Capacitative calcium entry in testosterone-induced intracellular calcium oscillations in myotubes.
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 Ca(2+)-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 Ca(2+)-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 Ca(2+)-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.
Estrada
M
M
Centro de Estudios Moleculares de la Célula and Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 6530499, Chile.
Espinosa
A
A
Gibson
C J
CJ
Uhlen
P
P
Jaimovich
E
E
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects*; D006614:Hindlimb; D007091:Image Processing, Computer-Assisted; D007424:Intracellular Fluid / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D018485:Muscle Fibers, Skeletal / Q000378:metabolism*; D018482:Muscle, Skeletal; D051381:Rats; D012815:Signal Processing, Computer-Assisted; D013739:Testosterone / Q000494:pharmacology*; D046509:Tissue Culture Techniques
D013739:Testosterone; D002118:Calcium