High Affinity Choline Uptake System Research Articles

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus.

Nitric oxide modulates intensity of non-quantal acetylcholine release in myocardium of the right atrium of rat

The main parasympathetic neurotransmitter acetylcholine (ACh) is released in the myocardium from the intramural postganglionic parasympathetic nerve endings. The mechanism of non-quantal ACh release has been recently demonstrated in these neurons. Non-quantal ACh release does not depend on exocytosis of ACh-containing vesicles in response to nerve impulse activity but is assumed to be mediated by the high-affinity choline uptake system. The intensity of non-quantal ACh release in the myocardium correlates with the degree of manifestation of the effects of acetylcholinesterase inhibitors inducing the accumulation of non-quantal ACh in the myocardium. The present study deals with the influence of putative modulators of non-quantal ACh release: nitric oxide (NO) and ATP, on the intensity of cholinergic effects induced by organophosphorous acetylcholinesterase inhibitor paraoxon. Intracellular registration of bioelectrical activity in isolated right atrium preparations from rats was used. Under normal conditions, paraoxon (10−7–10−5 M) induced a marked decrease in the action potential (AP) duration at a level of 50 and 90% repolarization in the working right atrial myocardium and slowed down the sinus rhythm. ATP, which is known to suppress nonquantal ACh release in the neuromuscular junction, did not induce significant reduction or augmentation of the effects of paraoxon (5 × 10−6 M). The NO donors, sodium nitroprusside (10−5 M) and SNAP (10−4 M), significantly reduced the paraoxon-induced AP shortening. Moreover, sodium nitroprusside decreased the negative chronotropic effect of paraoxon by 43.7%. On the contrary, NO synthase inhibitor L-NAME (10−4 M), which is known to suppress endogenous NO production, augmented the AP shortening caused by paraoxon. It may be deduced that NO is a universal regulator of non-quantal ACh release intensity both in the myocardium and in the neuromuscular junction.

Interactions of Amyloid β Peptide 1–40 and Cerebrosterol

Amyloid β peptides appear to play a role in physiological processes; however, they are also involved in the pathogenesis of Alzheimer disease. Their actions under normal conditions are probably mediated by soluble monomeric L-isoforms at low concentrations, perhaps via highly specific interactions. On the contrary, toxic effects of aggregated natural L-isoforms/synthetic D-isoforms on membranes are very similar, but synthetic reverse/random L: -isoforms without pronounced aggregation properties are not toxic. Our previous work reported interactions of non-aggregated/aggregated L-isoforms of amyloid β peptides 1-40/1-42 with racemic 24-hydroxycholesterol. In this study, stereospecificity in the interactions of natural 24(S)hydroxycholesterol (cerebrosterol) or synthetic 24(R)hydroxycholesterol with soluble fragment 1-40 was evaluated by means of an in vitro test based on increased vulnerability of the hemicholinium-3 sensitive high-affinity choline uptake system in rat hippocampal cholesterol-depleted synaptosomes to the actions of amyloid β; computational simulations were also performed. Our results suggest that: (1) 24(S)hydroxycholesterol interacts with L-peptide 1-40 but not with the reverse L-peptide 40-1, (2) 24(R)hydroxycholesterol does not interact with L-peptide 1-40 or reverse 40-1, and (3) both enantiomers can probably interact with D-peptide 1-40. Therefore, the binding of 24(S)hydroxycholesterol is not fully stereospecific and the interaction could not reflect a physiological mechanism. Data from the computational simulation indicate that the hydrophobic core of the amyloid β molecule interacts with the hydrophobic part of 24(S)hydroxycholesterol, but no hydrogen bonds with high stability were found. Using this procedure, globular amyloid β could retain 24(S)hydroxycholesterol and thus contribute to its pathological accumulation in the brains of patients with Alzheimer disease.

Substrate-Induced Internalization of the High-Affinity Choline Transporter

Cholinergic neurons are endowed with a high-affinity choline uptake system for efficient synthesis of acetylcholine at the presynaptic terminals. The high-affinity choline transporter CHT1 is responsible for choline uptake, the rate-limiting step in acetylcholine synthesis. However, endogenous physiological factors that affect CHT1 expression or function and consequently regulate the acetylcholine synthesis rate are essentially unknown. Here we demonstrate that extracellular substrate decreases the cell-surface expression of CHT1 in rat brain synaptosomes, primary cultures from the basal forebrain, and mammalian cell lines transfected with CHT1. Extracellular choline rapidly decreases cell-surface CHT1 expression by accelerating its internalization, a process that is mediated by a dynamin-dependent endocytosis pathway in HEK293 cells. Specific inhibitor hemicholinium-3 decreases the constitutive internalization rate and thereby increases cell-surface CHT1 expression. We also demonstrate that the constitutive internalization of CHT1 depends on extracellular pH in cultured cells. Our results collectively suggest that the internalization of CHT1 is induced by extracellular substrate, providing a novel feedback mechanism for the regulation of acetylcholine synthesis at the cholinergic presynaptic terminals.

Evaluation of [ 11C]hemicholinium-15 and [ 18F]hemicholinium-15 as new potential PET tracers for the high-affinity choline uptake system in the heart

[ 11C]Hemicholinium-15 ([ 11C]HC-15) and [ 18F]hemicholinium-15 ([ 18F]HC-15) have been synthesized as new potential PET tracers for the heart high-affinity choline uptake (HACU) system. [ 11C]HC-15 was prepared by N-[ 11C]methylation of the appropriate precursor, 4-methyl-2-phenyl-morpholin-2-ol, using [ 11C]CH 3OTf in 55–70% radiochemical yield decay corrected to end of bombardment (EOB) and 2–3 Ci/μmol specific activity at end of synthesis (EOS). [ 18F]HC-15 was prepared by N-[ 18F]fluoromethylation of the precursor using [ 18F]FCH 2OTf in 20–30% radiochemical yield decay corrected to EOB and >1.0 Ci/μmol specific activity at EOS. The biodistribution of both compounds was determined in rats at 20 min post-intravenous injection, and the results show the heart region uptakes 1.32 ± 0.75%ID/g in R-ventricle for [ 11C]HC-15 and 1.28 ± 0.81%ID/g in L-ventricle for [ 18F]HC-15, respectively. The dynamic PET imaging studies of [ 11C]HC-15 in rats were acquired 60 min post-intravenous injection of the tracer using the IndyPET-II scanner. For the blocking experiments, the rats were intravenously pretreated with 3.0 mg/kg of unlabeled HC-15 prior to [ 11C]HC-15 injection. [ 11C]HC-15 rat heart PET studies show rapid heart uptake to give clear heart images. The rat heart PET blocking studies found no significant blocking effect. The dynamic PET studies in normal and ablated dogs were performed using Siemens PET scanner with [ 13N]NH 3, [ 11C]HC-15, and [ 18F]HC-15. PET studies in dogs of both [ 11C]HC-15 and [ 18F]HC-15 also show significant heart uptake and give images of the heart. However, there is no significant change in [ 11C]HC-15 L-ventricle uptake following radiofrequency ablation in the dog. These results suggest that the localization of HC-15 tracers in the heart is mediated by non-specific processes, and the visualization of HC-15 tracers on the heart is related to non-specific binding of HACU.

Exposure of Postnatal Rats to a Static Magnetic Field of 0.14 T Influences Functional Laterality of the Hippocampal High-Affinity Choline Uptake System in Adulthood; In vitro Test with Magnetic Nanoparticles

Our previous experiments indicated an age- and sex-dependent functional lateralization of a high-affinity choline uptake system in hippocampi of Wistar rats. The system is connected with acetylcholine synthesis and also plays a role in spatial navigation. The current study demonstrates that a single in vivo exposure of 7- or 14-day-old males to a static magnetic field of 0.14 T for 60-120 min evokes asymmetric alterations in the activity of carriers in adulthood. Namely, the negative field (antiparallel orientation with a vertical component of the geomagnetic field) mediated a more marked decrease in the right hippocampus. The positive field (parallel orientation) was ineffective. Moreover, differences between the carriers from the right and the left hippocampi were observed on synaptosomes pretreated with superparamagnetic nanoparticles and exposed for 30 min in vitro. The positive field enhanced more markedly the activity of carriers from the right hippocampus, the negative that from the left hippocampus, on the contrary. Our results demonstrate functionally teratogenic risks of the alterations in the orientation of the strong static magnetic field for postnatal brain development and suggest functional specialization of both hippocampi in rats. Choline carriers could be involved as secondary receptors in magnetoreception through direct effects of geomagnetic field on intracellular magnetite crystals and nanoparticles applied in vivo should be a useful tool to evaluate magnetoreception in future research.

Ultrastructural localization of high-affinity choline transporter in the rat neuromuscular junction: enrichment on synaptic vesicles.

In cholinergic neurons, Na(+)- and Cl(-)-dependent, hemicholinium-3-sensitive, high-affinity choline uptake system is thought to be the rate-limiting step in acetylcholine (ACh) synthesis. The system is highly regulated by neuronal activity; the choline uptake is increased by a condition in which ACh release is favored. Here we analyzed the ultrastructural localization of the high-affinity choline transporter (CHT) in the rat neuromuscular junctions with two separate antibodies. The majority (>90%) of immunogold labeling of CHT was observed on synaptic vesicles rather than the presynaptic plasma membrane. Less than 5% of the gold-silver particles were associated with the plasma membrane, and more than 70% of such particles were localized within or in close vicinity to presynaptic active zones. Our morphological data support the recent hypothesis that trafficking of CHT from synaptic vesicles to the plasma membrane couples neuronal activity and choline uptake.

Age- and sex-dependent laterality of rat hippocampal cholinergic system in relation to animal models of neurodevelopmental and neurodegenerative disorders.

Studies suggest age- and sex-dependent structural and functional patterns of human cerebral lateralization underlie hemisphere specialization and its alterations in schizophrenia. Recent works report sexual dimorphism of neurons in the hippocampal formation and specialization of hemispheres in rats. Our experiments indicate for the first time functional lateralization of the high-affinity choline uptake (HACU) system directly associated with a synthesis of acetylcholine in the hippocampus of Wistar rats. The markedly increased HACU activity was found in the left compared to the right hippocampus of adult male but not female animals. Lineweaver-Burk plot analysis revealed a statistically significant increase of Vmax in the left hippocampus of 14-day-old when compared to 7-day-old males. It appears that laterality of HACU occurs during late postnatal maturation, and its degree is markedly enhanced after puberty and attenuated during aging. Quinolinic acid (QUIN), an endogenous agonist of N-methyl-D-aspartate type glutamate receptors, was used in this study to evaluate the neurodevelopmental hypothesis of schizophrenia. It is known that elevated levels of QUIN accompany viral infections, increasing the risk of developing schizophrenia. Bilateral intracerebroventricular application of QUIN (250 nmoles/ventricle) to pups aged 12 days significantly impaired the cholinergic hippocampal system of adolescent male and female rats and reversed lateralization of male HACU. Morphological analysis indicated marked changes in brain lesion sizes (extensive 24 h and moderate 38 days after the operation). Asymmetry of lesions was observed in the majority of cases, but the left hemisphere was not generally more vulnerable to QUIN effects than the right side. Moreover, no lateral differences were found between lesioned hippocampi in the specific binding of [3H]hemicholinium-3 (10%-15% loss of binding sites when compared to sham-operated animals). In summary, our results indicate a symmetrical drop in the number of choline carriers of lesioned male rats but a asymmetrical decrease in the activity of remaing carriers, suggesting defects in processes of sexual brain differentiation, leading under normal conditions to the higher activity of carriers in the left hippocampus. The data demonstrate viral infection-mediated alterations in normal patterns of brain asymmetry and are discussed in relation to animal models of neurodevelopmental and neurodegenerative diseases.

Neurochemical effects of repeated gestational exposure to chlorpyrifos in developing rats.

The neurochemical effects in developing rats exposed during gestation to the anticholinesterase organophosphorus insecticide chlorpyrifos (CPS) were determined. Pregnant rats were dosed daily with CPS (0, 3, or 7 mg/kg) in corn oil from gestation days (GD) 6-20. Pups were euthanized on postnatal days (PND) 1, 3, 6, 9, 12, and 30 for the determination of brain cholinesterase (ChE) and choline acetyltransferase (ChAT) activities, along with muscarinic receptor (mAChR) densities, the levels of the high-affinity choline uptake (HACU) system, and the vesicular acetylcholine transporter (VAChT). ChE activities were inhibited about 15 and 30% on PND 1, in the low- and high-dosage groups, respectively, and were not different from control values by PND 6. mAChR densities on PND 1 were reduced in the high-dosage group by about 18, 21, and 17%, using 3H-N-methylscopolamine, 3H-quinuclidinyl benzilate, and 3H-4-DAMP, respectively, as ligands, and were not different from control levels by PND 6. ChAT activity was decreased by approximately 12% in the high-dosage group on PND 9, 12, and 30. HACU levels, using 3H-hemicholinium-3 as the ligand, were reduced by approximately 25% on PND 6 in the low- and high-dosage groups, and by approximately 14 and 21% on PND 12 and 30, only in the high-dosage group. Levels of the VAChT were reduced by a range of 13-31% on PND 3 through 30 in the high-dosage group, using 3H-AH5183 (vesamicol) as the ligand. These data suggest that gestational exposure to 7 mg/kg/day CPS results in long-term alterations of presynaptic cholinergic neurochemistry.

Transport of choline and its relationship to the expression of the organic cation transporters in a rat brain microvessel endothelial cell line (RBE4)

The present study was undertaken to elucidate the functional characteristics of choline uptake and deduce the relationship between choline uptake and the expression of organic cation transporters in the rat brain microvessel endothelial cell line RBE4. Confluent RBE4 cells were found to express a high affinity choline uptake system. The system is Na +-independent and shows a Michaelis–Menten constant of approx. 20 μM for choline. The choline analogue hemicholinium-3 inhibits choline uptake in these cells with an inhibition constant of approx. 50 μM. The uptake system is also susceptible for inhibition by various organic cations, including 1-methyl-4-phenylpyridinium, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, clonidine, procainamide, and tetramethylammonium. The prototypical organic cation tetraethylammonium shows very little affinity for the choline uptake system in these cells. The inhibition of choline uptake by hemicholinium-3 is competitive. Northern analysis and RT-PCR show that these cells do not express the organic cation transporters OCT2 and OCT3. These cells do express, however, low levels of OCT1, but the functional characteristics of choline uptake in these cells are very different from the known properties of choline uptake via OCT1. The Na +-coupled high affinity choline transporter CHT1 is not expressed in these cells as evidenced by RT-PCR. This corroborates the Na +-independent nature of choline uptake in these cells. It is concluded that RBE4 cells express an organic cation transporter that is responsible for choline uptake in these cells and that this transporter is not identical to any of the organic cation transporters thus far identified at the molecular level in mammalian cells.

Synthesis of [18F]FA-4 and [11C]pipzA-4 as radioligands for the high affinity choline uptake system

We have prepared two radiolabelled analogues of hemicholinium-3 (HC-3) as potential in vivo tracers of the sodium dependent high affinity choline uptake (SDHAChU) system. Thus, 4-[1-hydroxy-2-(4-[18F]fluoromethylpiperidinyl)ethyl]-4′-[1-hydroxy-2-(4-methylpiperidinyl)ethyl]biphenyl ([18F]FA-4) and 4-[1-hydroxy-2-(4-[11C]methylpiperazinyl)ethyl]-4′-[1-hydroxy-2-(4-methylpiperidinyl)]biphenyl([11C] pipzA-4) have been synthesized. [18F]FA-4 was prepared by reaction of 4-[18F]fluoromethylpiperidine with 4-(α-bromoacetyl)-4′-(4-methylpiperidinylacetyl)-biphenyl followed by reduction of the keto groups to alcohols with NaBH4. The total synthesis time was 300 minutes and [18F]FA-4 was obtained with a specific activity of 5.6 GBq/μmol (EOS) and an overall decay corrected radiochemical yield of 3.1±0.6%. [11C]pipzA-4 was prepared by reaction of [11C]methyl triflate with 4-[1-hydroxy-2-piperazinyl)ethyl]-4′-[1-hydroxy-2-(4-methylpiperidinyl)ethyl]-biphenyl. The total synthesis time was 25 minutes and [11C]pipzA-4 was obtained with a specific activity of 13.7 GBq/μmol (EOS) and an overall decay corrected radiochemical yield of 19.5±2.2%. Copyright © 1999 John Wiley & Sons, Ltd.

Neurotransmitter and neuropeptide modulation of high affinity choline uptake in Limulus brain.

The role of neurotransmitters in the modulation of the sodium-dependent high affinity choline uptake system (HAChUS) of the horseshoe crab, Limulus polyphemus has been investigated utilizing a tissue slice preparation. Choline uptake was significantly decreased by carbachol but unaffected by atropine and d-tubocurarine. The muscarinic agonist oxotremorine decreased choline uptake by 30.4% while the muscarinic antagonist, pirenzepine, increased uptake by 29.6%. Applied in combination, pirenzepine and oxotremorine abolished their individual effects resulting in control values for choline uptake. The non-cholinergic transmitters octopamine and serotonin significantly enhanced choline uptake. The neuropeptide proctolin elicited a 20% increase in choline transport whereas Phe-Met-Arg-Phe (FMRF) amide was without effect. This study demonstrates that neurotransmitters and neuropeptides modulate the HAChUS, possibly through specific receptor-mediated second messenger systems.

Pyritinol facilitates the recovery of cortical cholinergic deficits caused by nucleus basalis lesions.

The effect of a nootropic, Pyritinol, on the recovery of cortical cholinergic deficits induced by injury of the nucleus basalis has been tested on two groups of unilateral quisqualic acid nbM-lesioned rats. The first group had a 30 nmol lesion producing a cortical cholinergic impairment at 21 days, with a spontaneous recovery at 45 days. The second group had a 50 nmol lesion that produced a deeper cholinergic deficit, which did not recover at 45 days. Pyritinol enhanced the recovery in the 30 nmol group of animals on the 21st day after surgery. The recovery was measured as an increase in the activities of acetylcholinesterase (AChE), choline acetyltransferase (ChAT) and the high affinity choline uptake system, and the histochemical densities of the cortical AChE network and the M2 receptor. Histochemical analysis of the nbM enabled cortical recovery to be related to the number of surviving neurons and also to their hypertrophy and AChE-ChAT hyperactivity. Pyritinol enhanced recovery in 30 nmol lesioned animals but in the other group, with a lower number of surviving neurons and a lower ability of the cells to become hypertrophic, the drug was unable to promote cortical recovery.

Effects of oxiracetam on organic brain syndrome following activation of the high-affinity choline uptake system: A neurochemical analysis of the cerebrospinal fluid of four patients

Nootropic drugs stimulate acetylcholine (ACh) synthesis by activating the high-affinity choline uptake system. Cerebrospinal fluid (CSF) samples were obtained from four patients with organic brain syndrome in order to gauge acetylcholinesterase activity and measure levels of ACh, 5-hydroxyindoleacetic acid, vanillylmandelic acid, and 5-hydroxytryptophan before and at the end of nootropic treatment. Patients were given oxiracetam for 30 days at dosages varying from 1600 mg/day to 2000 mg/day. Clinical evaluations conducted at the beginning and end of treatment were made by measuring the Sandoz Clinical Assessment—Geriatric (SCAG) score and reaction times to simple auditory and visual stimuli. Following oxiracetam treatment, average ACh levels in CSF decreased significantly, and there was a concomitant improvement in the clinical evaluation (SCAG).

Primary structure and functional expression of a choline transporter expressed in the rat nervous system

Synthesis of the neurotransmitter acetylcholine in cholinergic nerve terminals is regulated by a sodium-driven high-affinity choline uptake system in the plasma membrane. We have isolated cDNAs from rat spinal cord and brainstem which encode a choline transporter (CHOT1). The predicted protein shares considerable amino acid identity and several structural features including twelve putative transmembrane regions with other neurotransmitter transporters. Expression of in vitro transcribed CHOT1 RNA in Xenopus oocytes generated Na +-dependent choline uptake, which was not seen in control oocytes. Amplification by polymerase chain reaction (PCR) revealed significant amounts of CHOT1 mRNA in brain, cerebellum, spinal cord and, to a lesser extent, heart, but only very low expression in lung, kidney and muscle.

Further studies on the biochemical characterization and autoradiographic distribution of [ 3H]hemicholinium-3 binding sites in rat brain: a presynaptic cholinergic marker

Hemicholinium-3 (HC-3) is a potent inhibitor of the high-affinity choline uptake system (NACU). Here we report on the biochemical characterization and autoradiographic distribution of [3H]hemicholinium-3 binding sites in rat brain, confirming and expanding results from previous studies. The binding of [ 3H]HC-3 to striatal membranes was specific, to a single site, sodium-dependent, saturable, and of high-affinity, K d values being about 3 n m for striatum, 5 n m for the hippocampus and 12 n m for neocortex. [ 3H]HC-3 specific binding exhibited a pharmacological profile suggestive of physiologically relevant interactions and fully comparable to that reported for HACU. The uneven distribution of [ 3H]HC-3 binding sites exhibited a high degree of correspondence with the reported distribution of HACU and other enzymatic presynaptic cholinergic markers. The punctual differences between our study and previous works on [ 3H]HC-3 binding are analysed. We conclude that [3H]HC-3 labelling may be used as a selective and quantifiable marker of the cholinergic presynaptic terminals in close relationship with HACU.

An improved invertebrate synaptosomal preparation with cholinergic properties

Our laboratory has previously characterized a high affinity choline uptake system (HAChUS) in Limulus tissues and synaptosomes. We report here on the characterization of the HAChUS in synaptosomes prepared selectively from central nervous system tissues shown to be enriched for presumed cholinergic functions; namely the protocerebrum, corpora pedunculata and abdominal ganglia. Synaptosomes were prepared from these tissues by means of a modification of the subfractionation procedure developed by Dowdall and Whittaker. In our modification, we harvested a PP 2L fraction exclusively from the S 2 fraction. Compared to the P 2L fraction, the PP 2L was greater than three times more efficient in [ 3H]choline uptake and was significantly more sensitive to inhibition with micromolar concentrations of hemicholinium-3. The PP 2L fraction HAChUS was shown to have characteristics common to the HAChUS of identified cholinergic tissues.

Pharmacological Characteristics of Choline Transport System in Mouse Cerebral Cortical Neurons in Primary Culture

The characteristics of [3H]choline transport with high affinity were investigated using primary cultured neurons obtained from the mouse cerebral cortex. [3H]Choline uptake was saturable as a function of extracellular [3H]choline concentration. Analysis by Lineweaver-Burk plot revealed that [3H]choline was transported into neurons by a high affinity transport system with a Km value of 19.8 ± 0.8 μΜ and Vmax value of 0.334 ± 0.022 nmol/mg protein/min. This high affinity transport of [3H]choline was significantly inhibited by the withdrawal of sodium from the incubation medium, incubation at low temperature (4 C) and addition of metabolicinhibitors such as monoiodoacetate. These results indicate that the high affinity [3H]choline uptake in primary cultured neurons is sodium- and energy-dependent. Hemicholinium-3 also showed a competitive inhibition on the [3H]choline transport. Depolarization by high K+ induced an enhancement of the [3H]choline uptake in the presence of Ca2+. The crude synaptosomal fraction obtained from primary cultured neurons possessed approximately forty-fold higher synthesizing activity of [3H]acetylcholine from [3H]choIine than that found in the homogenate preparation of cultured neurons. The present results strongly suggest that the primary cultured neurons used in this study possess a sodium- and energy-dependent high-affinity choline uptake system as well as a synthesizing system for acetylcholine. Possible usefulness of these neurons for investigating neuronal uptake of choline and its functional role in the biosynthesis of acetylcholine are also suggested.

Pharmacological characteristics of choline transport system in mouse cerebral cortical neurons in primary culture.

The characteristics of [3H]choline transport with high affinity were investigated using primary cultured neurons obtained from the mouse cerebral cortex. [3H]Choline uptake was saturable as a function of extracellular [3H]choline concentration. Analysis by Lineweaver-Burk plot revealed that [3H]choline was transported into neurons by a high affinity transport system with a Km value of 19.8 +/- 0.8 microM and Vmax value of 0.334 +/- 0.022 nmol/mg protein/min. This high affinity transport of [3H]choline was significantly inhibited by the withdrawal of sodium from the incubation medium, incubation at low temperature (4 degrees C) and addition of metabolic inhibitors such as monoiodoacetate. These results indicate that the high affinity [3H]choline uptake in primary cultured neurons is sodium- and energy-dependent. Hemicholinium-3 also showed a competitive inhibition on the [3H]choline transport. Depolarization by high K+ induced an enhancement of the [3H]choline uptake in the presence of Ca2+. The crude synaptosomal fraction obtained from primary cultured neurons possessed approximately forty-fold higher synthesizing activity of [3H]acetylcholine from [3H]choline than that found in the homogenate preparation of cultured neurons. The present results strongly suggest that the primary cultured neurons used in this study possess a sodium- and energy-dependent high-affinity choline uptake system as well as a synthesizing system for acetylcholine. Possible usefulness of these neurons for investigating neuronal uptake of choline and its functional role in the biosynthesis of acetylcholine are also suggested.

Hemicholinium-3-resistant choline uptake system linked to acetylcholine synthesis in the rat hippocampus

Extracellular choline-dependency of acetylcholine (ACh) synthesis was examined in rat hippocampal slices. In the presence of hemicholinium-3 (HC-3, 5 μM), extracellular choline-dependent ACh synthesis appeared to consist of two different components. The first component (saturated at up to 50 μM choline) was dependent on the HC-3-resistant choline uptake system having a low capacity for choline in comparison with the high-affinity choline uptake system (HACU). The second component, observed in the presence of more than 50 μM choline, was considered due to competitive inhibition of HACU by HC-3. HACU-dependent ACh synthesis seemed to be saturated in the presence of up to 2 μM choline. These results indicate that both the HACU and HC-3-resistant choline uptake systems are linked to ACh synthesis in the hippocampus.