A method for the determination of the total number of³H-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 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.

Effect of strenuous strength training on the Na-K pump concentration in skeletal muscle of well-trained men

“…This conclusion should be drawn with some caution since possible variations in the techniques used by dierent laboratories have not been examined. However, since all studies have used the approach of Nùrgaard et al (1984) and Kjeldsen et al (1986) for optimal binding of 3 H-ouabain, these variations may probably be quite small.…”

“…3.6.1.37) in biopsies of 3±10 mg was determined as described elsewhere (Evertsen et al 1997). These authors used the method described by Nùrgaard et al (1983Nùrgaard et al ( , 1984 and Kjeldsen (1986) but used N-2-hydroxyethylpiperazine-N 0 -2-ethanesulphonic acid (HEPES) as buer rather than TRIS in agreement with the recommendations of Good et al (1966). Separate control experiments showed that incubation in HEPES gave as high ouabain binding as in TRIS but that the variability was reduced (not shown).…”

Human neuromuscular fatigue is associated with altered Na+-K+-ATPase activity following isometric exercise

“…The content of Na ϩ -K ϩ pumps in muscle biopsy samples was determined using the assay developed by Nørgaard et al (38) and employed previously in our laboratory (10). With the use of this procedure, two samples from each biopsy, weighing between 2 and 8 mg, are prewashed twice for 10-min periods in a Tris-sucrose buffer (10 mM Tris ⅐ HCl, 3 mM MgSO4, 1 mM Tris-vanadate, and 250 mM sucrose) containing sodium metavanadate (NaVO3) at 0°C.…”

Changes in Muscle Fiber Cross-Sectional Area and Concentrations of Na,K-ATPase in Deltoid Muscle in Patients With Impingement Syndrome of the Shoulder

“…This procedure might induce several sources of error, such as loss of tissue constituents during incubation, tissue swelling during incubation with ouabain, and unequal blotting and evaporation before and during weighing. However, experiments have shown that the procedure does not change the net wet weight of the samples ( 18). Determination of sample wet weight 1 or 2 minutes after blotting causes an overestimation of the ('H)ouabain binding site concentration by factors of 1.03 or 1.05, respectively.…”