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 for the mu-opioid receptor. The aims of this study are to characterize the binding of E-1/E-2 and the subsequent effects on cyclic AMP formation and [Ca2+]i levels in SH-SY5Y and Chinese hamster ovary (CHO) cells expressing endogenous and recombinant mu-opioid receptors. 2 E-1 displaced [3H]-diprenorphine ([3H]-DPN) binding in CHO micro and SH-SY5Y membranes with pKi values of 8.02+/-0.09 and 8.54+/-0.13 respectively. E-2 displaced [3H]-DPN binding in CHOmu and SH-SY5Y cells with pKi values of 7.82+/-0.11 and 8.43+/-0.13 respectively. E-1/E-2 bound weakly to CHOdelta and CHOkappa membranes, with IC50 values of greater than 10 microM. 3 In CHOmu cells, E-1/E-2 inhibited forskolin (1 microM) stimulated cyclic AMP formation with pIC50 values of 8.03+/-0.16 (Imax = 53.0+/-9. 3%) and 8.15+/-0.24 (Imax = 56.3+/-3.8%) respectively. In SH-SY5Y cells E1/E2 inhibited forskolin stimulated cyclic AMP formation with pIC50 values of 7.72+/-0.13 (Imax=46.9+/-5.6%) and 8.11+/-0.31 (Imax = 40.2+/-2.8%) respectively. 4 E-1/E-2 (1 microM) increased [Ca2+]i in fura-2 loaded CHOmu cell suspensions in a thapsigargin sensitive and naloxone reversible manner. Mean increases observed were 106+/-28 and 69+/-6.7 nM respectively. In single adherent cells E-1/E-2 (1 microM) increased [Ca2+]i with a mean 340/380 ratio change of 0.81+/-0.09 and 0.40+/-0.08 ratio units respectively. E-1/E-2 failed to increase intracellular calcium in CHOdelta, CHOkappa and SH-SY5Y cells. 5 These data show that E-1/E-2 bind with high affinity and selectivity to mu-opioid receptors and modulate signal transduction pathways typical of opioids. This provides further evidence that these two peptides may be endogenous ligands at the mu-opioid receptor.
Harrison
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C
University Department of Anaesthesia, Leicester Royal Infirmary, Leicester LE1 5WW.
McNulty
S
S
Smart
D
D
Rowbotham
D J
DJ
Grandy
D K
DK
Devi
L A
LA
Lambert
D G
DG
D000701:Analgesics, Opioid / Q000494:pharmacology*; D000818:Animals; D001667:Binding, Competitive / Q000187:drug effects; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000096:biosynthesis; D004174:Diprenorphine / Q000378:metabolism; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000494:pharmacology*; D051381:Rats; D017450:Receptors, Opioid, mu / Q000819:agonists / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis
D000701:Analgesics, Opioid; D009842:Oligopeptides; D017450:Receptors, Opioid, mu; D011994:Recombinant Proteins; C105293:endomorphin 1; D004174:Diprenorphine; D005576:Colforsin; C105294:endomorphin 2; D000242:Cyclic AMP; D002118:Calcium