of tacrine (tetrahydroaminoacridine) on the metabolism of acetylcholine and choline in brain cortical prisms

Summary The mechanism by which tacrine increases the content and synthesis of acetylcholine (ACh) in cerebrocortical prisms exposed to an irreversible inhibitor of cholinesterases and incubated under resting conditions (Doleal and Tuek, 1991) is not known. As found in the present experiments, this effect of tacrine is only apparent if its application had been preceded by a period of preincubation, but the preincubation is ineffective if it occurs in the presence of hemicholinium-3. Apparently, choline or a choline-containing compound accumulates in the slices during the preincubation and is then utilized for the enhanced synthesis of ACh in the presence of tacrine. Tacrine did not induce a decrease in the amount of radiolabel that had been incorporated from choline into acid-insoluble compounds, which suggests that the choline which is used for the synthesis of additional ACh does not originate from choline lipids. However, tacrine was found to diminish the efflux of choline from prisms which had been preincubated with an increased concentration of choline in the medium, and from prisms incubated in the presence of hemicholinium-3. It also diminished the efflux of radioactive choline that had accumulated in the prisms during preincubation with a very low concentration of tacrine, when the prisms were subsequently incubated with 4-aminopyridine. It is proposed that the potency of tacrine to increase the content and synthesis of ACh in cerebrocortical prisms whose cholinesterases had been inhibited is due to its ability to diminish the efflux of endogenous choline from the nerve terminals.     

Acetylcholine synthesis and release in NIH3T3 cells coexpressing the high‐affinity choline transporter and choline acetyltransferase

Acetylcholine (ACh) is known to be a key neurotransmitter in the central and peripheral nervous systems, but it is also produced in a variety of non‐neuronal tissues and cells, including lymphocytes, placenta, amniotic membrane, vascular endothelial cells, keratinocytes, and epithelial cells in the digestive and respiratory tracts. To investigate contribution made by the high‐affinity choline transporter (CHT1) to ACh synthesis in both cholinergic neurons and nonneuronal cells, we transfected rat CHT1 cDNA into NIH3T3ChAT cells, a mouse fibroblast line expressing mouse choline acetyltransferase (ChAT), to establish the NIH3T3ChAT 112‐1 cell line, which stably expresses both CHT1 and ChAT. NIH3T3ChAT 112‐1 cells showed increased binding of the CHT1 inhibitor [³H]hemicholinium‐3 (HC‐3) and greater [³H]choline uptake and ACh synthesis than NIH3T3ChAT 103‐1 cells, a CHT1‐negative control cell line. HC‐3 significantly inhibited ACh synthesis in NIH3T3ChAT 112‐1 cells but did not affect synthesis in NIH3T3ChAT 103‐1 cells. ACh synthesis in NIH3T3ChAT 112‐1 cells was also reduced by amiloride, an inhibitor of organic cation transporters (OCTs) involved in low‐affinity choline uptake, and by procaine and lidocaine, two local anesthetics that inhibit plasma membrane phospholipid metabolism. These results suggest that CHT1 plays a key role in ACh synthesis in NIH3T3ChAT 112‐1 cells and that choline taken up by OCTs or derived from the plasma membrane is also utilized for ACh synthesis in both cholinergic neurons and nonneuronal cholinergic cells, such as lymphocytes. © 2009 Wiley‐Liss, Inc.     

Precursor dependence of acetylcholine release from rat brain in vitro

These experiments were designed to test the extent to which the concentration of extracellular choline effects the synthesis and subsequent release of acetylcholine (ACh) by rat cortex in vitro. We found that the rate of potassium-depolarized ACh release from rat cortical minces was significantly accelerated when choline chloride was added to the incubation medium at concentrations of either 60 or 100 μM. The ACh content of the cortical minces was reduced by prolonged depolarization; this depletion was prevented by incubating minces with choline (100 μM). Raising the extracellular choline concentration of the incubation medium did not elevate the amount of ACh released spontaneously (4.7 mM K⁺) and had no effect on the accumulation of transmitter that occurs when cortical minces are incubated in physiologic buffer. A single dose of choline chloride administered orally to rats (20 mmol/kg) was without effect on the subsequent release of ACh and choline from cortical minces in vitro. The ACh and choline concentrations of rat cortex in vitro were similarly unaffected by in vivo choline administration. These results indicate that ACh release from rat cortex, in vitro, depends upon the direct availability of extracellular choline under conditions of prolonged neuronal depolarization.     

MKC-231, a choline uptake enhancer: (2) Effect on synthesis and release of acetylcholine in AF64A-treated rats

The effect of MKC-231 on acetylcholine (ACh) synthesis and release was studied in the hippocampus of normal and AF64A-treated rats. AF64A (3 nmol/brain, i.c.v.) produced significant reduction of high-affinity choline uptake (HACU) and high K+-induced ACh release in hippocampal synaptosomes. Treatments with MKC-231 (10(-8) and 10(-7) M) showed significant reverse of the decrease in both HACU and ACh release. In hippocampal slices superfused with choline-containing artificial cerebro-spinal fluid (ACSF), high K+-induced ACh release was gradually decreased by repeated alteration of resting and high K+ stimulations in AF64A-treated rats. However, addition of MKC-231 (10(-8) to 10(-7) M) in the superfusate reduces this decrease. In vivo microdialysis studies indicate MKC-231 (10 mg/kg, p.o.) significantly reversed reduction of basal ACh concentrations in AF64A-treated rats, measured by radioimmunoassay without a cholinesterase inhibitor in the perfusate. These results indicate MKC-231 improves AF64A-induced cholinergic hypofunction by enhancing HACU, subsequently facilitating ACh synthesis and release in vitro and in vivo.     

The effect of acetylcholine and dopamine on the caudate spindle in cats

Electrocortical activity was reacorded in cats whose caudate nuclei were perfused and electrically stimulated using a push-pull cannula. Electric stimulation invariably induced spindles (10-14 Hz) in the ipsilateral frontal cortex. Perfusions of acetylcholine together with physostigmine reduced the number of spontaneous spindles, the response to electric stimulation and induced behavioural arousal. This effect was atropine-sensitive. Perfusions of dopamine with or without tranylcypromine had no significant effect on the number of spontaneous spindles or the response to electric stimulation. Injections of both acetylcholine and dopamine into the cuadate nucleus invariably induced spontaneous spindles and slow waves. The significance of acetylcholine and dopamine in the caudate nucleus intiating caudate spindles and their significance in controlling arousal is discussed.     

Effects of trimethyltin (TMT) on choline acetyltransferase activity in the rat hippocampus

Trimethyltin (TMT) destroys specific subfields of the hippocampus in the rat. TMT also increases choline acetyltransferase (ChAT) activity in CA1 of Ammon’s horn and the outer molecular layer of the dentate gyrus. This observation suggests that axonal sprouting occurs in the cholinergic septohippocampal system in response to TMT. However, neither does-response nor time course data are available for the effects of TMT on this enzyme. The effects of three dose levels of TMT on ChAT activity in CA1 and the dentate gyrus were determined in Experiment 1 and ChAT activity in these two areas was measured at six time points following exposure to TMT in Experiment 2. Only the highest dose of TMT (6 mg/kg) significantly increased ChAT activity. ChAT activity in the dentate gyrus increased significantly by 3 d after administration and continued to increase until 21 d after exposure. A significant increase was not observed in CA1 until 7 d after exposure to TMT. Asymptotic levels were still reached at d 21. These results indicate a steep dose-response curve for TMT-induced changes in ChAT activity in the hippocampal formation and that this marker of cholinergic activity is more sensitive to perturbation by TMT in the dentate gyrus than Ammon’s horn.