Aage Nørgaard

Diabetes Decreases Na + -K + Pump Concentration in Skeletal Muscles, Heart Ventricular Muscle, and Peripheral Nerves of Rat

Na+-K+-ATPase or the Na+-K+ pump is essential for some specific properties of muscle and nerve tissue such as contractility and excitability. Previous studies have shown conflicting variations in Na+-K+-ATPase activity or Na+-K+ pump concentration of muscle cells in experimental diabetes. Our study demonstrates that early untreated diabetes in rats induced by injection of streptozocin is associated with decreases in [3H]ouabain binding-site concentration of 24-48% in various skeletal muscles and 16% in peripheral nerves as well as a decrease in K+-dependent 3-O-methylfluorescein phosphatase activity of 21% in the heart ventricle. These effects could be prevented by insulin treatment. They probably represent a decrease in the concentration of Na+-K+ pumps. There was no evidence for more than one population of Na+-K+ pumps in intact samples of skeletal muscle and nerves from normal, diabetic, and insulin-treated animals. The decrease in Na+-K+ pump concentration in nerve cells may be due to atrophy of the axons. In skeletal muscles, myocardium, and peripheral nerves, the observed decrease in Na+-K+ pump concentration may be important for the pathophysiology of diabetes. We emphasize that quantification of Na+-K+-ATPase or the Na+-K+ pump in muscle and nerve tissue from diabetic animals should preferably be performed with either intact samples or crude homogenates of whole tissue.

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.

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.

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.

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.

K+-dependent 3-O-methylfluorescein phosphatase activity in crude homogenate of rodent heart ventricle: Effect of K+ depletion and changes in thyroid status

The total Na+-K+-ATPase activity in rodent heart ventricle was quantified by determination of K+-dependent, ouabain suppressible 3-O-methylfluorescein phosphatase activity. A K+-dependent phosphatase activity of 0.80 mumol/min per g wet wt was obtained from crude homogenate of heart ventricle from 4-week-old guinea pigs. The anticipated association between K+-dependent phosphatase activity and Na+-K+-activated ATP hydrolysis could be clearly demonstrated in the crude homogenate. Based upon a molecular activity of 550/min the corresponding cardiac glycoside receptor concentration in the crude homogenate was 1450 pmol/g wet wt. In crude homogenate of heart ventricle from 3-month-old rats the K+-dependent phosphatase activity was 1.16 mumol/min per g wet wt. Following 4 weeks of K+ depletion of the rats, a decrease of 13% in total K+ content of the heart ventricle was seen. This was associated with a 14% decrease in K+-dependent phosphatase activity. The induction of hypo- and hyperthyroidism for 3 weeks in rats was followed by a decrease of 27% and an increase of 13% in K+-dependent phosphatase activity, respectively.

Heart Na, K-ATPase activity in cardiomyopathic hamsters as estimated from K-dependent 3-O-MFPase activity in crude homogenates

The hamster hereditary cardiomyopathy provides a unique model for the study of membrane abnormalities during chronic congestive heart failure. It is associated with intracellular calcium accumulation, mitochondrial calcification and cell necrosis. Previous studies have shown a decrease in Na,K-ATPase activity purified from ventricle sarcolemma. The present study demonstrates a decrease in K-dependent 3-O-methylfluorescein phosphatase (3-O-MFPase) activity from 1.93 to 1.30 mumol/g wet wt. or 33% in crude homogenates from the left ventricle of 7-months-old cardiomyopathic hamsters as compared to control animals. This represents an equivalent decrease in Na, K-ATPase activity. The values are several times higher than previously published for membrane fractions of myocardium from the hamster. Concomitantly, there was an increase in intracellular Na-concentration of the myocardium of 42% whereas the K-concentration was unchanged. The decrease in Na,K-pump concentration may be of importance for the increase in intracellular sodium and ensuing calcifying necrosis observed in the myocardium of cardiomyopathic hamsters. It is emphasized that quantification of the Na,K-ATPase or Na,K-pump should preferably be performed using crude homogenates.