Table 1

Binding affinities and stimulation of [35S]GTPγS binding by N/OFQ and peptides

Peptide[3H]N/OFQ BindingKi1-a[35S]GTPγS Binding
EC50 (nM)% Maximal Stimulation1-b
N/OFQ0.43  ± 0.054.0  ± 1.3100
1 Ac-RYYRWR-NH2 0.60  ± 0.072.2  ± 0.252  ± 2
2 Ac-RYYRWK-NH2 0.71  ± 0.112.1  ± 1.357  ± 5
3 Ac-RYYRIK-NH2 1.50  ± 0.115.1  ± 0.130  ± 5
4 Ac-RYYKWR-NH2 1.47  ± 0.647.8  ± 4.149  ± 1
5 Ac-RYYKWK-NH2 0.72  ± 0.216.7  ± 2.452  ± 6

Experiments were conducted on mouse ORL1 transfected into CHO cells. Data shown are the average ± SD of three experiments conducted in triplicate.

  • 1-a Ki values were calculated from the equation Ki = IC50/(1 + [L]/Kd). This equation appears valid because there seems to be a single binding site, with Hill coefficients close to 1.0. However, the reversibility of the synthetic peptides has not been determined, and the Ki values are based on the assumption that all components bind reversibly. The concentration of [3H]N/OFQ was 0.8 nM, and its Kd was determined to be 0.15 nM from direct saturation experiments.

  • 1-b The percent maximal stimulation was relative to N/OFQ as 100% in each experiment. The percent maximal stimulation of each peptide was significantly different than N/OFQ, P > .05 by analysis of variance with post hoc Newman-Keuls.