Abstract

Metabotropic glutamate receptors (mGluRs) are G protein-linked receptors that operate through the formation of different second messengers. Utilizing quantitative autoradiographic techniques, we have characterized [3H]glutamate binding to mGluRs in discrete regions of adult rat brain. [3H]Glutamate binding, in the presence of high concentrations of alpha-amino-3-hydroxymethyl-4-isoxazolepropionic acid (10 microM), N-methyl-D-aspartate (100 microM), and 2.5 mM calcium chloride (CaCl2), was saturable. Scatchard plots were linear in all regions examined and revealed similar affinity constants of about 500 nM. The largest number of sites was found in the outer cerebral cortical layers (10 pmol/mg of protein). [3H]Glutamate binding was displaced by quisqualate, trans-1-amino-1,3-cyclopentane dicarboxylic acid (t-ACPD) (racemic mixture), and (1S,3R)-ACPD but not by (1R,3S)-ACPD. The guanine nucleotide analogue guanosine-5'-O-(3-thio) triphosphate (100 microM) reduced the binding by affecting the affinity but not the total number of sites, as predicted for G protein-coupled receptor sites. Quisqualate displacement curves were always biphasic and resolved two binding sites, with Ki values in the low nanomolar (15 nM) and micromolar (63 microM) ranges. (1S,3R)-ACPD displaced [3H]glutamate binding both in the absence and in the presence of 2.5 microM quisqualate, suggesting that both high and low affinity quisqualate sites are linked to mGluRs. (1S,3R)-ACPD competition curves were broad (Hill coefficient = 0.73) but monophasic under both conditions, with Ki values in the micromolar range (14-116 microM), suggesting that (1S,3R)-ACPD acts on the two quisqualate sites with similar apparent affinities. The regional distributions of the two sites were different. The highest levels of the high affinity quisqualate binding site were found in the cerebellar molecular layer. The highest levels of the low affinity quisqualate binding sites were found in the outer cerebral cortex. The pharmacological profile and regional distribution suggest that the high and low affinity quisqualate-sensitive components of [3H]glutamate binding sites might correspond to the mGluR1/mGluR5 and mGluR2/mGluR3 subgroups of cloned mGluRs, respectively.