title: Effects of receptor density on Nociceptin/OrphaninFQ peptide receptor desensitisation: studies using the ecdysone inducible expression system.
abstract: Pretreatment of the G-protein coupled nociceptin receptor (NOP) with nociceptin/orphaninFQ (N/OFQ) produces desensitisation. The influences of receptor expression and genomic effects are largely unknown. We have used an ecdysone-inducible NOP expression system in a CHO line (CHO INDhNOP) to examine the effects of N/OFQ pretreatment upon receptor density, GTPgamma[35S] binding, cAMP formation and NOP-mRNA. CHO(INDhNOP) induced with 5 and 10 microM PonasteroneA (PonA) for 20 h produced NOP densities (Bmax) of 194 and 473 fmol. mg(-1) protein, respectively. This was accompanied by decreased NOP mRNA. The lower Bmax is typical of the central nervous system. Pretreatment with 1 microM N/OFQ significantly (p < 0.05) reduced Bmax at 5 and 10 microM PonA to 100 and 196 fmol. mg(-1) protein, respectively. There was no change in binding affinity. Along with the reduction in Bmax), potency and efficacy for N/OFQ-stimulated GTPgamma[35S] binding were also reduced (5 microM PonA: pEC50-control = 8.55 +/- 0.06, pretreated = 7.88 +/- 0.07; Emax-control = 3.52 +/- 0.43, pretreated = 2.48 +/- 0.10; 10 microM PonA: pEC50-control = 8.41 +/- 0.18, pretreated = 7.76 +/- 0.03; Emax-control = 5.07 +/- 0.17, pretreated = 3.38 +/- 0.19). For inhibition of cAMP formation, there was a reduction in potency (5 microM PonA: pEC50-control = 9.78 +/- 0.08, pretreated = 8.92 +/- 0.13; 10 microM PonA: pEC50-control = 9.99 +/- 0.07, pretreated = 9.04 +/- 0.14), but there was no reduction in efficacy. In addition, there were 39 and 31% reductions in NOP mRNA at 5 and 10 microM PonA, respectively, but these measurements were made following concurrent N/OFQ challenge and PonA induction. In CHO INDhNOP, we have shown a reduction in cell surface receptor numbers and a reduction in functional coupling after N/OFQ pretreatment. This was observed at pseudo-physiological and supraphysiological receptor densities. Moreover, we also report a reduction in NOP mRNA, but further studies are needed which include 'pulsing' PonA and desensitizing following wash-out.
authors: Department of Cardiovascular Sciences (Pharmacology and Therapeutics Group), Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester LE1 5WW, UK.
authors: T A
authors: TA
authors: Barnes
authors: J
authors: J
authors: McDonald
authors: D J
authors: DJ
authors: Rowbotham
authors: T L
authors: TL
authors: Duarte
authors: D G
authors: DG
authors: Lambert
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004440:Ecdysone / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D018847:Opioid Peptides / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011957:Receptors, Opioid / Q000187:drug effects / Q000378:metabolism*; D000094942:Nociceptin Receptor; D000097629:Nociceptin
chemical_list: D000242:Cyclic AMP; D004440:Ecdysone; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D018847:Opioid Peptides; D012333:RNA, Messenger; D011957:Receptors, Opioid; D000094942:Nociceptin Receptor; D000097629:Nociceptin