| pA2 online © Copyright 2004 The British Pharmacological Society |
181P
GKT, University of London Winter Meeting December 2003 |
|
Studies
on the desensitisation of the human nociceptin receptor (NOP). Use
of the ecdysone inducible expression system |
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Print abstractNociceptin/Orphanin FQ (N/OFQ) is the endogenous peptide ligand for the G-protein coupled nociceptin receptor (NOP) (Mogil et al., 2001). Activation of this receptor is involved in a variety of physiological responses including antinociception (Mogil et al., 2001). Prolonged activation of NOP produces desensitisation and we have demonstrated this in a stable high (~2pmol NOP/mg protein) expression recombinant system (Hashimoto et al., 2002). Here we use an inducible expression system in which Chinese Hamster Ovary cells express varying levels of NOP (CHOIDhNOP) by incubation with Ponasterone A (PonA) (McDonald et al., 2003).
For induction cells were cultured for 20h with 10µM PonA, 30µM amastatin, bestatin, captopril and phosphoramidon (peptidase inhibitor cocktail) and, where appropriate, 1µM N/OFQ. Cells were harvested and either membranes prepared for use in binding or whole cells used to determine cAMP inhibition. Binding of [3H]N/OFQ was performed in 0.5ml of 50mM Tris-HCl, 5mM MgSO4, 0.5% BSA buffer, pH 7.4 plus 10mM peptidase inhibitor cocktail and ~10µg of CHOIDhNOP membranes. Inhibition of forskolin (1µM) stimulated cAMP formation was performed in Krebs/HEPES buffer, pH 7.4 as described by McDonald et al., (2003).
In this batch of cells 10µM PonA induction resulted in a Bmax of 482 fmol/mg protein. Incubation of these cells with 1µM N/OFQ reduced receptor density by ~60%. There was no change in the affinity of the ligand for NOP (Table 1).
In functional studies (inhibition of cAMP formation by N/OFQ) there was a rightward shift in the concentration-response curve to N/OFQ with pre-treatment. In agreement with our previous study (at ~4-fold higher NOP density) there was no reduction in the maximum response. Following N/OFQ pre-treatment we also observed an increase in basal cAMP formation suggesting that even at 482fmol/mg protein, constitutive activity may occur. Studies aimed at defining desensitisation at lower induction and, hence, expression are currently underway.
Table 1. Effects of N/OFQ pre-treatment on NOP density and inhibition of cAMP formation at recombinant human NOP.
|
Control
|
N/OFQ
|
|
| Saturation Binding (Bmax, fmol/mg protein) | ||
| pKD |
9.90±0.10
|
9.85±0.10
|
| Bmax |
482±43
|
198±32*
|
| Inhibition of cAMP formation (Emax, %) | ||
| pEC50 |
9.84±0.12
|
8.92±0.09*
|
| Emax |
60±4
|
77±4*
|
Mean±s.e.mean
(n
4) for cells
induced with 10µM PonA. *p<0.05 compared to control, untreated
cells.
Hashimoto Y et
al., (2002) Eur. J. Pharmacol, 449, 17-22.
Kenakin T (2002) Ann. Rev. Pharmacol. Toxicol, 42, 349-379.
McDonald J et al., (2003) Br. J. Pharmacol, 140,
61-70.
Mogil JS & Pasternak GW (2001) Pharmacol Rev, 53, 381-415.
Supported by grants from BJA/RCA and UHL-NHS Trust.