Table 1

Binding of nonpeptide antagonists to R, RG, and R_O states of the CRF1 receptor in L-CRF₁ cell membranes

	vs ¹²⁵I-Sauvagine (RG)		vs ¹²⁵I-Sauvagine + GTPγS (R_O)		vs [³H]NBI 35965 + GTPγS (R)		vs ¹²⁵I-Astressin + GTPγS (R)
Antagonist Ligand	pK_i	% Maximal Displacement	pK_i	% Maximal Displacement	pK_i	% Maximal Displacement	pK_i	% Maximal Displacement	α
	nM		nM		nM		nM
Antalarmin	9.42 ± 0.03	101 ± 1	8.71 ± 0.08	92 ± 3	9.22 ± 0.26	101 ± 2	8.07 ± 0.15^a	20 ± 1	0.63 ± 0.03^a
	(0.38)		(2.0)		(0.61)		(8.6)
NBI 27914	9.02 ± 0.06	100 ± 1	8.70 ± 0.18	113 ± 13	8.68 ± 0.06	99 ± 2	8.95 ± 0.06^a	28 ± 1	0.59 ± 0.01^a
	(0.97)		(2.0)		(2.1)		(1.1)
NBI 35965	8.84 ± 0.10	99 ± 1	8.33 ± 0.10	94 ± 6	8.74 ± 0.06	100	8.87 ± 0.33^a	22 ± 1	0.65 ± 0.03^a
	(1.4)		(4.6)		(1.8)		(1.4)
DMP-696	8.63 ± 0.11	101 ± 1	8.38 ± 0.08	110 ± 3	8.71 ± 0.07	98 ± 2	8.89 ± 0.22^a	32 ± 2	0.54 ± 0.02^a
	(2.3)		(4.1)		(2.0)		(1.3)

Displacement of radioligand binding to L-CRF₁ cell membranes was measured as described under Materials and Methods. Inhibition of ¹²⁵I-sauvagine binding provides a measure of ligand affinity for the RG state of the CRF₁receptor. Ligand affinity for the R state was measured by displacement of ¹²⁵I-astressin or [³H]NBI 35965 binding in the presence of 30 μM GTPγS. A third state of the receptor was identified in L-CRF₁ cell membranes, which bound agonists with high affinity in a GTPγS-insensitive manner (R_O). Ligand affinity for R_O was measured by displacement of¹²⁵I-sauvagine binding with 30 μM GTPγS present. pK _i values were obtained by fitting the displacement data to a single affinity-state competition model, followed by conversion of IC₅₀ to K _i(Cheng and Prusoff, 1973). [In all cases, a two affinity-state model did not significantly improve the goodness of fit (p > 0.05)]. The K _d value used in theK _i calculation was 43 pM and 1.4 nM for¹²⁵I-sauvagine at RG and R_O, respectively, 0.56 nM for [³H]NBI 35965, and 70 pM for¹²⁵I-astressin.

^a The affinity of nonpeptide antagonists was determined using a model which assumes allosteric inhibition of¹²⁵I-astressin binding (the allosteric ternary model, eq.1; see , pK _i is equivalent to pK _N in eq. 1). This analysis estimates nonpeptide affinity, and the cooperativity between nonpeptide and¹²⁵I-astressin binding (α). Data are the mean ± S.E.M. (n = 3–4).