Vertebrate GABA receptors

Physiologic-pharmacologic studies in vivo and with tissue cultures have revealed that synaptic GABA receptors exist in the vertebrate CNS. The GABA antagonist, bicuculline, can be used to detect synaptic GABA receptors in both the presence and absence of Na⁺, even though GABA binding to cerebral subcellular fractions occurs mainly to transport (uptake) receptors in the presence of Na⁺.     

[3H]GABA binding in the cerebellum of the reeler murine mutant

In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [³H]GABA binding have been observed. This study shows decreased affinity of the Na⁺-independent, high affinity GABA binding component of synaptosomal membranes and an increased affinity of the Na⁺-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na⁺-dependent and Na⁺-independent binding sites was not significantly altered. The decreased affinity of the Na⁺-independent GABA binding and the increased affinity of the Na⁺-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.     

Relationship between synaptosomal uptake and release of [14C]gaba, [14C]diaminobutyric acid and [14C]beta-alanine.

[¹⁴C]GABA is taken up by rat brain synaptosomes via a high affinity, Na⁺-dependent process. Subsequent addition of depolarizing levels of potassium (56.2 MM) or veratridine (100 μM) stimulates the release of synaptosomal [¹⁴C]GABA by a process which is sensitive to the external concentration of divalent cations such as Ca²⁺, Mg²⁺, and Mn²⁺. However, the relatively smaller amount of [¹⁴C]GABA taken up by synaptosomes in the absence of Na⁺ is not released from synaptosomes by Ca²⁺ -dependent, K ⁺-stimulation. [¹⁴C]DABA, a competitive inhibitor of synaptosomal uptake of GABA (Iversen & Johnson , 1971) is also taken up by synaptosomal fractions via a Na ⁺ -dependent process; and is subsequently released by Ca²⁺ -dependent, K⁺-stimulation. On the other hand, [¹⁴C]β-alanine, a purported blocker of glial uptake systems for GABA (Schon & Kelly , 1974) is a poor competitor of GABA uptake into synaptosomes. Comparatively small amounts of [¹⁴C] β-alanine are taken up by synaptosomes and no significant amount is released by Ca²⁺ -dependent, K⁺-stimulation. These data suggest that entry of [¹⁴C]GABA into a releasable pool requires external Na⁺ ions and maximal evoked release of [¹⁴C]GABA from the synaptosomal pool requires external Ca²⁺ ions. The GABA analogue, DABA, is apparently successful in entering the same or similar synaptosomal pool. The GABA analogue, β-alanine, is not. None of the compounds or conditions studied were found to simultaneously affect both uptake and release processes. Compounds which stimulated release (veratridine) or inhibited release (magnesium) were found to have minimal effect on synaptosomal uptake. Likewise compounds (DABA) or conditions (Na⁺-free medium) which inhibited uptake, had little effect on release.     

gamma-Aminobutyric acid receptors in cerebellar membranes of rat brain after a treatment with Triton X-100.

Abstract— The treatment of cerebellar membranes of rat brain with a low concentration of Triton X-100 followed by sufficient washing results in an increase of the Na⁺-independent binding of [³H]GABA and a total loss of the Na ⁺-dependent binding of [³H]GABA. The Na⁺-independent binding of [³H]GABA was more abundant in membranes of cerebellum than in membranes of other rat brain regions and mainly localized in the synaptic membrane fraction of a cerebellar homogenate. In the Triton-treated membranes, the Na⁺-independent binding of [³H]GABA was a saturable process, which could be resolved into two components, a high and a low affinity component with dissociation constants of 4.5 and 30 nm , respectively. The neurophysiological agonists, muscimol, GABA, and imidazole acetic acid, and the antagonist, bicuculline, inhibited the high affinity Na⁺-independent binding of [³H]GABA by 50% at 0.003, 0.012, 0.3 and 10 μm respectively. These data suggest that the Na⁺-independent binding of [³H]GABA in the Triton-treated cerebellar membranes represents the synaptic receptors of GABA. It is emphasized that extensive washing of the membranes after a Triton treatment is necessary in order to detect the high affinity Na⁺-independent binding of [³H]GABA.     

GABA efflux from synaptosomes: Effects of membrane potential,and external GABA and cations

Summary Presynaptic GABAergic nerve terminals accumulate -aminobutyric acid (GABA) by a sodium-dependent carrier mechanism in which two Na⁺ are co-transported with one GABA. We have examined the influence of external GABA and cations on GABA efflux from³H-GABA loaded rat brain synaptosomes, to determine whether or not the carriers can also mediate GABA efflux. We observed that, in Ca-free media (to minimize Ca-dependent evoked release), external GABA promotes GABA efflux when the medium contains Na⁺, butinhibits GABA efflux in the absence of Na⁺. The efflux of GABA into Ca-free media is stimulated by depolarization (either with veratridine or increased external K⁺). These data, and published data on the internal Na⁺ dependence of GABA efflux into Ca-free media, indicate that exiting GABA is cotransported with Na⁺. The stimulatory effect of depolarization is consistent with efflux of Na⁺ along with the uncharged GABA. The (carrier-mediated) efflux is also stimulated when the carriers cycle inward with Na⁺+GABA. The inhibitory effect of GABA in Na⁺-free media implies that GABA can bind to unloaded carriers and that the carriers loaded only with GABA cycle very slowly, if at all. Our data, and data from the literature, can be fitted to a simple model with sequential binding of solutes: external GABA binds to the carrier first, and only the free or fully-loaded (with 2Na⁺+1GABA) carriers can cycle. Other binding sequences and random binding, do not fit the experimental observations.     

The Administration of Levocabastine,a NTS2 Receptor Antagonist,Modifies Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>-ATPase Properties

Neurotensin behaves as a neuromodulator or as a neurotransmitter interacting with NTS1 and NTS2 receptors. Neurotensin in vitro inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. This effect is prevented by administration of SR 48692 (antagonist for NTS1 receptor). The administration of levocabastine (antagonist for NTS2 receptor) does not prevent Na⁺, K⁺-ATPase inhibition by neurotensin when the enzyme is assayed with ATP as substrate. Herein levocabastine effect on Na⁺, K⁺-ATPase K⁺ site was explored. For this purpose, levocabastine was administered to rats and K⁺-p-nitrophenylphosphatase (K⁺-p-NPPase) activity in synaptosomal membranes and [³H]-ouabain binding to cerebral cortex membranes were assayed in the absence (basal) and in the presence of neurotensin. Male Wistar rats were administered with levocabastine (50 μg/kg, i.p., 30 min) or the vehicle (saline solution). Synaptosomal membranes were obtained from cerebral cortex by differential and gradient centrifugation. The activity of K⁺-p-NPPase was determined in media laking or containing ATP plus NaCl. In such phosphorylating condition enzyme behaviour resembles that observed when ATP hydrolyses is recorded. In the absence of ATP plus NaCl, K⁺-p-NPPase activity was similar for levocabastine or vehicle injected (roughly 11 μmole hydrolyzed substrate per mg protein per hour). Such value remained unaltered by the presence of 3.5 × 10^?6 M neurotensin. In the phosphorylating medium, neurotensin decreased (32 %) the enzyme activity in membranes obtained from rats injected with the vehicle but failed to alter those obtained from rats injected with levocabastine. Levocabastine administration enhanced (50 %) basal [³H]-ouabain binding to cerebral cortex membranes but failed to modify neurotensin inhibitory effect on this ligand binding. It is concluded that NTS2 receptor blockade modifies the properties of neuronal Na⁺, K⁺-ATPase and that neurotensin effect on Na⁺, K⁺-ATPase involves NTS1 receptor and -at least partially- NTS2 receptor.     

Na+-independent binding of GABA to the triton X-100 treated synaptic membranes from cerebellum of rat brain

The treatment of the membranes from cerebellum of rat brain with 0.5% Triton X-100 increases both the affinity and the density of the Na⁺-independent binding sites for ³H-GABA (γ-aminobutyric acid) from the values obtained from membranes of rat brain after an extensive freezing and thawing treatment (Young $\text{et al.}$, 1976). Upon repeated washings of the Triton-treated membranes, the binding of ³H-GABA is further increased and follows biphasic kinetics which indicates two binding components having dissociation constants of 5.9 and 27 nM and densities of 1.35 and 3.9 pmole/mg protein, respectively. GABA agonist, imidazoleacetic acid, and the GABA antagonists, bicuculline and d-tubocurarine, inhibit 50% of ³H-GABA binding at 1, 47 and 85 μM concentrations (IC₅₀ values), respectively. The IC₅₀ values for these compounds are unchanged by Na⁺. Thus, the Na⁺-independent binding of ³H-GABA to the Triton-treated membranes may represent binding to the synaptic GABA receptors.     

RAPID EFFECTS OF VERATRIDINE, TETRODOTOXIN, GRAMICIDIN D, VALINOMYCIN AND NaCN ON THE NA+, K+ AND ATP CONTENTS OF SYNAPTOSOMES

Abstract— The effects of brief exposures of a number of depolarizing agents on ²⁴Na⁺ influx and on the Na⁺, K⁺ and ATP contents of synaptosomes were studied using a Millipore filtration technique to terminate the reaction. When synaptosomes were incubated in normal medium, there was a rapid influx of ²⁴Na⁺ and a gain in Na’contents; neither the ²⁴Na⁺ influx nor the Na⁺ gain were blocked by tetrodotoxin suggesting that this Na⁺ entry did not involve Na⁺-channels. Veratridine markedly increased the rate of ²⁴Na⁺ influx into synaptosomes and also increased the Na⁺ content and decreased the K⁺ content of synaptosomes within the first 10s of exposure. The normal ion contents were reversed by 1 min. The effects of veratridine on Na⁺ influx and on synaptosomal ion contents were prevented by tetrodotoxin and required Na⁺ in the medium. The ionophores gramicidin D and valinomycin also rapidly reversed the Na⁺ and K⁺ contents of synaptosomes, but these effects could not be blocked by tetrodotoxin. The reducing effect of gramicidin D on synaptosomal K⁺ content required Na’in the medium, whereas valinomycin caused a fall in the K⁺ content of synaptosomes in a Na⁺-free medium. Veratridine and gramicidin D, at concentrations known to reverse the synaptosomal ion contents, did not affect synaptosomal ATP levels. In contrast, valinomycin and NaCN caused an abrupt fall in synaptosomal ATP levels. The above findings suggest that veratridine quickly alters synaptosomal Na⁺ and K⁺ contents by opening Na ⁺-channels in the presynaptic membrane, and provide direct evidence for the existence of Na⁺-channels in synaptosomes. In contrast, gramicidin D and valinomycin appear to act independently of Na ⁺-channels, possibly by their ionophoric effects and, in the case of valinomycin, by diminishing synaptosomal ATP contents and hence diminishing Na⁺-pump activity. The rapid reversals of Na⁺ and K⁺ contents by these drugs could affect the resting membrane potentials, Na⁺-Ca²⁺ exchange across the synaptosomal membrane, and the release, synthesis and uptake of neurotransmitters by synaptosomes.     

l-serine and GABA uptake by synaptosomes during postnatal development of rat

Postnatal development changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Specific uptake of radiolabeled l-serine was examined and compared with that of radiolabeled GABA using synaptosomes-enriched fractions freshly prepared from cerebral cortex at different postnatal days from the birth to young adulthood. The preparations were incubated with 10 nM of [³H]l-serine and 10 nM of [³H]-GABA in either the presence or absence of NaCl, KCl or choline chloride, at 2 and 30 °C, for different periods up to 30 min. The uptake of [³H]l-serine was temperature dependent in synaptosomal fractions prepared from cerebral cortex of rats in postnatal days 5, 7, 13 and 21, but stronger dependence was observed in adult brain, irrespective of the presence of Na⁺, K⁺ or choline ions. At all postnatal ages studied, [³H]-GABA uptake showed a high activity in the presence of Na⁺ ions and at 30 °C. The values of K_m were 90–489 μM in l-serine uptake. However, in the uptake of GABA the values of K_m were 80–150 μM. The highest values of V_max were obtained at 5 and 21 postnatal days for both transport systems. These results indicate that the uptake of l-serine and GABA are regulated differentially during postnatal development.     

GABA FLUXES IN PRESYNAPTIC NERVE ENDINGS FROM IMMATURE RATS

Abstract— Several parameters of GABA Auxes across the synaptosomal membrane were studied using synaptosomes prepared from the brain of immature (8-day-old) rats. The following aspects of GABA carrier-mediated transport were similar in immature and mature synaptosomes: (1) magnitude of [³H]GABA accumulation; (2) GABA homoexchange in normal ionic conditions; (3) GABA homoexchange in the presence of cationic fluxes (Na⁺ and Ca²⁺ influx, K⁺ efflux) characteristic of physiological depolarization. As in adult synaptosomes (Levi & Raiteri , 1978), in these conditions the stoichiometry of GABA homoexchange was in the direction of net outward transport (efflux > influx). The essential differences between the behaviour of 8-day-old and adult synaptosomes were the following: (1) β-alanine (a glial uptake inhibitor) inhibited GABA uptake in immature synaptosomes (the inhibition being greater in crude than in purified preparations) and was without a significant effect in adult synaptosomes. DABA and ACHC (two neuronal uptake inhibitors) depressed GABA uptake more efficiently in purified than in crude immature synaptosomes, but were as effective in crude and purified nerve endings from adult animals. The data suggest a greater uptake of GABA in the‘gliosomes’contaminating the synaptosomal preparations from immature animals. (2) In immature synaptosomes prelabelled with [³H]GABA the specific radioactivity of the GABA released spontaneously or by heteroexchange (with 300 μm -OH-GABA) was the same as that present in synaptosomes, while in adult synaptosomes OH-GABA released GABA with increased specific radioactivity. The data suggest a homogeneous distribution of the [³H]GABA taken up within the endogenous GABA pool in immature, but not in mature synaptosomes. (3) In immature synaptosomes the release of GABA (radioactive and endogenous) induced by depolarization with high KC was not potentiated by Ca²⁺, unless the synaptosomes had been previously depleted of Na⁺ These data suggest that, although a Ca²⁺ sensitive pool of GABA may be present, this pool is not susceptible to being released in normal conditions, probably because the high intrasynaptosomal Na⁺ level prevents a sufficient depolarization. The possible significance of these findings in terms of functional activity of GABAergic neurotransmission in the immature brain is discussed.     

In search of synaptosomal Na+, K+-ATPase regulators

The arrival of the nerve impulse to the nerve endings leads to a series of events involving the entry of sodium and the exit of potassium. Restoration of ionic equilibria of sodium and potassium through the membrane is carried out by the sodium/potassium pump, that is the enzyme Na⁺,K⁺-ATPase. This is a particle-bound enzyme that concentrates in the nerve ending or synaptosomal membranes. The activity of Na⁺,K⁺-ATPase is essential for the maintenance of numerous reactions, as demonstrated in the isolated synaptosomes. This lends interest to the knowledge of the possible regulatory mechanisms of Na⁺,K⁺-ATPase activity in the synaptic region. The aim of this review is to summarize the results obtained in the author's laboratory, that refer to the effect of neurotransmitters and endogenous substances on Na⁺,K⁺-ATPase activity. Mention is also made of results in the field obtained in other laboratories. Evidence showing that brain Na⁺,K⁺-ATPase activity may be modified by certain neurotransmitters and insulin have been presented. The type of change produced by noradrenaline, dopamine, and serotonin on synaptosomal membrane Na⁺,K⁺-ATPase was found to depend on the presence or absence of a soluble brain fraction. The soluble brain fraction itself was able to stimulate or inhibit the enzyme, an effect that was dependent in turn on the time elapsed between preparation and use of the fraction. The filtration of soluble brain fraction through Sephadex G-50 allowed the separation of two active subfractions: peaks I and II. Peak I increased Na⁺,K⁺- and Mg²⁺-ATPases, and peak II inhibited Na⁺,K⁺-ATPase. Other membrane enzymes such as acetylcholinesterase and 5′-nucleotidase were unchanged by peaks I or II. In normotensive anesthetized rats, water and sodium excretion were not modified by peak I but were increased by peak II, thus resembling ouabain effects.³H-ouabain binding was unchanged by peak I but decreased by peak II in some areas of the CNS assayed by quantitative autoradiography and in synaptosomal membranes assayed by a filtration technique. The effects of peak I and II on Na⁺,K⁺-ATPase were reversed by catecholamines. The extent of Na⁺,K⁺-ATPase inhibition by peak II was dependent on K⁺ concentration, thus suggesting an interference with the K⁺ site of the enzyme. Peak II was able to induce the release of neurotransmitter stored in the synaptic vesicles in a way similar to ouabain. Taking into account that peak II inhibits only Na⁺,K⁺-ATPase, increases diuresis and natriuresis, blocks high affinity³H-ouabain binding, and induces neurotransmitter release, it is suggested that it contains an ouabain-like substance.     

Effects of sodium and potassium ions on the uptake of noradrenaline and its precursor tyrosine

The effects of the monovalent cations Na⁺ and K⁺ were studied on the uptake of noradrenaline and tyrosine by a crude synaptosomal fraction in vitro. Sodium ions produced opposite effects on the uptake of noradrenaline and the uptake of tyrosine viz. an increase in noradrenaline uptake and a decrease in the uptake of its precursor tyrosine. A low concentration of K⁺ stimulated the uptake of noradrenaline in the presence of Na⁺, while in the absence of Na⁺ K⁺ had no effect. However, the uptake of tyrosine could be stimulated by low K⁺ in the absence of Na⁺. Besides the increased uptake in the absence of Na⁺, a second uptake was found which was Na⁺, K⁺ activated ATPase dependent. The contribution of this uptake system to the total uptake of tyrosine was about 20%. No evidence was obtained for the involvement of a Na⁺, K⁺ activated ATPase in noradrenaline uptake. It is suggested that another ATPase might be involved in the latter uptake system.     

Agonist Induced Conformation Alteration of Neurotensin Receptor and the Mechanism Behind Na+ Inhibition of 125I-NT Binding

Abstract

In the absence of Na⁺, ¹²⁵I-Neurotensin (¹²⁵I-NT) binding to the Neurotensin receptor (NTR) produces a stable noncovalent ¹²⁵I-NT-NTR complex whose dissociation rate is extremely low even after the addition of 1#M NT, 100#M SR48692 (antagonist), 100#M GPPNHP or 100mM NaCl. Lowering the medium pH to 4.5 enhances the process (～70% in 10 minutes). Labeling by photoactivatable ¹²⁵I-Tyr³-Azo⁴-NT identifies a ～50 KD Mr band along with several other minor components. Interestingly, the labeling intensity is drastically reduced when binding is performed in the presence of Na⁺ or GPPNHP. However, a minor reduction is noticed when Na⁺ or GPPNHP is added to the medium after binding. The binding kinetics indicates that Na⁺ lowers the rate of ¹²⁵I-NT association by acting as a noncompetitive inhibitor. On the contrary, Na⁺ favors the interaction of antagonist, SR48692 by lowering the value of K⁺. GTPγ³⁵S binding to membranes in the presence of 30mM NaCI suggests that Na⁺ inhibition of ¹²⁵I-NT binding is due to the uncoupling of NTR associated G protein(s). In order to explain the entire phenomenon, a two-step, binding model has been proposed. In Step-1, interaction between NT and NTR produces a transient complex, which attains a stable state in the absence of NaCI via step-2, thereby altering the native NTR conformation. The presence of Na⁺ prevents step-2 by dissociating the transition complex.     

RELATIONSHIP BETWEEN Ca2+-DEPENDENT AND INDEPENDENT RELEASE OF [3H]GABA EVOKED BY HIGH K+, VERATRIDINE OR ELECTRICAL STIMULATION FROM RAT CORTICAL SLICES

Abstract— It has been reported that the release of GABA by high K⁺ is Ca²⁺-dependent while release induced by veratridine or electrical stimulation has been frequently found to be independent of Ca²⁺. To see the source of Ca²⁺-dependent and independent release of GABA, cortical slices which had accumulated [³H]GABA were exposed to 50 mm -K⁺ or 50 μm -veratridine for 48min. In the presence of Ca²⁺ the 2 agents released approx the same amount of [³H]GABA but tetrodotoxin (TTX) abolished release induced only by veratridine, while omission of Ca²⁺ reduced release induced only by 50mm -K⁺. Pre-exposure of the slices for 48min to 50mm -K⁺ in the presence of Ca²⁺ reduced the second release by 50mm -K⁺ by 77% and that by veratridine by 74%, suggesting that in the presence of Ca²⁺ the 2 depolarizing agents release [³H]GABA from the same pool. Pre-exposure to 50mm -K⁺ in the absence of Ca²⁺ reduced the second release by 50mm -K⁺ or by veratridine only by 37 and 27% respectively, indicating that most of the reduction in release was the result of a depletion of releasable [³H]GABA stores. The second exposure to 50mm -K⁺ in the absence of Ca²⁺ reduced the evoked release further, while exposure to veratridine in the absence of Ca²⁺, after depletion of the stores, enhanced release 2.7 times. Electrical stimulation (64 Hz, 2 ms, 40 mA, alternating polarity) during 24min in the presence of Ca” caused an initial 5-fold increase in efflux, which declined subsequently. In the absence of Ca²⁺, instead of a rapid increase, a slow but smaller increase in the efflux of [³H]GABA was found. TTX almost completely abolished the electrically evoked increase in release. Pre-treatment with 50mm -K⁺ reduced the electrically evoked release by 94% but electrical stimulation in the absence of Ca²⁺ after depletion of releasable stores doubled this release. Results suggest that in the presence of Ca²⁺, high K⁺, veratridine and electrical stimulation release [³H]GABA from the same Ca²⁺-dependent store, but in the absence of Ca²⁺ veratridine and electrical stimulation enhance the release from a Ca²⁺-independent store, probably as a result of an increased influx of Na⁺.     

DEVELOPMENTAL TRANSITIONS IN UPTAKE OF AMINO ACIDS BY SYNAPTOSOMAL FRACTIONS ISOLATED FROM RAT CEREBRAL CORTEX

Developmental changes in mechanisms of synaptosomal amino acid transport have been studied in rat cerebral cortex. Well-defined changes over an age continuum could be observed in both the rates of amino acid accumulation and the effects of Na⁺ on the accumulation. The uptakes of five amino acids (threonine, serine and valine in Na⁺-free medium, aspartic acid and proline in Na⁺-containing medium) increased progressively with the age of the animal, whereas the uptakes of leucine and arginine (in Na⁺-free medium) decreased steadily. The uptake of serine or threonine by synaptosomal fractions prepared from newborn rats was markedly dependent on the presence of Na⁺in the incubation media. Na⁺exerted progressively less effect on the accumulation process with continuing postnatal development and to some extent inhibited uptake by fractions obtained from rats older than about 15 days. Na⁺significantly enhanced the accumulation of glycine in fractions from newborn and adult rats, but had only a slight effect in fractions prepared from 12 to 17-day old rats. A detailed study of the accumulation of glycine indicated that the synaptosomal transport of this amino acid proceeded by two independent systems, one of which was totally dependent on external Na⁺and the and adult animals than in fractions from 12 to 17-day-old rats, wheras the Na⁺-independent system was most active during this latter period of development. The decline in the Na⁺-independent accumulation of glycine from about the 15th day to adulthood was characterized by a decrease in the V_max. and an increase in the K_m.     

Nigericin-induced Na+/H+ and K+/H+ exchange in synaptosomes: Effect on [3H]GABA release

The effect of the putative K⁺/H⁺ ionophore, nigericin on the internal Na⁺ concentration ([Na _i ]), the internal pH (pH _i ), the internal Ca²⁺ concentration ([Ca _i ]) and the baseline release of the neurotransmitter, GABA was investigated in Na⁺-binding benzofuran isophtalate acetoxymethyl ester (SBFIAM), 2′,7′-bis(carboxyethyl)-5(6) carboxyfluorescein acetoxymethyl ester (BCECF-AM), fura-2 and [³H]GABA loaded synaptosomes, respectively. In the presence of Na⁺ at a physiological concentration (147 mM), nigericin (0.5 μM) elevates [Na _i ] from 20 to 50 mM, increases thepH _i , 0.16 pH units, elevates four fold the [Ca _i ] at expense of external Ca²⁺ and markedly increases (more than five fold) the release of [³H]GABA. In the absence of a Na⁺ concentration gradient (i.e. when the external Na⁺ concentration equals the [Na _i ]), the same concentration (0.5 μM) of nigericin causes the opposite effect on thepH _i (acidifies the synaptosomal interior), does not modify the [Na _i ] and is practically unable to elevate the [Ca _i ] or to increase [³H]GABA release. Only with higher concentrations of nigericin than 0.5 μM the ionophore is able to elevate the [Ca _i ] and to increase the release of [³H]GABA under the conditions in which the net Na⁺ movements are eliminated. These results clearly show that under physiological conditions (147 mM external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore, and all its effects are triggered by the entrance of Na⁺ in exchange for H⁺ through the ionophore itself. Nigericin behaves as a K⁺/H⁺ ionophore in synaptosomes just when the net Na⁺ movements are eliminated (i.e. under conditions in which the external and the internal Na⁺ concentrations are equal). In summary care must be taken when using the putative K⁺/H⁺ ionophore nigericin as an experimental tool in synaptosomes, as under standard conditions (i.e. in the presence of high external Na⁺) nigericin behaves as a Na⁺/H⁺ ionophore.     

Development of Na+-stimulated glucose oxidation in synaptosomes

Abstract— d -[¹⁴C]Glucose was oxidized to ¹⁴CO₂ by synaptosomes prepared from adult rat brain. Added Na⁺ stimulated glucose oxidation by 179%, but K⁺ and choline were without effect. Li⁺ stimulated glucose oxidation by 64%. Ouabain largely prevented the stimulatory effect of Na⁺ on glucose oxidation but had no effect in the absence of Na⁺. 2-Deoxy-d -glucose competitively inhibited glucose oxidation differently at two different ranges of deoxyglucose and glucose concentrations; the K_i was 0.54 and 16 mm , respectively. In the presence or absence of Na⁺ 2,4-DNP-stimulated glucose oxidation by 370% while iodoacetate inhibited glucose oxidation by 87–95%. There was a striking increase in Na⁺-stimulated glucose oxidation with development but glucose oxidation in the absence of Na⁺ did not change dramatically with age. Taken together the data suggest synaptosomes exhibit coupled respiration which can be modulated by Na⁺. In addition, the appearance of Na⁺-stimulated glucose oxidation with maturation probably is linked to the development of Na⁺-K⁺-ATPase acitivity in the synaptosomal membrane.     

COMPARISON OF THE BINDING’ OF β-ALANINE AND y-AMINOBUTYRIC ACID IN SYNAPTOSOMAL-MITOCHONDRIAL FRACTIONS OF RAT BRAIN1

Abstract— Na⁺-dependent ‘binding’ of β-alanine and GABA was examined with synaptosomal-mitochondrial fractions of rat brain incubated for 10 min at 0°C. GABA was bound to a much greater extent than β-alanine to particles of cerebral cortex, whole cerebellum and brain stem. For cerebral cortex, the binding capacity (B_max) for GABA was about 18 limes greater than that for β-alanine. and the affinity of the particles for GABA was about 2′ times greater than for β-alanine. The order of potency of GABA binding to brain regions was cerebral cortex > cerebellum > brain stem, whereas that for β-alanine was the reverse. If the binding of β-alanine is taken to indicate the glial component of the Na⁺-dependent binding process for GABA, then most of the GABA was bound to neuronal elements under the conditions employed.     

Adenosinetriphosphatase and nucleotide metabolism in synaptosomes of rat brain

—In the presence of synaptosomes prepared from rat brain, only ATP, dATP and ADP but not dADP were active as substrates of phosphatase (ATP phosphohydrolase; EC 3.6.1 4) in the presence of 150mm-Na⁺ and 20mm-K⁺. An active adenylate kinase (ATP:AMP phosphotransferase; EC 2.7.4.3.) was demonstrated in the synaptosomal fractions by means of paper chromatography, paper electrophoresis and enzymic reactions, so that the high activity with ADP as substrate could represent an activity of an ATPase. Apparently dADP was not a substrate for the kinase; no dATP was formed when dADP was incubated with the synaptosomal fraction in the presence of Na⁺, K⁺ and Mg²⁺. Small amounts of P₁ were liberated with dADP, IDP, GDP or CDP, but not UDP, as substrates, but none was produced in the presence of mononucleotides. The adenine-deoxyribose bond, but not the adenine-ribose bond, was hydrolysed upon the addition of 5% (w/v) TCA to the reaction mixture. The K_M for the hydrolysis of ATP but not ITP, in the presence of Mg²⁺, or of Na⁺, K⁺ and Mg²⁺, was lower for the synaptosomal ATPase than for the microsomal ATPase, and the values for V_max for synaptosomal ATPase were higher. The activation increment was generally higher for the synaptosomal ATPase and no distinct differences in the properties of the enzyme from either particulate fractions were observed. Mg²⁺ could be partially replaced by Mn²⁺ in the synaptosomal ATPase system, but there was little Na⁺-K⁺-activation observed in the presence of the latter. The effects of ouabain and of homogenization under various conditions suggested localization of the K⁺-sensitive site of the ATPase on the surface of the synaptosomal membrane. Activity of the Na⁺-K⁺-Mg²⁺ ATPase increased after freezing and thawing of the sonicated, sucrose or tris-treated preparations but decreased considerably in the synaptosomes treated with 001 m-deoxycholate. Activity of the Mg²⁺ ATPase in the latter preparation showed little change.     

Glutamate,aspartate, and γ-aminobutyrate transport by membrane vesicles prepared from rat brain

To prepare membrane vesicles, nerve terminal preparations (synaptosomes) isolated from rat cerebral cortex were first subjected to hypotonic lysis. After collecting the membranes contained in this fraction by centrifugation, membrane vesicles were then reconstituted during incubation in a potassium salt solution at 37 °C. The transport of glutamate, aspartate, or γ-aminobutyric acid (GABA) was measured by transferring vesicles to 10 vol of 0.1 m NaCl solution containing the radioactive substrate. Transport was temperature dependent and exhibited saturation kinetics with an apparent K_m of 2.5 μm. The rates and extent of l-glutamate and l-aspartate uptake were equivalent and were greater than those for GABA. Valinomycin increased the rate of uptake of each of these substances suggesting a role for an electrogenic component in transport. Consonant with this notion, external K⁺ and Rb⁺ decreased uptake of all three compounds. External thiocyanate also increases the rate of glutamate, aspartate, and GABA transport. Uptake of these neuroactive amino acids was absolutely dependent on external Na⁺; no other monovalent cation tested substitutes for it. Gramicidin D and nigericin inhibit glutamate transport by abolishing both the Na⁺ and K⁺ gradients. Monensin inhibits uptake by selectively dissipating the Na⁺ gradient. For both glutamate and GABA transport, the Na⁺ and K⁺ gradients are synergistic and not additive.