Injection of IP₃ does not lead to an increase in intracellular calcium in adult skeletal muscle fibers. (A) Microinjection of 50 µM IP₃ mediated by patch clamp triggers a rapid Ca²⁺ response in a C2C12 myotube. Note that an increase in [Ca²⁺]_i is seen in the myotube injected with IP₃ (outlined in red) but not in noninjected neighboring myotube (outlined in green). (B) Microinjection of intracellular solution alone does not elicit any [Ca²⁺]_i variation in C2C12 myotubes. (C) Microinjection of IP₃ in adult single fibers does not induce any changes in [Ca²⁺]_i. As a positive control for injection, the membrane-impermeable dye Alexa red was injected simultaneously. Microinjection of ionomycin leads to a marked increase in the Fura-2 signal. Similar results were obtained in three different experiments; a total of 10 fibers were examined. (D) Microinjection of the fluorescent dye calcein was used to monitor the velocity of diffusion in adult single fibers. Four regions of interest are shown in a single fiber (yellow, red, green, and blue squares), with their respective increases in fluorescence in time shown in the graph (color of the line corresponds to the color of the square). The top panels are taken at two time points (indicated by a and b). Note that in all these experiments (A–D), the resting ratios differ from those in the experiments of Fig. 1, because the light source and the permissivity of the filters for both Fura-2 wavelengths were not the same in the two setups.