Mechanisms of M3 muscarinic receptor regulation by wash-resistant xanomeline binding.
OBJECTIVE
Xanomeline has been shown to bind in a unique manner at M1 and M3 muscarinic receptors, with interactions at both the orthosteric site and an allosteric site. We have previously shown that brief exposure of Chinese hamster ovary cells that express the M3 receptor to xanomeline followed by removal of free agonist results in a delayed decrease in radioligand binding and receptor response to agonists. In the current study, we were interested in determining the mechanisms of this effect.


METHODS
Cells were treated with carbachol, pilocarpine or xanomeline for 1 h followed by washing and either used immediately or after waiting for 23 h. Control groups included cells that were not exposed to agonists and cells that were treated with agonists for 24 h. Radioligand binding and functional assays were conducted to determine the effects of agonist treatments.


RESULTS
The above treatment protocol with xanomeline resulted in similar effects of the binding of [(3)H]NMS and [(3)H]QNB. When receptor function is blocked using a variety of methods, the long-term effects of xanomeline binding were absent.


CONCLUSIONS
Our data indicate that xanomeline wash-resistant binding at the receptor allosteric site leads to receptor downregulation and that receptor activation is necessary for these effects.
Noetzel
Meredith J
MJ
Division of Neuroscience Research in Psychiatry, University of Minnesota Medical School, Minneapolis, MN 55455, USA.
Grant
Marianne K O
MK
El-Fakahany
Esam E
EE
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D002217:Carbachol / Q000494:pharmacology; D002461:Cell Line, Transformed; D006224:Cricetinae; D004347:Drug Interactions; D005260:Female; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000378:metabolism; D006801:Humans; D018721:Muscarinic Agonists / Q000493:pharmacokinetics* / Q000494:pharmacology*; D019832:N-Methylscopolamine / Q000493:pharmacokinetics; D010716:Phosphatidylinositols / Q000378:metabolism; D010862:Pilocarpine / Q000494:pharmacology; D011725:Pyridines / Q000493:pharmacokinetics* / Q000494:pharmacology*; D011813:Quinuclidinyl Benzilate / Q000493:pharmacokinetics; D043587:Receptor, Muscarinic M3 / Q000819:agonists* / Q000502:physiology*; D013830:Thiadiazoles / Q000493:pharmacokinetics* / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection
D018721:Muscarinic Agonists; D010716:Phosphatidylinositols; D011725:Pyridines; D043587:Receptor, Muscarinic M3; D013830:Thiadiazoles; D010862:Pilocarpine; D011813:Quinuclidinyl Benzilate; D001285:Atropine; D002217:Carbachol; C075257:xanomeline; D043802:GTP-Binding Protein alpha Subunits, Gq-G11; D019832:N-Methylscopolamine