Fig. 1.

Regulation of MLCK activity and muscle contraction by AMPK. Longitudinal muscle cells isolated from control mice were treated with 1 µM ACh in the presence or absence of CaMKK inhibitor STO609 (STO; 10 µM) for 30 seconds. Cells were preincubated with STO609 for 15 minutes before the addition of ACh for 30 seconds. (A) Phosphorylation of AMPK at Thr¹⁷² by CaMKK. Phosphorylation of AMPK was analyzed by Western blot using phospho-specific antibody. (B) Stimulation of ACh-induced AMPK activity by CaMKK. AMPK activity was measured by immunokinase assay using MLCK as substrate and [³²P]ATP. The results are expressed as cpm/mg protein. (C) Phosphorylation of MLCK by AMPK. Phosphorylation of MLCK was measured in cells labeled with ³²P. (D) Feedback inhibition of ACh-induced MLCK activity by AMPK. MLCK activity was measured by immunokinase assay using MLC₂₀ as substrate and [³²P]ATP, and the results are expressed as cpm/mg protein. (E) Attenuation of ACh-induced MLC₂₀ phosphorylation by AMPK. MLC₂₀ phosphorylation at Ser¹⁹ was measured by immunoblot using phospho-specific antibody. (F) Attenuation of ACh-induced muscle contraction by AMPK. Muscle contraction was measured by scanning micrometry and is expressed as the percentage of decrease in cell length from the basal length (108 ± 7 µm). Values are means ± S.E.M. of four experiments. **P < 0.05, significantly different from response to ACh alone.