UTP addition induces calcium release from cultured muscle cells but not from adult muscle fibers. Here, individual traces represent the [Ca²⁺]_i responses measured by Fura-2 video microscopy in single cells from the same field in a representative experiment. Similar results were obtained in at least three different experiments. Variations in [Ca²⁺]_i over time are represented by the ratio between the fluorescence intensities at 340- and 380-nm excitation wavelengths. (A) Changes in the 340/380 ratio of Fura-2 in C2C12 myotubes after UTP stimulation (100 µM). Top panels show the amount of calcium released at various time points (as indicated by a–d), with pseudocolors indicating the relative intensities. Each trace represents the calcium changes in an individual myotube (four different myotubes are shown, outlined in red, green, blue, and yellow). (B) Preincubation with 70 µM 2-APB inhibits [Ca²⁺]_i elicited by 100 µM UTP in C2C12 myotubes. The addition of ionomycin leads to a significant increase in intracellular calcium. (C) Response of adult fibers to the addition of 100 µM UTP. Traces of two individual fibers are shown. (D) Quantification of the response of C2C12 myotubes and adult single fibers (n = 25 for C2C12 and n = 10 for adult fibers). (E) Increase in [Ca²⁺]_i induced in two adult fibers by 10 mM caffeine. (F) The effect of caffeine is significantly reduced by the addition of 20 µM dantrolene, an inhibitor of RyR.