TABLE 1

Saturation and competition studies using [3H]DAMGO

Saturation and competition assays were performed using indicated membranes as described in Materials and Methods. In saturation assays, five conc. of [3H]DAMGO (0.125–2 nM) were used. KD values were calculated by nonlinear regression analyses (Prism 7). In competition assays, 1–1.5 nM of [3H]DAMGO was used against three to four conc. of unlabeled morphine and DAMGO (1–25 nM), M6G (5–100 nM), β-endorphin (10–200 nM), and dynorphin A (10–1000 nM). IC50 values were calculated by nonlinear regression analyses (Prism 7). Ki values were calculated by Ki = IC50/(1 + A/KD), in which A is the nM conc. of [3H]DAMGO, and KD value is obtained from saturation study. Results are the mean ± S.D. of three independent experiments. Significant differences between −Doxy and +Doxy groups were analyzed by two-tailed Student’s t test.

SaturationmMOR-1/mMOR-1G
+Doxy (100 ng/ml) No mMOR-1G−Doxy (0 ng/ml) With mMOR-1G95% Confidence Interval
KD (nM)0.53 ± 0.010.41 ± 0.06−0.00 to 0.25
Bmax (fmol/mg protein)33 ± 1.5104 ± 6.8**−84 to −58
Increase of Bmax315%
Competition Ki (nM)
Morphine1.0 ± 0.10.9 ± 0.8−1.8 to 1.6
DAMGO0.5 ± 0.00.3 ± 0.0**−0.26 to −0.14
M6G6.1 ± 0.12.3 ± 0.4**−5.1 to −2.5
Β-endorphin22 ± 2.84.6 ± 0.4*−26 to −10
Dynorphin A706 ± 15929 ± 24*−1121 to −233.1
  • * P < 0.05; **P < 0.01.