Pharmacology and autoradiography of human DP prostanoid receptors using [(3)H]-BWA868C, a DP receptor-selective antagonist radioligand.
1. 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. 2. Specific [(3)H]-BWA868C binding to human platelet membranes achieved equilibrium within 60 min at 23 degrees 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 nM (n=4). 3. Specific [(3)H]-BWA868C bound to DP receptors with a high affinity (K(D)=1.45+/-0.01 nM, n=3) and to a finite, saturable number of binding sites (B(max)=21.1+/-0.6 nmol g(-1) wet weight). 4. 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. 5. 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. 6. 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.
Sharif
N A
NA
Molecular Pharmacology Unit, Alcon Research Ltd., 6201 South Freeway, Fort Worth, Texas, TX 76134, USA. naj.sharif@alconlabs.com
Williams
G W
GW
Davis
T L
TL
D001345:Autoradiography; D001792:Blood Platelets / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D005123:Eye / Q000378:metabolism*; D006801:Humans; D006827:Hydantoins / Q000378:metabolism*; D010975:Platelet Aggregation Inhibitors / Q000378:metabolism*; D015230:Prostaglandin D2 / Q000378:metabolism*; D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin / Q000378:metabolism*
D006827:Hydantoins; D010975:Platelet Aggregation Inhibitors; D011971:Receptors, Immunologic; D011982:Receptors, Prostaglandin; C058848:BW A868C; D015230:Prostaglandin D2; C036293:prostaglandin D2 receptor