Mechanisms of
Na+-K+
pump regulation in cardiac myocytes during hyposmolar
swelling
N. L.
Bewick1,
C.
Fernandes1,2,
A. D.
Pitt1,
H. H.
Rasmussen1,2, and
D. W.
Whalley1,2
1 Cardiology Department, Royal
North Shore Hospital, St. Leonards, New South Wales 2065; and
2 University of Sydney, Sydney,
New South Wales 2006, Australia
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ABSTRACT |
We have previously demonstrated that the sarcolemmal
Na+-K+
pump current
(Ip) in cardiac
myocytes is stimulated by cell swelling induced by exposure to
hyposmolar solutions. However, the underlying mechanism has not been
examined. Because cell swelling activates stretch-sensitive ion
channels and intracellular messenger pathways, we examined their role
in mediating Ip
stimulation during exposure of rabbit ventricular myocytes to a
hyposmolar solution.
Ip was measured
by the whole cell patch-clamp technique. Swelling-induced pump
stimulation altered the voltage dependence of
Ip. Pump
stimulation persisted in the absence of extracellular
Na+ and under conditions designed
to minimize changes in intracellular Ca2+, excluding an indirect
influence on Ip
mediated via fluxes through stretch-activated channels. Pump
stimulation was protein kinase C independent. The tyrosine kinase
inhibitor tyrphostin A25, the phosphatidylinositol 3-kinase inhibitor
LY-294002, and the protein phosphatase-1 and -2A inhibitor okadaic acid
abolished Ip
stimulation. Our findings suggest that swelling-induced pump
stimulation involves the activation of tyrosine kinase,
phosphatidylinositol 3-kinase, and a serine/threonine protein
phosphatase. Activation of this messenger cascade may
cause activation by the dephosphorylation of pump units.
osmolarity; tyrosine kinase; ion transport; phosphatases
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INTRODUCTION |
THE SWELLING OF CARDIAC myocytes induced by exposure to
hyposmolar superfusates stimulates the activity of the membrane
Na+-K+
pump (27, 32). However, the cellular mechanisms involved in the
transduction of the osmotic stress into pump stimulation have not been
investigated. The swelling of myocytes activates stretch-sensitive ion
channels, including channels permeable to Na+ and
Ca2+ (3, 23). Pump stimulation
could therefore be secondary to an influx of extracellular
Na+ or
Ca2+, resulting in a rise in
cytosolic Na+ or
Ca2+ concentrations, known
determinants of pump activity (33). Cell swelling is also known to
rapidly activate a variety of intracellular messengers, including
protein tyrosine kinase and serine(threonine) protein
kinase (24, 26; see Ref. 25 for review). Serine(threonine) protein kinases can induce the phosphorylation of the
Na+-K+
pump, whereas tyrosine kinases are involved in inducing the
dephosphorylation of pump molecules via a mechanism involving
activation of protein serine/threonine phosphatases (21). Because the
activation of kinases and phosphatases can regulate the
Na+-K+
pump (2, 7), increased pump activity during exposure of cardiac
myocytes to hyposmolar superfusates could be mediated by protein
kinase- and/or protein phosphatase-dependent pathways.
Understanding the mechanism by which the
Na+-K+
pump is regulated by osmotic stress is desirable because the pump plays
a role in the maintenance of cell volume under physiological conditions and during pathophysiological states, including acute myocardial ischemia and reperfusion. Using the whole cell patch-clamp
technique, we studied
Na+-K+
pump current
(Ip) in
ventricular myocytes exposed to hyposmolar superfusates. We found that
swelling-induced
Na+-K+
pump stimulation is independent of the influx of extracellular Na+ and
Ca2+. However, stimulation was
dependent on tyrosine kinase and involved the activation of an okadaic
acid-inhibitable protein phosphatase. Pump stimulation induced by
exposure to hyposmolar superfusates was rapidly reversible on
reexposure to an isosmolar superfusate. This reversal of pump
stimulation could be blocked by inhibition of protein kinase. We
propose that the
Na+-K+
pump is regulated by cell volume via steps which involve the phosphorylation and dephosphorylation of the pump molecule by kinases
and phosphatases, respectively.
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MATERIALS AND METHODS |
Cells and solutions.
Single ventricular myocytes were isolated from male New Zealand White
rabbits weighing 2.5-3.5 kg as described previously (32). They
were stored at room temperature until used. For measurement of
Ip, myocytes were
suspended in superfusates flowing through a tissue bath mounted on an
inverted microscope. The superfusate used initially in all experiments
contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 0.44 NaH2PO4,
10 glucose, 10 HEPES, and 1 MgCl2.
The solution was titrated to pH 7.4 ± 0.01 at 35°C with 1 M
NaOH. Hyposmolar and isosmolar
Ca2+-free modified Tyrode
solutions, used subsequently when
Ip was measured,
contained (in mM) 105 NaCl, 5.6 KCl, 0.44 NaH2PO4,
10 glucose, 10 HEPES, 2 MgCl2, 0.2 CdCl2, and 2 BaCl2.
BaCl2 was added to block
K+ channels, and
CdCl2 was added to block the
Na+/Ca2+
exchanger. The solutions were titrated to pH 7.4 ± 0.01 at 35°C with 1 M NaOH. The isosmolar solution contained 70 mM sucrose, whereas
the hyposmolar solutions were sucrose free. We measured solution
osmolarity with a vapor pressure osmometer (model 5500 osmometer;
Wescor, Logan, UT). The osmolarities of the hyposmolar and isosmolar
solutions were 240 ± 3 and 305 ± 4 mosM, respectively (mean ± SD). Superfusates used at the time
Ip was measured
were warmed to 35 ± 0.5°C. For measurements of
Ip at a fixed
membrane voltage
(Vm) of
40 mV, the pipette filling solution contained (in mM) 70 potassium glutamate, 1 KH2PO4,
5 HEPES, 5 EGTA, and 2 MgATP. Unless otherwise indicated, the solution
also contained 10 mM sodium glutamate and 80 mM tetramethylammonium
chloride (TMA-Cl). It was titrated to pH 7.05 ± 0.01 at 35°C
with 1 M KOH. In experiments designed to examine the
Ip-Vm
relationship, the filling solution of the patch pipettes contained (in
mM) 10 sodium glutamate, 1 KH2PO4,
5 HEPES, 5 EGTA, 2 MgATP, 60 TMA-Cl, 20 tetraethylammonium chloride, 70 CsOH, and 50 aspartic acid. The solution was titrated to a pH of 7.05 ± 0.01 at 35°C with HCl. The resistances of pipettes filled
with either solution were 0.8-1.0 M
. The offset potential was
nulled by placing both the ground electrode and patch pipette in the
pipette filling solution (20). We used the computer program JPCalc (P. Barry, Univ. of New South Wales) to estimate junction potentials
between the ground electrode and superfusates. The differences in these
junction potentials were <0.3 mV between the different
superfusates used. They are not corrected for when Ip is reported.
Measurement of membrane currents.
We measured Ip in
freshly isolated cells 2-9 h after excision of the heart.
Ip was defined as
the shift in holding current induced by 100 µM ouabain (Sigma
Chemical, St. Louis, MO). For each cell
Ip was measured
during the superfusion of either isosmolar or hyposmolar
Ca2+-free Tyrode solutions.
Membrane currents were recorded by using the continuous
single-electrode voltage-clamp mode of an Axoclamp 2A amplifier and
AxoTape or pCLAMP software (Axon Instruments, Foster City, CA). The
Ip values
reported were normalized for membrane capacitance as described
previously (32). For measurement of the
Ip-Vm
relationship, we superfused myocytes with
Ca2+-free solutions having a
composition identical to that of solutions used to measure
Ip at the fixed
Vm of
40
mV with the exception that we added 1 mM anthracene-9-carboxylic acid
(9-AC; Sigma Chemical) to inhibit stretch-activated
Cl
channels (12).
Reagents.
In some experiments tyrphostin A25 or its inactive analog, tyrphostin
A63, okadaic acid or its inactive analog, okadaic acid methyl
ester, or staurosporine was added to the patch pipette. All were purchased from Calbiochem (La Jolla, CA). Tyrphostin A25 and
A63 were dissolved in DMSO. The final concentration of DMSO was 0.04%.
Staurosporine and okadaic acid methyl ester were also dissolved in
DMSO; the final concentrations of DMSO were 0.005 and 0.2%,
respectively. In experiments designed to examine the
Ip-Vm
relationship, 9-AC was dissolved in DMSO with a final concentration of
0.1%. It has previously been shown that DMSO in concentrations higher
than those used in the present study have no effect on the steady-state
holding current or
Ip in isolated cardiac myocytes (19). LY-294002, purchased from Calbiochem, was
dissolved in ethanol and added to the superfusates on the day of
experimentation. The final ethanol concentration in the superfusates
was 0.03%. Ethanol at this concentration has previously been shown in
control experiments to have no effect on basal
Na+-K+
pump activity (11).
Statistical analysis.
Results are expressed as means ± SE unless otherwise indicated.
Comparisons are made by using Student's
t-test for unpaired observations.
Statistical comparisons based on the same control experiments were made
with Dunnett's test. Comparisons between the voltage
dependence curves were made by two-way ANOVA for the interaction of
voltage and osmolarity. Differences between means were
regarded as statistically significant at
P < 0.05.
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RESULTS |
Role of Ca2+
influx in stimulation of Ip
by cell swelling.
We have previously shown that exposure of rabbit cardiac myocytes to
hyposmolar solutions stimulates
Ip when the patch
pipette Na+ concentration
([Na]pip) is near
physiological intracellular levels (32). In contrast, Sasaki et al.
(27) did not report a significant enhancement of the
Ip values for
patch-clamped guinea pig cardiac myocytes under similar experimental
conditions ([Na]pip 10 mM). Our experiments were performed in the presence of extracellular Ca2+, whereas the experiments of
Sasaki et al. (27) were performed with superfusates that were nominally
Ca2+ free. This raises the
possibility that the pump stimulation induced by exposure to hyposmolar
superfusates may be dependent on the influx of
Ca2+ via stretch-activated
channels. Because it cannot be ensured that the dialysis of the pipette
solution completely changes intracellular Ca2+ levels, such an
influx might cause changes in the intracellular concentrations of this
ion. We performed experiments to examine this possibility. The pipette
solution we used included 10 mM Na+, and, to buffer intracellular
Ca2+, 5 mM EGTA. The whole cell
configuration was first established while myocytes were being
superfused with isosmolar
Ca2+-containing Tyrode solution.
We then switched to a superfusate that contained 105 mM
Na+ and that was isosmolar (310 mosM) or hyposmolar (240 mosM). These solutions were nominally
Ca2+ free. The
Ca2+-free Tyrode solution was
superfused for 3 min before we measured Ip. We measured
Ip values for
nine cells superfused with isosmolar solution and for seven cells
superfused with hyposmolar solution. Representative recordings of
shifts in holding currents during the measurement of
Ip have been
published previously (32). The means of the
Ip values were
0.25 ± 0.03 pA/pF in isosmolar superfusates and 0.58 ± 0.06 pA/pF in hyposmolar superfusates. The difference in mean
Ip values was
statistically significant.
We previously found that swelling-induced stimulation of
Ip, measured by
using Ca2+-containing
superfusates, only occurred when
[Na]pip was near physiological levels of intracellular
Na+; there was no effect when
[Na]pip was high (32).
We wished to examine whether a similar
[Na]pip dependence of
the pump stimulation was apparent when
Ca2+-free superfusates were used.
We therefore performed experiments using an
[Na]pip of 80 mM, a
concentration that produces near-maximal pump activity. The mean
Ip was 1.68 ± 0.12 pA/pF (n = 12) in cells exposed
to isosmolar superfusate and 1.79 ± 0.16 pA/pF
(n = 12) in cells exposed to
hyposmolar superfusate. This difference was not statistically
significant. The results of experiments performed at
[Na]pip values of 10 and 80 mM are summarized in Fig.
1. Taken together, the data in
Fig. 1 indicate that the
[Na]pip-dependent stimulatory effect of the exposure of myocytes to hyposmolar
superfusates on
Ip is not
dependent on the influx of extracellular
Ca2+.

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Fig. 1.
Effect of osmotic swelling on sarcolemmal
Na+-K+
pump current
(Ip).
Superfusate was isosmolar (Iso) or hyposmolar (Hypo), and patch pipette
Na+ concentration
([Na+]pip)
was either 10 or 80 mM.
Ip has been
normalized for cell capacitance. No. in parentheses is no. of
experiments in each group.
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Role of Na+
influx in pump stimulation.
Cell swelling and mechanical stretch open nonselective
stretch-activated cation channels (23). An influx of
Na+ through such channels might
stimulate pump activity in the present study by increasing the
Na+ concentration at intracellular
binding sites in a restricted subsarcolemmal space to levels above
[Na]pip. To examine
this possibility, we measured
Ip while myocytes
were being superfused with
Na+-free isosmolar or hyposmolar
superfusates. Both superfusates were
Ca2+ free. We
replaced Na+ in the superfusates
with 105 mM
N-methyl-D-glucamine.
Isosmolar superfusates contained 70 mM sucrose, whereas hyposmolar
solutions were sucrose free.
[Na]pip was 10 mM. In
the absence of extracellular Na+,
the mean Ip of
eight myocytes superfused with hyposmolar solutions was 0.59 ± 0.04 pA/pF, whereas the mean
Ip of six
myocytes superfused with isosmolar solutions was 0.31 ± 0.03 pA/pF.
The difference was statistically significant
(P < 0.001). This indicates that the
stimulatory effect of cell swelling on
Ip is not due to
an increased Na+ influx. A direct
effect of cell swelling on the
Na+-K+
pump is suggested.
Effect of cell swelling on pump
Ip-Vm
relationship.
The experiments described above indicate that myocyte swelling
stimulates the
Na+-K+
pump at a fixed membrane voltage
(Vm) of
40 mV. Because pump function is voltage dependent, we examined
if stimulation also occurs at other voltages. After establishing the
whole cell configuration, we switched to a
Ca2+-free superfusate and
voltage-clamped myocytes at a holding potential of
40 mV. We
then applied 320-ms voltage steps to test potentials (Vm) from
100 to +60 mV in 20-mV increments. The command voltage was
returned to
40 mV for 2 s between each step to a test potential. To derive Ip at
each test potential, the membrane currents recorded during the
superfusion of ouabain were subtracted from the membrane currents at
corresponding voltages before the superfusion of ouabain. Details of
the protocol and representative current traces have been described
previously (10). Figure
2A
summarizes the
Ip-Vm relationships for six myocytes superfused with isosmolar solution and
six myocytes superfused with hyposmolar solution. Pump stimulation induced by cell swelling was most prominent at the most negative values
of Vm and
decreased with a shift of
Vm toward
positive levels.
Ip values
measured in these experiments using a
[Na]pip of 10 mM are
larger during the superfusion of both isosmolar and hyposmolar
solutions than those measured in experiments performed at a constant
Vm of
40
mV with 10 mM [Na]pip
(see Fig. 1). This difference can be attributed to differences in the
compositions of the pipette solutions used in the two experimental
protocols. In experiments examining the
Ip-Vm
relationship, we used a pipette solution containing
Cs+ as a substitute for
K+ to abolish time- and
voltage-dependent K+ conductances.
We have previously shown that the inhibitory effect of intracellular
Cs+ on
Ip is
significantly less than the inhibitory effect of intracellular K+. Such a reduction in inhibitory
effect is expected to cause an increase in
Ip (14). The
Ip-Vm
relationships have been normalized to the
Ip recorded at 0 mV in Fig. 2B to facilitate the
comparison of their slopes. The
Ip-Vm
relationship in isosmolar solutions was nearly linear and had a
positive slope throughout the entire range of test potentials. The
Ip-Vm
relationship for six myocytes superfused with hyposmolar solution
appeared less steep at negative potentials than that for myocytes
superfused with isosmolar solutions. It had a negative slope at
potentials more positive than
40 mV. The difference in these
curves was statistically significant
(P < 0.001; 2-way ANOVA). We
conclude that pump stimulation induced by myocyte swelling is voltage
dependent.

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Fig. 2.
A: voltage dependence of
Ip values for
myocytes superfused with isosmolar ( ) and hyposmolar superfusates
( ).
Ip-membrane
voltage (Vm)
relationships of 6 myocytes in each group have been summarized by
plotting mean ± SE
Ip measured at
each test potential. B: normalized
voltage dependence of
Ip. Plots were
obtained by first normalizing
Ip-Vm
relationship for each myocyte to
Ip recorded at 0 mV and then averaging normalized
Ip values at each
test potential.
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Effect of kinase inhibition on swelling-induced pump stimulation.
Both mechanical deformation of the cardiac sarcolemmal membrane and
osmotic swelling in a hyposmolar solution activate protein kinase C
(PKC) (24). Because PKC and other kinases, in turn, modulate
Na+-K+
pump activity in a variety of noncardiac tissues (2, 7), we examined
the effect of the potent but nonselective protein kinase inhibitor
staurosporine and the effect of bisindolylmaleimide I, an inhibitor
believed to be specific for PKC. We added 10 nM staurosporine to the
patch pipette solution.
[Na]pip was 10 mM in
all experiments. After the whole cell configuration had been established, we waited for 5 min to allow intracellular dialysis of the
staurosporine before myocytes were exposed to isosmolar or hyposmolar
superfusate, and then
Ip was
determined. The mean Ip of seven cells
exposed to isosmolar solution was 0.24 ± 0.02 pA/pF, and the mean
Ip of six cells
exposed to hyposmolar solution was 0.40 ± 0.04 pA/pF. The difference
was statistically significant. This indicates that nonselective kinase
inhibition does not prevent the stimulation of
Ip induced by
cell swelling. A similar result was obtained when we used 1 µM
bisindolylmaleimide I.
Effect of tyrosine kinase inhibition on pump stimulation in
hyposmolar solutions.
Swelling and mechanical stretch of cardiac myocytes rapidly activate
tyrosine kinase (24, 26). Because activation of the insulin receptor
tyrosine kinase has an effect on the
Ip-Vm
relationship (13) similar to the effect of swelling found in this
study, we examined the effect of the tyrosine kinase inhibitor
tyrphostin A25. In the first series of experiments, we examined the
effect of tyrphostin A25 on pump stimulation induced by myocyte
swelling at a fixed
Vm of
40
mV. We included 100 µM tyrphostin A25 in the patch pipette solution.
[Na]pip was 10 mM.
After the whole cell configuration was established, myocytes were
superfused with either isosmolar or hyposmolar
Ca2+-free solutions and
Ip was measured.
The mean Ip of
six myocytes superfused with isosmolar solution was 0.26 ± 0.03 pA/pF. This is similar to the mean
Ip measured under
similar conditions with tyrphostin A25-free pipette solutions (Fig. 1),
suggesting that there is no tonic effect of tyrosine kinase on the
Ip of myocytes in
isosmolar solutions. When tyrphostin A25 was included in pipette solutions, the mean
Ip of six
myocytes exposed to hyposmolar solutions was 0.29 ± 0.04 pA/pF. This
is similar to mean
Ip values of the myocytes exposed to isosmolar solutions, indicating that tyrphostin A25
completely blocked the pump stimulation induced by cell swelling. To
exclude a nonspecific effect of tyrphostin A25 on the
Na+-K+
pump, experiments were repeated with 100 µM tyrphostin A63, the inactive analog of tyrphostin A25, in the patch pipette. In the presence of tyrphostin A63, the mean
Ip of five
myocytes superfused with isosmolar solution and the mean
Ip of five
myocytes superfused with hyposmolar solutions were 0.20 ± 0.03 and
0.48 ± 0.07 pA/pF, respectively. The difference was statistically
significant. Results obtained with tyrphostin A25 and A63 in pipette
solutions have been summarized in Fig.
3A. We
conclude that the tyrosine kinase inhibitor tyrphostin A25 can abolish
the pump stimulation induced by cell swelling and that this is a
specific effect of the drug.

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Fig. 3.
A: effect of tyrosine kinase and
phosphatidylinositol 3-kinase (PI3K) inhibition. Tyrosine kinase
inhibitor tyrphostin A25 (Tyr A25) and its inactive analog, tyrphostin
A63 (Tyr A63), were included in patch pipette solutions. In a separate
group of experiments PI3K inhibitor LY-294002 was included in
superfusates. Inhibition of tyrosine kinase or PI3K abolished
swelling-induced pump stimulation. No. in parentheses is no. of
experiments in each group. B: effect
of tyrphostin A25 on
Ip-Vm
relationship during exposure to isosmolar ( ) and hyposmolar ( )
solutions.
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In a second series of experiments, we examined the effect of tyrphostin
A25 on the
Ip-Vm
relationship. Normalized
Ip-Vm
relationships obtained from six myocytes exposed to isosmolar solutions
and six myocytes exposed to hyposmolar solutions have been summarized in Fig. 3B. Their slopes were similar
to the slopes of
Ip-Vm
relationships for myocytes superfused with isosmolar solution and
voltage clamped with tyrphostin A25-free patch pipettes (Fig.
2B). Tyrphostin A25 completely
eliminated the effect of the hyposmolar solution on the slope of the
Ip-Vm
relationship. We also determined the Ip-Vm
relationship with the inactive analog tyrphostin A63 included in
pipette solutions. The shift in the
Ip-Vm
relationship induced by exposure to hyposmolar solutions (Fig.
2B) was unaffected by tyrphostin A63
(data not shown).
Role of phosphatidylinositol 3-kinase in mediating the stimulation
of Ip.
Activation of phosphatidylinositol 3-kinase (PI3K) is an essential step
in the transduction of cellular signals mediated via tyrosine
kinase-dependent processes (4, 15, 31). We investigated the role of
PI3K in the stimulation of
Ip by hyposmolar
solutions. Myocytes were superfused before and after establishment of
the whole cell configuration with solutions containing the specific PI3K inhibitor LY-294002 at a concentration of 5 µM. The mean Ip of six
myocytes superfused with LY-294002-containing isosmolar solutions was
0.29 ± 0.02 pA/pF, and the mean
Ip of six
myocytes superfused with LY-294002-containing hyposmolar solutions was 0.27 ± 0.02 pA/pF. The difference was not statistically significant. We have included the results of experiments performed with LY-294002 in
Fig. 3A. Because PI3K is strongly
linked to tyrosine kinase-mediated messenger pathways, the abolition of
swelling-induced pump stimulation by LY-294002 strongly supports the
conclusion derived from experiments using tyrphostin A25, i.e., that
pump stimulation is mediated by the activation of tyrosine kinase.
Role of serine/threonine protein phosphatase in
Ip stimulation.
Tyrosine kinase has been demonstrated to activate serine/threonine
protein phosphatase 1 (PP1) (6). PP1 and another protein phosphatase,
PP2A, may play a role in the regulation of the
Na+-K+
pump in the kidney (16). We examined the effect of okadaic acid, an
inhibitor of both PP1 and PP2A, on the stimulation of Ip by hyposmolar
solutions. In initial experiments we added 1 µM okadaic acid to the
patch pipette solution to completely inhibit both PP1 and PP2A (5). We
measured Ip in
isosmolar and hyposmolar superfusates. The mean
Ip in six
myocytes measured during exposure to isosmolar superfusate was 0.33 ± 0.03 pA/pF, whereas the mean Ip in eight
myocytes measured during exposure to hyposmolar superfusate was 0.37 ± 0.05 pA/pF. This difference was not statistically significant. This suggests that
Na+-K+
pump stimulation during exposure to hyposmolar solutions involves the
activation of PP1 and/or PP2A. To exclude a nonspecific effect of
okadaic acid on the pump, we repeated the experiments with the inactive
analog, okadaic acid methyl ester (1 µM) in pipette solutions. The mean Ip in seven myocytes
measured during exposure to isosmolar superfusate was 0.33 ± 0.02 pA/pF, whereas the mean Ip of seven
myocytes exposed to hyposmolar superfusates was 0.49 ± 0.03 pA/pF. The difference was statistically significant.
In multiple noncardiac tissues, 10 nM okadaic acid has been shown to
inhibit PP2A almost completely but to have no effect on PP1 (5). This
differential sensitivity of phosphatases to okadaic acid has also been
assumed to exist in cardiac myocytes (17). We used this presumed
differential sensitivity to examine which of the phosphatases might be
involved in the
Na+-K+
pump stimulation induced by exposure to hyposmolar solutions. We added
10 nM okadaic acid to patch pipette solutions and measured Ip in isosmolar
and hyposmolar solutions. The mean
Ip in eight myocytes measured during exposure to isosmolar solutions was 0.28 ± 0.03 pA/pF, whereas the mean
Ip in nine
myocytes measured during exposure to hyposmolar solutions was 0.51 ± 0.02 pA/pF. The difference was statistically significant. The results
of experiments performed with okadaic acid are summarized in Fig.
4. Figure 4 illustrates that
swelling-induced
Na+-K+
pump stimulation can be eliminated by the inhibition of protein phosphatases. The dependence of this inhibition on the concentration of
okadaic acid suggests that PP1 rather than PP2A is involved.

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Fig. 4.
Effect of serine/threonine phosphatase inhibition on swelling-induced
Ip stimulation.
Experiments were performed during exposure to isosmolar or hyposmolar
solution in presence of 1 µM or 10 nM okadaic acid. No. in
parentheses is no. of experiments in each group.
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Reversibility of swelling-induced pump stimulation.
We have previously found that changes in the intracellular
Na+ activity of intact papillary
muscles induced by exposure to hyposmolar superfusates are rapidly
reversible (32). We performed a series of experiments to examine if
this is reflected by reversible changes in the
Ip of
patch-clamped myocytes. After establishing the whole cell
configuration, we superfused isosmolar
Ca2+-free solution for 3 min. We
then switched to a hyposmolar
Ca2+-free superfusate to induce
cell swelling and pump stimulation. After 3 min we switched back to the
isosmolar superfusate for 5 min and measured
Ip. Figure
5 illustrates the changes in cell size
during the experimental protocol. Exposure to the hyposmolar superfusate induced a myocyte swelling that was completely reversible on switching back to the isosmolar superfusate for 5 min.
Ip was determined
after switching back to isosmolar superfusate at the end of the 5-min
period. To control for a possible time-dependent Na+-K+
pump run-down, control myocytes were superfused with isosmolar solution
for 11 min after the whole cell configuration was established before
Ip was
determined. The protocols for exposure to superfusates and measurement
of Ip are
illustrated in Fig. 6A.
In eight myocytes briefly exposed to hyposmolar solution, the mean
Ip
measured after switching back to isosmolar solution was 0.33 ± 0.02 pA/pF. The mean
Ip of nine
myocytes exposed to isosmolar solution only was 0.27 ± 0.03 pA/pF.
The difference was not statistically significant. These results suggest
that stimulation of
Ip by cell
swelling is rapidly reversible with restoration of extracellular
osmolarity and cell size.

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Fig. 5.
Reversibility of osmotically induced cell swelling in patch-clamped
cardiac myocyte. Shown is a photomicrograph of a patch-clamped myocyte
during sequence of exposure to isosmolar
(A) and hyposmolar solution
(B) and during reexposure to
isosmolar solution (C). Calibration
bars indicate myocyte width under basal isosmolar conditions.
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Fig. 6.
Reversibility of osmotically induced
Ip stimulation.
A: protocol used to examine role of
protein kinase C in reversing swelling-induced
Ip stimulation.
B: effect of staurosporine (Stauro) on
reversibility of swelling-induced
Ip stimulation.
Open bars and solid bars, results of protocol without and with
staurosporine, respectively, in patch pipette. Hypo, hyposmolar. No. in
parentheses is no. of experiments in each group.
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To explore the role of kinases in restoring
Ip to control
levels, we included 10 nM staurosporine in all pipette solutions. The
experimental protocols were otherwise identical to those illustrated in
Fig. 6A. The mean
Ip of eight
myocytes transiently exposed to the hyposmolar superfusate was 0.44 ± 0.03 pA/pF, whereas the mean
Ip of seven
myocytes exposed to isosmolar solution was 0.24 ± 0.02 pA/pF. The
difference was statistically significant. The experiments examining the
effect of transient swelling on
Ip are summarized
in Fig. 6B. Figure
6B illustrates that pump
stimulation is rapidly reversible and that the reversibility can be
blocked by a potent but nonspecific kinase inhibitor. A separate series of experiments performed with bisindolylmaleimide I in patch pipette solutions gave results similar to those with staurosporine, implicating PKC in the mechanism allowing the rapid reversal of
Ip on the reversal of cell swelling.
 |
DISCUSSION |
Role of transmembrane ion fluxes during myocyte swelling.
We have previously reported that
Ip in rabbit
cardiac myocytes is stimulated by exposure to hyposmolar superfusates
(32). This effect was observed when
[Na]pip was near the
physiological level of intracellular
Na+ concentration, whereas there
was no effect when
[Na]pip was high. We
concluded that the stimulation was due to an increase in the apparent
affinity of the pump for intracellular
Na+ rather than to an influx of
extracellular Na+ and secondary
pump stimulation. In the present study, we have directly ruled out any
effect of Na+ influx during
myocyte swelling by demonstrating that swelling activates
Ip even in the
absence of extracellular
Na+.
A study of patch-clamped guinea pig ventricular myocytes has found that
exposure to hyposmolar superfusates had no consistent effect on
Ip when
[Na]pip was near
physiological intracellular levels, whereas it increased
Ip when
[Na]pip was high (27). Because these findings were not compatible with our previously published results, we considered differences in experimental protocols that might account for the discrepancy. The
Ip of guinea pig
myocytes was measured by using superfusates that were nominally
Ca2+ free and by using pipette
solutions containing 10 mM EGTA to buffer cytosolic
Ca2+ (27), whereas we used
Ca2+-containing superfusates and 5 mM EGTA in pipette solutions in our earlier study (32). In the present
study we measured
Ip in nominally
Ca2+-free superfusates and we used
5 mM EGTA in pipette solutions. As in our previous study, exposure to
hyposmolar superfusates stimulated
Ip when
[Na]pip was low but
had no effect when
[Na]pip was high. We
conclude that transmembrane fluxes of
Ca2+ are not involved in the
mechanism activating the pumps of myocytes exposed to hyposmolar
solutions and that differences in extracellular Ca2+ and intracellular
Ca2+ buffering do not account for
the discrepancy between guinea pig and rabbit myocytes in the
[Na]pip dependence of
the pump response to swelling.
Site of effect of myocyte swelling on the
Na+-K+
pump.
Exposure of myocytes to hyposmolar solutions shifted the
Ip-Vm
relationship in a negative direction (Fig.
2B), suggesting that myocyte
swelling affects an electrogenic step in the pump cycle. Voltage
dependence is thought to arise, at least in part, from the interaction
of the transported ligands with the pump in access channels within the
electrical field of the membrane (see Ref. 22 for review). At
extracellular sites, the interaction of
Na+ and
K+ with the pump within access
channels contributes to the slope of the
Ip-Vm
relationship in a positive and a negative direction, respectively.
Experimentally, the voltage dependence of the pump arising from the
interaction of Na+ with
extracellular sites can be eliminated by using
Na+-free superfusates. Unless the
K+ concentration is very low, the
voltage dependence arising from the interaction of
K+ with extracellular sites is
also eliminated in such Na+-free
solutions (22). In the present study, swelling-induced pump stimulation
persisted when myocytes were superfused with Na+-free superfusates containing
5.6 mM K+, suggesting that it is
not one of the voltage-dependent steps at the extracellular sites that
is stimulated by cell swelling.
The voltage dependence of the
Na+-K+
pump cycle is also thought to arise at the intracellular sites for
Na+ binding. Two
Na+ are believed to bind in a
voltage-independent manner to sites near the cytoplasmic surface. At
these sites, Na+ competes with
K+ for binding. Voltage dependence
appears to arise from the highly selective binding of a third
Na+ in an access channel within
the membrane dielectric. At positive or negative membrane voltages, the
concentration of Na+ within the
channel is expected to increase or decrease, respectively. These
changes in Na+ concentrations will
cause an increase in
Ip, with a shift
of Vm in a
positive direction. It is conceivable that cell swelling alters the
depth of the Na+ access channel. A
decrease in depth might account for the shift in the
Ip-Vm
relationship in the negative direction, as shown in Fig. 2. The absence
of an effect of cell swelling on
Ip when [Na]pip was 80 mM
supports this speculation because the binding of
Na+ to intracellular sites is
expected to be independent of access channel depth when intracellular
Na+ concentration is
high (22).
Messenger cascade linking cell volume to pump activity.
We examined the effect of including staurosporine and
bisindolylmaleimide I in the pipette solution because a previous study had indicated that the mechanical stretch of cardiac myocytes activates
PKC (24). The drugs did not abolish the increase in Ip induced by
cell swelling. This might be taken to indicate that PKC does not
regulate sarcolemmal
Na+-K+
pump activity. However, it should be noted that total PKC activity, rather than the activity of specific isoforms, was measured in the
study of the effect of mechanical stretch of myocytes (24). Because the
effects of PKC are believed to be isoform specific (see Ref. 18 for
review), it is reasonable to think that the isoforms activated by
stretch have no effect on
Ip. Previous
studies have indicated that PKC is activated during osmotically induced cell shrinkage (29). We found that staurosporine and
bisindolylmaleimide I prevented the return of
Ip to control
values after reexposure of the osmotically swollen myocyte to the
isosmolar solution (Fig. 6B). These
findings suggest that the PKC isoform(s) activated by cell shrinkage
regulates the
Na+-K+
pump in cardiac tissue.
Tyrosine kinase has been demonstrated to be activated within seconds
when cardiac myocytes and intestinal epithelial cells are exposed to
osmotic swelling and mechanical stretch (24, 30). Such activation has
been implicated as an essential step in the activation of
volume-sensitive ion conductances and membrane transport pathways (28,
30). Because tyrphostin A25 and LY-294002 abolished pump stimulation
induced by cell swelling in the present study, a messenger pathway
involving tyrosine kinase is implicated. Tyrosine kinase-dependent
pathways have been reported to stimulate the pump in previous studies
(8, 21). Activation of receptor tyrosine kinase by insulin and
epidermal growth factor thus stimulates Na+-K+-ATPase
activity in rat proximal convoluted tubules (8) and in cultured rat
skeletal muscle cells (21). As in the present study, stimulation only
occurred at low intracellular Na+
levels and had no effect at high
Na+ levels. Finally, in support of
the role of tyrosine kinase in pump activation by cell swelling, it
should be noted that the insulin exposure of cardiac myocytes has an
effect on the
Ip-Vm relationship (13) virtually identical to the effect of swelling in the
present study (Fig. 2B).
The differential effect of 10 nM and 1 µM okadaic acid suggests that
PP1 rather than PP2A is involved in the messenger cascade linking cell
swelling to
Na+-K+
pump stimulation. Similar findings for the activation of the pump in
cultured rat skeletal muscle by insulin have been reported (21). PP1
induced the dephosphorylation of pump molecules, and it was suggested
that this dephosphorylation caused pump activation. It is reasonable to
think that a similar mechanism is involved in the activation of the
pump by cell swelling in the present study. However, this cannot be
firmly concluded because we did not directly measure the
phosphorylation of pump molecules. Figure 6 indicates that pump
stimulation develops within 3 min of the onset of swelling. If this
stimulation is mediated by dephosphorylation, we can conclude that
messenger mechanisms involving the activation of tyrosine kinase, PI3K,
and protein phosphatase in intact cardiac myocytes allow the
dephosphorylation of pump molecules within this time span. Our model,
however, does not allow a detailed kinetic analysis of the presumed
pump dephosphorylation.
Swelling-induced pump stimulation was rapidly reversible, as indicated
by Fig. 6. The reversal was blocked by kinase inhibition. Previous
studies have indicated that isolated
Na+-K+-ATPase
can be directly phosphorylated by protein kinase A and PKC (1, 9).
Direct phosphorylation of
Na+-K+
pump molecules by a protein kinase would offer the simplest explanation for the reversal of pump stimulation on the return to a normal cell
volume, although we cannot rule out the possibility that the
phosphorylation of intermediate pump-regulatory proteins is involved.
Taken together, the effect of the sequential exposure of cardiac
myocytes to solutions of different osmolarities, in combination with
the use of inhibitors of phosphatases and kinases, supports the
suggestion that
Na+-K+
pump activity in intact cells can be rapidly regulated by a balance between the effects of kinases and phosphatases on the pump molecules. The experimental model developed in the present study offers a convenient tool for the study of these processes.
 |
ACKNOWLEDGEMENTS |
This study was supported by a grant from the North Shore Heart
Research Foundation.
 |
FOOTNOTES |
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: D. Whalley, Cardiology Dept., Royal North Shore Hospital,
Pacific Highway, St. Leonards, NSW 2065, Australia.
Received 13 August 1998; accepted in final form 1 February 1999.
 |
REFERENCES |
1.
Beguin, P.,
A. T. Beggah,
A. V. Chibalin,
P. Burgener-Kairuz,
F. Jaisser,
P. M. Mathews,
B. C. Rossier,
S. Cotecchia,
and
K. Geering.
Phosphorylation of the Na,K-ATPase
-subunit by protein kinase A and C in vitro and in intact cells.
J. Biol. Chem.
269:
24437-24445,
1994[Abstract/Free Full Text].
2.
Bertorello, A. M.,
and
A. I. Katz.
Short-term regulation of renal Na-K-ATPase activity: physiological relevance and cellular mechanisms.
Am. J. Physiol.
265 (Renal Fluid Electrolyte Physiol. 34):
F743-F755,
1993[Abstract/Free Full Text].
3.
Bustamante, J. O., A. Ruknudin, and F. Sachs.
Stretch-activated channels in heart cells: relevance to cardiac
hypertrophy. J. Cardiovasc. Pharmacol.
17 Suppl. 2: S110-S113, 1991.
4.
Cantley, L. C.,
K. R. Auger,
C. Carpenter,
B. Duckworth,
A. Graziani,
R. Kapeller,
and
S. Soltoff.
Oncogenes and signal transduction.
Cell
64:
281-302,
1991[Medline].
5.
Cohen, P.,
S. Klumpp,
and
D. L. Schelling.
An improved procedure for identifying and quantitating protein phosphatases in mammalian tissues.
FEBS Lett.
250:
596-600,
1989[Medline].
6.
Dent, P.,
A. Lavoinne,
S. Nakielny,
F. B. Caudwell,
P. Watt,
and
P. Cohen.
The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle.
Nature
348:
302-307,
1990[Medline].
7.
Ewart, H. S.,
and
A. Klip.
Hormonal regulation of the Na+-K+-ATPase: mechanisms underlying rapid and sustained changes in pump activity.
Am. J. Physiol.
269 (Cell Physiol. 38):
C295-C311,
1995[Abstract/Free Full Text].
8.
Féraille, E.,
M. L. Carranza,
M. Rousselot,
and
H. Favre.
Modulation of Na+,K+-ATPase activity by a tyrosine phosphorylation process in rat proximal convoluted tubule.
J. Physiol. (Lond.)
498:
99-108,
1997[Abstract].
9.
Feschenko, M. S.,
and
K. J. Sweadner.
Conformation-dependent phosphorylation of Na,K-ATPase by protein kinase A and protein kinase C.
J. Biol. Chem.
269:
30436-30444,
1994[Abstract/Free Full Text].
10.
Gray, D. F.,
P. S. Hansen,
M. M. Doohan,
L. C. Hool,
and
H. H. Rasmussen.
Dietary cholesterol affects Na+-K+ pump function in rabbit cardiac myocytes.
Am. J. Physiol.
272 (Heart Circ. Physiol. 41):
H1680-H1689,
1997[Abstract/Free Full Text].
11.
Gray, D. F.,
A. S. Mihailidou,
P. S. Hansen,
K. A. Buhagiar,
N. L. Bewick,
H. H. Rasmussen,
and
D. W. Whalley.
Amiodarone inhibits the Na+-K+ pump in rabbit cardiac myocytes after acute and chronic treatment.
J. Pharmacol. Exp. Ther.
284:
75-82,
1998[Abstract/Free Full Text].
12.
Hagiwara, N.,
H. Masuda,
M. Shoda,
and
H. Irisawa.
Stretch-activated anion currents of rabbit cardiac myocytes.
J. Physiol. (Lond.)
456:
285-302,
1992[Abstract].
13.
Hansen, P. S.,
D. F. Gray,
and
H. H. Rasmussen.
Insulin regulates the voltage dependence of the sarcolemmal Na+-K+ pump (Abstract).
Circulation
92:
I-638,
1995.
14.
Hemsworth, P. D.,
D. W. Whalley,
and
H. H. Rasmussen.
Electrogenic Li+/Li+ exchange mediated by the Na+-K+ pump in rabbit cardiac myocytes.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1186-C1192,
1997[Abstract/Free Full Text].
15.
Kelly, K. L.,
and
N. B. Ruderman.
Insulin-stimulated phosphatidylinositol 3-kinase.
J. Biol. Chem.
268:
4391-4398,
1993[Abstract/Free Full Text].
16.
Li, D.,
A. Aperia,
G. Celsi,
E. F. da Cruz e Silva,
P. Greengard,
and
B. Meister.
Protein phosphatase-1 in the kidney: evidence for a role in the regulation of medullary Na+-K+-ATPase.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F673-F680,
1995[Abstract/Free Full Text].
17.
Light, P. E.,
B. G. Allen,
M. P. Walsh,
and
R. J. French.
Regulation of adenosine triphosphatase-sensitive potassium channels from rabbit ventricular myocytes by protein kinase C and type 2A protein phophatase.
Biochemistry
34:
7252-7257,
1995[Medline].
18.
Mochly-Rosen, D.,
and
L. M. Kauvari.
Modulating protein kinase C signal transduction.
Adv. Pharmacol.
44:
91-145,
1998[Medline].
19.
Nakao, M.,
and
D. C. Gadsby.
[Na] and [K] dependence of the Na/K pump current-voltage relationship in guinea pig ventricular myocytes.
J. Gen. Physiol.
94:
539-565,
1989[Abstract].
20.
Neher, E.
Correction for liquid junction potentials in patch clamp experiments.
Methods Enzymol.
207:
123-131,
1992[Medline].
21.
Ragolia, L.,
B. Cherpalis,
M. Srinivasan,
and
N. Begum.
Role of serine/threonine protein phosphatases in insulin regulation of the Na+/K+-ATPase activity in cultured rat skeletal muscle cells.
J. Biol. Chem.
272:
23653-23658,
1997[Abstract/Free Full Text].
22.
Rakowski, R. F.,
D. C. Gadsby,
and
P. De Weer.
Voltage dependence of the Na/K pump.
J. Membrane Biol.
155:
105-112,
1997[Medline].
23.
Ruknudin, A.,
F. Sachs,
and
J. O. Bustamante.
Stretch-activated ion channels in tissue-cultured chick heart.
Am. J. Physiol.
264 (Heart Circ. Physiol. 33):
H960-H972,
1993[Abstract/Free Full Text].
24.
Sadoshima, J.,
and
S. Izumo.
Mechanical stretch rapidly activates multiple signal transduction pathways in cardiac myocytes: potential involvement of an autocrine/paracrine mechanism.
EMBO J.
12:
1681-1692,
1993[Abstract].
25.
Sadoshima, J.,
and
S. Izumo.
The cellular and molecular response of cardiac myocytes to mechanical stress.
Annu. Rev. Physiol.
59:
551-571,
1997[Medline].
26.
Sadoshima, J.,
Z. Qui,
J. P. Morgan,
and
S. Izumo.
Tyrosine kinase activation is an immediate and essential step in hypotonic cell swelling-induced ERK activation and c-fos gene expression in cardiac myocytes.
EMBO J.
15:
5535-5546,
1996[Abstract].
27.
Sasaki, N.,
T. Mitsuiye,
Z. Wang,
and
A. Noma.
Increase of the delayed rectifier K+ and Na+-K+ pump currents by hypotonic solutions in guinea pig cardiac myocytes.
Circ. Res.
75:
887-895,
1994[Abstract].
28.
Sorota, S.
Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current.
Pflügers Arch.
431:
178-185,
1995[Medline].
29.
Terada, Y.,
K. Tomita,
M. K. Homma,
H. Nonoguchi,
T. Yang,
T. Yamada,
Y. Yuasa,
E. G. Krebs,
S. Sasaki,
and
F. Marumo.
Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells.
J. Biol. Chem.
269:
31296-31301,
1994[Abstract/Free Full Text].
30.
Tilly, B. C.,
N. van den Berghe,
L. G. J. Tertoolen,
M. J. Edixhoven,
and
H. R. de Jonge.
Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances.
J. Biol. Chem.
268:
19919-19922,
1993[Abstract/Free Full Text].
31.
Vlahos, C. J.,
W. F. Matter,
R. F. Brown,
A. E. Traynor-Kaplan,
P. G. Heyworth,
E. R. Prossnitz,
R. D. Ye,
P. Marder,
J. A. Schelm,
K. J. Rothfuss,
B. S. Serlin,
and
P. J. Simpson.
Investigation of neutrophil signal transduction using a specific inhibitor of phosphatidylinositol 3-kinase.
J. Immunol.
154:
2413-2422,
1995[Abstract/Free Full Text].
32.
Whalley, D. W.,
L. C. Hool,
R. E. Ten Eick,
and
H. H. Rasmussen.
Effect of osmotic swelling and shrinkage on Na+-K+ pump activity in mammalian cardiac myocytes.
Am. J. Physiol.
265 (Cell Physiol. 34):
C1201-C1210,
1993[Abstract/Free Full Text].
33.
Yingst, D. R.
Modulation of the Na,K-ATPase by Ca and intracellular proteins.
Annu. Rev. Physiol.
50:
291-303,
1988[Medline].
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