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Naunyn Schmiedebergs Arch Pharmacol. 2007 Nov;376(3):217-25. Epub 2007 Sep 25.

Effects of receptor density on Nociceptin/OrphaninFQ peptide receptor desensitisation: studies using the ecdysone inducible expression system.

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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.


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.

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