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

Methods

Measurement of force

Muscles were mounted in force transducers in thermostated chambers containing KR buffer with 4 mm K^{, exposed to direct field stimulation via platinum wire electrodes placed on either side of the central region, and adjusted to optimal length for measurement of isometric contractions. Trains of 0.2 or 1.0 ms pulses at supramaximal voltage (10–12 V) were applied at the frequency and duration indicated. To evaluate contractile endurance the initial force decline as recorded over the first 20 s of stimulation at 60 Hz was measured. From this the rate of force decline was calculated using the expression}

and given as per cent per second of peak force measured 1 s after the onset of 60 Hz stimulation. This definition of the rate of force decline was used in all experiments. Obviously, if the rate of force decline is increasing, contractile endurance is reduced.

Animals, preparation and incubation of muscles

All handling and use of rats complied with Danish animal welfare regulations. All experiments were performed using 4-week-old Wistar rats of own breed, weighing 65–75 g. The animals were fed ad libitum and kept in a thermostated environment at 21°C with a 12–12 h light–dark cycle. Animals were killed by cervical dislocation, followed by decapitation. Intact soleus muscles were dissected out during wash with 154 mm NaCl at room temperature and then incubated at 30°C in standard Krebs–Ringer bicarbonate (KR) buffer (pH 7.4 at 30°C), containing the following (mm): 120.1 NaCl, 25 NaHCO₃, 2.8 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.3 CaCl₂ and 5 d-glucose. In most experiments, the concentration of KCl was increased from 4 to 8 or 10 mm. In one instance, K ^{was omitted from the buffer and replaced by Na. This buffer was used to induce an increase in intracellular Na as previously described (}Clausen & Kohn, 1977). Isoosmolarity was maintained by an equivalent reduction or increase in NaCl. All incubations took place under continuous gassing with a mixture of 95% O₂ and 5% CO₂.

Measurement of force

Muscles were mounted in force transducers in thermostated chambers containing KR buffer with 4 mm K^{, exposed to direct field stimulation via platinum wire electrodes placed on either side of the central region, and adjusted to optimal length for measurement of isometric contractions. Trains of 0.2 or 1.0 ms pulses at supramaximal voltage (10–12 V) were applied at the frequency and duration indicated. To evaluate contractile endurance the initial force decline as recorded over the first 20 s of stimulation at 60 Hz was measured. From this the rate of force decline was calculated using the expression}

and given as per cent per second of peak force measured 1 s after the onset of 60 Hz stimulation. This definition of the rate of force decline was used in all experiments. Obviously, if the rate of force decline is increasing, contractile endurance is reduced.

Chemicals

All chemicals used were of analytical grade. Salbutamol was obtained from Sigma-Aldrich; rat calcitonin gene-related peptide (rCGRP) was from Bacherm, Switzerland; adrenaline was from the Pharmacies of Danish Hospitals; porcine insulin was from the Novo Nordic Company, Bagsværd, Denmark; and ascorbic acid was from Merck, Albertslund, Denmark.

Statistics

All data are presented as means with s.e.m. The statistical significance of a difference in rate of force decline between two groups was ascertained using Student's two-tailed t test. Unless otherwise noted, all other tests of significance were done with two-way ANOVA followed, where appropriate, by Bonferroni's post hoc test. Significance was accepted for P < 0.05.

Results

Figure 1 shows the time course of the effects of exposure to 10 mm K ^{on force development during continuous 60 Hz (tetanic) stimulation of soleus muscles. In all panels, the onset and cessation of force development are rapid as illustrated by the almost vertical time course of the force recordings.} Figure 1A shows the force development at 4 mm K^{, with an initial force of 45.5 ± 3.1 g and a final force of 30.2 ± 2.1 g. Force recording at 10 m}m K ^{was performed without pre-exposure to 10 m}m K ⁽Fig. 1B) or following the indicated periods of pre-exposure (10, 20 and 40 min). Muscles stimulated at 60 Hz immediately after the onset of exposure to 10 mm K ⁽Fig. 1B) showed the same initial force development (46.2 ± 1.7 g), final force (30.9 ± 1.1 g) and rate of force decline (0.49% s ^{as measured over the first 20 s of excitation) as the contralateral muscles stimulated at 4 m}m K ⁽Fig. 1A). However, following 10, 20 and 40 min of pre-exposure to 10 mm K^{, the initial force development decreased to 36.4 ± 0.3, 28.7 ± 2.4 and 17.1 ± 1.7 g, respectively, and the final force to 15.1 ± 0.6, 4.3 ± 0.8 and 3.9 ± 0.6 g, respectively (}Fig. 1C–E). Moreover, the loss of force occurred earlier in the presence of 10 mm K^{. Thus, after 20 min of exposure to 10 m}m K^{, the rate of force decline as measured during the first 20 s of stimulation had increased 7.2-fold (from 0.49% s to 3.5% s,} P < 0.001). As shown in Fig. 1F, in muscles that had been exposed to 10 mm K ^{for 40 min, the initial force (41.9 ± 1.3 g), the final force (27.7 ± 1.8 g) and the rate of force decline were almost restored to the control values shown in} Fig. 1A by a subsequent 60 min incubation at 4 mm K^{. This indicates that the inhibitory effect of 10 m}m K ^{was reversible.}

Figure 1

Time course of force decline in rat soleus during 60 s of continuous stimulation at 60 Hz at 4 mM K ⁽A), at 10 mM K ^{after 0, 10, 20 and 40 min of pre-exposure to 10 mM K (}B–E), and after 40 min of pre-exposure to 10 mM K^{, followed by 60 min of recovery at 4 mM K (}F)

Soleus muscles were mounted in force transducers, adjusted to optimal length for measurement of isometric contractions and stimulated with 60 Hz, 2 s trains of 0.2 ms pulses every 10 min until steady force production was reached. Then the muscles were exposed to KR buffer containing 10 mm K ^{and allowed to rest for the indicated intervals of time (0–40 min). Henceforth, the muscles were exposed to continuous stimulation at 60 Hz for 60 s. Each curve represents the mean of observations on 4–10 muscles with vertical bars denoting} s.e.m.*Force values significantly different from initial peak force (P < 0.05, one-way ANOVA).

The clearance of K ^{from the extracellular space of the muscles depends on the activity of the Na,K-pumps in the plasma membrane. Therefore, the effects of stimulating the Na,K-pumps by preloading the muscles with Na were examined. Previous studies have shown that in rat soleus, preincubation in a K-free KR buffer for 120 min increased intracellular Na 2.3-fold (}Clausen &amp; Kohn, 1977). When reexposed to the normal K ^{(4 m}m) for 20 min, the uptake of ^{K measured during that interval increased 1.9-fold. This effect was completely suppressed by ouabain (1 m}m), indicating that it could be attributed to stimulation of the Na^{,K-pumps. As shown in} Fig. 2, soleus muscles pre-exposed to K^{-free KR buffer for 120 min and then exposed to KR buffer containing 10 m}m K ^{for 10 min had a similar initial force (46.3 ± 2.2} versus 40.6 ± 3.4 g) to muscles pre-exposed to the standard KR buffer with 4 mm K^{, but the rate of force decline was significantly lower after pre-exposure to K-free KR buffer (0.87 ± 0.02} versus 2.15 ± 0.09% s^, P < 0.0001). Other experiments showed that after 120 min of pre-exposure to 0 K ^{followed by exposure to 10 m}m K ^{for 20 min, there was a slightly lower but also significantly improved contractile endurance as evidenced by a reduction in rate of force decline from 1.44 ± 0.04% s in control muscles to 0.70 ± 0.02% s after pre-exposure to K-free KR buffer (}P < 0.002, n= 3 and 4). These two sets of observations indicate that when the Na^{,K-pumps were stimulated by simple Na loading, the inhibitory effect of 10 m}m K ^{on endurance was reduced. The effects of stimulating the Na,K-pumps with salbutamol or adrenaline (10m}) were examined in parallel experiments. As shown in Fig. 3, after 20 min at 10 mm K^{, the initial force was reduced from 41.5 ± 1.5 to 32.4 ± 1.5 g (}P < 0.001). Salbutamol and adrenaline increased the initial force to 40.9 ± 1.1 and 45.7 ± 1.5 g, respectively (P < 0.05 and 0.001) and decreased the rate of force decline from 3.7 ± 0.23% s ^{to 0.95 ± 0.03 and 1.81 ± 0.16% s, respectively (}P < 0.0001 in both cases). Even though the force measured after 60 s of stimulation was still around 30% below that recorded at 4 mm K^{, the contractile performance was considerably improved by both β}₂-agonists.

Figure 3

Time course of force decline in rat soleus during 60 s of continuous stimulation at 60 Hz (0.2 ms pulses) at 10 mM K ^{after 20 min of pre-exposure to 10 mM K without (○) or with salbutamol (10 M) (⋄) or adrenaline (10 M) (□), compared to muscles exposed to the same stimulation protocol at 4 mM K (•)}

To the buffer containing adrenaline, 2 × 10^m ascorbic acid was added to avoid oxidation of adrenaline. Each curve represents the mean of observations on 4–12 muscles with bars denoting s.e.m. For all time points, the values for control muscles at 4 mm K ^{and adrenaline- or salbutamol-treated muscles at 10 m}m K ^{were significantly higher than for control muscles at 10 m}m K ⁽P < 0.05).

Figure 2

Time course of force decline in rat soleus during 60 s of continuous stimulation at 60 Hz (0.2 ms pulses) after 120 min of pre-exposure to either 0 mM K ^{(Na loaded) or 4 mM K, followed by 10 min of exposure to 10 mM K+}

Each curve represents the mean of observations on 7–8 muscles with bars denoting s.e.m. Pre-exposure to 0 mm K ^{(▵) led to significantly higher force during the 60 Hz stimulation (*}P < 0.05 **P < 0.01) than pre-exposure to 4 mm K ^(○).

At 10 mm K^{, rat CGRP (10m}), which is also known to stimulate the Na^{,K-pumps in rat soleus muscle (}Andersen &amp; Clausen, 1993), induced a highly significant recovery of initial contractile force (43.3 ± 2.3 g versus 29.9 ± 2.1 g, P < 0.001) and reduced the rate of force decline from 3.68 ± 0.23 to 0.771 ± 0.002% s ⁽P < 0.0001) (Fig. 4). These effects were closely similar to those observed with salbutamol, and again, the curve describing force decline almost coincided with that recorded during the first 30 s at 4 mm K^{. During the last 30 s, the force values were about 25% below those recorded at 4 m}m K^{, but markedly above the values recorded at 10 m}m K ^{without rCGRP.}

Figure 4

Time course of force decline in rat soleus during 60 s of continuous stimulation at 60 Hz (0.2 ms pulses) after 20 min of exposure to 10 mM K ^{without (○) or with rCGRP (10 M) (□) or intermittent stimulation during preincubation (1 s, 60 Hz trains of 1 ms pulses every 2 min) (▵), compared to the same stimulation protocol at 4 mM K (•)}

Each curve represents the mean of observations on 4–12 muscles with bars denoting s.e.m. For all time points, the values for control muscles at 4 mm K ^{and muscles exposed to intermittent stimulation or CGRP were significantly higher than for control muscles at 10 m}m K ⁽P < 0.01).

The effect of rCGRP is of particular interest, since repeated stimulation is known to release this peptide from endogenous stores in the nerve endings of skeletal muscle (Uchida et al. 1990; Nielsen et al. 1998). This release was shown to counterbalance the inhibitory effect of high [K^]_o on short tetanic contractions in rat soleus (Nielsen et al. 1998). We therefore examined the effects of pre-exposure to repeated stimulation on the contractile endurance at 10 mm K^{. As shown in} Fig. 4, repeated stimulation during the preincubation with 10 mm K ^{(60 Hz for 1 s every 2 min) induced a highly significant recovery of initial force (41.0 ± 2.2 g} versus 29.9 ± 2.1 g, P < 0.001) and reduced the rate of force decline from 3.68 ± 0.23 to 1.43 ± 0.29% s ⁽P < 0.0001). Compared to the experiment with 10^m rCGRP, however, the recovery elicited by repeated stimulation was somewhat smaller, possibly because the amount of CGRP released from endogenous stores in the muscle was not sufficient to produce the same stimulation of the Na^{,K-pumps as that elicited by 10m} rCGRP present in the buffer. Thus, the force reached during the last 40 s was only 50–80% of that reached at 4 mm K^.

Insulin also stimulates the Na^{,K-pumps in skeletal muscle but via a different pathway (}Clausen, 2003). Figure 5 shows the effect of the addition of insulin at a supramaximal concentration (10 mU ml^{) on contractile performance in muscles at 10 m}m K^{. In this experiment the initial force at 10 m}m K ^{was 34.7 ± 2.6 g, which was not significantly different from that measured at 4 m}m K ^{(39.6 ± 3.4 g) or that measured at 10 m}m K ^{with insulin (39.3 ± 2.1 g). At 10 m}m K ^{the addition of insulin reduced, however, the rate of force decline from 3.41 ± 1.19 to 2.23 ± 0.89% s (}P < 0.02), which led to an improved maintenance of force during the first 20 s of 60 Hz stimulation (P < 0.05). Since these effects might be related to a stimulating action of the hormone on glucose uptake, this experiment was performed in glucose-free Krebs–Ringer buffer. Additional experiments performed in the standard buffer containing 5 mm d-glucose gave similar results, showing a significant insulin-induced decrease of 60% in the rate of force decline as measured over the first 20 s of stimulation (n= 4 versus 4, P < 0.05, data not shown).

Figure 5

Time course of force decline in rat soleus during 60 s of continuous stimulation at 60 Hz (0.2 ms pulses) at 10 mM K ^{after 20 min of exposure to KR buffer with 10 mM K without glucose and without (○) or with porcine insulin (10 mU ml) (□), compared to the same stimulation protocol at 4 mM K (•)}

Each curve represents the mean of observations on 4–10 muscles with bars denoting s.e.m.*Values for muscles exposed to insulin significantly higher than for control muscles at 10 mm K^, P < 0.05.

Since maximum intensity of exercise increases plasma K ^{in arterial blood to around 8 m}m (Medbø &amp; Sejersted, 1990), it can be assumed that under such conditions most muscles in the body are exposed to this concentration of K^{. Moreover, in the working leg muscles releasing the K into the blood stream it is likely that the interstitial K was above 8 m}m. It was of particular interest therefore to examine the effects of 8 mm K ^{on force development and endurance.}

As can be seen in Fig. 6A, soleus muscles exposed to 8 mm K ^{for 40 min showed no reduction in the initial force development during 60 Hz stimulation, 39.0 ± 3.8 g} versus 41.6 ± 1.8 g, but the rate of force decline during the first 20 s of 60 Hz stimulation was increased 2.4-fold from 0.50 ± 0.01 to 1.15 ± 0.06% s ⁽P < 0.001). When the muscles were exposed to salbutamol (10^m), initial force was not changed (40.8 ± 3.3 g), but the rate of force decline was reduced to 0.40 ± 0.01% s^{, which was not different from the control value recorded at 4 m}m K^{, but significantly lower than the rate measured at 8 m}m K ⁽P < 0.0002) (Fig. 6A). As shown in Fig. 6B, similar effects were exerted by rCGRP (10^m), which had no effect on initial force (44.4 ± 3.6 g versus 46.3 ± 2.2 g) but reduced the rate of force decline during 60 Hz stimulation from 1.32 ± 0.12 to 0.68 ± 0.07% s ⁽P < 0.002).

Figure 6

Time course of force decline in rat soleus during 60 s of continuous stimulation at 60 Hz (0.2 ms pulses) at 4 mM K ^{(•) and at 8 mM K after 40 min of exposure to 8 mM K without (▪) or with salbutamol (10 M) (□) (}A) and without (▪) or with rCGRP (10 ^{M) (□) (}B)

Each curve represents the mean of observations on 4–8 muscles with bars denoting s.e.m.

The effect of [K^]_o on the rate of force decline during the first 20 s of stimulation was further examined by comparing the effect of K ^{concentrations from 4 to 10 m}m. Figure 7 shows that increasing [K^]_o induced a progressive rise in the rate of force decline, which became significant at 8 mm or more (P < 0.001). When pre-exposed to buffers containing the indicated concentrations of K ^{as well as salbutamol (10m}), the rate of force decline was significantly reduced at 8, 9 and 10 mm K ⁽P < 0.001).

Figure 7

Effect of exposure to varying concentrations of K ^{without (•) or with salbutamol (10 M) (▪) on the rate of force decline (0–20 s) in rat soleus during continuous stimulation at 60 Hz (0.2 ms pulses)}

The rate of force decline during that interval was expressed as per cent per second of the initial peak force development measured 1 s after the onset of stimulation, and values shown in Figs 3 and ​and66A were used as well as data from other experiments performed at 4 mm K^{. The preexposure to 10 m}m K ^{lasted for 20 min, and that of the other concentrations of K for 40 min. Each point represents the mean of observations on 4–14 muscles with bars denoting} s.e.m.*Values significantly different from value at 4 mm K ⁽P < 0.001, one way-ANOVA). **Values significantly different from corresponding values in control muscles not exposed to salbutamol (P < 0.001, two way-ANOVA).

Discussion

The major new observations of the present study are that when exposed to physiological increases in [K^]_o (to 8–10 mm), isolated rat soleus muscles show a pronounced reduction in contractile endurance and that this functional impairment is to a large extent counterbalanced by intracellular Na ^{loading or the addition of compounds known to stimulate the Na,K-pumps in the same muscle (}Clausen, 2003). Under intense exercise until fatigue, interstitial K ^{undergoes a progressive increase until it reaches concentrations of 10–12 m}m (Green et al. 2000; Nordsborg et al. 2003) where it can be assumed that excitability is lost and therefore the muscle cells cease to release K ^{into the extracellular space. In the present study, we have characterized the effects of increased [K]}_o on muscle endurance as such by exposing isolated muscles to elevated [K^]_o. In previous studies we demonstrated that the inhibitory effect of elevated [K^]_o on subtetanic contractions in isolated rat soleus is slow in onset, reflecting the diffusional delay for K ^{ions to reach all fibres in the muscle, the lumen of the T-tubules, as well as the clearance of interstitial K via the Na,K-pumps (}Clausen &amp; Everts, 1991). Thus, at a [K^]_o of 12.5 mm, full inhibition of subtetanic contractions (12 Hz) was not reached until after 23 min of exposure (Clausen &amp; Everts, 1991). Measurements of the resting membrane potential in deeply situated fibres showed that full depolarization was only reached after up to 18 min of exposure to a [K^]_o of 12.5 mm. To obtain representative information about the inhibitory effects of high [K^]_o, therefore, muscles have to be preequilibrated with K^{-enriched medium before measurements of contractile performance. The present results show that when exposed to a [K]}_o of 10 mm for 20 min, the rate of force decline, as measured during the first 20 s of continuous stimulation at 60 Hz, increases from 0.49% s ^{at 4 m}m K ^{to 3.52% s at 10 m}m K^.

In addition to the reduced endurance, exposure of muscles to 10 mm K ^{also led to a reduction in the initial force production. This effect of elevated [K]}_o has been related to the ensuing depolarization of the muscle fibres, leading to slow inactivation of the TTX-sensitive Na^{-channels and reduction in fibre excitability (}Ruff, 1996). In agreement with earlier studies that have demonstrated a considerable depolarization safety margin for the effect of elevated [K^]_o on force production (Cairns et al. 1995; Cairns et al. 1997; Yensen et al. 2002), incubation of muscles at 8 mm K ^{was without effect on initial force. Importantly, however, exposure to buffers containing 8 m}m K^{, i.e. within the physiological range for the exercise-induced increase in arterial plasma K in human subjects (}Medbø &amp; Sejersted, 1990), increased the rate of force decline 2.4-fold. The reason for the increased rate of force decline observed at 8 mm K ^{is most likely that for the same amount of cellular K released (e.g. corresponding to a rise in [K]}_o of 2 mm), an inhibitory level (10 mm) is reached, whereas when the muscles are exposed to 4 mm K ^{only 6 m}m is reached, a level causing no significant increase in the rate of force decline.

When soleus muscles were exposed to Na^{-loading, salbutamol, adrenaline or rCGRP, which stimulate the Na,K-pumps, the K-induced reduction in endurance was almost alleviated. Stimulation of the Na,K-pumps with insulin produced a somewhat smaller, but highly significant recovery of endurance. It is interesting that although the insulin-induced stimulation of the Na,K-pumps is exerted via a different mechanism, this hormone also improves endurance. The effects of insulin and salbutamol on force recovery in K inhibited rat soleus muscles have been shown to be additive (}Clausen, 2003).

The idea that a rise in [K^]_o should be an important cause of fatigue was recently questioned because incubation experiments showed no inhibitory effect of 10 mm K ^{on the contractile endurance of isolated whole mouse soleus muscles (}Zhang et al. 2006). It should be noted that in these experiments, the endurance test was preceded by 20 min of repeated stimulation every 2 min at 50–70 Hz. It cannot be excluded that this pretreatment, via excitation-induced release of CGRP from endogenous stores in the muscles (Nielsen et al. 1998), counterbalanced the inhibitory effect of 10 mm K ^{on contractile endurance, leading the authors to conclude that 10 m}m K ^{produces no inhibition. In the present study we found that under comparable conditions, repeated stimulation every 2 min caused a marked reduction in the inhibitory effect of 10 m}m K ^{on endurance in rat soleus muscle. The use of repeated stimulation may therefore conceal the inhibitory effect of high [K]}_o. Another objection to the idea that elevation of [K^]_o might cause fatigue is that the inhibitory effect of elevating [K^]_o on contractile performance of isolated intact muscles is slow in onset. It should be noted, however, that during excitation, the release of K ^{from the working muscle cells is very rapid. Thus, in isolated rat soleus muscles we measured that the excitation-induced net loss of K amounts to 1.6 nmol per gram wet weight per action potential (}Clausen et al. 2004). Due to diffusional delays, it can be assumed that in these muscles without circulation, the major part of this K ^{will be accumulated in the extracellular space, which is around 20% of the muscle volume. During stimulation at 60 Hz, this release of K is sufficient to produce a substantial and rapid increase in the average concentration of K in the extracellular space. In soleus this rise will amount to 0.48 m}m s ^{(60 × 1.6/0.2 = 480 nmol ml or 0.48 m}m). This means that within 8.3 s (4/0.48) [K^]_o will rise from 4 mm to reach 8 mm

During dynamic contractions in vivo, the accumulation of K ^{around the active fibres may by slowed down by a release of extracellular K to the blood stream. However, even in the blood stream, the concentration of K may increase to around 8 m}m within 1 min of intense exercise. The precipitation of fatigue in muscles exposed to 8 mm K ^{in the present study strongly suggests, therefore, that the exposure of muscles to elevated [K]}_o during exercise can accelerate the development of muscle fatigue. It should be noted, however, that the sensitivity of muscles to elevated [K^]_o has been shown to be modifiable by several parameters of relevance for the exercise setting. These include an exercise-induced increase in intracellular Na ^{of the muscle fibres. The ensuing reduction in the chemical gradient for Na has been shown to amplify the depressing effect of elevated [K]}_o (Bouclin et al. 1995). Probably more importantly, however, an increase in intracellular Na ^{leads to an activation of the Na,K-pump, which makes muscle more tolerant to elevated [K]}_o (Nielsen et al. 2004). As illustrated in the present study, this effect is probably reinforced by the increase in circulating catecholamines. In addition, it was lately shown that acidosis can improve the tolerance of muscles to elevated [K^]_o by reducing the Cl ^{conductance of the muscle fibres (}Nielsen et al. 2001; Pedersen et al. 2005). Since the importance of these mechanisms for the tolerance of muscles to elevated [K^]_o during exercise is difficult to determine from studies on isolated muscles, the exact concentration of [K^]_o at which fatigue is precipitated in vivo is difficult to estimate.

Perspectives

The use of preincubation in buffer containing elevated K ^{(8–10 m}m) allows the testing of agents influencing the excitability of skeletal muscle. Excitability depends on the transmembrane distribution of K^{, and the effects of agents or factors influencing excitation-induced K efflux or the intracellular reaccumulation of K can be detected and quantified. The present results show that using this approach, simple intracellular Na loading as well as three different hormones with a well-documented stimulating effect on the Na,K-pump-mediated uptake of K in skeletal muscle (}Clausen, 2003) improve contractile endurance. This further supports the idea that the activity of the Na^{,K-pumps is important for the acute maintenance of contractility in working skeletal muscle. Taken together, the observations strongly indicate that the exposure of muscles to the elevation of [K]}_o that takes place during exercise, can significantly reduce muscle endurance, not only in the muscle cells directly involved in the work, but also among the resting muscle cells in the body. This effect of K ^{is likely to be partly or completely counterbalanced by the concomitant increase in Na,K-pump activity, induced by the increase in intracellular Na as well as by the circulating catecholamines and by the CGRP released from nerve endings in the muscles. In conclusion, the data provide several different and new examples of how the Na,K-pumps can protect against fatigue and loss of endurance induced by the excitation-induced rundown of transmembrane gradients for Na and K.}