Elsevier

Brain Research

Volume 490, Issue 2, 26 June 1989, Pages 320-331
Brain Research

Uptake of γ-aminobutyric acid andl-glutamic acid by synaptosomes from postmortem human cerebral cortex: multiple sites, sodium dependence and effect of tissue preparation

https://doi.org/10.1016/0006-8993(89)90249-7Get rights and content

Abstract

The uptake of γ-aminobutyric acid (GABA) andl-glutamic acid by synaptosomes prepared from frozen postmortem human brain was shown to be affected via distinct high and low affinity sites. At approximately 17 h postmortem delay, the kinetic parameters for GABA uptake were: high affinity site, Km 7.1 ± 2.5 μM, Vmax 18.7 ± 4.8nmol·min−1 per 100 mg protein; low affinity site, Km 2 ± 1mM, Vmax 425 ± 250nmol·min−1 per 100 mg protein (means±S.E.M., n = 13). Kinetic parameters forl-glutamate uptake were: high affinity site, Km 7.5 ± 1.0 μM, Vmax 85 ± 8nmol·min−1 per 100 mg protein; low affinity site, Km 1.8 ± 1.2mM, Vmax 780 ± 175nmol·min−1 per 100 mg protein (n = 11). A detailed kinetic analysis of high affinity GABA uptake was performed over a range of sodium ion concentrations. The results were consistent with a coupling ratio of one Na+ ion to one GABA molecule; a similar results was found with rat brain synaptosomes. However, rat and human synaptosomes differed in the degree to which the substrate affinity of the high affinity GABA uptake site varied with decreasing Na+ ion concentration. High affinity GABA uptake was markedly affected by the method used to freeze and divide the tissue, but did not vary greatly in different cortical regions. There was some decline of high affinity GABA uptake activity with postmortem delay, apparently due to a loss of sites rather than a change in site affinity.

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      Our work and that of several other groups has shown that intact, metabolically active synaptosomes can be prepared from human brain obtained at autopsy that has been frozen and thawed; electron microscopic analysis showed that the preparations are morphologically comparable to those from fresh tissue (Hardy and Dodd, 1983). We optimized the preparation procedure (Dodd et al., 1986) and showed that the synaptosomes exhibit functions such as the uptake of transmitters against concentration gradients with appropriate kinetics (Dodd et al., 1989; Hardy et al., 1984) and stimulus-coupled Ca2+-dependent exocytotic transmitter release (Kuo and Dodd, 2011). Hahn et al. (2009) show electron micrographs of synaptosomes isolated from frozen human autopsy tissue together with immunoblots that demonstrate the enrichment of pre- and post-synaptic proteins including synaptophysin and PSD-95.

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