Sustained CGRP1 receptor stimulation modulates development of EC coupling by cAMP/PKA signalling pathway in mouse skeletal myotubes.

We investigated modulation of excitation-contraction (EC) coupling by calcitonin gene-related peptide (CGRP), which is released by motorneurons during neuromuscular transmission. Mouse skeletal myotubes were cultured either under control conditions or in the presence of 100 nm CGRP ( approximately 4-72 h). T- and L-type Ca(2+) currents, immobilization resistant charge movement, and intracellular Ca(2+) transients were characterized in whole-cell patch-clamp experiments. CGRP treatment increased the amplitude of voltage-gated Ca(2+) release ((DeltaF/F)(max)) approximately 75-350% and moderately increased both maximal L-current conductance (G(max)) and charge movement (Q(max)). In contrast, CGRP treatment did not affect their corresponding voltage dependence of activation (V(1/2) and k) or T-current density. CGRP treatment enhanced voltage-gated Ca(2+) release in approximately 4 h, whereas the effect on L-channel magnitude took longer to develop ( approximately 24 h), suggesting that short-term potentiation of EC coupling may lead to subsequent long-term up-regulation of DHPR expression. CGRP treatment also drastically increased caffeine-induced Ca(2+) release in approximately 4 h ( approximately 400%). Thus, short-term potentiation of EC coupling is due to an increase in sarcoplasmic reticulum Ca(2+) content. Both application of a phosphodiesterase inhibitor (papaverine) and a membrane-permeant cAMP analogue (Db-cAMP) produced a similar potentiation of EC coupling. Conversely, this potentiation was prevented by pretreatment with either CGRP1 receptor antagonist (CGRP(8-37)) or a PKA inhibitor (H-89). Thus, CGRP acts through CGRP1 receptors and the cAMP/PKA signalling pathway to enhance voltage-gated Ca(2+) release. Effects of CGRP on both EC coupling and L-channels were attenuated at later times during myotube differentiation. Therefore, we conclude that CGRP accelerates maturation of EC coupling.