Pharmacology and autoradiography of human DP prostanoid receptors using [ 3 H]‐BWA868C, a DP receptor‐selective antagonist radioligand

A potent and highly selective DP prostanoid receptor antagonist radioligand, [ 3 H]‐cyclohexyl‐N‐BWA868C (3‐benzyl‐5‐(6‐carboxyhexyl)‐1‐(2‐cyclohexyl‐2‐hydroxyethyl‐amino) hydantoin, ([ 3 H]‐BWA868C)), has been generated for receptor binding and autoradiographic studies. Specific [ 3 H]‐BWA868C binding to human platelet membranes achieved equilibrium within 60 min at 23°C and constituted up to 95% of the total binding. The association (K +1 ) and dissociation (K −1 ) rate constants of binding were 0.758±0.064 min −1 , mmol and 0.0042±0.0002 min −1 , respectively, yielding dissociation constants ( K D s) of 5.66±0.44 n M ( n =4). Specific [ 3 H]‐BWA868C bound to DP receptors with a high affinity ( K D =1.45±0.01 n M , n =3) and to a finite, saturable number of binding sites (B max =21.1±0.6 nmol g −1 wet weight). DP receptor class prostanoids (e.g. ZK118182, BW245C, BWA868C, PGD 2 ) exhibited high (nanomolar) affinities for [ 3 H]‐BWA868C binding, while prostanoids selective for EP, FP, IP and TP receptors showed a low (micromolar) affinity. Specific DP receptor binding sites were autoradiographically localized on the ciliary epithelium/process, longitudinal and circular ciliary muscles, retinal choroid and iris in human eye sections using [ 3 H]‐BWA868C. While [ 3 H]‐PGD 2 yielded similar quantitative distribution of DP receptors as [ 3 H]‐BWA868C, the level of non‐specific binding observed with [ 3 H]‐PGD 2 was significantly greater than that observed with [ 3 H]‐BWA868C. It is concluded that [ 3 H]‐BWA868C is a high‐affinity and very specific DP receptor radioligand capable of selectively labelling the DP receptor. [ 3 H]‐BWA868C may prove useful for future homogenate‐based and autoradiographic studies on the DP receptor.British Journal of Pharmacology (2000) 131 , 1025–1038; doi: 10.1038/sj.bjp.0703686