Possible role of sialocompounds in the uptake of choline into synaptosomes and nerve cell cultures

Incubation of primary nerve cell cultures and of crude synaptosomal preparations with neuraminidase released sialic acid from both gangliosides and sialoglycoproteins. After this treatment, the pattern of ganglioside distribution was severely modified with a decrease of polysialogangliosides (GD1b, GT1b, GT1L, GQ1) and a dramatic increase in monosialoganglioside GM1. The choline influx into neuraminidase treated cells and organelles was reduced by 30–50% but the efflux was unmodified. In particular the high affinity mechanism of choline uptake disappeared and the low affinity mechanism was modified in both cases. The disappearance of the high affinity uptake mechanism was not followed by a decreased acetylcholine synthesis as it should be if the current theories on choline uptake and acetylcholine synthesis are correct. Our present data thus confirm our previous hypothesis that choline metabolism regulates choline uptake rather than the other way round as is suggested by the theories most widely accepted at present. Choline uptake was unaffected by pretreatment of cells and organelles with tetanus toxin suggesting that the effect of neuraminidase on the choline uptake were either mediated through glycoproteins or through gangliosides other than those which bind to tetanus toxin (GD1b and GT1b). Several speculative models for explaining the effect of neuraminidase on choline uptake are proposed.     

NET UPTAKE OF L-GLUTAMATE AND GABA BY HIGH AFFINITY SYNAPTOSOMAL TRANSPORT SYSTEMS

Abstract— Reuptake of neuroactive amino acids by high affinity transport systems in the CNS is thought to terminate the neurotransmitter activity of these substances. This notion has been challenged since the homoexchange of synaptosomal and exogenous L-glutamate and the corresponding homoexchange of synaptosomal and exogenous GABA has been demonstrated. We reported that depolarizing media (56 mM-KCl, 1 mM-CaCl₂) lowers the GABA content of synaptosomes. In such synaptosomes, net and apparent (radioactive) GABA uptake are similar. When rat cortical synaptosomes (1 mg protein/ml) are incubated with 10μM-[¹⁴C] L-glutamate, net and apparent (radioactive) uptake are similar. When the synaptosome levels are decreased to 0.5 mg protein/ml or less, then net uptake becomes a fraction of radioactive uptake (exchange ensues). Net L-glutamate uptake is Na ⁺-dependent and temperature-dependent. Furthermore, a 1 mM concentration of KCl or RbCl supports net L-glutamate and GABA uptake. LiCl, NH₄Cl, CsCl and choline chloride are ineffective. In addition, diaminobutyric acid (but not β -alanine) inhibits net and apparent GABA uptake. The demonstration of net uptake of L-glutamate and GABA by their respective high affinity systems is consonant with the idea that these systems may play a role in neurotransmitter inactivation in the synaptic region.     

THE TOXIC EFFECT OF SODIUM GLUTAMATE AND DL-α-AMINOADIPIC ACID ON RAT RETINA: CHANGES IN HIGH AFFINITY UPTAKE OF PUTATIVE TRANSMITTERS

Subcutaneous administration of high doses of sodium glutamate to new born rats was used to destroy retinal interneurons and ganglion cells. Such treatment was accompanied by 90% reduction in the high affinity uptake of choline, 60–70% reductions in the uptakes of GABA, diamino-n-butyric acid and glycine and 30–40% reductions in the uptakes of asparatate and glutamate measured on retinal homogenates from 30-day-old rats. The high affinity uptakes of β-alanine and taurine were unchanged. Preincubation of retinal homogenates with 1 mM β-alanine or 100 μM diamino-n-butyric acid severely reduced the high affinity GABA uptake in control and experimental animals. In intact retinae, however, the glutamate treatment increased the high affinity uptake of β-alanine by 70%, whereas that of diamino-n-butyric acid was reduced by 40% and the high affinity uptakes of GABA and glutamate were unchanged. Four hours after injection of the gliotoxic compound DL-α-aminoadipic acid into the vitreous body of 30-day-old rats, the Müller cells could no longer be identified. This lesion was accompanied by 55% reduction in the high affinity uptake of β-alanine and 25% reduction in the uptakes of GABA and glutamate on intact retinae. The high affinity uptakes of diamino-n-butyric acid, choline and the enzyme activities of choline acetyltransferase and glutamate decarboxylase were unchanged under these conditions. After 24 h, however, the Müller cells could be recognized again, and the β-alanine uptake had normalized.     

Cortical modulation of cholinergic neurons in the striatum

Previous reports suggest the existence of a cortico-striatal pathway which might use glutamate as the transmitter. In the present study, the possible influence of this pathway on striatal cholinergic neurons was investigated. Two weeks following surgical destruction of the cerebral cortex, the high affinity uptake of glutamate and choline into striatal synaptosomes was significantly reduced whereas GABA uptake was unaffected. In acute experiments (1 hour following decortication), only choline uptake was significantly reduced while the uptake of glutamate and GABA were not altered. Acute injection (2 minutes) of kainic acid into the striatum, 1 hour after decortication, reversed the effect of the decortication on choline uptake, perhaps by simulating an excitatory input to the striatum which was presumably removed by the cortical ablation. These observations are consistent with the existence of a cortical input (perhaps glutamatergic) to the striatum and suggest that striatal cholinergic neurons can be influenced by this cortico-striatal pathway.     

Topography of synaptosomal high affinity uptake systems.

We have tested the hypothesis that the glycoproteins in the cell membrane of axonal terminals are involved in high affinity uptake of neurotransmitters by studying the effects of lectin binding and trypsin treatment on this process in synaptosomes. Binding of two lectins, Concanavalin A and a lectin isolated from the lentil Lens culinaris, to synaptosomes does not change the uptake of six putative transmitters: L-glutamate, norepinephrine (NE), 5-hydroxytryptamine (5-HT), dopamine, choline (Ch), and γ-aminobutyrate (GABA). While trypsin digestion of surface proteins of synaptosomes has no effect on the uptake of NE, 5-HT, dopamine, Ch and GABA, it reduces the rate of uptake of L-glutamate. This reduction is not due to synaptosomal lysis or a profound conformational change of the synaptic plasma membrane since the maximal velocity of high affinity uptake is reduced drastically with little attendant change in K_m.     

Synaptic Function of Cholinergic-Specific Chol-1α Ganglioside

The function of a cholinergic-specific ganglioside, Chol-1alpha, was investigated. The release of acetylcholine from synaptosomes was inhibited by anti-Chol-1alpha monoclonal antibody but not by monoclonal antibodies against other brain gangliosides tested. Chol-1alpha ganglioside stimulated the high-affinity choline uptake by synaptosomes and consequently enhanced acetylcholine synthesis, resulting in an increased release of acetylcholine from synaptosomes. The memory and learning abilities of rats given anti-Chol-1alpha antibody were remarkably suppressed. These in vitro and in vivo studies suggest that Chol-1alpha ganglioside plays a pivotal role in cholinergic synaptic transmission and participates in cognitive function.     

SODIUM-DEPENDENT HIGH AFFINITY CHOLINE UPTAKE: A REGULATORY STEP IN THE SYNTHESIS OF ACETYLCHOLINE

The sodium-dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50-80% reduction in sodium-dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal-hippocampal or habenuleinterpeduncular tracts by lesions (10 min-1 h) also caused a similar, large reduction in sodium-dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively. We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal-hippocampal tract. Stimulation (40 Hz) for 10-15 min completely reversed the depression in sodium-dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal. Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium-de-pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration. We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium-dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake. We examined the conversion of [³H]choline to [³H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60-70% of the total sodium-dependent tritium content was present as [³H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake. A kinetic analysis of sodium-dependent high affinity choline uptake was performed after all treatments. We found changes in V_max, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium-dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium-dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.     

Synaptic Chemistry Associated with Aberrant Neuronal Development in the Reeler Mouse

Abstract: Pre- and postsynaptic neurochemical markers for several afferent and intrinsic neuronal systems were measured in the mouse mutant, reeler. In the neocortex of the reeler, the relative positions of the polymorphic and pyramidal cells were inverted but this was not associated with alterations in the content/mg protein of synaptic markers for noradrenergic [tyrosine hydroxylase (TH), norepinephrine (NE), NE uptake], cholinergic [choline acetyltransferase (ChAT), quinuclidinyl benzilate (QNB) binding], γ-aminobutyric acid (GABA)ergic (glutamate decarboxylase, GABA uptake, GABA receptors, GABA) or glutamatergic (glutamate uptake, receptors, glutamate) neurons. The laminar distributions of the hippocampal neurons were disrupted and associated with mild hypoplasia; consistent with this alteration, the content/mg protein of some GABAergic (GABA uptake) and glutamatergic (glutamate receptors) markers were slightly increased. The reeler cerebellum was characterized not only by misalignment of neurons but also by a marked loss of granule cells. Commensurate with the degree of cerebellar hypoplasia, the total amount of glutamate content, [³H]l-glutamate uptake activity, [³H]muscimol, and [³H]QNB ligand binding were reduced in the reeler cerebellum. In contrast, presynaptic markers for the noradrenergic (TH, NE) climbing fibers and the cholinergic (ChAT) mossy fibers were significantly increased/mg protein but their total content/cerebellum was near normal. Our data support suggestions that cerebellar granule cells use glutamate as their neurotransmitter and contain GABA and cholinergic receptors. The findings also suggest that misplaced cortical and cerebellar neurons retain normal neurochemical characteristics and that the morphologic alterations do not markedly affect the quantitative development of aminergic afferent systems.     

Amino acid neurotransmission: Dynamics of vesicular uptake

Glutamate, GABA and glycine, the major neurotransmitters in CNS, are taken up and stored in synaptic vesicles by a Mg²⁺-ATP dependent process. The main driving force for vesicular glutamate uptake is the membrane potential, whereas both the membrane potential and the proton gradient contribute to the uptake of GABA and glycine. Glutamate is taken up by a specific transporter with no affinity for aspartate. Evans blue and related dyes are competitive inhibitors of the uptake of glutamate. GABA, β-alanine, and glycine are taken up by the same family of transporter molecules. Aspartate, taurine, and proline are not taken up by any synaptic vesicle preparations. It is suggested that vesicular uptake and release are characteristics that identify these amino acids as neurotransmitters. We also discuss that “quanta” in the brain are not necessarily related the content of neurotransmitter in the synaptic vesicles, but rather to postsynaptic events. Special issue dedicated to Dr. Herman Bachelard.     

Adult rat brain synaptic vesicles II. Lipid composition

The lipid composition of synaptic vesicles isolated from adult rat brain was determined. Vesicles contained cholesterol and phospholipid but very little ganglioside, galactolipid, free fatty acid and triglyceride was detected. Ethanolamine and choline phosphoglycerides were the dominant phospholipids. Lysophosphatidyl choline was present in very low amounts. The fatty acid composition of the phosphoglycerides was characterized by high levels of docosahexaenoic acid in the ethanolamine and serine phosphoglycerides, and the absence of long chain fatty acids from the sphingomyelins. All the characteristic features of the lipid composition of the synaptosomal plasma membrane (with the exception of the ganglioside content) were seen in the synaptic vesicle lipids. The results are discussed in terms of the exocytosis mechanism of transmitter release.     

Characterization of γ-Aminobutyric Acid Binding Sites on Crude Synaptic Membranes

[3H]GABA binding to crude synaptic membranes of rat brain was studied in an attempt to identify GABA binding to its synaptic receptor in the presence of Na+. Membrane vesicles prepared from crude synaptic membrane fractions were useful as a tool to differentiate synaptic GABA receptors from GABA uptake sites. The crude synaptic membranes treated with Triton X-100 [membranes (TX)] involved two classes of GABA binding sites (KD = 38.7 and 78.0 nM) in the absence of Na+, but the high-affinity sites disappeared in the presence of Na+ and a single class of GABA binding sites (KD = 75.0 nM) was detected. The failure to detect an active uptake of [3H]GABA into the vesicles prepared from membranes (TX) suggests that the [3H]GABA binding in the presence of Na+ was related to synaptic GABA receptors. It is probable that Na+ could mask the presence of the high-affinity class of GABA receptor.     

Synaptosomal phospholipid pool in rabbit brain and its effect on GABA uptake

Rabbit synaptosomes have been used to study the effect of the base-exchange reaction in membrane phospholipids on -aminobutyric acid (GABA) transport in vitro. The uptake of GABA was measured after a base-exchange reaction with ethanolamine, choline, orl-serine and after subsequent displacement of these exchanged moieties from lipid by bases of similar or different structures which were added to the synaptosomal medium. Serine incorporation stimulated GABA transport, but its displacement from membrane lipid by choline or ethanolamine induced an inhibition of GABA transport. Ethanolamine incorporation inhibited GABA transport, but its displacement by serine or choline resulted in stimulation of GABA uptake. Choline incorporation also inhibited GABA transport, although less than ethanolamine. The pool size of synaptosomal phospholipids, presumably involved in GABA uptake, accounted for 0.2 to 10% of the total content of membrane phospholipid. Thus, alteration of phospholipid compositior by exchange of the lipid hydrophilic head-groups influences the extent GABA uptake into rabbit synaptosomes.     

GABA and glycine in synaptic vesicles: storage and transport characteristics.

gamma-Aminobutyric acid (GABA) and glycine are major inhibitory neurotransmitters that are released from nerve terminals by exocytosis via synaptic vesicles. Here we report that synaptic vesicles immunoisolated from rat cerebral cortex contain high amounts of GABA in addition to glutamate. Synaptic vesicles from the rat medulla oblongata also contain glycine and exhibit a higher GABA and a lower glutamate concentration than cortical vesicles. No other amino acids were detected. In addition, the uptake activities of synaptic vesicles for GABA and glycine were compared. Both were very similar with respect to substrate affinity and specificity, bioenergetic properties, and regional distribution. We conclude that GABA, glycine, and glutamate are the only major amino acid neurotransmitters stored in synaptic vesicles and that GABA and glycine are transported by similar, if not identical, transporters.     

EFFECTS OF RETINAL ABLATION ON UPTAKE OF GLUTAMATE, GLYCINE, GABA, PROLINE AND CHOLINE IN PIGEON TECTUM

—The effect of retinal ablation on the high and low affinity uptake of choline, GABA, glutamate, glycine and proline into the crude mitochondrial fraction of the pigeon optic tectum was studied. After 4–8 weeks of degeneration the uptake for glutamate, and to a lesser degree the uptake for GABA, at both the high and the low affinity substrate concentration, decreased. In contrast, the uptake of glycine increased. Kinetic analysis showed that the reduced uptake of glutamate was due to a decrease in the V_max. By intraocular injection of [³H]proline the retinal endings in the optic tectum were labelled with the fast axoplasmic transport fraction. Optic terminals labelled in this way have the same sedimentation characteristics in continous sucrose gradients as the particles accumulating giutamate in the uptake assay.     

GABA accumulation by embryonic chick tectum.

Radioactive GBA accumulation by embryonic chick tectal tissue under conditions suitable for high affinity GABA uptake showed a marked increase with age. There was excellent correlation between the GABA accumulation and the GAD activity from the eighth to the twentieth embryonic day, suggesting that both may reflect primarily synaptic development.     

A vinblastine sensitive high affinity choline uptake system

1. The Limulus cardiac ganglion high affinity choline uptake system (HAChUS) was inhibited 40, 51 and 64% following pre-exposure to 10, 100 and 500 microM vinblastine, respectively. 2. In contrast, high affinity uptake of choline in the Limulus corpora pedunculata and abdominal ganglia, tissues in which a cholinergic function has been described, were unaffected. 3. In pulse-chase experiments, the cardiac ganglion was incubated in 0.1 microM [3H]choline for 60 min and then switched to an incubation medium containing 1 mM unlabelled choline for varying periods of time. 4. Under these conditions, a 3-fold increase of radiolabel above basal level was measured in the pellet fraction within 2 hr of post-labelling incubation. 5. Prior exposure of the ganglion to 500 microM vinblastine completely eliminated this increase of radioactivity in the pellet fraction. 6. Treatment of the radiolabelled pellet fraction with phospholipase C resulted in the solubilization of 72% of the radiolabel. 7. Ten (10) microM 5-hydroxytryptamine (5-HT), a concentration previously shown to inhibit spontaneous electrical activity within the cardiac ganglion, resulted in a 40% decrease in high affinity choline uptake in this tissue selectively. 8. These results are consistent with the view that a probable role of the Limulus cardiac ganglion HAChUS is the supply of choline subserving the synthesis of membrane phospholipid. 9. It is further speculated that this membrane phospholipid synthesis may be associated with synaptic vesicle turnover.     

SIMILARITIES OF β-BUNGAROTOXIN AND PHOSPHOLIPASE A2 AND THEIR MECHANISM OF ACTION

Abstract— β-Bungarotoxin, a presynaptic neurotoxin isolated from the venom of Bungarus multicinctus , has been shown to initially cause an increase in the frequency of miniature endplate potentials and subsequently block neuromuscular transmission by inhibiting nerve impulse induced release of acetylcholine. In rat brain synaptosomes it causes a Ca²⁺-dependent release of acetylcholine together, with a strong inhibition of the high affinity choline uptake system. In this report we demonstrate that β-bungarotoxin acts as a phospholipase A₂ (phosphatide 2-acyl hydrolase, EC 3.1.1.4), liberating fatty acids from synaptic membrane phospholipids. It also exhibits a striking similarity in a number of neurochemical properties with that of a purified phospholipase A₂ from Naja naja siamensis. In addition, both agents produce a marked depolarization of synaptosomal preparations as measured by a fluorescent dye. We propose that disruption of the membrane phospholipids by phospholipase activity can lead to depolarization of the synaptosomal preparation which promotes both transmitter release and inhibition of the energy-dependent high affinity choline uptake system. With this decreased supply of choline, the acetylcholine content of the cell would be gradually depleted leading to a decrease in transmission.     

CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN CORPUS STRIATUM OF RESERPINISED RATS1

Rats treated with reserpine show increased V_max for the high affinity uptake of choline into small slices of corpus striatum. The choline acetyltransferase activity of whole homogenates of striatum is also increased. These changes are consistent with increased cholinergic neuronal activity in the striatum and seem likely to be adaptations mediating increased rates of synthesis of acetylcholine. The maximal increases found occurred concurrently, consistent with coupling of the high affinity uptake of choline and its acetylation in cholinergic nerve terminals of the rat. That increased high affinity uptake is accompanied by increased choline acetyltransferase activity, suggests the input of choline is not the sole determinant of rates of synthesis of acetylcholine, in spite of the large Vmas for striatal choline acetyltransferase, compared with that for high affinity uptake. These results seem best explained by kinetic coupling, in the rat, of the high affinity uptake of choline with a limited pool of choline acetyltransferase preferentially localised at the nerve terminal plasma membrane.     

Release of acetylcholine and GABA,and activity of their synthesizing enzymes in the rat pontine reticular formation

The aim of this study was to obtain neurochemical information on the possible role of acetylcholine (ACh) and -aminobutyric acid (GABA) as neurotransmitters in the pontine reticular formation (PRF). We studied the uptake of labeled choline and GABA, as well as the release of this amino acid and of ACh, in PRF slices of the rat. In addition, choline acetyltransferase, acetylcholinesterase and glutamate decarboxylase activities were assayed in PRF homogenates. The uptake of GABA was strictly Na⁺-dependent, whereas choline uptake was only partially Na⁺-dependent. The release of both ACh and GABA was stimulated by K⁺-depolarization, but only the former was Ca²⁺-dependent. Choline acetyltransferase activity in the PRF was 74% of that in the striatum, whereas acetylcholinesterase activity was considerably lower. Glutamate decarboxylase activity in the PRF was about half that observed in the striatum. These findings support the possibility that both ACh and GABA may act as neurotransmitters in the rat PRF.     

MUSCIMOL UPTAKE, RELEASE AND BINDING IN RAT BRAIN SLICES

Abstract— The GABA analogue, muscimol, was taken up relatively inefficiently compared to GABA by slices of rat cerebral cortex at 37 C. Muscimol uptake followed saturation kinetics (K_m ImM. V_m 0.1 μmol g mini and showed an absolute dependence on sodium ions. The relative susceptibilities of muscimol uptake and GABA high affinity uptake to a variety of inhibitors, including (-)-nipecotic acid. (+)-2.4-diaminobutyric acid and arecaidine, and the stimulation of muscimol efflux by 50μM-GABA, suggest that muscimol and GABA share some common transport carriers. Since L-histidine inhibited muscimol uptake hut not GABA high affinity uptake, at least part of the observed muscimol uptake may be mediated by the 'small basic'amino acid transport system. Muscimol appeared to he taken up into nerve terminals, since uptake was inhibited by the neuronal uptake inhibitor cis -3-aminocyclohexanecarboxylic acid but not by the glial uptake inhibitor β-alanine. Muscimol efflux was stimulated in a calcium-dependent manner by an increased potassium ion concentration.
Sodium-independent binding of muscimol was observed in slices of rat cerebral cortex at 4 C. Binding could be inhibited by a variety of substances. including GABA, isoguvacine and (+)-bicuculline methochloride, which are known to inhibit the binding of muscimol to putative GABA receptors associated with synaptic membranes purified from rat brain.