Table 1

EC50 values and maximal effects of opioid ligands in stimulating [35S]GTPγS binding to CHO-hkor cell membranes and their K i values in inhibiting [3H]diprenorphine binding to the human kappareceptors expressed in CHO cells

Compound[35S]GTPγS BindingKi in [3H]Diprenorphine Binding
EC50Maximal effect[35S]GTPγS binding bufferTEL buffer
nM nM nM
(−)-U50,488H3.1  ± 0.11.004.1  ± 1.23.4  ± 1.0
Dynorphin 1–170.18  ± 0.041.00  ± 0.100.08  ± 0.010.07  ± 0.01
(±)-Ethylketocyclazocine0.57  ± 0.010.97  ± 0.070.34  ± 0.100.41  ± 0.06
β-FNA1.7  ± 0.11.03  ± 0.023.3  ± 0.33.7  ± 0.7
Tifluadom3.9  ± 0.50.93  ± 0.011.6  ± 0.31.5  ± 0.1
Nalorphine17.9  ± 2.01-a 0.68  ± 0.0412.2  ± 2.39.2  ± 1.3
Nalbuphine1-b 0.38  ± 0.041-b (at 30 μM)39.7  ± 8.130.9  ± 6.3
Pentazocine103  ± 301-a 0.23  ± 0.0316.8  ± 3.413.4  ± 0.9
Buprenorphine1-b 0.22  ± 0.021-b (at 30 μM)0.08  ± 0.010.07  ± 0.03
NaloxoneNo effect4.5  ± 1.14.0  ± 0.4
NorbinaltorphimineNo effect0.01  ± 0.010.02  ± 0.01
DAMGONo effect>1000
DPDPENo effect>1000

[35S]GTPγS binding was performed in the presence of 3 μM GDP, 5 mM Mg++ and 100 mM Na+ with ∼10 μg membrane proteins at 30°C for 60 min with various concentrations of each drug. The 100% maximal effect was designated as the level of binding in response to 10 μM U50,488H. Competitive inhibition of 0.3∼0.5 nM [3H]diprenorphine binding by opioid ligands was performed in [35S]GTPγS binding buffer as well as in TEL buffer at 25°C for 60 min as described under “Methods and Materials.” Each value represents mean ± S.E.M. of three independent experiments in duplicate.

  • 1-a Determined as the dose that elicited 50% of the maximal response of the drug.

  • 1-b Could not be determined.

  • 1-c No plateau was observed.