Functional coupling of the nociceptin/orphanin FQ receptor in dog brain membranes

Abstract

Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP) which is yet to be functionally characterized in dog brain. Ligand binding data reports low NOP density (29 fmol mg −1 protein) in dog. In this study using dog brain membranes, we have examined the effects of N/OFQ on [ leucyl- 3H]N/OFQ(1–17)OH ([ leucyl- 3H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 μM GTPγS. Data from standard [ 35S]GTPγS binding and immunoprecipitation (G αi1–3) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHO hNOP) cells. N/OFQ displaced [ leucyl- 3H]N/OFQ binding with p K i and slope values of 9.62±0.07 and 0.38±0.05, respectively. Addition of NaCl/GTPγS produced a steepening (slope 0.95±0.06, n=3) of the curve. N/OFQ stimulated [ 35S]GTPγS binding with pEC 50 and E max values of 8.21±0.17 and 1.17±0.01, respectively (in CHO hNOP, pEC 50 and E max values were 8.47±0.01 and 7.01±0.63). N/OFQ stimulated [ 35S]GTPγS binding in dog and CHO hNOP cell membranes could be immunoprecipitated with an anti-G αi1–3 antibody, indicating coupling to a pertussis toxin (PTx)-sensitive G-protein. N/OFQ actions were competitively antagonized by the selective NOP antagonists, 100 nM J-113397, 1 μM [Nphe 1]N/OFQ(1–13)NH 2 and 1 μM [Phe 1Ψ(CH 2-NH)Gly 2]N/OFQ(1–13)NH 2 (partial agonist) yielding p K B values of 8.58±0.21, 7.06±0.59 and 7.32±0.41, respectively (in CHO hNOP, a p K B for J-113397 of 8.33±0.02 was obtained). Despite relatively low receptor density, we were able to detect functional activity of native dog NOP, with pharmacology consistent with reports for other species.