Anatomical localisation and functional coupling of the nociceptin receptor in dog brain

Nociceptin (N/OFQ), the endogenous ligand for the nociceptin receptor, NOP, has many physiological roles, particularly in pain and cardiovascular modulation. There is an abundance of research for this system in humans and small laboratory species though none yet in dog. Radioligand binding showed significantly lower density of NOP (Bmax 28.7+/-2.8fmol mg -1 protein) in dog brain membranes compared to rat (Bmax 137.0+/-12.9). However the binding of various NOP and opioid ligands were similar indicating a pharmacologically identical receptor. Interestingly, there is a 3 fold higher density of total opioid receptors in dog compared to NOP although the significance of this is not fully understood. Functional studies show that NOP couples to G proteins and that N/OFQ stimulated GTPgamma 35S binding in dog (Emax 1.17+/-0.01, pEC50 8.21+/-0.17), which could be antagonised by the selective NOP antagonist, J-113397 with a pKB value of 8.58. Antibodies raised against full sequence N/OFQ showed no cross reactivity towards homologous opioid peptides and had an antibody titre of 1/3600. These antibodies were used in immunohistochemistry to localise N/OFQ in whole dog brain sections. N/OFQ's distribution was found to correlate with that reported for other species and for its precursor, preproN/OFQ. [ leucyl-3H]N/OFQ autoradiography showed extensive distribution for dog NOP in situ which was co-localised with N/OFQ. Highest binding was observed in the neocortex piriform cortex, hippocampus, caudate nuclei and putamen. Dog piriform cortex showed approximately 5 fold less specific binding compared to that observed in rat. This thesis describes a functional low density NOP in dog brain. Moreover peptide and receptor co-localise in areas of the brain involved in, for example, pain and cardiovascular modulation. Further in vivo studies of the role of N/OFQ in the dog are clearly warranted.