title: 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-(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.
authors.affiliation: University Department of Anaesthesia, Critical Care and Pain Management, Leicester Royal Infirmary, Leicester, LE15WW, UK.
authors.forename: Emma E
authors.initials: EE
authors.lastname: Johnson
authors.forename: John
authors.initials: J
authors.lastname: McDonald
authors.forename: Beverley
authors.initials: B
authors.lastname: Nicol
authors.forename: Remo
authors.initials: R
authors.lastname: Guerrini
authors.forename: David G
authors.initials: DG
authors.lastname: Lambert
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D016466:CHO Cells; D006224:Cricetinae; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D008297:Male; D009292:Narcotic Antagonists; D018847:Opioid Peptides / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding / Q000502:physiology; D011957:Receptors, Opioid / Q000819:agonists / Q000378:metabolism*; D000094942:Nociceptin Receptor; D000097629:Nociceptin
chemical_list: D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009292:Narcotic Antagonists; D018847:Opioid Peptides; D011957:Receptors, Opioid; D000094942:Nociceptin Receptor; D000097629:Nociceptin