Specific binding of L-[3H]-glutamic acid to rat substantia nigra synaptic membranes

The specific binding of L-[3H]-glutamic acid (GLU) was investigated in synaptic membranes from rat substantia nigra. L-[3H]-GLU binding to the membrane preparations occurred in a reversible and saturable way. The specific binding was stimulated by the presence of CaCl2 and was reduced by freezing and thawing the membranes. Scatchard analysis of the saturation isotherms yielded a non-linear plot suggesting that the binding reaction does not occur through a simple bimolecular association. Assuming non-interacting binding sites, a high (KD1, 139 nM; Bmax1, 3.5 pmoles/mg protein) and a low (KD2, 667 nM; Bmax2, 15.1 pmoles/mg protein) affinity L-[3H]-GLU binding site were obtained. The kinetics of dissociation of bound L-[3H]-GLU was biphasic; the respective dissociation rate constant (k-1) being 0.20 min-1 and 0.013 min-1. A series of amino acid receptor agonists and antagonists were tested as inhibitors of L-[3H]-GLU specific binding. Quisqualic acid, L-GLU and D-alpha-aminoadipate (D-alpha-AA) were the most potent inhibitors. DL-2-amino-4-phosphonobutyrate (APB), N-Methyl-D-aspartate (NMDA) and D-GLU were moderate inhibitors, whereas diaminopimelic acid (DAPA) and glutamate diethyl ester (GDEE) exhibited the lowest relative potency. Kainic acid (KA), gamma-aminobutyric acid (GABA) and bicuculline were not able to modify at any concentration used the specific binding of L-[3H]-GLU. These data demonstrate the presence of specific GLU binding sites in synaptic structures at substantia nigra level and support the idea that excitatory amino acids may play a role in synaptic transmission in this brain region.