Angiotensin regulates the selectivity of the
Na+-K+ pump
for intracellular Na+
Kerrie A.
Buhagiar1,2,
Peter S.
Hansen1,2,
David F.
Gray1,2,
Anastasia S.
Mihailidou1, and
Helge H.
Rasmussen1,2
1 Department of Cardiology,
Royal North Shore Hospital, St. Leonards 2065; and
2 University of Sydney, Sydney,
New South Wales 2006, Australia
 |
ABSTRACT |
Treatment of rabbits with angiotensin-converting enzyme (ACE)
inhibitors increases the apparent affinity of the
Na+-K+
pump for Na+. To explore the
mechanism, we voltage clamped myocytes from control rabbits and rabbits
treated with captopril with patch pipettes containing 10 mM
Na+. When pipette solutions were
K+ free, pump current
(Ip) for
myocytes from captopril-treated rabbits was nearly identical to that
for myocytes from controls. However, treatment caused a significant
increase in Ip
measured with pipettes containing
K+. A similar difference was
observed when myocytes from rabbits treated with the ANG II receptor
antagonist losartan and myocytes from controls were compared.
Treatment-induced differences in Ip were
eliminated by in vitro exposure to ANG II or phorbol 12-myristate 13-acetate or inclusion of the protein kinase C fragment composed of
amino acids 530-558 in pipette solutions. Treatment
with captopril had no effect on the voltage dependence of
Ip. We conclude
that ANG II regulates the pump's selectivity for intracellular
Na+ at sites near the cytoplasmic
surface. Protein kinase C is implicated in the messenger cascade.
cardiac myocytes; sodium; protein kinase C
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INTRODUCTION |
CYTOSOLIC LEVELS of
Na+ in ventricular papillary
muscle isolated from rabbits that have been treated with
angiotensin-converting enzyme-inhibiting drugs (ACE inhibitors) are
lower than levels in papillary muscle from untreated control rabbits
(18). This is due to a treatment-induced enhancement of the activity of
the sarcolemmal
Na+-K+
pump. The enhanced pump activity can be demonstrated as an increase in
electrogenic
Na+-K+
pump current
(Ip) in
ventricular myocytes voltage clamped with patch pipettes containing
Na+ in a concentration
([Na+]pip)
that is near physiological intracellular levels. In contrast, treatment
has no effect on
Ip when
[Na+]pip
is high, indicating that there is no effect on maximal pump rate (18).
These findings are likely to be important both for our understanding of
the mechanism of action of ACE inhibitors and, by inference, for our
understanding of the role of the renin-angiotensin system in
cardiovascular homeostasis (17). In addition, the findings are of
interest because they indicate that the
Na+-K+
pump's apparent affinity for intracellular
Na+ can be modulated in vivo.
Intracellular binding of three Na+
to the pump in each cycle is thought to occur at two negatively charged
sites near the cytoplasmic surface and at an uncharged site located
inside the membrane dielectric. Na+ competes with
K+ for binding at the negatively
charged sites, and because of the location of the sites near the
cytoplasmic surface, this binding is voltage independent. In contrast,
interaction with the uncharged site is highly selective for
Na+ and dependent on membrane
voltage (Vm)
(16, 25, 26, 28).
The increase in the overall apparent affinity of the pump for
intracellular Na+ induced by
treatment with ACE inhibitors could be due to an increase in the
affinity for Na+ relative to the
affinity for K+ at the pump sites
near the cytoplasmic surface. One would expect that such a change would
be dependent on the presence of intracellular K+ and independent of
Vm. The overall
increase in apparent Na+ affinity
could also be due to an increase in the intrinsic affinity for
Na+ at the site inside the
membrane dielectric. Binding of
Na+ at this site is expected to be
voltage dependent. A change in the affinity of the site for binding of
Na+ is therefore expected to cause
a change in the voltage dependence of steady-state pump activity if the
binding can be a rate-limiting step in the pump cycle. It follows that
an analysis of the effect of treatment with ACE inhibitors on the
dependence of pump activity on intracellular
K+ and
Vm may provide
insight into the mechanism for the treatment-induced increase in the
apparent affinity of the pump for
Na+. Such an analysis was
performed in this study.
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MATERIALS AND METHODS |
Treatment protocols.
A group of male New Zealand White rabbits weighing 2.5-3.0 kg were
given captopril in their drinking water for 8 days as described previously (18). Another group given captopril-free water served as
controls. The effects of the treatment protocol on systemic blood
pressure and serum renin activity have been described previously (18).
A third group of rabbits were given losartan at 25 mg/kg body wt once
daily by gavage. After completion of treatment protocols, rabbits were
anesthetized with ketamine (50 mg/kg) and xylazine hydrochloride (20 mg/kg) given intramuscularly, and the heart was excised when deep
anesthesia was achieved.
Measurement of Ip.
Single ventricular myocytes were isolated as described previously (18).
The isolation procedure typically lasted ~2 h. Unless indicated
otherwise, cells were stored at room temperature in Krebs-Henseleit
buffer (KHB) solution (17) until
Ip was measured. Cells were used for experimentation on the day of isolation only, and
Ip was typically
measured within 2.5-8 h after cell isolation. In some experiments
myocytes were preincubated for a period of 45 min at 35°C in KHB
solution containing 10 nM ANG II or for 60 min in KHB solution
containing 160 nM phorbol 12-myristate 13-acetate (PMA) before
Ip was measured.
For incubation with ANG II we used polypropylene test tubes to prevent
the loss of ANG II by adhesion to glass. ANG II was dissolved in water,
and PMA was dissolved in DMSO. The final concentration of DMSO was
0.01%. DMSO in this concentration has no effect on
Ip (14).
Myocytes were suspended in a tissue bath mounted on an inverted
microscope for measurement of
Ip. The bath was
perfused with modified Tyrode solution warmed to 35 ± 0.5°C. The
solution contained (in mM) 140 NaCl, 5.6 KCl, 2.16 CaCl2, 1 MgCl2, 0.44 NaH2PO4, 10 glucose, and 10 HEPES. It was titrated with NaOH to a pH of 7.40 ± 0.01 at 35°C. This solution was used in all experiments until the
whole cell configuration had been established and membrane capacitance
had been measured. In most experiments we then switched to a
superfusate that was identical except that it was nominally Ca2+ free and contained 0.2 mM
CdCl2 and 2 mM
BaCl2. In some experiments this
superfusate was replaced with a
Na+-free solution that contained
(in mM) 14.56 KCl, 140 N-methyl-D-glucamine chloride, 0.44 KH2PO4,
10 HEPES, 1 MgCl2, 10 glucose, 2 BaCl2, and 0.2 CdCl2. The solution was titrated
with HCl to a pH of 7.4 ± 0.01 at 35°C. In experiments designed
to examine the effect on the apparent affinity for extracellular
K+, the superfusate was similar to
the Na+-containing,
Ca2+-free superfusate described
above except the K+ concentration
was varied between 0 and 15 mM. The osmolality of these superfusates
was maintained constant with tetramethylammonium (TMA) chloride.
Myocytes were patch clamped with wide-tipped (4-5 µm) pipettes made
from borosilicate glass as described previously (31). 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). 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 previously (31).
For measurement of
Ip at a fixed
Vm of
40
mV we used pipette filling solutions that contained (in mM) 9 sodium
glutamate, 1 NaH2PO4,
5 HEPES, 2 MgATP, 5 EGTA, and 0-140 KCl. The osmolality was
maintained constant by the addition of 10-150 mM TMA chloride. The
solutions were titrated with TMA hydroxide to a pH of 7.05 ± 0.01 at 35°C. In some experiments we included the protein
kinase C (PKC) fragment composed of amino acids 530-558 (PKCF) in
these solutions to activate PKC. PKCF was dissolved in a 0.05 M acetic acid stock solution for storage at
20°C and added to pipette solutions on the day of experimentation to a final concentration of 4 nM. Addition of the quantity of stock solution required to achieve this
concentration did not alter the pH of pipette solutions.
For determination of the voltage dependence of
Ip we used a
filling solution that contained (in mM) 10 sodium glutamate, 1 KH2PO4,
5 HEPES, 5 EGTA, 2 MgATP, 60 TMA chloride, 20 tetraethylammonium chloride, 70 CsOH, and 50 aspartic acid. The solution was titrated with
HCl to a pH of 7.05 ± 0.01 at 35°C. In experiments designed to
determine the apparent affinity for extracellular
K+, filling solutions contained
(in mM) 80 sodium glutamate, 70 potassium glutamate, 1 KH2PO4,
5 HEPES, 5 EGTA, 2 MgATP, and 10 TMA chloride. The solution
was titrated with KOH to a pH of 7.05 ± 0.01 at 35°C. Patch
pipettes filled with the solutions used in this study had resistances
of 0.8-1.1 M
.
Ip was usually
identified as the shift in holding current induced by superfusion of
100 µM ouabain. We exposed myocytes to ouabain with the same latency
of 10-12 min after the whole cell configuration had been
established in all experiments to eliminate variability, which would
arise if rundown of
Ip were to occur. Ip was identified
as the shift in holding current induced by switching from a
K+-free to a
K+-containing superfusate in one
series of experiments. The shifts induced by ouabain or
K+ were measured by sampling
stable currents with an electronic cursor every 5-10 s five times
before and five times after the onset of exposure, and the mean values
of the samples were used to determine the effect of ouabain or
K+. Unless specified otherwise,
currents were normalized for membrane capacitance. When the voltage
dependence of Ip
was determined, we voltage clamped myocytes at
40 mV and applied
320-ms voltage steps to test
Vm levels in
20-mV increments from
100 to +60 mV. Details of the experimental
protocol used to determine the voltage dependence of
Ip have been
described previously (15).
Reagents and chemicals.
TMA chloride was "purum" grade and purchased from Fluka. All
other chemicals were analytical grade and purchased from BDH. ANG II,
ouabain, and PKCF were purchased from Sigma, and PMA was purchased from
Calbiochem. Captopril was donated by Bristol-Myers Squibb
Pharmaceuticals, and losartan was donated by Merck.
Statistical analysis.
Results are expressed as means ± SE. One-way ANOVA was used for
statistical comparisons. Dunnett's test was used when the same control
group was used in more than one comparison. An empirical equation
describing the pipette K+ concentration
([K+]pip)-Ip
relationship was fitted with nonlinear regression, and parameters were
compared by Student's t-test for
unpaired data. Ip-Vm
relationships were compared by both linear regression and two-way
ANOVA. P < 0.05 is regarded as
significant in all comparisons.
 |
RESULTS |
Ip of right and left
ventricular myocytes.
We have previously found that treatment of rabbits with captopril for 8 days induces an increase in the
Ip of myocytes
isolated from the right ventricle (17, 18). In initial experiments in
the present study, we wished to see if this observation can be extended
to myocytes from the left ventricle. We isolated myocytes separately
from the right and left ventricles of rabbits treated with captopril
and from those of untreated controls. They were voltage clamped at
40 mV with pipettes containing
K+ at a
[K+]pip of
70 mM. The mean
Ip values for
right and left ventricular myocytes isolated from rabbits in the two
groups are summarized in Fig. 1. Treatment
with captopril caused a significant increase in
Ip in both right
and left ventricular myocytes.
Ip and the effect
of treatment were similar for right and left ventricular myocytes. We
therefore used either right or left ventricular myocytes in all
subsequent experiments in this study.

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Fig. 1.
Pump current
(Ip) in left
ventricular (LV) and right ventricular (RV) myocytes. Myocytes were
isolated separately from LVs and RVs of control rabbits and rabbits
treated with captopril. Pipette solutions contained 10 mM
Na+ and 70 mM
K+. Numbers of myocytes used are
in parentheses. Treatment with captopril induced a significant increase
in Ip values for
myocytes from both LVs and RVs.
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Dependence of Ip on
[K+]pip.
To determine if the effect of treatment with captopril on
Ip is dependent
on intracellular K+, we measured
Ip with patch
pipettes containing either 0, 35, 70, or 140 mM
K+. Experiments were performed on
28 myocytes from 6 control rabbits and 41 myocytes from 11 rabbits
treated with captopril. We used different
[K+]pip
values in experiments on myocytes from the same rabbit to reduce
effects that might arise from interrabbit variability. Mean
Ip values
are shown in Fig. 2. Mean
Ip values for
myocytes from control rabbits and rabbits treated with captopril
were similar when
[K+]pip
was 0 mM. In contrast, the mean
Ip was
significantly greater in myocytes from rabbits treated with captopril
than in myocytes from controls when
[K+]pip
was 35, 70, or 140 mM.

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Fig. 2.
Effect of pipette K+ concentration
([K+]pip)
on Ip.
Ip was measured
in myocytes from control rabbits, from rabbits treated with captopril
or losartan, and from rabbits treated with captopril and subsequently
exposed to ANG II in vitro. Median number of myocytes for each of the
data points shown was 7 (range 5-15). Equation 1 was fitted to
[K+]pip-Ip
relationship for myocytes from control rabbits and from rabbits treated
with captopril.
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It appears from Fig. 2 that the inhibitory effect of
[K+]pip
is greater in myocytes from control rabbits than in myocytes from rabbits treated with captopril. To compare the
[K+]pip
values that cause half-maximal inhibition
(K1/2 values), we fitted the
[K+]pip-Ip
relationships for myocytes from controls and from captopril-treated rabbits to an equation of the form
|
(1)
|
where
Imaxp is the
Ip measured at a
[K+]pip
of 0 mM. This equation is empirical and not intended to have mechanistic implications. The derived values for
K1/2 were 55 ± 5 and
84 ± 8 mM for myocytes from control rabbits and myocytes from
captopril-treated rabbits, respectively. The difference was statistically significant. Values for
n were 1.7 ± 0.3 for myocytes from
control and captopril-treated rabbits.
[K+]pip
and effect of ANG II.
Because treatment with captopril inhibits the synthesis of both ANG II
and kinins (33), we examined if treatment of rabbits with the specific
ANG II receptor antagonist losartan had an effect similar to that of
treatment with captopril. We treated five rabbits with losartan.
Because the method of administration differed from the protocol we used
previously (17), we measured blood pressure by direct cannulation of an
ear artery in anesthetized rabbits to ascertain the treatment effect.
Satisfactory recordings were obtained for four rabbits. The mean
systolic blood pressures measured before treatment was started and
immediately before sacrifice were 97.5 ± 4.5 and 83.8 ± 1.9, respectively. The difference was statistically significant.
Administration of placebo capsules not containing losartan had no
effect on Ip
(data not shown). The
Ip values
measured at
[K+]pip
values of 0, 35, 70, and 140 mM have been included in Fig. 2. The mean
Ip of myocytes
from rabbits treated with losartan and the mean
Ip of myocytes
from control rabbits were similar when
[K+]pip
was 0 mM. ANOVA indicated that mean
Ip was
significantly greater in myocytes from rabbits treated with losartan
than in myocytes from controls when
[K+]pip
was 35, 70, or 140 mM. It should be noted that
Ip values for
myocytes from losartan-treated rabbits were similar to
Ip values for
myocytes from rabbits treated with captopril.
The similarity in the effect of treatment with captopril and ANG II
receptor blockade on the
[K+]pip-Ip
relationship suggests that an effect of captopril on ANG II metabolism
rather than on kinin metabolism induces the increase in
Ip. To obtain
independent support for this, we incubated myocytes from
captopril-treated rabbits with 10 nM ANG II at 35°C as described previously (17). During the 45-min incubation period myocytes settled
on the bottoms of the test tubes. The supernatant was then aspirated,
and the cells were resuspended in ANG II-free solution and stored at
room temperature until used for patch-clamp studies. The superfusates
used for measurement of
Ip were also ANG
II free.
We measured Ip
values for 26 myocytes isolated from rabbits treated with captopril and
exposed to ANG II in vitro. The
Ip values
measured at
[K+]pip
values of 0, 35, 70, and 140 mM have been included in Fig. 2. The mean
Ip for myocytes
from the captopril-treated rabbits was significantly lower for myocytes
exposed than for those not exposed to ANG II in vitro when
[K+]pip
was 35, 70, or 140 mM. In contrast, the mean
Ip values for such myocytes were similar when
[K+]pip
was 0 mM. Figure 2 indicates that ANG II induced a reduction in
Ip for myocytes
from the captopril-treated rabbits to a level similar to that measured
in myocytes that were isolated from the untreated controls and not
subsequently exposed to ANG II.
Effect of PKC activation on
Ip.
Exposure of myocytes isolated from captopril-treated rabbits to the PKC
activator PMA has an effect similar to the effect of ANG II (17). To
examine if the effect of PMA is dependent on
[K+]pip,
we incubated myocytes from captopril-treated rabbits with 160 nM PMA
for 60 min at 35°C as described previously (17). After the
incubation we aspirated the supernatant and resuspended the cells in
PMA-free solution at room temperature until they were used for
patch-clamp experiments. The superfusates used for these experiments
were also PMA free.
We measured Ip at
a
[K+]pip
of 0 mM in nine myocytes that had been isolated from captopril-treated
rabbits and subsequently exposed to PMA. The mean
Ip was 0.88 ± 0.08 pA/pF. This was similar to the mean
Ip for myocytes
isolated from captopril-treated rabbits and not subsequently exposed to
PMA (Fig. 2). Thus, as was the case for exposure to ANG II, exposure to
PMA had no effect on Ip when
[K+]pip
was 0 mM. We also measured mean
Ip at a
[K+]pip
of 70 mM. The mean
Ip for seven
myocytes isolated from captopril-treated rabbits and subsequently
exposed to PMA was significantly lower than the mean
Ip for similar
myocytes not exposed to PMA (Fig. 3).

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Fig. 3.
Effect of PKC activation and ANG II on
Ip. Myocytes were
isolated from rabbits treated with captopril (capt) or losartan (los),
and protein kinase C (PKC) was activated by incubation with phorbol
12-myristate 13-acetate (PMA) or by inclusion of PKC fragment composed
of amino acids 530-538 (PKCF) in pipette solutions. A
[K+]pip
of 70 mM was used in all experiments. Number of myocytes in each group
is in parentheses. * Significantly different vs. captopril
treatment alone; ** significantly different vs. losartan
treatment alone. Data in 1st, 2nd, and 5th bars from left, also shown
in Fig. 2, have been included here to facilitate
comparison.
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PMA, although it is a rapid and potent activator of PKC, can also bind
to receptors other than the PKC family of enzymes (32). We therefore
performed a series of experiments in which myocytes from
losartan-treated rabbits were dialyzed with pipette filling solution
containing 4 nM PKCF to selectively activate PKC (19, 30). The mean
Ip for seven
myocytes measured at a
[K+]pip
of 0 mM was 0.96 ± 0.07 pA/pF. This is similar to the mean Ip shown in Fig.
2 for myocytes isolated from losartan-treated rabbits and not
subsequently exposed to PKCF. We also examined the effect of PKCF at a
[K+]pip
of 70 mM. As shown in Fig. 3, the mean
Ip was
significantly lower than the mean
Ip for myocytes
isolated from losartan-treated rabbits and not exposed to PKCF. This
[K+]pip
dependence of the effect of PKCF on
Ip is similar to
the [K+]pip
dependence we observed when we exposed myocytes isolated from rabbits
treated with captopril to ANG II and to PMA.
Exposure to PMA and PKCF had an effect on
Ip similar to the
effect of ANG II. This suggests that the effect of ANG II on
Ip is mediated
via PKC. We performed additional experiments to obtain support for
this. Myocytes were isolated from rabbits treated with captopril and
then exposed to both ANG II and PKCF. We used a
[K+]pip
of 70 mM in these experiments. The mean
Ip, shown in Fig. 3, was similar to the mean
Ip for myocytes
exposed to ANG II only. The absence of an additive effect of ANG II and
PKCF is consistent with the involvement of the two peptides in the same
messenger pathway.
Effect of captopril treatment on voltage dependence of
Ip.
The
[K+]pip
dependence of the effect of treatment with captopril suggests that the
treatment alters the interaction of
Na+ with pump sites near the
cytoplasmic surface because it is at these sites that
K+ competes with
Na+ for binding. Any change in
pump function at these sites should be independent of
Vm. To examine
the effect of captopril treatment on the pump's voltage dependence we
determined the
Ip-Vm
relationships for myocytes from rabbits treated with captopril and for
myocytes from controls.
Myocytes were patch clamped with pipettes containing a filling solution
designed to eliminate time-dependent currents through ion channels (see
MATERIALS AND METHODS for details).
The voltage-clamp protocol and details of data analysis have been
reported previously (15). To facilitate a comparison of the voltage
dependence of the pump for myocytes from captopril-treated rabbits with
that for myocytes from controls, we normalized
Ip for each
myocyte to its Ip
recorded at 0 mV. Summaries of the normalized
Ip values for six
myocytes from captopril-treated rabbits and seven myocytes from
controls are shown in Fig.
4A. The
relationships were nearly linear and had a positive slope over the
voltage range examined. There was no significant difference between the
slopes of linear regression models fitted to the
Ip-Vm
relationships or between curves compared by using a two-way ANOVA.


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Fig. 4.
A: voltage
(Vm) dependence
of
Na+-K+
pump current. Mean
Ip values for
myocytes isolated from control rabbits ( ) and rabbits treated with
captopril ( ) are shown. The
Ip-Vm
relationships were normalized to the
Ip values
recorded at 0 mV
(Ip%) to
facilitate comparison of their slopes.
B:
Vm dependence of
Ip recorded in
high-K+,
Na+-free superfusates.
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Release of Na+ and binding of
K+ at extracellular sites are
thought to be the most important steps generating the voltage
dependence of the pump (24, 27), whereas binding of
Na+ at intracellular sites only
exhibits a modest voltage dependence (7, 16, 26). Variability between
cells arising from the steps at extracellular pump sites might
therefore make it difficult to detect a change in the voltage
dependence of intracellular binding of
Na+. We performed additional
experiments using superfusates designed to eliminate the voltage
dependence arising at extracellular pump sites.
The patch pipette filling solution and the voltage- clamp protocol were
identical to those used for the experiments illustrated in Fig.
4A. However the superfusate used at
the time Ip was
measured was Na+ free and
contained 15 mM K+ (see
MATERIALS AND METHODS for details).
The normalized
Ip-Vm relationships for seven myocytes from captopril-treated rabbits and for
eight myocytes from controls are summarized in Fig.
4B. The
Ip-Vm
relationships for myocytes from the two groups of rabbits were similar.
We conclude that treatment with captopril does not alter
voltage-dependent binding of Na+
at cytosolic
Na+-K+
pump sites.
Effect of captopril treatment on the pump's apparent affinity for
extracellular K+.
To examine if the apparent affinity of the
Na+-K+
pump for extracellular K+ is
affected by treatment with captopril, we voltage clamped myocytes from
rabbits treated with captopril with patch pipettes containing 80 mM
Na+. This concentration was used
to cause near-maximal pump activation to allow detection of small pump
currents at low extracellular K+
concentrations. After stable holding currents were
recorded at a holding potential of
40 mV, we inactivated the
pump by switching to a K+-free
superfusate. Ip
was then identified as the shift in holding current induced by
reexposure to solutions containing
K+ in concentrations ranging from
0.5 to 15 mM in random order. We have previously established that such
K+-induced shifts in holding
currents are not contaminated by other K+-sensitive currents (15).
Each exposure to K+ was bracketed
by exposure to K+-free
superfusate, and the same nonzero
K+ concentration was used for the
first and the last exposures to detect if rundown of pump current had
occurred. Figure 5 has been included to
illustrate that K+-induced shifts
in membrane currents were stable during long periods of recording.
Figure 5 shows a tracing of the holding current of a myocyte exposed to
K+-free and
K+-containing superfusates over a
period of >1 h. There was no evidence of rundown of the
K+-activated current in this or 11 other experiments in the present study. We also found no evidence for
rundown in 17 experiments in a previous study using a similar protocol
(15).

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Fig. 5.
K+-induced shifts in holding
current (Ih)
during prolonged recording in a myocyte isolated from a rabbit treated
with captopril. K+-induced shifts
in Ih increased
progressively with each increase in concentration
([K]o) over a period
of ~1 h. The shift, measured with an electronic cursor, induced by
the first exposure to 0.5 mM K+
(52 pA) was similar to shift induced by
K+ in same concentration at
completion of experimental protocol (54 pA). Experiment shown was not
included in analysis of apparent
K+ affinity of pump because
different K+ concentrations were
not used in random order.
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Ip at each
K+ concentration was normalized to
the Ip recorded
when superfusates contained 7 mM
K+. The Hill equation was fitted
to the relationship between the K+
concentration in the superfusate and the normalized
Ip to derive K1/2. Details of data analysis
have been reported previously (15). The mean
K1/2 was 2.6 ± 0.2. This is
similar to the value of 2.7 mM we have found previously for myocytes
from control rabbits (15).
 |
DISCUSSION |
Modulation of apparent Na+
affinity.
The increase in the apparent Na+
affinity of the sarcolemmal
Na+-K+
pump induced by treatment with captopril (18) is not a unique mechanism
by which activity of the pump is regulated. Both short-term in vitro
and long-term in vivo interventions have previously been reported to
alter the pump's apparent Na+
affinity. A change in the affinity with in vitro interventions has been
reported for exposure of rat adipocytes and renal cells to insulin
(8-10, 22), epidermal growth factor (10), and the
-adrenergic
agonist oxymetazoline (1, 20) and for osmotically induced swelling or
shrinkage of rabbit cardiac myocytes (31). Changes can also be induced
in vivo by dietary cholesterol supplementation (15).
Although it is well established that the pump's apparent affinity for
Na+ can be regulated, the
mechanism for this regulation is poorly understood. To explore the
mechanism for the captopril-induced changes, we examined the effect of
[K+]pip
and Vm on
Ip at a fixed
[Na+]pip
of 10 mM. There was no effect of treatment with captopril when
Na+ was the only monovalent cation
expected to interact with intracellular pump sites. This suggests that
a change in the intrinsic binding affinity for
Na+ at the selective site within
the membrane dielectric is unlikely. This conclusion is supported by
the absence of an effect of treatment with captopril on the voltage
dependence of Ip
because interaction of Na+ with
the pump at the selective pump site is expected to be voltage dependent
(25, 26).
Effect of intracellular
K+.
The increase in
Ip induced by
treatment with captopril was dependent on
[K+]pip
(Fig. 2) and consistent with a captopril-induced decrease in inhibition
of Ip by
intracellular K+. The
K1/2 values for
Na+-K+-ATPase-rich
membrane fragments (29) and
Na+-K+-ATPase
reconstituted into liposomes (6) have been reported to be ~10-20
and 40 mM, respectively. The inhibitory effect of K+ at cytoplasmic pump sites is
highly dependent on experimental conditions (28), and meaningful
comparisons of values for K1/2 between studies are difficult.
The ability of K+ to act as a
competitive inhibitor of Na+
activation of the pump is reflected by the ratio of affinities for K+ and
Na+ at intracellular sites (29).
We used a fixed
[Na+]pip
of 10 mM, and the absence of an effect of treatment with captopril or
losartan when pipette solutions were
K+ free suggests that treatment
alters the affinity for K+ rather
than for Na+. However, a definite
distinction between the effects of treatment on the apparent affinities
at intracellular pump sites for
Na+ and
K+ based on a separate kinetic
analysis for the interaction of the two ligands with intracellular pump
sites cannot be made from the data.
The competition for Na+ and
K+ exhibited by
1-,
2-, and
3-isoforms of
Na+-K+
ATPase obtained from different sources has been examined in a previous
study (29). Identical experimental techniques were used to
measure ATPase activity. The apparent affinity of
K+ as a competitive inhibitor of
Na+ binding was tissue rather than
isoform specific. It was concluded that the primary structure of the
-isoform is not the sole determinant of intracellular cation
selectivity, and it was speculated that a tissue-specific pump
modulator regulates the inhibition of the pump by
K+ at intracellular sites (29).
The present study indicates that the inhibition within the same organ
can be modulated by pharmacological intervention. An effect of
treatment on a tissue-specific pump modulator might account for this.
PKC and the
Na+-K+
pump.
Isolated, purified
Na+-K+-ATPase
can be a substrate for PKC in vitro (2, 3, 5, 11, 21), and in situ
pumps can be phosphorylated when PKC is activated by exposing a variety
of intact cells to phorbol esters (5, 12, 13, 23). The functional significance of PKC-mediated pump phosphorylation has not been firmly
established. Stimulation, inhibition, and a complete absence of any
effect have been reported (see Refs. 4 and 12 for review). We found
that the exposure of myocytes from rabbits treated with captopril to
PMA had no effect on
Ip when patch
pipette solutions were K+ free. In
contrast, a decrease in
Ip was induced
when pipettes contained 70 mM K+
(Fig. 3). These findings suggest that exposure of myocytes to PMA
reduced the selectivity for Na+
relative to K+ at intracellular
pump sites, a change that is expected to reduce the
apparent affinity of the pump for
Na+. Such
K+ dependence of the effect of a
phorbol ester on the pump has not been reported previously and has not
been taken into account in previous studies. This is likely to have
contributed to the controversy regarding the effects of phorbol
ester-induced phosphorylation of the
Na+-K+ pump.
Phorbol esters are convenient to use for activating PKC in intact cells
because they are membrane permeable. We used one of these, PMA, in the
present study because an extensive literature documenting the effect of
phorbol esters on the
Na+-K+
pump exists. However, it is widely recognized that they are not specific activators of PKC. The patch-clamp technique allowed the
access of PKCF to the intracellular compartment in our study. This
peptide is a highly specific activator of the PKC family of kinases
(19), and the feasibility of the effective dialysis of it into
patch-clamped myocytes has been demonstrated previously (30). The
effect of PKCF was similar to that of PMA. This supports the
conclusion, reached by previous studies using phorbol esters, that PKC
can regulate the
Na+-K+
pump in intact cells.
We have previously reported that an ANG II-induced decrease in
Ip of myocytes
from rabbits treated with captopril is abolished by inhibitors of PKC
(17). Available inhibitors of PKC are not absolutely specific. However,
the similar, nonadditive effects of ANG II and PKCF on
Ip in the present
study (Fig. 3) provide additional support for the involvement of PKC in
the regulation of the
Na+-K+
pump by ANG II. When one considers the evidence available from our
studies and from studies by other groups, it is reasonable to think
that an ANG II-induced protein kinase-dependent phosphorylation of the
pump regulates competitive inhibition by
K+ of
Na+ binding to the pump at the
sites near the cytoplasmic surface. Such a specific functional effect
of a hormone-induced phosphorylation on the pump has not been
implicated previously. Definitive proof for this scheme would require
direct demonstration of changes in the phosphorylation of cytoplasmic
Na+ binding sites accompanying the
functional changes we demonstrated. Such a degree of resolution in the
analysis of the pump's structure-function relationship is not possible
at present.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Heart Foundation of Australia
Grant G 96S 4589 and by 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: H. H. Rasmussen,
Dept. of Cardiology, Royal North Shore Hospital, Pacific
Highway, St. Leonards, New South Wales 2065, Australia
(E-mail: helger{at}mail.med.usyd.edu.au).
Received 24 September 1998; accepted in final form 1 June 1999.
 |
REFERENCES |
1.
Aperia, A.,
F. Ibarra,
L.-B. Svensson,
C. Klee,
and
P. Greengard.
Calcineurin mediates
-adrenergic stimulation of Na+,K+-ATPase activity in renal tubule cells.
Proc. Natl. Acad. Sci. USA
89:
7394-7397,
1992[Abstract].
2.
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].
3.
Bertorello, A. M.,
A. Aperia,
S. I. Walaas,
A. C. Nairn,
and
P. Greengard.
Phosphorylation of the catalytic subunit of Na+,K+-ATPase inhibits the activity of the enzyme.
Proc. Natl. Acad. Sci. USA
88:
11359-11362,
1991[Abstract].
4.
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].
5.
Chibalin, A. V.,
L. A. Vasilets,
H. Hennekes,
D. Pralong,
and
K. Geering.
Phosphorylation of Na,K-ATPase
-subunits in microsomes and in homogenates of Xenopus oocytes resulting from the stimulation of protein kinase A and protein kinase C.
J. Biol. Chem.
267:
22378-22384,
1992[Abstract/Free Full Text].
6.
Cornelius, F.
Cis-allosteric effects of cytoplasmic Na+/K+ discrimination at varying pH. Low-affinity multisite inhibition of cytoplasmic K+ in reconstituted Na+/K+-ATPase engaged in uncoupled Na+-efflux.
Biochim. Biophys. Acta
1108:
190-200,
1992[Medline].
7.
Cornelius, F.
Hydrophobic ion interaction on Na+ activation and dephosphorylation of reconstituted Na+,K+-ATPase.
Biochim. Biophys. Acta
1235:
183-196,
1995[Medline].
8.
Féraille, E.,
M. L. Carranza,
B. Buffin-Meyer,
M. Rousselot,
A. Doucet,
and
H. Favre.
Protein kinase C-dependent stimulation of Na+-K+-ATPase in rat proximal convoluted tubule.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1277-C1283,
1995[Abstract/Free Full Text].
9.
Féraille, E.,
M. L. Carranza,
M. Rousselot,
and
H. Favre.
Insulin enhances sodium sensitivity of Na-K-ATPase in isolated rat proximal convoluted tubule.
Am. J. Physiol.
267 (Renal Fluid Electrolyte Physiol. 36):
F55-F62,
1994[Abstract/Free Full Text].
10.
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].
11.
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].
12.
Feschenko, M. S.,
and
K. J. Sweadner.
Phosphorylation of Na,K-ATPase by protein kinase C at Ser18 occurs in intact cells but does not result in direct inhibition of ATP hydrolysis.
J. Biol. Chem.
272:
17726-17733,
1997[Abstract/Free Full Text].
13.
Fisone, G.,
G. L. Snyder,
J. Fryckstedt,
M. J. Caplan,
A. Aperia,
and
P. Greengard.
Na+,K+-ATPase in the choroid plexus.
J. Biol. Chem.
270:
2427-2430,
1995[Abstract/Free Full Text].
14.
Gadsby, D. C.,
and
M. Nakao.
Steady-state current-voltage relationship of the Na/K pump in guinea pig ventricular myocytes.
J. Gen. Physiol.
94:
511-537,
1989[Abstract].
15.
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].
16.
Heyse, S.,
I. Wuddel,
H.-J. Apell,
and
W. Stürmer.
Partial reactions of the Na,K-ATPase: determination of rate constants.
J. Gen. Physiol.
104:
197-240,
1994[Abstract].
17.
Hool, L. C.,
D. F. Gray,
B. G. Robinson,
and
H. H. Rasmussen.
Angiotensin-converting enzyme inhibitors regulate the Na+-K+ pump via effects on angiotensin metabolism.
Am. J. Physiol.
271 (Cell Physiol. 40):
C172-C180,
1996[Abstract/Free Full Text].
18.
Hool, L. C.,
D. W. Whalley,
M. M. Doohan,
and
H. H. Rasmussen.
Angiotensin-converting enzyme inhibition, intracellular Na+, and Na+-K+ pumping in cardiac myocytes.
Am. J. Physiol.
268 (Cell Physiol. 37):
C366-C375,
1995[Abstract/Free Full Text].
19.
House, C.,
P. J. Robinson,
and
B. E. Kemp.
A synthetic peptide analog of the putative substrate-binding motif activates protein kinase C.
FEBS Lett.
249:
243-247,
1989[Medline].
20.
Ibarra, F.,
A. Aperia,
L.-B. Svensson,
A.-C. Eklöf,
and
P. Greengard.
Bidirectional regulation of Na+,K+-ATPase activity by dopamine and an
-adrenergic agonist.
Proc. Natl. Acad. Sci. USA
90:
21-24,
1993[Abstract].
21.
Logvinenko, N. S.,
I. Dulubova,
N. Fedosova,
S. H. Larsson,
A. C. Nairn,
M. Esmann,
P. Greengard,
and
A. Aperia.
Phosphorylation by protein kinase C of serine-23 of the
-1 subunit of rat Na+,K+-ATPase affects its conformational equilibrium.
Proc. Natl. Acad. Sci. USA
93:
9132-9137,
1996[Abstract/Free Full Text].
22.
Lytton, J.
Insulin affects the sodium affinity of the rat adipocyte (Na+,K+)-ATPase.
J. Biol. Chem.
260:
10075-10080,
1985[Abstract/Free Full Text].
23.
Middleton, J. P.,
W. A. Khan,
G. Collinsworth,
Y. A. Hannun,
and
R. M. Medford.
Heterogeneity of protein kinase C-mediated rapid regulation of Na/K-ATPase in kidney epithelial cells.
J. Biol. Chem.
268:
15958-15964,
1993[Abstract/Free Full Text].
24.
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].
25.
Or, E.,
P. David,
A. Shainskaya,
D. M. Tal,
and
S. J. D. Karlish.
Effects of competitive sodium-like antagonists on Na,K-ATPase suggest that cation occlusion from the cytoplasmic surface occurs in two steps.
J. Biol. Chem.
268:
16929-16937,
1993[Abstract/Free Full Text].
26.
Or, E.,
R. Goldshleger,
and
S. J. D. Karlish.
An effect of voltage on binding of Na+ at the cytoplasmic surface of the Na+-K+ pump.
J. Biol. Chem.
271:
2470-2477,
1996[Abstract/Free Full Text].
27.
Sagar, A.,
and
R. F. Rakowski.
Access channel model for the voltage dependence of the forward-running Na+/K+ pump.
J. Gen. Physiol.
103:
869-894,
1994[Abstract].
28.
Schulz, S.,
and
H.-J. Apell.
Investigation of ion binding to the cytoplasmic binding sites of the Na,K-pump.
Eur. Biophys. J.
23:
413-421,
1995[Medline].
29.
Therien, A. G.,
N. B. Nestor,
W. J. Ball,
and
R. Blostein.
Tissue-specific versus isoform-specific differences in cation activation kinetics of the Na,K-ATPase.
J. Biol. Chem.
271:
7104-7112,
1996[Abstract/Free Full Text].
30.
Watson, C. L.,
and
M. R. Gold.
Modulation of Na+ current inactivation by stimulation of protein kinase C in cardiac cells.
Circ. Res.
81:
380-386,
1997[Abstract/Free Full Text].
31.
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].
32.
Wilkinson, S. E.,
and
T. J. Hallam.
Protein kinase C: is its pivotal role in cellular activation overstated?
Trends Pharmacol. Sci.
15:
53-57,
1994[Medline].
33.
Zusman, R. M.
Eicosanoids: prostaglandins, thromboxane, and prostacyclin.
In: The Heart and Cardiovascular System, edited by H. A. Fozzard,
R. B. Jennings,
E. Haber,
A. M. Katz,
and H. E. Morgan. New York: Raven, 1991, p. 1797-1815.
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