The effects of endomorphin-1 and endomorphin-2 in CHO cells expressing recombinant mu-opioid receptors and SH-SY5Y cells

1 Endomorphin-1 and -2 (E-1/E-2) have been proposed as endogenous ligands f or the mu-opioid receptor. The aims of this study are to characterize the b inding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [C a2+](i) levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant mu-opioid receptors. 2 E-l displaced [H-3]-diprenorphine ([H-3]-DPN) binding in CHO mu and SH-SY 5Y membranes with pK(i) values of 8.02+/-0.09 and 8.54+/-0.13 respectively. E-2 displaced [3H]-DPN binding in CHO mu and SH-SY5Y cells with pK(i), val ues of 7.82+/-0.11 and 8.43+/-0.13 respectively. E-1/E-2 bound weakly to CH O delta and CHO kappa membranes, with IC50 values of greater than 10 mu M. 3 In CHO mu cells, E-1/E-2 inhibited forskolin (1 mu M) stimulated cyclic A MP formation with pIC(50) values of 8.03+/-0.16 (I-max=53.0+/-9.3%) and 8.1 5+/-0.24 (I-max=56.3+/-3.8%) respectively. Tn SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC(50), values of 7.72+/-0 .13 (I-max=46.9+/-5.6%) and 8.11+/-0.31 (I-max=40.2+/-2.8%) respectively. 4 E-1/E-2 (1 mu M) increased [Ca2+](i) in fura-2 loaded CHO mu cell suspens ions in a thapsigargin sensitive and naloxone reversible manner. Mean incre ases observed were 106+/-28 and 69+/-6.7 nM respectively. In single adheren t cells E-1/E-2 (1 mu M) increased [Ca2+](i) with a mean 340/380 ratio chan ge of 0.81+/-0.09 and 0.40+/-0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHO delta, CHO kappa and SH-SY5Y cells . 5 These data show that E-1/E-2 bind with high affinity and selectivity to m u-opioid receptors and modulate signal transduction pathways typical of opi oids. This provides further evidence that these two peptides may be endogen ous ligands at the mu-opioid receptor.