Search Results

We studied the effect of IGF-I and insulin on intracellular Ca(2+) in primary cultured myotubes. IGF-I induced a fast and transient Ca(2+) increase, measured as fluo-3 fluorescence. This response was blocked by both genistein and AG538. IGF-I induced a fast inositol-1,4,5-trisphosphate (IP(3)) increase, kinetically similar to the Ca(2+) rise. The Ca(2+) signal was blocked by inhibitors of the IP(3) pathway. On the other hand, insulin produced a fast (<1 s) and transient Ca(2+) increase. Insulin-induced Ca(2+) increase was blocked in Ca(2+)-free medium and by either nifedipine or ryanodine. In the normal muscle NLT cell line, the Ca(2+ )signals induced by both hormones resemble those of primary myotubes. GLT cells, lacking the alpha1-subunit of dihydropyridine receptor (DHPR), responded to IGF-I but not to insulin, while GLT cells transfected with the alpha1-subunit of DHPR reacted to both hormones. Moreover, dyspedic muscle cells, lacking ryanodine receptors, responded to IGF-I as NLT cells, however they show no insulin-induced calcium increase. Moreover, G-protein inhibitors, pertussis toxin (PTX) and GDPbetaS, blocked the insulin-induced Ca(2+) increase without major modification of the response to IGF-I. The different intracellular Ca(2+) patterns produced by IGF-I and insulin may improve our understanding of the early action mechanisms for these hormones in skeletal muscle cells.

Ca²⁺ oscillations are one of the most important signals within the cell. The mechanism for generation of Ca²⁺ oscillations is still not yet fully elucidated. We studied the role of capacitative Ca²⁺ 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 in a majority of myotubes. Spectral analysis of the Ca²⁺ oscillations revealed a periodicity of 20.3 ± 1.8 s (frequency of 49.3 ± 4.4 mHz). 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. This intracellular Ca²⁺ release was previously shown in myotubes to be dependent on inositol-1,4,5-trisphosphate (IP₃). Intracellular Ca²⁺ store depletion in Ca²⁺-free medium, using a sarcoplasmic/endoplasmic reticulum calcium ATPase-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-aminoethyl diphenylborate and La³⁺, as well as perturbation of the cytoskeleton by cytochalasin D, inhibited testosterone-induced Ca²⁺ oscillations and CCE. The steady increase in Ca²⁺ induced by testosterone was not, however, affected by either La³⁺ or cytochalasin D. These results demonstrate testosterone-induced Ca²⁺ oscillations in myotubes, mediated by the interplay of IP₃-sensitive Ca²⁺ stores and Ca²⁺ influx through CCE.