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Na⁺-K⁺ Pump Regulation and Skeletal Muscle Contractility

Department of Physiology, University of Aarhus, Århus, Denmark

ABSTRACT

I. INTRODUCTION

II. THE PLASMA MEMBRANE AND ITS Na+-K+ TRANSPORT PATHWAYS

III. LOCALIZATION OF THE Na+-K+ PUMPS

A. Sarcolemma

B. T Tubules

C. Intracellular Pools

IV. STRUCTURE, IDENTIFICATION, AND QUANTIFICATION OF Na+-K+ PUMPS

A. General Structure and Isoforms of Na+-K+-ATPase

B. Measurement of Na+-K+-ATPase Activity

C. Measurement of [3H]ouabain Binding

D. Measurement of Transport Capacity

V. ACUTE REGULATION OF Na+-K+ PUMP ACTIVITY

A. Excitation

1. Stimulatory effects

2. Mechanisms for excitation-induced stimulation of the Na+-K+ pump

3. Excitation-induced inhibition of Na+-K+-ATPase activity

B. Catecholamines

C. Peptide Hormones

1. Insulin

2. IGF-I

3. CGRP and calcitonins

4. Amylin and other related peptides

5. Inhibitory agents

D. Energy Depletion/Repletion

E. General Mechanisms for Acute Regulation of the Na+-K+ Pump

VI. LONG-TERM REGULATION OF Na+-K+ PUMP CONTENT

A. Training and Inactivity

1. Effects on the content of Na+-K+ pumps in skeletal muscle

2. Correlations to K+ clearance

3. Correlation to energy turnover

4. Mechanisms for training-induced upregulation of Na+-K+ pumps

B. Thyroid Hormones, Starvation, and Diabetes

1. Thyroid hormones

2. Starvation

3. Diabetes

C. Steroid Hormones

D. Growth, Differentiation, and Fiber Type

E. K+ Deficiency and K+ Overload

1. K+ deficiency

2. K+ overload

F. Heart Failure and Hypoxia

1. Heart failure

2. Hypoxia

G. Muscular Dystrophy and McArdle Disease

1. Muscular dystrophy

2. McArdle disease

H. Digitalis and Amiodarone

1. Digitalis

2. Amiodarone

I. Implications of Acute Plus Long-Term Regulation for Na+-K+ Pump Capacity

VII. EXCITATION, PASSIVE Na+-K+ FLUXES, AND CONTRACTILITY

A. Excitation-Induced Na+-K+ Leaks May Exceed Na+-K+ Pump Activity/Capacity

B. Excitation-Induced Na+-K+ Leaks and Endurance Depend on Fiber Type

C. Effects of Na+ Channel Modulation on Contractile Endurance

D. Effects of K+ Channel Modulation on Contractile Endurance

E. Are the Processes of Excitation in Skeletal Muscle Self-limiting?

F. Role of t Tubules

VIII. THE Na+-K+ PUMP AND CONTRACTILE PERFORMANCE

A. Effects of Excitation-Induced Na+-K+ Pump Stimulation

B. Effects of Acute Hormonal Na+-K+ Pump Stimulation

C. Effects of Temperature, Extracellular Ca2+, and pH

D. Effects of Na+-K+ Pump Inhibition or Downregulation

E. Energy Depletion, Loss of Excitability, and Contractility

F. Training, Na+-K+ Pump Upregulation, and Contractile Performance

G. Effects of Thyroid Hormones

IX. MAJOR CONCLUSIONS AND GENERAL PERSPECTIVES

	ABSTRACT

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	I. INTRODUCTION

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The Na⁺-K⁺-ATPase mediating the active coupled transport of Na⁺ and K⁺ across cell membranes was first identified in 1957 by Skou (392), who in 1997 was awarded the Nobel Prize in chemistry for this discovery. His review defining the role of this transport system, the Na⁺-K⁺ pump, as a regulator of the transport and distribution of Na⁺ and K⁺ across cell membranes was published in this journal (393). This raised the question whether and how this transport regulator itself was regulated. Later reviews have described the regulation of the Na⁺-K⁺ pumps in skeletal muscle (62), kidney (139), and heart (166). Many early or more general references may be found in these reviews. A related area, the K⁺ homeostasis in skeletal muscle and the heart during exercise, was recently reviewed (384). The detailed molecular mechanisms and specific signaling pathways involved in the regulation of the Na⁺-K⁺ pumps in a wide variety of tissues have been reviewed (134, 408). The present review is written with the specific purpose of analyzing how regulation of the activity and the capacity of the Na⁺-K⁺ pumps influences excitability and contractile performance in skeletal muscle. With the growing realization that the Na⁺-K⁺ pumps undergo large regulatory changes both in their transport activity and capacity (tissue content of Na⁺-K⁺-ATPase), the functional implications of such changes are gaining interest; in particular for the understanding of the frequently occurring pathophysiological and pharmacological modifications of Na⁺-K⁺ pump function. Therefore, the more general homeostatic role of Na⁺-K⁺ pump regulation in skeletal muscle will also be illustrated by some clinical examples (for a more general review on the clinical significance of the Na⁺-K⁺ pump in various tissues, see Ref. 65).

	II. THE PLASMA MEMBRANE AND ITS Na+-K+ TRANSPORT PATHWAYS

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Skeletal muscle cells are surrounded by an outer plasma membrane (sarcolemma), which is covered by a glycocalyx layer. From the cell surface, invaginations of the sarcolemma extend into the cell interior, forming a grid of tubules running transversely across the cell. The lumina of these t tubules are open to the extracellular space, allowing ions and metabolites by simple diffusion to reach transport systems in the walls of t tubules in most parts of the muscle cell. This system not only offers a large membrane surface for the exchange of various compounds between the cytoplasm and the extracellular phase, but also allows the propagation of action potentials from the sarcolemma to the terminal cisternae of the sarcoplasmic reticulum (SR) in every part of the cell (89, 218). This ensures prompt and coordinated release of Ca²⁺ from the SR to the muscle fibrils in the entire cell.

In skeletal muscle, Na⁺ and K⁺ are exchanged across the plasma membrane of sarcolemma and t tubules via a number of specific transport systems. As illustrated in Figure 1, the passive movements of Na⁺ and K⁺ are counterbalanced by one single active transport system, the Na⁺-K⁺ pump. The major passive fluxes are mediated by the voltage-sensitive Na⁺ channels and at least four different categories of K⁺ channels (208). During excitation, the action potentials are elicited by a rapid and marked influx of Na⁺ via the Na⁺ channels, immediately followed by an almost equivalent efflux of K⁺. These fluxes may exceed the capacity of the Na⁺-K⁺ pumps (basal or stimulated) for restoring Na⁺-K⁺ distribution (see sect. VIIA). Studies on frog and snake skeletal muscle indicate that at variance with many other membrane proteins, the Na⁺ channels and most of the K⁺ channels are immobile, possibly due to an anchoring to the cell membrane (367). This might limit lateral movements in the membrane or between t tubules and sarcolemma.

View larger version (22K): [in this window] [in a new window]   FIG. 1. Na+-K+ transport pathways in skeletal muscle. The transmembrane Na+-K+ concentration gradients are generated by the continuous operation of the Na+-K+ pumps. The Na+ gradient drives the cotransport of amino acids and chloride and the antiport of H+ and Ca2+. By far the major influx pathways for Na+ are the voltage-sensitive Na+ channels. Passive K+ fluxes are mediated by the voltage-sensitive inward rectifier (inw.) and delayed rectifyer (del.), the Ca2+-sensitive and the ATP-sensitve K+ channels. [Modified from Clausen (64).]

In resting rat soleus muscle, ²²Na uptake is inhibited by a wide variety of membrane stabilizing agents, such as local anesthetics, barbiturates, phenothiazines, propranolol (78, 98), as well as the Na⁺ channel blocker tetrodotoxin (TTX) (112), indicating that part of the resting uptake of Na⁺ is mediated by Na⁺ channels. This type of inhibition leads to a decrease in intracellular Na⁺ ([Na⁺]_i). In rat soleus, TTX reduced ²²Na uptake by 17%. This effect was additive to that of the NaK2Cl symport inhibitor bumetanide, which produced an inhibition of 23% and the Na⁺/H⁺ antiport inhibitor amiloride, causing 19% inhibition. When exposed to all three inhibitors, ²²Na uptake was reduced by 54%, which is in good agreement with the sum of the observed effects of the three inhibitors (59%).

Bumetanide as well as furosemide clearly inhibited ²²Na uptake and induced progressive reduction in intracellular Na⁺ content. This was not associated with any change in ⁴²K uptake or K⁺ content. Neither was there any effect of bumetanide on ⁴²K efflux or net K⁺ loss during excitation, nor on the reaccumulation of K⁺ following stimulation. The conclusion of these studies is that in skeletal muscle, there is no functional NaK2Cl symport system, but apparently a NaCl symporter that can be activated under hyperosmolar conditions and suppressed by bumetanide or furosemide.

In addition, the transmembrane Na⁺ gradient is driving the uptake of a variety of amino acids, inorganic phosphate (238), and the Na⁺/Ca²⁺ antiporter (17). Finally, a NaHCO₃ cotransport system has been identified in skeletal muscle (358). These processes and systems may all contribute to the uptake of Na⁺, but little is known about their transport properties or capacity.

In resting rat soleus, the influx and efflux of ⁴²K are markedly inhibited by the above-mentioned membrane stabilizors (98, 78), and by Ba²⁺ (83), which also inhibits ⁸⁶Rb efflux in frog semitendinosus muscle (53). This indicates that a large part of the resting passive K⁺ fluxes are mediated by K⁺ channels. The membrane stabilizors or Ba²⁺ caused no change in the Na⁺-K⁺ pump-mediated Na⁺-K⁺ fluxes.

In frog semitendinosus muscle, metabolic poisoning with cyanide and iodoacetate induced a modest increase in K⁺ efflux. Subsequent complete metabolic exhaustion elicited by electrical stimulation at 1 Hz for a few minutes induced a five- to sixfold increase in both ⁴²K and ⁸⁶Rb efflux. This increase was suppressed by Ba²⁺, glibenclamide, tolbutamide, TEA, or local anesthetics, but not by inhibitors of the delayed rectifier or Ca²⁺-activated K⁺ channels (53). This indicates that during metabolic exhaustion, the ATP-sensitive K⁺ channels mediate a large K⁺ efflux.

There is substantial evidence that the Na⁺-K⁺ pump is electrogenic and therefore contributes to the maintenance of the membrane potential in skeletal muscle (2, 62, 158). In isolated rat soleus, blocking the Na⁺-K⁺ pump with ouabain (10^-3 M) caused 8-mV depolarization in 10 min. Conversely, stimulating the Na⁺-K⁺ pump with epinephrine caused 7.5-mV hyperpolarization in 10 min (72). It cannot be excluded that Na⁺-K⁺ pump stimulation, by reducing [K⁺]_o, might induce hyperpolarization by increasing the K⁺ equilibrium potential. Conversely, indirect depolarizing effects of elevated [K⁺]_o could be envisaged in tissues exposed to Na⁺-K⁺ pump inhibition. In frog extensor digitorum longus (EDL) muscles, rapid increase or decrease in temperature of 17°C caused, respectively, hyperpolarization and depolarization of 10 mV within 2 s. About half of these changes were attributed to changes in the K⁺ concentration in the t-tubular lumen, and the other half were directly related to the electrogenic action of the Na⁺-K⁺ pump (158).

On the other hand, in isolated rat skeletal myotubes, where diffusional limitations for K⁺ are negligible, ouabain or cooling from 37 to 13°C induced up to 20- to 25-mV depolarization (35). Studies with chick myotubes showed that ouabain induced a comparable depolarization within 2-5 s, which was not accompanied by any change in input resistance (40). When exposed to K⁺-free buffer, which prevents the operation of the Na⁺-K⁺ pump, the cells showed the same depolarization as in the presence of ouabain (10^-3 M).

In conclusion, the Na⁺-K⁺ pumps generate steep transmembrane gradients for Na⁺ and K⁺, allowing the maintenance of the membrane potential, excitability, and the operation of several secondary active transport processes. In addition, the Na⁺-K⁺ pump has a direct and rapid (within seconds) electrogenic action, arising from its 3:2 exchange of Na⁺ against K⁺.

	III. LOCALIZATION OF THE Na+-K+ PUMPS

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A. Sarcolemma

In skeletal muscle, the Na⁺-K⁺ pumps are located in the sarcolemma and the t tubules. This has been documented using isolation of membrane preparations by differential centrifugation of muscle homogenates combined with electron microscopic identification of membrane vesicles (55) as well as by immunofluorescence labeling (136, 435). Although it has been possible to isolate and purify sarcolemma preparations from muscle homogenates, most procedures reported are complicated and inadequate from a quantitative point of view (see sect. IVB). Measurements of [³H]ouabain binding indicate that in sarcolemma, the density of Na⁺-K⁺ pumps is 3,350 molecules/µm² in the soleus of 4-wk-old rats (75), 2,500 molecules/µm² in frog sartorius (422), and 1,000-1,800 molecules/µm² in muscles from 8-wk-old pigs (196). However, these values are based on the assumption that all [³H]ouabain binding sites are located in the sarcolemma. Because most of the [³H]ouabain binding sites may be located in the t tubules, the density of Na⁺-K⁺ pumps in sarcolemma is probably much lower (see sect. IIIB).

B. T Tubules

Already long ago, the t tubules were shown to constitute an extremely branched system. Because the visualization of the structure has not been surpassed since the publication of Peachey and Eisenberg in 1978 (348), their figure was selected to illustrate the extraordinary complexity (Fig. 2). Later studies using other techniques have demonstrated a network of similar complexity in muscles of the guinea pig (155) and the rat (435).

View larger version (89K): [in this window] [in a new window]   FIG. 2. T-tubular network in a frog sartorius muscle fiber. Reconstruction of the t-tubular system over the whole cross section of a single muscle fiber stained using the horseradish peroxidase method to visualize the t tubules. [From Peachey and Eisenberg (348), copyright Biophysical Society.]

Single fibers prepared from frog semitendinosus muscles maintained contractile response even after the sarcolemma had been removed and the t tubules sealed (90), implying that the t-tubular membranes maintain concentration gradients for Na⁺ and K⁺ similar to those across the sarcolemma. This requires Na⁺-K⁺ pumps, and the contractile response was abolished by inhibiting the Na⁺-K⁺ pumps with strophanthidin, added before the removal of sarcolemma so as to allow access of this cardiac glycoside to the lumen of the t tubules. Thus, even when the connections between sarcolemma and the t tubules have been disrupted, the tubular membranes can carry excitatory action potentials, and this ability depends on functional Na⁺-K⁺ pumps. Another important implication is that the propagation of the excitatory signals along the t-tubular network does not depend on diffusional Na⁺-K⁺ exchange between the t-tubular lumen and the interstitial water space. It is possible that most of the excitation-induced passive Na⁺-K⁺ fluxes are directly counterbalanced by the Na⁺-K⁺ pumps situated in the t-tubular walls and that this localized circuitry for Na⁺ and K⁺ is adequate for the maintenance of signal propagation into the interior of the cell.

T-tubular membranes isolated from rabbit sacrospinalis skeletal muscle and identified by their morphological association with the terminal cisternae bind [³H]ouabain (55) and contain Na⁺-K⁺-ATPase (266). When [³H]ouabain was injected directly into the muscles 20 min before homogenization, or intravenously 2 h before homogenization, this tracer was trapped inside the lumen of resealed t-tubular vesicles and could be recovered in the microsomal fraction. Isolated t tubules prepared from rabbit sacrospinalis muscle were permeable to digitoxin, but not to [³H]ouabain (266). When pretreated with deoxycholate, however, the t tubules took up [³H]ouabain and bound it with high affinity (37 pmol/mg protein, K_D = 5. 4 x 10^-8 M). The tubules also accumulated Na⁺ by an ATP-dependent process, suppressible by digitoxin. Thus it is rather directly documented that the Na⁺-K⁺ pumps in the t tubules operate to maintain the transmembrane concentration gradient for Na⁺ required for action potential propagation.

In intact frog sartorius muscle, treatment with hyperosmolar glycerol disconnected 90% of the t tubules from the sarcolemma, as estimated from the accessability of the ttubulular lumen to lanthanum as an extracellular marker. In this preparation, when incubated for 6-8 h, strophanthidinsuppressible ²²Na efflux was halved (421), indicating that the Na⁺-K⁺ pumps in the t tubules are major contributors to the active Na⁺-K⁺ transport in skeletal muscle.

An attempt was also made to quantify the amount of [³H]ouabain binding to the t tubules in the intact frog sartorius muscle by comparing untreated muscles with glycerolpretreated muscles (422). The glycerol pretreatment only reduced [³H]ouabain binding by 20%, indicating that the density of ouabain binding sites in the t-tubular membrane is only around 5% of that of the sarcolemma (125 vs. 2,500 sites/µm², respectively). This estimate for the density of [³H]ouabain binding sites in the t-tubular membrane is in good agreement with that which can be calculated from the above-mentioned data of Lau et al. (266) (180 sites/µm²). However, these values for t-tubular Na⁺-K⁺ pump density are likely to be much too low, because in a whole muscle, the glycerol pretreatment procedure may not give complete disruption of the t-tubular connections to sarcolemma. Even if only 10% of these connections are preserved, [³H]ouabain may gain access to the tubular lumen via the recently described longitudinal connections (356). Therefore, the measurements will not detect much of a decrease in [³H]ouabain binding after the glycerol pretreatment. More importantly, measurements of the content of [³H]ouabain binding sites in t-tubular and sarcolemmal membranes obtained from frog skeletal muscle showed similar values; 215 and 163 pmol/mg protein, respectively (222). The same study showed that the t tubules from rabbit skeletal muscle contained 169 pmol/mg protein. These rather high values would correspond to a Na⁺-K⁺-ATPase activity of 80 µmol ATP split · h^-1 · mg protein^-1, which is not too far from the values of 39 and 50 µmol · h^-1 · mg protein^-1 reported for t tubules from rabbit muscle (299, 206). In the sarcolemma of rabbit skeletal muscle, Mitchell et al. (299) obtained a Na⁺-K⁺-ATPase activity of 57 µm · h^-1 · mg protein^-1, rather close to that of t-tubular membranes (39 µmol · h^-1 · mg protein^-1). In rat skeletal muscle, the Na⁺-K⁺-ATPase activity in a membrane fraction containing primarily t tubules was 58% of that in the fraction containing sarcolemma (20). Thus, as already pointed out by Hidalgo et al. (206), the contents of Na⁺-K⁺-ATPase in t tubules and sarcolemma are not as different as suggested by the [³H]ouabain binding studies by Venosa and Horowicz (422).

Following sarcolemmal leakage due to micropuncture or cuts, the t tubules in the adjacent segments of the muscle fiber within minutes underwent considerable swelling leading to vacuolation (52). Because this swelling could be prevented by ouabain, cooling, the omission of Na⁺ from the incubation medium, or by inhibiting energy metabolism, it was attributed to the Na⁺ leaking into the cytoplasm being pumped into the t-tubular lumen and favoring the entry of water from the surrounding cytoplasm. These structural studies indicate that the Na⁺-K⁺ pumps in the t tubules possess a considerable transport capacity and are important for the exchange of Na⁺ and water.

Immunofluorescence labeling showed that in the human soleus muscle, the ₁-subunit isoform was mainly located in the sarcolemma, whereas the ₂-subunit iso-form was observed both in the sarcolemma and diffusely distributed in the muscle fibers, possibly located in the t tubules (217). The plasma membrane seems to contain the ₃-subunit isoform. In the guinea pig heart, the t tubules and sarcolemma contain only the ₁-subunit isoform of the Na⁺-K⁺ pump, whereas in the rat heart, t tubules contained both ₁- and ₂-subunit isoforms (291). More recent immunofluorescence and immunoprecipitation studies of rat and mouse skeletal muscle showed that whereas the sarcolemma contains both the ₁- and the ₂-subunit, the t-tubular membranes contain only the ₂-subunit and no ₁-subunit (435). The same study showed that both the ₁- and ₂-isoform subunits were associated with -spectrin and ankyrin. This was proposed to concentrate both isoform subunits in costameres present at the sarcolemma over Z and M lines and in longitudinal strands. Thus, in mutant mice lacking -spectrin, the Na⁺-K⁺-ATPase subunit isoforms were not concentrated in costameres, but seemed to be released from the anchoring to become more uniformly distributed in the sarcolemma.

In conclusion, the preponderant evidence indicates that the content of Na⁺-K⁺ pumps/µm² of t-tubular wall is not far below that of sarcolemma. Because the area of the t-tubular membranes is severalfold larger than that of the sarcolemma, this implies that in the entire muscle cell, most of the Na⁺-K⁺ pumps are situated in the t tubules, allowing rapid clearance of K⁺ from the t-tubular lumen during excitation. This transport mechanism may be more efficient than the diffusion of K⁺ out of the t-tubular network and therefore decisive for the maintenance of excitability.

C. Intracellular Pools

In cell cultures prepared from chick embryo leg muscles, immunolabelling showed that the Na⁺-K⁺-ATPase located in an intracellular pool corresponded to 60% of the total amount of the enzyme present in the cells (136, 436). Unfortunately, the precise localization of this pool was not identified, and it cannot be excluded that due to the use of saponin and formaldehyde during the procedures, the t tubules had been permeabilized so as to allow binding to the Na⁺-K⁺ pumps situated in the t tubules. In this and some other studies, part of the pool of Na⁺-K⁺ pumps termed "intracellular" may be residing in the t tubules (136). Several studies have described an intracellular pool, from where Na⁺-K⁺ pumps may be recruited to the sarcolemma by hormones or excitation. This pool is not homogeneous and may include subsarcolemmal vesicles, triad junctions, and t tubules, and the relative distribution of Na⁺-K⁺ pumps among these structures has not been quantified (216, 267, 268, 285). Another uncertainty in quantitative comparisons to the plasma membrane pool is that the protein recovery of intracellular membranes is eightfold larger than that of plasma membrane (216).

In conclusion, the intracellular pool, from where Na⁺-K⁺ pumps are recruited to the sarcolemma, may in part be situated in the t tubules, but more precise identification and quantification is needed.

	IV. STRUCTURE, IDENTIFICATION, AND QUANTIFICATION OF Na+-K+ PUMPS

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A. General Structure and Isoforms of Na⁺-K⁺-ATPase

As shown in Figure 3, the Na⁺-K⁺-ATPase is a heterodimer, composed of two protein subunits, a catalytic -subunit involved in the splitting of ATP (molecular mass 112 kDa) and a -subunit (35 kDa). The -subunit which actually pumps Na⁺ and K⁺ contains binding sites for Na⁺, K⁺, ATP, and digitalis glycosides. It has 10 transmembrane segments and depends on the -subunit for transport activity. The -subunit is a glycoprotein, necessary for the transfer of the entire enzyme molecule from its site of synthesis in the endoplasmic reticulum to its site of insertion in the plasma membrane. A -subunit (not shown) has been described in several tissues and seems to have regulatory function.

View larger version (49K): [in this window] [in a new window]   FIG. 3. A planar model of the Na+-K+-ATPase showing membrane topology and secondary structure. Each point represents one amino acid (numbered from 1 to 1,000 in the -subunit and from 1 to 300 in the -subunit) with linear sequences representing loops, zig-zag alternations representing -strands and stacked sequences of three or four residues representing -helices. The -subunit has 10 transmembrane segments, a phosphorylation site (P) at amino acid 369, and as indicated an extracellular area for the binding of ouabain or other cardiac glycosides. The -subunit has one transmembrane segment and three glycosylated amino acids in its mainly extracellular loops. [Modified from Jørgensen et al. (230).]

Like many other proteins, the subunits of the Na⁺-K⁺-ATPase are expressed in various isoforms, which can be detected using specific antibodies (403). Four isoforms of the -subunit (₁-₄) and three isoforms of the -subunit (₁-₃) have been identified. In skeletal muscle, ₁, ₂, ₁, and ₂ are expressed, only minor amounts of ₃ have been found, and the -subunit has not been detected. Rat skeletal muscle in vivo expresses both the ₁-, ₂-, ₁-, and ₂-subunits (216, 267, 403). The ratio between ₂ and ₁ was reported to amount to 1.6 in the surface membranes and to approach 7 in the internal membranes (267), and ₁ seems to be mostly confined to sarcolemma (216). Primary cultures of rat skeletal muscle, however, express only the ₁-, ₁-, and ₂-subunit isoforms, even though they contain mRNA for ₂ (387) and in L8 rat myogenic cells, only ₁- and ₁-isoform protein and mRNA are expressed (388). In avian skeletal muscle cells, only the ₁-isoform is expressed (405).

In a cell line obtained from mouse skeletal muscle (C2C12) the mRNA and protein levels of the ₁-subunit isoform remained constant during differentiation. Concomitantly, the ₂-subunit isoform underwent a marked increase, but this was found to contribute little to ionic homeostasis (207). In contrast, in the intact rat skeletal muscle advancing age from 6 to 30 mo was associated with marked increase in ₁- and ₁-subunit and a decrease in ₂- and ₂-subunit (396).

Cultured myotubes derived from chicken were found to bind 4.8 x 10⁵ molecules [³H]ouabain/cell. Using a monoclonal ³⁵S-labeled antibody, an almost identical number of antigenic sites was measured (135). A more general use of such comparisons would allow for a "translation" of specific antibody binding data to [³H]ouabain binding data. This might solve some of the discrepancies arising from isoform studies.

Thus there seems to be little consensus with respect to the relative content of ₁- and ₂-subunit isoforms in skeletal muscle. With one exception (188), the content of isoform ₁- or ₂-subunits as expressed per gram tissue wet weight has unfortunately not been quantified. Using an ₁-specific antibody to chicken kidney Na⁺-K⁺-ATPase, the content of ₁-subunit isoform in soleus and EDL of 4-wk-old rats was determined. The content of -subunit varied from 15 to 30% of the content of the ₂-isoform quantified using [³H]ouabain binding, thus representing 14-21% of the total content of Na⁺-K⁺-ATPase. Although these results seem more accurate than most other estimates of isoform abundancy, they leave some questions unanswered. Thus the antibody used was raised against the ₁-subunit from another species (chicken), and it cannot be excluded that it will interact differently with the ₁-subunits present in the muscles and the purified rat kidney ₁-subunit used as a reference. Moreover, the antibody may interact with newly synthesized -peptide molecules, not necessarily representing functional Na⁺-K⁺ pumps situated in the membranes. Another recent study indicated that in the EDL muscle of mice the ₂-subunit isoform accounted for 87% of the total amount of -subunit isoform of Na⁺-K⁺-ATPase, whereas the -isoform accounted for only 13% (202).

The research on subunit isoforms has been motivated by the expectation that the different isoforms would be linked to specific tissues and cell structures or serve specific functions. In the rat, this expectation has been fulfilled by the observation of a preponderance of ₂-subunit isoform in the t tubules. An early suggestion was that the ₁-subunit isoform might be a "housekeeping version" of the Na⁺-K⁺ pump, involved in the maintenance of basic transport function, whereas the ₂-isoform would be the regulated version. This seems unlikely, however, since in the kidney, mainly the ₁-isoform is found, which would then preclude the well-documented regulation of renal Na⁺-K⁺-ATPase (139). Likewise, in avian muscle, only the ₁-isoform has been found (135, 405), with the same unfortunate implications for the regulation of Na⁺-K⁺ pumps in birds. In the rat heart, the ₁-subunit isoform is by far the most abundant. Still, the Na⁺-K⁺ pumps in the heart are definitely subject to regulation by hormones (166). In cultured human fibroblasts containing only the ₁-subunit isoform, insulin stimulates ⁸⁶Rb uptake (279). Moreover, in K⁺-deficient rats, where the ratio between ₂- and ₁-subunit isoform was reduced from 2.1 to 0.3, there was still a full stimulating effect of insulin and epinephrine on Na⁺-K⁺ pump mediate ⁸⁶Rb uptake (214). In rat skeletal muscle, exercise caused the same relative increase in plasma membrane content of the ₁-and ₂-subunit isoform (414).

Finally, the concept of regulation being exerted on a specific subunit isoform has not been causally linked to any specific structural or functional properties of the Na⁺-K⁺-ATPase molecule. In brief, we have to know why the ₂-subunit isoform should be regulated and not the ₁-subunit isoform.

In conclusion, the detection and description of subunit isoforms has added much detail to the information about Na⁺-K⁺ pumps, their localization, and regulation. However, the quantitative analysis is still inaccurate and the functional significance of the different isoforms is not yet precisely explained.

B. Measurement of Na⁺-K⁺-ATPase Activity

Crude homogenates of skeletal muscle contain very high concentrations of various ATPases, and only a minor fraction can be identified as specifically activated by Na⁺, K⁺, and Mg²⁺ or suppressed by cardiac glycosides. It is difficult, therefore, to obtain accurate values for the total content of Na⁺-K⁺-ATPase activity. Because most of the early studies on Na⁺-K⁺-ATPase activity were focused on the identification and molecular characterization of the enzyme, the strategy was to obtain highly purified membrane preparations containing high concentrations of Na⁺-K⁺-ATPase rather than to gain information about the total content of the enzyme in a given tissue. If these classical procedures developed for purification should be applied for quantification, detailed and precise information about the recovery of Na⁺-K⁺-ATPase activity is required. However, analysis of 17 papers with information on the yield of Na⁺-K⁺-ATPase activity showed that the methods published only allow between 0.02 and 8.9% recovery of the total content of Na⁺-K⁺-ATPase activity in skeletal muscle (189). Later estimates of the recovery have given values of 4% (336) and 1% (3) (see comment in Ref. 190). This is likely to give misleading information about the regulation of the total content of Na⁺-K⁺-ATPase activity in skeletal muscle, also because it is difficult to ascertain that such small samples of the total pool of Na⁺-K⁺-ATPase are representative for the plasma membrane. Thus the Na⁺-K⁺-ATPase obtained in the final steps of the purification procedures may have originated from contaminating nervous and vascular tissue, adipocytes, and fibrocytes, and not from the plasma membrane (190).

In crude homogenates of skeletal muscle, the content of Na⁺-K⁺-ATPase activity can be measured by a highly sensitive fluorometric assay using 3-O-methylfluorescein phosphate as substrate (215). This enzyme activity (3-O-MFPase) is stimulated by K⁺, completely inhibited by ouabain, and reflects the amount of Na⁺-K⁺-ATPase (329, 330). On the basis of measurements of the molecular 3-O-MFPase activity (620 min^-1), the content of Na⁺-K⁺-ATPase activity in muscle homogenates could be quantified and was in satisfactory agreement with measurements of [³H]ouabain binding capacity of the intact muscle (329). Modified versions of this procedure were used for quantification of 3-O-MFPase activity in rodent heart (330, 381) and human skeletal muscle (152, 154, 295). In biopsies of human vastus lateralis muscle, a significant correlation was found between [³H]ouabain binding site content and 3-O-MFPase activity (153).

3-O-MFPase activity of muscle homogenates only reached its optimum value when deoxycholate was added. This was attributed to the opening of inside-out vesicles of sarcolemma, formed during the homogenization. Later studies showed that repeated freeze-thaw cycles produced a similar effect, possibly by opening vesicles or t tubules (152). It is interesting that the binding of [³H]ouabain to crude muscle homogenate was increased 10-fold by deoxycholate (266). It is possible that both phenomena reflect the opening of t tubules and that most of the Na⁺-K⁺-ATPase in skeletal muscle resides in the lumen of these structures (see sect. IIIB).

In conclusion, the use of sophisticated purification procedures has yielded much valuable information about the localization, kinetics, molecular biology, and structure of the Na⁺-K⁺-ATPase in skeletal muscle. For the quantification of the enzyme, however, measurements of [³H]ouabain binding or K⁺-activated 3-O-MFPase activity are more accurate than estimates based on purified plasma membranes, corrected by the recovery.

C. Measurement of [³H]ouabain Binding

Studies on Na⁺-K⁺-ATPase purified from various sources show that cardiac glycosides bind to the -subunit of the enzyme with a 1:1 molecular stoichiometry (186). This is the basis for the widespread use of [³H]ouabain for the quantification of Na⁺-K⁺ pumps in tissues, cells, and isolated membrane preparations.

Intact skeletal muscle preparations bind [³H]ouabain to the sarcolemma and the luminal surface of the t-tubular membranes. In rat soleus muscles that had been equilibrated with [³H]ouabain (10^-6 M) for 120 min to reach complete saturation, the fractional loss of [³H]ouabain during a subsequent washout in an ice-cold buffer was only marginally affected by cutting the fibers. This argues against any major intracellular accumulation of [³H]ouabain (75).

Also in vivo, 15 min after intraperitoneal injection of a saturating dose of [³H]ouabain to the rat, this ligand was bound to skeletal muscles and the content of [³H]ouabain binding sites could be quantified after removing the unbound [³H]ouabain by subsequent washout in ice-cold buffer (77). Over an eightfold range of values there was no significant difference between the content of [³H]ouabain binding sites as determined by incubating intact muscles with the ligand in vitro or by injection of [³H]ouabain into the intact animal. This indicates that the access and binding of [³H]ouabain in the isolated muscles in vitro is as complete as in the intact rat with preserved circulation and oxygenation. Furthermore, the in vivo studies showed that the method can be used for quantification of changes in [³H]ouabain binding site content under different conditions (K⁺ deficiency, ageing, denervation), yielding the same results as in vitro measurements. It should be recalled, though, that other animals than the rat might not tolerate being injected with ouabain at the required concentration. The reason why rats tolerate being injected with ouabain is that part of the Na⁺-K⁺ pumps in the heart, kidney, and brain contain the ₁-subunit isoform and therefore have a low affinity for ouabain. This allows the animals to avoid severe blockade of Na⁺-K⁺ pumps and functional impairment in these vital organs. This also implies that in the rat the [³H]ouabain binding assay cannot be used for these tissues.

In rat skeletal muscle, a minor fraction of the Na⁺-K⁺ pumps contain the ₁-subunit isoform, which may not be detected at the concentrations of [³H]ouabain used in the standard assay (10^-6 to 5 x 10^-6 M). Measurements of the maximum rate of ouabain-suppressible active Na⁺-K⁺ transport in intact rat muscles and 3-O-MFPase activity in crude homogenates have given values in reasonable agreement with those obtained in the standard [³H]ouabain binding assay (71, 329), indicating that this method quantifies the major part of the total content of Na⁺-K⁺ pumps in rat skeletal muscle.

The initial rate of [³H]ouabain binding can easily be quantified by exposing a muscle to [³H]ouabain for 15 min at 30°C, followed by four 30-min washouts in ice-cold buffer to remove [³H]ouabain not bound. Several in vitro studies have shown that when active Na⁺-K⁺ transport in intact muscles is stimulated by Na⁺ loading, insulin, insulin-like growth factor I (IGF-I), catecholamines, amylin, and excitation, the rate of [³H]ouabain binding increases (66, 76, 111, 127). Also in the intact organism, the rate of [³H]ouabain binding to rat soleus and EDL muscles is considerably accelerated (66-82%) by the ₂-agonist salbutamol (242). Conversely, pretreatment with tetracaine decreased Na⁺-K⁺ pump-mediated ⁴²K uptake and reduced [³H]ouabain binding rate (76). A close correlation between ouabain-suppressible ⁴²K uptake and the rate of [³H]ouabain was seen over a wide range of values (76). This correlation reflects that during increased rate of pumping, the Na⁺-K⁺-ATPase will more frequently exist in the conformation optimal for the binding of ouabain. An increase in the rate of [³H]ouabain binding can be taken as rather specific evidence that the pumping rate is accelerated.

As originally observed by Hansen et al. (191), vanadate (VO₄) can replace ATP in facilitating the binding of [³H]ouabain to the Na⁺-K⁺-ATPase. VO₄ isaPO₄ analog, and to exert this action, it must bind to the phosphorylation site of the Na⁺-K⁺ pumps on the intracellular surface of the plasma membrane. This high-affinity binding of VO₄ maintains the Na⁺-K⁺-ATPase in a configuration capable of binding [³H]ouabain to the extracellular surface of the molecule. This formed the basis for the development of a VO₄-facilitated binding assay for quantification of the content of [³H]ouabain binding sites in cut specimens of skeletal muscle. The assay is based on the concept that VO₄ via the cut ends of the muscle fibers gains access to the phosphorylation sites on the inner surface of the Na⁺-K⁺-ATPase. When incubated in a VO₄-containing buffer, cut muscle specimens bound the same amount of [³H]ouabain per gram wet weight as intact muscles obtained from the contralateral leg of the same rat and incubated in K⁺-free Krebs-Ringer bicarbonate buffer. Over a ninefold range of values, the two procedures gave the same results (331). Several methodological studies showed that to get accurate results, muscle specimens weighing 5 mg should be equilibrated for 120 min at 37°C in a Tris buffer containing VO₄ and [³H]ouabain (10^-6 M). Following a washout for 4 x 30 min in ice-cold buffer, to remove the [³H]ouabain not bound, the tissue specimens are blotted and taken for counting of [³H]ouabain. The values should be corrected for nonspecific uptake of [³H]ouabain, loss of specifically bound [³H]ouabain during washout, weight loss due to evaporation from the muscle specimens, impurity of the [³H]ouabain, and incomplete saturation of [³H]ouabain binding sites (241, 251). For measurements on rat skeletal muscle, these correction factors are usually lumped into one of around 1.3, by which the counted value for [³H]ouabain binding is multiplied. If the washout in the cold is omitted, and the results are corrected for the [³H]ouabain residing in the extracellular water space, the same values are obtained, albeit with a larger scatter (243). Measurements performed over a wide range of ouabain concentrations showed no evidence of heterogeneity of the [³H]ouabain binding sites in rat soleus muscle specimens, and the apparent K_D was 5 x 10^-8 M (243). Neither in biopsies of human vastus lateralis muscle, nor in guinea pig skeletal muscle, Scatchard plots showed any evidence of heterogeneity (251, 328). The same procedure was developped and tested for guinea pig skeletal muscle and heart (251) as well as for the heart of dogs and pigs (381).

The VO₄-facilitated assay for [³H]ouabain binding has been widely used for measurement on biopsy specimens from the human vastus lateralis muscle and several other muscles. As shown in Table 1, the values obtained for control subjects are rather similar in 15 studies performed in 6 different laboratories.

Some of the values were higher because they were measured in rather well-trained control subjects (133, 298). It is important that the method can be used for frozen samples, allowing long-distance transport or long-term storage of the frozen samples for at least 11 wk (328), or, as found later, for up to 4 yr (Clausen, unpublished observations). Furthermore, the VO₄-facilitated [³H]ouabain binding assay has been adapted for measurements on specimens from the human heart (324, 325), porcine and canine heart (381), and uterine smooth muscle (132).

A comparison of the binding kinetics of [³H]ouabain in a variety of human tissues showed that the affinity of ₁-, ₂-, and ₃-subunit isoforms for ouabain is similar (306, 426). This confirms repeated observations that in biopsies of human skeletal muscle and myocardium, only a single population of high-affinity sites can be detected (242, 328). This implies that in human skeletal muscle and myocardium, Na⁺-K⁺ pumps can be quantified by measuring the content of [³H]ouabain binding sites.

The VO₄-facilitated binding assay also allows measurements to be performed on muscle samples taken post mortem (332). A comparison of the values obtained in fresh rat skeletal muscle with those determined after storage at 20°C for 12 h showed that the loss of the ability to bind [³H]ouabain was surprisingly slow, 1%/h. Studies on vastus lateralis muscle samples obtained from 10 human subjects 0.5-6 h after death showed that the content of [³H]ouabain binding sites declined by only 8% in 6 h (332). Another post mortem study on vastus lateralis in nine subjects (380) showed a value of 274 ± 26 pmol/g wet wt, which is well within the range reported for in vivo biopsies (Table 1).

Measurements performed on post mortem samples from four different human muscles with considerable variation in the ratio between type I (slow-twitch) and type II (fast-twitch) fibers showed rather modest differences in the content of [³H]ouabain binding sites. Again, the values were not significantly lower than those obtained in parallel measurements on in vivo needle biopsies obtained from vastus lateralis of six normal subjects (115). Although more observations are needed, these results indicate that with respect to [³H]ouabain binding, the vastus lateralis is representative for a substantial part of human skeletal muscles.

In conclusion, [³H]ouabain binds stoichiometrically to a specific receptor on the -subunit of the Na⁺-K⁺-ATPase, allowing accurate quantification of the content of Na⁺-K⁺ pumps in intact skeletal muscle, biopsies, and membranes. The method can be applied to frozen or post mortem tissue samples, and the values obtained over a 20-yr period in several laboratories around the world are fairly consistent, allowing the detection of numerous regulatory changes.

D. Measurement of Transport Capacity

The function of the Na⁺-K⁺ pumps in intact skeletal muscle can be characterized by measurements of Na⁺-K⁺ contents and isotope fluxes of Na⁺ and K⁺. The ouabainsuppressible components of the basal and hormone-stimulated uptake of ⁴²K and ⁸⁶Rb are rather similar, and both isotopes seem to be reliable tracers for the Na⁺-K⁺ pump-mediated K⁺ transport (110). For other transport pathways for K⁺, however, ⁸⁶Rb is unreliable, yielding very different or even opposite results to those obtained with ⁴²K or in measurements of K⁺ content.

Due to diffusional limitations, the quantification of the maximum transport capacity of the Na⁺-K⁺ pumps in intact skeletal muscle preparations is difficult. On the basis of a procedure developed by Sejersted et al. (385), an attempt was made to circumvent this problem by increasing the concentration of K⁺ in the incubation medium so as to compensate for the diffusional delay of the access of K⁺ to the Na⁺-K⁺ pumps in intact isolated rat soleus (71). Following preloading with Na⁺ so as to increase [Na⁺]_i to 126 mM, measurements of ouabain-suppressible ⁴²K and ⁸⁶Rb uptake, ²²Na efflux, and the net changes in Na⁺-K⁺ contents (by flame photometry) performed in a K⁺-rich buffer gave values corresponding to 90% of the theoretical maximum predicted from measurements of the content of [³H]ouabain binding sites. In rat soleus, where the content of [³H]ouabain binding sites had undergone regulatory changes over a 4.5-fold range as a result of differentiation, K⁺ depletion or pretreatment with thyroid hormone, there was a close correlation between the content of [³H]ouabain binding sites and the maximum ouabain-suppressible ⁸⁶Rb uptake. These maximal transport rates were suppressed by cooling to 0°C or the metabolic inhibitor dinitrophenol and were not affected by K⁺ channel blockers (71). It seems reasonable to conclude, therefore, that the major part of the population of [³H]ouabain binding sites measured in rat soleus represents functional Na⁺-K⁺ pumps capable of operating close to their expected maximum theoretical transport rate. As described below, when stimulated at high frequency, the rates of active Na⁺-K⁺ transport of isolated muscles may reach this theoretical maximum (314).

In conclusion, there seems to be satisfactory agreement between the total content of [³H]ouabain binding sites in intact rat muscles and muscle segments and the maximum capacity for active Na⁺-K⁺ transport measured in the same muscles.

	V. ACUTE REGULATION OF Na+-K+ PUMP ACTIVITY

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This section and section VI describe the major factors involved in the acute and long-term regulation of the Na⁺-K⁺ pump in skeletal muscle, illustrated in the diagram shown in Figure 4. The largest single acute stimulus to the activity of the Na⁺-K⁺ pump seems to be excitation.

View larger version (34K): [in this window] [in a new window]   FIG. 4. Diagram of regulatory factors controlling the activity and contents of Na+-K+ pumps in skeletal muscle. Left: factors eliciting acute stimulation of the Na+-K+ pumps via second messengers. Right: factors influencing the content of Na+-K+ pumps by modifying their synthesis or degradation. IGF-I, insulin-like growth factor I. [Modified from Clausen (63).]

A. Excitation

1. Stimulatory effects

The action potentials causing excitation are elicited by an influx of Na⁺, followed by an efflux of K⁺. This leads to rapid increases in the concentrations of Na⁺ and K⁺ on the inner and the outer surfaces of sarcolemma, respectively, a strong stimulus for the activity of the Na⁺-K⁺ pumps. Indeed, electrical stimulation induced a marked increase in the rate of active Na⁺-K⁺ transport, both in young and adult animals (127, 200, 232, 296, 314). Ion-sensitive microelectrode recordings of the net recovery of intracellular K⁺ after electrical stimulation of isolated mouse soleus showed values in accordance with the theoretical maximum as estimated from the content of [³H]ouabain binding sites (232). Concomitant measurements of the net extrusion of Na⁺, however, showed values corresponding to only 30% of the theoretical maximum. Since the activity coefficient for [Na⁺]_i is relatively low, however, this value for Na⁺ efflux is likely to represent an underestimate. The postexcitatory net recovery of intracellular K⁺ and Na⁺ activity was suppressed by ouabain. Also in the perfused hindlimb muscles of adult dogs (30 kg), the reuptake of K⁺ taking place after electrical stimulation was suppressed by ouabain (200).

In isolated rat soleus mounted for unloaded contractions, continuous stimulation for 10 s at 60 Hz increased [Na⁺]_i by 58%. In the following resting period, reextrusion of Na⁺ to the resting level was complete in 2 min and could be prevented by ouabain (10^-4 M) or cooling to 0°C (127). The net reextrusion of Na⁺ as measured by flame photometry amounted to 4,430 nmol · g^-1 · min^-1, corresponding to an 12-fold increase in Na⁺-K⁺ pump-mediated Na⁺ efflux or 60% of the theoretical maximum rate. When the muscles were stimulated for 60 s at 10 Hz, the net Na⁺ reextrusion as measured over the following minute amounted to 3,850 nmol · g^-1 · min^-1, indicating that also low frequencies may elicit large stimulation of the Na⁺-K⁺ pumps. Stimulation for 10 s at 120 Hz produced a 22-fold increase in net Na⁺ extrusion as measured over the following 30 s, corresponding to the theoretical Na⁺ maximum transport rate (314).

It should be noted that in these experiments with muscles allowed to shorten freely without any external load, excitation-induced Na⁺ influx was fourfold larger than in muscles undergoing isometric contractions (see Table 3 and Ref. 314). In soleus muscles mounted for isometric contraction, stimulation at 60-120 Hz for 30 s increased net Na⁺ extrusion rate to 50% of the theoretical maximum.

Electrical stimulation also increases ²²Na efflux and the uptake of ⁴²K or ⁸⁶Rb. All of these effects can be blocked by ouabain, indicating that they are the outcome of activation of the Na⁺-K⁺ pump (125-127). This interpretation is further supported by the observation that electrical stimulation also increases the rate of [³H]ouabain binding (127), a specific reflection of an increased rate of active Na⁺-K⁺ transport (see sect. IVC and Ref. 76).

In rat soleus, direct electrical stimulation at 2 Hz increased the ouabain-suppressible ⁸⁶Rb uptake 2.4-fold more than in EDL (126). In soleus, the large stimulation of ⁸⁶Rb uptake was not associated with any detectable increase in [Na⁺]_i, whereas in EDL, the much more modest increase in ⁸⁶Rb uptake was associated with an increase in [Na⁺]_i of 63%. Also when [Na⁺]_i was increased using monensin or veratridine, soleus showed a larger increase in ⁸⁶Rb uptake than EDL. Thus, in muscles containing predominantly type I fibers, the Na⁺-K⁺ pump is considerably more sensitive to increases in [Na⁺]_i than in muscles containing mainly type II fibers. Because the EDL muscles used contain 20% more [³H]ouabain binding sites than soleus, this phenomenon cannot be accounted for by differences in the content of Na⁺-K⁺ pumps.

In rat soleus, the early phase of electrical stimulation in vivo induced hyperpolarization and increased M-wave area, effects that were suppressed by ouabain (204, 262). This hyperpolarization is likely to reflect the electrogenic action of the Na⁺-K⁺ pump. M waves arise by summation of the action potentials generated in the muscle cells, and recordings of the amplitude or area of these waves provide information about the number of muscle cells undergoing excitation as well as the intensity and degree of synchronization of action potentials. It is widely used to assess changes in excitability. In human biceps brachii muscle, indirect stimulation via the nerve at 10 or 20 Hz induced a doubling of the M-wave area within 120 or 40 s, respectively (97). Also in human soleus and tibialis anterior muscles, repetitive stimulation caused an early enlargement of the M-wave area, which was ascribed to hyperpolarization due to increased activity of the Na⁺-K⁺ pumps (160, 289). This hypothesis was supported by the observation that cooling of the abductor pollicis brevis muscle to 20°C before and during contraction significantly reduced the M-wave amplitude (369). A lowering of the temperature from 30 to 20°C reduces the rate of active Na⁺-K⁺ transport in isolated rat soleus by almost 60% (79). In the human brachioradial muscle, the increase in muscle fiber conduction velocity during recovery from repeated ischemic isometric exercise was suppressed by intra-arterial injection of ouabain, indicating that it reflected activation of the Na⁺-K⁺ pump (368). Also in human subjects, excitation seems to stimulate active electrogenic Na⁺-K⁺ transport, leading to an initial increase in the intensity of the action potentials.

In conclusion, there is strong evidence that in intact skeletal muscle preparations, excitation induces a rapid stimulation of the Na⁺-K⁺ pumps which, dependent on frequency, mode of contraction, and fiber type, may activate anything up to all of the available Na⁺-K⁺ pumps so as to reach full utilization of the entire active transport capacity. There is evidence that excitation stimulates the Na⁺-K⁺ pump also in vivo and in human subjects, favoring excitability.

2. Mechanisms for excitation-induced stimulation of the Na⁺-K⁺ pump

The excitation-induced stimulation of the Na⁺-K⁺ pump has generally been seen as the result of an increase in [Na⁺]_i, which in turn activates the Na⁺-K⁺-ATPase. The following observations indicate that also other mechanisms are important for the activation of the Na⁺-K⁺ pump.

1) In rat soleus, electrical stimulation at 60 Hz for 1 s is sufficient to elicit highly significant stimulation (22%) of ²²Na efflux (127) and ⁸⁶Rb influx (40%), even though this caused no increase in [Na⁺]_i (43).

2) Stimulation of rat soleus at 2 Hz more than doubles ouabain-suppressible ⁸⁶Rb uptake without significant increase in [Na⁺]_i (127, 130).

3) In soleus and EDL muscles mounted for isometric contractions, stimulation at frequencies from 30 to 90 Hz for 5-30 s elicits an initial short-lasting increase in [Na⁺]_i, followed by a 23-32% decrease below the resting control level, lasting up to 30 min (43, 314, 317) (Fig. 5).