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

Abstract

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...

Activation of the Voltage‐Sensitive Sodium Channel by a β‐Scorpion Toxin in Rat Brain Nerve‐Ending Particles

“…4a. From the time courses of uptake measured at different concentrations of 13H]GABA, a K , of 6.5 pA4 and a V,,, of 400 pmoliminlmg of protein were calculated, in good agreement with values measured by other authors in rat synaptosomes (Martin, 1973;Hitzemann and Loh, 1978;Pastuszko et al, 1981). The addition of 0.5 p M p-ScTx did not modify the K , value but reduced V,,,, to 210 pmoliminimg of protein.…”

“…It is well known that neurotoxins that activate voltage-dependent N a + channels modify the uptake and the release of neurotransmitters in synaptosomes (Blaustein, 1975). GABA, one of the primary inhibitory neurotransmitters in the mammalian CNS, is actively taken up by nerve endings (Martin, 1973;Hitzemann and Loh, 1978;Pastuszko et al, 1981), and this specific uptake is inhibited by veratridine (Blaustein and King, 1976) or a-ScTx (Romey et al, 1976). In the present study, we have confirmed that the inhibition is total with veratridine and almost total with the a-ScTx AaH 11.…”

Release of Glutamate, Aspartate, and γ‐Aminobutyric Acid from Isolated Nerve Terminals

“…These results were based on the observation that external GABA promotes the release of [3H]GABA (Levi and Raiteri, 1978). However, this is due to exchange rather than net efflux ; indeed, on addition of external GABA, there is a net uptake of HPLC-detected GABA, rather than a release (Roskoski, 1978;Pastuszko et al, 1981). Figure 2A illustrates the distinctive kinetics of KCIinduced L-glutamate release from synaptosomes by Ca2+-dependent and -independent mechanisms as monitored by continuous fluorometric assay.…”

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

“…In order to fulfill these functions, these nerve terminal transport systems must be able to mediate net transmitter uptake. Indeed, Roskoski (1978; and see Ryan & Roskoski, 1977;Pastuszko, Wilson & Erecinska, 1981) has shown that synaptosomes can engage in net accumulation of 7-aminobutyric acid (GABA) and L-glutamate.…”