Choline Uptake by Cerebral Capillary Endothelial Cells in Culture

A passage of choline from blood to brain and vice versa has been demonstrated in vivo. Because of the presence of the blood-brain barrier, such passage takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, the choline transport properties of cerebral capillary endothelial cells have been studied in vitro. Bovine endothelial cells in culture were able to incorporate [3H]choline by a carrier-mediated mechanism. Nonlinear regression analysis of the uptake curves suggested the presence of two transport components in cells preincubated in the absence of choline. One component showed a Km of 7.59 +/- 0.8 microM and a maximum capacity of 142.7 +/- 9.4 pmol/2 min/mg of protein, and the other one was not saturable within the concentration range used (1-100 microM). When cells were preincubated in the presence of choline, a single saturable component was observed with a Km of 18.5 +/- 0.6 microM and a maximum capacity of 452.4 +/- 42 pmol/2 min/mg of protein. [3H]Choline uptake by endothelial cells was temperature dependent and was inhibited by the choline analogs hemicholinium-3, deanol, and AF64A. The presence of ouabain or 2,4-dinitrophenol did not affect the [3H]choline transport capacity of endothelial cells. Replacement of sodium by lithium and cell depolarization by potassium partially inhibited choline uptake. When cells had been preincubated without choline, recently transported [3H]choline was readily phosphorylated and incorporated into cytidine-5'-diphosphocholine and phospholipids; however, under steady-state conditions most (63%) accumulated [3H]choline was not metabolized within 1 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of [3H]Hemicholinium-3 to the High-Affinity Choline Transporter in Electric Organ Synaptosomal Membranes

Sodium-dependent binding of [3H]hemicholinium-3 was observed to be 10-fold higher with presynaptic membranes from the electric organ than with electroplaque membranes and this binding site copurified with synaptosomal membranes. The KD for specific [3H]hemicholinium-3 binding was found to be 31 +/- 4 nM and the Bmax, 5.0 +/- 0.2 pmol/mg protein; a Ki of 16 nM was estimated for hemicholinium-3 as a competitive inhibitor of high-affinity choline transport in electric organ synaptosomes. Choline and choline analogues were equally potent as inhibitors of [3H]choline uptake and [3H]hemicholinium-3 binding. Tubocurarine and oxotremorine also inhibited uptake and binding, but carbachol was without effect in both tests. These findings suggest that [3H]hemicholinium binds to the high-affinity choline transporter present at the cholinergic nerve terminal membrane. A comparison of maximal velocities for choline transport and the maximal number of hemicholinium-3 binding sites indicated that the high-affinity choline transporter has an apparent turnover number of about 3s-1 at 20 degrees C under resting conditions. The high transport rates observed in electric organ synaptosomes are likely due to the high density of high-affinity choline transporters in this tissue, estimated on the basis of [3H]hemicholinium-3 binding to be of the order of 100/micron2 of synaptosomal membrane.     

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.     

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.     

Regulation of Rat Brain Synaptosomal [3H]Hemicholinium-3 Binding and [3H]Choline Transport Sites Following Exposure to Choline Mustard Aziridinium Ion

Abstract: Choline uptake by cholinergic nerve terminals is increased by depolarization; the literature suggests that this results from either the appearance of occult transporters or the increased activity of existing ones. The present experiments attempt to clarify the mechanism by which choline transport is regulated by testing if the preexposure of synaptosomes to choline mustard aziridinium ion prevents the stimulation-induced appearance of hemicholinium-3 binding sites and/or choline transport activity. Choline mustard inhibited irreversibly most of the “ground-state” (basal) high-affinity choline transport but only 50% of “ground-state” hemicholinium-3 binding sites. Exposure of both striatal and hippocampal synaptosomes to the mustard, before stimulation, inhibited K⁺-stimulated increases in choline transport and of [³H]hemicholinium-3 binding. We conclude that the mechanism by which choline transport is regulated involves the increased activity of a pool of transport sites that are occluded to hemicholinium-3 but are available to choline mustard aziridinium ion, and presumably to choline, before stimulation. However, the concentration of mustard needed to inhibit the stimulation-induced increase of [³H]hemicholinium-3 binding and choline transport was lower for striatal synaptosomes than for hippocampal synaptosomes. In the absence of extracellular Ca²⁺ or presence of high Mg²⁺ levels, the choline mustard did not prevent the appearance of extra striatal hemicholinium-3 binding sites. Also, high Mg²⁺ levels removed the ability of the mustard to inhibit K⁺-stimulated increases of either [³H]hemicholinium-3 binding or choline transport by hippocampal synaptosomes. In contrast, the preexposure of hippocampal synaptosomes to the mustard in the presence of a calcium ionophore (A23187) reduced the concentration of inhibitor needed to prevent the activation of [³H]hemicholinium-3 binding and choline uptake. Thus, we conclude that the ability of the choline mustard to alkylate the pool of choline transporters that are activated by stimulation appears dependent on the entry of extracellular Ca²⁺.     

Effects of choline augmentation on acetylcholine synthesis and release in rat atrial minces

The uptake and acetylation of [3H]-choline, as well as the calcium-dependent release of a newly synthesized [3H]-ACh, was studied in a new rat atrial mince preparation. The hemicholinium-3-sensitive uptake and acetylation of [3H]-choline increased as [3H]-choline concentrations were elevated to 100 microM in atrial minces. In contrast, hemicholinium-3-sensitive [3H]-choline uptake was saturated with 15 microM [3H]-choline in brain synaptosomes. The increased atrial [3H]-ACh synthesized in the presence of [3H]-choline augmentation was releasable by 50 mM K+-depolarization in a 1 mM cobalt-sensitive manner. These results suggest that atrial parasympathetic activity may be more sensitive to circulating choline concentrations than brain cholinergic neurons are.     

High-Affinity [3H]Choline Accumulation in Cultured Human Skin Fibroblasts

[3H]Choline can be transported across cell membranes by high-affinity (KT less than 5 microM) and low-affinity (KT much greater than 5 microM) systems. High-affinity choline accumulation (HACA) has been demonstrated in synaptosomes made from cholinergic brain regions such as the hippocampus and caudate-putamen. In cell culture, HACA has been demonstrated in glia and avian telencephalon, dissociated spinal cord, and muscle fibroblasts. We examined [3H]choline accumulation in a single normal human fibroblast line cultured from skin biopsy. [3H]Choline accumulation was temperature-dependent and linear with incubation time up to 6 min at 0.125 microM-choline. The apparent KT for [3H]choline was 5 microM, which is similar to that observed in avian fibroblasts. Isoosmotic replacement of Na+ with either Li+ (144 mM) or sucrose (288 mM) severely reduced [3H]choline accumulation (by 70-90%). Pre-incubation with ouabain (100 microM), sodium orthovanadate (100 microM), or 2,4-dinitrophenol (100 microM), or replacement of Ca2+ by Mg2+ had little or no effect on subsequent [3H]choline accumulation. [3H]Choline accumulation was inhibited by hemicholinium-3 (HC-3); after pre-incubation in HC-3 at 37 degrees C for 10 min, the IC50 (at 0.125 microM-choline) was 5.6 microM. The HC-3 sensitivity, Na+ dependence, and low KT suggest that human skin fibroblasts have a high-affinity transport system for choline.     

An in vivo cholinergic influence on the retention of [3H]noradrenaline in regional brain synaptosomes

Rats were intraventricularly (icv) injected with [³H]noradrenaline and the retention of the amine was determined in synaptosomes obtained from cerebral cortex, hypothalamus and brain stem. Previous icv administration of hemicholinium-3, effective enough to markedly decrease brain acetylcholine levels, increased the retention of synaptosomal [³H]noradrenaline in hypothalamus and cerebral cortex; this increased retention did not occur in the brain stem. The increased retention of [³H]noradrenaline, produced by hemicholinium-3, was reversed by a concomitant icv dose of choline, which in turn reversed the decrease of acetylcholine caused by hemicholinium-3. These results are interpreted as brain cholinergic activity having an influence on the turnover of noradrenaline in some brain regions.     

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.     

A comparative study of high affinity choline uptake and choline utilization in cholinergic and non-cholinergic tissues

Comparative studies of [3H]choline accumulation were done in the Limulus corpora pedunculata, abdominal ganglia and cardiac ganglion. Dual uptake processes for choline were found in all three tissues. In acute experiments, the corpora pedunculata high affinity choline uptake system showed exclusive sensitivity to ouabain. Prolonged exposure to ouabain revealed that the HAChUS of all three tissues were significantly inhibited. The metabolism of [3H]choline transported via the high affinity process in the three tissues was studied. [3H]Acetylcholine was a major product of the [3H]choline taken up by the corpora pedunculata and the abdominal ganglia. Phosphorylcholine was the major product seen in cardiac ganglion extracts and occurred in significant proportions in abdominal ganglia extracts. [3H]Acetylcholine was not detected in cardiac ganglion extracts. Treatment with either lithium chloride or hemicholinium-3 markedly inhibited high affinity uptake of [3H]choline in all three tissues.     

The incorporation of solubilized choline-transport activity into liposomes.

The choline-transport system has been solubilized from synaptic plasma membrane by using either sodium cholate or Triton X-100, and re-incorporated into unilamellar liposomes by using the technique of cholate dialysis. The criteria of choline-transport activity were saturability by excess choline, inhibition by hemicholinium-3, and trans-activation (i.e. stimulation of the uptake of [3H]choline into liposomes by preloading them with non-radioactive choline). Liposomes prepared from detergent extracts of synaptic plasma membrane and added lipid showed uptake of [3H]choline fulfilling these three criteria. Data on choline-transport activity of liposomes at various choline concentrations could be interpreted as implying that the transport system has two apparent Km values (2-5 microM and 50-100 microM), or alternatively that the system is composed of two or more negatively co-operating subunits (or units). It was shown by t.l.c. that the transported radioactivity was choline and that it was not significantly acetylated. Replacing Na+ by K+ on the outside of these liposomes partially inhibited uptake, and the formation of a potential gradient (inside negative) with valinomycin increased the total but not the saturable components of uptake when liposomes were prepared in a K+ medium, and transferred to an Na+ medium.     

The characterization of a sodium-dependent high affinity choline uptake system unassociated with acetylcholine biosynthesis

The cardiac ganglion of the horseshoe crab, Limulus polyphemus, was incubated in Chao's solution containing 0.01 microM [3H]choline at room temperature (25 +/- 2 degrees C) and the ganglion readily accumulated the radiolabel. The ganglion uptake of [3H]choline was linear over 60 min. Kinetic analysis revealed dual choline uptake systems within the cardiac ganglion, a high affinity uptake system (Km = 2.2 microM, Vmax = 0.16 pmoles/mg/min) and a low affinity system (Km = 92.3 microM, Vmax = 3.08 pmoles/mg/min). The high affinity uptake system was sodium-dependent and inhibited by micromolar concentrations of hemicholinium-3. A 15 min pre-exposure of the ganglion to Chao's solution containing 90 mM potassium stimulated a significant increase in choline uptake. There was no detectable synthesis of [3H]acetylcholine from the [3H]choline taken up by the cardiac ganglion. The major portion of the extractable label appeared in a fraction which co-electrophoresed with phosphorylcholine. These results suggest that the sodium-dependent high affinity [3H]choline uptake system of the cardiac ganglion subserves a specific requirement for choline which is unrelated to a cholinergic function.     

The involvement of protein kinase C in the regulation of choline cotransport in Limulus.

The involvement of protein kinase C (PKC) in the regulation of [3H]choline cotransport was studied in Limulus brain hemi-slice preparations. The PKC activators, phorbol 12-myristate 13-acetate (PMA) or phorbol 12,13-dibutyrate (PDBu), significantly decreased [3H]choline cotransport. Conversely, the PKC inhibitors, staurosporine (STAURO) and polymyxin B (PMB), each increased [3H]choline cotransport. These PKC inhibitors prevented the phorbol ester-induced reduction of transport. Both the PMA induced decrease and the STAURO induced increase in [3H]choline cotransport were paralleled by respective and comparable changes in [3H]hemicholinium-3 (HC-3) specific binding. Pre-exposure of brain hemi-slices to elevated potassium chloride (120 mM KCl) resulted in a doubling of [3H]choline cotransport and [3H]HC-3 binding. The enhancement of [3H]choline cotransport by STAURO and antecedent 120 mM KCl treatment were additive. PMA did not significantly alter elevated potassium stimulated transport. Moreover, arachidonyltrifluoromethyl ketone (AACOCF3) and quinacrine (QUIN), both phospholipase A2 (PLA2) inhibitors, markedly decreased enhanced [3H]choline transport and [3H]HC-3 binding induced by antecedent exposure to depolarizing concentrations of potassium. These results suggest that PKC and PLA2 are involved in the regulation of [3H]choline cotransport but at different regulatory sites.     

Stereoselectivity of the Inhibition of [3H]Hemicholinium-3 Binding to the Sodium-Dependent High-Affinity Choline Transporter by the Enantiomers of a- and β-Methylcholine

Abstract: In a previous report, we showed that the enantiomers of α- and β-methylcholine inhibited choline uptake with Stereoselectivity, but that their transport by the choline carrier of nerve terminals showed stereospecificity. The present experiments used the same choline analogues to determine if either of the above characteristics pertains to their ability to interact with the [³H]-hemicholinium-3 binding site present on striatal membranes and synaptosomes. [³H]Hemicholinium-3 binding to striatal membranes could be inhibited stereoselectively by the enantiomers of β-methylcholine, but R (+)-α-methyl-choline was little better than its enantiomer in this test. However, [³H]hemicholinium-3 binding to striatal synaptosomes was inhibited stereoselectively by the enantiomers of both α- and β-methylcholine. This difference between the properties of [³H]hemicholinium-3 binding to membranes or to synaptosomes appears related to the presence of two ligand binding states. The [³H]hemicholinium-3 binding site could be shifted to a low-affinity state by ATP treatment and to a high-affinity state by EDTA washing. When the [³H]hemicholinium-3 binding site existed in its low-affinity state, binding was inhibited stereoselectively by the enantiomers of both a- and β-methylcholine, but when shifted to its high-affinity state, it was inhibited stereoselectively only by the enantiomers of β–methylcholine. We conclude that hemicholinium-3 interacts with the substrate recognition site of the high-affinity choline transporter, but that the Stereoselectivity of this site changes depending on its affinity state.     

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.     

Sodium-dependent high-affinity binding of [3H]hemicholinium-3 in the rat brain: a potentially selective marker for presynaptic cholinergic sites

This report describes the membrane binding properties of [3H]hemicholinium-3 ([3H]HC-3), a selective inhibitor of sodium-dependent high-affinity choline uptake (SDHACU) in cholinergic nerve terminals. Under the described assay conditions, [3H]HC-3 bind with a saturable population of high-affinity (apparent Kd = 1.9 nM) CNS membrane sites having the regional distribution: striatum much greater than hippocampus greater than cerebral cortex greater than cerebellum. High-affinity [3H]HC-3 binding is entirely dependent upon the presence of sodium chloride (EC50 = 35-50 mM) and is markedly reduced when other salts of sodium or monovalent ions are substituted. [3H]HC-3 binding is inhibited by choline (Ki = 6 microM) and acetylcholine (Ki = 35 microM) but markedly less sensitive to other cholinergic agents and metabolic inhibitors. In light of the similar ionic dependencies, regional distributions and pharmacological specificities of [3H]HC-3 binding and SDHACU, closely associated sites may be involved in both processes.     

Ouabain-Sensitive Choline Transport System in Capillaries Isolated from Bovine Brain

In physiological conditions, there is a net transport of choline from brain to blood, despite the fact that the choline concentration is higher in plasma than in CSF. Because of the blood-brain barrier characteristics, such passage against the concentration gradient takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, [3H]choline uptake properties have been analyzed in capillaries isolated from bovine brain. [3H]Choline uptake was linear with time for up to 1 h. Nonlinear regression analysis of the uptake rates at different substrate concentrations gave the best fit to a system of two components, one of which was saturable (Km = 17.8 +/- 4.8 microM; Vmax = 11.3 +/- 3.4 pmol/min/mg of protein) and the other of which was nonsaturable at concentrations up to 200 microM. The [3H]choline transport was significantly reduced in the absence of sodium and after incubation with 10(-4) M ouabain for 30 min. Ouabain also inhibited choline uptake in purified cerebral endothelial cells, but not in the endothelium isolated from bovine aorta. Accordingly, cerebral endothelial cells were able to concentrate [3H]choline, with this effect being abolished by ouabain, whereas in aortic endothelial cells the [3H]choline intracellular concentration was never higher than that of the incubation medium. These results suggest that the blood-brain barrier endothelium is specifically provided with an energy-dependent choline transport system, which may explain the choline efflux from the brain and the maintenance of a low choline concentration in the cerebral extracellular space.     

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.     

Molecular cloning and functional characterization of a neuronal choline transporter from Trichoplusia ni

A cDNA encoding a high-affinity Na(+)-dependent choline transporter (TrnCHT) was isolated from the CNS of the cabbage looper Trichoplusia ni using an RT-PCR-based approach. The deduced amino acid sequence of the CHT cDNA predicts a 594 amino acid protein of 64.74 kDa prior to glycosylation. TrnCHT has 80%, 79%, 76%, and 58% amino acid identity to putative CHTs from Anopheles gambiae, Drosophila melanogaster and Apis mellifera, and a cloned CHT from Limulus polyphemus, respectively. In situ hybridization of TrnCHT cRNA in whole-mount preparations of caterpillar CNS revealed that TrnCHT mRNA is expressed by hundreds of presumably cholinergic neurons present in both the brain and cortex of all segmental ganglia. Na(+)-dependent [(3)H]-choline uptake was induced in Sf9 cells in vitro following infection with a TrnCHT-expressing recombinant baculovirus. Virally induced [(3)H]-choline uptake was found to approximately equal the endogenous rate of choline uptake in insect cells, seen either after infection with a control virus or in TrnCHT-infected cells exposed to [(3)H]-choline in the absence of Na(+). The Na(+)-dependent component of [(3)H]-choline uptake by TrnCHT-infected cells was saturable with a K(m) for choline transport of 8.4 microM. Several compounds reported to be potent blockers of [(3)H]-choline uptake by cloned vertebrate choline transporters proved to be relatively weak inhibitors of choline uptake by Sf9 cells expressing TrnCHT. Hemicholinium-3 (K(i)=4.1 microM) and two oxoquinuclidium analogues of choline, quireston-A (K(i) approximately 10 microM) and quireston (K(i) approximately 100 microM) inhibited 50% of control uptake only at micromolar concentrations. The endogenous low-affinity Na(+)-independent uptake of [(3)H]-choline was also inhibited by high micromolar concentrations of hemicholinium-3.     

Release of Acetylcholine from Tissue Slices of the Rat Nucleus Basalis Magnocellularis

We investigated the release of acetylcholine (ACh) from tissue slices obtained from the nucleus basalis magnocellularis (nbM) of the rat brain. Potassium (35 mM) depolarization produced a 10- to 12-fold increase in the release of endogenous ACh above spontaneous release. Potassium-evoked ACh release was Ca2+ dependent. Injection of the excitotoxin quinolinic acid into the nbM produced a 72.8 +/- 13.0% decrease in spontaneous ACh release and a 60.4 +/- 8.2% decrease in potassium-evoked release. A fourfold increase in ACh release was observed following perfusion of the tissue with 1 mM 3,4-diaminopyridine (3,4-DAP) whereas 10 mM 3,4-DAP caused a sevenfold increase. The increase in ACh release caused by 3,4-DAP was inhibited by tetrodotoxin. Tissue slices accumulated [3H]choline by high-affinity choline uptake and this could be inhibited by hemicholinium-3. These results indicate that ACh can be released from tissue slices of the nbM by a calcium-dependent process and that a part of this release appears to be from the cholinergic neurons of the nbM.