A radioreceptor assay of luteinizing hormone-releasing hormone receptor and characterization of LHRH binding to pituitary receptors in Shao duck

The properties of Shao duck pituitary luteinizing hormone-releasing hormone (LHRH) receptors were analyzed in pituitary membrane preparation and isolated pituitary cells prepared by enzymatic dispersion with collagenase and trypsin, by using a super-agonist analog of (D-Lys6) LHRH. High binding of 125I-(D-Lys6) LHRH to 106 cultured cells of Shao duck was observed after a 90 minute incubation at 4 degree C, while binding was significantly reduced after a 24h incubation. Binding of the radioligand was a function of tissue concentration of Shao duck pituitary membrane preparation, with a positive correlation over the range of 1-2 pituitary per-tube. Specific binding for 125I-(D-Lys6) LHRH increased with the increase in the amount of 125I-(D-Lys6) LHRH. The Scatchard analysis of data revealed a linear relationship between the amount of specific binding and the ratio of specific binding to free 1'25I(D-Lys6)LHRH, indicating a single class of high affinity sites. Equilibrium dissociation constant (Kd) was 0.34 nM in pituitary membrane preparation and 0.43 nM in isolated pituitary cells. Both Kd values were near and the maximum binding capacity (Bmax) was great in isolated cells, suggesting no significant loss of the LHRH receptor population caused by the enzymatic procedure employed for cell dispersion in the present study. Addition of 9D-Lys6) LHRH displaced bound 125I-(D-Lys6) LHRH. These results demonstrated the presence and provided characterization of LHRH receptors in Shao duck pituitary