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Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  • 1
    The present experiments were performed to assess the role of K+ channels in hormonal stimulation of the Na+-K+ pump and to determine the contribution of Na+-K+ pumps to the recovery of excitability and contractility in depolarized skeletal muscle.
  • 2
    In soleus muscle, Ba2+ (0.02 and 1 mm) was found to inhibit 42K+ efflux and 42K+ influx. Both in the absence and the presence of Ba2+ (1 mm), salbutamol and calcitonin gene-related peptide (CGRP) induced a marked decrease in intracellular Na+ and stimulation of 42K+ uptake.
  • 3
    In soleus muscles Ba2+ (0.1 and 1.0 mm) decreased twitch and tetanic force. Subsequent stimulation of the Na+-K+ pumps by salbutamol, CGRP or repeated electrical stimulation produced a highly significant restoration of force development, which was suppressed by ouabain, but not by glibenclamide. Also, in extensor digitorum longus muscles Ba2+ (0.1 mm) produced a considerable force decline, which was partly restored by salbutamol and CGRP.
  • 4
    The area of compound action potentials (M-waves) elicited by indirect stimulation was decreased by Ba2+ (0.1 mm). This was associated with a concomitant decrease in tetanic force and depolarization. Salbutamol, CGRP or repeated electrical stimulation all elicited marked recovery of M-wave area, force and membrane potential. All recordings showed close correlations between these three parameters.
  • 5
    The data add further support to the concept that due to its electrogenic nature and large transport capacity, the Na+-K+ pump is a rapid and efficient mechanism for the maintenance of excitability in skeletal muscle, acting independently of Ba2+- or ATP-sensitive K+ channel function.

It is well-documented that catecholamines, β2-adrenoceptor agonists and/or CGRP induce hyperpolarization in skeletal muscle (Tashiro, 1973; Clausen & Flatman, 1977; Andersen & Clausen, 1993; van Mil et al. 1995), cardiac myocytes (Gadsby, 1983; Glitsch et al. 1989) and vascular smooth muscle cells (for review, see Standen & Quayle, 1998). In skeletal muscle, this effect has often been attributed to cAMP-mediated stimulation of the electrogenic Na+-K+ pump (Clausen & Flatman, 1977; Andersen & Clausen, 1993; Li & Sperelakis, 1993). In heart and smooth muscle, however, the hyperpolarization induced by catecholamines and CGRP is thought to reflect the opening of K+ channels, mediated by cyclic AMP (Glitsch et al. 1989; Standen & Quayle, 1998). It has been proposed that this is also the mechanism in skeletal muscle (van Mil et al. 1995).

In skeletal muscles where contractility had been inhibited by exposure to high extracellular K+ concentration ([K+]o) (10–12.5 mm), acute activation of the Na+-K+ pump by hormones or electrical stimulation leads to a considerable force recovery (Clausen et al. 1993; Andersen & Clausen, 1993; Cairns et al. 1995; Nielsen et al. 1998). This was seen as the result of hyperpolarization induced by stimulation of the Na+-K+ pump. Since the force recovery is less complete at 12.5 mm K+ than at 10 mm K+ and abolished at 15 mm K+ (Clausen et al. 1993), it cannot be excluded, however, that it partly depends on activation of K+ channels.

The present study has three main purposes: (1) to determine the role of K+ channels in the stimulating effects of salbutamol and CGRP on the Na+-K+ pump in skeletal muscle; (2) to determine whether in depolarized muscles K+ channels are required for the restoration of excitability and contractility elicited by salbutamol, CGRP or repetitive electrical stimulation; and (3) to assess the potential of the Na+-K+ pumps in the maintenance of excitability and contractility by analysing for possible correlations between membrane potential (Vm), compound action potentials (M-waves) and contractile force.

Ba2+ blocks K+ channels in skeletal muscle, leading to reduced transmembrane K+ fluxes, depolarization, loss of excitability and paralysis (Roza & Berman, 1971; Layzer, 1982; Gallant, 1983; Castle & Haylett, 1987). We established in addition that, in rat soleus, blocking the K+ channels with Ba2+ induced inhibition of passive K+ fluxes, depolarization, suppression of M-waves and contractile force. In the presence of Ba2+, we tested the effects of hormonal and non-hormonal stimulation of the Na+-K+ pump and observed repolarization, restoration of M-waves and contractile force. Force restoration was also obtained in the presence of glibenclamide, which specifically blocks ATP-sensitive K+ channels (Castle & Haylett, 1987). The experiments indicate that Ba2+-sensitive K+ channels are not involved in the effects of salbutamol or CGRP on K+ influx, intracellular Na+, Vm, M-waves and contractile force.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Animals

All handling and use of animals complied with Danish animal welfare regulations. All experiments were performed using fed 4-week-old female or male Wistar rats weighing 60–75 g (own breed). The animals had free access to food (Altromin pellets No. 1314, Spezialfutterwerke, Lage, Germany) and water and were kept in a temperature-controlled environment (21°C) with a constant light-dark cycle (12–12 h).

Muscle preparation and incubations

Animals were killed by decapitation, and intact soleus or EDL muscles (wet wt 20–25 mg) dissected out. In muscles where nerve stimulation was applied, approximately 10 mm of the nerve was left attached. The standard incubation medium was Krebs-Ringer bicarbonate buffer (NKR; pH 7.4 at 30°C) containing (mm): 120.1 NaCl, 25.1 NaHCO3, 4.7 KCl, 1.2 KH2PO4, 1.2 MgSO4, 1.3 CaCl2, and 5 D-glucose. In experiments where Ba2+ was added, MgSO4 was replaced by MgCl2 in order to avoid precipitation of BaSO4. Before tension recordings, all muscles were equilibrated for at least 30 min in the standard buffer containing 1.2 mm SO42- and then transferred into SO42--free buffer. All experiments were carried out at 30°C to reduce metabolic requirements and thus ensure sufficient oxygenation of the central muscle fibres. During all experiments, the buffer was continuously gassed with a mixture of 95 % O2 and 5 % CO2. This procedure was shown to allow the maintenance of a high intracellular K+/Na+ ratio, constant membrane potential (Clausen & Flatman, 1977) and tetanic force for several hours in vitro (Clausen & Everts, 1991).

Force development

This was recorded as described in detail elsewhere (Clausen & Everts, 1991). Soleus or EDL muscles with intact tendons were mounted in thermostatically controlled chambers (volume, 23 ml; temperature, 30°C) containing Krebs-Ringer bicarbonate buffer and in most experiments stimulated directly via two platinum electrodes positioned close to the muscle surface. Supramaximal pulses (12 V and 1 ms duration) were obtained from a pulse generator, and force development measured with a force displacement transducer (Grass FTO3) using a chart recorder calibrated with standard weights. After adjustment to optimal length, active force of single twitches and tetanic contractions (soleus, 30 Hz for 2 s; EDL, 60 Hz for 0.5 s) were tested. This was followed by a 30 min rest period. During the subsequent experiment changes in contractile performance were followed by applying 2 s pulse trains of 30 Hz every 10 min. In some experiments the effects of shortening the intervals between these 2 s pulse trains to 1 min was explored.

42K+ and 86Rb+ exchange

The uptake and washout of 42K+ and 86Rb+ were measured using procedures described in detail elsewhere (Dørup & Clausen, 1994) and in the legend to Table 1.

Table 1.  Effects of Ba2+, ouabain, salbutamol and CGRP on 42K+ uptake and intracellular Na+ content in rat soleus
 42K+uptake (nmol (g wet wt)−1 min−1)PNa+ content (μmol (g wet wt)−1)P
  1. Soleus muscles were equilibrated for 30 min in SO42−-free buffer, preincubated for 15 min in the same buffer without or with Ba2+ or ouabain and then incubated for 20 min in buffer containing 42K+ (0.1 7mu;Ci ml−1) with the indicated additions. After this, the muscles were washed 4 × 15 min in icecold Na+-free Trissucroseto remove isotope and Na+ from the extracellular phase, blotted on dry filter paper, weighed and taken for counting and flame photometric determination of Na+. On the basis of the specific activity of 42K+ in the incubation medium, the uptake of 42K+ was calculated and expressed as nmol (g wet wt)−1 min−1 (means ±s.e.m.). Intracellular Na+ content was corrected for loss of Na+ during the washout in the cold (Everts & Clausen, 1992) and given in μmol (g wet wt)−1 (means ±s.e.m.). The number of observations is given in parentheses. Within each of the three sections, P values denote the significance of the difference between the upper group of values and the other values.

Controls558 ± 12 (12) 13.9 ± 0.1 (12) 
10−5m salbutamol723 ± 21 (4)< 0.0016.3 ± 0.9 (4)< 0.001
10−7m CGRP673 ± 23 (8)< 0.0016.3 ± 0.3 (8)< 0.001
1 mm BaCl2265 ± 5 (11) 10.8 ± 0.3 (11) 
10−5m salbutamol+ 1 mm BaCl2420 ± 12 (8)< 0.0016.0 ± 0.4 (8)< 0.001
10−7m CGRP+1 mm BaCl2435 ± 4 (8)< 0.0015.8 ± 0.4 (8)< 0.001
1 mm ouabain386 ± 12 (4) 42.9 ± 0.6 (4) 
1 mm ouabain+1 mm BaCl286 ± 2 (12)< 0.00131.2 ± 0.4 (12)< 0.001
10−5m salbutamol+1 mm ouabain+1 mm BaCl2109 ± 4 (8)< 0.00131.0 ± 1.0 (8)< 0.001
10−7m CGRP+1 mm ouabain+1 mm BaCl2114 ± 4 (4)< 0.00129.6 ± 1.9 (4)< 0.001

Na+ content

Intracellular Na+ content was measured as described in detail elsewhere (Everts & Clausen, 1992) and in the legend to Table 1.

Nerve stimulation, M-wave recordings and membrane potentials

Nerve stimulation and M-wave recording was performed essentially as described elsewhere (Overgaard et al. 1999) with the following exceptions. Fixed current pulses of 5 mA were given through a stimulus isolator (ISU 165, Cibertec, Spain). This was found to be supramaximal for stimulation of the nerve without producing any direct stimulation of muscle fibres. In these experiments a 0.2 ms pulse duration and 1.5 s 30 Hz tetanic trains were used. Amplification, data processing and analysis of M-wave signals were performed as previously described (Overgaard et al. 1999).

The resting membrane potential (Vm) of surface muscle fibres was measured at 30°C by standard electrophysiological technique essentially as described previously (Clausen et al. 1993) except for the following details. The microelectrodes were filled with 1 M potassium citrate (resistance 15–35 MΩ). The muscles were placed in a thermostatically controlled 30 ml flow chamber with buffer flowing through at a rate of 15 ml min−1 from a reservoir. The muscle bath was grounded with a Ag-AgCl electrode placed a few centimetres from the muscle. Vm was determined as the average of the values obtained in a series of 10 impalements. In order to avoid measuring from the same fibre twice, the electrode was moved a small distance across the muscle between each impalement in a series.

Statistics

The statistical significance of any difference was ascertained using Student's two-tailed t test for non-paired observations. All values are given as means ±s.e.m. Correlations between variables were analysed with linear regression.

Chemicals and isotopes

All chemicals were of analytical grade. Rat CGRP was obtained from Peninsula Laboratories Inc., Belmont, CA, USA. Glibenclamide, ouabain and salbutamol were obtained from Sigma Chemicals, St Louis, MO, USA. 42K+ (40 Ci mol−1) and 86Rb+ (0.4 Ci mol−1) were obtained from the Danish Atomic Energy Commission Isotope Laboratory (Risø, Denmark).

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Effects of Ba2+, salbutamol and CGRP on K+ fluxes and Na+ content

The first series of experiments served to identify the effects of Ba2+ on K+ transport and Na+ content in rat soleus. Ba2+, at concentrations of 0.02 and 1 mm, induced a progressive and highly significant inhibition (P < 0.001,n= 3 in each group) of 42K+ efflux, reaching a maximum decrease of 29 and 65 %, respectively, within 20 min.

The same concentrations of Ba2+ were found to inhibit 42K+ uptake by 17 and 53 %, respectively. As shown in Table 1, the decrease in 42K+ uptake induced by 1 mm Ba2+ (293 nmol (g wet wt)−1 min−1) and 1 mm ouabain (172 nmol (g wet wt)−1 min−1) were strictly additive, and in combination the two agents produced an inhibition of 472 nmol (g wet wt)−1 min−1. In the absence of Ba2+, salbutamol (10−5 M) and CGRP (10−7 M) increased 42K+ uptake by 165 nmol (g wet wt)−1 min−1 and 115 nmol (g wet wt)−1 min−1, respectively. In the presence of 1 mm Ba2+, salbutamol (10−5 M) and CGRP (10−7 M) increased 42K+ uptake by 155 nmol (g wet wt)−1 min−1 and 170 nmol (g wet wt)−1 min−1, respectively, indicating that these effects are not dependent on the function of K+ channels. In the presence of both ouabain (1 mm) and Ba2+ (1 mm) the effects of salbutamol and CGRP were considerably reduced (to 15 and 16 %, respectively, of the absolute increase in 42K+ uptake elicited in the presence of Ba2+ alone), but still statistically significant (P < 0.001). This indicates that in the presence of Ba2+ both agents stimulate K+ uptake mediated by the Na+-K+ pump. Since 1 mm Ba2+ might be inadequate to produce complete suppression of K+ channel function, the effect of 5 mm Ba2+ was also tested. At that concentration, salbutamol (10−5 M) and CGRP (10−7 M) increased 86Rb+ uptake by 35 and 26 %, respectively (n= 8vs. 8 muscles, P < 0.001 in both instances).

Table 1 shows that salbutamol and CGRP both induced a significant decrease in intracellular Na+ content. Ba2+ (1 mm) also produced a minor (22 %) decrease in intracellular Na+ content (P < 0.001). In the presence of 1 mm Ba2+, salbutamol and CGRP produced a further decrease in intracellular Na+ (44 % and 46 %, respectively). Also, in the presence of ouabain (1 mm), Ba2+ (1 mm) decreased intracellular Na+, but the effects of salbutamol and CGRP were abolished.

Other experiments showed that in the presence of 5 mm Ba2+, salbutamol (10−5 M) and CGRP (10−7 M) decreased intracellular Na+ by 25 and 20 %, respectively (n= 8vs. 8, P < 0.005 and P < 0.025), again indicating that even at a very high concentration of Ba2+, the stimulating effect of these agents on the Na+-K+ pump was preserved.

Effects of Ba2+, salbutamol, CGRP and repeated electrical stimulation on contractile force

Figure 1 shows the time course of the effect of Ba2+, salbutamol and CGRP on tetanic force development in rat soleus. Ba2+ (0.1 mm) clearly decreased force development and after 120 min, a value corresponding to 50 ± 3 % of the control level was still maintained. Twitch force showed a similar decrease (to 40 ± 6 % (n= 4,P < 0.002) of the control level, not shown). When Ba2+ was washed out by repeated changes to Ba2+-free buffer, force recovery was complete within 30 min (3 muscles, data not shown). In another experiment, where a muscle had been exposed to 1 mm Ba2+ and then washed with Ba2+-free buffer, full force recovery was reached in 40 min, indicating that the effect of Ba2+ is completely reversible. It should be noted that, as shown in the early phase of the curves, the omission of SO42- from the buffer caused no change in force development.

image

Figure 1. Effects of salbutamol and CGRP on tetanic force in the presence of Ba2+

Soleus muscles were equilibrated for 30 min in standard buffer and isometric tetanic contractions were elicited by direct stimulation using 30 Hz pulse trains of 2 s duration (1 ms pulses, 12 V). The buffer (NKR, Krebs-Ringer bicarbonate buffer) was then changed to SO42--free buffer and after a further 15 min of equilibration, BaCl2 (0.1 mm) was added. After 120 min, salbutamol (10−5 M) or rat CGRP (10−7 M) were added. Each point represents the mean of observations on 3–5 muscles with bars indicating s.e.m.

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In buffer containing 0.1 mm Ba2+, salbutamol (10−5 M) induced a substantial and highly significant (P < 0.01) recovery of tetanic force, reaching 84 ± 8 % of the control level within 40 min. Twitch force increased to 87 ± 9 % (n= 4,P < 0.003) of the control level (not shown). In the presence of ouabain (5 mm), the salbutamol-induced force recovery at 0.1 mm Ba2+ was decreased by 84 % (n= 2). Other experiments showed that 1 mm Ba2+ produced a more pronounced reduction in tetanic force (to 31 ± 3 % of the control level, n= 5,P < 0.001; not shown). The subsequent addition of salbutamol (10−5 M) produced a recovery to 67 % of the control level within 30 min (n= 2).

As shown in Fig. 1, at 0.1 mm Ba2+, CGRP (10−7 M) produced a force recovery similar to that of salbutamol (to 87 ± 5 % of the control level, n= 5,P < 0.001). In the presence of ouabain (1 mm), the CGRP-induced force recovery was suppressed by 90 % (n= 2, not shown). At 1 mm Ba2+, CGRP (10−7 M) induced a force recovery from 31 ± 3 % to 56 ± 4 % of the control level (n= 3,P < 0.01, not shown). In the presence of ouabain (1 mm) this force recovery was suppressed by 91–93 % (n= 2).

We have previously shown that the inhibitory effect of high [K+]o can be alleviated by shortening the intervals between the pulse trains from 10 to 1 min. This effect could be attributed to stimulation of the Na+-K+ pump, partly mediated by CGRP released from nerve endings in the muscle (Nielsen et al. 1998).

In the present study, when force was tested at 10 min intervals Ba2+ (0.1 mm) produced a considerable inhibition. When the intervals were then reduced to 1 min, however, force increased progressively, reaching 82 ± 3 % of the control level within 25 min (n= 3,P < 0.001; not shown). In the presence of ouabain (1 mm or 5 mm), this force recovery was suppressed by 86 % (n= 1) and 89–90 % (n= 2), respectively.

It might be envisaged that the recovery of contractility elicited by salbutamol, CGRP or repeated electrical stimulation were the outcome of activation of ATP-sensitive K+ channels. Although 1 mm Ba2+ produces near maximum inhibition of the increase in 86Rb+ efflux produced by metabolic exhaustion in frog semitendinosus muscle (Castle & Haylett, 1987), a fraction of operative K+ channels may still be left. This possibility was tested by repeating the experiments with 0.1 mm Ba2+, adding the K+ channel blocker glibenclamide (10−4 M) 30 min prior to the application of the above-mentioned three Na+-K+ pump-stimulating treatments. Even at this concentration, which was shown to produce maximum inhibition of 86Rb+ efflux (Castle & Haylett, 1987), glibenclamide caused no detectable change in force development or the force recovery induced by Na+-K+ pump stimulation. Thus, the force recovery seems independent of ATP-sensitive K+ channels (data not shown).

In order to examine whether the effects of Na+-K+ pump stimulation were restricted to soleus muscles, similar experiments were performed using EDL muscles. Ba2+ (0.1 mm) was found to reduce tetanic force to 30 ± 8 % (n= 5) of the control level. Salbutamol (10−5 M) and CGRP (10−7 M) were found to elicit a force recovery reaching 53–87 % (n= 2) and 51 ± 3 % (n= 3), respectively, of the control level within 20 min (data not shown).

Effects of Ba2+, salbutamol, CGRP and repeated electrical stimulation on Vm, M-waves and force development

Figure 2 shows that in soleus muscles stimulated via the nerve, 0.1 mm Ba2+ induced a marked decrease of both tetanic force and M-wave area to 21 ± 4 % and 16 ± 5 % of the control level, respectively. Addition of CGRP (10−7 M) induced a recovery of both tetanic force and M-wave area which was maximal (73 ± 6 % and 68 ± 17 % of the control level, respectively; n= 4,P < 0.05) within 40 min. In a similar set of experiments (not shown), incubation with 0.1 mm Ba2+ decreased tetanic force and M-wave area to 43 ± 10 % and 32 ± 10 % of the control level, respectively. Addition of salbutamol (10−5 M) induced a recovery of tetanic force and M-wave area, which was maximal (87 ± 1 % and 73 ± 6 % of the control level, respectively; n= 4,P < 0.05) within 30 min. In a third series of experiments (not shown) 0.1 mm Ba2+ reduced the tetanic force and M-wave area to 27 ± 6 % and 17 ± 7 %, respectively, of the control value. Stimulating with tetanic pulse trains (1.5 s at 30 Hz) at 1 min intervals induced a recovery of tetanic force and M-wave area to 59 ± 5 % and 47 ± 6 %, respectively, of control level (n= 3,P < 0.05) within 30–40 min. In muscles that had been exposed to 0.1 and 1 mm Ba2+, the return to a Ba2+-free buffer caused a recovery of force and M-wave of the same order of magnitude as elicited by the stimulation of the Na+-K+ pump described above (not shown).

image

Figure 2. Effects of CGRP on tetanic force, M-wave area and Vm in the presence of Ba2+

Upper panel: muscles were stimulated tetanically (30 Hz, 1.5 s) via the nerve at the time points indicated. Each point represents the mean of observations on 4 muscles with bars indicating ±s.e.m. M-wave area, ○. Tetanic force, •. Lower panel: recordings of membrane potential were taken at the time points indicated. Each point represents the mean observation on 4 muscles (total of 40 fibres) with bars indicating ±s.e.m. (n= 4).

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As shown in Fig. 2, the membrane potential was measured at several times during the time course of the experiments. Incubation in buffer containing 0.1 mm Ba2+ led to a progressive depolarization from −70 ± 2 to −56 ± 2 mV (P < 0.01,n= 4) after 150 min. A significant repolarization to −65 ± 2 mV (P < 0.05,n= 4) was seen 40 min after addition of CGRP (10−7 M). In another group of muscles, 0.1 mm Ba2+ induced a depolarization from −73 ± 1 mV to −59 ± 2 mV (P < 0.01,n= 4), and salbutamol induced a repolarization to −67 ± 1 mV (P < 0.01,n= 4). In the final group a Ba2+-induced depolarization from −71 ± 1 mV to −56 ± 2 mV (P < 0.01,n= 3) was seen. Stimulating with tetanic pulse trains (1.5 s at 30 Hz) at 1 min intervals induced a repolarization to −63 ± 2 mV (P < 0.05,n= 3). Washout of Ba2+ and return to a Ba2+-free buffer caused a similar repolarization (not shown).

A set of experiments were done to examine whether a higher concentration of Ba2+ could interfere with the hyperpolarizing effect of salbutamol. Muscles were incubated in SO42--free buffer with a Ba2+ concentration of 5 mm for 120 min. This led to a membrane depolarization from −70 ± 2 mV to −50 ± 0.2 mV (n= 3). When salbutamol (10−5 M) was subsequently added, a significant repolarization to −57 ± 2 mV (n= 3,P < 0.05) was seen within 30–50 min. In the presence of 5 mm Ba2+, tetanic force and M-waves were reduced to 6 % and 7 % of the control level, respectively. Following addition of salbutamol only marginal recovery was observed.

The changes induced by Ba2+ and Na+-K+ pump stimulation in tetanic force, M-wave area and membrane potential had time courses that were closely similar (see Fig. 2). This led us to further examine the relations between these three parameters in the muscles that were incubated at 0.1 mm Ba2+ and subsequently exposed to salbutamol, CGRP or repeated electrical stimulation. To illustrate the possible correlations, corresponding data points for tetanic force, M-wave area and Vm obtained during the entire time course were plotted in a three-dimensional diagram (Fig. 3). When the relations between the three variables were examined separately, the following significant linear correlations were found.

image

Figure 3. Relationships between membrane potential, M-wave area and tetanic force

Corresponding data points for simultaneous recordings of M-wave area, tetanic force and Vm. Data points were collected from 11 individual muscles in 3 groups subjected to experiments with a time course similar to that shown in Fig. 2. The data points included correspond to the times 0, 60, 150 and 180 min in Fig. 2. After incubation in SO42--free buffer containing 0.1 mm Ba2+ muscles were given either CGRP (10−7 M) (as in Fig. 2) or salbutamol (10−5 M), or were stimulated at 1 min intervals to induce recovery (n= 3–4 in each group). A regression plane was fitted to the data points in the cube; the points situated above the plane are represented by open circles, and those below the plane by shaded circles.

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Tetanic force vs. wave area: r= 0.95,P < 0.001,

Tetanic force vs. Vm: r= 0.85,P < 0.001,

M-wave area vs. Vm: r= 0.83,P < 0.001.

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

Hormonal stimulation of the Na+-K+ pump

Ba2+ (1 mm) was found to produce a marked inhibition of 42K+ uptake in soleus muscles. In the presence of 1 mm Ba2+, ouabain inhibits 42K+ uptake by almost the same absolute amount as in its absence. This indicates that the basal rate of Na+-K+ pump-mediated K+ influx is almost unaffected by Ba2+.

As previously shown, salbutamol and CGRP stimulate the uptake of 42K+ and decrease intracellular Na+. These effects are not reduced by Ba2+ (1 mm), but the further addition of ouabain (1 mm) almost completely (84–85 %) suppressed the increase in 42K+ uptake and abolished the effect on intracellular Na+. This strongly indicates that in the presence of Ba2+, salbutamol and CGRP stimulate the Na+-K+ pump. Moreover, even in the presence of 5 mm Ba2+, both salbutamol and CGRP induced highly significant stimulation of 86Rb+ uptake and a decrease in intracellular Na+. Finally, in the presence of Ba2+ (0.1 mm or even 5 mm), where the muscles were depolarized by 14–20 mV, salbutamol induced repolarizations of 7–9 mV. This effect is unlikely to reflect activation of K+ channels. Taken together the data indicate that in rat soleus muscles, hormonal or excitation-induced stimulation of the Na+-K+ pump does not depend on the functional state of the K+ channels. This conclusion is not in keeping with the observation that in mouse lumbrical muscles fibres, the hyperpolarizing effect of the β-adrenoceptor agonist isoprenaline is blocked by 0.6 mm Ba2+ (van Mil et al. 1995). It cannot be excluded, therefore, that in certain types of skeletal muscle, K+ channels contribute to β-adrenoceptor agonist-induced hyperpolarization.

Role of Na+-K+ pump in force recovery in Ba2+-depolarized muscles

In muscles, where contractility had been reduced by the addition of Ba2+ (0.1 or 1.0 mm), both salbutamol, CGRP and repeated electrical stimulation produce a highly significant force recovery. This is considerably suppressed by ouabain, indicating that activation of the Na+-K+ pump is instrumental in eliciting the effect, probably due to its electrogenic action.

It is surprising, however, that in the presence of ouabain (1 mm or even up to 5 mm), salbutamol or CGRP still elicit a minor force recovery. This is in keeping with the persistance of a small stimulating effect of salbutamol or CGRP on 42K+ uptake observed in the presence of Ba2+ and ouabain (see Table 1). A possible explanation is that in rat skeletal muscle, part of the Na+,K+-ATPase is the α1-isoform, which in rats has a low affinity for ouabain and therefore may not be completely inhibited (McDonough & Thompson, 1996). The force recovery elicited by salbutamol and CGRP was also observed in EDL muscles exposed to Ba2+, indicating that the response is not restricted to muscles containing predominantly slow-twitch fibres.

Ba2+ blocks the inwardly rectifying K+ channels with a relatively high affinity (Standen & Stanfield, 1978) and ATP-sensitive K+ channels with a somewhat lower affinity. The experiments with glibenclamide indicate that ATP-sensitive K+ channels are not involved in the force recovery induced by salbutamol, CGRP or electrical stimulation.

Membrane potential, M-waves and force development

We observed close correlations between Vm, M-wave area and tetanic force in muscles exposed to Ba2+ and subjected to different Na+-K+ pump-stimulating treatments. This is in keeping with the previous findings in isolated rat muscles exposed to high [K+]o that even modest changes in Vm, within a critical range, could produce a steep drop in tetanic force (Cairns et al. 1995). Furthermore, a tight correlation between M-wave area and tetanic force in rat soleus muscles exposed to a combination of high [K+]o and low [Na+]o was recently observed (Overgaard et al. 1999).

Depolarization has been shown to induce slow inactivation of voltage-gated Na+ channels, a mechanism that is active at normal resting Vm in mammalian skeletal muscle (Ruff et al. 1988). This would reduce the size of action potentials and interfere with their propagation. Furthermore, depolarization entails a reduced electrochemical gradient for Na+ and thus may reduce the size of action potentials.

It is well established that membrane depolarization inactivates the excitation-contraction (EC) coupling in skeletal muscle (Hodgkin & Horowicz, 1960; Caputo et al. 1984). More recently, it was shown that, in rat soleus fibres, the contractile response to high [K+]o contractures declined when fibres were exposed to conditioning depolarizations to −40 mV or less negative values (Dulhunty, 1991). Since the membrane potentials in our Ba2+-treated muscles never depolarized beyond −50 mV, it seems unlikely that the reduced contractility in the presence of Ba2+ was caused by inactivation of EC coupling. It rather can be attributed to reduced sarcolemmal excitability due to inactivation of Na+ channels – a conclusion further supported by the strong correlation between M-wave area and tetanic force.

Following long exposure to 5 mm Ba2+, salbutamol caused only marginal recovery of M-wave area and tetanic force. Under these conditions, however, Vm reached only −57 mV, which is not sufficient to allow recovery of excitability. This is in line with the observation that in rat soleus muscle, depolarization to around −56 mV leads to almost 90 % loss of peak tetanic force (Cairns et al. 1995).

In conclusion, the results indicate that in rat skeletal muscle, activation of the Na+-K+ pump by hormones or electrical stimulation contributes substantially to the maintenance of excitability and contractile performance. Moreover, three different stimuli gave the same close correlations between Vm, M-wave area and force development. These stimulating effects on the Na+-K+ pump and excitability can be exerted even during severe suppression of K+ channel function, including that of ATP-sensitive K+ channels.

Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

The study was supported by the Danish Biomembrane Research Centre and the Danish Medical Research Council (j. nr. 9802488). The technical assistance of Ann-Charlotte Andersen, Tove Lindahl Andersen, Marianne Stürup-Johansen and Vibeke Uhre is gratefully acknowledged. We thank Ole Bækgaard Nielsen for helpful comments on the manuscript.