(R)-N-[4,4-Bis(3-Methyl-2-Thienyl)but-3-en-1-yl]Nipecotic Acid Binds with High Affinity to the Brain γ-Aminobutyric Acid Uptake Carrier

(R)-N-4,4-Bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid (NO 328) has previously been shown to be a potent anticonvulsant in both mice and rats. Here, we report that NO 328 is a potent inhibitor of gamma-3H]aminobutyric acid ( 3H]GABA) uptake in a rat forebrain synaptosomal preparation (IC50 = 67 nM) and in primary cultures of neurons and astrocytes. Inhibition of 3H]GABA uptake by NO 328 is apparently of a mixed type when NO 328 is preincubated before 3H]GABA uptake; the inhibition is apparently competitive without preincubation. NO 328 itself is not a substrate for the GABA uptake carrier, but NO 328 is a selective inhibitor of 3H]GABA uptake. Binding to benzodiazepine receptors, histamine H1 receptors, and 5-hydroxytryptamine1A receptors was inhibited by NO 328 at 5-30 microM, whereas several other receptors and uptake sites were unaffected. 3H]NO 328 showed saturable and reversible binding to rat brain membranes in the presence of NaCl. The specific binding of 3H]NO 328 was inhibited by known inhibitors of 3H]GABA uptake; GABA and the cyclic amino acid GABA uptake inhibitors were, however, less potent than expected. This indicates that the binding site is not identical to, but rather overlapping with, the GABA recognition site of the uptake carrier. The affinity constant for binding of 3H]NO 328 is 18 nM, and the Bmax is 669 pmol/g of original rat forebrain tissue. The regional distribution of NaCl-dependent 3H]NO 328 binding followed that of synaptosomal 3H]GABA uptake.(ABSTRACT TRUNCATED AT 250 WORDS)