Effects of β₂-agonists on force during and following anoxia in rat extensor digitorum longus muscle

Electrical stimulation of isolated muscles may lead to membrane depolarization, gain of Na(+), loss of K(+) and fatigue. These effects can be counteracted with β(2)-agonists possibly via activation of the Na(+)-K(+) pumps. Anoxia induces loss of force; however, it is not known whether β(2)-agonists affect force and ion homeostasis in anoxic muscles. In the present study isolated rat extensor digitorum longus (EDL) muscles exposed to anoxia showed a considerable loss of force, which was markedly reduced by the β(2)-agonists salbutamol (10(-6) M) and terbutaline (10(-6) M). Intermittent stimulation (15-30 min) clearly increased loss of force during anoxia and reduced force recovery during reoxygenation. The β(2)-agonists salbutamol (10(-7)-10(-5) M) and salmeterol (10(-6) M) improved force development during anoxia (25%) and force recovery during reoxygenation (55-262%). The effects of salbutamol on force recovery were prevented by blocking the Na(+)-K(+) pumps with ouabain or by blocking glycolysis with 2-deoxyglucose. Dibutyryl cAMP (1 mM) or theophylline (1 mM) also improved force recovery remarkably. In anoxic muscles, salbutamol decreased intracellular Na(+) and increased (86)Rb uptake and K(+) content, indicating stimulation of the Na(+)-K(+) pumps. In fatigued muscles salbutamol induced recovery of excitability. Thus β(2)-agonists reduce the anoxia-induced loss of force, leading to partial force recovery. These data strongly suggest that this effect is mediated by cAMP stimulation of the Na(+)-K(+) pumps and that it is not related to recovery of energy status (PCr, ATP, lactate).     

Effects of calcitonin gene-related peptide on rat soleus muscle excitability: mechanisms and physiological significance

Intense exercise causes a large loss of K(+) from contracting muscles. The ensuing elevation of extracellular K(+) ([K(+)](o)) has been suggested to cause fatigue by depressing muscle fiber excitability. In isolated muscles, however, repeated contractions confer some protection against this effect of elevated K(+). We hypothesize that this excitation-induced force-recovery is related to the release of the neuropeptide calcitonin gene-related peptide (CGRP), which stimulates the muscular Na(+)-K(+) pumps. Using the specific CGRP antagonist CGRP-(8-37), we evaluated the role of CGRP in the excitation-induced force recovery and examined possible mechanisms. Intact rat soleus muscles were stimulated to evoke short tetani at regular intervals. Increasing extracellular K(+) ([K(+)](o)) from 4 to 11 mM decreased force to approximately 20% of initial force (P < 0.001). Addition of exogenous CGRP (10(-9) M), release of endogenous CGRP with capsaicin, or repeated electrical stimulation recovered force to 50-70% of initial force (P < 0.001). In all cases, force recovery could be almost completely suppressed by CGRP-(8-37). At 11 mM [K(+)](o), CGRP (10(-8) M) did not alter resting membrane potential or conductance but significantly improved action potentials (P < 0.001) and increased the proportion of excitable fibers from 32 to 70% (P < 0.001). CGRP was shown to induce substantial force recovery with only modest Na(+)-K(+) pump stimulation. We conclude that the excitation-induced force recovery is caused by a recovery of excitability, induced by local release of CGRP. The data suggest that the recovery of excitability partly was induced by Na(+)-K(+) pump stimulation and partly by altering Na(+) channel function.     

Ionic mechanisms of excitation-induced regulation of Na+-K+-ATPase mRNA expression in isolated rat EDL muscle

This study investigated the effects of electrical stimulation on Na+-K+-ATPase isoform mRNA, with the aim to identify factors modulating Na+-K+-ATPase mRNA in isolated rat extensor digitorum longus (EDL) muscle. Interventions designed to mimic exercise-induced increases in intracellular Na+ and Ca2+ contents and membrane depolarization were examined. Muscles were mounted on force transducers and stimulated with 60-Hz 10-s pulse trains producing tetanic contractions three times at 10-min intervals. Ouabain (1.0 mM, 120 min), veratridine (0.1 mM, 30 min), and monensin (0.1 mM, 30 min) were used to increase intracellular Na+ content. High extracellular K+ (13 mM, 60 min) and the Ca2+ ionophore A-23187 (0.02 mM, 30 min) were used to induce membrane depolarization and elevated intracellular Ca2+ content, respectively. Muscles were analyzed for Na+-K+-ATPase alpha1-alpha3 and beta1-beta3 mRNA (real-time RT-PCR). Electrical stimulation had no immediate effect on Na+-K+-ATPase mRNA; however at 3 h after stimulation, it increased alpha1, alpha2, and alpha3 mRNA by 223, 621, and 892%, respectively (P = 0.010), without changing beta mRNA. Ouabain, veratridine, and monensin increased intracellular Na+ content by 769, 724, and 598%, respectively (P = 0.001) but did not increase mRNA of any isoform. High intracellular K+ concentration elevated alpha1 mRNA by 160% (P = 0.021), whereas A-23187 elevated alpha3 mRNA by 123% (P = 0.035) but reduced beta1 mRNA by 76% (P = 0.001). In conclusion, electrical stimulation induced subunit-specific increases in Na+-K+-ATPase mRNA in isolated rat EDL muscle. Furthermore, Na+-K+-ATPase mRNA appears to be regulated by different stimuli, including cellular changes associated with membrane depolarization and increased intracellular Ca2+ content but not increased intracellular Na+ content.     

Activity-induced recovery of excitability in K(+)-depressed rat soleus muscle

Increased extracellular K(+) concentration ([K(+)](o)) can reduce excitability and force in skeletal muscle. Here we examine the effects of muscle activation on compound muscle action potentials (M waves), resting membrane potential, and contractility in isolated rat soleus muscles. In muscles incubated for 60 min at 10 mM K(+), tetanic force and M wave area decreased to 23 and 24%, respectively, of the control value. Subsequently, short (1.5 s) tetanic stimulations given at 1-min intervals induced recovery of force and M wave area to 81 and 90% of control levels, respectively, within 15 min (P < 0.001). The recovery of force and M wave was associated with a partial repolarization of the muscle fibers. Experiments with tubocurarine suggest that the force recovery was related to activation of muscle Na(+)-K(+) pumps caused by the release of some compound from sensory nerves in response to muscle activity. In conclusion, activity produces marked recovery of excitability in K(+)-depressed muscle, and this may protect muscles against fatigue caused by increased [K(+)](o) during exercise.     

Effects of carbamylcholine on membrane potential and Na-K pump activity of cultured rat skeletal myotubes

1. We measured changes in resting membrane potential (Em) and Na-K pump activity, assayed by ouabain-sensitive 86Rb uptake, in response to carbamylcholine (CCh) and its continued presence in single rat skeletal myotubes in culture. 2. CCh caused immediate depolarization from control Em (-80 to -85 mV) to near 0 followed by repolarization of varying degrees depending on the age of the culture and temperature of the recording medium; repolarization of Em was most apparent by culture age 8-9 days in vitro (DIV), Em reaching values as high as -60 mV by 5-10 min after peak depolarization at 37 degrees C. 3. Input resistance, which decreased during CCh depolarization, increased only slightly during the initial phase of repolarization and then remained essentially unchanged during the major component of membrane repolarization in the presence of CCh. 4. Ouabain, given before CCh, prevented repolarization of Em and, when given after repolarization had begun, reversed it and caused Em to return to about -7 mV. 5. Na-K pump activity was decreased in myotubes in which Em did not repolarize or did so only slightly, and was increased by over 40-50% in myotubes whose Em repolarized by 40-60 mV, even though CCh was still present in the medium. Inhibition of pump activity in non repolarizing myotubes was related to Na influx, inhibition being reversed to stimulation when CCh was administered to myotubes in Na-free medium. 6. Repeated (three or four times) or prolonged (up to 60-min) administration of CCh to myotubes in which repolarization was hardly expressed (age 6-7 DIV) caused increases both in the amount of repolarization and in 86Rb uptake, both being related to the number or duration of CCh exposures. 7. We conclude that repolarization of Em following CCh-induced depolarization of cultured rat skeletal myotubes depends to a large extent on an increase in activity of the electrogenic Na-K pump.     

Wheel-running exercise alters rat diaphragm action potentials and their regulation by K+ channels.

Endurance exercise modifies regulatory systems that control skeletal muscle Na+ and K+ fluxes, in particular Na+-K+-ATPase-mediated transport of these ions. Na+ and K+ ion channels also play important roles in the regulation of ionic movements, specifically mediating Na+ influx and K+ efflux that occur during contractions resulting from action potential depolarization and repolarization. Whether exercise alters skeletal muscle electrophysiological properties controlled by these ion channels is unclear. The present study tested the hypothesis that endurance exercise modifies diaphragm action potential properties. Exercised rats spent 8 wk with free access to running wheels, and they were compared with sedentary rats living in conventional rodent housing. Diaphragm muscle was subsequently removed under anesthesia and studied in vitro. Resting membrane potential was not affected by endurance exercise. Muscle from exercised rats had a slower rate of action potential repolarization than that of sedentary animals (P = 0.0098), whereas rate of depolarization was similar in the two groups. The K+ channel blocker 3,4-diaminopyridine slowed action potential repolarization and increased action potential area of both exercised and sedentary muscle. However, these effects were significantly smaller in diaphragm from exercised than sedentary rats. These data indicate that voluntary running slows diaphragm action potential repolarization, most likely by modulating K+ channel number or function.     

The importance of limitations in aerobic metabolism, glycolysis, and membrane excitability for the development of high-frequency fatigue in isolated rat soleus muscle

We investigated the role of limitations in aerobic metabolism, glycolysis, and membrane excitability for development of high-frequency fatigue in isolated rat soleus muscle. Muscles mounted on force transducers were incubated in buffer bubbled with 5% CO(2) and either 95% O(2) (oxygenated) or 95% N(2) (anoxic) and stimulated at 60 Hz continuously for 30-120 s or intermittently for 120 s. Cyanide (2 mM) and 2-deoxyglucose (10 mM) were used to inhibit aerobic metabolism and both glycolysis and aerobic metabolism, respectively. Excitability was reduced by carbacholine (10 microM), a nicotinic ACh receptor agonist, or ouabain (10 microM), an Na(+)-K(+) pump inhibitor. Membrane excitability was measured by recording M waves. Intracellular Na(+) and K(+) contents and membrane potentials were measured by flame photometry and microelectrodes, respectively. During 120 s of continuous stimulation, oxygenated and anoxic muscles showed the same force loss. In oxygenated muscles, cyanide did not alter force loss for up to 90 s, whereas 2-deoxyglucose increased force loss (by 19-69%; P < 0.01) from 14 s of stimulation. In oxygenated muscles, 60 s of stimulation reduced force, M wave area, and amplitude by 70-90% (P < 0.001). Carbacholine or ouabain increased intracellular Na(+) content (P < 0.001), induced a 7- to 8-mV membrane depolarization (P < 0.001), and accelerated the rate of force loss (by 250-414%) during 30 s of stimulation (P < 0.001). Similar effects were seen with intermittent stimulation. In conclusion, limitations in glycolysis and subsequently also in aerobic metabolism, as well as membrane excitability but not aerobic metabolism alone, appear to play an important role in the development of high-frequency fatigue in isolated rat soleus muscle.     

Cationic concentrations and transmembrane fluxes in erythrocytes of humans during exercise

The effect of exercise on the intraerythrocyte cationic concentrations and transmembrane fluxes such as the Na+-K+-adenosinetriphosphatase (ATPase) pump, the Na+-K+ cotransport, and the Na+-Li+ countertransport system was studied in 11 normal male volunteers. All subjects performed an uninterrupted incremental exercise test on a bicycle ergometer, starting at an initial work load of 20% of the subjects' maximal exercise capacity, as determined in a pretest. The work rate was increased with an additional 20% each 6 min up to a final work load of 80%. Blood samples were taken at rest, at 60 and 80% of maximal exercise capacity, and 1, 2, 3, 4, 5, and 30 min after cessation of exercise. At moderate exercise (60% of maximal exercise capacity) the intraerythrocyte potassium concentration was not changed, but at severe exercise (80% of maximal exercise capacity) it was decreased. After exercise the intraerythrocyte potassium concentration returned to base line within 2 min. Exercise did not affect the intraerythrocyte concentrations of sodium and magnesium. The activity of the Na+-K+-ATPase pump and the Na+-K+ cotransport in the erythrocytes during and after exercise was no different from the resting level. The activity of the Na+-Li+ countertransport system on the contrary tended to decrease during exercise. It is concluded that exercise is accompanied by a leakage of potassium out of the erythrocytes without major alterations in the active red cell cationic fluxes.     

Membrane leakage and increased content of Na+ -K+ pumps and Ca2+ in human muscle after a 100-km run.

During prolonged exercise, changes in the ionic milieu in and surrounding the muscle fibers may lead to fatigue or damage of the muscle and thereby impair performance. In 10 male subjects, we investigated the effects of 100 km running on muscle and plasma electrolyte contents, muscle Na+ -K+ pump content, and plasma concentrations of creatine kinase (CK) and lactate dehydrogenase (LDH). After completion of a 100-km run, significant increases were found in plasma K+ (from 4.0 +/- 0.1 to 5.5 +/- 0.2 mM, P < 0.001), muscle Na+ -K+ pump content (from 334 +/- 11 to 378 +/- 17 pmol/g, P < 0.05), and total muscle Ca2+ content (from 0.84 +/- 0.03 to 1.02 +/- 0.04 micromol/g, P < 0.001). There was also a large increase in the plasma levels of the muscle-specific enzymes CK and LDH, which reached peak values at the end of the run and lasted several days after the run, indicating that a significant degree of muscle membrane leakage was present. The simultaneous occurrence of raised cellular Ca2+ content and muscle membrane leakage supports the theory that Ca2+ plays a role in the initiation of degenerative processes in muscles after severe exercise.     

Epinephrine stimulates the Na+-K+ ATPase in isolated rat jejunal crypt cells

The effect of epinephrine on the activity of the Na+-K+ ATPase was studied in isolated rat jejunal cells. The activity of the pump was assessed by measuring the ouabain inhibitable K+ accumulation by the enterocytes using 86Rb as a tracer. Epinephrine stimulated significantly the Na+-K+ ATPase in crypt cells but not in villus cells. This effect was still apparent in presence of propranolol and prazocin but disappeared in presence of yohimbine. Amiloride did not affect the epinephrine-induced stimulation. Calcium channel blockers and dibutyryl cAMP enhanced the activity of the pump, and exerted respectively overlapping and additive effects with epinephrine, when added simultaneously. The calcium ionophore A23187 inhibited the basal activity of the ATPase and the stimulatory effect of epinephrine disappeared in its presence. These results suggest that epinephrine stimulates the Na+-K+ ATPase in jejunal crypt cells by activating alpha2 receptors and decreasing intracellular calcium, and not by altering cAMP levels.     

Insulin and exercise stimulate muscle alpha-aminoisobutyric acid transport by a Na+-K+-ATPase independent pathway

Sodium ions are required for the active transport of amino acids such as alpha-aminoisobutyric acid (AIB) into skeletal muscle. To examine the role of Na+-K+-ATPase in this phenomenon, studies were carried out using the isolated perfused rat hindquarter preparation. Perfusion for 30 min with ouabain at a dose sufficient to inhibit the Na+-K+ pump (10(-4) M) inhibited the basal rate of AIB uptake in all muscles studied by up to 80%. However, it failed to inhibit the stimulation of AIB uptake, either by insulin (200 microU/ml) or electrically-induced muscle contractions. The increase in K+ release by the hindquarter in the presence of ouabain was the same under all conditions suggesting comparable inhibition of the Na+-K+ pump. These studies suggest that the basal, but not insulin or exercise-stimulated AIB transport into muscle is acutely dependent on a functional Na+-K+ pump. They also suggest that stimulated and basal uptake of AIB involve different mechanisms.     

Role of skeletal muscle Na+-K+ ATPase activity in increased lactate production in sub-acute sepsis

Bacterial sepsis is frequently accompanied by increased blood concentration of lactic acid, which traditionally is attributed to poor tissue perfusion, hypoxia and anaerobic glycolysis. Therapy aimed at improving oxygen delivery to tissues often does not correct the hyperlactatemia, suggesting that high blood lactate in sepsis is not due to hypoxia. Various tissues, including skeletal muscle, demonstrate increased lactate production under well-oxygenated conditions when the activity of the Na+-K+ ATPase is stimulated. Although both muscle Na+-K+ ATPase activity and muscle plasma membrane content of Na+, K+-ATPase subunits are increased in sepsis, no studies in vivo have demonstrated correlation between lactate production and changes in intracellular Na+ and K+ resulting from increased Na+-K+ pump activity in sepsis. Plasma concentrations of lactate and epinephrine, a known stimulator of the Na+-K+ pump, were increased in rats made septic by E. coli injection. Muscle lactate content was significantly increased in septic rats, although muscle ATP and phosphocreatine remained normal, suggesting oxygen delivery remained adequate for mitochondrial energy metabolism. In septic rats, muscle intracellular ratio of Na+:K+ was significantly reduced, indicating increased Na+-K+ pump activity. These data thus demonstrate that increased muscle lactate during sepsis correlates with evidence of elevated muscle Na+-K+ ATPase activity, but not with evidence of impaired oxidative metabolism. This study also further supports a role for epinephrine in this process.     

Electrophysiological examination of transmitter release in non-quantal form in the mouse diaphragm and the activity of membrane ATP-ase.

The subsynaptic area of mouse diaphragm fibres was hyperpolarized by 1--2 mV during local curarization of the junctional zone in the presence of the reversible anticholinesteraze prostigmine (6 X 10(-6) M), or after treatment of the muscle with organophosphate cholinesterase inhibitor Soman. In a solution containing 5 mM K+ the mean hyperpolarization was 1.1 +/- 0.27 mV at mean resting potential--70 mV. After adding 2 X 10(-5) M ouabain the hyperpolarization increased to 1.5 +/- 0.25 mV. Removal of potassium ions from the bathing medium also increased curare induced hyperpolarization to 1.80 +/- 0.40 mV. Reactivation of membrane ATP-ase by addition of K+ after a period in K+-free medium reduced the hyperpolarization to zero, where measurements were performed 10--20 min after the readdition. It was concluded that spontaneous non-quantal leakage of acetylcholine occurs at the mouse neuromuscular junction, as it does in the frog (ref. Katz and Miledi 1977). Conditions which block the Na+-K+-dependent ATP-ase of nerve terminals increased the continuous leakage of ACh and activation of the pump decreased it.     

Tension development of normal and hypertrophied-failing papillary muscles following rapid stimulation

Recent studies have shown that numerous cellular alterations exist in hypertrophied-failing (HF) cardiac muscle. Of particular interest is the finding of an altered ability of the Na-K pump to regulate membrane potential in this tissue during periods of transient stimulation. The present study was designed to determine if this altered Na-K pump function is in any way related to the ability of this tissue to develop force. Along these lines the rate of stimulation (6/min) of normal and hypertrophied-failing right ventricular papillary muscles from cats was increased to 60/min for 90 sec. This procedure was repeated in solutions with low Na+, low Na+ and Ca++, and Ouabain. These solutions were utilized to vary the ionic load on the Na-K pump and the Na-Ca exchanger. The results demonstrate that the pattern of changes in tension in HF papillary muscles seen following periods of rapid stimulation are significantly different from those of normal muscles. The pattern of changes in mechanical performance were found to be similar to the membrane potential changes described in previous studies. In addition, lowering the Na+ load presented to HF muscles returned the characteristic pattern of changes in tension, following drive, toward normal. Ouabain was found to inhibit the changes in tension development following increased rates of stimulation that are thought to be produced by activation of the Na-K pump. The results suggest that the ability of the Na-K pump to maintain normal transmembrane ionic gradients may be altered in HF muscles. This alteration appears to be capable of affecting cellular Ca++ possibly through the Na-Ca exchange system.     

Changes in resting membrane potential induced by active sensitization in the guinea-pig vas deferens

Smooth muscles hyperresponsiveness is a common feature in anaphylaxis and allergic diseases. The aim of the present work was to investigate whether the enhanced reactivity of sensitized guinea-pig vas deferens was associated with changes in the resting membrane potential (Er) of the smooth muscle cells. Active sensitization was performed by subcutaneous injection of egg albumen. Er was measured in vitro in isolated vas deferens with conventional KCl-filled microelectrodes. Quantification of [3H]ouabain binding sites, measurements of 86Rb efflux, and measurements of Na and K contents were also performed. In normal physiological solution, at 35 degrees C, Er was a mean of -54.1+/-0.3 mV (mean +/- SEM) in control vas deferens. Sensitization resulted in depolarizing Er by about 7 mV. In control and sensitized preparations, the 3H-ouabain binding site concentration, the efflux of 86Rb, and the K content were similar. In guinea-pig vas deferens, active sensitization induced a partial depolarization of the resting membrane potential of the smooth muscle cells, which did not result from a downregulation of Na+ -K+ pump sites.     

beta-Adrenergic effect on Na+-K+ transport in rat skeletal muscle.

1. Intact rat extensor digitorum longus muscles soaked in L-isoproterenol plus 10(-5) M ouabain gained less sarcoplasmic Na+ than did muscles soaked in ouabain alone. Half maximal effect was produced by 10(-8) M L-isoproterenol. 2. D-Isoproterenol and oxidized L-isoproterenol were only 3 and 1%, respectively, as potent as L-isoproterenol. Other catechols tested had no effect. 3. The effect of L-isoproterenol on sarcoplasmic Na+ content appears to be a beta-adrenergic function in that it was blocked by propranolol, but not by phentolamine, and could be mimicked by dibutyryl cyclic AMP or by caffeine. 4. Reduced gain in sarcoplasmic Na+ was accompanied by reduced loss of sarcoplasmic K+. 5. L-Isoproterenol increased loss of sarcoplasmic Na+ in the absence of ouabain, in muscles recovering from cold treatment. 6. Results suggest that the beta-adrenergic system stimulates a coupled Na-K+ pump. 7. A model is proposed in which stimulation of the Na+-K+ pump in response to beta-adrenergic agents involves a number of intermediate steps, identified tentatively.     

Conformational studies of Escherichia coli pyruvate oxidase

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3'-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from -51 mV to -36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using 86Rb+ as a radioactive tracer for K+, demonstrated that the depolarization was not caused by changes in (Na+ + K+)-ATPase-mediated K+ influx or in the transmembrane K+ gradient. The permeability ratio (PNa/PK), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of PNa/PK was shown by 24Na+ and 86Rb+ flux measurements to be due to both an increase of the Na+ permeability and a decrease of the K+ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Participation of electrogenic Na+-K+-ATPase in the membrane potential of earthworm body wall muscles

The effect of Na+-K+-ATPase inhibitor ouabain on the resting membrane potential (Vm) was studied by glass microelectrodes in isolated somatic longitudinal muscles of the earthworm Lumbricus terrestris and compared with frog sartorius muscle. In earthworm muscle, Vm was -49 mV (inside negative) in a reference external solution with 4 mmol/l K+. The electrogenic participation of Na+-K+-ATPase was absent in solutions with very low concentrations of 0.01 mmol/l K+, higher in 4 and 8 mmol/l K+ (4-5 mV) and maximal (13 mV) in solutions containing 12 mmol/l K+ where Vm was -46 mV in the absence and -33 mV in the presence of 1 x 10(4) M ouabain. The electrogenic participation of Na+-K+-ATPase was much smaller in m. sartorius of the frog Rana temporaria bathed in 8 and 12 mmol/l K+. The results indicate that the Na+-K+-ATPase is an important electrogenic factor in earthworm longitudinal muscle fibres and that its contribution to Vm depends directly on the concentration of K+ in the bathing solution.     

Inactivation of human muscle Na+-K+-ATPase in vitro during prolonged exercise is increased with hypoxia.

This study investigated the effects of prolonged exercise performed in normoxia (N) and hypoxia (H) on neuromuscular fatigue, membrane excitability, and Na+-K+ -ATPase activity in working muscle. Ten untrained volunteers [peak oxygen consumption (Vo2peak) = 42.1 +/- 2.8 (SE) ml x kg(-1) x min(-1)] performed 90 min of cycling during N (inspired oxygen fraction = 0.21) and during H (inspired oxygen fraction = 0.14) at approximately 50% of normoxic Vo2peak. During N, 3-O-methylfluorescein phosphatase activity (nmol x mg protein(-1) x h(-1)) in vastus lateralis, used as a measure of Na+-K+-ATPase activity, decreased (P < 0.05) by 21% at 30 min of exercise compared with rest (101 +/- 53 vs. 79.6 +/- 4.3) with no further reductions observed at 90 min (72.8 +/- 8.0). During H, similar reductions (P < 0.05) were observed during the first 30 min (90.8 +/- 5.3 vs. 79.0 +/- 6.3) followed by further reductions (P < 0.05) at 90 min (50.5 +/- 3.9). Exercise in N resulted in reductions (P < 0.05) in both quadriceps maximal voluntary contractile force (MVC; 633 +/- 50 vs. 477 +/- 67 N) and force at low frequencies of stimulation, namely 10 Hz (142 +/- 16 vs. 86.7 +/- 10 N) and 20 Hz (283 +/- 32 vs. 236 +/- 31 N). No changes were observed in the amplitude, duration, and area of the muscle compound action potential (M wave). Exercise in H was without additional effect in altering MVC, low-frequency force, and M-wave properties. It is concluded that, although exercise in H resulted in a greater inactivation of Na+-K+-ATPase activity compared with N, neuromuscular fatigue and membrane excitability are not differentially altered.     

Increased excitability of acidified skeletal muscle: role of chloride conductance

Generation of the action potentials (AP) necessary to activate skeletal muscle fibers requires that inward membrane currents exceed outward currents and thereby depolarize the fibers to the voltage threshold for AP generation. Excitability therefore depends on both excitatory Na+ currents and inhibitory K+ and Cl- currents. During intensive exercise, active muscle loses K+ and extracellular K+ ([K+]o) increases. Since high [K+]o leads to depolarization and ensuing inactivation of voltage-gated Na+ channels and loss of excitability in isolated muscles, exercise-induced loss of K+ is likely to reduce muscle excitability and thereby contribute to muscle fatigue in vivo. Intensive exercise, however, also leads to muscle acidification, which recently was shown to recover excitability in isolated K(+)-depressed muscles of the rat. Here we show that in rat soleus muscles at 11 mM K+, the almost complete recovery of compound action potentials and force with muscle acidification (CO2 changed from 5 to 24%) was associated with reduced chloride conductance (1731 +/- 151 to 938 +/- 64 microS/cm2, P < 0.01) but not with changes in potassium conductance (405 +/- 20 to 455 +/- 30 microS/cm2, P < 0.16). Furthermore, acidification reduced the rheobase current by 26% at 4 mM K+ and increased the number of excitable fibers at elevated [K+]o. At 11 mM K+ and normal pH, a recovery of excitability and force similar to the observations with muscle acidification could be induced by reducing extracellular Cl- or by blocking the major muscle Cl- channel, ClC-1, with 30 microM 9-AC. It is concluded that recovery of excitability in K(+)-depressed muscles induced by muscle acidification is related to reduction in the inhibitory Cl- currents, possibly through inhibition of ClC-1 channels, and acidosis thereby reduces the Na+ current needed to generate and propagate an AP. Thus short term regulation of Cl- channels is important for maintenance of excitability in working muscle.