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-(3)H]N/OFQ(1-17)OH ([leucyl-(3)H]N/OFQ) binding in the presence and absence of 120 mM NaCl and 100 microM GTPgammaS. Data from standard [(35)S]GTPgammaS binding and immunoprecipitation (G(alphai1-3)) 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-(3)H]N/OFQ binding with pK(i) and slope values of 9.62+/-0.07 and 0.38+/-0.05, respectively. Addition of NaCl/GTPgammaS produced a steepening (slope 0.95+/-0.06, n=3) of the curve. N/OFQ stimulated [(35)S]GTPgammaS 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 [(35)S]GTPgammaS binding in dog and CHO(hNOP) cell membranes could be immunoprecipitated with an anti-G(alphai1-3) 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 microM [Nphe(1)]N/OFQ(1-13)NH(2) and 1 microM [Phe(1)Psi(CH(2)-NH)Gly(2)]N/OFQ(1-13)NH(2) (partial agonist) yielding pK(B) values of 8.58+/-0.21, 7.06+/-0.59 and 7.32+/-0.41, respectively (in CHO(hNOP), a pK(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.
Subject(s)
Brain/metabolism , Receptors, Opioid/metabolism , Animals , Brain/drug effects , CHO Cells , Cricetinae , Dogs , Dose-Response Relationship, Drug , Female , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Male , Narcotic Antagonists , Opioid Peptides/metabolism , Opioid Peptides/pharmacology , Protein Binding/physiology , Receptors, Opioid/agonists , Nociceptin Receptor , NociceptinABSTRACT
Nociceptin/orphanin FQ (N/OFQ) is the endogenous ligand for the N/OFQ receptor (NOP), whose characteristics in the dog are unknown. We therefore compared [(3)H]N/OFQ binding in dog and rat brain membranes. Radioligand saturation/competition studies with these membranes and leucyl-[(3)H]N/OFQ(1-17)OH or the novel radioligand [(3)H]N/OFQ(1-13)NH(2) were performed to determine receptor density and ligand affinity. The density of classic opioid receptors was determined by using [(3)H]diprenorphine. Leucyl-[(3)H]N/OFQ(1-17)OH binding was concentration dependent and saturable in dog (maximum binding capacity [B(max)], 28.7 +/- 2.8 fmol/mg of protein; equilibrium dissociation constant as negative log [pK(d)], 10.27 +/- 0.11) and rat (B(max), 137.0 +/- 12.9 fmol/mg of protein; pK(d), 10.41 +/- 0.05). In comparison, the B(max) and pK(d) of [(3)H]diprenorphine were, respectively, 77.7 +/- 5.3 fmol/mg of protein and 9.74 +/- 0.09 in dog and 79.1 +/- 18.2 fmol/mg of protein and 9.51 +/- 0.04 in rat. In dog, [(3)H]N/OFQ(1-13)NH(2) binding to NOP receptors was also saturable (B(max), 23.7 +/- 2.0 fmol/mg of protein; pK(d), 10.16 +/- 0.12). In both species, leucyl-[(3)H]N/OFQ(1-17)OH was displaced by various NOP ligands. Dynorphin A, N/OFQ(1-5)NH(2), and nocistatin were essentially inactive. There was a significant positive correlation (r(2) = 0.95; P < 0.0001) between pK(i) values (an estimate of affinity) obtained in displacement studies in rat and dog. We have demonstrated a low density of NOP receptors, measured with two radioligands, in dog, and these receptors display a high degree of pharmacological similarity with those natively expressed in the rat.
