Excitation of skeletal muscle is a self-limiting process, due to run-down of Na+, K+ gradients, recoverable by stimulation of the Na+, K+ pumps.

The general working hypothesis of this study was that muscle fatigue and force recovery depend on passive and active fluxes of Na(+) and K(+). This is tested by examining the time-course of excitation-induced fluxes of Na(+) and K(+) during 5-300 sec of 10-60 Hz continuous electrical stimulation in rat extensor digitorum longus (EDL) muscles in vitro and in vivo using (22)Na and flame photometric determination of Na(+) and K(+). 60 sec of 60 Hz stimulation rapidly increases (22)Na influx, during the initial phase (0-15 sec) by 0.53 µmol(sec)(-1)(g wet wt.)(-1), sixfold faster than in the later phase (15-60 sec). These values agree with flame photometric measurements of Na(+) content. The progressive reduction in the rate of excitation-induced Na(+) uptake is likely to reflect gradual loss of excitability due to accumulation of K(+) in the extracellular space and t-tubules leading to depolarization. This is in keeping with the concomitant progressive loss of contractile force previously demonstrated. During electrical stimulation rat muscles rapidly reach high rates of active Na(+), K(+)-transport (in EDL muscles a sevenfold increase and in soleus muscles a 22-fold increase), allowing efficient and selective compensation for the large excitation-induced passive Na(+), K(+)-fluxes demonstrated over the latest decades. The excitation-induced changes in passive fluxes of Na(+) and K(+) are both clearly larger than previously observed. The excitation-induced reduction in [Na(+)]o contributes considerably to the inhibitory effect of elevated [K(+)]o. In conclusion, excitation-induced passive and active Na(+) and K(+) fluxes are important causes of muscle fatigue and force recovery, respectively.

Relationship between intracellular Na+ concentration and reduced Na+ affinity in Na+,K+-ATPase mutants causing neurological disease.

The neurological disorders familial hemiplegic migraine type 2 (FHM2), alternating hemiplegia of childhood (AHC), and rapid-onset dystonia parkinsonism (RDP) are caused by mutations of Na(+),K(+)-ATPase α2 and α3 isoforms, expressed in glial and neuronal cells, respectively. Although these disorders are distinct, they overlap in phenotypical presentation. Two Na(+),K(+)-ATPase mutations, extending the C terminus by either 28 residues ("+28" mutation) or an extra tyrosine ("+Y"), are associated with FHM2 and RDP, respectively. We describe here functional consequences of these and other neurological disease mutations as well as an extension of the C terminus only by a single alanine. The dependence of the mutational effects on the specific α isoform in which the mutation is introduced was furthermore studied. At the cellular level we have characterized the C-terminal extension mutants and other mutants, addressing the question to what extent they cause a change of the intracellular Na(+) and K(+) concentrations ([Na(+)]i and [K(+)]i) in COS cells. C-terminal extension mutants generally showed dramatically reduced Na(+) affinity without disturbance of K(+) binding, as did other RDP mutants. No phosphorylation from ATP was observed for the +28 mutation of α2 despite a high expression level. A significant rise of [Na(+)]i and reduction of [K(+)]i was detected in cells expressing mutants with reduced Na(+) affinity and did not require a concomitant reduction of the maximal catalytic turnover rate or expression level. Moreover, two mutations that increase Na(+) affinity were found to reduce [Na(+)]i. It is concluded that the Na(+) affinity of the Na(+),K(+)-ATPase is an important determinant of [Na(+)]i.

Quantification of Na+,K+ pumps and their transport rate in skeletal muscle: functional significance.

During excitation, muscle cells gain Na(+) and lose K(+), leading to a rise in extracellular K(+) ([K(+)]o), depolarization, and loss of excitability. Recent studies support the idea that these events are important causes of muscle fatigue and that full use of the Na(+),K(+)-ATPase (also known as the Na(+),K(+) pump) is often essential for adequate clearance of extracellular K(+). As a result of their electrogenic action, Na(+),K(+) pumps also help reverse depolarization arising during excitation, hyperkalemia, and anoxia, or from cell damage resulting from exercise, rhabdomyolysis, or muscle diseases. The ability to evaluate Na(+),K(+)-pump function and the capacity of the Na(+),K(+) pumps to fill these needs require quantification of the total content of Na(+),K(+) pumps in skeletal muscle. Inhibition of Na(+),K(+)-pump activity, or a decrease in their content, reduces muscle contractility. Conversely, stimulation of the Na(+),K(+)-pump transport rate or increasing the content of Na(+),K(+) pumps enhances muscle excitability and contractility. Measurements of [(3)H]ouabain binding to skeletal muscle in vivo or in vitro have enabled the reproducible quantification of the total content of Na(+),K(+) pumps in molar units in various animal species, and in both healthy people and individuals with various diseases. In contrast, measurements of 3-O-methylfluorescein phosphatase activity associated with the Na(+),K(+)-ATPase may show inconsistent results. Measurements of Na(+) and K(+) fluxes in intact isolated muscles show that, after Na(+) loading or intense excitation, all the Na(+),K(+) pumps are functional, allowing calculation of the maximum Na(+),K(+)-pumping capacity, expressed in molar units/g muscle/min. The activity and content of Na(+),K(+) pumps are regulated by exercise, inactivity, K(+) deficiency, fasting, age, and several hormones and pharmaceuticals. Studies on the α-subunit isoforms of the Na(+),K(+)-ATPase have detected a relative increase in their number in response to exercise and the glucocorticoid dexamethasone but have not involved their quantification in molar units. Determination of ATPase activity in homogenates and plasma membranes obtained from muscle has shown ouabain-suppressible stimulatory effects of Na(+) and K(+).

Excitation-induced exchange of Na+, K+, and Cl- in rat EDL muscle in vitro and in vivo: physiology and pathophysiology.

In skeletal muscle, excitation leads to increased [Na(+)](i), loss of K(+), increased [K(+)](o), depolarization, and Cl(-) influx. This study quantifies these changes in rat extensor digitorum longus (EDL) muscles in vitro and in vivo using flame photometric determination of Na(+) and K(+) and (36)Cl as a tracer for Cl(-). In vitro, 5-Hz stimulation for 300 s increased intracellular Na(+) content by 4.6 ± 1.2 µmol/g wet wt (P < 0.002) and decreased intracellular K(+) content by 5.5 ± 2.3 µmol/g wet wt (P < 0.03). This would increase [K(+)](o) by 28 ± 12 mM, sufficient to cause severe loss of excitability as the result of inactivation of Na(+) channels. In rat EDL, in vivo stimulation at 5 Hz for 300 s or 60 Hz for 60 s induced significant loss of K(+) (P < 0.01), sufficient to increase [K(+)](o) by 71 ± 22 mM and 73 ± 15 mM, respectively. In spite of this, excitability may be maintained by the rapid and marked stimulation of the electrogenic Na(+),K(+) pumps already documented. This may require full utilization of the transport capacity of Na(+),K(+) pumps, which then becomes a limiting factor for physical performance. In buffer containing (36)Cl, depolarization induced by increasing [K(+)](o) to 40-80 mM augmented intracellular (36)Cl by 120-399% (P < 0.001). Stimulation for 120-300 s at 5-20 Hz increased intracellular (36)Cl by 100-188% (P < 0.001). In rats, Cl(-) transport in vivo was examined by injecting (36)Cl, where electrical stimulation at 5 Hz for 300 s or 60 Hz for 60 s increased (36)Cl uptake by 81% (P < 0.001) and 84% (P < 0.001), respectively, indicating excitation-induced depolarization. Cl(-) influx favors repolarization, improving K(+) clearance and maintenance of excitability. In conclusion, excitation-induced fluxes of Na(+), K(+), and Cl(-) can be quantified in vivo, providing new evidence that in working muscles, extracellular accumulation of K(+) is considerably higher than previously observed and the resulting depression of membrane excitability may be a major cause of muscle fatigue.

Muscle phenotype in patients with myotonic dystrophy type 1.

INTRODUCTION: The pathogenesis of muscle involvement in patients with myotonic dystrophy type 1 (DM1) is not well understood. In this study, we characterized the muscle phenotype in patients with confirmed DM1. METHODS: In 38 patients, muscle strength was tested by hand-held dynamometry. Myotonia was evaluated by a handgrip test and by analyzing the decrement of the compound muscle action potential. Muscle biopsies were assessed for morphological changes and Na(+)-K(+) pump content. RESULTS: Muscle strength correlated with a decline in Na(+)-K(+) pump content (r = 0.60, P < 0.001) and with CTG expansion. CTG expansion did not correlate with severity of myotonia, proximal histopathological changes, or Na(+)-K(+) pump content. Histopathologically, we found few centrally placed nuclei (range 0.2-6.9%). CONCLUSIONS: The main findings of this study are that muscle weakness correlated inversely with CTG expansion and that central nuclei are not a prominent feature of proximal muscles in DM1.

Na+,K+-pump stimulation improves contractility in isolated muscles of mice with hyperkalemic periodic paralysis.

In patients with hyperkalemic periodic paralysis (HyperKPP), attacks of muscle weakness or paralysis are triggered by K(+) ingestion or rest after exercise. Force can be restored by muscle work or treatment with ß(2)-adrenoceptor agonists. A missense substitution corresponding to a mutation in the skeletal muscle voltage-gated Na(+) channel (Na(v)1.4, Met1592Val) causing human HyperKPP was targeted into the mouse SCN4A gene (mutants). In soleus muscles prepared from these mutant mice, twitch, tetanic force, and endurance were markedly reduced compared with soleus from wild type (WT), reflecting impaired excitability. In mutant soleus, contractility was considerably more sensitive than WT soleus to inhibition by elevated [K(+)](o). In resting mutant soleus, tetrodotoxin (TTX)-suppressible (22)Na uptake and [Na(+)](i) were increased by 470 and 58%, respectively, and membrane potential was depolarized (by 16 mV, P < 0.0001) and repolarized by TTX. Na(+),K(+) pump-mediated (86)Rb uptake was 83% larger than in WT. Salbutamol stimulated (86)Rb uptake and reduced [Na(+)](i) both in mutant and WT soleus. Stimulating Na(+),K(+) pumps with salbutamol restored force in mutant soleus and extensor digitorum longus (EDL). Increasing [Na(+)](i) with monensin also restored force in soleus. In soleus, EDL, and tibialis anterior muscles of mutant mice, the content of Na(+),K(+) pumps was 28, 62, and 33% higher than in WT, respectively, possibly reflecting the stimulating effect of elevated [Na(+)](i) on the synthesis of Na(+),K(+) pumps. The results confirm that the functional disorders of skeletal muscles in HyperKPP are secondary to increased Na(+) influx and show that contractility can be restored by acute stimulation of the Na(+),K(+) pumps. Calcitonin gene-related peptide (CGRP) restored force in mutant soleus but caused no detectable increase in (86)Rb uptake. Repeated excitation and capsaicin also restored contractility, possibly because of the release of endogenous CGRP from nerve endings in the isolated muscles. These observations may explain how mild exercise helps locally to prevent severe weakness during an attack of HyperKPP.

The secretory response of parathyroid hormone to acute hypocalcemia in vivo is independent of parathyroid glandular sodium/potassium-ATPase activity.

The involvement of sodium/potassium-ATPase in regulating parathyroid hormone (PTH) secretion is inferred from in vitro studies. Recently, the α-klotho-dependent rapid recruitment of this ATPase to the parathyroid cell plasma membrane in response to low extracellular calcium ion was suggested to be linked to increased hormone secretion. In this study, we used an in vivo rat model to determine the importance of sodium/potassium-ATPase in PTH secretion. Glands were exposed and treated in situ with vehicle or ouabain, a specific inhibitor of sodium/potassium-ATPase. PTH secretion was significantly increased in response to ethylene glycol tetraacetic acid-induced acute hypocalcemia and to the same extent in both vehicle and ouabain groups. The glands were removed, and inhibition of the ATPase was measured by (86)rubidium uptake, which was found to be significantly decreased in ouabain-treated parathyroid glands, indicating inhibition of the ATPase. As ouabain induced systemic hyperkalemia, the effect of high potassium on hormone secretion was also examined but was found to have no effect. Thus, inhibition of the parathyroid gland sodium/potassium-ATPase activity in vivo had no effect on the secretory response to acute hypocalcemia. Hence, the suggested importance of this ATPase in the regulation of PTH secretion could not be confirmed in this in vivo model.

In isolated skeletal muscle, excitation may increase extracellular K+ 10-fold; how can contractility be maintained?

During excitation, isolated rat muscles undergo a net loss of cellular K(+), which cannot be cleared via capillaries and can only be cleared to a small extent via diffusion into the surrounding buffer. Therefore, K(+) accumulates in the extracellular space and can be quantified using flame photometry and compared with contractile performance in extensor digitorum longus (EDL) and soleus muscles. During electrical stimulation, extracellular potassium concentration ([K(+)](o)) shows a rapid early rise, followed by a slower increase, reflecting progressive loss of excitability. Thus, after 30 s of 60 Hz stimulation in EDL, where [K(+)](o) reaches 50 mm, increasing the pulse duration from 0.2 to 1.0 ms augments force by 172%. Excitation-induced cellular loss of K(+) coincides with an equivalent gain of Na(+), in keeping with the one-for-one exchange of Na(+) and K(+). This Na(+)-K(+) exchange is completely reversible and is not accompanied by changes in the (14)C-sucrose space or release of intracellular enzyme activity, indicating that it is not due to unspecific cellular leakage. In EDL, 60 s of 20 Hz stimulation induced force elevation and increased [K(+)](o) to 43-47 mm. The force elevation was suppressed by ouabain (10(-5)m), indicating that the Na(+)-K(+) pump contributes to maintenance of excitability. After 15 s of 60 Hz stimulation, resting net re-extrusion of Na(+) was 7.3-fold faster than basal Na(+) efflux. Bumetanide, which blocks Na(+)-K(+)-2Cl(-) cotransport, caused no change in force and K(+) contents, excitation-induced loss of K(+) or postexcitatory reaccumulation of K(+). Omission of Cl(-) increased the rate of force decline 14-fold. In conclusion, during repeated excitation, isolated rat muscles undergo a much greater increase in [K(+)](o) than previously reported, sufficient to explain loss of force. This K(+) is not cleared via Na(+)-K(+)-2Cl(-) cotransport, but rather via Na(+)-K(+) pumps and by processes depending on Cl(-) exchange. These mechanisms are essential for the maintenance of force during intense contractions in vivo, where the clearance of K(+) via the capillaries may be suppressed.

Hormonal and pharmacological modification of plasma potassium homeostasis.

Human skeletal muscles contain the largest single pool of K+ in the body (2600 mmol, 46 times the total K+ content of the extracellular space). Intense exercise may double arterial plasma K+ in one min. This is because of excitation-induced release of K+ from the working muscle cells via K+ channels. This hyperkalemia is rapidly corrected by reaccumulation of K+ into the muscle cells via Na+,K+ pumps, often leading to hypokalemia. Hyperkalemia may also arise from muscle cell damage, excessive oral or intravenous administration of K+, acidosis, renal failure, depolarization of muscle cells with succinyl choline, activation of K+ channels by fluoride poisoning, hyperkalemic periodic paralysis, malignant hyperthermia, inhibition of the Na+,K+ pumps by digitalis glycosides or treatment with nonselective beta blockers. Hyperkalemia may cause arrhythmia and can be treated with beta2 agonists, insulin or hemodialysis. Hypokalemia may be induced by the stimulation of the Na+,K+ pumps in skeletal muscles seen postexercise, or by catecholamines, beta2 agonists, pheochromocytoma, theophylline, caffeine or insulin, by sepsis, myocardial infarction, trauma, burns and heart failure. Rare causes are hypokalemic periodic paralysis, inhibition of K+ channels by barium, chloroquine or barbiturates. Hypokalemia often reflects dietary K+ deficiency, alkalosis, renal or gastrointestinal loss of K+. Hypokalemia is more likely to cause arrhythmia than hyperkalemia and can be treated by oral or intravenous administration of K+ under frequent control of electrocardiogram and plasma K+. Because of their size and high contents of K+, Na+,K+ pumps and K+ channels, the skeletal muscles play a central role in the acute, from min-to-min ongoing regulation of plasma K+. This is decisive for the maintenance of muscle contractility and heart function.

Effects of step exercise on muscle damage and muscle Ca2+ content in men and women.

Eccentric exercise often produces severe muscle damage, whereas concentric exercise of a similar load elicits a minor degree of muscle damage. The cellular events initiating muscle damage are thought to include an increase in cytosolic Ca. It was hypothesized that eccentric muscle activity in humans would lead to a larger degree of cell damage and increased intracellular Ca accumulation in skeletal muscle than concentric activity would. Furthermore, possible differences between men and women in muscle damage were investigated following step exercise. Thirty-three healthy subjects (18 men and 15 women) participated in a 30-minute step exercise protocol involving concentric contractions with 1 leg and eccentric contractions with the other leg. Muscle Ca content, maximal voluntary contraction (MVC), and muscle enzymes in the plasma were measured. In a subgroup of the subjects, T2 relaxation time was measured by magnetic resonance imaging. No significant changes were found in muscle Ca content in vastus lateralis biopsy specimens in women or in men. Following step exercise, MVC decreased in both legs of both genders. The women had a significantly larger strength decrease in the eccentric leg than the men had on postexercise day 2 (p < 0.01). Plasma creatine kinase increased following step exercise, with a sevenfold higher response in women than in men on day 3 (p < 0.001). The women, but not the men, had an increase in T2 relaxation time in the eccentrically working adductor magnus muscle, peaking on day 3 (75%) (p < 0.001). In conclusion, step exercise does not lead to Ca accumulation in the vastus lateralis but does induce muscle damage preferentially in the eccentrically working muscles, considerably more in women than in men. This indicates that gender-specific step training programs may be warranted to avoid excessive muscle damage.

Clearance of extracellular K+ during muscle contraction--roles of membrane transport and diffusion.

Excitation of muscle often leads to a net loss of cellular K + and a rise in extracellular K+([K+]o), which in turn inhibits excitability and contractility. It is important, therefore, to determine how this K+ is cleared by diffusion into the surroundings or by reaccumulation into the muscle cells. The inhibitory effects of the rise in [K+] o may be assessed from the time course of changes in tetanic force in isolated muscles where diffusional clearance of K+ is eliminated by removing the incubation medium and allowing the muscles to contract in air. Measurements of tetanic force, endurance, and force recovery showed that in rat soleus and extensor digitorum longus (EDL) muscles there was no significant difference between the performance of muscles contracting in buffer or in air for up to 8 min. Ouabain-induced inhibition of K+ clearance via the Na+,K+ pumps markedly reduced contractile endurance and force recovery in air. Incubation in buffer containing 10 mM K+ clearly inhibited force development and endurance,and these effects were considerably reduced by stimulating Na+,K+ pumps with the 2 -agonist salbutamol. Following 30-60 s of continuous stimulation at 60 Hz, the amount of K + released into the extracellular space was assessed from washout experiments. The release of intracellular K+ per pulse was fourfold larger in EDL than in soleus,and in the two muscles, the average [K+] o reached 52.4 and 26.0 mM, respectively, appreciably higher than previously detected. In conclusion, prevention of diffusion of K+ from the extracellular space of isolated working muscles causes only modest interference with contractile performance. The Na+,K+ pumps play a major role in the clearance of K+ and the maintenance of force. This new information is important for the evaluation of K+ -induced inhibition in muscles, where diffusional clearance of K+ is reduced by tension development sufficient to suppress circulation.

Potassium, Na+,K+-pumps and fatigue in rat muscle.

During contractile activity, skeletal muscles undergo a net loss of cytoplasmic K(+) to the interstitial space. During intense exercise, plasma K(+) in human arterial blood may reach 8 mm, and interstitial K(+) 10-12 mm. This leads to depolarization, loss of excitability and contractile force. However, little is known about the effects of these physiological increases in extracellular K(+) ([K(+)](o)) on contractile endurance. Soleus muscles from 4-week-old rats were mounted on transducers for isometric contractions in Krebs-Ringer bicarbonate buffer containing 4-10 mm K(+), and endurance assessed by recording the rate of force decline during continuous stimulation at 60 Hz. Increasing [K(+)](o) from 4 to 8 or 10 mm and equilibrating the muscles for 40 or 20 min augmented the rate of force decline 2.4-fold and 7.2-fold, respectively (P < 0.001). The marked loss of endurance elicited by exposure to 8 or 10 mm K(+) was alleviated or significantly reduced by stimulating the Na(+),K(+)-pumps by intracellular Na(+) loading, the beta(2)-agonist salbutamol, adrenaline, calcitonin gene related peptide, insulin or repeated excitation. In conclusion, excitation-induced increase in [K(+)](o) is an important cause of high-frequency fatigue, and the Na(+),K(+)-pumps are essential for the maintenance of contractile force in the physiological range of [K(+)](o). Recordings of contractile force during continuous stimulation at 8-10 mm K(+) may be used to analyse the effects of agents or conditions influencing the excitability of working isolated muscles.

Causes of excitation-induced muscle cell damage in isometric contractions: mechanical stress or calcium overload?

Prolonged or unaccustomed exercise leads to muscle cell membrane damage, detectable as release of the intracellular enzyme lactic acid dehydrogenase (LDH). This is correlated to excitation-induced influx of Ca2+, but it cannot be excluded that mechanical stress contributes to the damage. We here explore this question using N-benzyl-p-toluene sulfonamide (BTS), which specifically blocks muscle contraction. Extensor digitorum longus muscles were prepared from 4-wk-old rats and mounted on holders for isometric contractions. Muscles were stimulated intermittently at 40 Hz for 15-60 min or exposed to the Ca2+ ionophore A23187. Electrical stimulation increased 45Ca influx 3-5 fold. This was followed by a progressive release of LDH, which was correlated to the influx of Ca2+. BTS (50 microM) caused a 90% inhibition of contractile force but had no effect on the excitation-induced 45Ca influx. After stimulation, ATP and creatine phosphate levels were higher in BTS-treated muscles, most likely due to the cessation of ATP-utilization for cross-bridge cycling, indicating a better energy status of these muscles. No release of LDH was observed in BTS-treated muscles. However, when exposed to anoxia, electrical stimulation caused a marked increase in LDH release that was not suppressed by BTS but associated with a decrease in the content of ATP. Dynamic passive stretching caused no increase in muscle Ca2+ content and only a minor release of LDH, whereas treatment with A23187 markedly increased LDH release both in control and BTS-treated muscles. In conclusion, after isometric contractions, muscle cell membrane damage depends on Ca2+ influx and energy status and not on mechanical stress.

Excitation-induced cell damage and beta2-adrenoceptor agonist stimulated force recovery in rat skeletal muscle.

Intensive exercise leads to a loss of force, which may be long lasting and associated with muscle cell damage. To simulate this impairment and to develop means of compensating the loss of force, extensor digitorum longus muscles from 4-wk-old rats were fatigued using intermittent 40-Hz stimulation (10 s on, 30 s off). After stimulation, force recovery, cell membrane leakage, and membrane potential were followed for 240 min. The 30-60 min of stimulation reduced tetanic force to approximately 10% of the prefatigue level, followed by a spontaneous recovery to approximately 20% in 120-240 min. Loss of force was associated with a decrease in K+ content, gain of Na+ and Ca2+ content, leakage of the intracellular enzyme lactic acid dehydrogenase (10-fold increase), and depolarization (13 mV). Stimulation of the Na+-K+ pump with either the beta2-adrenoceptor agonist salbutamol, epinephrine, rat calcitonin gene-related peptide (rCGRP), or dibutyryl cAMP improved force recovery by 40-90%. The beta-blocker propranolol abolished the effect of epinephrine on force recovery but not that of CGRP. Both spontaneous and salbutamol-induced force recovery were prevented by ouabain. The salbutamol-induced force recovery was associated with repolarization of the membrane potential (12 mV) to the level measured in unfatigued muscles. In conclusion, in muscles exposed to fatiguing stimulation leading to a considerable loss of force, cell leakage, and depolarization, stimulation of the Na+-K+ pump induces repolarization and improves force recovery, possibly due to the electrogenic action of the Na+-K+ pump. This mechanism may be important for the restoration of muscle function after intense exercise.

Magnesium supplementation and muscle function in patients with alcoholic liver disease: a randomized, placebo-controlled trial.

OBJECTIVE: The study was undertaken in order to evaluate the effect of magnesium (Mg) supplementation on muscle contents of Mg, muscle strength, muscle mass and sodium, potassium pumps (Na,K-pumps) in patients with alcoholic liver disease. Retrospectively, patients were also stratified according to spironolactone treatment. MATERIAL AND METHODS: The study comprised a placebo-controlled, randomized trial in which 59 consecutive patients with alcoholic liver disease were treated with Mg intravenously and orally (12.5 mmol daily) or placebo for 6 weeks. Muscle content of Mg, maximum isokinetic muscle strength, skeletal muscle mass and muscle content of Na,K-pumps were measured before and after Mg supplementation. RESULTS: Muscle Mg did not increase during the trial (paired t-test), but Mg supplementation and the duration of pre-study spironolactone treatment were independent predictors of muscle Mg (multiple regression). Muscle strength increased by 14% during the trial (p<0.001) and muscle mass increased by 11% (p=0.05), but with no difference between placebo and Mg treatment. Spironolactone treatment was associated with a 33% increase in the content of Na,K-pumps (p<0.001). CONCLUSIONS: Six weeks of Mg supplementation did not increase muscle Mg, although Mg supplementation and spironolactone treatment were independent predictors of muscle Mg. The intervention had no effect on muscle strength and mass, but both increased during the study, probably owing to the general care and attendance to the patients.

Anoxia induces Ca2+ influx and loss of cell membrane integrity in rat extensor digitorum longus muscle.

Anoxia can lead to skeletal muscle damage. In this study we have investigated whether an increased influx of Ca2+, which is known to cause damage during electrical stimulation, is a causative factor in anoxia-induced muscle damage. Isolated extensor digitorum longus (EDL) muscles from 4-week-old Wistar rats were mounted at resting length and were either resting or stimulated (30 min, 40 Hz, 10 s on, 30 s off) in the presence of standard oxygenation (95% O2, 5% CO2), anoxia (95% N2, 5% CO2) or varying degrees of reduced oxygenation. At varying extracellular Ca2+ concentrations ([Ca2+]o), 45Ca influx and total cellular Ca2+ content were measured and the release of lactic acid dehydrogenase (LDH) was determined as an indicator of cell membrane leakage. In resting muscles, incubated at 1.3 mM Ca2+, 15-75 min of exposure to anoxia increased 45Ca influx by 46-129% (P<0.001) and Ca2+ content by 20-50% (P<0.001). Mg2+ (11.2 mM) reduced the anoxia-induced increase in 45Ca influx by 43% (P<0.001). In muscles incubated at 20 and 5% O2, 45Ca influx was also stimulated (P<0.001). Increasing [Ca2+]o to 5 mM induced a progressive increase in both 45Ca uptake and LDH release in resting anoxic muscles. When electrical stimulation was applied during anoxia, Ca2+ content and LDH release increased markedly and showed a significant correlation (r2=0.55, P<0.001). In conclusion, anoxia or incubation at 20 or 5% O2 leads to an increased influx of 45Ca. This is associated with a loss of cell membrane integrity, possibly initiated by Ca2+. The loss of cell membrane integrity further increases Ca2+ influx, which may elicit a self-amplifying process of cell membrane leakage.

Effects of lactic acid and catecholamines on contractility in fast-twitch muscles exposed to hyperkalemia.

Intensive exercise is associated with a pronounced increase in extracellular K+ ([K+]o). Because of the ensuing depolarization and loss of excitability, this contributes to muscle fatigue. Intensive exercise also increases the level of circulating catecholamines and lactic acid, which both have been shown to alleviate the depressing effect of hyperkalemia in slow-twitch muscles. Because of their larger exercise-induced loss of K+, fast-twitch muscles are more prone to fatigue caused by increased [K+]o than slow-twitch muscles. Fast-twitch muscles also produce more lactic acid. We therefore compared the effects of catecholamines and lactic acid on the maintenance of contractility in rat fast-twitch [extensor digitorum longus (EDL)] and slow-twitch (soleus) muscles. Intact muscles were mounted on force transducers and stimulated electrically to evoke short isometric tetani. Elevated [K+]o (11 and 13 mM) was used to reduce force to approximately 20% of control force at 4 mM K+. In EDL, the beta2-agonist salbutamol (10(-5) M) restored tetanic force to 83 +/- 2% of control force, whereas in soleus salbutamol restored tetanic force to 93 +/- 1%. In both muscles, salbutamol induced hyperpolarization (5-8 mV), reduced intracellular Na+ content and increased Na+-K+ pump activity, leading to an increased K+ tolerance. Lactic acid (24 mM) restored force from 22 +/- 4% to 58 +/- 2% of control force in EDL, an effect that was significantly lower than in soleus muscle. These results amplify and generalize the concept that the exercise-induced acidification and increase in plasma catecholamines counterbalance fatigue arising from rundown of Na+ and K+ gradients.

Hyperthyroidism and cation pumps in human skeletal muscle.

Skeletal muscle constitutes the major target organ for the thermogenic action of thyroid hormone. We examined the possible relation between energy expenditure (EE), thyroid status, and the contents of Ca2+-ATPase and Na+-K+-ATPasein human skeletal muscle. Eleven hyperthyroid patients with Graves' disease were studied before and after medical treatment with methimazole and compared with eight healthy subjects. Muscle biopsies were taken from the vastus lateralis muscle, and EE was determined by indirect calorimetry. Before treatment, the patients had two- to fivefold elevated total plasma T3 and 41% elevated EE compared with when euthyroidism had been achieved. In hyperthyroidism, the content of Ca2+-ATPase was increased: (mean +/- SD) 6,555 +/- 604 vs. 5,212 +/- 1,580 pmol/g in euthyroidism (P = 0.04) and 4,523 +/- 1,311 pmol/g in healthy controls (P = 0.0005). The content of Na+-K+-ATPase showed 89% increase in hyperthyroidism: 558 +/- 101 vs. 296 +/- 34 pmol/g (P = 0.0001) in euthyroidism and 278 +/- 52 pmol/g in healthy controls (P < 0.0001). In euthyroidism, the contents of both cation pumps did not differ from those of healthy controls. The Ca2+-ATPase content was significantly correlated to plasma T3 and resting EE. This provides the first evidence that, in human skeletal muscle, the capacity for Ca2+ recycling and active Na+-K+ transport are correlated to EE and thyroid status.