Sodium‐dependent Efflux and Exchange of Gaba in Synaptosomes

Simon, Jay R.; Martin, David L.; Kroll, Marie

doi:10.1111/j.1471-4159.1974.tb10750.x

Cited by 92 publications

(39 citation statements)

References 25 publications

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“…Direct measurements ofthe concentrations of GABA with an amino acid analyzer confirmed our suggestion that net uptake of [I4C]GABA was primarily responsible for the observed accu-'c2000- (26), who reported an increase in the concentration ofthis amino acid in the supernatant when preloaded synaptosomes were diluted in a medium containing unlabeled GABA. The inability of these authors to show net uptake of GABA may be attributed either to the quality of their synaptosomal preparations or to the methods used to separate the synaptosomes from the medium or to a combination ofboth.…”

Section: Resultssupporting

confidence: 61%

Net uptake of gamma-aminobutyric acid by a high-affinity system of rat brain synaptosomes.

Pastuszko¹,

Wilson²,

Erecińska³

1981

Proc. Natl. Acad. Sci. U.S.A.

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Rat brain synaptosomes isolated on. discontin- 'using different and more sensitive methods for measuring GABA concentration. In this paper we show that synaptosomes carry out rapid net uptake of GABA and that this net-uptake occurs through a high-affinity system. Homoexchange is shown to account for less than 10% ofthe measured [14C]GABA uptake. MATERIALS AND METHODSMale Sprague-Dawley rats (180-220 g) were used throughout the study. Synaptosomes were isolated from the forebrains of rats essentially as described (24). The final synaptosomal pellet was suspended in Krebs-=Henseleit Hepes buffer (140 mM NaCl/5 mM KCV1.3 mM MgSO4/5 mM NaHCOj2.5 mM CaCl2/1 mM Tris phosphate/10 mM Tris Hepes, pH 7.4), containing 10 mM glucose.Synaptosomes, suspended at about 6 mg of protein per ml, were preincubated for 6 min with constant shaking at 250C. The uptake of GABA was measured in three different experimental systems after dilution of the incubation mixture in the Krebs-Henseleit Hepes buffer to a final protein concentration of 1-2 mg/ml (unless indicated otherwise). (i) The synaptosomal suspensions were supplemented with 2.4 AM [14C]GABA (Amersham: specific activity, 224 Ci/mol; 1 -Ci = 3.7 X 10'°b ecquerels), and the radioactivity of the supernatant and of the pellet was measured after rapid centrifugation (Beckman microfuge) of the aliquots of the incubation mixture through a layer of silicone oil (specific gravity 1.03) at appropriate time intervals. (ii) The synaptosomal suspension was supplemented with 2 ,uM nonradioactive GABA, and aliquots were centrifuged through silicone oil into a 3% (wt/vol) solution of perchloric acid. The supernatants were removed, acidified with perchloric acid (3% final concentration), and centrifuged. The perchloric acid-soluble fractions (i.e., synaptosomal pellets and postsynaptosomal supernatants) were diluted 1:2 in 50 mM citrate buffer (pH 2.0), and concentrations of GABA were measured in a Perkin-Elmer amino acid analyzer. (iii) Synaptosomes (5-6 mf of protein per ml) were preincubated for 6 min with 2 juM [ 4C]GABA and then diluted 1:10 into KrebsHenseleit Hepes buffer containing various concentrations of nonradioactive GABA (0-10 AM). Radioactivity was then measured in the supernatants and in the pellets at appropriate time intervals after separation of the synaptosomes from the medium by rapid centrifugation through silicone oil. Radioactivity was determined in a Searle Delta 300 liquid scintillation counter with aqueous counting scintillant (Amersham).Protein was measured by a biuret reaction, with bovine serum albumin as the standard. RESULTS AND DISCUSSIONRadioactive GABA was rapidly taken up by suspensions of synaptosomes, and the initial rate ofuptake was proportional to the protein concentration in the range 0.5-2 mg of protein per ml (Fig. 1). From the initial~linearrates ofGABA uptake at various external concentrations of this amino acid, kinetic constants were calculated by a double-reciprocal plot analysis of the results (Fig. 2). The'Km was found to be 6.25 ,AM a...

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Section: Resultssupporting

confidence: 61%

Net uptake of gamma-aminobutyric acid by a high-affinity system of rat brain synaptosomes.

Pastuszko¹,

Wilson²,

Erecińska³

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…As in ganglia, BALA is an effective substrate for the glial carrier and causes an accelerated efflux of [3H1-GABA from olfactory cortical slices (Brown & Scholfield, unpublished observations). L-2,4-Diaminobutyric acid (DABA) is a good substrate for the synaptosomal (presumed neural) carrier (Iversen & Johnston, 1971;Simon & Martin, 1973;Kelly, Dick & Schon, 1975), and accelerates GABA-efflux from brain synaptosomes (de Belleroche & Bradford, 1972;Simon et al, 1974). BABA has also been reported to accelerate the release of GABA from synaptosome preparations (de Belleroche & Bradford, 1972).…”

Section: Discussionmentioning

confidence: 99%

“…(b) Addition of carrier-substrates has been observed to accelerate the efflux of GABA from central nervous system preparations (Cutler, Hammerstad, Cornick & Murray, 1971;de Belleroche & Bradford, 1972;Simon, Martin & Kroll, 1974;Levi & Raiteri, 1974;Raiteri, Federico, Colethi & Levi 1975). When measured under comparable experimental conditions the rate coefficients for resting and substratestimulated efflux of [3H]-GABA from isolated mammalian olfactory cortex slices are equal to or exceed those for the corresponding release from ganglia (Brown & Scholfield, unpublished observations).…”

Section: Discussionmentioning

confidence: 99%

Indirect Effects of Amino‐acids on Sympathetic Ganglion Cells Mediated Through the Release of Γ‐aminobutyric Acid From Glial Cells

Bowery

Brown

Collins

et al. 1976

British J Pharmacology

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All experiments were performed on rat isolated desheathed superior cervical ganglia maintained in Krebs solution containing amino‐oxyacetic acid (10 μm) at 25°C. Influx rates of γ‐amino‐n‐butyric acid (GABA) were measured by incubating ganglia in 0.5 μm [3H]‐GABA for 30 minutes. Influx was inhibited by 50% on adding 14.3 μm unlabelled GABA, 59.2 μm β‐alanine (BALA) or 424 μm β‐amino‐n‐butyric acid (BABA). Efflux of [3H]‐GABA into non‐radioactive solution superfused over ganglia previously incubated for 60 min in 1 μm [3H]‐GABA was measured. The mean resting efflux rate coefficient (k) was 0.64 ±0.05 × 10−3 min−1. Addition of high concentrations of unlabelled GABA, BABA or BALA to the superfusing solution increased k by (maximally) 3.6–4.3 times; half‐maximal increases occurred at the following concentrations: GABA, 16 μm; BALA, 85 μm; BABA, 606 μm. Replacement of external Na+ with Li+ or TRIS increased the resting value of k and inhibited acceleration by external amino acids. Prior incubation in 1 μm [3H]‐GABA with 1 mm unlabelled GABA increased resting k 1.5 times;, but did not alter the peak rate coefficient produced by external amino acids. Neuronal depolarization produced by the amino acids was measured with surface electrodes. Preincubation in 1 mm GABA for 60 min potentiated low‐amplitude responses to BALA or BABA but not those to GABA or 3‐aminopropanesulphonic acid (a potent agonist with low affinity for the GABA carrier). Omission of external Na+ reduced responses to BABA but increased those to GABA. Incubation in 1 mm GABA for 60 min (as required to potentiate BABA or BALA actions) increased the amount of GABA in the tissue from 0.21 to 0.73 mmol/kg wet weight. Autoradiographs in which labelled GABA was used indicated that uptake into neuroglial cells was responsible for this accumulation. It is suggested that: (i) BALA and BABA are substrates for the inward GABA carrier responsible for GABA entry into ganglionic glial cells; (ii) they accelerate efflux by inhibiting carrier‐mediated re‐accumulation of effluent GABA by the glial cells; (iii) interstitial GABA concentrations are thereby increased to a level capable of depolarizing adjacent neurones; and (iv) this, rather than direct GABA‐receptor activation, accounts for the depolarization produced by low concentrations of BALA and BABA. Potentiation of their depolarizing action after pre‐incubation in 1 mm GABA is suggested to result from the increased amount of intracellular GABA available for release, and is quantitatively compatible with this increase; inhibition in Na+‐free solution is due to their inability to inhibit re‐accumulation of GABA under these conditions. A model for the action of carrier substrates is described in an Appendix. Calculations based thereon yield increments in interstitial GABA concentration in the presence of carrier substrates compatible with those determined experimentally (up to 1 μm at rest or 3.4 μm after pre‐incubation in GABA).

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“…For example, for 'y-aminobutyric acid (GABA), no evidence for an exocytotic release has been provided, and the mechanism of reuptake is still under discussion (8)(9)(10)(11)(12)(13)(14)(15). In fact, several recent studies in vitro have shown that GABA transport consists essentially of a 1:1 homoexchange process in nerve endings incubated in standard media in the presence of low concentrations of the amino acid (9)(10)(11)(12)(13)(14). This finding does not seem to fit with the high-affinity reuptake theory for inactivation of synaptically released GABA (4,6,16) and raises the question of the functional significance of a transport system that does not result in a net translocation of the substrate in one or the other direction.…”

mentioning

confidence: 99%

Modulation of γ-aminobutyric acid transport in nerve endings: Role of extracellular γ-aminobutyric acid and of cationic fluxes

Levi

Raiteri

1978

Proc. Natl. Acad. Sci. U.S.A.

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The aim of the present study was to elucidate the possible functional significance of 'y-aminobutyric acid (GABA) homoexchange at nerve endings. Using synaptosomes from adult rat cerebrum, we found that a number of conditions altering cationic fluxes produced a concomitant change in the stoichiometry of GABA homoexchange, In fact, exogenous GABA (10 AM), while not causing net release of intrasynaptosomal GABA in standard conditions, triggered a large net GABA release in the presence of veratridine, Na+-K+-ATPase inhibitors, or the ionophore A23187, superimposed on that due to the various agents tested alone. This extra release was mediated by the membrane carrier, being largely inhibited by the GABA carrier-blocker L-diaminobutyric acid. The altered stoichiometry of GABA homoexchange observed under these conditions (efflux> influx) appeared to be coupled to the influx of Na+ (or of Ca2+), rather than determined by the establishment of a high intrasynaptosomal [Na+J Under conditions of reversed Na+ flux (Na+ efflux), the GABA outward/inward flux ratio was also reversed, and the stoichiometry of GABA homoexchange was in favor of net influx. The possible contribution of K+ to the effects observed is also discussed. It is concluded that the GABA transport system of nerve endings is susceptible to fine modulation by changes in cationic fluxes similar to those occurring in vivo during depolarization and repolarization. These fluxes may have a prominent role in determining the direction of net GABA transport in GABA-ergic nerve terminals of the living brain.The two essential steps of chemical neurotransmission are the depolarization-induced release of the transmitter from a presynaptic nerve ending and the rapid inactivation of the transmitter by specific biochemical mechanisms. The release of biogenic amines is generally considered to occur through an exocytotic-like process (1-5), and their inactivation mainly through specific reuptake mechanisms present in presynaptic nerve terminals (4, 6, 7). This scheme cannot be easily applied to neurotransmitter amino acids. For example, for 'y-aminobutyric acid (GABA), no evidence for an exocytotic release has been provided, and the mechanism of reuptake is still under discussion (8-15). In fact, several recent studies in vitro have shown that GABA transport consists essentially of a 1:1 homoexchange process in nerve endings incubated in standard media in the presence of low concentrations of the amino acid (9-14). This finding does not seem to fit with the high-affinity reuptake theory for inactivation of synaptically released GABA (4, 6, 16) and raises the question of the functional significance of a transport system that does not result in a net translocation of the substrate in one or the other direction.In the present study we have analyzed the relationship between GABA homoexchange and the processes of release and reuptake of the amino acid. On the basis of our results, we submit a hypothesis for GABA release and reuptake mechanisms and, in particular, we suggest...

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Sodium‐dependent Efflux and Exchange of Gaba in Synaptosomes

Cited by 92 publications

References 25 publications

Net uptake of gamma-aminobutyric acid by a high-affinity system of rat brain synaptosomes.

Net uptake of gamma-aminobutyric acid by a high-affinity system of rat brain synaptosomes.

Indirect Effects of Amino‐acids on Sympathetic Ganglion Cells Mediated Through the Release of Γ‐aminobutyric Acid From Glial Cells

Modulation of γ-aminobutyric acid transport in nerve endings: Role of extracellular γ-aminobutyric acid and of cationic fluxes

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