A method for the determination of the total number of 3H-ouabain binding sites in biopsies of human skeletal muscle.

A new method based on vanadate facilitated binding of 3H-ouabain has been applied for the quantitative determination of the number of 3H-ouabain binding sites (Na-K-pumps) in needle biopsies of human skeletal muscle. Samples of the vastus lateralis muscle weighing 2-8 mg showed specific and saturable binding of 3H-ouabain with an apparent KD of 1.9 X 10(-8) mol/l. In 20 healthy human subjects in the age range 25-80 years, the number of 3H-ouabain binding sites was 278 +/- 15 pmol/g wet weight with no relation to age or sex. In samples of the intercostal and rectus abdominis muscles, the number of 3H-ouabain binding sites varied from 225 to 280 pmol/g wet weight. These values are at least 2 times higher than those previously reported for human skeletal muscle. The number of 3H-ouabain and 3H-digoxin binding sites were identical, and ouabain (10(-3) mol/l) completely displaced specifically bound 3H-digoxin. When biopsies were frozen in liquid N2 immediately after withdrawal, storage at -20 degrees C for up to 11 weeks caused no significant change in the number of 3H-ouabain binding sites. The method allows quantitative determination of the number of 3H-ouabain binding sites in standard biopsies of human skeletal muscle to be performed by simple procedures within a few hours. This can be used for the study of conditions where the number of Na-K-pumps is known to undergo fluctuations.