Endomorphin‐1 induced desensitization and down‐regulation of the recombinant μ ‐opioid receptor

Endomorphin‐1 (E1) is a peptide with high affinity and selectivity for the μ‐opioid receptor. The aim of this study was to determine if endomorphin‐1 caused desensitization and down‐regulation of the μ‐opioid receptor expressed in Chinese hamster ovary cells. Following 10 μ M E1 pre‐treatment, desensitization was assessed by measuring cyclic AMP inhibition, down‐regulation was assessed by [ 3 H]‐diprenorphine ([ 3 H]‐DPN) binding and immunoblotting. Pre‐treatment of CHOμ cells with 10 μ M E1 for 11 and 18 h caused significant reduction in cyclic AMP inhibition. (11 h=39.0±16.7%, 18 h 47.0±11.1% reduction). At 18 h E1 pre‐treatment there was an enhancement (4.5 fold) of cyclic AMP production under forskolin stimulated conditions accompanied by a small rightward shift in the concentration‐response curve (pEC 50 control=7.8±0.3, pEC 50 E1=7.3±0.2) when cells were re‐challenged with E1. In membranes prepared from untreated and 0.5 h E1 pre‐treated cells, addition of GTPγS produced a significant rightward shift in the concentration response curves for E1 displacement of [ 3 H]‐DPN (0 h K i control=7.86±0.11, GTPγS=7.37±0.15; 0.5 h K i control=7.92±0.12, GTPγS=7.36±0.08) This was not observed in membranes prepared from cells that had been treated with E1 for 18 h (18 h K i control=7.69±0.11, GTPγS=7.75±0.08). In whole cells E1 treatment caused a rapid loss of cell surface receptors such that at 0.5 h there was a 30.5±1.5 reduction (this was unchanged for 18 h). In crude membranes a loss of receptors was also observed using radioligand binding or immuno‐blotting protocols. These data show that E1 causes desensitization and down‐regulation of the rat μ‐opioid receptor expressed in CHO cells. However, these two responses appear temporally distinct.British Journal of Pharmacology (2000) 131 , 1220–1226; doi: 10.1038/sj.bjp.0703683