Developing skeletal muscle cells express functional muscarinic acetylcholine receptors coupled to different intracellular signaling systems

Abstract

This study analyzed the expression of muscarinic acetylcholine receptors (mAChRs) in the rat cultured skeletal muscle cells and their coupling to G protein, phospholipase C and adenylyl cyclase (AC). Our results showed the presence of a homogeneous population of [ 3H]methyl-quinuclidinyl benzilate-binding sites in the membrane fraction from the rat cultured muscle (K D = 0.4 nM, B max = 8.9 fmol mg protein -1). Specific muscarinic binding sites were also detected in denervated diaphragm muscles from adult rats and in myoblasts isolated from newborn rats. Activation of mAChRs with carbachol induced specific [ 35S]GTPγS binding to cultured muscle membranes and potentiated the forskolin-dependent stimulation of AC. These effects were totally inhibited by 0.1-1 μM atropine. In addition, mAChRs were able to stimulate generation of diacylglycerol (DAG) in response to acetylcholine, carbachol or selective mAChR agonist oxotremorine-M. The carbachol-dependent increase in DAG was inhibited in a concentration- dependent manner by mAChR antagonists atropine, pirenzepine and 4-DAMP mustard. Finally, activation of these receptors was correlated with increased synthesis of acetylcholinesterase, via a PKC-dependent pathway. Taken together, these results indicate that expression of mAChRs, coupled to G protein and distinct intracellular signaling systems, is a characteristic of noninnervated skeletal muscle cells and may be responsible for trophic influences of acetylcholine during formation of the neuromuscular synapse. © 2005 Nature Publishing Group All rights reserved.