[ 125 I]‐GR231118: a high affinity radioligand to investigate neuropeptide Y Y 1 and Y 4 receptors

GR231118 (also known as 1229U91 and GW1229), a purported Y 1 antagonist and Y 4 agonist was radiolabelled using the chloramine T method. [ 125 I]‐GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [ 125 I]‐GR231118 binds with very high affinity ( K d of 0.09–0.24 n M ) in transfected HEK293 cells with the rat Y 1 and Y 4 receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y 2 or Y 5 receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [ 125 I]‐GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y 1 , but not rat Y 4 , receptor cDNA. Autoradiographic studies demonstrated that [ 125 I]‐GR231118 binding sites were fully inhibited by the Y 1 antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [ 125 I]‐GR231118/BIBO3304‐insensitive binding sites were detected in few areas. These [ 125 I]‐GR231118/BIBO3304‐insensitive binding sites likely represent labelling to the Y 4 receptor subtype. In summary, [ 125 I]‐GR231118 is a new radiolabelled probe to investigate the Y 1 and Y 4 receptors; its major advantage being its high affinity. Using highly selective Y 1 antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [ 125 I]‐GR231118 to the Y 1 receptor allowing for the characterization and visualization of the purported Y 4 subtype.British Journal of Pharmacology (2000) 129 , 37–46; doi: 10.1038/sj.bjp.0702983