Pharmacological characterisation of recombinant nociceptin receptors

The heptadecapeptide Nociceptin/OrphaninFQ (N/OFQ) is the endogenous ligand for the G-protein coupled receptor, NOP, activation of which is involved in a plethora of physiological functions, including pain. Studies in main are hampered by a relative lack of suitable, well characterised ligands, especially antagonists and an understanding of the effects of prolonged receptor activation.;The N/OFQ sequence can be divided into a message domain, associated with receptor activation and an address domain, associated with receptor binding. In a series of biochemical assays, using Chinese Hamster Ovary (CHO) cells stably expressing human NOP, a number of ligands suitable for in vivo animal testing were characterised including a high affinity, high potency agonist ([(iF)Phe4, Arg14, Lys15]N/OFQ-NH2), a high affinity antagonist ([Nphe 1, Arg114, Lys15]N/OFQ-NH 2 or UFP-101) and several partial agonist molecules.;In a CHO cell line expressing NOP at a range of levels via the ecdysone inducible expression system, and behaviour of the partial agonist [F/G]N/OFQ(1-13)-NH 2 could be modulated to encompass full agonism and antagonism, using the previous assays, underscoring the need to assess activity in a range of models and steps in the signal transduction cascade.;Receptor desensitisation, by prolonged exposure of the inducible cells to N/OFQ, led to a decrease in NOP density, a reduction in N/OFQ stimulated GTPgamma[35S] binding with a reduction in functional potency and a reduction in potency of cAMP inhibition. More interestingly, a reduction in NOP mRNA was also observed.;Collectively this thesis has made a significant contribution to the N/OGQ-NOP field in that: (1) several novel molecules have been characterised and are now available to other researchers; (2) a system in which pharmacological behaviour can be accurately defined has been characterised; (3) the first example of genomic desensitisation of NOP has been described. The N/OFQ-NOP system is ready to leave the pre-clinical laboratory and full clinical evaluation is eagerly awaited.