Agonist-induced down-regulation and antagonist-induced up-regulation of m2- and m3-muscarinic acetylcholine receptor mRNA and protein in cultured cerebellar granule cells.
Cerebellar granule cells express m2- and m3-muscarinic acetylcholine receptors (mAChRs) and their corresponding mRNA with m3-mAChR being the predominant receptor subtype. After stimulation with the mAChR agonist, carbachol, m2- and m3-mAChR mRNA levels were decreased in a time- and concentration-dependent manner with the maximal down-regulation at 2 and 8 hr, respectively. Immunoprecipitation studies revealed that amounts of m2- and m3-mAChR protein also decreased at 8 and 24 hr, respectively. The carbachol-induced down-regulation of m3-mAChR mRNA was associated with a decrease in the transcription rate, but a substantial enhancement of the mRNA stability. Upon removal of carbachol after treatment for 8 hr, the levels of m3-mAChR mRNA and mAChR binding sites returned to their original values with a t1/2 of approximately 80 min and 6 hr, respectively. The carbachol-elicited loss of m2- and m3-mAChR mRNA was blocked by their corresponding receptor subtype-specific antagonists, AF-DX 116 (m2-selective) and 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) (m3-selective), and was concurrent with an increase in c-fos mRNA levels. Exposure of granule cells to the nonselective mAChR antagonist, atropine, caused a time- and concentration-dependent increase in the level of both m2- and m3-mAChR mRNA and mAChR binding sites. At 24 hr, immunoprecipitable m3-mAChR protein was predominantly increased. The atropine-induced up-regulation of m3-mAChR mRNA was concurrent with a marked enhancement of the mRNA stability and its transcription rate. The elevated levels of m3-mAChR mRNA and binding sites declined to their untreated values after the removal of atropine. Treatment with AF-DX 116 and 4-DAMP also produced an increase in the level of m2- and m3-mAChR mRNA and their corresponding immunoprecipitable receptor protein. These results demonstrate that the mAChR agonist and antagonist induce a down- and up-regulation of mAChR expression, respectively, through receptor-mediated mechanisms in cerebellar granule cells. Moreover, at least for m3-mAChR mRNA, the agonist- and antagonist-induced effects are reversible and associated with corresponding changes in the transcription rate of this receptor mRNA species.
Fukamauchi
F
F
Section on Molecular Neurobiology, National Institute of Mental Health, Bethesda, Maryland 20892.
Saunders
P A
PA
Hough
C
C
Chuang
D M
DM
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000378:metabolism*; D015536:Down-Regulation; D010276:Parasympatholytics / Q000494:pharmacology*; D010277:Parasympathomimetics / Q000494:pharmacology*; D010880:Piperidines / Q000494:pharmacology; D010890:Pirenzepine / Q000031:analogs & derivatives / Q000494:pharmacology; D012333:RNA, Messenger / Q000187:drug effects* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D010276:Parasympatholytics; D010277:Parasympathomimetics; D010880:Piperidines; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D010890:Pirenzepine; D001285:Atropine; C042375:4-diphenylacetoxy-1,1-dimethylpiperidinium; D002217:Carbachol; C048593:otenzepad