Changes in ryanodine receptor-mediated calcium release during skeletal muscle differentiation

We have observed a disparity between the actions of caffeine and ryanodine, two agents known to affect the same site of intracellular calcium (Ca2+) r elease in muscle. The site of intracellular Ca2+ release, the ryanodine rec eptor (RyR), is established as the route of Ca2+ movement from the sarcopla smic reticulum (SR) to the cytosol during excitation-contraction coupling. We measured Ca2+ release fluorimetrically in both saponin-permeabilized and intact L6 cells, in response to known modulators (i.e., caffeine and ryano dine), during differentiation in vitro. The undifferentiated L6 cells showe d little response to caffeine. However, a substantial caffeine-induced calc ium release (caffCR) was evident by Day 3 of differentiation, and was nearl y maximal by Day 7 of differentiation. By contrast, ryanodine failed to sti mulate Ca2+ release until Day 4, lagging behind the caffeine response. Ryan odine-stimulated Ca2+ release was also maximal by Day 7. Higher concentrati ons of ryanodine, known to inhibit Ca2+ release, only began to effect caffC R at Day 4, indicating that cells were insensitive to both ryanodine stimul ation and ryanodine inhibition prior to this time. Most of the results coul d be obtained both in permeabilized and intact cells. Using Intact cells, w e measured the time course of K+-dependent (i.e., depolarization-induced) C a2+ release. This time course matched caffeine and not ryanodine induced Ca 2+ release suggesting the action of caffeine was not due to Ca2+ release un related to excitation-contraction coupling. These findings suggest that rya nodine binding sites on the RyR may not be functional at early stages of mu scle development, that ryanodine sensitivity is a poor indicator of Ca2+ fl ux through the RyR, or that other proteins are involved in Ca2+ release und er certain circumstances.