AMP-activated protein kinase activator A-769662 is an inhibitor of the Na+-K+-ATPase

Abstract

Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AMPK). AMPK restores energy balance by activating processes that produce energy while inhibiting those that consume energy. The role of AMPK in the regulation of active ion transport is unclear. Our aim was to determine the effect of the AMPK activator A-769662 on Na+-K+-ATPase function in skeletal muscle cells. Short-term incubation of differentiated rat L6 myotubes with 100 μM A-769662 increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in parallel with decreased Na+-K+-ATPase α1-subunit abundance at the plasma membrane and ouabain-sensitive86Rb+uptake. Notably, the effect of A-769662 on Na+-K+-ATPase was similar in muscle cells that do not express AMPK α1- and α2-catalytic subunits. A-769662 directly inhibits the α1-isoform of the Na+-K+-ATPase, purified from rat and human kidney cells in vitro with IC5057 μM and 220 μM, respectively. Inhibition of the Na+-K+-ATPase by 100 μM ouabain decreases sodium pump activity and cell surface abundance, similar to the effect of A-769662, without affecting AMPK and ACC phosphorylation. In conclusion, the AMPK activator A-769662 inhibits Na+-K+-ATPase activity and decreases the sodium pump cell surface abundance in L6 skeletal muscle cells. The effect of A-769662 on sodium pump is due to direct inhibition of the Na+-K+-ATPase activity, rather than AMPK activation. This AMPK-independent effect on Na+-K+-ATPase calls into question the use of A-769662 as a specific AMPK activator for metabolic studies.