CHARACTERIZATION OF A NOVEL IODINATED LIGAND, IPMPP, FOR HUMAN DOPAMINE D4 RECEPTORS EXPRESSED IN CHO CELLS

A novel radioiodinated ligand with a high specific activity (2,200 Ci/ mmol), iodophenyl)piperazin-1-yl]methyl-1H-pyrrolo(2,3-b) pyridine ([I -125]IPMPP), was successfully prepared. Binding characteristics of [I- 125]IPMPP were evaluated using human dopamine D4 (D4.2 variant) recept ors expressed in Chinese hamster ovary (CHO) cells. Saturation analysi s revealed high affinity binding sites for [I-125]IPMPP (K-d = 0.39 +/ - 0.18 nM). The number of D4 receptors labeled with [I-125]IPMPP at ro om temperature was four times higher than that labeled with [I-125]S(- )5-OH-PIPAT, a radioiodinated agonist ligand (572 fmol/mg protein vs. 125 fmol/mg protein). A significant decrease in the number of binding sites was observed with [I-125]S(-)5-OH-PIPAT when assays were carried out at a higher temperature (37 degrees C vs. 25 degrees C). In contr ast to [I-125]S(-)5-OH-PIPAT, [I-125]IPMPP labeled more D4 sites at 37 degrees C. Neither magnesium ion nor guanylimidodiphosphate (Gpp(NH)p ) affected [I-125]IPMPP binding. These data support the conclusion tha t [I-125]IPMPP is an antagonist ligand. The potency of various compoun ds, including clozapine, to inhibit [I-125]IPMPP binding is consistent with the rank order measured with other radioligands for D4 receptors . In addition, measuring D4 receptor stimulation of [S-35]GTP gamma S binding further demonstrated the antagonist property of IPMPP.