Proceedings of The Physiological Society

Kings College London (2005) J Physiol 565P, PC132

Communications

Unique patterns of calcium transients associate to hormone response in skeletal muscle cells

Espinosa, Alejandra ; Estrada, Manuel ; Jaimovich, Enrique ;

1. Centro de Estudios Moleculares de la Celula, ICBM, Universidad de Chile, Facultad de Medicina, Santiago, Chile.


Both K+ depolarization and tetanic electrical stimulation have been shown to produce slow Ca2+ signals, unrelated to contraction and associated to regulation in gene expression in muscle cells (Carrasco et al. 2003; Eltit et al. 2004). Several hormones are known to produce anabolic effects in skeletal muscle. We studied the effect of IGF-I, insulin and testosterone on intracellular Ca2+ in primary cultured myotubes and cell lines. The fluorescent dye Fluo-3 AM was used for temporal and spatial characterization of Ca2+ signals. Insulin produced a fast (<1 s) and transient Ca2+ increase lasting less than 10 s. IGF-I induced a transient Ca2+ increase, reaching a fluorescence peak 6 s after stimulus, to return to basal values after 60 s (Espinosa et al. 2004). Testosterone induced delayed (35 s) and long lasting (100-200 s) signals, frequently associated to oscillations (Estrada et al. 2003). We studied the role of capacitative calcium entry (CCE) on intracellular Ca2+ oscillations induced by testosterone at the single cell level in primary myotubes. Testosterone (100 nM) rapidly induced an intracellular Ca2+ rise, accompanied by Ca2+ oscillations with a periodicity of 20.3 ± 1.8 s (mean ± SD) in the majority (76%) of myotubes. In Ca2+-free medium, an increase in intracellular Ca2+ was still observed, but no oscillations. Neither nifedipine nor ryanodine affected the testosterone-induced Ca2+ response. Intracellular Ca2+ store depletion in Ca2+-free medium, using a SERCA-pump inhibitor, followed by re-addition of extracellular Ca2+ gave a fast rise in intracellular Ca2+, indicating that CCE was present in these myotubes. Application of either testosterone or albumin-bound testosterone induced Ca2+ release and led to CCE after re-addition of Ca2+ to Ca2+-free extracellular medium. The CCE blockers 2-APB and La3+, inhibited testosterone-induced Ca2+ oscillations and CCE. The steady increase in Ca2+ induced by testosterone was not, however, affected by La3+. These results demonstrate testosterone-induced Ca2+ oscillations in myotubes, mediated by the interplay of IP3-sensitive Ca2+ stores and Ca2+ influx through CCE. G protein inhibitors, PTX and GDPbS, only slightly modified the response to IGF-1, but both testosterone and insulin-induced Ca2+ increase was blocked. The different intracellular Ca2+ patterns produced by testosterone, IGF-1 and insulin, may help to understand the role of intracellular calcium kinetics in the regulation of gene expression by hormones in skeletal muscle cells.

Where applicable, experiments conform with Society ethical requirements