Keld Kjeldsen

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.

Quantification of the maximum capacity for active sodium-potassium transport in rat skeletal muscle.

1. Intact skeletal muscle fibres have been shown to contain a high concentration of [3H]ouabain binding sites (100‐800 pmol g wet wt.‐1). Under resting conditions, however, it seems that in isolated muscles only 2‐6% of the corresponding expected capacity for active Na+‐K+ transport is utilized. 2. In order to determine whether all [3H]ouabain binding sites in rat soleus muscle represent functional Na+‐K+ pumps, we have measured the maximum rates of the ouabain‐suppressible components of isotopic fluxes of Na+ and K+ as well as the net changes in Na+‐K+ contents. 3. Experiments with soleus muscles isolated from 4‐week‐old rats showed that following Na+ loading (I.C. Na+, 126 mmol l‐1), the ouabain‐suppressible 86Rb+ uptake and 22Na+ efflux as measured during 3 min of exposure to K+‐rich buffer were 5800 and 6500 nmol g wet wt.‐1 min‐1, respectively. 4. These initial high rates of isotopic fluxes were confirmed by flame photometric measurements of Na+‐K+ contents. The ouabain‐suppressible 86Rb+ uptake had a temperature coefficient of 2.1, was inhibited by 2,4‐dinitrophenol, but showed no response to tetracaine, BaCl2, Ca2+‐free buffer or tetraethylammonium chloride. 5. In soleus muscles, where the total population of [3H]ouabain binding sites had undergone changes as a result of differentiation, K+ depletion or pre‐treatment with thyroid hormone, there was a significant correlation (r = 0.95, P less than 0.005) between the concentration of [3H]ouabain binding sites (260‐1170 pmol g wet wt.‐1) and the maximum ouabain‐suppressible 86Rb+ uptake (2300‐10,900 nmol g wet wt.‐1 min‐1). 6. It is concluded that by the combination of Na+ loading and high extracellular K+, the available Na+‐K+ pumps as quantified by the [3H]ouabain binding capacity can be activated to reach a transport rate around 90% of the theoretical maximum at 30 degrees C.

A simple and rapid method for the determination of the number of 3H-ouabain binding sites in biopsies of skeletal muscle

Summary Based on vanadate facilitation of 3H-ouabain binding to Na-K-ATPase, a method has been developed allowing the measurement of the number of 3H-ouabain binding sites in small rat muscle biopsies (2–14 mg). Comparison with results obtained using intact fibres showed close agreement over the wide range of values for 3H-ouabain binding sites (100–900 pmol per g wet weight) associated with variations in age, thyroid status and K-deficiency.

Training increases the concentration of [3H]ouabain-binding sites in rat skeletal muscle.

Exercise is associated with a net loss of K+ from the working muscles and an increased plasma K+ concentration, indicating that the capacity for intracellular reaccumulation of K+ is exceeded. Training reduces the exercise-induced rise in plasma K+, and an increased plasma [K+] may interfere with physical performance. Since the clearing of K+ from the extracellular space depends on the capacity for active K+ uptake in skeletal muscle, the effects of training and inactivity on the total concentration of (Na+ + K+)-ATPase was determined. Following 6 weeks of swim training, the concentration of [3H]ouabain-binding sites in rat hindlimb muscles was up to 46% (P less than 0.001) higher than in those obtained from age-matched controls. Whereas muscle Na+, K+ contents remained unchanged, the concentration of citrate synthase increased by up to 76% (P less than 0.001). Training induced no change in the [3H]ouabain-binding-site concentration in the diaphragm, but in the heart ventricles, the K+-dependent 3-O-methylfluorescein phosphatase activity increased by 20% (P less than 0.001). Muscle inactivity induced by denervation, plaster immobilisation or tenotomy reduced the [3H]ouabain-binding-site concentration by 20-30% (P less than 0.02-0.001) within 1 week. In conclusion, training leads to a significant and reversible rise in the concentration of (Na+ + K+)-ATPase in muscle cells. This may be of importance for the beneficial effects on physical performance by improving the maximum capacity for K+ clearance.

Relation of left ventricular function and Na,K-pump concentration in suspected idiopathic dilated cardiomyopathy

The possible relation between Na-K-pump concentration and left ventricular (LV) function was studied in 24 patients with suspected idiopathic dilated cardiomyopathy. This was done by measurement of 3H-ouabain binding to biopsies obtained during left-sided heart catheterization. In all patients light microscopy of biopsies was compatilel with dilated cardiomyopathy. Nineteen patients had impaired LV function as defined by NYHA/WHO and a Na,K-pump concentration of 331 +/- 19 pmol/g wet weight, whereas 5 patients had normal LV function and a Na,K-pump concentration of 559 +/- 62 pmol/g wet weight (p less than 0.001). The correlation between Na,K-pump concentration and ejection fraction was highly significant n = 24, r = 0.81, p less than 0.001). There was no correlation between volume fraction of collagen tissue and Na,K-pump concentration in the biopsies (n = 24, r = -0.08, p less than 0.80), indicating that the decrease in Na,K-pump concentration with dilated cardiomyopathy is not the simple outcome of increased fibrosis in the myocardium. The results indicate that the decrease in Na,K-pump concentration may be of importance for myocardial dysfunction and suggest a simple biochemical assessment of dilated cardiomyopathy by measurement of 3H-ouabain binding.

The age-dependent changes in the number of3H-ouabain binding sites in mammalian skeletal muscle

The influence of age on the binding of3H-ouabain in skeletal muscle has been characterized in rats, mice and guinea pigs. Measurements performed using biopsies and intact fibers obtained from different types of rat muscles showed that from birth to the 4th week of life, the number of3H-ouabain binding sites per unit weight increases up to 5-fold, followed by almost the same relative decrease to a plateau around 250 pmol/g wet wt at an age of 22 weeks. These changes were not associated with any major alterations in apparentKD (1.7–3.1×10−7M) dissociation rate or heterogeneity in binding characteristics. Measurements of 3-O-methylfluorescein phosphatase activity, an enzyme activity which is closely correlated to the Na−K-ATPase activity, confirmed the3H-ouabain binding data.In mice, the number of3H-ouabain binding sites showed similar, albeit less pronounced changes with age, a maximum being reached at the 4th week of life. In guinea pigs, the number of3H-ouabain binding sites per unit weight decreased by 60% from birth to maturity.The results indicate that the early development and differentiation of individual skeletal muscles is associated with a marked increase in the number of Na−K-pumps (when expressed as pmol/muscle), until at maturity a plateau is reached. However, when expressed as pmol/g wet wt the increase is followed by a decrease to a plateau. This may in part account for the relatively low digitalis sensitivity seen in infants as compared to newborn and mature individuals.

(Na+ + K+)-ATPase activity of crude homogenates of rat skeletal muscle as estimated from their K+-dependent 3-O-methylfluorescein phosphatase activity

A highly sensitive fluorimetric assay using 3-O-methylfluorescein phosphate as substrate was used in the determination of K+-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K+-dependent phosphatase activities in the range 0.19-0.37 mumol X (g wet weight)-1 X min-1 were obtained, the value decreasing with age and K+-deficiency. Complete inhibition of the K+-dependent phosphatase was obtained with 10(-3) M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K+-dependent phosphatase activity and (Na+ + K+)-activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min-1 was estimated from simultaneous determination of K+-dependent phosphatase activity and [3H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [3H]ouabain binding sites measured in intact muscles or biopsies hereof.

The effects of thyroid hormones on3H-ouabain binding site concentration, Na, K-contents and86Rb-efflux in rat skeletal muscle

Using a recently developed method based upon vanadate facilitated3H-ouabain binding, the total concentration of3H-ouabain binding sites was determined in biopsies of rat skeletal muscles containing varying proportions of slow-twitch fibres. In extensor digitorum longus, diaphragm, gastrocnemius and soleus muscles from mature (12-week-old) hyperthyroid rats the values obtained were respectively 2.6, 3.5, 5.1 and 9.8 times higher than those found in the same muscles from hypothyroid animals. This indicates that the effect of thyroid hormones is more pronounced on slow-twitch than on fast-twitch fibres. The changes in3H-ouabain binding site concentration with thyroid status could not be accounted for by differences in affinity or the rate of3H-ouabain binding.In young (4–5 week old) rats, where the K-content and the3H-ouabain binding site concentration in muscle had been reduced by K-depletion, T3-pretreatment produced an even larger relative increase in the3H-ouabain binding site concentration than in age-matched controls, but no increase in K-content. Therefore, the downregulation of3H-ouabain binding sites seen during K-depletion cannot be attributed to a decreased response to thyroid hormones. In normal rats the marked stimulating effect of thyroid hormone on the synthesis of3H-ouabain binding sites was not associated with any significant change in K-content, but clearly preceded by a significant (P<0.001) rise in the efflux of86Rb.

Effect of age, potassium depletion and denervation on specific displaceable [3H]ouabain binding in rat skeletal muscle in vivo

1. Following intraperitoneal injection of [3H]ouabain in rats, the isotope is rapidly distributed in blood plasma available for binding to the Na‐K‐ATPase in the plasma membranes of most tissues. In skeletal muscle tissue excised and washed 4 × 30 min in ice‐cold buffer, 95% of the 3H activity retained was shown to be [3H]ouabain using a specific binding assay.

The concentration of the Na,K-pump in skeletal and heart muscle in congestive heart failure

Na,K-ATPase (or the Na,K-pump) is essential for excitability and contractility of muscle tissue. Previous studies have shown a decrease in the concentration of this pump in endomyocardial biopsies from patients with dilated cardiomyopathy. The effect of congestive heart failure on the concentration of Na,K-ATPase in skeletal muscle was assessed in 16 patients by measurement of binding of 3H-ouabain to biopsies of the vastus lateralis muscle. Ten patients had impaired left ventricular function with an ejection fraction of 0.32 +/- 0.03 and a concentration of the Na,K-pump of 229 +/- 15 pmol/g wet weight in the skeletal muscle, whereas 6 patients had an ejection fraction of 0.66 +/- 0.05 (P less than 0.001) and a concentration of 307 +/- 17 pmol/g wet weight (P less than 0.01). In endomyocardial biopsies, the concentration of Na,K-ATPase was 340 +/- 37 and 500 +/- 39 pmol/g wet weight (P less than 0.025) in patients with impaired and normal ventricular function, respectively. There was a significant correlation between the concentration of the Na,K-pump in the biopsies of the skeletal muscle and ejection fraction, as well as between its concentration in the endomyocardial and skeletal muscular biopsies (r = 0.56, P less than 0.025 and r = 0.72, P less than 0.005, respectively). The decrease in concentration of the pump in skeletal muscle may contribute to the limitation of exercise capacity in congestive heart failure.