INSULIN INCREASES THE NA+-K+-ATPASE ALPHA-2-SUBUNIT IN THE SURFACE OF RAT SKELETAL-MUSCLE - MORPHOLOGICAL EVIDENCE

The cellular localization of the alpha2-subunit of the Na+-K+-ATPase was defined by immunoelectron microscopy, and the effect of insulin on the amount Of alpha2-immunoreactive subunits on the cell surface was quantitated. Two protocols were used for tissue fixation and immunolocalization. Protocol 1 was characterized by fixation with 2% paraformaldehyde, use of a monoclonal antibody, and detection with 3-nm-diameter gold-labeled Fab fragments or 10-nm gold-labeled immunoglobulin G. Protocol 2 was characterized by fixation with 4% paraformaldehyde plus 0.1% glutaraldehyde, use of a polyclonal antibody, and detection with 10-nm gold-labeled protein A. In control muscle, the alpha2-Subunit of the Na+-K+-ATPase was present at the plasma membrane and in intracellular tubular and vesicular structures located in subsarcolemmal and triadic regions. Acute insulin stimulation increased the number of immunolabeled alpha2-subunits in the plasma membrane after both fixation protocols. The gain in the plasma membrane ranged from 1.5- to 3.7-fold and was significant at the level of P < 0.005. These results provide morphological quantitative evidence that the alpha2-subunit of the Na+-K+-ATPase is present both at the plasma membrane and intracellularly in mammalian skeletal muscle and that insulin acutely increases its abundance in the muscle surface.