Alterations of Amino Acid Transmitter Systems in Spinal Cords of Chronic Paraplegic Dogs

The high-affinity uptake of [3H]serotonin, [3H]glutamate, and [3H]gamma-aminobutyric acid [( 3H]GABA) and the Na+-independent binding of [3H]glutamate and [3H]GABA were studied using spinal cord preparations obtained from normal mongrel dogs and from dogs made paraplegic by midthoracic spinal cord crush. Lumbosacral regions of the spinal cord were removed either before (1 week) or after (3 to 8 weeks) onset of spasticity. A myelin-free synaptosomal fraction was obtained by centrifugation and used for studying high-affinity uptake and for preparing synaptic plasma membranes for Na+-independent binding experiments. For the paraplegic groups, the uptake of 30 nM [3H]serotonin was 66 and 18% of control values after 1 and 3 weeks, respectively. Eadie-Hofstee analysis of [3H]serotonin uptake showed a 90% reduction in Vmax for the paraplegic group relative to control values, thereby indicating the expected loss of descending serotonergic pathways. The high-affinity uptakes of 1 microM [3H]glutamate and [3H]GABA were the same in both the control and nonspastic paraplegic groups after 1 week. However, after 3 weeks, the uptakes of [3H]glutamate and [3H]GABA were 60-70% higher for the spastic group than for the control animals. For both amino acids, Eadie-Hofstee plots revealed no difference in Km and higher Vmax for the spastic group relative to control values. After 1 and 3 weeks, the Na+-independent binding of 5 nM [3H]glutamate was 40-85% higher and the binding of 10 nM [3H]GABA was 40-60% lower for the paraplegic groups relative to the values for the control animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium-Dependent Proline Uptake in the Rat Hippocampal Formation: Association with Ipsilateral-Commissural Projections of CA3 Pyramidal

Na+-dependent uptake of L-[3H]proline was measured in a crude synaptosomal preparation from the entire rat hippocampal formation or from isolated hippocampal regions. Among hippocampal regions, Na+-dependent proline uptake was significantly greater in areas CA1 and CA2-CA3-CA4 than in the fascia dentata, whereas there was no marked regional difference in the distribution of Na+-dependent gamma-[14C]aminobutyric acid ([14C]GABA) uptake. A bilateral kainic acid lesion, which destroyed most of the CA3 hippocampal pyramidal cells, reduced Na+-dependent proline uptake by an average of 41% in area CA1 and 52% in area CA2-CA3-CA4, without affecting the Na+-dependent uptake of GABA. In the fascia dentata, neither proline nor GABA uptake was significantly altered. Kinetic studies suggested that hippocampal synaptosomes take up proline by both a high-affinity (KT = 6.7 microM) and a low-affinity (KT = 290 microM) Na+-dependent process, whereas L-[14C]glutamate is taken up predominantly by a high-affinity (KT = 6.1 microM) process. A bilateral kainic acid lesion reduced the Vmax of high-affinity proline uptake by an average of 72%, the Vmax of low-affinity proline uptake by 44%, and the Vmax of high affinity glutamate uptake by 43%, without significantly changing the affinity of the transport carriers for substrate. Ipsilateral-commissural projections of CA3 hippocampal pyramidal cells appear to possess nearly as great a capacity for taking up proline as for taking up glutamate, a probable transmitter of these pathways. Therefore proline may play an important role in transmission at synapses made by the CA3-derived Schaffer collateral, commissural, and ipsilateral associational fibers.     

Depolarization Increases Chloride-Dependent Glutamate Sequestration in Synaptic Membranes of Rat Cerebral Cortex

To assess the functions of Cl- -dependent glutamate "binding" (Cl- -dependent glutamate uptake) in synaptic membranes, possible effects of depolarization on the uptake were examined. When rat cerebral cortical slices were preincubated with depolarizing agents such as veratrine (7 micrograms/ml), 10 microM aconitine, 56 mM K+, and 50 microM monensin, [3H]glutamate uptake by the crude synaptic membranes, which were subsequently prepared from the pretreated slices, was increased by 60-85%. Stimulation of the glutamate uptake by predepolarization was dependent on Na+ but not on Ca2+. The bindings of gamma-[3H]aminobutyric acid and 5-[3H]hydroxytryptamine were not significantly affected by the predepolarization. Veratrine pretreatment increased the maximal density of the glutamate uptake sites without affecting the affinity for glutamate. Several characteristics of the uptake sites increased by the veratrine pretreatment coincided with those of Cl- -dependent glutamate uptake sites. Na+-dependent glutamate binding (Na+-dependent glutamate uptake) to the membranes was not affected by pretreatment with veratrine. The content of endogenous glutamate and the noninulin space in the membrane fractions were not changed by the predepolarization. The increase in the glutamate uptake induced by pretreatment with high K+ was reversible: it returned to the control level after a second incubation of the slices in control medium. These results suggest that the Cl- -dependent glutamate sequestration system in synaptic membranes is regulated by the membrane potential.     

Strychnine-Sensitive, Glycine-Induced Release of [3H]Norepinephrine from Rat Hippocampal Slices

The amino acid glycine is the primary inhibitory neurotransmitter of the mammalian spinal cord. Glycine has also been shown to facilitate the excitatory actions of glutamate at the N-methyl-D-aspartic acid receptor subtype. In this article, glycine is shown to increase the Ca2(+)-dependent release of [3H]norepinephrine from preloaded slices of the rat hippocampus. This effect was inhibited noncompetitively by nanomolar concentrations of strychnine, which differentiates it from the glycine site associated with the N-methyl-D-aspartate receptor. Glycine also released [3H]acetylcholine, but was without effect on the efflux of [3H]serotonin or gamma-[3H]aminobutyric acid from the same tissue preparation. The release of [3H]norepinephrine was reversibly blocked by tetrodotoxin, indicating the effect is not initiated at the noradrenergic terminals, but requires propagation of an action potential. The results suggest that a glycine site that is pharmacologically similar to that found in the spinal cord exists in the rat hippocampus. We suggest that this site may participate in modulating the release of specific neurotransmitters in the brain.     

Ubiquitin-Directed Antibodies Inhibit Neuronal Transporters in Rat Brain Synaptosomes

Affinity-purified antibodies specific for ubiquitin were found to inhibit the sodium-dependent uptake of [3H]choline, gamma-[3H]aminobutyric acid [( 3H]GABA), [3H]glutamate, [3H]norepinephrine, [3H]aspartate, and [3H]serotonin in rat cerebral cortical synaptosomes at a low concentration (10 micrograms/ml). These antibodies (termed anti-Ub) had no effect on the sodium-independent uptake of these substances or their calcium-dependent efflux. Synaptosomal [3H]deoxyglucose uptake was not affected in normal Krebs Ringer buffer containing 10 mM glucose, but was inhibited in glucose-free medium. Other nonneuronal sodium-dependent transport processes were found to be unaffected by 10 micrograms/ml anti-Ub, suggesting that anti-Ub does not bind indiscriminantly to sodium-binding sites on sodium-dependent organic solute transporters. Finally, anti-Ub inhibited sodium-dependent [3H]GABA and [3H]glutamate uptake in plasma membrane ghosts, devoid of membrane potential, which were derived from rat cerebral cortical synaptosomes. These results suggest that neuronal transporters or sites proximal to them may be ubiquitinylated on the plasma membrane surface.     

Lack of Selectivity Between the Uptake of [3H] Adrenaline and [3H]Noradrenaline into Rat Hypothalamic Slices

The uptake of [3H]adrenaline and [3H]noradrenaline into rat hypothalamic slices was compared for determination of whether adrenaline uptake was independent of uptake into noradrenergic neurones. Kinetic analysis revealed a similar high-affinity uptake process for both adrenaline and noradrenaline, with Km and Vmax values within similar ranges. These uptakes were inhibited by desipramine and maprotiline in a dose-dependent manner, but the selective dopamine and 5-hydroxytryptamine uptake inhibitors benztropine and fluoxetine, respectively, were without effect. Competition for uptake sites by unlabelled adrenaline with [3H]adrenaline and [3H]-noradrenaline and by unlabelled noradrenaline with [3H]-adrenaline and [3H]noradrenaline was very similar. Lesioning of the major adrenaline-containing cell group (C1 cell group) decreased the hypothalamic adrenaline concentration but had no effect on hypothalamic [3H]adrenaline or [3H]noradrenaline uptake. The results suggest that exogenous adrenaline is largely taken up by high-affinity sites on noradrenergic nerve terminals.     

Comparison of the Properties of γ-Aminobutyric Acid and L-Glutamate Uptake into Synaptic Vesicles Isolated from Rat Brain

Rat brain synaptic vesicles exhibit ATP-dependent uptake of gamma-[3H]amino-n-butyric acid ([3H]GABA) and L-[3H]glutamate. After hypotonic shock, the highest specific activities of uptake of both L-glutamate and GABA were recovered in the 0.4 M fraction of a sucrose gradient. The uptakes of L-glutamate and GABA were inhibited by similar, but not identical, concentrations of the mitochondrial uncoupler carbonyl cyanide m-chlorophenylhydrazone and the ionophores nigericin and gramicidin, but they were not inhibited by the K+ carrier valinomycin. N,N'-Dicyclohexyl-carbodiimide and N-ethylmaleimide, Mg2+-ATPase inhibitors, inhibited the GABA and L-glutamate uptakes similarly. Low concentrations of Cl- stimulated the vesicular uptake of L-glutamate but not that of GABA. The uptakes of both L-glutamate and GABA were inhibited by high concentrations of Cl-. These results indicate that the vesicular GABA and L-glutamate uptakes are driven by an electrochemical proton gradient generated by a similar Mg2+-ATPase. The vesicular uptake mechanisms are discussed in relation to other vesicle uptake systems.     

D-Aspartate Uptake and Release in the Guinea Pig Spinal Cord After Partial Ablation of the Cerebral Cortex

This study attempts to determine if L-glutamate and L-aspartate may be transmitters of the guinea pig corticospinal tract. Unilateral ablations were made of the frontal and parietal neocortex which destroyed most of the motor and somatosensory areas in the right cerebral hemisphere. In lesioned animals, transverse sections of the cervical enlargement of the spinal cord (segments C6--T1) were stained to reveal degenerating fibers. Degeneration of axons first appeared 4 days after surgery, reached a maximum on the seventh day, and began to wane by the ninth day. The most prominent loss of axons appeared deep in the dorsal funiculus and in laminae IV-IX of the gray matter contralateral to the cortical lesion. Ipsilaterally, there was very sparse degeneration of fibers in the dorsal and ventral funiculi and in the spinal gray matter. The uptake and release of D-[3H]aspartate, a putative nonmetabolizable marker for L-glutamate and L-aspartate, were measured in dissected quadrants of the cervical enlargement taken from intact and lesioned animals. The uptake and the electrically evoked, Ca2+-dependent release of D-[3H]aspartate were depressed by 29-35% at 4 and 7 days after surgery, but only in tissue that was contralateral to the cortical ablation. The lesion had no effect on the uptake and release of exogenous gamma-[14C]aminobutyric acid, which were measured as indices of the postlesion integrity of neurons in the spinal gray matter. These findings suggest that the synaptic endings of corticospinal fibers probably mediate the uptake and release of D-[3H]aspartate and, therefore, may use L-glutamate and/or L-aspartate as a transmitter.     

Basic fibroblast growth factor promotes the survival and development of mesencephalic neurons in culture

Neuronotrophic effects of basic fibroblast growth factor (bFGF) have been reported for some central nervous system neurons. Here we report that also rat mesencephalic neurons are responsive to bFGF. bFGF produces a significant increase in the number of neurite-bearing cells, as well as in the degree of their fiber network. The present findings also provide the first evidence that bFGF can affect, in a concentration-dependent manner, at least two defined CNS neuronal populations, i.e., dopaminergic and gabaergic neurons. This effect was quantified by assessing the specific [3H]dopamine and [gamma-14C]aminobutyric acid uptakes with time in culture. Stimulation of uptake was more pronounced for dopaminergic neurons, suggesting a relative specificity in the actions of bFGF. These effects of bFGF were completely blocked by affinity-purified polyclonal antibodies. The possibility that bFGF plays a key role in normal nervous system development or function is discussed.     

[3H]Imipramine Labels Sites on Brain Astroglial Cells Not Related to Serotonin Uptake

Brain astroglial cells, whether from a bulk isolated preparation or in culture, have been shown to take up serotonin actively. [3H]imipramine has been proposed as a specific label for serotonin uptake sites in brain. We therefore studied the binding of [3H]imipramine to C6 astroglial cells in culture to determine if some of the binding of this radioligand in brain homogenates is actually to serotonin transporting sites on glia. [3H]Imipramine binds saturably (Bmax = 202 fmol/mg protein) and with high affinity (KD = 1.72 nM) to C6 cells. This binding is competitively inhibited by other tricyclic antidepressants. The C6 cells actively transport [3H]serotonin with a Km of 2 microM and a Vmax of 1080 fmol/10(6) cells/min. However, the pharmacological profile for inhibition of serotonin uptake does not correlate with the pharmacological profile for inhibition of [3H]imipramine binding. These results suggest that the binding of [3H]imipramine to astroglial cells is not related to their capacity for active uptake of serotonin. Further, in brain homogenates, some of the binding of [3H]imipramine may not be to neuronal uptake sites but rather may be to sites on astroglial cells.     

(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)-N-[4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of gamma-[3H]aminobutyric acid [( 3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50 = 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of [3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before [3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of [3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5-hydroxytryptamine1A receptors was inhibited by NO 328 at 5-30 microM, whereas several other receptors and uptake sites were unaffected. [3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCl. The specific binding of [3H]NO 328 was inhibited by known inhibitors of [3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of [3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl-dependent [3H]NO 328 binding followed that of synaptosomal [3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sodium Dependence of [3H]]Paroxetine Binding and 5- [3H]]Hydroxytryptamine Uptake in Rat Diencephalon

The sodium dependence of binding of [3H]-paroxetine, a selective serotonin uptake inhibitor, to the serotonin transporter in rat diencephalon was studied in both brain membranes and tissue sections and compared with that of 5-[3H]hydroxytryptamine ([3H]5-HT) uptake by synaptosomes from the same region. Binding of [3H]-paroxetine in both the membranes and sections displayed clear sodium dependence until a plateau occurring at 60 nM NaCl, the EC50 for sodium being 8 and 25 mM, respectively. The affinity (1/KD) of [3H]paroxetine binding was a simple hyperbolic function of sodium concentration. In contrast, the density of [3H]paroxetine sites was not affected by external Na+ concentration. The uptake of [3H]5-HT showed a similar pattern of sodium dependence with an EC50 for Na+ of 25 mM. Both the affinity (1/Km) and the rate (Vmax) of [3H]5-HT uptake were dependent on external [Na+] with sodium-dependence curves fitting a rectangular hyperbola. The kinetic analysis of results indicates that one sodium ion is required for the binding of [3H]paroxetine as well as for the binding and translocation of each [3H]5-HT molecule. The results concur with a single-site model of the sodium-dependent serotonin transporter with common or overlapping domains for 5-HT and 5-HT uptake inhibitors.     

Release of γ-[3H]Aminobutyric Acid from Rat Olfactory Bulb and Substantia Nigra: Differential Modulation by Glutamic Acid

We have studied the glutamate modulation of gamma-[3H]aminobutyric acid ([3H]GABA) release from GABAergic dendrites of the external plexiform layer of the olfactory bulb and from GABAergic axons of the substantia nigra. In the olfactory bulb, [3H]GABA release was induced by high K+ and kainate, and not by aspartate and glutamate alone. However, when the tissue was conditioned by a previous K+ depolarization, glutamate and aspartate caused [3H]GABA release. The effect of glutamate was significantly enhanced when the GABA uptake mechanism was blocked by nipecotic acid. N-Methyl-D-aspartate and quisqualate did not cause [3H]GABA release under the same conditions. The acidic amino acid receptor antagonist 2-amino-4-phosphonobutyric acid and the N-methyl-D-aspartate receptor antagonist 2-amino-5-phosphonovaleric acid significantly inhibited the K+-glutamate- and the kainate-induced [3H]GABA release. Mg2+ (5 mM), which blocks the N-methyl-D-aspartate receptors, significantly inhibited the K+-glutamate-induced but not the kainic acid-induced [3H]GABA release. The K+-glutamate-stimulated release, but not the K+-stimulated [3H]GABA release, was strongly inhibited by Na+-free solutions or by 300 nM tetrodotoxin. Apparently the glutamate-induced release of [3H]GABA occurs through an interneuron because it is dependent on the presence of nerve conduction. In the substantia nigra no [3H]GABA release was elicited by any of the glutamate agonists tested. The present results clearly differentiate between the effects of glutamate on the release of [3H]GABA from the substantia nigra and from the olfactory bulb.(ABSTRACT TRUNCATED AT 250 WORDS)     

An Excitant Amino Acid Projection from the Medial Prefrontal Cortex to the Anterior Part of Nucleus Accumbens in the Rat

High-affinity uptake of neurotransmitter substrates in synaptosome-containing homogenates and tissue concentrations of amino acids were examined in subcortical areas 5-6 days after bilateral N-methyl-D-aspartate lesions confined to rat medial prefrontal cortex. D-[3H]Aspartate (32% of control) and [3H] gamma-aminobutyric acid ( [3H]GABA) (60% of control) uptakes were significantly reduced in medial prefrontal cortex, whereas [3H]choline (110% of control) uptake was unchanged, suggesting the production of axon-sparing lesions. The uptake of D-[3H]aspartate (76% of control), but not of [3H]GABA or [3H]choline, was significantly reduced in nucleus accumbens, with no concomitant reduction in amino acid concentrations. When examined in serial coronal sections, reduced D-[3H]aspartate uptake was confined to the most anterior 500 micron of nucleus accumbens (67% of contralateral sample). No significant reductions of uptake or amino acid concentrations were observed in caudate putamen or ventral tegmental area. These results suggest a role for glutamate or aspartate as neurotransmitters in projections from medial prefrontal cortex to anterior nucleus accumbens. Medial prefrontal cortex may represent the major excitatory cortical input to the nucleus accumbens.     

Guanosine Enhances Glutamate Transport Capacity in Brain Cortical Slices

1. The effect of guanosine on L-[3H] glutamate uptake was investigated in brain cortical slices within physio-pathological range of glutamate(1-1000 microM). In these conditions, glutamate uptake was significantly enhanced in slices treated with 100 microM guanosine only at 100 and 300 microM glutamate (44 and 52%, respectively). 2. Evaluation of kinetic parameters showed that guanosine affected significantly only uptake Vmax (23%). 3. The guanosine withdrawal did not abolish its significant effect on glutamate uptake when 100 or 300 microM glutamate were used (an increase of 66 and 35%, respectively). 4. These results support the hypothesis of a protective role for guanosine during excitotoxic conditions when glutamate levels are enhanced (e.g. brain ischemia and seizures), possibly by activating glutamate uptake. Moreover, our results may contribute to understand the antiexcitotoxic mechanism of guanosine on glutamate transport, giving new information concerning its mechanism of action.     

γ-Aminobutyric Acid Function in the Rat Striatum Is Under the Double Influence of Nigrostriatal Dopaminergic and Thalamostriatal Inputs: Two Modes of Regulation?

Glutamic acid decarboxylase (GAD), gamma-[3H]-aminobutyric acid [( 3H]GABA) high-affinity uptake into synaptosomes, and endogenous GABA content were measured in the rat striatum 2-3 weeks following 6-hydroxydopamine injection in the ipsilateral substantia nigra to destroy the nigrostriatal dopaminergic pathway and after kainic acid injection into the centromedial-parafascicular complex of the ipsilateral thalamus to lesion the thalamostriatal input. Both lesions resulted in apparent GAD increase concomitant with a decreased [3H]GABA uptake into striatal synaptosomes. GABA content was increased selectively following the dopaminergic lesion. Kinetic analysis of the uptake process for [3H]GABA showed selectively a decreased Vmax following the dopaminergic lesion; in animals with thalamic lesion, however, the change only concerned the Km, which showed a decreased affinity of the transport sites for [3H]GABA. Determination of Km and Vmax for GAD action on its substrate glutamic acid showed an increased affinity of GAD for glutamic acid in the case of the dopaminergic lesion without any change in Vmax, whereas the thalamic lesion resulted in GAD increase concomitant with a selective increase in Vmax. These data suggest that striatal GABA neurons are under the influence of nigrostriatal dopaminergic neurons which may reduce the GABA turnover, whereas the exact nature of the powerful control also revealed on these neurons following thalamic lesion remains to be determined. Both lesions induced adaptive neurochemical responses of striatal GABA neurons, possibly reflecting in the case of the dopaminergic deprivation an increased GABA turnover.     

β-N-Oxalylamino-l-Alanine: Action on High-Affinity Transport of Neurotransmitters in Rat Brain and Spinal Cord Synaptosomes

beta-N-Oxalylamino-L-alanine (BOAA) is a dicarboxylic diamino acid present in Lathyrus sativus (chickling pea). Excessive oral intake of this legume in remote areas of the world causes humans and animals to develop a type of spastic paraparesis known as lathyrism. BOAA is one of several neuroactive glutamate analogs reported to stimulate excitatory receptors and, in high concentrations, cause neuronal vacuolation and necrosis. The present study investigates the action of BOAA in vitro on CNS high-affinity transport systems for glutamate, gamma-aminobutyric acid (GABA), aspartate, glycine, and choline and in the activity of glutamate decarboxylase (GAD), the rate-limiting enzyme in the decarboxylation of glutamate to GABA. Crude synaptosomal fractions (P2) from rat brain and spinal cord were used for all studies. [3H]Aspartate transport in brain and spinal cord synaptosomes was reduced as a function of BOAA concentration, with reductions to 40 and 30% of control values, respectively, after 15-min preincubation with 1 mM BOAA. Under similar conditions, transport of [3H]glutamate was reduced to 74% (brain) and 60% (spinal cord) of control values. High-affinity transport of [3H]GABA, [3H]glycine, and [3H]choline, and the enzyme activity of GAD, were unaffected by 1 mM BOAA. While these data are consistent with the excitotoxic (convulsant) activity of BOAA, their relationship to the pathogenesis of lathyrism is unknown.     

Tryptamine, a Substrate for the Serotonin Transporter in Human Platelets, Modifies the Dissociation Kinetics of [3H]Imipramine Binding: Possible Allosteric Interaction

Tricyclic antidepressants and nontricyclic serotonin (5-hydroxytryptamine) uptake blockers monophasically inhibit [3H]imipramine binding in human platelets. Similarly, serotonin and tryptamine inhibit the binding of [3H]imipramine in the low micromolar range and with a pseudo-Hill coefficient near unity. Dissociation of the [3H]imipramine receptor complex in the presence of uptake inhibitors follows first-order kinetics with a half-life of approximately 60 min. Although serotonin and tryptamine do not decrease [3H]imipramine binding when added under equilibrium conditions, simultaneous addition of serotonin or tryptamine with serotonin uptake inhibitors decreases the rate of ligand-receptor dissociation in a concentration-dependent manner. These data suggest a common site of action for serotonin, which is the substrate of the transporter system, and of tryptamine, its nonhydroxylated analog. This hypothesis is supported by the identification of a high-affinity (Km = 0.55 microM), saturable, and temperature-dependent uptake of [3H]tryptamine in human platelets. Uptake of [3H]tryptamine was inhibited potently by imipramine and nontricyclic serotonin uptake inhibitors with a potency similar to that observed for [3H]serotonin uptake. These data support the hypothesis that in platelets, [3H]imipramine, tricyclic, and nontricyclic serotonin uptake inhibitors bind to a common recognition site that is associated with the serotonin transporter but that differs from the substrate recognition site of the carrier through which serotonin and tryptamine exert a heterotropic allosteric modulation on [3H]imipramine binding.     

Biosynthesis of NAAG by an enzyme-mediated process in rat central nervous system neurons and glia

The peptide transmitter N-acetylaspartylglutamate (NAAG) is present in millimolar concentrations in mammalian spinal cord. Data from the rat peripheral nervous system suggest that this peptide is synthesized enzymatically, a process that would be unique for mammalian neuropeptides. To test this hypothesis in the mammalian CNS, rat spinal cords were acutely isolated and used to study the incorporation of radiolabeled amino acids into NAAG. Consistent with the action of a NAAG synthetase, inhibition of protein synthesis did not affect radiolabel incorporation into NAAG. Depolarization of spinal cords stimulated incorporation of radiolabel. Biosynthesis of NAAG by cortical astrocytes in cell culture was demonstrated by tracing incorporation of [3H]-glutamate by astrocytes. In the first test of the hypothesis that NAA is an immediate precursor in NAAG biosynthesis, [3H]-NAA was incorporated into NAAG by isolated spinal cords and by cell cultures of cortical astrocytes. Data from cerebellar neurons and glia in primary culture confirmed the predominance of neuronal synthesis and glial uptake of NAA, leading to the hypothesis that while neurons synthesize NAA for NAAG biosynthesis, glia may take it up from the extracellular space. However, cortical astrocytes in serum-free low-density cell culture incorporated [3H]-aspartate into NAAG, a result indicating that under some conditions these cells may also synthesize NAA. Pre-incubation of isolated spinal cords and cultures of rat cortical astrocytes with unlabeled NAA increased [3H]-glutamate incorporation into NAAG. In contrast, [3H]-glutamine incorporation in spinal cord was not stimulated by unlabeled NAA. These results are consistent with the glutamate-glutamine cycle greatly favoring uptake of glutamine into neurons and glutamate by glia and suggest that NAA availability may be rate-limiting in the synthesis of NAAG by glia under some conditions.     

21-Aminosteroids Interact with the Dopamine Transporter to Protect Against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 microM. Non-specific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U-78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of gamma-[3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 microM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP(+)-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.