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A filtration binding assay using [3H]dichlorokynurenic acid to label the glycine binding site on the N-methyl-D-aspartic acid receptor has been evaluated on rat cortical membranes. This ligand binds to a single population of binding sites following mass action kinetics with a KD of 29 nM and a capacity of 5.73 pmol (mg protein)-1. The pharmacological specificity of the binding site is identical to that previously reported for this binding site using [3H]glycine as a radioligand. Agonists showed lower affinity and antagonists higher affinity when [3H]dichlorokynurenic acid was used compared with [3H]glycine. The higher affinity of [3H]dichlorokynurenic acid compared with [3H]glycine make it the more suitable compound with which to label the glycine site.

The effects of different potassium channel blockers on tritiated dopamine [( 3H]DA) release were investigated in rat striatal slices in the presence of pargyline and nomifensine (10 microM each). 4-Aminopyridine (4-AP; 10 and 30 microM) and 3,4-diaminopyridine (3,4-DAP; 30 microM) markedly increased the basal tritium outflow, whereas tetraethylammonium (TEA; 100-1000 microM) was without effect. The facilitating effect of 4-AP (10 microM) on spontaneous release was Ca(2+)- and K(+)-dependent. Moreover, the 4-AP-induced increase in spontaneous release was abolished in the presence of tetrodotoxin, indicating that voltage-dependent Na+ channels were involved in the release mechanism. 4-AP (10 and 30 microM) induced a dose-dependent decrease in K(+)-evoked [3H]DA release. This effect was confirmed with 3,4-DAP (30 microM). When striatal slices were depolarized with veratridine (5 microM), these two aminopyridines increased the evoked release of [3H]DA. TEA increased both K(+)- and veratridine-evoked [3H]DA release. These biochemical results are consistent with electrophysiological differences between the mechanism of action of aminopyridines and that of TEA.

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