Transport of G AB A, β-Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high-affinity carrier-mediated transport systems for [³H]GABA ( K _t=1.9 μM, V = 1.8 pmol/10⁶ cells/min) and [³H]glutamate ( K _t= 10 μM, V = 7.9 pmol/10⁶ cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [³H]β-alanine (which is taken up specifically by the glial GABA transport system) and [³H]glutamate were concentrated by glial-enriched fractions. However [³H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high-affinity [³H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high-affinity glutamate transport is a property of glial cells but not granule neurons.     

An Analysis of γ-Aminobutyrate Receptors and Uptake by Isolated Purkinje Cells

Abstract: An isolated fraction of Purkinje perikarya was prepared. This fraction had [³H]GABA receptor binding and [³H]muscimol binding analogous to that reported for heterogeneous cerebellar membranes. The finding of two binding components with each ligand suggests that these components do not represent two binding sites, one presynaptic and the other postsynaptic, since both are clearly seen on purified Purkinje cell bodies. Subcellular fractionation indicates that synaptic endings and plasma membranes are enriched in GABA receptors compared with intracellular organelles. Purkinje cell bodies were also found to possess a high-affinity transport system for GABA, which was sensitive to inhibition by diaminobutyric acid (DABA) but not by β-alanine. They showed no evidence of homoexchange (the movement of label without net transport). This supports our suggestion that homoexchange is an artifact of synaptic particle formation.     

THE SELECTIVE NEURONAL UPTAKE AND RELEASE OF [3H]DL-2,4-DIAMINOBUTYRIC ACID BY RAT CEREBRAL CORTEX

Abstract— At 25°C the accumulation of [³H] dl -2,4-diaminobutyric acid (DABA) into small rat cortical slices was linear with time and a tissue: medium ratio of 35:1 was attained after 60 min. At 37°C the uptake was no longer linear and the tissue: medium ratio at 60 min was 66:1. Uptake was unaffected by the addition of 10 μ m -AOAA and dependent on the presence of Na⁺ in the incubation media. The uptake was shown to have a high affinity component with a K _m of 20.7 μ m and a V _max of 28.6 nmol/g/min. IC50's for the inhibition of [³H]DABA uptake by dl -DABA, l -DABA and GABA were 80, 40 and 17 μ m respectively. Two m m β -alanine, however, caused less than 13% inhibition of [³H]DABA uptake. Electron microscopic autoradiographs showed the [³H]DABA to be accumulated by 22% of the identifiable nerve terminals and, after 14 days exposure, the density of silver grains over nerve terminals was 36–38 times higher than that over the rest of the electron micrograph. On the other hand, [³H]DABA was not taken up into rat sensory ganglia and light level autoradiography showed the small amount of [³H]DABA accumulated by the ganglia to be evenly distributed throughout the tissue. Both electrical stimulation for 30 s and exposure of the tissue to a medium containing 47 m m -K⁺ for 2 min caused a marked increase in the efflux of [³H]DABA from the tissue. Both these effects were abolished by a reduction in Ca²⁺ concentration and an increase in the Mg²⁺ concentration of the superfusing medium. These results suggest that l -DABA acts as a 'false transmitter' for the neuronal uptake, storage and release of GABA.     

STUDIES OF THE UPTAKE AND RELEASE OF [3H]β-ALANINE BY FROG SPINAL SLICES

Abstract— [³H]β-Alanine was accumulated by frog spinal cord slices by two transport components with estimated K_m values of 31 M ('high-affinity') and 11 HIM ('low affinity') respectively. The high affinity uptake exhibited sodium ion and energy dependence, temperature sensitivity, had a very low V_max (10.4 nmol/g/min) compared to GABA and glycine, was competitively inhibited by GABA (K_t 2 M), and was significantly reduced by the presence of glycine and of taurine in the incubating medium.
When slices preloaded with [³H]β-alanine were superfused with medium containing depolarizing concentrations of potassium ions, there was a small, but consistent, increase in [³H]β-alanine efflux: 1.4 times prestimulation rates in 40 mM potassium. When the superfusate was altered by omission of calcium and addition of concentrations of magnesium (10 mm), manganese (1 mM), and cobalt (1 mM) ions sufficient to block reflex transmission in the isolated in vitro frog cord, the potassium-evoked release was not blocked. Release was decreased by lanthanum ions (1 mM). Release of [³H]GABA and [³H]glycine in parallel experiments was inhibited by magnesium, manganese, cobalt and lanthanum. Veratridine significantly increased the release of [³H]GABA and [³H]glycine but not of [³H]β-alanine.
These observations demonstrate the non-specificity of β-alanine uptake and the unconventional nature of the calcium-dependence of β-alanine release and therefore do not lend support to the hypothesis that β-alanine functions as a neurotransmitter in frog spinal cord.     

THE EFFECT OF ELECTRICAL STIMULATION AND HIGH POTASSIUM CONCENTRATIONS ON THE EFFLUX OF [3H] γ-AMINOBUTYRIC ACID FROM BRAIN SLICES

Abstract— Brain slices were incubated with [³H]GABA in a medium containing aminooxyacetic acid to prevent metabolism of [³H]GABA by GABA-glutamate transaminase. The slices, which rapidly accumulated radioactivity, were then continuously perfused and the efflux of [³H]GABA from the tissue was measured. The spontaneous efflux of [³H]GABA consisted of an initial rapid phase followed by a much slower release of [³[H]GABA. After 40 min perfusion 90 per cent of the radioactivity remained in the tissue.
The slices were depolarized by electrical stimulation or by perfusion with a medium containing a high potassium concentration (40 mM). These procedures caused a striking increase in the efflux of [³H]GABA. The increased efflux produced by potassium, but not that produced by electrical stimulation, was dependent on calcium ions in the medium. The effect of electrical stimulation on [³H]GABA release was considerably reduced by a raised concentration (10 mM) of magnesium in the medium.
High potassium concentrations and electrical stimulation did not cause an increase in the efflux of [¹⁴C]urea, L-[³H]leucine or [¹⁴C]α-amino-isobutyric acid from brain slices. These results are consistent with the suggestion that GABA may be an inhibitory transmitter in the cerebral cortex.     

RELEASE OF [3H]GAMMA-AMINOBUTYRIC ACID FROM GLIAL CELLS IN RAT DORSAL ROOT GANGLIA.

Abstract— Desheathed rat dorsal root ganglia were incubated in a medium containing amino-oxyacetic acid and [³H]GABA. Under these conditions, [³H]GABA is taken up exclusively by the satellite glial cells in the ganglia. Efflux of [³H]GABA from the tissue was measured after passing the ganglia through a series of wash solutions. The spontaneous efflux of radioactivity, mostly [³H]GABA, was more rapid in the absence of amino-oxyacetic acid in the incubation and wash media.
Raising the potassium concentration in the wash media caused an increase in the efflux of [³H]GABA. This increase was sigmoidally related to the potassium concentration in the wash media, reaching a maximum at 64 m m -K⁺. The releasing effect of K⁺ was inhibited by removing calcium from the media. Reducing the calcium and raising the magnesium concentration in the wash solutions inhibited the increased efflux of [³H]GABA due to 64 m m -K⁺ by 48 per cent, while 5 mM-La³⁺ and diphenylhydantoin (0·005 and 0·5 m m ) had no effect on this increase.
Only a small increase in the efflux of [¹⁴C]glutamate was produced by 64 m m -K⁺ and it had no effect upon the effluxes of [³H]glycine, [³H]alanine or [³H]leucine. The efflux of lactate dehydrogenase was similarly unaffected by 64 mM-K⁺. The results suggest that glial cells in spinal ganglia can respond to depolarizing concentrations of potassium by releasing GABA in a calcium-dependent process.     

Nitric Oxide-Induced [3H]GABA Release from Cerebral Cortical Neurons Is Mediated by Peroxynitrite

Abstract: The functional significance of peroxynitrite in the release of [³H]GABA induced by nitric oxide (NO) liberated from NO generators was investigated using cerebral cortical neurons in primary culture. NO generators such as sodium nitroprusside (SNP) and S -nitroso- N -acetylpenicillamine (SNAP) increased [³H]GABA release in a dose-dependent manner. These increases in [³H]GABA release were significantly inhibited by hemoglobin, indicating that those NO generators evoke the release of [³H]GABA by the formation of NO. Two types of superoxide scavengers, Cu²⁺/Zn²⁺ superoxide dismutase and ceruloplasmin, significantly reduced the increase in [³H]GABA release induced by both SNP and SNAP, which assumes that NO requires superoxide to induce [³H]GABA release from the neurons. In addition, synthesized peroxynitrite induced a dose-dependent increase in [³H]GABA release from the neurons. These results indicate that NO-induced [³H]GABA release is mediated by peroxynitrite formed by the reaction of NO with superoxide.     

EFFECTS OF AMINO-OXYACETIC ACID ON [3H]GABA UPTAKE BY RAT BRAIN SLICES

Abstract— The effect of amino-oxyacetic acid on the uptake of [³H]GABA by rat brain slices was studied. When added simultaneously with [³H]GABA, amino-oxyacetic acid had no significant effect on [³H]GABA uptake. However, preincubation of brain slices with amino-oxyacetic acid prior to addition of [³H]GABA produced inhibition of uptake, which increased with longer duration of preincubation. The inhibitory effect of amino-oxyacetic acid was maximal at 2 mM concentration and concentrations sufficient to inhibit significantly GABA:glutamate transaminase (10^--⁶ M) had no effect on [³H]GABA uptake. D-Cycloserine and β-hydrazino-propionic acid also inhibited [³H]GABA uptake, but the amounts required were considerably in excess of those needed to inhibit GABA:glutamate transaminase. 4-Deoxypyridoxine inhibited [³H]GABA uptake, whether given in vivo or in vitro , and the inhibitory effect of amino-oxyacetic acid was reversed with pyridoxine. GABA transport appears to be dependent on pyridoxal phosphate and interference with this function of the vitamin is suggested as the basis for the inhibitory effect of amino-oxyacetic acid on [³H]GABA uptake.     

Measurement of the Membrane Potential of Isolated Nerve Terminals by the Lipophilic Cation [3H]Triphenylmethylphosphonium Bromide

Abstract: The lipophilic cation [³H]triphenylrnethylphosphonium bromide ([³H]TPMP⁺) was investigated as a measure of the membrane potential of synaptosomes. Conditions under which [³H]TPMP⁺ achieved an equilibrium distribution were tested. The toxicity of TPMP has been studied relative to its inhibitory effects on [³H]y-aminobutyric acid ([³H]GABA) transport. In some experiments the distribution of ⁸⁶RbZ⁺ and [³H]TPMP⁺ was changed upon incubation in the presence of elevated levels of K⁺, ouabain, or KCN, or at 0°C in a way that would be expected from the membrane potential. In normal incubation conditions a membrane potential of ∼−60 mv was calculated.     

Phosphatidylserine Enhancement of [3H]γ-Aminobutyric Acid Uptake by Rat Whole Brain Synaptosomes

Abstract: [³H] γ -Aminobutyric acid ([³H]GABA) binding to purified lipids was examined in an organic solvent-aqueous partition system. In addition, the [³H]GABA binding capacity in the partition system was compared with the capacity of lipids to alter sodium-dependent [³H]GABA uptake into synaptosomes isolated from rat whole brains. [³H]GABA was found to bind to all of the lipids studied in the organic solvent-aqueous partition system [phosphatidic acid (PA), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), gangliosides, and sulfatide], although PS exhibited the greatest binding capacity. [³H]GABA uptake into synaptosomes was enhanced by PS (48.0%) but was not altered by any other lipid. PS enhancement of [³H]GABA uptake required the presence of sodium and was blocked by nipecotic acid (10 μ m ). These results suggest that PS may play a role in the sodium-dependent GABA reuptake process in the presynaptic nerve end.     

Increase in Chloride-Dependent l-Glutamate Transport Activity in Synaptic Membrane After In Vitro Ischemic Treatment

Abstract: The effect of energy failure on Cl⁻-dependent l -glutamate ( l -Glu) transport was examined with an in vitro preparation. Rat brain slices were incubated in low oxygen and glucose-deprived medium (in vitro ischemia), and a synaptic membrane fraction was prepared from the slices. Cl⁻-dependent l -[³H]Glu uptake into vesicles increased about twofold after 20 min of in vitro ischemia. The increased l -[³H]Glu uptake was inhibited by l -Glu, dl -2-amino-4-phosphonobutyrate, l -homocysteic acid, l -cystine, 4,4'-diisothiocyano-2,2'-disulfonic stilbene, and removal of Cl⁻. Uptakes of Na⁺-dependent l -[³H]-Glu, [³H]GABA, and [³H]taurine were not changed by the in vitro ischemia. In vitro ischemia increased the V _max value without affecting the K _m value. The increased l -[³H]Glu uptake by in vitro ischemia was reduced by subsequent incubation in a normoxic glucose-containing solution. ATP content in brain slices decreased to <10% of control values by in vitro ischemia for 10 min. The decrease in ATP content was restored by subsequent incubation in normoxic glucose-containing solution. Treatment with veratrine, 2,4-dinitrophenol, carbonyl cyanide m -chlorophenylhydrazone, and NaCN in normoxic conditions increased l -[³H]Glu uptake with a concomitant decrease in ATP content in slices. These results suggest that Cl⁻-dependent l -Glu transport activity in synaptic membranes increases in ischemia- or hypoxia-induced brain energy failures.     

Classical Noncholinergic Neurotransmitters and the Vesicular Transport System for Acetylcholine

Abstract: The acetylcholine transporter exhibits such low affinity and specificity for acetylchoiine that it appeared possible it could fail to select against other neurotransmitters. Potential interactions of classical noncholinergic neurotransmitters with cholinergic synaptic vesicles purified from electric organ were studied. No active transport of [³H]serotonin, [³H]noradrenaline, or [³H]glutamate occurred. Serotonin, noradrenaline, and N -acetylaspartyl glutamate inhibited active transport of [³H]acetylcholine by the vesicles. Dopamine previously had been shown to inhibit transport. Glutamate and γ-aminobutyric acid were shown here not to inhibit active transport of [³H]-acetylcholine. Noradrenaline was competitive with respect to [³H]acetylcholine in this effect. Serotonin, noradrenaline, and dopamine inhibited binding of [³H]vesamicol to the vesicles, and dopamine was a competitive inhibitor of the binding of this allosteric ligand of the acetylcholine transporter. The results indicate that the acetylcholine transporter does not transport any other classical neurotransmitter, but serotonin, noradrenaline, and dopamine bind to the acetylcholine site.     

Differential Regulation by Guanine Nucleotides of Opiate Agonist and Antagonist Receptor Interactions

Abstract: Guanine nucleotides differentiate binding of tritium-labeled agonists and antagonists to rat brain membranes. In the absence of sodium, GTP (50 μM) decreased binding of [³H]-labeled agonists by 20–60% and [³H]-labeled antagonists by 0–20%. In the presence of 100 mM-NaCl, GTP had no effect on antagonist binding, but decreased agonist binding by 60–95%. GMP was less potent than either GTP or GDP in decreasing agonist binding. GTP (50 μM) reduced high-affinity [³H]dihydromorphine sites by 52% and low-affinity sites by 55%. Without sodium, GTP reduced high-affinity [³H]-naloxone sites by 36%; in the presence of 100 mM-NaCl, GTP had no effect on either high- or low-affinity [³H]naloxone sites. GTP increased the association rate of [³H]dihydromorphine twofold and the dissociation rate by fourfold, while having no effect on association or dissociation rates of the antagonist [³H]diprenorphine. The affinities of uniabeled antagonists in inhibiting [³H]-diprenorphine binding were not affected by GTP or sodium, but the affinities of agonists were reduced 40- 120-fold, with met- and leu-enkephalin affinities reduced by the greatest degree. GTP and sodium lowered [³H]dihydromorphine binding in an additive fashion, while divalent cations, especially manganese, reversed the effects of GTP on [³H]-labeled agonist binding by stimulating membrane-bound phosphatases that hydrolyze GTP to GMP and guanosine. These results suggest that by affecting binding of agonists, but not antagonists, GTP may regulate opiate receptor interactions with their physiological effectors.     

Neurochemical Studies of the Mesolimbic Dopaminergic Pathway: Somatodendritic Mechanisms and GABAergic Neurones in the Rat Ventral Tegmentum

Abstract: The rat ventral tegmentum (containing dendrites and somata of mesolimbic neurones) contained 1.3 μg/g of dopamine, which was reduced to 40% of the control level by reserpine. Slices of ventral tegmentum were able to accumulate and release (elevated potassium or protoveratrine A) exogenous [³H]dopamine. In parallel studies the uptake mechanism in ventral tegmentum was shown to be virtually identical to the nerve terminal uptake of [³H]dopamine by slices of nucleus accumbens. The release of [³H]dopamine was indistinguishable from that observed in substantia nigra, where there is substantial evidence for dendritic mechanisms. Basal adenylate cyclase activity was present, but dopamine-stimulated activity was not detected. A high GABA concentration (7.7 μmol/g) was present in ventral tegmentum, in conjunction with an uptake and a release mechanism for [³H]GABA. GABA and muscimol elicited a small, reproducible efflux of [³H]dopamine, but an interaction between dopamine and [³H]GABA efflux was not observed. The results are in accord with transmitter roles for dopamine and GABA in the somatoden-dritic area of mesolimbic dopaminergic neurons.     

Enhancement of diazepam and gamma-aminobutyric acid binding by (+)etomidate and pentobarbital

Abstract: (+) Etomidate and pentobarbital enhance [³H]diazepam and [³H]γ-aminobutyric acid ([³H]GABA) binding to cerebral cortex membranes. Both (+)etomidate and pentobarbital increase the affinity of [³H]diazepam for its binding sites. In contrast, they increase the B _max of both the high- and low-affinity GABA receptor sites. The enhancement of [³H]diazepam and [³H]GABA by (+)etomidate and pentobarbital is blocked by GABA antagonists. These results indicate that hypnotic drugs such as (+)etomidate and pentobarbital, which are not structurally related, modulate diazepam and GABA binding sites via similar mechanisms.     

SUBCELLULAR DISTRIBUTION OF ENDOGENOUS AND [3H] γ-AMINOBUTYRIC ACID IN RAT CEREBRAL CORTEX

Abstract— Slices of rat cerebral cortex were labelled by incubation with [³H]γ-aminobutyric acid (GABA) and homogenized in isotonic sucrose. The subcellular distributions of endogenous GAB A, [³H]GABA and glutamate decarboxylase (GAD) were studied by density gradient centrifugation. The subcellular distributions of the labelled and endogenous amino acid were remarkably similar, indicating that [³H]GABA is taken up into the endogenous GABA pool. About 40 per cent of both endogenous and [³H]GABA were recovered in particles which were tentatively identified as synaptosomes from their equilibrium density and sensitivity to osmotic shock. In slices labelled with [³H]GABA and [¹⁴C]α-aminoisobutyric (AIB) acid, significantly more [³H]GABA was recovered in paniculate fractions than [¹⁴C]AIB. About 80 per cent of the enzyme GAD was also recovered in the same particle fractions which contained [³H]GABA and endogenous GABA. Evidence is presented which suggests that a loss of particle-bound GABA occurs during subcellular fractionation procedures.     

4-Aminobutyraldehyde as a Substance Convertible In Vivo to GABA

Abstract: [2,3-³H]4-Aminobutyraldehyde ([³H]ABAL) was injected subcutaneously into mice, which were sacrificed at various intervals following injection. [³H]γ-Aminobutyric acid ([³H]GABA) synthesized in vivo from [³H]ABAL was extracted from the brains, separated, and quantitated. The results showed that in the brain, injected [³H]ABAL was rapidly transformed into [³H]GABA. [³H]ABAL may penetrate the blood-brain barrier into the central nervous system and then be oxidized to [³H]GABA.     

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.     

Comparison of the Kinetics of [3H]Diazepam and [3H]Flunitrazepam Binding to Cortical Synaptosomal Membranes

Abstract: [³H]Diazepam and [³H]flunitrazepam ([³H]FNP) binding to washed and frozen synaptosomal membranes from rat cerebral cortex were compared. In Tris-citrate buffer, γ -aminobutyric acid (GABA) and NaCl both increased [³H]diazepam binding more than [³H]FNP binding. GABA and pentobarbital both enhanced this effect of NaCl. Because of the extremely rapid dissociation of [³H]diazepam in the absence of NaCl and GABA, the B_max (maximal binding capacity) was smaller by the filtration assay than by the centrifugation assay. [³H]FNP, which dissociates more slowly, had the same B_max in both assays. [³H]Diazepam association had two components, and was faster than [³H]FNP association. [³H]Diazepam dissociation, which also had two components, was faster than that of [³H]FNP, and also had a greater fraction of rapidly dissociating species. [³H]FNP dissociation was similar when initiated by diazepam, flunitrazepam, clonazepam, or Ro15-1788, which is a benzodiazepine antagonist. [³H]Diazepam dissociation with Ro15-1788, flunitrazepam, or clonazepam was slower than with diazepam. GABA and NaCl, but not pentobarbital, increased the percentage of slowly dissociating species. This effect of NaCl was potentiated by GABA and pentobarbital. The results support the cyclic model of benzodiazepine receptors existing in two interconvertible conformations, and suggest that, distinct from their binding affinity, some ligands (like flunitrazepam) are better than others (like diazepam) in inducing the conversion of the receptor to the higher-affinity state.     

GABA UPTAKE IN RAT CENTRAL NERVOUS SYSTEM: COMPARISON OF UPTAKE IN SLICES AND HOMOGENATES AND THE EFFECTS OF SOME INHIBITORS

Abstract— Fifty-two substances were tested as inhibitors of the uptake of [³H]GABA in slices of rat cerebral cortex. Among GABA analogues tested, only the 2-fluoro, 3-hydroxy and 2-amino compounds had affinities for the uptake mechanism comparable to that of GABA. [³H]GABA uptake was also potently inhibited by p -chloromercuriphenylsulphonate, N -ethylmaleimide, chlorpromazine and haloperidol. No inhibitors were found to act in a competitive manner with respect to GABA. [³H]GABA uptake was also examined in homogenates of cerebral cortex and other regions of CNS. There was a rapid uptake of [³H]GABA into particles when homogenate samples were incubated with the labelled amino acid; this uptake had similar kinetic properties and inhibitor sensitivity to that observed in slices of intact tissue. Density gradient centrifugation experiments indicated that the particles responsible for the uptake of [³H]GABA in homogenates were probably synaptosomes. Uptake of [³H]GABA also occurred in slices and homogenates of rat spinal cord, and evidence was obtained by the simultaneous labelling of homogenates with [¹⁴C]glycine and [³H]GABA that these two amino acids were taken up by different nerve terminals in this region.