Quantitative autoradiographic studies of dopamine D3 receptors in rat cerebellum using [125I]S(-)5-OH-PIPAT.

Recently, [125I]S(-)5-OH-PIPAT (5-hydroxy-2-(N-n-propyl-N-3'-iodo-2'-propenyl)amino-tetralin) was reported to be a selective radioiodinated ligand for dopamine D2-like receptors. This ligand displayed a high binding affinity (Kd = 0.3-0.4 nM) and an agonist binding profile to dopamine D2 and D3 receptors expressed in HEK293 cells and dopamine D4 receptors expressed in CHO cells. Herein, a series of studies to characterize D3 receptors in native tissues is presented. Based on studies of the distribution of receptor mRNA, D3, but not D2, receptors are present in the rat cerebellum. Quantitative autoradiographic experiments using [125I]S(-)5-OH-PIPAT to label molecular layers 9 and 10 of rat cerebellum were conducted. Saturation experiments demonstrated that [125I]S(-)5-OH-PIPAT bound with high affinity (Kd = 0.1 nM) to a low density (approximately 3 fmol/mg protein) of sites in molecular layers 9 and 10 of rat cerebellum. Increasing concentrations of Gpp(NH)p, but not ATP, decreased the specific binding of [125I]S(-)5-OH-PIPAT in rat cerebellum slices. In comparison studies, binding of [125I]NCQ298, a dopamine D2/D3 receptor antagonist, with a similar affinity (Kd = 0.2 nM) for D3 receptors as [125]S(-)5-OH-PIPAT, was not sensitive to Gpp(NH)p. Analysis of inhibition by S(-)5-OH-PIPAT of [125I]NCQ298 binding to rat cerebellum resulted in two-site binding with IC50 values of 0.07 nM and 6.0 nM. In the presence of GTP (300 microM), the data best fit a one-site model with an IC50 value of 1.6 nM. Agonists and antagonists inhibited the binding of [125I]S(-)5-OH-PIPAT in the cerebellum with a rank order of potency consistent with an interaction at D3 receptors. These results indicate that [125I]S(-)5-OH-PIPAT binds to D3 receptors in rat cerebellum. Furthermore, [125I]S(-)5-OH-PIPAT binds to GTP sensitive and GTP insensitive states of D3 receptors with distinctive high and low affinity states, respectively.