Search of a l-glutamate receptor related to modulation of neurotransmission in the rat corpus striatum

Abstract

The specific binding of l-[(3)H]glutamic acid to crude membrane preparations obtained from rat striatum was studied with the aid of radioligand binding techniques. Saturation isotherms and binding of 40 nM l-[(3)H]glutamate in the presence of increasing concentrations of unlabeled l-glutamate revealed an anomalous kinetic pattern which suggested the presence of three populations of l-[(3)H]glutamate affinity binding sites in fresh striatal membranes. The presence of the three binding sites became more evident in frozen membranes and in membranes prepared from striatal slices previously subjected to K(+)-depolarization; in addition, these experimental manipulations increased the specific binding of l-[(3)H]glutamate. A number of glutamate analogs were tested with the aim to discriminate among the three binding sites. Of the analogs tested, d-aspartate displaced membrane-bound l-[(3)H]glutamate from only two of the three binding sites. This finding permitted to investigate the characteristics of the d-aspartate-insensitive affinity site, independently to the other sites, by just including 1 mM d-aspartate in the binding assays. Specific binding of l-[(3)H]glutamate to the d-aspartate-insensitive site occurred in a reversible manner. A saturation curve with sigmoidal characteristic was generated when fresh, frozen and depolarized membranes were incubated with increasing concentrations of l-[(3)H]glutamate. Hill plots revealed slopes close or higher than 2 in all these membrane preparations. K(D), and Hill slopes values were found not to differ significantly among the different membrane preparations used. However, frozen membranes as well as membranes obtained from striatal slices previously depolarized by high K(+) (55 mM), showed a significant increase in B(max) as compared to fresh and non-depolarized membranes, respectively. Only l-glutamate, quisqualate and l-glutamate diethylester were relatively potent in inhibiting the specific binding of l-[(3)H]glutamate. l-Aspartate and N- methyl- d,l -aspartate competed partially and with relatively less potency whereas d-glutamate and kainate, even when tested at 0.5 mM, produced only a slight displacement of membrane-bound radioligand. The characteristics and pharmacological specificity of this binding site correlates very well with previous observations from us (Rudolph and Bustos, 1983; Rudolph et al., 1983) when studying the effects that l-glutamate and structural analogs produce upon depolarization-evoked release of [(3)H]dopamine from rat striatal slices. The proposal is made that the d-aspartate-insensitive binding site, reported in this work, might be related to a quisqualate-type receptor which becomes predominantly exposed following depolarization and might exert then a modulation of neuronal transmission in the rat striatum.