Evidence for high affinity prostacyclin binding sites in vascular tissue: Radioligand studies with a chemically stable analogue

Prostacyclin-specific binding sites are described in the muscularis of pig aorta using [ 3H]ZK 36374, a chemically stable prostacyclin analogue, as radioligand. Under standard incubation conditions [300 μg membrane protein in 350 μl Tris buffer (50 mmoles/l., pH 7.4) containing 3 mM Ca 2+ at 37° for 10 min] both association and dissociation were complete within 30 sec, thus not allowing the determination of association or dissociation rate constants. The Scatchard plot was upward convex, whereas the Hill plot was linear, having a slope of 1.9. The equilibrium dissociation constant ( K D) was 22.4 nmoles/l. and the specific binding was saturated at 360 fmoles [ 3H]ZK 36374/mg protein. The reversibility of binding was demonstrated by displacement of bound ligand with ZK 36374, its 5-( Z)-stereoisomer (ZK 36375), PGI 2 and PGE 1, but not with PGF 2α. The data suggest high affinity binding sites for ZK 36374 in the smooth muscle cells of pig aorta for which PGI 2 may be the physiological ligand. They also demonstrate a possible co-operativity with two molecules binding simultaneously to two interacting sites.