GNRH INTERACTION WITH ANTERIOR-PITUITARY .1. DETERMINATION OF THE AFFINITY AND NUMBER OF RECEPTORS FOR GNRH IN OVINE ANTERIOR-PITUITARY

The specificity of the interaction of [125I]-GNRH and its receptor in ovine anterior pituitary tissue was evaluated. Hormone and receptor were found to be stable during incubation for at least 4 h at 4° C in the presence of Aprotinin (100 KIU/ml). The association rate constant (K1) was 1.3 x 107 M-1 sec-1 and the dissociation rate (k-1) was 3 x 10-3 sec-1. Of the substances tested only synthetic GNRH and (D-Leu)6-(des-Gly-NH2)10-GNRH ethylamide were capable of inhibiting the binding of [125I]-GNRH to the anterior pituitary preparation. Vasopressin, melatonin, thyrotropin releasing hormone, LH, FSH, prolactin and inactive analogs of GNRH did not inhibit binding of [125I]-GNRH to its receptor. Scatchard plot analysis of saturation experiments indicated that a single class of high affinity sites (KA = 2.33 ± 0.31 x 1010 M-1) were present. It should be emphasized that the relative affinity of GNRH for receptors in anterior pituitary tissue is only 3% that of [125I]-GNRH. A constant concentration of available sites (4.85 ± 0.27 x 10-11 moles/g fresh anterior pituitary tissue) were present in ewes 10-12 days after superovulation. The number of receptors present in anterior pituitaries of anestrous ewes was not different at 0, 5, 10 or 20 h after injection of 100 μg estradiol-17β even though serum levels of LH increased ~100-fold 13 h after treatment.