Insulin increases the Na(+)-K(+)-ATPase alpha 2-subunit in the surface of rat skeletal muscle: morphological evidence.
The cellular localization of the alpha 2-subunit of the Na(+)-K(+)-ATPase was defined by immunoelectron microscopy, and the effect of insulin on the amount of alpha 2-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 alpha 2-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 alpha 2-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 alpha 2-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.
Marette
A
A
Division of Cell Biology, Hospital for Sick Children, Toronto, Ontario, Canada.
Krischer
J
J
Lavoie
L
L
Ackerley
C
C
Carpentier
J L
JL
Klip
A
A
D000818:Animals; D000911:Antibodies, Monoclonal; D002462:Cell Membrane / Q000187:drug effects / Q000201:enzymology; D004790:Enzyme Induction; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007328:Insulin / Q000494:pharmacology*; D046911:Macromolecular Substances; D016253:Microscopy, Immunoelectron / Q000379:methods; D009132:Muscles / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000494:pharmacology; D012508:Sarcolemma / Q000201:enzymology / Q000648:ultrastructure; D000254:Sodium-Potassium-Exchanging ATPase / Q000032:analysis / Q000096:biosynthesis*
D000911:Antibodies, Monoclonal; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D007328:Insulin; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D000254:Sodium-Potassium-Exchanging ATPase