Excitation- and beta(2)-agonist-induced activation of the Na(+)-K(+) pump in rat soleus muscle

Abstract

In rat skeletal muscle, Na(+)-K(+) pump activity increases dramatically in response to excitation (up to 20-fold) or beta(2)-agonists (2-fold), leading to a reduction in intracellular Na(+). This study examines the time course of these effects and whether they are due to an increased affinity of the Na(+)-K(+) pump for intracellular Na(+). Isolated rat soleus muscles were incubated at 30 (o)C in Krebs-Ringer bicarbonate buffer. The effects of direct electrical stimulation on (86)Rb(+) uptake rate and intracellular Na(+) concentration ([Na(+)](i)) were characterized in the subsequent recovery phase. [Na(+)](i) was varied using monensin or buffers with low Na(+). In the [Na(+)](i) range 21-69 mM, both the beta(2)-agonist salbutamol and electrical stimulation produced a left shift of the curves relating (86)Rb(+) uptake rate to [Na(+)](i). In the first 10 s after 1 or 10 s pulse trains of 60 Hz, [Na(+)](i) showed no increase, but (86)Rb(+) uptake rate increased by 22 and 86 %, respectively. Muscles excited in Na(+)-free Li(+)-substituted buffer and subsequently allowed to rest in standard buffer also showed a significant increase in (86)Rb(+) uptake rate and decrease in [Na(+)](i). Na(+) loading induced by monensin or electroporation also stimulated (86)Rb(+) uptake rate but, contrary to excitation, increased [Na(+)](i). The increase in the rate of (86)Rb(+) uptake elicited by electrical stimulation was abolished by ouabain, but not by bumetanide. The results indicate that excitation (like salbutamol) induces a rapid increase in the affinity of the Na(+)-K(+) pump for intracellular Na(+). This leads to a Na(+)-K(+) pump activation that does not require Na(+) influx, but possibly the generation of action potentials. This improves restoration of the Na(+)-K(+) homeostasis during work and optimizes excitability and contractile performance of the working muscle.

Figure 1. Time course of the effects of salbutamol on [Na⁺]_i and ⁸⁶Rb⁺ uptake rate in rat soleus muscle

Groups of four muscles in polyethylene baskets were preincubated for 2 × 30 min in KR buffer. At time zero they were transferred into KR buffer containing ⁸⁶Rb⁺ (0.1 μCi ml⁻¹) ± 10⁻⁵m salbutamol and incubated for 1–5 min. After incubation the muscles were washed for 4 × 15 min in ice-cold Na⁺-free Tris-sucrose buffer, blotted and taken for counting of ⁸⁶Rb⁺ activity and determination of [Na⁺]_i (see Methods for details). •, control muscles; ○, incubated with 10⁻⁵m salbutamol. Each point represents the mean value ± s.e.m. of observations on 4 muscles.

Figure 2. Effects of duration of electrical stimulation on undershoot in [Na⁺]_i after 10 min of recovery in rat soleus muscle

Muscles mounted isometrically on electrodes were preincubated for 2 × 30 min in KR buffer. Then they were stimulated for 0.5-10 s at 60 Hz (1 ms and 10 V) and incubated for another 10 min without stimulation. Finally the muscles were washed 4 × 15 min in ice-cold Na⁺-free Tris-sucrose buffer, blotted and taken for determination of [Na⁺]_i (see Methods for details). Each column represents the mean value with bars denoting s.e.m. of observations on 3–6 muscles.

Figure 3. Time course of the effects of 10 s of electrical stimulation on [Na⁺]_i and⁸⁶Rb⁺ uptake rate in rat soleus muscle

Muscles mounted isometrically on electrodes were preincubated for 2 × 30 min in KR buffer. Then they were stimulated for 10 s at 60 Hz (1 ms pulses at 10 V), immediately transferred into KR buffer containing ⁸⁶Rb⁺ (0.1 μCi ml⁻¹) and incubated for 1–4 min. In a few experiments, muscles were incubated for 10–20 s in a buffer containing 1 μCi ml⁻¹⁸⁶Rb⁺. Finally the muscles were washed 4 × 15 min in ice-cold Na⁺-free Tris-sucrose buffer, blotted and taken for counting of ⁸⁶Rb⁺ activity and determination of [Na⁺]_i (see Methods for details). •, resting control muscles; ○, muscles stimulated electrically. Each point represents the mean value ± s.e.m. of observations on 3–8 muscles.

Figure 4. Effects of salbutamol on the relation between [Na⁺]_i and ⁸⁶Rb⁺ uptake rate in rat soleus muscle

Groups of 4 muscles in polyethylene baskets were preincubated in KR buffer for 30–120 min. Then they were transferred to KR buffer containing ⁸⁶Rb⁺ (0.1 μCi ml⁻¹) and incubated for 10 min. Finally, the muscles were washed for 4 × 15 min in ice-cold Na⁺-free Tris-sucrose buffer, blotted and taken for counting of ⁸⁶Rb⁺ activity and determination of [Na⁺]_i (see Methods for details). In some experiments [Na⁺]_i was manipulated by preincubating for 30–120 min in buffer where half of the Na⁺ was replaced with N-methyl-d-glucamine or in standard KR buffer where 10⁻⁶-10⁻⁴m monensin was added. •, control muscles; ○, 10⁻⁵m salbutamol added 30 min before determination of ⁸⁶Rb⁺ uptake rate. Each point represents the mean value ± s.e.m. of observations on 4 muscles. The curves were fitted using a computer program.

Figure 5. Effects of electrical stimulation on the relation between [Na⁺]_i and ⁸⁶Rb⁺ uptake rate in rat soleus muscle

Muscles mounted isometrically on electrodes were preincubated in KR buffer. Then they were stimulated for 10 s at 60 Hz (1 ms pulses at 10 V). After a resting period of 2 min they were transferred into tubes containing ⁸⁶Rb⁺ (0.1 μCi ml⁻¹) and incubated for 2 min. Finally the muscles were washed 4 × 15 min in ice-cold Na⁺ -free Tris-sucrose buffer, blotted and taken for counting of ⁸⁶Rb⁺ activity and determination of [Na⁺]_i (see Methods for details). [Na⁺]_i was manipulated by preincubating for 30–120 min in buffer where half of the Na⁺ was replaced with N-methyl-d-glucamine or in standard KR buffer with 10⁻⁶-10⁻⁴m monensin. •, resting control muscles; ○, muscles stimulated electrically. Each point represents the mean value ± s.e.m. of observations on 3 muscles. The curves were fitted using a computer program.

Figure 6. Effects of electrical stimulation during incubation in Li⁺-KR buffer on [Na⁺]_i and ⁸⁶Rb⁺ uptake rate in rat soleus muscle

Muscles mounted isometrically on electrodes were preincubated for 30 min in KR buffer followed by 10 min of preincubation in Li⁺-KR buffer. Then they were stimulated for 10 s at 60 Hz (1 ms pulses at 10 V) and immediately transferred into tubes containing standard KR buffer with ⁸⁶Rb⁺ (0.1 μCi ml⁻¹) and incubated for 1 min. Finally the muscles were washed 4 × 15 min in ice-cold Na⁺-free Tris-sucrose buffer, blotted and taken for counting of ⁸⁶Rb⁺ activity and determination of [Na⁺]_i (see Methods for details). Each column represents the mean value with bars denoting s.e.m. of observations on 5 muscles.

Figure 7. Effects of electroporation on total Na⁺ content, intracellular Na⁺ content and ⁸⁶Rb⁺ uptake rate in rat soleus muscle

Isolated muscles in groups of 3 or 4 were placed in polyethylene baskets and incubated at 30 °C in KR buffer. Then the groups of muscles were transferred to an electroporation cuvette containing KR buffer and given three square pulses at 125 V (313 V cm⁻¹) and for a 200 μs duration followed by resting periods lasting from 0 to 15 min. Afterwards they were incubated for 5 min in KR buffer containing ⁸⁶Rb⁺ (0.1 μCi ml⁻¹), blotted and taken for counting of ⁸⁶Rb⁺ activity and determination of Na⁺ content (see Methods for details). In some experiments muscles were preincubated for 75 min in KR buffer containing [¹⁴C]sucrose (0.1 μCi ml⁻¹) and 1 mm unlabelled sucrose before they were transferred to the electroporation cuvette containing KR buffer with the same concentrations of [¹⁴C]sucrose and unlabelled sucrose as in the incubation medium and stimulated 3 times for 200 μs at 125 V (313 V cm⁻¹). Subsequently the muscles were incubated for 5–20 min in KR containing [¹⁴C]sucrose. Each point represents the mean value ± s.e.m. of observations on 4–6 muscles.