Riding the Tides: K+ Concentration and Volume
Regulation by Muscle Na+-K+-2Cl Cotransport
Activity
Aidar R. Gosmanov,1 Michael I. Lindinger,2 and Donald B. Thomason1
1
Department of Physiology, The University of Tennessee Health Science Center, Memphis, Tennessee 38163; and 2Department of Human Biology and Nutritional Sciences, The University of Guelph, Guelph, Ontario N1G 2W1, Canada
S
keletal muscle cells contain 7075% of the body’s K+ and
45% of the body’s water. Consequently, K+ and water transport by muscle has the potential to affect whole body K+ (12)
and water balance. The electrogenic Na+-K+-ATPase is well
known for its inward transport of K+ and net efflux of water.
Recent evidence indicates that Na+-K+-2Cl cotransporter
(NKCC) expression and activity in skeletal muscle also provides
inward transport of K+ and, in contrast to the Na+-K+-ATPase,
provides a net influx of water (Fig. 1). In many cell types, the
inward transport of solute by the NKCC, with water following,
helps to maintain the cell volume in the face of a hyperosmotic
challenge (3, 7, 13). Thus skeletal muscle NKCC activity may
have multifunctional roles for K+ and water transport depending on specific conditions. In the following paragraphs, we discuss the role and regulation of NKCC activity in skeletal muscle under different osmotic conditions and the potential physiological importance of this transport mechanism.
NKCC activity in isosmotic conditions
The NKCC couples the inward transport of K+ and Cl to the
Na+ concentration ([Na+]) gradient. As such, the NKCC provides a K+ transport pathway that could complement the Na+K+-ATPase (Fig. 1). For muscle at rest, the bumetanide- and
furosemide-sensitive Cl transport in skeletal muscle produces
a plasma membrane depolarization (19); bumetanide specifically inhibits the NKCC, and furosemide inhibits the K+-Cl
cotransporter and, at high concentrations, the NKCC. The tendency of increased NKCC activity to increase intracellular
[Na+] and Cl concentration ([Cl]) and decrease the membrane potential would necessitate increased Na+-K+-ATPase
activity to maintain low intracellular [Na+] and [Cl]. This combination of NKCC and Na+-K+-ATPase activities would effectively increase the rate at which K+ is transported (unidirectional inward flux, or JinK) compared with a sole reliance on the
Na+-K+-ATPase. However, the involvement of the Na+-K+ATPase in maintaining the activity of the cotransporter has been
controversial. Over the long term, the driving force for NKCC
activity is clearly the Na+ electrochemical gradient established
196
News Physiol Sci 18: 196200, 2003;
10.1152/nips.01446.2003
by the Na+-K+-ATPase. However, in cultured skeletal muscle
myocytes, NKCC-mediated JinK is dependent on extracellular
[Na+] and [Cl] but is reported to be ouabain insensitive during
short incubations (17); these data indicate that ouabain-sensitive Na+-K+-ATPase activity may not be needed. A striking distinction of these cultured muscle cells is that they appear to
exhibit an altered density of NKCC and Na+-K+-ATPase such
that NKCC accounts for two-thirds of total JinK (18), compared
with 1520% of JinK in muscle in situ (8). Similarly, in vitro
muscle preparations demonstrate that bumetanide inhibits
basal ouabain-insensitive JinK over short periods of time. Conversely, in skeletal muscle in situ, ouabain inhibition of Na+-K+ATPase activity abolishes NKCC-mediated JinK, whereas barium, a nonspecific blocker of cation channels, does not affect
NKCC activity (7, 8). Although Na+-K+-ATPase activity must, in
the long run, provide the driving force for NKCC activity, the
NKCC also apparently provides a inwardly directed Na+ transport mechanism that affects Na+-K+-ATPase activity by its ability to change intracellular [Na+]. Inhibition of basal NKCC
activity by bumetanide diminishes ouabain-sensitive K uptake
into muscle by 35% (7). Interaction between cotransporter
activity and Na+-K+-ATPase has been described in several different muscle preparations (as noted in Ref. 7).
An important question is whether NKCC-mediated transport
is actively regulated and, if so, is the regulation independent
of Na+-K+-ATPase regulation? The answers to these questions
are apparent under conditions that stimulate K+ uptake by
muscle. In contrast to the low NKCC activity in muscle under
basal conditions [where muscle water and K+ balance are in
steady state (5, 7)], NKCC activity is increased by several stimuli of K+ uptake by muscle. Adrenergic receptor activation
causes a robust stimulation of total 86Rb uptake in rat muscle
under isosmotic conditions, and NKCC activation accounts for
up to one-third of the total stimulated 86Rb transport (7). Muscle contraction produced by either in vitro electrical stimulation or treadmill running also stimulates total 86Rb uptake and
NKCC activity independent of cellular shrinkage (3). It is of
note that NKCC inhibition by bumetanide does not change
muscle cell volume when NKCC activity is stimulated by the
0886-1714/03 5.00 © 2003 Int. Union Physiol. Sci./Am. Physiol. Soc.
www.nips.org
Downloaded from http://physiologyonline.physiology.org/ by 10.220.33.6 on June 18, 2017
Until recently, the existence of a Na+-K+-2Cl cotransporter (NKCC) in skeletal muscle was unclear.
Recent evidence shows that the NKCC is strongly expressed and provides both K+ and water
transport functions in resting and contracting skeletal muscle. The contribution of NKCC activity to K+ and volume regulation in skeletal muscle has potential consequences for muscle
contractility and metabolism.
-adrenergic agonist isoproterenol or by the adenylyl cyclase
activator forskolin (3), indicating that NKCC activity does not
result in a net transport of solute under isosmotic conditions.
Although insulin stimulates Na+-K+-ATPase activity in skeletal
muscle (2), insulin in fact inhibits NKCC activity (4). Therefore,
although Na+-K+-ATPase activity provides the necessary driving force for NKCC-mediated transport, there is independent
regulation of the activity of these two transport processes. A
summary scheme of possible interactions between the NKCC
and the Na+-K+-ATPase is shown in Fig. 1.
The mechanism for NKCC regulation under isosmotic conditions requires the extracellular signal-regulated kinase (ERK)
arm of the mitogen-activated protein kinase (MAPK) path-
ways. Blockade of the ERK MAPK pathway abolishes isoproterenol-, phenylephrine-, and contraction-stimulated NKCC
activity (4, 5). However, ERK MAPK pathway activation by the
NKCC stimuli is complex, showing apparent compartmentalization and muscle fiber phenotype-specific features. Evidence for compartmentalization comes from experiments in
which muscle is stimulated with insulin. Insulin stimulates
Na+-K+-ATPase activity and the ERK MAPK pathway but does
not stimulate NKCC activity (4). Moreover, insulin inhibits
NKCC activity by stimulating phosphatidylinositol 3kinase/Akt and p38 MAPK pathways that, in turn, apparently
inhibit a specific ERK MAPK pathway necessary for NKCC
stimulation (4). The importance of the ERK MAPK pathway as
News Physiol Sci • Vol. 18 • October 2003 • www.nips.org
197
Downloaded from http://physiologyonline.physiology.org/ by 10.220.33.6 on June 18, 2017
FIGURE 1. Although the Na+-K+-2Cl cotransporter (NKCC) and the Na+-K+-ATPase act in concert for the inward transport of K+, they act in opposition for the
balance of water (A) and Na+ (B) content. Experimental data indicate that muscle strikes a balance between the extremes of water and Na+ balance (C).
NKCC activity in hyperosmotic conditions
A well-established function of the NKCC in epithelial cells is
to protect cells from water loss during hyperosmotic challenge
(a process called a regulatory volume increase, or RVI) (15).
Until recently, evidence for a volume-regulatory function of the
NKCC in striated muscle was scarce. However, recent ex vivo
and in vivo experiments demonstrate that skeletal muscle activates a bumetanide-sensitive RVI in response to hyperosmotic
challenge. Intracellular water content of ex vivo skeletal muscle preparations is not affected by 10-min hyperosmotic challenge (indicative of activation of volume-regulatory mechanisms) unless the NKCC is blocked by bumetanide, in which
case the muscle loses water (3). Similarly, in perfused rat skeletal muscle inhibition of the NKCC activity before perfusion
with hypertonic medium (still containing bumetanide) results
in a transient net loss of water from muscle that is gradually followed by a net gain of water after ~20 min (7). These are characteristics of the NKCC in other tissues (13) and indicate that
the bumetanide-sensitive JinK and net water uptake can be
attributed to cotransporter activity in skeletal muscle also.
FIGURE 2. Putative scheme of signal transduction pathways regulating skeletal muscle NKCC activity under isotonic conditions. Activation of NKCC
activity requires the extracellular signal-regulated kinase (ERK) arm of the
mitogen-activated protein kinase (MAPK) signal transduction pathways. Inhibition of the ERK MAPK pathway can occur through several different mechanisms, depending on muscle fiber phenotype. MEK, MAPK kinase kinase.
198
News Physiol Sci • Vol. 18 • October 2003 • www.nips.org
Hyperosmotic challenge appears to produce coordinate regulation of NKCC activity and K+ channel conductivity in skeletal muscle. Approximately 30% of total JinK is attributed to passive K+ entry through K+ channels in resting, isotonic muscle
(8). However, total JinK does not increase during perfusion of
hindlimbs with hypertonic medium despite a doubling of the
bumetanide-sensitive component of JinK (Fig. 3) (7). The lack of
increase in JinK is attributed to a simultaneous decrease in passive influx of K+ through K+ channels, such that a transient
increase in JinK in response to cell shrinkage becomes apparent
when these cation channels are blocked with barium (Fig. 3).
Closure of K+ channels is consistent with the report that hypertonic challenge of muscle has a depolarizing influence that is
furosemide sensitive (18). Temporally, these data indicate that
hypertonic challenge causes a decrease in cellular volume
such that cell shrinkage in turn decreases passive K+ flux into
the cells via K+ channels in concert with increases in the
NKCC-mediated K+ influx. This makes sense with respect to
cell volume regulation because the reduction in passive K+
flux, both into and out of the cells, through K+ channels serves
to retain osmolytes within the cells. The transience in JinK when
K+ channels are blocked (Fig. 3) may result from subsequent
deactivation of the NKCC-mediated transport in response to
cell volume stabilization that occurs with additional net solute
(primarily Na+ and Cl) and water flux into the cell (7).
Independent regulation of NKCC and Na+-K+-ATPase activities in muscle under hyperosmotic conditions appears to be
necessary because the Na+-K+-ATPase could actually counteract the consequences of NKCC activation. Na+-K+-ATPase
activity in isolation causes a net efflux of solute, counteracting
a preservation of cell volume (7) (Fig. 1). Indeed, the increase
in NKCC-mediated JinK observed with hyperosmotic challenge
(3) accounts for the entire increase in total JinK (3). Figure 1
illustrates that NKCC and Na+-K+-ATPase, despite their coordinate inward transport of K+, act in opposition for Na+ and
water balance. Na+ content in steady state or water content in
steady state are the two extremes for these transport mechanisms in isolation. On the basis of experimental measurements of the relative contribution by each transport mechanism to JinK (5, 7, 8), the balance of activity lies in between the
two extremes. This would poise the NKCC and Na+-K+-ATPase
transport mechanisms to adjust accordingly for regulation of
either Na+ or water content. We can see, then, how these two
mechanisms can compensate for resting K+ leakage and the
high Cl conductivity present in muscle while maintaining
water content without the accumulation of Na+.
The mechanism(s) for NKCC activation under hyperosmotic
conditions is markedly different from isosmotic conditions (3).
Inhibition of the ERK MAPK pathway or insulin treatment does
not affect hyperosmolarity-stimulated NKCC activity. In addition, insulin has no effect on muscle cell volume, supporting
the hypothesis that the distinct roles of the NKCC (K uptake
vs. volume regulation) use different intracellular signal transduction pathways to activate the NKCC. Paradoxically,
whereas isoproterenol and forskolin stimulate NKCC activity
in isosmotic conditions, these agents inhibit NKCC activation
under hyperosmotic conditions (3). An increase in protein
kinase A activity stimulated by these agents can affect the
Downloaded from http://physiologyonline.physiology.org/ by 10.220.33.6 on June 18, 2017
an “integrating point” for NKCC regulatory signals is emphasized by fiber phenotype differences in signaling. In slowtwitch fibers, but not fast-twitch fibers, pertussis toxin abrogates isoproterenol-stimulated NKCC activity by allowing activation of Akt and, in turn, inhibition of ERK MAPK pathway
(5). Thus different muscle phenotypes contain functionally distinct G protein-coupled signal transduction pathways for
NKCC regulation. Also unique to slow-twitch muscle is a p38
MAPK pathway-mediated inhibition of NKCC activity, again
through inhibition of the ERK MAPK pathway (5). A summary
for known signaling pathways regulating NKCC activity under
isosmotic conditions is shown in Fig. 2. Because slow-twitch
fibers have approximately twice the K transport activity as
fast-twitch fibers (4, 5), phenotype-specific differences have
important physiological consequences. For example, individuals that are physically inactive, obese, or have diabetes mellitus exhibit both an increased reliance on fast-twitch muscle
fibers and hyperkalemia during periods of activity. Upregulation of NKCC-mediated JinK in exercise-trained rats demonstrates that regular physical activity may help maintain a slowtwitch phenotype that has a greater capacity for K buffering.
organization of actin cytoskeleton and may affect intracellular
translocation or functional characteristics of sarcolemmal proteins. One may speculate that cytoskeletal mechanotransduction events associated with cell shrinkage rapidly activate
cotransporter activity (14), increasing JinK. Lionetto and
coworkers (10) have recently implicated cytoskeletal elements
together with protein kinase C and myosin light-chain kinase
for hyperosmotic activation of NKCC activity in eel intestinal
epithelium. Therefore, protein kinase A activity may provide a
counterregulatory mechanism to inhibit NKCC activity under
appropriate hyperosmotic conditions. Counterregulatory inhibition of muscle NKCC activity under hyperosmotic conditions might be appropriate when preservation of plasma volume and blood pressure must override the preservation of
muscle volume.
ing plasma osmolality (Fig. 4). The classic study of Lundvall and
coworkers (11) demonstrates that the increase in plasma osmolality during exercise is due to “the delivery of osmoles from
the exercising muscle.” Because the net loss of K+ and lactate
from muscle approximately balances the net accumulation of
lactate within contracting skeletal muscle (9), the increase in
Physiological consequences of skeletal muscle NKCC
activity
Because the NKCC carries K+ in addition to Na+ and Cl, the
potential volume-regulatory role and K+-regulatory role for
NKCC are intimately linked. The impact of these dual roles in
skeletal muscle is twofold: an effect on composition and volume of fluid compartments and an effect on muscle contractile function.
Exercise studies provide a good illustration of the integrative
nature of skeletal muscle’s impact on fluid compartments.
Within contracting muscle cells, the rapid hydrolysis of phosphocreatine consumes water and results in the production of
two osmotically active molecules: creatine and inorganic
phosphate. At the same time, hydrolysis of glycogen results in
the production of lactate, another osmolyte, and the release of
bound water. Contractile activity causes muscle to lose osmotically active K+ and lactate, which accumulate within the interstitial fluid compartment and enter the venous circulation, rais-
FIGURE 4. Metabolic events accompanying exercise results in fluid shifts
within working and nonworking muscles. Osmolytes accumulating in the
working muscle cause water to move from the interstitium to the working
muscle. Osmolyte release from the muscle increases plasma osmolality and
draws water from the nonworking tissue. NKCC activation helps to defend the
nonworking muscle from shrinkage through a regulatory volume increase
(RVI). PCr, phosphocreatine; Pi, inorganic phosphate.
News Physiol Sci • Vol. 18 • October 2003 • www.nips.org
199
Downloaded from http://physiologyonline.physiology.org/ by 10.220.33.6 on June 18, 2017
FIGURE 3. Hyperosmotic challenge produces coordinate upregulation of NKCC activity and downregulation of channel conductivity in skeletal muscle. Total inward K+ flux (JinK) does not
increase during perfusion of hindlimbs with hypertonic medium
despite a doubling of the bumetanide-sensitive component of JinK
(squares). In contrast, when K+ channels are first blocked with
barium (circles), there is a transient increase in JinK during hyperosmotic challenge, indicating a decrease in K+ channel-mediated
JinK along with a transient increase in the NKCC-mediated JinK.
200
News Physiol Sci • Vol. 18 • October 2003 • www.nips.org
We are grateful to our colleagues for their work that we were unable to cite.
M. I. Lindinger’s research was supported by grants from the National Sciences and Engineering Research Council, Canada. D. B. Thomason’s research
was supported by grants from the American Diabetes Association and the
American Heart Association.
References
1. Clausen T, Dahl-Hansen AB, and Elbrink J. The effect of hyperosmolarity
and insulin on resting tension and calcium fluxes in rat soleus muscle. J
Physiol 292: 505526, 1979.
2. Clausen T and Everts ME. Regulation of the Na,K-pump in skeletal muscle. Kidney Int 35: 113, 1989.
3. Gosmanov AR, Schneider EG, and Thomason DB. NKCC activity restores
muscle water during hyperosmotic challenge independent of insulin,
ERK, and p38 MAPK. Am J Physiol Regul Integr Comp Physiol 284: R655
R665, 2003.
4. Gosmanov AR and Thomason DB. Insulin and isoproterenol differentially
regulate mitogen-activated protein kinase-dependent Na+-K+-2Cl
cotransporter activity in skeletal muscle. Diabetes 51: 615623, 2002.
5. Gosmanov AR, Wong JA, and Thomason DB. Duality of G protein-coupled mechanisms for -adrenergic activation of NKCC activity in skeletal
muscle. Am J Physiol Cell Physiol 283: C1025C1032, 2002.
6. Gulati J and Babu A. Tonicity effects on intact single muscle fibers: relation between force and cell volume. Science 215: 11091112, 1982.
7. Lindinger MI, Hawke TJ, Lipskie SL, Schaefer HD, and Vickery L. K+ transport and volume regulatory response by NKCC in resting rat hindlimb
muscle. Cell Physiol Biochem 12: 279292, 2002.
8. Lindinger MI, Hawke TJ, Vickery L, Bradford L, and Lipskie SL. An integrative, in situ approach to examining K+ flux in resting skeletal muscle.
Can J Physiol Pharmacol 79: 9961006, 2001.
9. Lindinger MI, Spriet LL, Hultman E, Putman T, McKelvie RS, Lands LC,
Jones NL, and Heigenhauser GJF. Plasma volume and ion regulation during exercise after low- and high-carbohydrate diets. Am J Physiol Regul
Integr Comp Physiol 266: R1896R1906, 1994.
10. Lionetto MG, Pedersen SF, Hoffmann EK, Giordano ME, and Schettino T.
Roles of the cytoskeleton and of protein phosphorylation events in the
osmotic stress response in EEL intestinal epithelium. Cell Physiol Biochem
12: 163178, 2002.
11. Lundvall J, Mellander S, Westling H, and White T. Fluid transfer between
blood and tissues during exercise. Acta Physiol Scand 85: 258269, 1972.
12. McDonough AA, Thompson CB, and Youn JH. Skeletal muscle regulates
extracellular potassium. Am J Physiol Renal Physiol 282: F967F974,
2002.
13. O’Neill WC. Physiological significance of volume-regulatory transporters.
Am J Physiol Cell Physiol 276: C995C1011, 1999.
14. Orlov SN, Kolosova IA, Cragoe EJ, Gurlo TG, Mongin AA, Aksentsev SL,
and Konev SV. Kinetics and peculiarities of thermal inactivation of volumeinduced Na+/H+ exchange, Na+,K+,2Cl cotransport and K+,Cl cotransport
in rat erythrocytes. Biochim Biophys Acta 1151: 186192, 1993.
15. Russell JM. Sodium-potassium-chloride cotransport. Physiol Rev 80: 211
276, 2000.
16. Sejersted OM and Sjogaard G. Dynamics and consequences of potassium
shifts in skeletal muscle and heart during exercise. Physiol Rev 80: 1411
1481, 2000.
17. Sen CK, Hanninen O, and Orlov SN. Unidirectional sodium and potassium flux in myogenic L6 cells: mechanisms and volume-dependent regulation. J Appl Physiol 78: 272281, 1995.
18. Urazaev AK, Naumenko NV, Nikolsky EE, and Vyskocil F. The glutamate
and carbachol effects on the early post-denervation depolarization in rat
diaphragm are directed towards furosemide-sensitive chloride transport.
Neurosci Res 33: 8186, 1999.
19. Van Mil HG, Geukes Foppen RJ, and Siegenbeek van Heukelom J. The
influence of bumetanide on the membrane potential of mouse skeletal
muscle cells in isotonic and hypertonic media. Br J Pharmacol 120: 39
44, 1997.
20. Vaughan PC, Bressler BH, Dusik LA, and Trotter MJ. Hypertonicity and
force development in frog skeletal muscle fibres. Can J Physiol Pharmacol
61: 847856, 1983.
Downloaded from http://physiologyonline.physiology.org/ by 10.220.33.6 on June 18, 2017
intracellular osmolality can be approximated from the increase
in intracellular creatine concentration. The study by Lundvall et
al. (11) demonstrates that the increase in the volume of active
muscle is primarily “caused by osmosis resulting from
increased muscle tissue osmolality”; also, capillary hydrostatic
pressure plays only a minor role in fluid accumulation of active
muscles. The increase in fluid accumulation within active muscle greatly exceeds the fluid loss from the vascular compartment (9), further contributing to the increase in plasma osmolality. Accordingly, fluid from noncontracting tissues moves,
primarily by osmotic forces, into the vascular compartment, in
part defending vascular volume (Fig. 4). It can now be appreciated that an increase in NKCC activity within noncontracting
muscle would counteract the net loss of water from these cells,
preventing excessive cellular dehydration with impairment of
force-producing ability (6). When modeled in situ, perfusion of
skeletal muscle with hypertonic medium simulating that seen
in blood during high-intensity exercise results in a rapid loss of
water from muscle, followed within 2 min by a more prolonged increase in net water and solute uptake that is sensitive
to NKCC inhibition with bumetanide (7). Activation of the
NKCC by contractile activity also would help the contracting
muscle accumulate osmolytes and preserve cell volume (3).
A muscle cell’s ability to maintain cell volume and K+ content has contractile consequences. Although hypertonic challenge leads to an increase in resting tension (1), maximum isometric force production is dramatically reduced (6). An
increase in cytosolic Ca2+ concentration resulting from
increased entry of extracellular (1) and sarcoplasmic reticulum
Ca2+ into the cytoplasm may increase resting tension. The
decreased magnitude of isometric force development is
apparently due to a slowed rate of myosin-actin cross-bridge
formation resulting from increased intracellular ionic strength
(20). Impaired contractile activity also occurs with loss of K+
from contracting muscle (16). As outlined in Fig. 1, NKCC
activity could contribute significantly to JinK to maintain or
restore intracellular K+ concentration and indeed appears to
do so under isosmotic and hypertonic conditions (3, 7, 8).
However, a significant loss of K+ could also trigger a regulatory volume increase, making it difficult to assign separate volume- and K-regulatory roles for the NKCC in isolation.
In summary, the skeletal muscle NKCC appears to have
multifunctional roles for K+ and water transport. Mechanisms
regulating NKCC activity are complex and largely unexplored
but depend on muscle phenotype, local conditions, and
whole body conditions. The multifunctional roles of the NKCC
in skeletal muscle may occur simultaneously within an animal. For example, in humans and other animals, exercise
results in a redistribution of water and ions among the tissues
in response to rapid and pronounced changes in fluid osmolality, ion concentrations, and capillary hydrostatic pressure.
The NKCC in working muscle could act to help restore intracellular K+ concentration and volume, whereas in the nonworking muscle the NKCC would preserve cell volume
against hyperosmotic plasma and interstitial fluid. Given the
importance of skeletal muscle on whole body K+ and water
balance, the coordinated control of NKCC activity to meet
multiple cellular demands is of physiological significance.