Calcium release induced by high K⁺ and caffeine in cultured skeletal muscle cells of embryonic chicken

Abstract.

To further understand the function of excitation–contraction coupling in skeletal muscle cells developing in vitro, Ca²⁺ transients elicited by high-K⁺ depolarization in the presence and absence of extracellular Ca²⁺ were compared with Ca²⁺ release induced by caffeine in cultured skeletal muscle cells isolated from 9-day-old chicken embryos (E9). Almost all myoblasts and myotubes cultured for 1 (E9I1) to 8 (E9I8) days responded to 80 mM [K⁺]_O with an elevation of [Ca²⁺]_i. Although all myotubes cultured for more than 4 days exhibited Ca²⁺ release independent of extracellular Ca²⁺, only about 50% of E9I1 and E9I2 cells maintained their response to Ca²⁺-free high-[K⁺]_O solution. Strikingly, a considerable proportion of cells of short-term culture were insensitive to 10 mM caffeine. Moreover, 46.8% of the caffeine-insensitive E9I1 and E9I2 cells, 29 out of 62, was still responsive to 80 mM [K⁺]_O in the absence of extracellular Ca²⁺. Western blot and immunocytochemistry showed that ryanodine receptor (RyRs) expression increases with culture. The Ca²⁺ release from caffeine-insensitive cells induced by Ca²⁺-free high-[K⁺]_O solution could be blocked by 100–200 µM ryanodine, which suggests the involvement of RyRs. Evidence is presented to show that a low resting [Ca²⁺]_i may be one factor responsible for the caffeine insensitivity of RyRs in cells of short-term culture.