title: Muscarinic acetylcholine M4receptors play a critical role in oxotremorine-induced DARPP-32 phosphorylation at threonine 75 in isolated medium spiny neurons.
abstract: Dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32) play essential roles in dopamine (DA) transmission in the striatum. It is suggested that a link exists between muscarinic acetylcholine receptors (mAChRs) and DA/DARPP-32 signaling, but the molecular mechanisms mediating this relationship have not been elucidated. The predominant mAChRs subtypes in the striatum are M1and M4. In this study, we investigated the functions of these two receptors, particularly M4, in regulating cAMP production and DARPP-32 phosphorylation in rat striatal medium spiny neurons (MSNs). We used time-resolved fluorescence resonance energy transfer, immunofluorescence confocal microscopy, and western blot assays. In cultured intact MSNs, we confirmed that muscarinic M1and M4receptors were highly expressed. Notably, M4receptors were co-expressed with D1receptors in only a portion of the cultured MSNs. The nonselective muscarinic agonist oxotremorine M (OX) slightly enhanced cAMP production, but this effect was independent of M1or M4receptors. However, OX directly participated in DARPP-32 phosphorylation, phosphorylating DARPP-32 at Thr75 (the CDK5 site) and concomitantly de-phosphorylating DARPP-32 at Thr34 (the PKA site) in virtually cultured MSNs, whereas APO phosphorylated DARPP-32 at both Thr34 and Thr75. The OX-induced time-dependent increase in DARPP-32 phosphorylation at Thr75 was accompanied by increased p35 and CDK5 activity. Specifically, elevated immunoreactivity for phospho-DARPP-32-Thr75 and p35 was detected in M4receptor-expressing MSNs. Both genetic knockdown and pharmacologic inhibition of M4receptors with MT3, an M4receptor-selective antagonist, decreased the OX-induced DARPP-32-Thr75 phosphorylation in MSNs. These results indicate that the M4muscarinic receptor plays a critical role in modulating phosphorylation of DARPP-32-Thr75 in MSNs. The results suggest that M4receptor activation acts antagonistically with dopamine D1-like receptors within the striatum, and indicate that M4receptors may be a potential target for the treatment of Parkinson's disease and other relevant central nervous system disorders.
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, PR China.
authors.forename: Liqun
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Pharmacology, Ningxia Medical University, 1160 Shengli Street, Yinchuan, Ningxia 750004, PR China.
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Huang
authors.affiliation: Department of Pharmacology, Ningxia Medical University, 1160 Shengli Street, Yinchuan, Ningxia 750004, PR China.
authors.forename: Qing
authors.initials: Q
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, PR China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Department of Pharmacology, Ningxia Medical University, 1160 Shengli Street, Yinchuan, Ningxia 750004, PR China.
authors.forename: Jianqiang
authors.initials: J
authors.lastname: Yu
authors.affiliation: State Key Laboratory of Toxicology and Medical Countermeasures, Beijing Institute of Pharmacology and Toxicology, Beijing 100850, PR China. Electronic address: lylywang_1103@hotmail.com.
authors.forename: Liyun
authors.initials: L
authors.lastname: Wang
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D003342:Corpus Striatum / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32 / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D018721:Muscarinic Agonists / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010095:Oxotremorine / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D017207:Rats, Sprague-Dawley; D043643:Receptor, Muscarinic M1 / Q000378:metabolism; D043588:Receptor, Muscarinic M4 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017447:Receptors, Dopamine D1 / Q000378:metabolism
chemical_list: D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; C497930:Ppp1r1b protein, rat; D043643:Receptor, Muscarinic M1; D043588:Receptor, Muscarinic M4; D017447:Receptors, Dopamine D1; D010095:Oxotremorine; D000242:Cyclic AMP
keywords: Basal ganglia circuitry; Dopamine and cAMP-regulated phosphoprotein (DARPP-32); Dopamine receptor; Medium spiny neuron (MSN); Muscarinic acetylcholine receptor 4 (M(4))