TABLE 2

Receptor binding and stimulation of [³⁵S]GTPγS binding by [Dmt¹]DALDA and DAMGO in CHO cells transfected with MOR-1 splice variants

K_D values for [³H][Dmt¹]DALDA were determined using saturation analysis on membranes prepared from CHO cells stably transfected with the indicated clone. Each value represents the mean ± S.E.M. of three independent replications. The K_D values for [³H]DAMGO are from the literature (Pan et al., 1999, 2000), with the exception of MOR-1, which was assayed in the same membranes [³H][Dmt¹]DALDA. Stimulation of [³⁵S]GTPγS binding by [Dmt¹]DALDA was performed as described under Materials and Methods on membranes prepared from CHO cells stably transfected with the indicated clones. Values for DAMGO were from the literature (E. A. Bolan and G. W. Pasternak, manuscript submitted for publication). All stimulations were normalized to DAMGO (10 μM), which was assigned the value of 100%. The stimulation by DAMGO at 10 μM was 180% of basal levels in the spinal cord and 150% basal levels in brain. Values in the transfected cell lines varied from 135 to 240% basal levels. This variation may be due to the different levels of expression from line to line. EC₅₀ values and normalized maximal stimulations calculated using GraphPad Prism. Each value represents the mean ± S.E.M. for at least three independent determinations. The EC₅₀ ratio represents the ratio between the two drugs. The relatively potency of stimulation normalizes the EC₅₀ value for differences in affinity in binding assays by dividing the EC₅₀ ratio by the ratio of the K_D values).