[3H]Dopamine Labeling of D3Dopaminergic Sites in Human, Rat, and Calf Brain

Abstract

The binding of [3H]dopamine to brain regions of calf, rat and human was investigated. The calf caudate contained the highest density of [3H]dopamine binding sites, with a Bmax [maximum bound concentration] value of 185 fmol/mg protein, whereas rat and human striatum contained 1/3 this number of sites. The Kd values for [3H]dopamine in all tissues were 2-3 nM. Dopaminergic catecholamines (dopamine, apomorphine, 6,7-dihydroxy-2-aminotetralin [ADTN] and N-propylnorapomorphine) inhibited the binding of [3H]dopamine in all 3 species, at low concentrations, with IC50 [median inhibitory concentration] values of 1.5-6 nM. Neuroleptics inhibited the binding at high concentrations (with IC50 values of 200-40,000 nM). The [3H]dopamine binding sites were saturable, heat-labile and detectable only in dopamine-rich brain regions; these sites differed from D2 dopamine sites (labeled by [3H]butyrophenone neuroleptics), and from D1 dopamine sites (labeled by [3H]thioxanthene neuroleptics) associated with the dopamine-stimulated adenylate cyclase. These high-affinity [3H]dopamine binding sites are called D3 sites. [3H]Apomorphine and [3H]ADTN also appeared to label D3 sites. These ligands were less selective than [3H]dopamine, and labeled sites other than D3 as well. Assay conditions were important in determining the parameters of [3H]dopamine binding. The optimum conditions for selective labeling of the D3 dopaminergic sites, using [3H]dopamine, required the presence of EDTA and ascorbate.