Bovine iris-sphincter muscle lacks FP receptor binding sites

Abstract

PURPOSE: To determine the affinity, density, and specificity of the binding sites of tritium-labeled prostaglandin F 2a in membrane preparations of bovine iris-sphincter muscle and corpus luteum. METHODS: Membrane preparations were incubated with 0.312-40.0 nM 3 H-prostaglandin F 2a in saturation experiments, or 8 nM 2a H-prostaglandin F 2a in competition studies, in the presence or absence of varying concentrations of unlabeled prostaglandin F 2a or other prostaglandin receptor agonists. The affinity (K 2a ) and the density of the binding sites (B 2a ) of 3 H-prostaglandin F 2a in the bovine iris-sphincter muscle were determined by Scatchard analysis. Reverse transcription polymerase chain reaction was performed with bovine iris-sphincter muscle and corpus luteum total RNA and the PCR products were hybridized with specific 32 P-labeled probe for further confirmation of FP receptor expression. RESULTS : Specific binding sites of 3 H-prostaglandin F 2a in the membranes of bovine iris-sphincter muscle are saturable with an affinity of 9.5 nM and a density of 596 fmoles/mg of protein. Prostaglandin E 2a , 17-phenyl trinor prostaglandin E 2a , and GR63799 (EP, EP 2a , and EP 2a receptor agonists, respectively) inhibited 3 H-prostaglandin F 2a binding with an IC 2a of 0.0048 µM, 0.0038 µM, and 0.044 µM, respectively. Fluprostenol, a specific FP receptor agonist did not inhibit 3 H-prostaglandin F 2a binding. In contrast, prostaglandin F 2a and fluprostenol effectively inhibited 3 H-prostaglandin F 2a binding in the bovine corpus luteum with an IC 2a of 0.031 µM and 0.037 µM, respectively. CONCLUSIONS: Our results demonstrate that in the bovine iris-sphincter muscle, FP receptors are not expressed and 3 H-prostaglandin F 2a binds to EP 2a and EP 2a receptor sites. RT-PCR results demonstrated that FP receptor mRNA, which is present in bovine corpus luteum, is probably absent in iris-sphincter muscle.