Effects of naloxone benzoylhydrazone on native and recombinant nociceptin/orphanin FQ receptors

We have studied the effects of naloxone benzoylhydrazone (NalBzoH) at recombinant human OP4 receptors expressed in Chinese hamster ovary (CHO) cells (CHOhOP4) and native OP4 sites in isolated tissues from various species. In CHOhOP4 membranes, nociceptin (NC) and NalBzoH displaced [I-125]Tyr(14)-NC with pK(i) values of 10.1 and 7.3. In the presence of 100 muM GDP, NC stimulated GTPgamma(35)S binding (pEC(50) = 8.5). NalBzoH was ineffective but antagonized the effects of NC (pA(2) = 6.9). At 5 muM GDP, there was an increase in potency (pEC(50) = 9.3) and efficacy (4.3-fold) of NC. NalBzOH was a partial agonist (pEC(50) = 7.0, E-max = 13% relative to NC). In CHOhOP4 cells, NC and NalBzoH inhibited cAMP formation with pEC(50) and E-max values of 9.8 and 100% and 6.0 and 44%, respectively. In the rat vas deferens, NalBzoH (10 muM) did not modify electrically induced twitches but competitively antagonized the inhibitory action of NC (pA(2) = 6.2). In the mouse vas deferens (mVD) and guinea pig ileum (gpI), NalBzoH inhibited twitches with pEC(50) and E-max values of 7.6 and 78% and 8.5 and 77%, respectively. The effect of 3 muM NalBzoH was fully inhibited by 3 muM naloxone in mVD and 30 muM in gpI. Under these conditions, NalBzoH antagonized the actions of NC in both preparations with pA(2) values of 6.3 and 6.8, respectively. Collectively, these data demonstrate that NalBzoH is a nonselective OP4 ligand with system-dependent behaviour.