title: Muscarinic drugs regulate the PKG-II-dependent phosphorylation of M3 muscarinic acetylcholine receptors at plasma membranes from airway smooth muscle.
abstract: Muscarinic agonists induce the activation of the airway smooth muscle (ASM) leading to smooth muscle contraction, important in asthma. This activation is mediated through M2/M3 muscarinic acetylcholine receptors (mAChRs). Muscarinic receptor activity, expressed as [(3)H]QNB binding at plasma membranes from bovine tracheal smooth muscle (BTSM), increased with cGMP and was augmented significantly cGMP plus ATP but diminished with the PKG-II inhibitor, Sp-8-pCPT-cGMPS. The [(3)H]-QNB binding was accelerated by okadaic acid, (OKA), a protein phosphatase (PPase) inhibitor. These two results indicated the involvement of a membrane-bound PPase. Moreover, a cGMP-dependent-[(32)P]γATP phosphorylation of plasma membranes from BTSM was stimulated at low concentrations of muscarinic agonist carbamylcholine (CC). However, higher amounts of CC produced a significant decrement of [(32)P]-labeling. A selective M3mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [(32)P]-labeling. The [(32)P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M2/M3mAChR antibodies. The M3mAChR immuno-precipitates exhibited the highest cGMP-dependent [(32)P]-labeling, indicating it is a PKG-II substrate. Experiments using synthetic peptides from the C-terminal of the third intracellular loop (i3) of both M2mAChR (356-369) and M3mAChR (480-493) as external PKG-II substrates resulted in the i3M3-peptide being heavily phosphorylated. These results indicated that PKG-II phosphorylated the M3mAChR at the i3M3 domain ((480)MSLIKEKK(485)), suggesting that Ser(481) may be the target. Finally, this phosphorylation site seems to be regulated by a membrane-bound PPase linked to muscarinic receptor. These findings are important to understand the role of M3mAChR in the patho-physiology of ASM involved in asthma and COPD.
authors.affiliation: a Facultad de Medicina, Sección de Biomembranas , Instituto de Medicina Experimental (IME), Universidad Central de Venezuela , Sabana Grande, Caracas , Venezuela.
authors.forename: Marcelo J
authors.initials: MJ
authors.lastname: Alfonzo
authors.affiliation: a Facultad de Medicina, Sección de Biomembranas , Instituto de Medicina Experimental (IME), Universidad Central de Venezuela , Sabana Grande, Caracas , Venezuela.
authors.forename: Ramona González de
authors.initials: RG
authors.lastname: Alfonzo
authors.affiliation: a Facultad de Medicina, Sección de Biomembranas , Instituto de Medicina Experimental (IME), Universidad Central de Venezuela , Sabana Grande, Caracas , Venezuela.
authors.forename: Marcelo
authors.initials: M
authors.lastname: Alfonzo González
authors.affiliation: a Facultad de Medicina, Sección de Biomembranas , Instituto de Medicina Experimental (IME), Universidad Central de Venezuela , Sabana Grande, Caracas , Venezuela.
authors.forename: Itala Lippo
authors.initials: IL
authors.lastname: de Becemberg
mesh_terms: D000818:Animals; D001249:Asthma / Q000209:etiology / Q000503:physiopathology; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D017869:Cyclic GMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D025461:Feedback, Physiological; D006801:Humans; D066298:In Vitro Techniques; D018721:Muscarinic Agonists / Q000378:metabolism / Q000494:pharmacology*; D018727:Muscarinic Antagonists / Q000378:metabolism / Q000494:pharmacology*; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011813:Quinuclidinyl Benzilate / Q000378:metabolism / Q000493:pharmacokinetics; D043587:Receptor, Muscarinic M3 / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013873:Thionucleotides / Q000494:pharmacology; D014132:Trachea / Q000187:drug effects / Q000378:metabolism
chemical_list: C090860:8-(4-chlorophenylthio)guanosine 3',5'-cyclic monophosphorothioate; D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D047428:Protein Kinase Inhibitors; D043587:Receptor, Muscarinic M3; D013873:Thionucleotides; D011813:Quinuclidinyl Benzilate; D017869:Cyclic GMP-Dependent Protein Kinases; D006152:Cyclic GMP
keywords: cGMP; carbamylcholine; muscarinic receptors; tracheal smooth muscle