The fibers that compose mammalian skeletal muscles may differ greatly in both twitch speed and levels of enzymes of energy metabolism. Evidence suggests that this heterogeneity is caused by differences in the patterns of contractile activity among the fibers. Increased 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. I propose to use muscle cell culture to define ways in which muscle activity and differentiation control enzyme levels. 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. Pharmacological evidence suggests that the effects of muscle activity on enzyme levels are mediated by calcium. To investigate further, we will determine the effects of electrical stimulation and the divalent cation ionophore, A23187, on the rates of synthesis and degradation of selected enzymes. These experiments should provide information concerning the mechanisms by which calcium and muscle activity change enzyme levels. Under appropriate conditions, cultured myotubes contract spontaneously, so that the effects of activity may be investigated by comparing active cells to cells paralyzed with tetrodotoxin. Using this strategy, we have shown that muscle 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 regulating phosphorylase activity is phosphorylase kinase. The enzyme is composed of 4 nonidentical subunits (Alpha, Beta, Gamma, Delta)4. Red and white skeletal muscles contain isozymes that differ in the molecular weight of the Alpha subunits. Preliminary experiments indicate that both isozymes exist in cultured myotubes, and that interconversion of isozymes can be accomplished by modifying the contractile activity of these cells. I now propose to investigate the influences of differentiation and muscle activity on the synthesis, degradation, and assembly of the kinase subunits.
| Lawrence Jr, J C; Piper, R C; Robinson, L J et al. (1992) GLUT4 facilitates insulin stimulation and cAMP-mediated inhibition of glucose transport. Proc Natl Acad Sci U S A 89:3493-7 |
| Lawrence Jr, J C; Smith, R L (1990) Phosphorylase kinase isozymes and phosphorylase in denervated skeletal muscles. Muscle Nerve 13:133-7 |
| James, D E; Hiken, J; Lawrence Jr, J C (1989) Isoproterenol stimulates phosphorylation of the insulin-regulatable glucose transporter in rat adipocytes. Proc Natl Acad Sci U S A 86:8368-72 |
