The Na+-K+-ATPase and its regulation is important for maintaining membrane potential and transmembrane Na+ gradient in all skeletal muscle cells and thus is essential for cell survival and function. In our previous study, cyclic stretch activated the Na pump in cultured skeletal muscle cells. Presently, we investigated whether this stimulation was the result of translocation of Na+-K+-ATPase from endosomes to the plasma membrane, and also evaluated the role of phosphatidylinositol 3-kinase (PI 3-kinase), the activation of which initiated vesicular trafficking and targeting of proteins to specific cell compartments. Skeletal muscle cells were stretched at 25% elongation continuous for 24 h using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na+-K+-ATPase alpha(1)- and alpha(2)-subunit protein. The results showed stretch increased Na+-K+-ATPase alpha(1)-and alpha(2)-subunit protein expression in plasma membrane fractions and decreased it in endosomes. The alpha(2)-subunit had a more dynamic response to mechanical stretch. PI 3-kinase inhibitors (LY294002) blocked the stretch-induced translocation of the Na+-K+-ATPase alpha(2)-subunit, while LY294002 had no effect on the transfer of alpha(1)-subunit. We concluded that cyclic stretch mainly stimulated the translocation of the alpha(2)-subunit of Na+-K+-ATPase from endosomes to the plasma membrane via a PI 3-kinase-dependent mechanism in cultured skeletal muscle cells in vitro, which in turn increased the activity of the Na pump. (c) 2006 Elsevier Inc. All rights reserved.