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

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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 (CHOINDhNOP) to examine the effects of N/OFQ pretreatment upon receptor density, GTPγ[35S] binding, cAMP formation and NOP-mRNA. CHOINDhNOP induced with 5 and 10 μM PonasteroneA (PonA) for 20 h produced NOP densities (B max) of 194 and 473 fmol. mg-1 protein, respectively. This was accompanied by decreased NOP mRNA. The lower B max is typical of the central nervous system. Pretreatment with 1 μM N/OFQ significantly (p < 0.05) reduced B max at 5 and 10 μM PonA to 100 and 196 fmol. mg-1 protein, respectively. There was no change in binding affinity. Along with the reduction in B max, potency and efficacy for N/OFQ-stimulated GTPγ[35S] binding were also reduced (5 μM PonA: pEC50-control = 8.55 ± 0.06, pretreated = 7.88 ± 0.07; E max-control = 3.52 ± 0.43, pretreated = 2.48 ± 0.10; 10 μM PonA: pEC50-control = 8.41 ± 0.18, pretreated = 7.76 ± 0.03; E max-control = 5.07 ± 0.17, pretreated = 3.38 ± 0.19). For inhibition of cAMP formation, there was a reduction in potency (5 μM PonA: pEC50-control = 9.78 ± 0.08, pretreated = 8.92 ± 0.13; 10 μM 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 μM PonA, respectively, but these measurements were made following concurrent N/OFQ challenge and PonA induction. In CHOINDhNOP, 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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Acknowledgements

Supported in part by British Journal of Anaesthesia/Royal College of Anaesthetists and UHL-NHS Trust. We would like to thank Drs. G. Calo and R. Guerrini (University of Ferrara, Italy) for providing N/OFQ.

Conflict of interest stament

We have no conflict of interests to declare.

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Authors and Affiliations

  1. 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

    T. A. Barnes, J. McDonald & D. G. Lambert

  2. Department of Health Sciences, Division of Anaesthesia, Critical Care and Pain Management, University of Leicester, Leicester Royal Infirmary, Leicester, LE1 5WW, UK

    D. J. Rowbotham

  3. Department of Cancer Studies and Molecular Medicine, Clinical Sciences Building, University of Leicester, Leicester Royal Infirmary, Leicester, LE2 7LX, UK

    T. L. Duarte

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  1. T. A. Barnes
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  2. J. McDonald
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  3. D. J. Rowbotham
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  4. T. L. Duarte
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  5. D. G. Lambert
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Correspondence to D. G. Lambert.

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Barnes, T.A., McDonald, J., Rowbotham, D.J. et al. Effects of receptor density on Nociceptin/OrphaninFQ peptide receptor desensitisation: studies using the ecdysone inducible expression system. Naunyn-Schmied Arch Pharmacol 376, 217–225 (2007). https://doi.org/10.1007/s00210-007-0189-z

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  • DOI: https://doi.org/10.1007/s00210-007-0189-z

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