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 [³H]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 [³H]-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-[³²P]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 [³²P]-labeling. A selective M₃mAChR antagonist, 4-DAMP produced a dramatic inhibition of the basal and CC-dependent [³²P]-labeling. The [³²P] labeled membrane sediments were detergent solubilized and immunoprecipitated with specific M_2/M₃mAChR antibodies. The M₃mAChR immuno-precipitates exhibited the highest cGMP-dependent [³²P]-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 (⁴⁸⁰MSLIKEKK⁴⁸⁵), suggesting that Ser⁴⁸¹ 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.

• cGMP
• carbamylcholine
• muscarinic receptors
• tracheal smooth muscle

Acknowledgements

The authors thank Dr. Coral Wynter for revision of the English version of this article.

Declaration of interest

This work was supported by grants from CDCH-UCV # PG -09-7401-2008/2 (R. G. A.); CDCH-UCV # PI-09-7726.2009/2 (I. L. B.); CDCH-UCV # PG 09-7772-2009/1 (M. J. A.). The authors declare no conflicts of interests.

Supplementary information available online