Functional consequences of nociceptin receptor activation

Nociceptin orphanin FQ (N/OFQ) is the 17 amino acid endogenous ligand for the G icoupled N/OFQ-receptor (NOP). In vivo administration produces a wide range of physiological responses including; analgesia, hyperalgesia and anti-opioid actions. In a series of in vitro assays including [/ewcy/-3H]N/OFQ binding, GTPy[35S] binding and inhibition of cAMP formation the following linked studies were performed; (l)N/OFQ structure-activity relationships (SAR) in cells (CHO) stably expressing human NOP (2)evaluation of receptor density on efficacy using the ecdysone inducible expression system and native tissues, (3)an investigation of NOP/G-protein coupling efficiency. SAR studies can be summarized as combining arginine14,lysine15 repeat in N/OFQ (increase affinity/potency) with C-terminal [F/G]N/OFQ(l-13)NH2([F/G]) and [Nphe1]N/OFQ(l-13)NH2([Nphe1]) modifications (reduce/eliminate efficacy). Arg14/Lys15 increased the affinity (pIQ: 10.31-11.16) and potency (pECso:8.98-9.85) of N/OFQ. [F/G] and [Nphe1] reduced the efficacy of N/OFQ from 1.0 to 0.44 and 0. Combination of Arg14/Lys 5 and [Nphe1] to UFP-101 produced the highest affinity peptide antagonist available (pA2:9.13, [Nphe1] alone:7.54). Using the ecdysone inducible expression system it was possible to vary the efficacy o f [F/G], between antagonist (a:0 upstream/low density), partial agonist (a:0.3-0.75) and full agonist (a:l). In the mouse vas deferens and colon [F/G] displayed varying degrees o f partial agonism possibly indicating differences in NOP receptor density. The binding of GDP and GTP in CHOhNOP was to high and low affinity sites. The fraction of GDP binding to the high affinity site was reduced by N/OFQ (77% under basal to 32%). The reduction in high affinity binding of GDP appeared dependent on the efficacy of the ligand. This thesis has identified several new peptides o f varying efficacy/potency for use in defining the pathophysiological role(s) of N/OFQ-NOP. Efficacy is not just a property of the ligand but of the assay system and its input receptor density. A cautious approach to new ligands characterized at a single endpoint is advocated. Acknowledgements While this work carries only my name it truly is a team effort. To my friend Dave (Professor Lambert), the greatest thanks are extended. Professionally and academically the assistance, support, guidance, wealth of knowledge and encouragement offered has been invaluable. On a personal level the support you and your family have extended to me has been colossal. I have so much gratitude for what you have given and continue to give me. For all my friends who I have worked with, Tim (you take an extra one), John Williams (a great landlord and even better friend), Jim, Chris, Anton, Emma, Trish (you made it interesting! ), Dr Carra, Massimo, Masato, Eddy, Paul, Kathleen, Wei, I thank you all for help, encouragement and love. Thanks to my Italian buddies, Giro, Remo and all the lovely ladies! I am sure there is many, many more and I thank you all. My gratitude list would not be complete without thanks to, Janet Harris, Bob Chamberlain and Margaret Crawford. I am indebted to you all for giving me yet another chance. To the Living Room, specifically Danny, Alex, AJfie, Rochelle, Nancy, Janis and all the other counsellors and clients, who have loved me back to life! I am so, so grateful for all the empathy, altruism and love you have extended and continue to extend to me. You have made an atheist into an agnostic and given me a new way of living, all I had to bring was HOW. To everybody in the “Fellowship” and all my peeps in recovery, I love you all! "For, to these people, I am truly related. First, through mutual pain and despair, and later through mutual objectives and new-found faith and hope. And, as the years go by, working together, sharing our experiences with one another, and also sharing a mutual trust, understanding and love-without strings, without obligation-we acquire relationships that are unique and priceless. " -Alcoholics Anonymous, p. 312" To Dr Bob, I am truly blessed to have you in my life and privileged to call you a friend. So much thanks for all that you give me, you are a very special person in my world. May we continue to trudge the road of happy destiny! Last and by no means list I thank my family, Mum, Dad, Sis, Dave, Liam and Kieran. To Bev and Jenifer (not forgetting Si) 1 love you all. Not forgetting the Boyz, Stu (AKA “The Big Fella”), Arun, Dave, Pete and Keith, let there be many more years of drink and debauchery. "Now there is a sense of belonging, of being wanted and needed and loved. In return for a bottle and a hangover, we have been given the Keys of the Kingdom. " —Alcoholics Anonymous, p. 276