Endomorphin-1 induced desensitization and down-regulation of the recombinant mu-opioid receptor

1 Endomorphin-1 (E1) is a peptide with high affinity and selectivity for th e mu -opioid receptor. The aim of this study was to determine if endomorphi n-1 caused desensitization and down-regulation of the mu -opioid receptor e xpressed ill Chinese hamster ovary cells. 2 Following 10 muM E1 pre-treatment, desensitization was assessed by measur ing cyclic AMP inhibition, down-regulation was assessed by [H-3]-diprenorph ine ([H-3]-DPN) binding and immunoblotting. 3 Pre-treatment of CHO mu cells with 10 muM E1 for 11 and 18 h caused signi ficant reduction in cyclic AMP inhibition. (11 h = 39.0 +/- 16.7%, 18 h 47. 0 +/- 11.1% reduction). 4 At 18 h E1 pre-treatment there was an enhancement (4.5 fold) of cyclic AM P production under forskolin stimulated conditions accompanied by a small r ightward 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. 5 In membranes prepared from untreated and 0.5 h El pre-treated cells, addi tion of GTP gammaS produced a significant rightward shift in the concentrat ion response curves for Fl displacement of [H-3]-DPN (0 h K-i control = 7.8 6 +/- 0.11, GTP gammaS = 7.37 +/- 0.15; 0.5 h K-i control = 7.92 +/- 0.12, GTP gammaS = 7.36 +/- 0.08) This was not observed in membranes prepared fro m cells that had been treated with Fl for 18 h (18 h K-i control = 7.69 +/- 0.11, GTP gammaS = 7.75 +/- 0.08). 6 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 r adioligand binding or immune-blotting protocols. 7 These data show that E1 causes desensitization and down-regulation of the rat mu -opioid receptor expressed in CHO cells. However, these two respons es appear temporally distinct.