Torben Clausen

THE EFFECT OF CATECHOLAMINES ON NA-K TRANSPORT AND MEMBRANE POTENTIAL IN RAT SOLEUS MUSCLE

1. The action of catecholamines on the transport and the distribution of Na and K and the resting membrane potential (EM) has been investigated in soleus muscles isolated from fed rats.

TREATMENT OF ATTACKS IN HYPERKALÆMIC FAMILIAL PERIODIC PARALYSIS BY INHALATION OF SALBUTAMOL

In fifteen patients with hyperkalaemic familial periodic paralysis, inhalation of salbutamol alleviated hyperkaleamia and paralysis precipitated by exercise or oral administration of potassium chloride. In-vitro studies with rat soleus muscles indicated that the hypokalaemic effect of salbutamol is related to stimulation of the active coupled transport of sodium and potassium in muscle cells. Follow-up studies proved that the inhalation of salbutamol is a simple and adequate method for the treatment of the paralytic episodes in these patients.

The relationship between the transport of glucose and cations across cell membranes in isolated tissues.

1. 1. The permeability of adipocytes to a nonmetabolizable sugar was estimated by measuring the washout of 3-O-[14C]methylglucose from preloaded whole epididymal fat pads into a sugar-free environment. 2. 2. After an initial rapid phase, the release of 3-O-[14C]methylglucose was found to proceed with a rate coefficient of 0.0122 for up to 120 min. 3. 3. The release of 3-O-[14C]methylglucose was promptly stimulated by insulin (10–1000 μunits/ml) and inhibited by phlorizin (5 mM) and phloretin (1 mM). 4. 4. In a K+-free medium or in the presence of ouabain, the release of 3-O-[14C]methylglucose was gradually augmented, maximal effects being obtained after around 50 min. 5. 5. Other factors (2,4-dinitrophenol, CN−, epinephrine and ACTH), which could also be shown to reduce the K+ content of the tissue, stimulated 3-O-[14C]methylglucose release with a similar time-lag.

Relations between excitability and contractility in rat soleus muscle: role of the Na+-K+ pump and Na+/K+ gradients

1 The effects of reduced Na+/K+ gradients and Na+‐K+ pump stimulation on compound action potentials (M waves) and contractile force were examined in isolated rat soleus muscles stimulated through the nerve. 2 Exposure of muscles to buffer containing 85 mM Na+ and 9 mM K+ (85 Na+/9 K+ buffer) produced a 54 % decrease in M wave area and a 50 % decrease in tetanic force compared with control levels in standard buffer containing 147 mM Na+ and 4 mM K+. Subsequent stimulation of active Na+‐K+ transport, using the β2‐adrenoceptor agonist salbutamol, induced a marked recovery of M wave area and tetanic force (to 98 and 87 % of the control level, respectively). Similarly, stimulation of active Na+‐K+ transport with insulin induced a significant recovery of M wave area and tetanic force. 3 During equilibration with 85 Na+/9 K+ buffer and after addition of salbutamol there was a close linear correlation between M wave area and tetanic force (r= 0·92, P< 0·001). Similar correlations were found in muscles where tetrodotoxin was used to reduce excitability and in muscles fatigued by 120 s of continuous stimulation at a frequency of 30 Hz. 4 These results show a close correlation between excitability and tetanic force. Furthermore, in muscles depressed by a reduction in the Na+/K+ gradients, β‐adrenergic stimulation of the Na+‐K+ pump induces a recovery of excitability which can fully explain the previously demonstrated recovery of tetanic force following Na+‐K+ pump stimulation. Moreover, the data indicate that loss of excitability is an important factor in fatigue induced by high‐frequency (30 Hz) stimulation.

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.

Active Na—K transport and the rate of ouabain binding. The effect of insulin and other stimuli on skeletal muscle and adipocytes

1. The effect of stimulation or inhibition of active Na—K transport on [3H]ouabain binding has been investigated in isolated soleus muscles and adipocytes.

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.

Effect of thyroid function on number of Na-K pumps in human skeletal muscle.

To evaluate the effect of thyroid function on the number of Na-K pumps in skeletal muscle, the number of 3H-ouabain binding sites was measured in biopsy specimens from the vastus lateralis muscle of euthyroid subjects and patients with hypothyroidism or hyperthyroidism. In hypothyroidism there was a decrease of 50% in 3H-ouabain binding sites, and in hyperthyroidism there was an increase of 68% in 3H-ouabain binding sites. When thyroid status became normal after treatment the number of 3H-ouabain binding sites also became normal. There were significant correlations between several thyroid function tests and the number of 3H-ouabain binding sites. Free T4-index gave the highest r value (0.87). These changes may account for the variations in digitalis sensitivity associated with thyroid disorders.