QUANTITATIVE AUTORADIOGRAPHIC STUDIES OF DOPAMINE D-3 RECEPTORS IN RAT CEREBELLUM USING [I-125] S(-)5-OH-PIPAT

Recently, [I-125]S(-)5-OH-PIPAT -(N-n-propyl-N-3'-iodo-2'-propenyl)ami no-tetralin) was reported to be a selective radioiodinated ligand for dopamine D-2-like receptors. This ligand displayed a high binding affi nity (K-d = 0.3-0.4 nM) and an agonist binding profile to dopamine D-2 and D-3 receptors expressed in HEK293 cells and dopamine D-4 receptor s expressed in CHO cells. Herein, a series of studies to characterize D-3 receptors in native tissues is presented. Based on studies of the distribution of receptor mRNA, D-3, but not D-2, receptors are present in the rat cerebellum. Quantitative autoradiographic experiments usin g [I-125]S(-)5-OH-PIPAT to label molecular layers 9 and 10 of rat cere bellum were conducted. Saturation experiments demonstrated that [I-125 ]S(-)5-OH-PIPAT bound with high affinity (K-d = 0.1 nM) to a low densi ty (similar to 3 fmol/mg protein) of sites in molecular layers 9 and 1 0 of rat cerebellum. Increasing concentrations of Gpp(NH)p, but not AT P, decreased the specific binding of [I-125]S(-)5-OH-PIPAT in rat cere bellum slices. In comparison studies, binding of [I-125]NCQ298, a dopa mine D-2/D-3 receptor antagonist, with a similar affinity (K-d = 0.2 n M) for D-3 receptors as [I-125]S(-)5-OH-PIPAT, was not sensitive to Gp p(NH)p. Analysis of inhibition by S(-)5-OH-PIPAT of [I-125]NCQ298 bind ing 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 mu M), the data best fit a one-site model with an IC50 value of 1.6 nM. Agonists and antago nists inhibited the binding of [I-125]S(-)5-OH-PIPAT in the cerebellum with a rank order of potency consistent with an interaction at D-3 re ceptors. These results indicate that [I-125]S(-)5-OH-PIPAT binds to D- 3 receptors in rat cerebellum. Furthermore, [I-125]S(-)5-OH-PIPAT bind s to GTP sensitive and GTP insensitive states of D-3 receptors with di stinctive high and low affinity states, respectively. (C) 1997 Elsevie r Science B.V.