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Muscle development involves a series of complex cell-cell interactions that are mediated, at least in part, by several different cell adhesion molecules. Previous work from this lab showed that the different isoforms of NCAM and its level of polysialylation are developmentally regulated during chick myogenesis in vivo and that this regulation is important for normal muscle development. Using developing chick secondary myotubes grown in culture, we show here that both the polysialylation of NCAM and the developmental switch in isoform expression are regulated by activity and that Ca2+ entry through voltage-gated channels and the subsequent activation of protein kinase C are required for the developmental changes in NCAM isoform synthesis. Specifically, PSA expression was shown to be developmentally regulated with high expression being temporally correlated with the onset of spontaneous contractile activity. Furthermore, blocking contractile activity caused a decrease in PSA expression, while increasing activity with electrical stimulation resulted in its up-regulation. Immunoblot and metabolic labeling studies indicated that dividing myoblasts synthesize primarily 145-kD NCAM, newly formed, spontaneously contracting myotubes synthesize 130-, 145-, and 155-kD NCAM isoforms, while older, more mature myotubes primarily synthesize the glycosylphosphatidylinositol-anchored 130-kD isoform which, in contrast to the other three isoforms, had a high rate of turnover. This developmental switch in NCAM isoform expression could be inhibited with Ca2+ channel blockers and inhibitors of protein kinase C. Taken together, these results suggest that Ca2+ ions and protein kinase C are involved in a second messenger cascade coupling membrane depolarization with transcriptional factors that regulate NCAM isoform synthesis and polysialylation.