Increased skeletal muscle activity in vivo is associated with increases in enzymes of oxidative energy metabolism and decreases in glycolytic enzymes. The mechanisms involved have not been established, due in part to experimental limitations imposed by working with whole animals. An alternative is muscle cell culture which provides a model system for investigating mechanisms involved in both differentiation and muscle activity. In the absence of nerve, cultured muscle cells develop high levels of glycolytic enzymes and low levels of oxidative enzymes. Primary cultures of rat myotubes will be electrically stimulated to determine if increased activity (in the absence of neural influences) is sufficient to promote conversion to a more oxidative enzymatic complement. Phamacological evidence suggests that the effects of muscle activity on enzyme levels are mediated by calcium. To investigate further, the effects of electrical stimulation and the divalent cation ionophore, A23187, on the rates of synthesis and degradation of selected enzymes will be determined. These experiments should provide information with respect to the mechanisms by which calcium and muscle activity change enzyme levels. Under appropriate conditions cultured myotubes contract spontaneously. This activity leads to an increase in phosphorylase degradation and a fall in the level of the enzyme. The mechanism of the activity-dependent control of degradation will be investigated in both cultured myotubes and skeletal muscle in organ culture. A key enzyme in regulation phosphorylase activity is phosphorylase kinase. The enzyme is composed of 4 nonidentical subunits (Alpha, Beta, Gamma, Delta)4. Red and white skeletal muscle contain isozymes that differ in the molecular weight of the Alpha subunits. Experiments will be performed to determine which of the isozymes exist in uninnervated cultured myotubes, and to find out if interconversion of isozymes can be accomplished by modifying the contractile activity of these cells. In addition, the possible influences of differentiation and muscle activity on the synthesis, degradation, and assembly of the 4 nonidentical subunits of the kinase will be investigated.