BINDING-KINETICS OF THE LONG-ACTING GONADOTROPIN-RELEASING-HORMONE (GNRH) ANTAGONIST ANTIDE TO RAT PITUITARY GNRH RECEPTORS

The GnRH antagonist Antide has been shown to produce prolonged inhibit ion of gonadotropin secretion in ovariectomized monkeys and other anim al models. The reasons for such a long duration of action have not yet been clarified. To understand the mode of action of this new antagoni st, we have performed association and dissociation binding kinetics us ing either crude rat pituitary homogenates as source of GnRH receptors or dispersed pituitary cells in culture. The binding characteristics of the radioiodinated Antide analog I-125-labeled [D-Tyr(0)] Antide to GnRH receptors in rat pituitary homogenates were comparable to those of the first generation GnRH antagonist I-125-labeled [Ac(3)Pro(1),pFD -Phe(2),D-Trp(3,6)] GnRH or the GnRH agonist I-125-labeled [D-Trp(6),( N-Et)Pro(9),Des,Gly(10)]GnRH, with an affinity constant (K-a) in the 1 0(10) M(-1) range. The maximum binding capacity was consistently highe r with the antagonist tracers than with the [I-125]GnRH agonist. Both antagonists dissociated at a slower rate at 4 C (similar to 4 times) t han the [I-125]GnRH agonist. Incubation at 23 C of I-125-labeled [D-Ty r(0)] Antide previously bound at 4 C resulted in complete dissociation within 8 h after the addition of an excess amount of any of the GnRH analogs; in addition, simple dilution of the incubation medium produce d spontaneous dissociation at this temperature. Using rat pituitary ce lls, Antide was found to inhibit the LH response to native GnRH (10(-8 ) M) in a dose-related manner. To test whether the binding of Antide i s normally reversible at 37 C, Antide (10(-7) M) was added to the cult ure medium 3 days after cell plating, and the initial preincubation wa s resumed for 24 h. Cells were then washed twice, and dissociation was allowed to take place. Bound Antide was shown to dissociate rapidly a t 37 C, as cells previously treated with Antide produced a full LH res ponse within 24 h if challenged with native GnRH. In conclusion, the b inding kinetics of I-125-labeled [D-Tyr(0)]Antide to GnRH receptors, w hich should reflect those of Antide, did not present abnormal features . Although this antagonist, similar to other GnRH antagonists, dissoci ated from pituitary receptors at a slower rate than GnRH analogs, rapi d and spontaneous dissociation was achieved at 23 C with simple diluti on, and dissociation of unmodified Antide occurred at 37 C. Taken toge ther, our results support the concept that the long duration of action of Antide is not due to any toxic effect of Antide at the receptor si te and could derive only marginally from the slow dissociation rate of this antagonist.