Na⁺–K⁺ pump stimulation restores carbacholine-induced loss of excitability and contractility in rat skeletal muscle

Abstract

Intense exercise results in increases in intracellular Na⁺ and extracellular K⁺ concentrations, leading to depolarization and a loss of muscle excitability and contractility. Here, we use carbacholine to chronically activate the nicotinic acetylcholine (nACh) receptors to mimic the changes in membrane permeability, chemical Na⁺ and K⁺ gradients and membrane potential observed during intense exercise. Intact rat soleus muscles were mounted on force transducers and stimulated electrically to evoke short tetani at regular intervals. Carbacholine produced a 2.6-fold increase in Na⁺ influx that was tetrodotoxin (TTX) insensitive, but abolished by tubocurarine, resulting in a significant 36% increase in intracellular Na⁺, and 8% decrease in intracellular K⁺ content. The mid region, near the motor end plate, had much larger alterations than the more distal regions of the muscle, and showed a larger membrane depolarization from −73 ± 1 to −60 ± 1 mV compared with −64 ± 1 mV. Carbacholine (10⁻⁴m) significantly reduced tetanic force to 31 ± 3% of controls, which underwent significant recovery upon application of Na⁺–K⁺ pump stimulators: salbutamol (10⁻⁵m), adrenaline (10⁻⁵m) and calcitonin gene-related peptide (CGRP; 10⁻⁷m). The force recovery with salbutamol was accompanied by a recovery of intracellular Na⁺ and K⁺ contents, and a small but significant 4–5 mV recovery of membrane potential. Similar results were obtained using succinylcholine (10⁻⁴m), indicating that Na⁺–K⁺ pump stimulation may prevent or restore succinylcholine-induced hyperkalaemia. The stimulation of the Na⁺–K⁺ pump allows muscle to partially recover contractility by regaining excitability through electrogenically driven repolarization of the muscle membrane.

Acetylcholine (ACh) released at the motor end plate binds to nicotinic ACh (nACh) receptors, resulting in a membrane depolarization of sufficient magnitude to activate the voltage-gated Na⁺ channels of the muscle membrane. The subsequent Na⁺ influx across the membrane initiates the propagation of an action potential along the muscle membrane, eventually leading to muscle contraction. The Na⁺ and K⁺ fluxes associated with each action potential lead to a small, transient increase in intracellular Na⁺ concentration ([Na⁺]_i) and a concomitant loss in intracellular K⁺ concentration ([K⁺]_i). Since the Na⁺–K⁺ pump compensates for these perturbations in Na⁺ and K⁺ gradients, this transport system is essential for maintaining muscle membrane excitability.

However, during high-frequency fatiguing stimulation, the Na⁺–K⁺ pump is unable to fully compensate for the accelerated Na⁺ and K⁺ fluxes, leading to significant increases in [Na⁺]_i (Nielsen & Clausen, 1997) and net loss of [K⁺]_i (Sejersted & Sjøgaard, 2000), reducing the chemical Na⁺ and K⁺ gradients, and thus inducing membrane depolarization. The diffusional limitations between the opening of the T-system and the interstitium would further exacerbate the reduction in chemical Na⁺ and K⁺ gradients in the T-system (Bezanilla et al. 1972). To assess the significance of these changes on muscle function and muscle fatigue, investigators have previously altered extracellular Na⁺ concentration ([Na⁺]_o) and extracellular K⁺ concentration ([K⁺]_o) (Bouclin et al. 1995; Overgaard et al. 1997, 1999; Cairns et al. 1997, 2003). However, this approach differs from the exercise-induced changes in chemical Na⁺ and K⁺ gradients, making the comparison between exercise-induced and experimentally induced perturbations in membrane potential and chemical Na⁺ and K⁺ gradients difficult. In particular, an experimental reduction in [Na⁺]_o tends to cause a reduction in [Na⁺]_i (Overgaard et al. 1997), which is in contrast to the elevated [Na⁺]_i observed during fatiguing stimulation in frogs and rats (Juel, 1986; Balog & Fitts, 1996; Nielsen & Clausen, 1996), and during exercise in humans (Sahlin et al. 1978; Sjøgaard et al. 1985). In experiments examining the effect of stimulation of the Na⁺–K⁺ pump, this discrepancy is exaggerated by a further reduction in [Na⁺]_i, mediated by an increase in Na⁺–K⁺ pump activity (Overgaard et al. 1997). Since [Na⁺]_i is an important determinant for the activity of the Na⁺–K⁺ pump (Clausen, 2003), such a reduction in [Na⁺]_i could potentially affect interpretations regarding the importance of the Na⁺–K⁺ pump for the maintenance of excitability in contracting muscle.

To better mimic the changes in [Na⁺]_i during intensive exercise, here we use carbacholine to increase membrane permeability to Na⁺ and K⁺, and thus induce depolarization and a reduction in the chemical Na⁺ and K⁺ gradients. Carbacholine chronically opens the nACh receptors (Hille, 2001), allowing both Na⁺ and K⁺ to move along their electrochemical gradients, resulting in an increase in [Na⁺]_i (Creese et al. 1977) and a reduction in [K⁺]_i (Zaimis & Head, 1976). Using this approach we show here that carbacholine, via its effects on membrane function, causes a decrease in muscle excitability and contractility, which can be restored by Na⁺–K⁺ pump stimulation.

Methods

Animals, preparation and incubation of muscles

All handling and use of animals complied with Danish animal welfare regulations. Experiments were performed using 4-week-old Wistar rats of own breed, weighing 65–75 g, which were kept in a thermostated environment at 21°C with a 12 h light/12 h dark cycle and fed ad libitum. The animals were killed by cervical dislocation, followed by decapitation. Intact soleus and extensor digitorum longus (EDL) muscles were prepared and incubated in standard Krebs–Ringer bicarbonate buffer (KR) containing the following (mm): 122.1 NaCl, 25.1 NaHCO₃, 2.8 KCl, 1.2 KH₂PO₄, 1.2 MgSO₄, 1.3 CaCl₂ and 5.0 d-glucose (pH 7.4). All incubations took place at 30°C under continuous gassing with a mixture of 95% O₂ and 5% CO₂.

Measurement of force and membrane potentials

Muscles were mounted for isometric contractions in thermostated chambers containing standard KR, adjusted to optimal length for force production, and exposed to field stimulation across the central region through platinum electrodes using 2 s trains of 0.2 ms 12 V pulses at 60 Hz every 10 min. Force was measured using force displacement transducers, and recorded with a chart recorder and/or digitally on a computer. The mean absolute force produced under control conditions was 0.39 ± 0.01 N, with results expressed as a percentage of the control force produced directly prior to the addition of carbacholine or other relevant agents. The resting membrane potential was recorded by single penetrations of muscle fibres at two distinct regions of the muscle: the mid region of the muscle near the motor end plate, and at a region approximately 6–8 mm distal. Previously described standard electrophysiological techniques were used (Overgaard & Nielsen, 2001).

Measurements of Na⁺ and K⁺ contents, Na⁺ influx, ⁸⁶Rb⁺ influx and Na⁺–K⁺ pump activity

Passive Na⁺ influx was determined by exposing muscles to the standard KR containing ²²Na⁺ (0.5 μCi ml⁻¹) for 5 min. At the end of the exposure, the muscles were immediately transferred to ice-cold Na⁺-free Tris-sucrose buffer, and then they underwent a 4 × 15 min washout to remove extracellular ²²Na⁺. The muscles were then blotted, weighed, and the activity of the ²²Na⁺ retained in the muscles was determined by γ-counting. Following washout, some muscles were cut into three distinct sections, corresponding to the origin, the motor end plates and the insertion (Achilles tendon) regions of the muscle, in order to determine the location of the ²²Na⁺ influx and changes in Na⁺ and K⁺ content. After correction for the loss of intracellular ²²Na⁺ during the washout (see Buchanan et al. 2002), the Na⁺ influx was calculated from the specific ²²Na⁺ activity in the incubation buffer.

Previous studies have shown that ⁸⁶Rb⁺ is a reliable tracer for determination of K⁺ transport via the Na⁺–K⁺ pump (Clausen et al. 1987). The activity of the Na⁺–K⁺ pump was determined from the ouabain-sensitive ⁸⁶Rb⁺ uptake, as previously described by Buchanan et al. (2002). Briefly, muscles were preincubated for 15 min with or without 10⁻³m ouabain, followed by a further 10 min incubation in KR containing ⁸⁶Rb⁺ (0.5 μCi ml⁻¹) with or without ouabain. Finally the muscles were washed for 4 × 15 min in ice-cold Na⁺-free Tris-sucrose buffer to remove extracellular ⁸⁶Rb⁺ and Na⁺. The muscles were then blotted, weighed, and taken for counting of ⁸⁶Rb⁺ activity by Cerenkov radiation in a β-counter. The amount of ⁸⁶Rb⁺ activity retained after washout was calculated, and the uptake of K⁺ was then determined by converting the relative uptake of ⁸⁶Rb⁺ to K⁺ using the concentration of K⁺ in the incubation medium (Buchanan et al. 2002).

Muscles from the Na⁺ and ⁸⁶Rb⁺ influx experiments were retained to measure intracellular Na⁺ and K⁺ contents, soaked overnight in 0.3 m trichloroacetic acid, which gives complete extraction of Na⁺ and K⁺ from the muscle tissue (Clausen et al. 1993), before Na⁺ and K⁺ concentrations were measured using flame photometry (FLM3, Radiometer, Copenhagen).

Chemicals and isotopes

All chemicals were of analytical grade. Carbamylcholine chloride (carbacholine), succinylcholine, ouabain, salbutamol, tetrodotoxin (TTX) and tubocurarine were obtained from Sigma-Aldrich; rat calcitonin gene-related peptide (rCGRP) was from Bacherm, Switzerland; and adrenaline was from SAD, Denmark. ²²Na⁺ and ⁸⁶Rb⁺ were from Amersham International (Aylesbury, Buckinghamshire, UK).

Statistics

All data are expressed as means ± s.e.m. The statistical significance of any difference between groups was accepted at P < 0.05, as determined using Student's two-tailed t test for nonpaired observations or ANOVA, where appropriate.

Results

Effect of carbacholine on muscle contractility

Carbacholine reduced steady-state control force of soleus muscle in a concentration- and time-dependent manner as depicted in Fig. 1A. Exposure of muscles to 10⁻⁶m carbacholine had no deleterious effect on force, whereas 10⁻⁵m carbacholine initially decreased force to 93 ± 2% (P < 0.05) of the control force after 10 min, followed by a steady-state level of around 75% of the control force. Further increasing the carbacholine to 10⁻⁴ and 10⁻³m reduced the steady-state force to approximately 30% of controls (P < 0.05). Only the initial responses after 10 min in the presence of 10⁻⁴ or 10⁻³m carbacholine were significantly different from each other (22 ± 4 and 10 ± 4% of controls, respectively, P < 0.05), suggesting that at 10⁻⁴m, carbacholine has already reached saturating concentration.

Figure 1. Effect of carbacholine on tetanic force.

A, time course of tetanic force development in soleus muscles during 60 min exposure to Krebs–Ringer bicarbonate buffer (KR) containing 10⁻⁶ (▪), 10⁻⁵ (□), 10⁻⁴ (○) and 10⁻³m (•) carbacholine. Data points are means ± s.e.m., with n = 10 for all points, except 10⁻⁶m carbacholine where n = 4. B, effect of 10⁻⁴m tubocurarine on tetanic force before (•) and after (○) exposure to 10⁻⁴m carbacholine. Data points are means ± s.e.m., n = 4.

Pre-treatment of the muscle with the nACh receptor blocker tubocurarine (10⁻⁴m), prior to carbacholine exposure, prevented any suppressing effect of carbacholine on muscle contractility (Fig. 1B). Application of tubocurarine to muscles already depressed by carbacholine produced complete recovery of force to control levels, which taken together suggest that the action of carbacholine is mediated by the nACh receptor. Since the nACh receptors are associated with the neuromuscular junction, which is situated in the mid region of the muscle, a series of experiments was performed to compare the effect of carbacholine on force in muscles stimulated with an electrical field across the end-region to muscles stimulated via an electrical field across on the mid region. The experiments showed no effect of the site of electrical stimulation on force production (data not shown).

Recovery of contractility after exposure to carbacholine was also obtained by the application of various Na⁺–K⁺ pump stimulators, including the β₂-agonist salbutamol (Fig. 2A), adrenaline (Fig. 2B) and the neuropeptide CGRP, which is released locally from motor nerves, and has been shown to stimulate the Na⁺–K⁺ pump in rat skeletal muscle (Andersen & Clausen, 1993) (Fig. 2C). All three agents produced a full recovery of contractility in muscles suppressed by 10⁻⁵m carbacholine. In muscles suppressed by 10⁻⁴m carbacholine, the three agents led to a significant (P < 0.05) although incomplete improvement of contractility, with salbutamol recovering force to 82 ± 1% of controls (P < 0.05), while adrenaline and rCGRP recovered force to 70 ± 3 and 63 ± 2% (P < 0.05), respectively. The upper trace in Fig. 2A shows that Na⁺–K⁺ pump stimulation only augments force production in already suppressed muscles, as those that maintained full contractility showed no force enhancement upon application of salbutamol. Further experiments shown in Fig. 3 demonstrated that 10⁻⁴m carbacholine significantly reduced (P < 0.05) twitch force in soleus muscles in a similar manner to that of tetanic force (34 ± 6% of control force), and that twitch force could be markedly recovered (P < 0.05) to 94 ± 5% of controls by Na⁺–K⁺ pump stimulation with salbutamol.

Figure 2. Restoration of tetanic force in carbacholine depressed muscles.

Time course of the recovery of tetanic force development in carbacholine-suppressed soleus muscles during 60 min exposure to 10⁻⁵m salbutamol (A), 10⁻⁵m adrenaline (B) and 10⁻⁷m rat calcitonin gene-related peptide (rCGRP) (C). Concentrations of carbacholine are 10⁻⁶m (▪); 10⁻⁵m (□) and 10⁻⁴m (○), and data points are means ± s.e.m., n = 4 in all cases except for 10⁻⁵ and 10⁻⁴m carbacholine in A, where n = 6.

Figure 3. Effect of carbacholine on twitch force.

Time course of the recovery of twitch force development in 10⁻⁴m carbacholine-suppressed soleus muscles during 60 min exposure to 10⁻⁵m salbutamol. Data points are means ± s.e.m., n = 4.

Other experiments were performed on fast-twitch EDL muscles, where 10⁻⁴m carbacholine produced a similar significant (P < 0.05, n = 4) reduction in tetanic force (steady-state level of 17 ± 4% of controls) and subsequent recovery, albeit incomplete, with salbutamol (to 63 ± 8% of controls, data not shown), to that observed in slow-twitch soleus muscle. For simplicity, all subsequent experiments were performed on soleus muscles.

Effect of carbacholine on Na⁺ influx

As the binding of carbacholine to nACh receptors increases muscle membrane permeability (Hille, 2001), it was important to measure the Na⁺ influx in the presence of carbacholine. Figure 4A shows that carbacholine significantly increased ²²Na⁺ influx by 2.6-fold compared with controls (P < 0.05). This increased Na⁺ influx was abolished by preincubation for 15 min with tubocurarine (Fig. 4A), while tubocurarine alone had no significant effect on Na⁺ influx. Pre-incubation of the muscles with TTX (which completely blocks force production, data not shown) did not alter the carbacholine-induced increase in Na⁺ influx (Fig. 4B). It therefore appears that the increased influx of Na⁺ of the muscle after exposure to carbacholine was not related to opening of the voltage-gated Na⁺ channels of the muscle membrane. Thus the effect of carbacholine on Na⁺ influx (and the membrane potential) was selectively restricted to the nACh receptors.

Figure 4. Effect of carbacholine on Na⁺ influx.

²²Na⁺ influx in soleus muscles during 5 min exposure to 10⁻⁴m carbacholine with or without 10⁻⁵m tubocurarine (A), or 10⁻⁶m tetrodotoxin (B). Each column represents a mean value, with bars denoting s.e.m. of 4 muscles. *Significant difference (P < 0.05) compared with controls.

Intracellular Na⁺ and K⁺ contents

The intracellular Na⁺ and K⁺ contents of muscles exposed to carbacholine are shown in Table 1, and they demonstrate a biphasic response with length of incubation. A short 10 min incubation with carbacholine caused a significant (P < 0.05) 36% increase in intracellular Na⁺, and a concomitant 8% decrease in intracellular K⁺. Increasing incubation time to 60 min gave a significant (P < 0.05) but smaller (24%) increase in intracellular Na⁺ content, but the same (9%) decrease in intracellular K⁺ content, when compared to controls.

Table 1.

Carbacholine-induced alteration to intracellular Na⁺ and K⁺ contents, total and ouabain-sensitive ⁸⁶Rb⁺ uptake

		Carbacholine incubation
	Control	10 min	60 min	60 min + salbutamol
Na+ content (μmol (g wet weight)−1)	15.2 ± 0.2	20.8 ± 0.3*	18.8 ± 0.7*	15.1 ± 0.4†
K+ content (μmol (g wet weight)−1)	87.7 ± 0.8	81.5 ± 1.0*	81.0 ± 1.3*	86.2 ± 0.4†
Total 86Rb+ uptake (nmol (g wet weight)−1 min−1)	514 ± 13	581 ± 8*	523 ± 16	704 ± 13*†
Ouabain-sensitive 86Rb+ uptake (nmol (g wet weight)−1 min−1)	296 ± 9	404 ± 12*	323 ± 16	516 ± 12*†

Muscles were mounted for isometric contraction and incubated with or without 10⁻⁴m carbacholine for 10 or 60 min ±salbutamol, with the final 10 min in the presence of ⁸⁶Rb⁺. After incubation, the muscles were washed for 4 × 15 min in ice-cold Na⁺-free Tris-sucrose, blotted, weighed, and prepared for flame photometric analysis of Na⁺ and K⁺ contents and counting of ⁸⁶Rb⁺. All values are given as means ± s.e.m. (n = 6–9).

^*Significant difference (P < 0.05) compared with control;

^†significant difference (P < 0.05) compared with 60 min carbacholine incubation.

Na⁺–K⁺ pump activity

It is well known that increasing [Na⁺]_i results in stimulation of the Na⁺–K⁺ pump (Clausen, 2003), and it was therefore necessary to investigate the effect of carbacholine on the Na⁺–K⁺ pump activity. Exposure to carbacholine for 10 min significantly increased (P < 0.05) the total ⁸⁶Rb⁺ uptake compared with control muscles; however, after 60 min exposure to carbacholine it returned to the control level. Further experiments were performed where ouabain was used to block the activity of the Na⁺–K⁺ pump (Table 1), with subtraction of the ouabain-insensitive ⁸⁶Rb⁺ uptake from the total ⁸⁶Rb⁺ uptake, giving the ouabain-sensitive ⁸⁶Rb⁺ uptake. Exposure to carbacholine for 10 min significantly increased (P < 0.05) ouabain-sensitive ⁸⁶Rb⁺ uptake by 36% compared with controls (Table 1). Interestingly, prolonging the time of incubation to 60 min significantly (P < 0.05) reduced the ouabain-sensitive ⁸⁶Rb⁺ uptake back to control levels, despite both the intracellular Na⁺ and K⁺ contents still being significantly altered (Table 1). The large 60% increase in ouabain-sensitive ⁸⁶Rb⁺ uptake (P < 0.05) during exposure to salbutamol, confirms that salbutamol does indeed stimulate the Na⁺–K⁺ pump, as indicated by the significant restoration of intracellular Na⁺ and K⁺ contents also shown in Table 1.

Location of the Na⁺ influx

As the nACh receptors in normal skeletal muscle are primarily located at the motor end plate, muscles that had been exposed to carbacholine in the presence of ²²Na⁺ for 5 min were cut into three sections to determine the location of the carbacholine-induced Na⁺ influx. Figure 5A shows that in the mid region of the muscle, near the motor end plate, carbacholine (10⁻⁴m) induced a significant (P < 0.05) 3.2-fold increase in Na⁺ influx, while in the distal regions, there was a twofold increase compared with the corresponding regions in the control muscles. This increase in permeability of the membrane was further shown in the muscle Na⁺ and K⁺ contents for the corresponding regions. Thus, the Na⁺ content of the mid region was significantly (P < 0.05) increased by 70% compared with controls, whereas both distal regions showed a significant (P < 0.05) but smaller 20% increase in Na⁺ content (Fig. 5B). The K⁺ content of the mid region after exposure to carbacholine was significantly (P < 0.05) reduced, whereas that of the distal regions was unchanged compared with controls (Fig. 5C). All of these effects of carbacholine could be completely abolished by pretreatment with tubocurarine.

Figure 5. Site of the carbacholine-induced Na⁺ influx.

Effect of 10⁻⁴m carbacholine (filled columns) and carbacholine with 10⁻⁴m tubocurarine (open columns) versus control (grey columns) on ²²Na⁺ influx (A), Na⁺ content (B), and K⁺ content (C) at the origin (or), mid region (mid) and insertion (in) regions of rat soleus muscles. Values are means with bars denoting s.e.m. (n = 4–8), with * and # representing a significant difference (P < 0.05) compared with controls and with distal regions of the muscle, respectively.

Membrane potential

Carbacholine induced a significant depolarization of resting membrane potential (Fig. 6), although the degree of increase differed depending on the region of the muscle. In the mid region of the muscle the membrane potential was significantly depolarized from −73 ± 1 to −60 ± 2 mV (P < 0.05) after 5 min incubation with carbacholine, whereas at the distal regions the membrane potential changed from −73 ± 1 to −67 ± 1 mV (P < 0.05). Over the following 55 min, still in the presence of carbacholine, the membrane potential did not change at the mid region, but significantly further depolarized at the distal region to −64 ± 1 mV. Upon the addition of salbutamol to the muscle, membrane potential was significantly (P < 0.05) repolarized to −63 ± 1 and −66 ± 1 mV, and further repolarized to −65 ± 1 and −68 ± 1 mV over the next 55 min at the mid region and distal regions of the muscle, respectively.

Figure 6. Carbacholine-induced membrane depolarisation.

Effect of 10⁻⁴m carbacholine and 10⁻⁵m salbutamol on muscle resting membrane potential at the mid region (•) and distal (○) regions of rat soleus muscles. Values are means ± s.e.m. (n = 5–8) with a total of 50–80 measurements on 5–8 individual muscles for each value.

Succinylcholine

In clinical situations succinylcholine, a compound similar to carbacholine, is used for the blockade of motor end plate function, so additional experiments were performed using succinylcholine (10⁻⁴m) rather than carbacholine, with the results shown in Fig. 7. After a 10 min exposure to succinylcholine, force was reduced to 23 ± 8% of controls, which then partially recovered to a steady-state level of approximately 40% of initial control force. This effect of succinylcholine could be abolished by pretreatment with tubocurarine, and markedly recovered to 77 ± 5% of control force by Na⁺–K⁺ pump stimulation with salbutamol. These results are very similar to those observed using carbacholine (Figs 1 and 2).

Figure 7. Effect of succinylcholine on tetanic force.

Effect of succinylcholine (10⁻⁴m) on tetanic force development (•) in soleus muscles, which is prevented by pretreatment with tubocurarine (10⁻⁵m; ▪), or significantly recovered by addition of salbutamol (10⁻⁵m; ○) to stimulate Na⁺–K⁺ pump activity. Data points are means ± s.e.m., n = 4.

Discussion

The major new observation of this study is that chronic activation of the nACh receptor by carbacholine mimics the reduction in the membrane potential and the chemical Na⁺ and K⁺ gradients observed during intense exercise, and that the subsequent loss of muscle excitability and contractility can be restored by Na⁺–K⁺ pump stimulation.

Site of the carbacholine-induced Na⁺ influx

The site of the carbacholine-induced Na⁺ influx was exclusively through the nACh receptors, as it could be completely abolished by pretreatment with tubocurarine (Figs 1B and 4A). As the nACh receptors are located primarily in the motor end plate region in normal skeletal muscle (Kuffler & Yoshikami, 1975; Bekoff & Betz, 1977), the 50% greater Na⁺ influx in the mid region of the muscle compared with the distal regions seems reasonable (Fig. 4). A previous study by Creese et al. (1977) found that carbacholine induced a Na⁺ influx that was three times larger in the motor end plate region than in more distal regions of rat diaphragm muscle. The smaller increase in Na⁺ influx observed in this study may be a result of the longer 5 min incubation time in ²²Na⁺ as opposed to the 15 s incubation with ²⁴Na⁺ used by Creese et al. (1977). Indeed, Fig. 5 shows that a fraction of the ²²Na⁺ has spread along the length of the muscle fibres to the periphery, a scenario unlikely to occur to such an extent after just 15 s incubation. Along with an increase in Na⁺ influx, it has been previously shown that carbacholine induces an increase in [Ca²⁺] around the neuromuscular junction (Evans, 1974). However, no change in baseline force was observed in the present study, indicating that the amount of Ca²⁺ entering the muscle must be relatively small. Rather surprisingly, TTX had no effect on the Na⁺ influx, despite the significant membrane depolarization to −60 mV, suggesting that a depolarization of this magnitude is not large or fast enough to open the voltage-gated Na⁺ channels.

Loss of muscle contractility

Opening of the nACh receptors by the addition of carbacholine increases the permeability of the muscle fibres to Na⁺ and K⁺, leading to depolarization, net influx of Na⁺ and net efflux of K⁺ with ensuing accumulation of intracellular Na⁺ and loss of intracellular K⁺. Similar changes in membrane potential and muscle Na⁺ and K⁺ contents have been implicated in high-frequency muscle fatigue, in which the loss of excitability is related to a depolarization caused by accumulation of extracellular K⁺ (for review see Sejersted & Sjøgaard, 2000). In this context, Cairns et al. (1997) suggested that the loss of excitability in depolarized muscles was via two mechanisms that are related to the extent of depolarization of the membrane. The first was associated with a limited reduction in contractility when the membrane potential was depolarized to −60 mV and related to altered action potential profile. The second occurred when force was severely depressed and was associated with a membrane potential between −60 and −55 mV, and the result of total inexcitability of some fibres. Similar effects of depolarization have been demonstrated by Rich & Pinter (2003) who showed that at a membrane potential of around −55 mV, several fibres were totally inexcitable. The magnitude of the carbacholine-induced membrane depolarization depicted in Fig. 6, and concomitant decrease in tetanic force (Fig. 1A) and twitch force (Fig. 2), concur with this observation, and suggest that the loss of force induced by carbacholine was predominantly related to the depolarization of the muscles. Ruff (1996) proposed that the depolarization-induced loss of excitability was due to slow inactivation of a proportion of the voltage-gated Na⁺ channels, rendering the membrane incapable of propagating action potentials.

The finding that addition of salbutamol to carbacholine-exposed muscles led to complete recovery of the intracellular contents of Na⁺ and K⁺ (Table 1), but only produced a partial recovery of membrane potential (Fig. 6), indicates that the depolarization induced by carbacholine was mainly related to the increase in Na⁺ permeability of the muscles. The loss of force in the carbacholine-exposed muscles may, however, have been influenced by the concomitant increase in intracellular Na⁺, which depending on the concomitant reduction in the chemical gradient for Na⁺, may increase or further decrease excitability of depolarized muscles. Thus, in mechanical skinned fibres depolarized by lowered chemical gradient for K⁺, an increase in cytosolic Na⁺ from 10 to 20 mm led to an increase in the excitation-induced force production, which was related to stimulation of the Na⁺–K⁺ pump (Nielsen et al. 2004). At more pronounced increases in cytosolic Na⁺, however, the reduction in the chemical gradient for Na⁺ tended to reduce excitability, especially at high motor frequencies (Nielsen et al. 2004). In concord with this, it has been shown in both frog sartorius muscle (Bouclin et al. 1995) and rat soleus muscle (Overgaard et al. 1999) that a reduction in the chemical gradient for Na⁺ by lowering extracellular [Na⁺] (i.e. at constant intracellular Na⁺) exacerbates the loss of force induced by depolarizing the muscles by increasing [K⁺]_o.

The membrane depolarization and increases in the Na⁺ and K⁺ permeabilities induced by carbacholine were clearly more pronounced in the region of the muscle close to the motor end plates than in the distal parts of the muscle. This could indicate that the loss of excitability after addition of carbacholine was more severe close to the motor end plates. Despite this, the contractile force elicited by field stimulation across the central part of the muscle was similar to the force response when stimulated across the ends of the muscles. One possible explanation for this could be that the density of the voltage-gated Na⁺ channels per membrane area is threefold higher in the mid portion of the muscle than at the ends (Ruff, 1996). This distribution of Na⁺ channels is suggested to function as a safety factor for the neuromuscular transmission (Ruff, 1996), but may in addition make the excitability of the central region less sensitive to slow inactivation of Na⁺ channels.

Recovery of contractility with Na⁺–K⁺ pump stimulation

Na⁺–K⁺ pump stimulation has previously been shown to restore the reduced contractility in muscles depressed by elevated [K⁺]_o (Tomita, 1975; Clausen et al. 1993; Cairns et al. 1995), lowered [Na⁺]_o (Overgaard et al. 1997), the presence of Ba²⁺ (Clausen & Overgaard, 2000) and following membrane permeabilization elicited by electroporation (Clausen & Gissel, 2005). The force recovery was attributed to either an increase in transmembrane Na⁺ gradient (Cairns et al. 1995; Overgaard et al. 1997), membrane repolarization (Clausen & Gissel, 2005) or a combination of both of these mechanisms (Clausen & Overgaard, 2000). An important difference between the results from the current study and those that have increased [K⁺]_o to depolarize muscles (e.g. Clausen et al. 1993) is the alteration in the time course of salbutamol-induced force recovery. Clausen et al. (1993) observed a maximal recovery of force after approximately 10 min, which then declined to pre-salbutamol levels. This subsequent loss of contractility is likely to be due to the accelerated Na⁺–K⁺ pump activity reducing intracellular Na⁺ (Overgaard et al. 1997; Buchanan et al. 2002), which itself will decrease the activity and the electrogenic effect of the Na⁺–K⁺ pump. Our results here show a force recovery mediated by Na⁺–K⁺ pump stimulation that requires 30 min for maximum effect, which is then maintained (Fig. 2). The ability to sustain force after addition of salbutamol (in excess of 2 h, data not shown) must be due to the intracellular Na⁺ content being maintained sufficiently high in the presence of carbacholine, which ensures that the Na⁺–K⁺ pump is significantly activated. Since this situation more closely resembles the intracellular Na⁺ status in muscle during intensive contractile activity, where an increase, rather than a decrease in intracellular Na⁺ takes place (Juel, 1986; Balog & Fitts, 1996; Nielsen & Clausen, 1996), the maintained force recovery observed in this study suggests that catecholamine-induced stimulation of the Na⁺–K⁺-pump is more important for muscle function during exercise than indicated by previous studies.

In Table 1 it can be seen that addition of salbutamol to a carbacholine-depressed muscle re-instates the chemical Na⁺ and K⁺ gradients. Despite the recovery of the chemical Na⁺ and K⁺ gradients, the preparation was still significantly depolarized compared to controls (Fig. 6). As carbacholine was still present, the permeabilities for Na⁺ and K⁺ would be higher than control muscles, which may explain the long-lasting membrane depolarization and inability to completely recover force. Others have shown that only a small hyperpolarization was sufficient to enable significant recovery of contractility in depolarized muscle (Overgaard et al. 1997), presumably by re-activating a percentage of the voltage-gated Na⁺ channels, which undergo slow in-activation due to depolarization of the membrane (Ruff, 1996). Additionally, Cairns et al. (1997) showed that depolarization of the membrane potential to −60 mV reduced tetanic force by only 20%, whereas a further depolarization to between −60 and −55 mV was related to an 80% reduction in force. Taking this into consideration, the small hyperpolarizing effect of salbutamol on carbacholine-depressed muscles (membrane potential from −60 to −65 mV) would be sufficient to cause significant force recovery to approximately 80% of controls, as shown in Fig. 2.

The recovery of force in carbacholine-treated muscles could also be induced by addition of CGRP (Fig. 2C). In contrast to catecholamines, which during exercise are released to the blood stream, CGRP is released locally from nerve endings in the contracting muscles (Sakaguchi et al. 1991). As such, the exercise-induced increase in the circulating levels of catecholamines will stimulate Na⁺–K⁺ pump activation in all muscles of the body, and hence protect both resting and active muscles against the depolarizing effect of increased [K⁺]_o. In active muscles, this protection may be reinforced by local release of CGRP, which could further stimulate the Na⁺–K⁺ pump.

Spontaneous recovery of contractility

An interesting observation from this study was the partial spontaneous recovery of force after the initial 10 min exposure to high concentrations of carbacholine (Fig. 1A), which appears to be accompanied by a small recovery of chemical Na⁺ and K⁺ gradients as shown in Table 1. Creese et al. (1987) also observed a spontaneous recovery of membrane potential in rat diaphragm muscles exposed to carbacholine depolarization, and suggested that this may be due to an accelerated Na⁺–K⁺ pump activity. The increased [Na⁺]_i upon increased membrane permeability would lead to Na⁺–K⁺ pump stimulation (Clausen, 2003) as outlined by the increase in ouabain-sensitive ⁸⁶Rb⁺-uptake from Table 1, partially re-establishing the chemical Na⁺ and K⁺ gradients. De-sensitization of the nACh receptor by depolarizing drugs is well known (Hille, 2001), which would lead to a reduction in the carbacholine-induced Na⁺ influx. Creese et al. (1987) found that the initial carbacholine-induced motor end plate current of 100 nA was reduced to just 11 nA after 30 min preincubation with carbacholine, and had previously shown, using ²⁴Na⁺ as a tracer, that the Na⁺ influx was significantly reduced after preincubation with carbacholine prior to exposure to the isotope (Creese et al. 1977). Indeed, Table 1 shows that the ouabain-sensitive ⁸⁶Rb⁺ uptake is returned to control levels after 60 min carbacholine exposure. The spontaneous recovery of force is therefore most likely to be due to a combination of Na⁺–K⁺ pump activation and de-sensitization of the nACh receptor to carbacholine.

Perspectives

In association with surgical procedures, succinylcholine is widely used for the blockade of motor end plate function, and it has been repeatedly shown to induce hyperkalaemia, in some cases sufficient to cause cardiac arrest (for review see Huggins et al. 2003). A subset of patients administered succinylcholine show an upregulation of skeletal muscle nACh receptors (Fung et al. 1991), which is thought to be the source of the hyperkalaemia. These patients are deprived of neural activity, which stimulates the synthesis of new nACh receptors (Huggins et al. 2003), not only around the motor end plate region of the muscle, but across the whole muscle membrane (Martyn et al. 1992). Thus, there is a large increase in the succinylcholine-sensitive area, which allows more ion channels to become available to release K⁺ from the muscle. Furthermore, the newly synthesized nACh receptors containing the γ subunit have a longer open time than that of the regular ε subunit (Martyn et al. 1992), which may further exacerbate the hyperkalaemia. Additionally, downregulation of the Na⁺–K⁺ pump in skeletal muscle also occurs with inactivity (Kjeldsen et al. 1986; Leivseth & Reikas, 1994), which further compromises the ability of the muscles to compensate for the elevated K⁺ loss. Indeed, Gronert & Theye (1975) showed that denervated, paraplegic and immobilized canine muscle produced a significantly larger K⁺ efflux in response to succinylcholine administration compared with normal muscle. The current study highlights the need for caution when administering depolarizing drugs to patients that have undergone long periods of inactivity, such as extended bed rest, and that stimulation of the Na⁺–K⁺ pump using a β₂-agonist, such as salbutamol, may counteract the succinylcholine-induced hyperkalaemia.