Binding of luteinizing hormone-releasing hormone analogues to dispersed rat pituitary cells

Abstract

The binding of luteinizing hormone-releasing hormone (LHRH) to dispersed rat pituitary cells was studied by using 125I-labeled analogues of the neurohormone: a superactive agonist [D Ser (Bu t) 6]LHRH(1–9) ethylamide and an antagonist DpGlu 1, DPhe 2, DTrp 3,6-LHRH. Although these cells were exposed to proteolytic enzymes, their ability to respond to LHRH stimulation by gonadotropin release, is preserved. The time course of binding of the two analogues at different temperatures has demonstrated that highest specific binding is evident at 4°C and that equilibrium is reached after 90 min of incubation at this temperature. Incubation of pituitary cells with the labeled analogues together with increasing concentrations of LHRH or unlabeled analogues exhibited parallel competition curves, suggesting binding to the same receptor sites but with different affinities. Biologically inactive analogues of LHRH or unrelated peptides such as TRH did not compete for binding sites. K a values for the agonist, LHRH and the antagonist were 2.1 × 10 9M −1, 0.92 × 10 8M −1 and 0.76 × 10 9M −1, respectively, and the binding capacity was 116 fmoles/10 6 pituitary cells.