THE JOURNAL OF PHARMACOLOGY ANt) EXPERIMENTAL THERAPEUTICS

RYAN, LESLIE D. AND ROBERT RosKosKI, JR.: Net uptake of y-aminobutyric acid by a high affinity synaptosomal transport system. J. Pharmacol. Exp. Ther. 200: 285-291, 1977. Reuptake of y-aminobutyric acid (GABA) by a high affinity transport system in nerve endings in the central nervous system is thought to terminate the action of this postulated neurotransmitter. This hypothesis has been challenged since the demonstration of exchange between synaptosomal and exogenous GABA (G. Levi and M. Raiteri, Nature 250: 735, 1974). In our studies, rat cortical synaptosomes were incubated (25#{176}C) in various media containing 10 M ‘4C-GABA. After the synaptosomes were removed by centrifugation, ‘4C and total GABA (fluorometric assay) in the resulting supernatant were measured. Uptake of labeled GAB A, detected by a decrease in medium radioactivity, is Na’F and K’ -dependent. Net GABA uptake, however, does not parallel ‘4C-GABA translocation. Exchange accounts for 20 to 70% of radiolabeled GABA accumulation depending upon the experimental conditions. On the other hand, GABA-deficient synaptosomes (prepared by treatment with 56 mM KC1 and 1 mM CaCl2) show equivalent net and radiolabeled GABA uptake in Ringer’s solution containing 1 to 4 mM KC1 and 60 to 150 mM NaCl (average 4.6 nmol of GABA accumulated per mg of synaptosomal protein). Net and ‘4C-GABA uptake by GABAdeficient synaptosomes are identical at various pH values (6.0-8.5), synaptosomal protein concentrations (0.4-3.5 mg/ml) and temperatures (5-37#{176}C). Although GABA homoexchange may contribute significantly to radiolabel accumulation by synaptosomes containing higher GABA levels (9.5-9.9 nmol/mg), homoexchange is limited in GABA-depleted synaptosomes. Our results are consistent with the proposal that presynaptic GABA capture by a high affinity system in vivo may terminate the action of this neuroactive amino acid. y-Amino butynic acid (GABA) is a postulated inhibitory neurotransmitter in the vertebrate central nervous system and an established neurotransmitter at the crustacean neuromuscular junction (Krnjevi#{233}, 1970). Nerve terminal prepReceived for publication June 7, 1976. ‘This work was supported by U.S. Public Health Service Grant NS-11310 and a Training Grant GM550 to the University of Iowa. ’ arations (synaptosomes) accumulate radiolabeled GABA by two classes of transport systems (Levi and Raiteri, 1974; Weinstein et a! ., 1965; Martin, 1973). Two similar classes of transport systems occur for other neuroactive amino acids (Logan and Snyder, 1971; Iversen and Johnston, 1971). The low affinity transport system, which is temperatureand Na’ ’-independent, is associated with both neuroactive and inactive amino acids (Logan and Snyder, 1971). The high affinity transport system, which is Na’ and temperature-dependent, is 286 RYAN AND ROSKOSKI Vol. 200 Synaptosome preparation. Male Sprague-Dawley rats (150-200 g) were decapitated and cerebral corassociated with the neuroactive amino acids and GABA. It has been suggested that the high affinity systems are specifically involved with the termination of neurotransmitter action by uptake (Iversen, 1971). Before this can be concluded for GABA (or other transmitter agents) it must be demonstrated that the high affinity system will mediate net concentrative trans-