Short‐ and long‐term influences of calcitonin gene‐related peptide on the synthesis of acetylcholinesterase in mammalian myotubes

Abstract

1 The present study analyses the short-(15 min ± 2 h) and long-term (24 ± 48 h) in¯uences of calcitonin gene-related peptide (CGRP) on acetylcholinesterase (AChE) expression in the rat cultured skeletal muscle and the signal transduction events underlying CGRP actions. 2 To assess the eect of CGRP on AChE synthesis, myotubes were pre-exposed to the irreversible AChE inhibitor diisopropyl¯uorophosphate (DFP) and treated with CGRP or forskolin, an adenylyl cyclase (AC) activator. Treatment of myotubes with 1 ± 100 nM CGRP for 2 h increased by up to 42% the synthesis of catalytically active AChE with a parallel increase in the intracellular cyclic AMP. 3 The stimulation of AChE synthesis induced by CGRP was mimicked by direct activation of AC with 3 ± 30 mM forskolin. In contrast, pre-treatment of cultures with 100 nM CGRP for 20 h reduced by 37% the subsequent synthesis of AChE, resulting in a 15% decrease in total AChE activity after 48 h CGRP treatment. 4 Moreover, 24 h treatment of myotubes with 100 nM CGRP reduced by 54% the accumulation of cyclic AMP induced by a subsequent CGRP treatment. 5 These ®ndings indicate that, in skeletal muscle cells, CGRP modulates the AChE expression in a time-dependent manner, initially stimulating the enzyme synthesis through a cyclic AMP-dependent mechanism. The decreased AChE synthesis observed after long-term CGRP treatment suggests that CGRP signalling system is subject to desensitization or down-regulation, that might function as an important adaptative mechanism of the muscle ®bre in response to long-term changes in neuromuscular transmission.

CGRP, a Vasodilator Neuropeptide that Stimulates Neuromuscular Transmission and EC Coupling

“…Typically, an increase on intracellular levels of cAMP translates CGRP binding into physiological responses, such as vasodilatation of blood vessels [1] and trophic effects in muscle, including the synthesis and function of skeletal muscle acetylcholine receptors (AChR) and acetylcholinesterase (AChE) [26][27][28]. Nonetheless, other studies report that CGRP receptor activation is sequentially coupled to activation of phospholipase C, phosphatidilylinositol turnover, and calcium release from intracellular stores [29][30][31].…”

“…The trophic effects of CGRP on muscle cells include regulation of AChRs and acetylcholinesterase in rat neuromuscular junctions [26,28]. Specifically, application of CGRP in adult rat soleo in vivo induces AChR expression and accumulation at the neuromuscular junction [94].…”

Skeletal muscle expresses the extracellular cyclic AMP–adenosine pathway

“…For example, when calcitonin gene‐related peptide is released by motor neurons at the synaptic cleft, there is a postsynaptic increment of intracellular cAMP through activation of adenylyl cyclase (AC; Poyner et al , 2002 ). Intracellular increase of cAMP induced by activation of AC accelerates desensitization of muscle nicotinic receptors and β‐adrenoceptors through mechanisms involving cyclic AMP‐dependent protein kinase ( Paradiso and Brehm, 1998 ; Swope et al , 1999 ; Lanuza and Gizaw, 2006 ; Vaughan et al , 2006 ), modulates acetylcholinesterase expression ( da Costa et al , 2001 ; Rossi et al , 2003 ; Xie et al , 2007 ) and regulates glucose uptake after stimulation of β2‐adrenoceptors ( Nevzorova et al , 2006 ).…”

Ectopic development of skeletal muscle induced by subcutaneous transplant of rat satellite cells

“…Briefly, the animals were killed under CO 2 anesthesia, the muscles were removed and the cells dissociated for 3 h in Hanks' balanced salt solution (HBSS), pH 7.4, containing collagenase type IA (200 U/ml) as described by da Costa et al (13). After mechanical dissociation, the cell suspension was centrifuged at 500 g for 5 min and the resulting pellet was resuspended in Dulbecco's Modified Eagle's Medium (DMEM, Gibco-BRL, Life Technologies, Grand Island, NY, USA) containing 40 mg/l gentamicin.…”