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

Abstract

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 K_D of 1.9 × 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 ³ H-ouabain and ³H-digoxin binding sites were identical, and ouabain (10³ mol/l) completely displaced specifically bound ³H-digoxin. When biopsies were frozen in liquid N₂ immediately after withdrawal, storage at 20 °C for up to II 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.

Key Words:

• ³H-ouabain binding
• Na-K-pumps
• digitalis glycosides
• human skeletal muscle
• muscle biopsies