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

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, GTP gamma[S-35] binding, cAMP formation and NOP-mRNA. CHOINDhNOP induced with 5 and 10 mu M 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 mu M N/OFQ significantly (p < e0.05) reduced B (max) at 5 and 10 mu M 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 GTP gamma[S-35] binding were also reduced (5 mu M PonA: pEC(50)-control=8.55 +/- 0.06, pretreated=7.88 +/- 0.07; E (max)-control=3.52 +/- 0.43, pretreated=2.48 +/- 0.10; 10 mu M PonA: pEC(50)-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 mu M PonA: pEC(50)-control=9.78 +/- 0.08, pretreated=8.92 +/- 0.13; 10 mu M PonA: pEC(50)-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 mu M 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 Cypulsing' PonA and desensitizing following wash-out.