Calcium transients in 1B5 myotubes lacking ryanodine receptors are related to inositol trisphosphate receptors

Abstract

Potassium depolarization of skeletal myotubes evokes slow calcium waves that are unrelated to contraction and involve the cell nucleus (Jaimovich, E., Reyes, R., Liberona, J. L., and Powell, J. A. (2000) Am. J. Physiol. 278, C998-C1010). Studies were done in both the 1B5 (Ry53-/-) murine "dyspedic" myoblast cell line, which does not express any ryanodine receptor isoforms (Moore, R. A, Nguyen, H., Galceran, J., Pessah, I. N., and Alien, P. D. (1998) J. Cell Biol. 140, 843-851), and C2C12 cells, a myoblast cell line that expresses all three isoforms. Although 1B5 cells lack ryanodine binding, they bind tritiated inositol (1,4,5)-trisphosphate. Both type 1 and type 3 inositol trisphosphate receptors were immune-located in the nuclei of both cell types and were visualized by Western blot analysis. After stimulation with 47 mM K+, inositol trisphosphate mass raised transiently in both cell types. Both fast calcium increase and slow propagated calcium signals were seen in C2C12 myotubes. However, 1B5 myotubes (as well as ryanodine-treated C2C12 myotubes) displayed only a long-lasting, non-propagating calcium increase, particularly evident in the nuclei. Calcium signals in 1B5 myotubes were almost completely blocked by inhibitors of the inositol trisphosphate pathway: U73122, 2-aminoethoxydiphenyl berate, or xestospongin C. Results support the hypothesis that inositol trisphosphate mediates slow calcium signals in muscle cell ryanodine receptors, having a role in their time course and propagation.