Intracerebroventricular injection of mu- and delta-opiate receptor antagonists block 60 Hz magnetic field-induced decreases in cholinergic activity in the frontal cortex and hippocampus of the rat

In previous research, we have found that acute exposure to a 60 Hz magnetic field decreased cholinergic activity in the frontal cortex and hippocampus of the rat as measured by sodium-dependent high-affinity choline uptake activity. We concluded that the effect was mediated by endogenous opioids inside the brain because it could be blocked by pretreatment of rats before magnetic field exposure with the opiate antagonist naltrexone, but not by the peripheral antagonist naloxone methiodide. In the present study, the involvement of opiate receptor subtypes was investigated. Rats were pretreated by intracerebroventricular injection of the mu-opiate receptor antagonist, β-funaltrexamine, or the delta-opiate receptor antagonist, naltrindole, before exposure to a 60 Hz magnetic field (2 mT, 1 hour). It was found that the effects of magnetic field on high-affinity choline uptake in the frontal cortex and hippocampus were blocked by the drug treatments. These data indicate that both mu- and delta-opiate receptors in the brain are involved in the magnetic field-induced decreases in cholinergic activity in the frontal cortex and hippocampus of the rat. Bioelectromagnetics 19:432–437, 1998. © 1998 Wiley-Liss, Inc.     

High-affinity choline uptake in regions of rat brain and the effect of antidepressants.

The effect of tricyclic antidepressants, chlorpromazine, and some monoamine oxidase inhibitors on the accumulation of [14C]choline by crude synaptosomal (P2) fraction from different regions of rat brain (cortex, striatum, and hippocampus) was investigated. Analysis of choline uptake kinetics resulted in high- and low-affinity components with different Michaelis constants. All tricyclic antidepressants tested inhibited in a dose-dependent manner the high-affinity choline uptake in the three regions, amitriptyline being the most potent. The IC50 values correlated significantly with the relative potencies of imipramine congeners in binding to muscarinic receptors in the brain. Neither tranylcypromine nor pargyline in concentrations up to 0.1 mM had any effect on choline transport. Concentrations of tricyclic antidepressants effective in inhibiting the uptake of choline failed to influence significantly the activity of choline acetyltransferase in brain regions examined. The results suggest that the effect of imipramine congeners on high-affinity choline uptake may be reflected in the anticholinergic properties of these compounds.     

Arachidonic Acid Inhibits Choline Uptake and Depletes Acetylcholine Content in Rat Cerebral Cortical Synaptosomes

The effects of arachidonic acid on [3H]choline uptake, on [3H]acetylcholine accumulation, and on endogenous acetylcholine content and release in rat cerebral cortical synaptosomes were investigated. Arachidonic acid (10-150 microM) produced a dose-dependent inhibition of high-affinity [3H]choline uptake. Low-affinity [3H]choline uptake was also inhibited by arachidonic acid. Fatty acids inhibited high-affinity [3H]choline uptake with the following order of potency: arachidonic greater than palmitoleic greater than oleic greater than lauric; stearic acid (up to 150 microM) had no effect. Inhibition of [3H]choline uptake by arachidonic acid was reversed by bovine serum albumin. In the presence of arachidonic acid, there was an increased accumulation of choline in the medium, but this did not account for the inhibition of [3H]choline uptake produced by the fatty acid. Arachidonic acid inhibited the synthesis of [3H]acetylcholine from [3H]choline, and this inhibition was equal in magnitude to the inhibition of high-affinity [3H]choline uptake produced by the fatty acid. A K+-stimulated increase in [3H]acetylcholine synthesis was inhibited completely by arachidonic acid. Arachidonic acid also depleted endogenous acetylcholine stores. Concentrations of arachidonic acid and hemicholinium-3 that produced equivalent inhibition of [3H]choline uptake also produced equivalent depletion of acetylcholine content. In the presence of eserine, arachidonic acid had no effect on acetylcholine release. The results suggest that arachidonic acid may deplete acetylcholine content by inhibiting high-affinity choline uptake and subsequent acetylcholine synthesis. This raises the possibility that arachidonic acid may play a role in the impairment of cholinergic transmission seen in cerebral ischemia and other conditions in which large amounts of the free fatty acid are released in brain.     

Low-Level Microwave Irradiations Affect Central Cholinergic Activity in the Rat

Sodium-dependent high-affinity choline uptake was measured in various regions of the brains of rats irradiated for 45 min with either pulsed or continuous-wave low-level microwaves (2,450 MHz; power density, 1 mW/cm2; average whole-body specific absorption rate, 0.6 W/kg). Pulsed microwave irradiation (2-microseconds pulses, 500 pulses/s) decreased choline uptake in the hippocampus and frontal cortex but had no significant effect on the hypothalamus, striatum, and inferior colliculus. Pretreatment with a narcotic antagonist (naloxone or naltrexone; 1 mg/kg i.p.) blocked the effect of pulsed microwaves on hippocampal choline uptake but did not significantly alter the effect on the frontal cortex. Irradiation with continuous-wave microwaves did not significantly affect choline uptake in the hippocampus, striatum, and hypothalamus but decreased the uptake in the frontal cortex. The effect on the frontal cortex was not altered by pretreatment with narcotic antagonist. These data suggest that exposure to low-level pulsed or continuous-wave microwaves leads to changes in cholinergic functions in the brain.     

Purification and Characterization of a Central Cholinergic Enhancing Factor from Rat Brain: Its Identity as Phosphoethanolamine

A compound that can enhance the apparent synthesis of acetylcholine in cultured explants of the medial septal nucleus has been purified from rat brain and identified as phosphoethanolamine. Acetylcholine synthesis is stimulated two- to threefold in cultures grown for 5 days in the presence of phosphoethanolamine, ethanolamine, or cytidine 5'-diphosphoethanolamine at concentrations above 100 microM. This effect appears to result from an increase in the accumulation of choline via the high-affinity, sodium-dependent uptake mechanism. The development of choline acetyltransferase activity is not affected. Phosphoethanolamine and ethanolamine seem to enhance the ability of developing cholinergic neurons to utilize choline accumulated via the sodium-dependent high-affinity choline uptake mechanism for the preferential production of acetylcholine without increasing the general metabolism of the cultures. Choline itself and its related derivatives are not stimulatory for these effects.     

Cortical choline transporter function measured in vivo using choline-sensitive microelectrodes: clearance of endogenous and exogenous choline and effects of removal of cholinergic terminals

The capacity of the high-affinity choline transporter (CHT) to import choline into presynaptic terminals is essential for acetylcholine synthesis. Ceramic-based microelectrodes, coated at recording sites with choline oxidase to detect extracellular choline concentration changes, were attached to multibarrel glass micropipettes and implanted into the rat frontoparietal cortex. Pressure ejections of hemicholinium-3 (HC-3), a selective CHT blocker, dose-dependently reduced the uptake rate of exogenous choline as well as that of choline generated in response to terminal depolarization. Following the removal of CHTs, choline signal recordings confirmed that the demonstration of potassium-induced choline signals and HC-3-induced decreases in choline clearance require the presence of cholinergic terminals. The results obtained from lesioned animals also confirmed the selectivity of the effects of HC-3 on choline clearance in intact animals. Residual cortical choline clearance correlated significantly with CHT-immunoreactivity in lesioned and intact animals. Finally, synaptosomal choline uptake assays were conducted under conditions reflecting in vivo basal extracellular choline concentrations. Results from these assays confirmed the capacity of CHTs measured in vivo and indicated that diffusion of substrate away from the electrode did not confound the in vivo findings. Collectively, these results indicate that increases in extracellular choline concentrations, irrespective of source, are rapidly cleared by CHTs.     

Alterations of Acetylcholine and Choline Metabolism in Mammalian Preparations Treated with β-Bungarotoxin

Abstract: We have studied the effects of β-bungarotoxin on acetylcholine and choline metabolism in central and peripheral cholinergic preparations using a gas chromatographic-mass spectrometric assay for acetylcholine and choline. In contrast with previous reports, β-bungarotoxin did not inhibit the high-affinity uptake of labeled choline or the synthesis of acetylcholine in rat brain synaptosomal fractions. However, the toxin did cause a significant increase of medium choline when it was incubated with synaptosomal fractions. This increase of endogenous choline in the medium may account for the previously reported inhibition of choline uptake because of a dilution of the specific activity of the labeled choline in the medium. Several experiments are reported in which a further characterization was made of the effect of β-bungarotoxin on medium choline. β-Bungarotoxin was also shown to cause a large increase of acetylcholine release from rat brain minces and a depletion of the acetylcholine content of minces. A similar phenomenon was found in diaphragm preparations that were exposed continuously to β-bungarotoxin. However, diaphragms that were treated for only 30 min with toxin showed the previously reported increase of acetylcholine content. β-Bungarotoxin did not have any measurable effect on acetylcholine turnover in smooth muscle preparations from guinea pig ileum. These results help to explain certain inconsistencies in the literature regarding the action of β-bungarotoxin.     

Identification of Sodium-Dependent, High-Affinity Choline Uptake Sites in Rat Brain with [3H]Hemicholinium-3

[3H]Hemicholinium-3 (HC-3) was used to label sodium-dependent, high-affinity choline uptake sites in regions of rat brain. Autoradiography revealed a high density of [3H]HC-3 binding sites in brain regions with a high density of cholinergic terminals, such as the interpeduncular nucleus, caudate-putamen, and olfactory tubercle. This distribution of [3H]HC-3 binding sites was in close agreement with the amounts of choline acetyltransferase in specific nuclei and subregions of rat brain. Destruction of presynaptic cholinergic projections in the cerebral cortex and the basal ganglia by injection of excitotoxins reduced [3H]HC-3 binding by 40-50%. These data indicate that sodium-dependent [3H]HC-3 binding sites are related to the choline transport system present in cholinergic neurons.     

Regulation of Phospholipid Metabolism in Differentiating Cells from Rat Brain Cerebral Hemispheres in Culture: Ontogenesis of Carrier-specific Transport of Choline and N-Methyl-substituted Choline Analogs

Abstract: Dimethylaminoethanol was studied both as a substrate and as an inhibitor of choline uptake in long-term cultures of foetal rat cerebral hemispheres. A saturable component with an apparent K_m of 28 μM and V_max of 11 pmol/min/μg DNA for dimethylaminoethanol, was observed. Like choline, dimethylaminoethanol was also taken up by a second, low-affinity component, the apparent V_max of which was about 102 pmol/min/μg DNA. Dimethylaminoethanol inhibited the high-affinity but not the low-affinity choline uptake in a competitive manner with an apparent inhibition constant of 6.0 μM. Monomethylaminoethanol (K₁# 60 μM) competitively inhibited high-affinity choline transport. At low concentrations hemicholinium-3, but not ethanolamine, effectively inhibited high-affinity uptake of choline and to a lesser degree the uptake of the dimethylaminoethanol. While the high-affinity uptake of both substrates was inhibited by high concentrations of hemicholinium-3 or ethanolamine, the low-affinity system was not affected by hemicholinium-3. From the kinetics of uptake and inhibition patterns of choline and its related analogs, the methyl group seems to play a major role in determining the affinity rate constants for these substrates. The maximum rate of choline uptake via the high-affinity component increases about sixfold during a period of 2 weeks. In the absence of serum the maximum velocity of the high-affinity component is greatly reduced. These observations suggest that the high-affinity choline uptake component is an integral property and a useful marker, of the developing cerebral cells.     

Deficits in acetylcholine homeostasis, receptors and behaviors in choline transporter heterozygous mice

Cholinergic neurons elaborate a hemicholinium-3 (HC-3) sensitive choline transporter (CHT) that mediates presynaptic, high-affinity choline uptake (HACU) in support of acetylcholine (ACh) synthesis and release. Homozygous deletion of CHT (-/-) is lethal shortly after birth (Ferguson et al. 2004), consistent with CHT as an essential component of cholinergic signaling, but precluding functional analyses of CHT contributions in adult animals. In contrast, CHT+/- mice are viable, fertile and display normal levels of synaptosomal HACU, yet demonstrate reduced CHT protein and increased sensitivity to HC-3, suggestive of underlying cholinergic hypofunction. We find that CHT+/- mice are equivalent to CHT+/+ siblings on measures of motor co-ordination (rotarod), general activity (open field), anxiety (elevated plus maze, light/dark paradigms) and spatial learning and memory (Morris water maze). However, CHT+/- mice display impaired performance as a result of physical challenge in the treadmill paradigm, as well as reduced sensitivity to challenge with the muscarinic receptor antagonist scopolamine in the open field paradigm. These behavioral alterations are accompanied by significantly reduced brain ACh levels, elevated choline levels and brain region-specific decreased expression of M1 and M2 muscarinic acetylcholine receptors. Our studies suggest that CHT hemizygosity results in adequate baseline ACh stores, sufficient to sustain many phenotypes, but normal sensitivities to physical and/or pharmacological challenge require full cholinergic signaling capacity.     

Characterization of [3H]Hemicholinium-3 Binding Sites in Human Brain Membranes: A Marker for Presynaptic Cholinergic Nerve Terminals

We report here on the binding properties of [3H]hemicholinium-3, a selective inhibitor of the high-affinity choline uptake process, to human brain membranes. Under the assay conditions described, the binding of [3H]hemicholinium-3 exhibited a dependency of physiological conditions on pH, temperature, and NaCl concentrations. Striatal binding proved to be specific, to a single site, saturable, and reversible, with an apparent KD of 10 nM and a Bmax of 82 fmol/mg of protein. [3H]Hemicholinium-3 specific binding exhibited a pharmacological profile and an ionic dependency suggestive of physiologically relevant interactions and comparable with those reported for the high-affinity choline uptake. Moreover, specific [3H]hemicholinium-3 binding exhibited an uneven regional distribution: striatum much greater than nucleus basalis greater than spinal cord much greater than midbrain = cerebellum greater than or equal to hippocampus greater than neocortex = anterior thalamus greater than posterior thalamus much much greater than white matter. This distribution closely corresponds to the reported activity of both enzymatic cholinergic presynaptic markers and high-affinity choline uptake in mammalian brain. There are no significant differences between these results and those previously found in the rat brain using this radioligand. Our results demonstrate, for the first time, the presence of [3H]hemicholinium-3 binding sites in human brain and strongly support the proposal that this radioligand binds to the carrier site mediating the high-affinity choline uptake process on cholinergic neurons. Thus, [3H]hemicholinium-3 binding may be used in postmortem human brain as a selective and quantifiable marker of the presynaptic cholinergic terminals.     

Effect of Quinolinic Acid in the Nucleus Basalis Magnocellularis on Cortical High-Affinity Choline Uptake

A transient 45% increase in cortical high-affinity choline uptake (HACU) was observed after an injection of quinolinic acid (QUIN) into the nucleus basalis magnocellularis (nbM) of the rat. This was followed by a steady decline in choline uptake, which resulted in a 46% decrease by day 7. Specific [3H]hemicholinium-3 binding to coronal brain sections showed a similar pattern following injections of QUIN into the nbM. The increase in cortical HACU elicited by QUIN appeared to be dose dependent.     

Stereospecificity of High-and Low-Affinity Transport of Choline Analogues into Rat Cortical Synaptosomes

The present experiments used methylcholines to examine the stereoselectivity of choline transport into rat synaptosomes. R(+)-alpha-methylcholine and S(+)-beta-methylcholine were significantly better inhibitors of the high-affinity choline transport system than were their enantiomers. Although both enantiomers of alpha- and of beta-methylcholine inhibited [3H]choline transport, only R(+)-alpha-methylcholine and S(+)-beta-methylcholine could be transported by the high-affinity choline uptake mechanism. Therefore, we conclude that the chiral requirements for recognition of and for transport by the high-affinity transporter are clearly different. In addition to high-affinity choline transport, Na(+)-independent low-affinity transport was measured. This process transported R(+)-alpha-methylcholine, but not S(-)-alpha-methylcholine; however, it showed no stereoselectivity for the enantiomers of beta-methylcholine. Thus, high- and low-affinity choline transport mechanisms exhibit distinct differences in their substrate selectivities. We suggest that the stereoselective properties of choline transport might present a unique opportunity to study choline uptake and metabolism.     

Inhibition of the rat blood–brain barrier choline transporter by manganese chloride

Choline transport has been characterized by multiple mechanisms including the blood-brain barrier (BBB), and high- and low-affinity systems. Each mechanism has unique locations and characteristics yet retain some similarities. Previous studies have demonstrated cationic competition by monovalent cations at the BBB and cation divalent manganese in the high-affinity system. To evaluate the effects of divalent manganese inhibition as well as other cationic metals at the BBB choline transporter, brain choline uptake was evaluated in the presence of certain metals of interest in Fischer-344 rats using the in situ brain perfusion technique. Brain choline uptake was inhibited in the presence of Cd(2+) (73 +/- 2%) and Mn(2+) (44 +/- 6%), whereas no inhibition was observed with Cu(2+) and Al(3+). Furthermore, it was found that manganese caused a reduction in brain choline uptake and significant regional choline uptake inhibition in the frontal and parietal cortex, the hippocampus and the caudate putamen (45 +/- 3%, 68 +/- 18%, 58 +/- 9% and 46 +/- 15%, respectively). These results suggest that choline uptake into the CNS can be inhibited by divalent cationic metals and monovalent cations. In addition, the choline transporter may be a means by which manganese enters the brain.     

Uptake and Storage of Choline by Rat Brain: Influence of Dietary Choline Supplementation

In order to elucidate the regulation of the levels of free choline in the brain, we investigated the influence of chronic and acute choline administration on choline levels in blood, CSF, and brain of the rat and on net movements of choline into and out of the brain as calculated from the arteriovenous differences of choline across the brain. Dietary choline supplementation led to an increase in plasma choline levels of 50% and to an increase in the net release of choline from the brain as compared to a matched group of animals which were kept on a standard diet and exhibited identical arterial plasma levels. Moreover, the choline concentration in the CSF and brain tissue was doubled. In the same rats, the injection of 60 mg/kg choline chloride did not lead to an additional increase of the brain choline levels, whereas in control animals choline injection caused a significant increase; however, this increase in no case surpassed the levels caused by chronic choline supplementation. The net uptake of choline after acute choline administration was strongly reduced in the high-choline group (from 418 to 158 nmol/g). Both diet groups metabolized the bulk (greater than 96%) of newly taken up choline rapidly. The results indicate that choline supplementation markedly attenuates the rise of free choline in the brain that is observed after acute choline administration. The rapid metabolic choline clearance was not reduced by dietary choline load. We conclude that the brain is protected from excess choline by rapid metabolism, as well as by adaptive, diet-induced changes of the net uptake and release of choline.     

Effect of Unilateral Perinatal Hypoxic-Ischemic Brain Injury in the Rat on Striatal Muscarinic Cholinergic Receptors and High-Affinity Choline Uptake Sites: A Quantitative Autoradiographic Study

The binding characteristics and distribution of M1 and M2 muscarinic cholinergic receptors and high-affinity choline uptake sites were studied in the striatum of the rat at 3-4 and 9-12 weeks of age after exposure to unilateral perinatal hypoxic-ischemic brain injury. High-affinity choline uptake sites were labeled with [3H]hemicholinium-3, M1 receptors with [3H]pirenzepine, and M2 receptors with [3H]AF-DX 116. Saturation experiments revealed a significant decrease in the maximal binding capacity (Bmax) for [3H]pirenzepine-labeled M1 receptors in the lesioned caudate/putamen complex in immature rats with moderate brain injury, in comparison with controls. In contrast, the Bmax value for [3H]hemicholinium-3-labeled high-affinity choline uptake sites was significantly increased. No changes in dissociation constants (KD) were observed. These changes were most pronounced in the dorsolateral region of striatum. Striatal regional distribution of [3H]AF-DX 116 was not affected. In mature rats, binding of [3H]pirenzepine returned to control values, whereas [3H]hemicholinium binding showed a persistent increase (23%). The increase in [3H]hemicholinium-3 binding, as a specific marker of cholinergic nerve terminals, is consistent with our prior morphologic studies demonstrating relative preservation of cholinergic neurons and neuropil, and supports the concept that striatal cholinergic systems are resistant to hypoxic-ischemic injury.     

Effect of ethanolamine on choline uptake and incorporation into phosphatidylcholine in human Y79 retinoblastoma cells

The effect of physiological concentrations of ethanolamine on choline uptake and incorporation into phosphatidylcholine was investigated in human Y79 retinoblastoma cells, a multipotential, undifferentiated retinal cell line that has retained many neural characteristics. These cells have a high-affinity uptake system for choline, and the majority of the choline taken up was incorporated into phosphatidylcholine via the CDP-choline pathway. The presence of extracellular ethanolamine significantly decreased high-affinity choline uptake and, subsequently, the amount of choline incorporated into phosphatidylcholine. When 100 mumol/L ethanolamine was added, there was a decrease of about 8% in the phosphatidylcholine content. Ethanolamine had no effect on choline incorporation into phosphatidylcholine, however, once choline was taken up by the cell. The K'M and V'max for high-affinity choline uptake was increased from 0.93 to 9.74 microM and 19.60 to 79.25 pmol/min per mg protein, respectively, by the presence of 25 mumol/L ethanolamine. In contrast, 25 mumol/L choline had no effect on the kinetic parameters of high-affinity ethanolamine uptake. Therefore, the reduction in high-affinity choline transport by ethanolamine apparently is not simply due to competitive inhibition. 2,2-Dimethylethanolamine and 2-methylethanolamine both reduced choline uptake to a greater extent than ethanolamine. However, because these compounds exist at much lower concentrations than ethanolamine, they probably have little physiological influence. These results suggest that changes in ethanolamine concentration within the physiologic range can regulate the synthesis and content of phosphatidylcholine in a neural cell by influencing the uptake of choline.     

CHOLINE: HIGH AFFINITY UPTAKE IN VIVO BY RAT HIPPOCAMPUS

Abstract— [³H]Choline uptake has been measured in vivo in the rat hippocampus. Pharmacological agents and lesions which profoundly affect sodium-dependent, high-affinity [³H]choline uptake in vivo similarly affect [³H]choline uptake measured in vitro. Pentobarbital (65 mg/kg) and oxotremorine (0.75 mg kg) cause a decrease in [³H]choline uptake. Scopolamine (5 mg/kg) and iontophoretically applied extracellular potassium cause an increase in [³H]choline uptake. Septal lesions cause a decrease in [³H]choline uptake. Application of the general method may allow direct examination of presynaptic function and neural integration in the undisrupted living mammalian brain.     

Expression of the high-affinity choline transporter CHT1 in rat and human arteries.

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.     

Phosphoethanolamine Enhances High-Affinity Choline Uptake and Acetylcholine Synthesis in Dissociated Cell Cultures of the Rat Septal Nucleus

Dissociated rat septal nucleus cells cultured in defined medium exhibited twofold increases in the maximal rates of sodium-dependent, high-affinity choline uptake and acetylcholine formation when grown in the presence of phosphoethanolamine. The effect was concentration-dependent (EC50 = 15 microM) and appeared to be associated with in vitro maturation of cholinergic neurons rather than with enhanced survival. Choline acetyltransferase, acetylcholinesterase, and choline kinase activities were unaffected by this treatment. The effect of phosphoethanolamine was specific for cholinergic neurons, because treatment with this compound did not alter the kinetic constants for high-affinity neuronal uptake of gamma-aminobutyric acid or dopamine. The action appeared to be mediated primarily through activation of the sodium-dependent, high-affinity transport mechanism for choline as opposed to alterations in the storage and release of acetylcholine.