Abstract
Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.
Highlights
Contractions activate the sodium pump, Naϩ,Kϩ-ATPase, and the energy sensor, AMP-activated protein kinase (AMPK), in skeletal muscle
AMPK Activation Increases Transport Activity and Cell Surface Abundance of Naϩ,Kϩ-ATPase in L6 Muscle Cells—Ouabain-sensitive 86Rbϩ uptake and cell surface biotinylation was measured to assess the activity and abundance of the sodium pump in the plasma membrane in the presence or absence of AMPK activators (AICAR or cyanide). Both AICAR and cyanide increased AMPK phosphorylation at Thr172 in L6 cells (Fig. 1A), concomitant with increased cell surface expression of ␣1-subunit of the sodium pump (Fig. 1B) and pump activity (Fig. 1C). These data provide evidence to suggest that AMPK activation increases expression of the ␣1-subunit Naϩ,Kϩ-ATPase at the plasma membrane, as well as the activity of the pump in L6 cells
Cell surface expression of the Naϩ,Kϩ-ATPase ␣1-subunit was quickly normalized during the washout and was markedly increased after a 2-h incubation with starvation medium, which was followed by return to the basal level (Fig. 2C)
Summary
Contractions activate the sodium pump, Naϩ,Kϩ-ATPase, and the energy sensor, AMP-activated protein kinase (AMPK), in skeletal muscle. Contraction stimulates Na؉,K؉-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. AMPK stimulation leads to Na؉,K؉-ATPase ␣1-subunit dephosphorylation at Ser, which may prevent endocytosis of the sodium pump. AICAR-triggered dephosphorylation of the Na؉,K؉-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A1⁄7PME-1 complex in AMPK-mediated regulation of Na؉,K؉ATPase. Contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle
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