Voltage-dependent stimulation of the
Na+-K+ pump by insulin in rabbit cardiac
myocytes
Peter S.
Hansen,
Kerrie A.
Buhagiar,
David F.
Gray, and
Helge H.
Rasmussen
Department of Cardiology, Royal North Shore Hospital, and Department
of Medicine, University of Sydney, St. Leonards, New South Wales
2065, Australia
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ABSTRACT |
Insulin enhances
Na+-K+ pump activity in various noncardiac
tissues. We examined whether insulin exposure in vitro regulates Na+-K+ pump function in rabbit ventricular
myocytes. Pump current (Ip) was measured using the
whole-cell patch-clamp technique at test potentials
(Vms) from
100 to +60 mV. When the
Na+ concentration in the patch pipette
([Na]pip) was 10 mM, insulin caused a
Vm-dependent increase in Ip.
The increase was ~70% when Vm was at near
physiological diastolic potentials. This effect persisted after
elimination of extracellular voltage-dependent steps and when
K+ and K+-congeners were excluded from the
patch pipettes. When [Na]pip was 80 mM, causing
near-maximal pump stimulation, insulin had no effect, suggesting that
it did not cause an increase in membrane pump density. Effects of
tyrphostin A25, wortmannin, okadaic acid, or bisindolylmaleimide I in
pipette solutions suggested that the insulin-induced increase in
Ip involved activation of tyrosine kinase,
phosphatidylinositol 3-kinase, and protein phosphatase 1, whereas
protein phosphatase 2A and protein kinase C were not involved.
sodium-potassium-adenosinetriphosphatase; whole-cell voltage clamp; second messengers
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INTRODUCTION |
STUDIES ON RAT ADIPOCYTES and isolated renal proximal
convoluted tubule indicate that insulin stimulates the membrane
Na+-K+ pump by increasing its sensitivity to
intracellular Na+ (Nai), although there is no
effect on maximal pump rate (5, 14, 16). Binding of Nai to
the pump occurs at two relatively nonselective negatively charged sites
at the cytoplasmic surface and at a third, highly selective, uncharged
site located some distance inside the electrical field of the membrane
(20, 21). Because Na+ competes with K+ for
binding to the sites at the cytoplasmic surface, insulin might increase
the overall sensitivity of the Na+-K+ pump to
Na+ by increasing the Na+/K+
selectivity ratio. Detection of this should be dependent on
intracellular K+ (Ki) and independent of
membrane voltage. In contrast, because binding of Na+ to
the third pump site is highly selective for Na+, an effect
of insulin at these sites should be independent of Ki.
However, because binding occurs within the membrane dielectric, such an
effect should depend on membrane voltage.
The techniques used in the previous studies in adipocytes and renal
tubule cells (5, 14, 16) do not allow selective experimental control of
Ki and membrane voltage, and no information is available
regarding the Ki and voltage dependence of the effect of
insulin on the pump in these cells. We have used the whole-cell patch-clamp technique to study electrogenic
Na+-K+ pump current (Ip) in
single isolated cardiac myocytes. When wide-tipped patch pipettes are
used, cardiac myocytes are small enough to allow dialysis of the
intracellular compartment and accurate control of membrane voltage.
However, because of a high pump density of cardiac myocytes, the
Ip is large enough to be identified with high
resolution (7). We found that insulin increases Ip
when pipette solutions contain Na+ in a concentration near
physiological intracellular levels. This effect is dependent on
membrane voltage but is independent of Ki. Pump stimulation
was abolished by including pharmacological blockers of the insulin
tyrosine kinase receptor, phosphatidylinositol 3-kinase (PI 3-kinase)
and serine/threonine protein phosphatase 1 (PP-1) in the patch pipette solution.
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MATERIALS AND METHODS |
Preparation of single ventricular myocytes.
Single ventricular myocytes were isolated from male New Zealand White
rabbits as described previously (10). After isolation they were
maintained at room temperature and used on the day of isolation only.
Pump currents were always measured within 10 h of excising the heart.
Solutions.
After isolation, myocytes were stored in Krebs-Henseleit buffer (KHB)
solution containing the following (in mM): 130 NaCl, 4.8 KCl, 1.2 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, 12.5 glucose, 0.5 CaCl2, and 1.0% BSA.
The solution was bubbled with 5% CO2-95% O2
for 1 h before use to achieve a pH of 7.40 ± 0.05 at 35°C. Myocytes were transferred to a 350-µl tissue bath for patch-clamp studies. The bath was perfused at a rate of 5 ml/min with modified Tyrode solution warmed to 35°C. The solution contained the
following (in mM): 140 NaCl, 5.6 KCl, 2.16 CaCl2, 0.44 NaH2PO4, 10 glucose, 1 MgCl2, and
10 HEPES. It was titrated with 1 M NaOH to a pH of 7.40 ± 0.01 at
35°C. This solution was used in all experiments until the
whole-cell configuration was established and membrane capacitance had
been measured. For measurement of Ip we switched to
a solution that was identical to the solution used while the whole-cell
configuration was established, except that it was nominally Ca2+-free and contained 0.2 mM CdCl2 to limit
Na+-Ca2+ exchange. It also contained 2 mM
BaCl2 to reduce membrane K+ conductance.
Additional variations in the composition of superfusates are indicated
in RESULTS.
Myocytes were voltage clamped with wide-tipped patch pipettes (4-5
µm) made as described previously (33). For the measurement of
Ip at a fixed holding potential of
40 mV we
filled pipettes with a solution containing the following (in mM): 70 potassium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA,
2 MgATP, and 90 sodium glutamate plus tetramethylammonium chloride
(TMA-Cl). The solution was titrated with 1 M KOH to pH 7.05 ± 0.01 at
35°C. In experiments designed to examine the relationship between
Ip and test potential (Vm) we
blocked time-dependent K+ currents by including
tetraethylammonium chloride (TEA-Cl) in pipette solutions and
replacing potassium glutamate with CsCl. The solution contained the
following (in mM): 10 sodium glutamate, 1 KH2PO4, 5 HEPES, 5 EGTA, 2 MgATP, 60 TMA-Cl, 20 TEA-Cl, 70 CsCl, and 50 aspartic acid. The solution was titrated with 1 M HCl to a pH of 7.05 ± 0.01 at 35°C. To examine the
Ip-Vm relationship with a high
pipette Na+ concentration
([Na]pip), the solution contained 80 mM sodium glutamate, 65 mM CsCl, and 45 mM aspartic acid. We omitted TMA-Cl in
this solution to maintain osmotic balance. To examine the
Ip-Vm relationship using
K+- and K+-congener (Cs+)-free
pipettes we eliminated K+- and Cs+-containing
compounds. Osmotic balance was maintained by including TMA-Cl.
Insulin exposure protocols.
In initial studies myocytes were exposed to insulin in the tissue bath
both before and after we achieved the whole-cell configuration. The
duration of this exposure was 15-40 min. Ca2+ was
included in the superfusate during the initial period of exposure to
insulin. We used insulin in a nominal concentration of 100 mU/ml.
Because it is expected to adhere to glass we measured the concentration
in the perfusion chamber. It was 20 ± 3 mU/ml (n = 12), a
concentration reported to cause near-maximal pump stimulation (31). A
second protocol was developed to examine the role of Ca2+
in insulin-induced pump stimulation and to allow blockade of intracellular messengers by drugs included in pipette solutions before
cells were exposed to insulin. In these experiments insulin was only
superfused for the period between establishment of the whole-cell
configuration and measurement of Ip (10-12
min). The superfusate was Ca2+-free during that period.
Measurement of Ip.
Ip was identified as the shift in holding current
induced by 100 µM ouabain, a concentration known to completely block
the Na+-K+ pump in rabbit ventricular myocytes
(10). Membrane currents were recorded through the use of the continuous
single-electrode voltage-clamp mode of an Axoclamp-2A amplifier and
Axotape and pCLAMP software (Axon Instruments, Foster City, CA).
Voltage-clamp protocols were generated with pCLAMP. Details of the
experimental protocols used to measure membrane capacitance and
Ip, and details of the electronic recording system,
have been described (10, 33). Ip is reported
normalized for membrane capacitance unless specified otherwise.
Drugs and chemicals.
"Purum" grade TMA-Cl was purchased from Fluka (Switzerland). All
other chemicals were analytical grade and purchased from Sigma (St.
Louis, MO). Tyrphostin A25, tyrphostin A63, wortmannin, bisindolylmaleimide I, sodium salt okadaic acid, and methyl ester okadaic acid were purchased from Calbiochem (La Jolla, CA). Sodium salt
okadaic acid was dissolved in water. All other second-messenger inhibitors and their inactive controls were dissolved in
0.01-0.04% DMSO. Ouabain was purchased from Sigma.
Statistical analysis.
Results are expressed as mean ± SE. Student's t-test for
paired and unpaired data was used. Dunnett's test was used when the same control group was used for more than one comparison. P < 0.05 is regarded as significant in all comparisons. Slopes of Ip-Vm relationships were
compared using linear regression. Nonlinear regression was used to fit
the Hill equation to data.
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RESULTS |
Effect of insulin on Ip.
In an initial series of experiments we examined the effect on
Ip when myocytes were exposed to 100 mU/ml insulin
after they were transferred to the tissue bath. Insulin was included in
the Ca2+-containing superfusate we used in all experiments
in this study while we sought to establish the whole-cell
configuration, and in the Ba2+-containing solution we used
at the time Ip was measured. The latter solution
was Ca2+-free and contained Cd2+. The duration
of exposure to insulin before Ip was measured
depended on the time required to achieve the whole-cell configuration. The range of exposure was 15-40 min. To reduce variability in Ip arising if rundown of pump activity were to
occur, we always exposed myocytes to ouabain with the same latency of
10-12 min after the whole-cell configuration had been established.
Myocytes were voltage clamped at
40 mV during this period and
during the subsequent exposure to ouabain. We have previously published
representative recordings of ouabain-induced shifts in holding currents
(33). Mean Ip, measured using a
[Na]pip of 10 mM, was 0.33 ± 0.01 pA/pF in 6 control myocytes and 0.43 ± 0.01 pA/pF in 13 myocytes exposed to
insulin. The difference was statistically significant.
To examine whether insulin can stimulate the pump in a patch-clamped
myocyte we included insulin only in the Ca2+-free,
Ba2+- and Cd2+-containing superfusates used
after the whole-cell configuration had been established. In these
experiments myocytes were exposed to insulin for ~10-12 min.
Control myocytes not exposed to insulin were maintained in the
whole-cell configuration and superfused with Ca2+-free,
Ba2+- and Cd2+-containing solution for the same
period before Ip was measured. Mean
Ip, measured using a
[Na]pip of 10 mM, was 0.31 ± 0.01 pA/pF in
six control myocytes and 0.41 ± 0.03 pA/pF in six myocytes exposed to insulin. The difference was statistically significant. This
indicates that the messenger pathways linking the insulin receptor to
the Na+-K+ pump can be activated within
10-12 min and that the pathways are functional despite the nominal
absence of extracellular Ca2+ and buffering of cytosolic
Ca2+ by the EGTA contained in pipette filling solutions.
Effect of insulin on voltage dependence of the
Na+-K+
pump.
To examine whether insulin affects the voltage dependence of
Ip, we patch-clamped myocytes with the use of a
[Na]pip of 10 mM. Pipette filling solutions
included CsCl and TEA-Cl in these experiments. After we achieved the
whole-cell configuration myocytes were superfused for 10-12 min
with Ca2+-free solution that contained insulin or was
insulin-free. The myocytes were voltage clamped at
40 mV during
this time. We then applied voltage steps of 320-ms duration in 20-mV
increments to test potentials ranging from
100 to +60 mV. Each
test potential was bracketed by a return to the
40-mV holding
potential for 2 s. The voltage-clamp protocol was applied three times
before and three times after myocytes were exposed to 100 µM ouabain and averaged steady-state holding currents were determined. Figure 1A shows an example of currents
recorded in a myocyte exposed to insulin. The criteria for identifying
ouabain-induced shifts in currents at each test potential have been
described previously (9). The
Ip-Vm relationships for
myocytes exposed to insulin and for control myocytes have been
summarized in Fig. 1B. The Ip-Vm relationships for both
groups of cells were nearly linear throughout the voltage range
examined. Control myocytes had a clearly positive slope of the
Ip-Vm relationship, whereas the slope appeared much less steep for myocytes exposed to insulin. The
relationships were compared by linear regression. There was a
statistically significant difference between their slopes. We conclude
that insulin stimulates the Na+-K+ pump and
reduces its voltage dependence when [Na]pip is
10 mM.

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Fig. 1.
A: holding currents pre- and post-ouabain exposure of a myocyte
superfused with insulin-containing superfusates for ~10 min when
Na+ concentration in the pipette
([Na]pip) was 10 mM. The ouabain-induced shift
in currents was used to identify pump current (Ip)
at each test potential (Vm). B: effect of
insulin on Vm dependence of absolute
Ip using [Na]pip of 10 mM.
Ip-Vm relationships are
averaged for 16 myocytes (5 rabbits) exposed to insulin-free
superfusates (open circles) and 14 myocytes (5 rabbits) exposed to
insulin-containing superfusates (filled circles).
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We next examined whether insulin can be shown to stimulate
Ip when [Na]pip is high.
Pipette filling solutions were identical to those used in the
experiments shown in Fig. 1 except that [Na]pip was increased to 80 mM, whereas the concentration of TMA-Cl was decreased by 60 mM and CsCl and aspartic acid by 5 mM each to maintain
osmotic balance. The Ip-Vm
relationships for myocytes exposed to insulin and for control myocytes
have been summarized in Fig. 2. The mean
levels of Ip for the two groups of cells were similar at all test potentials. This indicates that the
voltage-dependent effect of insulin on Ip is
eliminated when intracellular Na+ is at levels expected to
nearly saturate the pump's binding sites.

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Fig. 2.
Effect of insulin on Vm dependence of absolute
Ip using [Na]pip of 80 mM.
Ip-Vm relationships are
averaged for 8 myocytes (3 rabbits) exposed to insulin-free
superfusates (open circles) and 7 myocytes (3 rabbits) exposed to
insulin-containing superfusates (filled circles). Comparison with Fig.
1A indicates that increase in [Na]pip
from 10 to 80 mM caused an approximately four- to sevenfold increase in
mean Ip in control myocytes over the voltage range
examined. There was no effect of insulin on Ip.
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The interaction of Na+ and K+ with
extracellular pump sites is the major contributor to the voltage
dependence of Ip. This voltage dependence is
eliminated if the Na+ concentration in the superfusate is
low and the K+ concentration ([K]o)
is maintained at or above physiological levels (see Ref. 25 for
review). To examine whether the effect of insulin on the pump's
voltage dependence is due to an effect at extracellular sites we
determined the Ip-Vm
relationship of myocytes through the use of a superfusate that was
Ca2+-free and contained 1.5 mM Na+, 140 mM
N-methyl-D-glucamine chloride (NMGC), 5.6 mM
K+, and 0.2 mM Cd2+. The composition of the
superfusate was adopted from a previous study on the voltage dependence
of the Na+-K+ pump in cardiac myocytes (18).
The pipette filling solution included 10 mM Na+ and 71 mM
Cs+. Figure 3 shows the
Ip-Vm relationship of control
myocytes and myocytes exposed to insulin. Insulin stimulated
Ip at negative test potentials, whereas it had no
effect at positive potentials. The
Ip-Vm relationships were
compared by linear regression. There was a statistically significant
difference between their slopes. We also normalized the
Ip-Vm relationships in Fig. 3
to the Ip recorded at 0 mV (not shown) and compared
the normalized relationships. There was a significant difference
between their slopes. We conclude that a functional change in the
interaction of Na+ and K+ with extracellular
sites does not account for the effect of insulin on the pump's voltage
dependence.

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Fig. 3.
Effect of insulin on Vm dependence of
Ip when extracellular Na+ concentration
([Na]o) was 1.5 mM, extracellular
K+ concentration ([K]o) 5.6 mM, and
[Na]pip 10 mM.
Ip-Vm relationships are
averaged for 10 myocytes (3 rabbits) exposed to insulin-free
superfusates (open circles) and 15 myocytes (3 rabbits) exposed to
insulin-containing superfusates (filled circles).
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Ki and effect of insulin on Ip.
To examine the effect of Ki on the insulin-induced pump
stimulation we used K+- and K+-congener
(Cs+)-free pipette filling solutions. We maintained osmotic
balance by replacing CsCl with TMA-Cl. The solution contained 10 mM
Na+ and 20 mM TEA-Cl. The
Ip-Vm relationship was
determined in a Ca2+-free superfusate in which NaCl had
been replaced with NMGC. [K]o was 15 mM. Figure
4 shows the
Ip-Vm relationships for control myocytes and myocytes exposed to insulin. Insulin significantly stimulated Ip at negative test potentials and
reduced the slope of the Ip-Vm
relationship. The slope of the normalized
Ip-Vm relationship (not shown)
was also significantly altered by insulin.

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Fig. 4.
Effect of insulin on Vm dependence of
Ip when pipette solution was K+- and
K+-congener (Cs+)-free and contained 10 mM
Na+. Superfusate composition eliminated an effect from
extracellular voltage-dependent steps of the pump cycle.
Ip-Vm relationships are
averaged for 11 myocytes (3 rabbits) exposed to insulin-free
superfusates (open circles) and 10 myocytes (3 rabbits) exposed to
insulin-containing superfusates (filled circles).
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Effect of insulin on the apparent
K+ affinity.
Because in vivo insulin deficiency is reported to affect the
K+ affinity of the Na+-K+ pump
(19), we next examined whether exposure of myocytes to insulin in vitro
has an effect on the pump's sensitivity to extracellular K+. We used a [Na]pip of 80 rather
than 10 mM in these experiments for two reasons. Insulin increases the
turnover rate of the Na+-K+ pump when
[Na]pip is 10 mM (Fig. 1B). The
dependence of the pump's apparent K+ affinity on turnover
rate (11) is therefore expected to induce a change in affinity even if
insulin has no effect on the pump's interaction with extracellular
K+. Use of a [Na]pip of 80 mM was
also expected to facilitate detection of small pump currents when the
[K]o was low.
After obtaining the whole-cell configuration we voltage clamped
myocytes at
40 mV and switched to a superfusate that was Ca2+ free and contained 2 mM Ba2+, 0.2 mM
Cd2+, and 5.6 mM K+. We maintained the myocyte
in this solution for 10 min. If the holding currents were stable we
then switched to a K+-free superfusate to inactivate the
Na+-K+ pump. Ip was
subsequently identified as the shift in holding current induced by
reexposure to K+. We have previously shown that such a
K+-induced shift in holding current is free from
contamination by non-pump K+-sensitive currents (9). Each
myocyte was exposed in random order to Ca2+-free
superfusates with different K+ concentrations ranging from
0.5 to 15 mM. Each exposure to K+ was bracketed by exposure
to the K+-free superfusate to ensure a return to the
baseline holding current. To detect rundown of Ip
we exposed eight randomly selected myocytes to the first
[K]o used in the protocol and measured the
K+-induced shift in holding current. There was no evidence
for rundown in the time needed to complete the experimental protocol
(25-32 min). An illustration of the experimental protocol and
representative traces of holding currents have been published
previously (9). We included insulin in all Ca2+-free
superfusates used after the whole-cell configuration was established.
The relationship between [K]o and
Ip is shown in Fig. 5.
We fitted the Hill equation to the data as described previously (9).
The [K]o for half-maximal pump activation was
2.3 mM for control myocytes and 2.7 mM for myocytes exposed to insulin,
whereas Hill coefficients were 1.17 and 1.28, respectively. There were no significant differences between these.

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Fig. 5.
Effect of [K]o on Ip in 12 myocytes (4 rabbits) exposed to insulin-free superfusates (open
circles) or 7 myocytes (3 rabbits) exposed to insulin-containing
superfusates (filled circles). Ip at each
[K]o has been normalized to the current
recorded when [K]o was 5.6 mM rather than to
cell capacitance. This eliminates variability of data arising from cell
capacitance determination.
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Messenger pathway for the effect of insulin on Ip.
Because all cellular effects of insulin are mediated by the insulin
receptor tyrosine kinase (1), we first examined the effect of tyrosine
kinase inhibition. We measured Ip at a fixed holding potential of
40 mV through the use of a
Ca2+-free superfusate that included 140 mM Na+
and 5.6 mM K+. Pipette filling solutions included 10 mM
Na+ and 71 mM K+. We inhibited tyrosine kinase
by including 100 µM tyrphostin A25 in pipette filling solutions.
Figure 6 summarizes mean
Ip measured in myocytes through the use of drug-
and solvent-free control filling solutions containing tyrphostin A25,
its inactive analog tyrphostin A63, or DMSO used only to dissolve the
drugs. Myocytes were either exposed or not to insulin. To allow time for dialysis of the compounds into the intracellular compartment we did
not expose myocytes to insulin until the whole-cell configuration had
been established for ~3-5 min. Figure 6 shows that DMSO and tyrphostin A63 had no effect on mean Ip, whereas
tyrphostin A25 caused a small but statistically significant decrease in
mean Ip compared with mean Ip
of other cells not exposed to insulin. Tyrphostin A25 completely
abolished the insulin-induced increase in mean Ip
while this increase persisted in the presence of the inactive analog,
tyrphostin A63.

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Fig. 6.
Effect of tyrosine kinase inhibition on response of
Ip to insulin. Mean Ip from
myocytes exposed to drug-free, tyrphostin A25- (100 µM), tyrphostin
A63- (100 µM), or DMSO-containing (0.04%) pipette solutions and
insulin-free superfusates are compared with mean Ip
from myocytes exposed to drug-free, tyrphostin A25- (100 µM) or
tyrphostin A63-containing (100 µM) pipette solutions and
insulin-containing superfusate (con, control; ins, insulin). Numbers in
parentheses indicate number of myocytes in each group. Experiments were
obtained from three or more rabbits in each group.
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Because the insulin receptor tyrosine kinase activates PI 3-kinase we
next examined the effect of inhibition of PI 3-kinase on the
insulin-induced increase in Ip. We included 1 µM
wortmannin in patch pipette solutions and measured
Ip in the presence or absence of insulin. The
results are summarized in Fig. 7. Mean Ip of myocytes exposed to wortmannin was
significantly lower than mean Ip of myocytes
voltage clamped using wortmannin-free pipette solutions and wortmannin
completely abolished the insulin-induced increase in mean
Ip. The results summarized in Figs. 6 and 7 suggest that the increase in Ip induced by insulin is a
specific insulin receptor-mediated effect.

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Fig. 7.
Effect of phosphatidylinositol 3-kinase inhibition on response of
Ip to insulin. Mean Ips from
myocytes exposed to drug-free or wortmannin-containing (wort, 1 µM)
pipette solutions in insulin-free superfusates are compared with mean
Ip from myocytes exposed to identical pipette
solutions and insulin-containing superfusate (con, control; ins,
insulin). Numbers in parentheses indicate number of myocytes in each
group. Experiments were obtained from three rabbits in each group.
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Because both protein kinase C (PKC) and serine/threonine phosphatases
have been reported to mediate cellular effects of insulin, we also
examined the effect of the PKC inhibitor bisindolylmaleimide I and the
phosphatase inhibitor okadaic acid. In one series of experiments we
included 1 µM bisindolylmaleimide I in pipette solutions. As shown in
Fig. 8 this had no effect on basal
Ip and it did not block the insulin-induced
increase in mean Ip.

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Fig. 8.
Effect of protein kinase C inhibition on response of
Ip to insulin. Mean Ip from
myocytes exposed to drug-free or bisindolylmaleimide I-containing (B-I,
1 µM) pipette solutions in insulin-free superfusates are compared
with mean Ip from myocytes exposed to identical
pipette solutions and insulin-containing superfusate (con, control;
ins, insulin). Numbers in parentheses indicate number of myocytes in
each group. Experiments were obtained from three rabbits in each
group.
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To examine the effect of protein phosphatase inhibition we initially
included 1 µM okadaic acid in pipette solutions. Okadaic acid in this
concentration inhibits both PP-1 and serine/threonine protein
phosphatase 2A (PP-2A). As shown in Fig. 9,
1 µM okadaic acid abolished the insulin-induced increase in mean
Ip. The inactive okadaic acid analog, okadaic acid
methyl ester, had no effect on Ip of control cells
nor on the insulin-induced increase in Ip. Because
PP-1 and PP-2A exhibit a differential sensitivity to okadaic acid we
also examined the effect of including 10 nM okadaic acid in pipette
solutions. This had no effect on the insulin-induced increase in mean
Ip (Fig. 9).

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Fig. 9.
Effect of protein phosphatase 1 and 2A inhibition on response of
Ip to insulin. Mean Ip from
myocytes exposed to drug-free, okadaic acid sodium salt- (OA-N, 1 µM
or 10 nM) or okadaic acid methyl ester-containing (OA-M, 1 µM)
pipette solutions in insulin-free (con) superfusates are compared with
mean Ips from myocytes exposed to identical pipette
solutions and insulin-containing superfusate (ins). Numbers in
parentheses indicate number of myocytes in each group. Experiments were
obtained from three or more rabbits in each group.
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DISCUSSION |
The effect of insulin on the cardiac Na+-K+
pump has been examined previously by measuring ouabain-sensitive
86Rb+-uptake in ventricular slices or isolated
myocytes. Some studies have reported that insulin stimulates
86Rb+ uptake (30, 32), whereas others have
reported that insulin has no effect (4, 12). The membrane potential was
not measured in any of the studies and depolarization induced by the
tissue or cell isolation procedures or induced by the experimental
solutions used to study 86Rb+ uptake cannot be
ruled out. As illustrated by Fig. 1B, an effect of insulin on
pump function might be difficult to demonstrate in depolarized cells.
Conversely, if insulin does cause an increase in
86Rb+ uptake the increase does not necessarily
indicate that insulin directly stimulates pump function. Insulin can
enhance Na+-influx and cause an increase in Nai
at least in noncardiac cells. The increase in Nai, in turn,
stimulates pump function (27). Such indirect pump stimulation is
difficult to rule out using the 86Rb+ uptake
technique. In our study insulin-induced pump stimulation could be
demonstrated even when the transmembrane electrochemical gradient for
Na+ was directed outward (Figs. 3 and 4). This clearly
indicates that the effect of insulin on the pump is not secondary to a
rise in Nai.
Insulin and voltage dependence of the
Na+-K+
pump.
We are aware of only one previous study that has examined whether
insulin-induced Na+-K+ pump stimulation is
voltage dependent (6). Insulin-induced Na+-K+
pump stimulation was studied in rat renal tubular cells with a
physiological membrane potential and in cells depolarized by exposure
to 20 mM extracellular Rb+ or 3 mM Ba2+. It was
concluded that the effect of insulin was voltage independent. The range
of membrane voltage achieved in the experiments was not measured (6).
However, the resting membrane potential of renal tubular cells is only
approximately
50 to
60 mV and a substantial component of
this is generated by the electrogenic Na+-K+
pump current (22). Interference with ion channel currents by extracellular Rb+ or Ba2+ should cause a shift
in the membrane potential much smaller than the range from
100
to +60 mV used in this study and a voltage-dependent effect of insulin
on pump activity is expected to be difficult to detect.
The voltage dependence of the insulin-induced pump stimulation we
demonstrated has implications for the site of action of insulin on the
pump. Release of Na+ and binding of K+ at
extracellular sites are believed to be the major steps contributing to
the voltage dependence in the Na+-K+ pump cycle
(17, 23). The effect of insulin on the
Ip-Vm relationship in our study
persisted after voltage dependence arising at extracellular pump sites
was expected to have been eliminated (Figs. 3 and 4), indicating that
insulin influences some other voltage-dependent step in the pump cycle.
Studies on isolated Na+-K+ ATPase in
noncellular experimental systems have indicated that binding of
cytosolic Na+ is weakly voltage dependent, a finding
consistent with binding within a shallow access channel (3, 8, 21, 28).
The concentration of Na+ within such a channel should
increase with a shift of Vm in a positive direction
and this increase should stimulate pump activity. It follows that
binding of Na+ to pump sites within the channel contributes
to a positive slope of the
Ip-Vm relationship.
The effect of insulin on the
Ip-Vm relationship was
demonstrated when we used a [Na]pip of 10 mM.
According to an access channel model there is kinetic equivalence
between ligand concentration and membrane voltage (26). The effect of
insulin on the Ip-Vm relationship might therefore be eliminated when the intracellular Na+ concentration is at a level expected to saturate
binding sites at all membrane voltages. In agreement with this
expectation insulin had no effect on the
Ip-Vm relationship when
[Na]pip was increased to 80 mM (Fig. 2).
Definitive support for an effect of insulin on binding of
Na+ within an access channel might in principle be obtained
by studying transient currents arising from charge movements in the
Na+ limb of the Na+-K+ pump cycle
in response to membrane voltage steps. However, transient currents
arising from shallow internal access channels have not been reported
(25), and are probably impossible to record without contamination by
larger currents arising from interaction of Na+ with the
pump in an external channel.
Mechanism for the effect of insulin on Ip.
Insulin has been reported to induce translocation of a latent pool of
Na+-K+ pumps to the cell membrane in some
tissues (see Ref. 15 for example). However, the absence of an effect of
insulin on Ip, measured when
[Na]pip is expected to nearly saturate
intracellular binding sites, in the present study suggests that insulin
does not induce an increase in the membrane
Na+-K+ pump density in cardiac myocytes.
Similar conclusions have been reached from studies on rat skeletal
muscle (2) and renal tubular cells (6). An effect of insulin on
functional properties of preexisting pumps must be invoked to account
for the increase in Ip in our study.
The intracellular messenger pathways linking the insulin receptor to
effector molecules are incompletely understood, in part because
available pharmacological blockers of the pathways are not absolutely
specific (29). We used such blockers to examine whether the
pharmacological profile of the effect of insulin in our study is
similar to that reported in previous studies on cellular responses to
insulin. The effect of insulin on Ip was blocked by
an inhibitor of tyrosine kinase. We are not aware of evidence indicating that the Na+-K+ pump can be
phosphorylated on tyrosine residues, and the effect of tyrosine kinase
inhibition on Ip almost certainly reflects interruption of the upstream part of the messenger cascade only. The
effect of insulin was also blocked by wortmannin. Wortmannin inhibits
PI 3-kinase and mitogen-activated protein kinase (29). Because
insulin-induced activation of mitogen-activated protein kinase has
nuclear effects (29), it is unlikely to be involved in the
short-latency response in our study. This suggests that PI 3-kinase is
involved, a kinase implicated in most cellular responses to insulin.
Bisindolylmaleimide I had no effect on the insulin-induced increase in
Ip. This indicates that classical and novel PKC
isoforms are not involved. However, the atypical isoform PKC
is
relatively insensitive to pharmacological blockers (13), and we cannot
rule out a role of PKC
. Unfortunately this isoform can only be
blocked when PKC inhibitors are used in very high, nonspecific
concentrations. The concentration-dependent effect of okadaic acid
suggests that PP-1 may be involved in mediating the effect of insulin
on Ip. Studies on rat myotubes have suggested that
insulin causes activation of PP-1 and that PP-1 in turn
dephosphorylates Na+-K+ pump
-subunits on
serine/threonine residues (24). PP-1 may induce dephosphorylation of
pump units in cardiac myocytes exposed to insulin in a similar manner.
Our findings are consistent with a functional change in the binding
site believed to be located within the membrane dielectric. However,
this scheme is speculative, and currently available techniques do not
allow the resolution in structure-function relationships required to
obtain definitive support for it.
 |
ACKNOWLEDGEMENTS |
This study was supported by the North Shore Heart Research
Foundation. P. S. Hansen received a Postgraduate Medical Research Scholarship from the National Heart Foundation of Australia, K. A. Buhagiar was supported by National Heart Foundation of Australia Grant
No. G96S4589, and D. F. Gray received a National Health & Medical
Research Council Medical Postgraduate Research Scholarship.
 |
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: H. H. Rasmussen,
Dept. of Cardiology, Royal North Shore Hospital, Pacific Highway, St.
Leonards, NSW 2065, Australia (E-mail: helger{at}mail.med.usyd.edu.au).
Received 3 May 1999; accepted in final form 18 October 1999.
 |
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