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 AM GTPgS. Data from standard [35S]GTPgS binding and immunoprecipitation (Gai1 – 3) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHOhNOP) cells. N/OFQ displaced [leucyl-3H]N/OFQ binding with pKi and slope values of 9.62F0.07 and 0.38F0.05, respectively. Addition of NaCl/GTPgS produced a steepening (slope 0.95F0.06, n = 3) of the curve. N/OFQ stimulated [35S]GTPgS binding with pEC50 and Emax values of 8.21F0.17 and 1.17F0.01, respectively (in CHOhNOP, pEC50 and Emax values were 8.47F0.01 and 7.01F0.63). N/OFQ stimulated [35S]GTPgS binding in dog and CHOhNOP cell membranes could be immunoprecipitated with an anti-Gai1 – 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 AM [Nphe1]N/OFQ(1–13)NH2 and 1 AM [Phe1C(CH2-NH)Gly2]N/OFQ(1–13)NH2 (partial agonist) yielding pKB values of 8.58F0.21, 7.06F0.59 and 7.32F0.41, respectively (in CHOhNOP, a pKB for J-113397 of 8.33F0.02 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.
Functional coupling of the nociceptin/orphanin FQ receptor in dog brain membranes
Guerrini R.;
2004
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 AM GTPgS. Data from standard [35S]GTPgS binding and immunoprecipitation (Gai1 – 3) assays are also presented, along with data from a limited number of control experiments with human NOP expressed in Chinese hamster ovary (CHOhNOP) cells. N/OFQ displaced [leucyl-3H]N/OFQ binding with pKi and slope values of 9.62F0.07 and 0.38F0.05, respectively. Addition of NaCl/GTPgS produced a steepening (slope 0.95F0.06, n = 3) of the curve. N/OFQ stimulated [35S]GTPgS binding with pEC50 and Emax values of 8.21F0.17 and 1.17F0.01, respectively (in CHOhNOP, pEC50 and Emax values were 8.47F0.01 and 7.01F0.63). N/OFQ stimulated [35S]GTPgS binding in dog and CHOhNOP cell membranes could be immunoprecipitated with an anti-Gai1 – 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 AM [Nphe1]N/OFQ(1–13)NH2 and 1 AM [Phe1C(CH2-NH)Gly2]N/OFQ(1–13)NH2 (partial agonist) yielding pKB values of 8.58F0.21, 7.06F0.59 and 7.32F0.41, respectively (in CHOhNOP, a pKB for J-113397 of 8.33F0.02 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.I documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
