Quantitative autoradiographic characterization of the binding of [3H]tiagabine (NNC 05-328) to the GABA uptake carrier.

The kinetic properties and regional distribution of [3H]tiagabine ([3,4-3H]N-[4,4-bis(3-methyl-2-thienyl)but-3-en-1-yl]nipecotic acid) binding to the central GABA uptake carrier was examined in the rat brain using quantitative receptor autoradiography. In slide mounted sections of frontal cortex, the binding of [3H]tiagabine was saturable, reversible and sodium dependent. The kinetics of association and dissociation of [3H]tiagabine were monophasic, and Scatchard transformation of saturation isotherms resulted in a linear plot with a Kd = 58 +/- 7 nM and a Bmax = 58.9 +/- 0.9 pmol/mg protein. The autoradiographic distribution of [3H]tiagabine binding sites in rat brain was heterogeneously distributed. The highest density of [3H]tiagabine binding sites was present in the cerebral cortex, mammillary body, globus pallidus, substantia nigra pars reticulata, hippocampus, dorsal raphé, superior colliculus (outer layer), and cerebellum. The distribution of GABA uptake sites, as measured by [3H]tiagabine binding, in the rat brain is highly consistent with the organization of GABAergic terminals and cell bodies. The present investigation characterized the use of [3H]tiagabine as a novel radioligand for the GABA uptake carrier using quantitative receptor autoradiography. [3H]Tiagabine has several major advantages over the currently utilized radioligand for the GABA uptake carrier [3H]nipecotic acid, in that [3H]tiagabine has an increased affinity, specificity, and is not transported intracellularly via the GABA uptake carrier. These data suggest that [3H]tiagabine represents a novel and highly useful ligand for studying the GABA uptake carrier using quantitative receptor autoradiography.