GNRH Interaction with Anterior Pituitary. I. Determination of the Affinity and Number of Receptors for GNRH in Ovine Anterior Pituitary1

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Abstract
The specificity of the interaction of [125I]-GNRH [gonadoliberin] and its receptor in ovine anterior pituitary tissue was evaluated. Hormone and receptor were stable during incubation for at least 4 h at 4.degree. C in the presence of Aprotinin (100 kIU/ml). The association rate constant (k1) was 1.3 .times. 107/M per s and the dissociation rate (k-1) was 3 .times. 10-3/s. 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 [lutropin], FSH [follitropin], 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 [rate constant] = 2.33 .+-. 0.31 .times. 1010/M) were present. 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 .times. 10-11 mol/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 .mu.g estradiol-17.beta. even though levels of LH increased .apprx. 100-fold 13 h after treatment.