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A Espinosa, M Estrada, and E Jaimovich

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.

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M Estrada, A Espinosa, C J Gibson, P Uhlen, and E Jaimovich

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.