Agonist-induced functional desensitization of the mu-opioid receptor is mediated by loss of membrane receptors rather than uncoupling from G protein.
The effects of acute exposure of the opioid peptide [D-Ala2,N-MePhe4, Gly-ol5]enkephalin (DAMGO) on the mu-opioid receptor were examined in Chinese hamster ovary (CHO) K-1 and baby hamster kidney stable transfectants. In the CHO cell line, acute 1-hr treatment with DAMGO decreased the density of receptors without affecting the affinity or proportion of agonist-detected sites and attenuated the ability of the agonist to inhibit forskolin-stimulated cAMP accumulation. In contrast, similar 1-hr treatment of baby hamster kidney cells did not affect receptor density or agonist ability to inhibit cAMP accumulation, but longer duration of agonist exposure resulted in a reduction in membrane receptor, identical to the CHO cells. These results suggested that for the mu-opioid receptor, alteration in receptor density was the major determinant for the observed agonist-induced desensitization. Consistent with this notion, the ratio of the DAMGO concentration yielding half-maximal occupation of the mu receptor to that yielding half-maximal functional response was < 1. This suggests the necessity for a high mu receptor occupancy rate for maximal functional response, so that any loss of cell surface opioid-binding sites was a critical determinant in reducing the maximal response. This hypothesis was further supported by the observation that irreversible inactivation of fixed proportions of opioid-binding sites with beta-chlorn-altrexamine demonstrated that there were few spare receptors, which is in contrast to what has been reported for other G protein-coupled receptors, including the delta-opioid receptor. Taken together, these data suggest that the opioid agonist DAMGO has a high affinity for the mu receptor but must occupy > 70% of the available receptors to generate the maximal second messenger-linked response.
Pak
Y
Y
Department of Pharmacology, University of Toronto, Ontario, Canada.
Kouvelas
A
A
Scheideler
M A
MA
Rasmussen
J
J
O'Dowd
B F
BF
George
S R
SR
D000700:Analgesics / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells / Q000378:metabolism / Q000648:ultrastructure; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004745:Enkephalins / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D007668:Kidney / Q000378:metabolism / Q000648:ultrastructure; D007700:Kinetics; D009271:Naltrexone / Q000031:analogs & derivatives / Q000494:pharmacology; D009292:Narcotic Antagonists / Q000494:pharmacology; D017450:Receptors, Opioid, mu / Q000819:agonists* / Q000378:metabolism / Q000502:physiology; D012680:Sensitivity and Specificity; D013268:Stimulation, Chemical; D014162:Transfection
D000700:Analgesics; D004745:Enkephalins; D009292:Narcotic Antagonists; D017450:Receptors, Opioid, mu; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D005576:Colforsin; D009271:Naltrexone; C017120:chlornaltrexamine; D000242:Cyclic AMP; D019204:GTP-Binding Proteins